38 - pc_1_1_Jul_Sep_2011

Newly added Journal from Phcog.Net

Pharmacognosy Communications An Official Publication of Pharmacognosy Network Worldwide [Phcog.Net] www.phcogcommn.org | www.phcog.net Editor-in-Chief Dr Ian Cock Biomolecular and Physical Sciences Griffith University, Nathan campus, 170 Kessels Rd, Nathan, Queensland 4111 Australia Editorial Board Members Dr. William N. Setzer, Professor and Chair Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35899, USA

Dr. Michał Tomczyk Medical University of Białystok, Faculty of Pharmacy, Department of Pharmacognosy, ul. Mickiewicza 2a, 15-089 Białystok, Poland

Dr. Khuraman MUSTAFAYEVA Pharmaceutical faculty Azerbaijan Medical University

Prof. Ameenah Gurib-Fakim, CEPHYR Ltd (Centre for Phytotherapy and Research) 7th Floor, Cyber Tower 2 Ebene, Mauritius

Dr David Ruebhart HydroTox Services Melbourne Australia Dr. Omayma A. El Dahshan, Ph D Pharmacognosy Dept., Faculty of pharmacy, Ain shams University, Cairo, Egypt

Dr. Philip G. Kerr PhD School of Biomedical Sciences Charles Sturt University Wagga Wagga NSW 2678 Australia Prof. Dr. Rimantas Venskutonis Department of Food Technology Kaunas University of Technology Radvilenu pl. 19, Kaunas LT-50254, Lithuania Editor - Publications Dr. Mueen Ahmed KK

Aim and Scope Phcog Commn. is aimed at a broad readership, publishing articles on all aspects of pharmacognosy, and related fields. The journal aims to increase understanding of pharmacognosy as well as to direct and foster further research through the dissemination of scientific information by the publication of manuscripts. Subscription Rates for the year 2012 (4 issues) Rs. 2000 1000/year (National) | USD : 350 200 USD (International) DD/Cheque should be in favour of “Phcog.Net” payable at Bangalore, INDIA All correspondence regarding subscription should be addressed to the following address: Phcog.Net, 1713, 41 A Cross, 18 Main, Jayanagar 4 T blk, Bangalore 560041 INDIA. Email: [email protected] This issue is being sent as complimentary copy for library recommendation.

Pharmacognosy Communications

www.phcogcommn.org

Volume 1 | Issue 1 | Jul-Sep 2011

Contents Editorial

Pharmacognosy Communications: The Scope of Pharmacognosy I.E. Cock

1

Invited Review

Plant Drugs Used to Combat Menace of Anxiety Disorders Reecha Madaan, Suresh Kumar, Gundeep Bansal, Anupam Sharma

4

Review Article

Problems of Reproducibility and Efficacy of Bioassays Using Crude Extracts, with Reference to Aloe vera I.E. Cock

52

Research Article

Cassane-type diterpenoids from the genus Caesalpinia R. A. Dickson, T. C. Fleischer, P. J. Houghton

63

Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves Dineshkumar B, Analava Mitra, Manjunatha M

78

Research Letter

Antimicrobial and anti-inflammatory activities of the leaves of Clerodendrum splendens leaves Fleischer, TC, Mensah, AY, Oppong, AB, Mensah, MLK, Dickson, RA, Annan, K

85

Chemical Examination and Hair Growth studies on the Rhizomes of Hedychium spicatum Buch.-ham G. Venkateswara Rao, T. Mukhopadhyay, M. S. L. Madhavi, S. Lavakumar

90

World Wide Web

Inside Pharmacognosy: A Blog [Pharmanocognosy.in]

94

Medicinal Plant Images

Eucalyptus ficifolia and Chondrodendron tomentosum

95

Department Profile

Biomolecular and Physical Sciences, Griffith University, Australia.

96

Upcoming Events

99

Instructions



100

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Pharmacognosy Communications

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Volume 1 | Issue 1 | Jul-Sep 2011

Editorial Pharmacognosy Communications: The Scope of Pharmacognosy I. E. Cocka,b* a b

Biomolecular and Physical Sciences, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia. Environmental Futures Centre, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia

Pharmacognosy is the branch of pharmacology that studies drugs in their crude and/or natural states.[1] In general, when we describe pharmacognosy, we are usually referring to plant based medicinal systems. However, it is important to note that medicinal preparations may also be derived from animal sources as well as from fungi and microorganisms. Indeed, the discovery of the fungal antibiotic agent penicillin (from Penicillinum spp.)‌[2] is one of the most important medicinal findings to date. Many other useful medicinal products are also derived from fungi including the immunosuppressant mycophenolic acid (also from Penicillinum spp.)[3] and purgative anthraquinone emodin (from Penicillium islandicum).[4] Also, numerous hallucinogenic substances (eg. psilocin and psilocybin) are produced by Psilocybe spp. (family Tricholometaceae) of fungi.[5] Similarly, numerous medicinal agents are produced by bacteria, especially further antibiotic agents. Very early studies demonstrated the antibiotic potential of bacteria towards other bacterial species. In 1887 it was accidently discovered that prior injection of Streptococcus erysipelatis protected guinea pigs from developing cholera when injected with Vibrio cholera.[6] Furthermore, it was also shown that previous injection of either Streptococcus erysipelatis or Pseudomonas aeruginosa also prevented the development of anthrax in experimental animals injected with Bacillus anthracis[6] and that pre-injection of sterilised cultures of the protective bacteria have the same protective effect as live bacteria.[7] This discovery stimulated further studies into the antibiotic activity of bacteria, resulting in the discovery of streptomycin, chloramphenicol, chlortetracycline, tetracycline, erythromycin, neomycin and numerous other antibiotics, especially from Streptomyces spp. (family Streptomycetaceae). Other bacteria, particularly Bacillus spp., are noted for their production of antibiotic polypeptides such as actinomycin,[8] bacitracin,[9] tyrothrycin[10] and polymixin.[10] These antibiotic polypeptides were initially not widely used as they also display strong cytotoxic *Correspondence: Tel.: +61 7 37357637; fax: +61 7 37355282 E-mail: [email protected] (I. E. Cock). DOI: 10.5530/pc.2011.1.1

properties. More recently, there is renewed interest in their use due to their antitumor potential. Indeed, the bacterial antibiotic polypeptides doxorubicin, daunorubicin and actinomycin D are now routinely used in the treatment of a variety of cancers.[11,12] Although the number of animal derived pharmacognostical agents is small when compared to fungi, bacteria and plants, there has recently been an increase in interest in marine creatures as a source of new drugs. Marine invertebrates in particular, account for much of the recent publications describing animal pharmacognosy. Some species of sponges have been found to have antibacterial, antifungal, antimalarial, cytotoxic and anticancer bioactivities.[13] Furthermore, sponges produce interesting metabolites including bromophenols, cyclic peroxides, peroxyketals and modified sesquiterpenes which warrant further investigation.‌[13] The soft coral Sarcophyton glaucum produces the diterpenoids sarcophytol A and sarcophytol A, which have tumour inhibiting bioactivity.[14] Whilst marine animals are receiving much recent interest, there are also many examples of pharmacognostical agents derived from terrestrial animals. For examples, bees (Apis mellifica) provide us with multiple useful medicinal properties. The antimicrobial activity of honey produced by bees feeding on some plant species is known to be exceptionally good. Manuka honey (made by bees feeding on the Eastern Australian/New Zealand plant Leptospermum scoparium) is an especially good antimicrobial agent.‌[15] Additionally, beeswax and royal jelly are also reported to have therapeutic properties.[16] Toad skins contain cardioactive agents and were used to treat oedema prior to the development of more effective agents.[17] Pharmacognostic agents produced by vertebrates include lanolin from wool, gelatine and musk. In my own region of the world (Australia) there is also much interest in oils obtained from emu for its many therapeutic properties.‌[18] Inorganic chemicals may also have important medicinal properties. Silver is particularly well known for its antibacterial activity[19] and  has been used since the times of ancient Greece. Silver nanoparticles have also been shown to have a potent cytoprotective bioactivity towards HIV infected cells.[20] Gold thiolates have been

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1

Cock: The Scope of Pharmacognosy

used in the treatment of rheumatoid arthritis and as anti-tumour agents (as reviewed in Parish and Cottrill).[21] A variety of copper and iron complexes demonstrate potent cytotoxic activities against human cancer cells.[22] Recent studies have highlighted the importance of selenium in blocking the production of reactive oxygen species (ROS) and thus blocking oxidative stress and its associated disease states and medical conditions.‌[23] A variety of other inorganic molecules and ions also have medicinal promise, possibly also through their maintenance of cellular redox state. Despite the importance of pharmacognostic agents from fungi, microorganisms and animals, plants provide us with the greatest variety of medicinal agents and arguably hold the most promise for future drug discovery. Asian medicinal botany in particular has been especially well documented. Traditional Chinese Medicinal (TCM) systems and Indian Ayuverda are widely practiced with approximately 85% of Indians regularly using crude plant formulations for the treatment of various diseases and ailments.[24] Similarly, African and Middle Eastern medicinal ethnobotanies are also widely practiced well documented. Even allopathic/Western medicine practiced in developed countries owes much to our understanding of plant based remedies. Indeed, it has been estimated that approximately 25% of all prescription drugs currently in use are originally derived from plants.[26,27] Furthermore, approximately 75% of new anticancer drugs marketed between 1981 and 2006 are derived from plant compounds.[26] Recently, there has been an increase in interest in pharmacognosy and natural therapies due to the perception that natural therapeutics offer a safer alternative than synthetic formulations due to their organic origin. This is reflected in the dramatic increase in publications in pharmacognosy journals over the period 2005-2010.[28] It is evident that a further publication outlet is required to accommodate this expanding field. Pharmacognosy Communications is a new journal published by Pharmacognosy Network Worldwide [www.phcog.net]. We aim to publish high quality original research articles, methods, techniques and evaluation reports, critical reviews, short communications, commentaries and editorials of all aspects of pharmacognosy research. The journal is aimed at a broad readership, publishing articles on all aspects of pharmacognosy, and related fields. The journal aims to increase understanding of pharmacognosy as well as to direct and foster further research through the dissemination of scientific information by the publication of manuscripts. The submission of original contributions in all areas of pharmacognosy are welcomed. The journal aims to cater the latest outstanding developments in the field of pharmacognosy and natural products and drug design covering but not limited to the following topics: • Pharmacognosy and pharmacognistic investigations • Research based ethnopharmacological evaluations • Biological evaluation of crude extracts, essential oils and pure isolates 2

• Natural product discovery and evaluation • Mechanistic studies • Method and technique development and evaluation • Isolation, identification and structural elucidation of natural products • Synthesis and transformation studies We look forward to receiving your valuable pharmacognosy communications.

References 1. 2.

3. 4.

5.

6.

7.

8.

9. 10. 11.

12. 13. 14.

15.

16. 17. 18.

19.

20.

21. 22.

The American Heritage Medical Dictionary, 2007, Houghton Mifflin Company, USA. Fleming A, 1928, On the antibacterial action of cultures of a Penicillium with special reference to their use in the isolation of B. Influenza. British Journal of Experimental Pathology, 10, 216-226. Florey HW, Gilliver K, Jennings MA, Sanders AG, 1946, Mycophenolic acid, an antibiotic from Penicillium brevi-campactum Dierckx. Lancet, 1, 46-49. Ghosh AC, Manmade A, Demain AL, 1977, Toxins from Penicillium islandicum Sopp. In Mycotoxins in Human and Animal Health, Edited by Rodricks JV, Hesseltine CW, Mehlman MA, Pathotox, Chicago, USA, 625-638. Hofmann A, Heim R, Brack A, Kobel H, 1958, Psilocybin ein psychotroper Wirkstoff aus dem moscikanischen Rauschpilz Psilocybe mexicana Heim. Experientia, 14, 107. Bouchard C, 1889, Influence qu’exerce sur la maladie charbonneuse l’inoculation du bacilli pyocyanique, Comptes Rendus de l’Académie des Sciences, 108, 713‑714. Woodhead GS, Wood C, 1889, De l’action antidotique exercée par les liquids pyocyaniques sur le cours de la maladie charbonneuse. Comptes Rendus de l’Académie des Sciences, 109, 985-988. Waksman SA, Woodruff HB, 1940, Bacteriostatic and bactericidal substances produced by soil actinomycetes. Proceedings of the Society for Experimental Biology and Medicine, 45, 609-614. Johnson BA, Anker H, Meleney FL, 1945, Bacitracin: a new antibiotic produced by a member of the B. Subtilise group. Science, 102, 376-377. Dubos RJ, Hotchkiss RD, 1941, The production of bactericidal substances by aerobic sporulating Bacilli. Journal of Experimental Medicine, 73, 5, 629-640. Lasek W, Giermasz A, Kuc K, Wańkowicz A, Feleszko W, Golab J, Zagozdzon R, Stoklosa T, Jakobisiak M, 1996, Potential of the anti-tumor effect of actinomycin D by tumor necrosis factor α in mice: Correlation between in vitro and in vivo results. International Journal of Cancer, 66, 374-379. Weiss RB, 1992, The anthracyclines: will we ever find a better doxorubicin? Seminars in Oncology, 19, 6, 670-686. Fusetani N, Matsunaga S, 1993, Bioactive sponge peptides. Chemistry Reviews, 93, 1793-1806. Wei H, Frenkel K, 1992, Suppression of tumor promoter-induced oxidative events and DNA damage in vivo by sarcophytol A: A possible mechanism of antipromotion. Cancer Research, 52, 2298-2303. Brophy JJ, Goldsack RJ, Bean AR, Forster PI, Lepschi BJ, 1991, Leaf essential oils of the genus Leptospermun (Mytaceae) in Eastern Australia. Part 5, Leptospermum continentale and its allies. Flavour and Fragrance Journal, 14, 98-104. Fujii A, 1995, Pharmacological effect of royal jelly. Honeybee Science, 16, 97-104. Chen KK, Kovariková A, 1967, Pharmacology and toxicology of toad venom. Journal of Pharmaceutical Sciences, 56, 12, 1535-1541. Whitehouse MW, Turner Ag, Davis CKC, Roberts MS, 1998, Emu oil(s): A source of non-toxic transdermal anti-inflammatory agents in Aboriginal medicine, Inflammopharmacology, 6, 1-8. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO, 2000, A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus, Journal of Biomedical Materials Research, 52, 662-668. Sun RWY, Chen R, Chung NPY, Ho CM, Lin CLS, Che CM, 2005, Silver nanoparticles fabricated in Hepes buffer exhibit cytoprotective activities towards HIV-1 infected cells. Chemistry Communications, 40, 5059-5061. Parish RV, Cottrill SM, 1987, Medicinal gold compounds, Gold Bulletin, 20, 3-12. Easmon J, Pürstinger G, Heinisch G, Roth T, Fiebig HH, Holzer W, Jäger W, Jenny M, Hofmann J, 2001, Synthesis, cytotoxicity, and antitumor activity of copper(II)

Cock: The Scope of Pharmacognosy

23.

24.

and iron(II) complexes of 4N-azabiclo[3.2.2]nonane thiosemicarbazones derived from acyl diazines, Journal of Medicinal Chemistry, 44, 13, 2164-2171. Venardos K, Harrison G, Headrick J, Perkins A, 2004, Effects of dietary selenium on glutathione peroxidise and thioredoxin reductase activity and recovery from cardiac ischemia-reperfusion, Journal of Trace Elements in Medicine and Biology, 18, 1, 81-88. Kamboj VP, 2000, Herbal medicine. Current Science, 78, 35-39.

25. 26.

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Newman DJ, Cragg GM, Snader KM, 2000, The influence of natural products on drug discovery. Natural Product Reports, 17, 215-234. Hostettmann K, Hamburger M, 1993, Search for new lead compounds of natural origin. In Perspectives in Medical Chemistry, Testa B, Kyburz E, Fuhrer W, Giger R (eds), Verlag Helvitica Acta, Basel. Ahmed MKK, 2011, New challenges in the new year for Pharmacog Mag.: 5 years of quality publication. Pharmacognosy Magazine, 7, 25, 1-3.

About journal Pharmacognosy Communications [Phcog Commn.] www.phcogcommn. org is a new journal published by Pharmacognosy Network Worldwide [www.phcog.net]. It is a peer reviewed journal aiming to publish high quality original research articles, methods, techniques and evaluation reports, critical reviews, short communications, commentaries and editorials of all aspects of medicinal plant research. The journal is aimed at a broad readership, publishing articles on all aspects of pharmacognosy, and related fields. The journal aims to increase understanding of pharmacognosy as well as to direct and foster



further research through the dissemination of scientific information by the publication of manuscripts. The submission of original contributions in all areas of pharmacognosy are welcome. The journal aims to cater the latest outstanding developments in the field of pharmacognosy and natural products and drug design covering but not limited to the following topics: • Pharmacognosy and pharmacognistic investigations • Research based ethnopharmacological evaluations • Biological evaluation of crude extracts, essential oils and pure isolates • Natural product discovery and evaluation • Mechanistic studies • Method and technique development and evaluation • Isolation, identification and structural elucidation of natural products • Synthesis and transformation studies

3

Pharmacognosy Communications

www.phcogcommn.org

Volume 1 | Issue 1 | Jul-Sep 2011

Invited Review Plant Drugs Used to Combat Menace of Anxiety Disorders Reecha Madaan*1, Suresh Kumar2, Gundeep Bansal2, Anupam Sharma3 Chitkara College of Pharmacy, Chitkara University, Rajpura, Punjab, India ([email protected]). 2Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala- 147 002, Punjab, India ([email protected]). 3Pharmacognosy Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160 014, India ([email protected]) 1

ABSTRACT: In present era, a sudden holocaust of mental disorders, and recognition of severe side effects and addiction liabilities associated with long term administration of widely prescribed synthetic drugs have aroused the attention of researchers towards natural resources. This review includes 351 references, and emphasizes pharmacological reports on anxiolytic plant products and formulations. Various chemical constituents (with structures), isolated from different plants, responsible for antianxiety activity, and their possible mechanism of actions have been incorporated in this review.The review has been compiled using references from major databases like Chemical Abstracts, Medicinal and Aromatic Plants Abstracts, PubMed, Scirus, Science Direct and Online Journals. It has been concluded that preliminary antianxiety activity studies have been carried out on crude extracts of most of traditonally used and clinically potential plants. Such plantsneed to be explored properly with a view to isolate anxiolytic constituents, and to evaluate their possible mode of actions. KEY WORDS: Antianxiety activity, chemical constituents, mechanism of action, pharmacology

INTRODUCTION Anxiety Disorders: An Overview

Global scenario of persons afflicted by mental disorders is alarming.[1] About 500 million people suffer from neurotic, stress related and somatoform problems, 200 million from mood disorders, 83 million from mental retardation, 30 million from epilepsy, 22 million from dementia, and 16 million from schizophrenia. Anxiety disorders are serious medical illnesses that have affected 1/8th of total population worldwide irrespective of gender, age, religion, nationality and profession.[2] Anxiety Disorders Association of America (ADAA) described anxiety disorders as the most common mental illness in the US, that have affected 19.1 million (13.3%) of the adult (18-54 years) US population.[3] A study commissioned by ADAA on ‘The Economic Burden of Anxiety Disorders’ revealed that anxiety disorders cost the US more than $42 billion a year, almost one-third of the $148 billion total mental health bill for the US. In India, prevalence rate for all mental disorders is 65.4 per 1000 population, and that for anxiety neurosis is 18.5 per 1000 population.[4] The Global Research on Anxiety and Depression (GRAD) network, a consortium of world’s leading psychiatric epidemiologists and clinical researchers, during the 154th annual meeting of ‘American *Correspondence: [email protected]; [email protected] Tel.: +91-9872981142, +91-9815916142 DOI: 10.5530/pc.2011.1.2

4

Psychiatric Association’ (APA) has observed that, “a significant number of world’s population is plagued by chronic and excessive anxiety, also known as generalized anxiety disorder (GAD), which is more serious than those of lung disease, sleep disorders and major depression, and affects more than 5% of the world population”.[5] Following is the categories of anxiety disorders.‌[3,6] 1. Panic disorder (PD) is characterized by panic attacks, sudden feeling of terror that strike repeatedly and without warning. Physical symptoms include chest pain, heart palpitations, sweating, trembling, shortness of breath, dizziness, abdominal discomfort, fear of losing control, fear of dying, tingling sensations, and hot flushes. Panic disorders have affected 6 million (2.7%) adult US population. Women are twice more likely to be afflicted than men. 2. Obsessive–compulsive disorder (OCD) is characterized by uncontrollable obsessions (recurring thoughts or impulses that are intrusive or inappropriate and cause the sufferer anxiety) and compulsions (repetitive behaviours or rituals). It has affected 2.2 million (1%) adult US population. It is equally common among men and women. 3. Post-traumatic stress disorder (PTSD) is characterized by persistent symptoms (nightmares, flashbacks, numbing of emotions, depression, feeling angry and irritable) that occur after experiencing a traumatic event such as war, rape, child abuse and natural disaster. It has affected 7.7 million (3.5%) adult US population. Women are more likely to be afflicted by this disorder.

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Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

4. Social phobia or Social anxiety disorder (SAD) is characterized by an intense fear of situations where embarrassment may occur. Physical symptoms include palpitations, tremors, sweating, diarrhoea, confusion and blushing. It has affected 15 million (6.8%) US adult population. It is equally common among men and women. 5. Specific phobia (SP) is characterized by the excessive fear of an object or a situation, exposure to which causes an anxious response. Specific phobias affect an estimated 19 million (8.7%) US adult population and are twice as common in women as in men. 6. Generalized anxiety disorders (GAD) are characterized by chronic, exaggerated worry about everyday routine life events and activities, lasting at least six months. Physical symptoms include fatigue, trembling, muscle tension, headache or nausea. It has affected an estimated 6.8 million (3.1%) US adult population and is twice as common in women as in men. Though, GAD is the most frequent anxiety disorder, yet only 20% of patients receive proper treatment.[7] GAD results loss of 6 for every 30 work-impairment days. Causes of Anxiety Disorders

Various factors causing anxiety disorders are described below.‌[8-9] Heredity/Genetic factors

Anxiety disorders (PD and OCD) tend to run in families. Studies have shown that if one of the twins has an anxiety disorder, the second is more likely to have an anxiety disorder.

Thought patterns

Negative thoughts can actually create physical symptoms of anxiety. Management of anxiety disorders

Such a horrid emergence of mental disorders has attracted the attention of researchers towards various pharmacotherapeutic approaches for the management of these ‘modernization borne diseases’.[10] Barbiturates, benzodiazepines (BZDs), azaspirones, norepinephrine and serotonin-reuptake inhibitors, monoamine oxidase inhibitors and phenothiazines are some of the commonly used psychotropic drugs.[10] Among these, BZDs are the most widely prescribed synthetic chemical drugs for the treatment of anxiety, insomnia, epilepsy, and stress. Regular use of BZDs causes deterioration of cognitive functioning, addiction, physical dependence and tolerance.[10-12] Abrupt cessation of chronic treatment with BZDs causes the appearance of withdrawal effects comprising re-bound anxiety, restlessness, epilepsy, and motor agitation.[13,14] In the light of adverse effects associated with the synthetic drugs, researchers have been exploring natural resources to find out safer and effective drugs. Investigating plants, based on their use in traditional systems of medicine, is a sound, viable and cost effective strategy to develop new drugs.[15] Plants like Valeriana officinalis, Nardostachys jatamansi, Withania somnifera and Panax ginseng have been used extensively in various traditional systems of therapy because of their adaptogenic and psychotropic properties. Inclusion of these well-established CNS affecting plants in the arsenal of modern therapeutics has revived the faith of researchers in the plants.[16]

Brain chemistry

The symptoms of long term social anxiety disorder can be attributed to the improper chemical balance in the brain. Several neurotransmitters namely serotonin, norepinephrine, gammaamino butyric acid (GABA), which are produced in the brain, directly affect one’s feelings about a given situation. Thus brain, too, appears to play a role in the onset of anxiety disorders because symptoms of anxiety disorders are often relieved by medications that alter the level of chemicals in the brain. Personality

People with low self-esteem and poor coping skills are more prone to anxiety disorders. Conversely, an anxiety disorder that begins in childhood may itself contribute to the development of low self-esteem. Life experiences

Long term exposure to abuse, violence, poverty or stressful experiences (the early death of a parent, bad marital or family relationships, or traumatic experiences) may affect individual’s susceptibility to anxiety disorders. Stress overload/Lifestyle factors

Excessive stress over time, and poor lifestyle habits such as overwork, lack of sleep, poor diet and lack of regular exercise promote anxiety. 

Targets for Treatment of Anxiety

With anxiety, various brain neurotransmitters and hormones levels change immediately. In particular, monoamines, such as norepinephrine, serotonin and dopamine, are involved in mood, stress and other physical homeostasis.[17] Serotonin and norepinephrine mainly regulate stress and negative mood in the mammalian brain, and their dysfunctions cause various mood disorders, such as social anxiety disorder and depression.[18] Dopamine also regulates mood and emotion-related behaviors and has a motivation/reward function and conditional fear responses.[19,20] Various anxiolytics and antidepressants aim at  monoamine neurocircuitry, such as their receptors and transporters.[21] The 5-hydroxytryptamine 1A (5-HT1A) receptor is viewed as a relevant target for the treatment of psychiatric disorders, notably anxiety and depression.[22] 5-HT1A receptors are located at the presynaptic and postsynaptic sites.[23] The somatodendritic autoreceptor, when activated by systemic stimulation, is believed to exert anxiolytic-like effects and to reduce 5-HT release both in the cell body and in the terminal regions of the serotonergic neurons.[24] The other 5-HT1A receptor is localized postsynaptically to the serotonergic neurons in the hippocampus, septum, amygdala, and cortex, where it increases signal transfer, which leads to an inhibition of the firing activity.[25] 5

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

GABA is a major inhibitory transmitter in the central nervous system. The γ-aminobutyric acid type A (GABAA) receptor, the chloride ion channel complex and the central benzodiazepine receptors located on the neuronal membranes within this complex have been suggested to play an important role in the regulation of the stress and anxiety states.[26,27] The benzodiazepine binding site and GABAA receptor are structurally and functionally coupled.‌[28] Benzodiazepines (BZDs) have become the primary pharmacological treatment for generalized anxiety disorder. However, BZDs are often associated with tolerance development and withdrawal symptoms, which pose a risk of relapse upon discontinuation.[29,30]

In brain, Nitric oxide synthase (NOS) has been localized in regions involved with anxiety, such as hypothalamus, amygdala and hippocampus.[36,37] Inhibition of NOS by nonselective or by relatively selective inhibitors of nNOS produced antianxietylike effect. Neurosteroids can rapidly alter the excitability of   central nervous system by modulating neurotransmittergated ion channels such as GABAA and N-methyl-D-aspartate ‌ receptors.[38] Anxiolytic, anticonvulsant and anaesthetic effects of neuroactive steroids are mediated by their capacity to positively modulate GABAA receptor. 5-alpha reductase, the enzyme that converts into 5-alpha-reduced metabolites like the GABAA positive neuroactive steroid 3-alpha-hydroxy-5-alphapregnan-20-one, thus, few drugs exhibits anxiolytic action via an indirect activation of the GABA-ergic system through neuroactive steroids.[39]

Monoamine oxidase (MAO) catalyzes the oxidative deamination of a variety of monoamines such as dopamine, norepinephrine and serotonin. The MAO reaction yields aldehydes and hydrogen peroxide (H2O2), which induces apoptosis.[31] Increased endogenous MAO inhibitory activity (tribulin activity) is associated with conditions associated with stress and anxiety, both in animals and in man.‌[32] Rat brain tribulin activity is significantly augmented by anxiogenic agents like pentylenetetrazole, and this effect can be prevented by anxiolytic agents.[33] Inhibition of MAO and subsequent H2O2 generation effectively prevents depression and various oxidative stresses in the brain.[34] The presence of plant-derived MAO inhibitors suggests that such plant extracts could be useful as potential neuroprotectants in the treatment or prevention of depression.[35]

PLANTS HAVING ANTIANXIETY ACTIVITY Antianxiety activity reports of various plants, and plant constituents and formulations have been presented in tables 1 and 2. Various patented formulations of anxiolytic plant drugs have been depicted in table 3. Various review articles published on anxiolytic plants are shown in table 4.

O O

O

O O

OH

O

(1)

(2)

H

R2

N R1

O R1

R2

(3)

OH

OH

(4)

H

H

O (5)

6

O

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

OH OH

OH HO

HO

O

O

OH

OH O

OH

O

OH

(6)

(7)

NH2

OH

HO

H

H

N O

O

HO

(8)

(9)

CH3 H3C O C

HO CH3

HO

CH2

HOH2C

CH3

O

O

CH2OH O

OH

OH OH

OH O HO

O

O

OH

OH

CH3

O

OHOH

OH

(11)

(10)

CH2OOCCH2CH(CH3)2

CHO (12)



7

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

O

R

R

O

O O

(13), R = β-Gentiobiosyl OH HO

O

OH OH

O OH

(15)

(14)

OH

HO

CH3 H3CO

OCH3

H3CO O

HO (16)



OH (17)



R' CH3O N CH3O

RO (19) (20) (21)

O (18)

R CH3 H H

R’ OH H OH

NH2

RO

N

N

N H 3CO

N

N HO

O

H 3CO R

8

(22)

H

(23)

CH3

OH

HO (24)

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

H 3CO 2C H

H

OH

OH

O

O O R 3O R1

R2

R3

(25)

H

H

H

(26)

H

OH

H

OH

(27)

R 1 C(CH ) 3 3 OH

R2

O

OR 2

R1

O

O

O

OCH3 H 3C

O

OH

(28)

O O

R1

R2

N

H

H

(29)

O

OH

OH

HO HO R

HO HO

HO

CH3

CH2OH O

HO

OH

O

HO

OH

O (32)

O OH

O

CH3O HO

R1

O

R1

R2

(33) β-Glc

H β-Glc

O O

O

OH



O

OH

R2

H

OH

OH

(30), R=CH3 (31), R =CH2OH

(34)

OCH3

O (35)

9

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

OH

H2C

OH O

OH

OH

CH2

(37)

(36) H3CO

N O

H

HO

OH

CH3

OCH3 H

O

HO

H3C

OH

OCH3

N

OCH3

O (39)

(38)

OH OH

HO

O

HO O

OH H OH

OH

HO O

HO H

O

H

H O

H O OH

HO OH (40)

10

H

O O HO

OH

OH

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

HO OH

OH O

O HO HO

OR R (41)

D-glucose

(42)

H OH

HO

O

HO

O

O

OH

O

OH

OH

(43)

(44) OH OH

O

OH

O

CH

CH

COOH

O HO

OH

OH

OH

(45)

(46)

OCH 3 4

R5 R1

11

5

6

9 7

14

R2

3

8

10

O

1

2

O

O

N

12 13

R4

O

R3





(47) (48) (49) (50) (51) (52)

R1 H H H H H H

R2 R3 OCH 2OH OCH 2OH H H H H H H OCH 3 H

R4 H H H H H H

R5 C5-C6 C7-C8 = H H H H

O

H = = =

H H

O O



(53)

11

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

OCH3 R5 R1

6

9

O

7

14

R2

3

8

10

11

4

5

2

1

O

12 13

R4

H

R3 R1



(54) (55) (56) (57) (58) (59) (60)

R2

OCH2O OCH2O OCH2O OCH2O OCH2O OCH2O OCH3O CH3

R3

R4

R5

C5-C6 C7-C8

H H H H H H H

H OCH3 H H OCH3 H H

H H OCH3 H H H H

= = = = = = =

H = = = OCH3

HO

H (61)

H OH

H HO

HOOC

H

OH

OH

O

OH

OH

(63)

(62)

O H O

H

O

O

H O

O O

O

O

O

(64)

(65)

OCH3 HO

COOH O

OH OH

H OH

H

H

OH

O

O H

HO OH

O

(66)

12

O

H

(67)

O

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

O

HO

HO OH

HO

O (68)

(69) OH OCH3

OH

O

HO

H3 C

H3C

O

HO OH

HO

O

OH

O

OH

O

OH OH

OH

(70)

HO H3C

O

(71)

OH OH O

H

OCH3

O HO HO

O

O

O

CH3

O

O

OH

CH3

CH3 OH

O

COOH

(72)

(73)

O O O HN O N

N H

N H

CH3

CH3

(74)



O (75)

13

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

CH3 O

O

CH3 CH3

(76)

(77)

CH3

O

H2C

H

H

CH3 CH3 (78)

(79)

OH

OH

(81)

(80)

N

H

N O

H

H

O OH

(82)

CONCLUSION In present era, a sudden holocaust of mental disorders, and recognition of severe side effects and addiction liabilities associated with long term administration of widely prescribed synthetic drugs have aroused the attention of researchers towards natural resources. Plants like Valeriana officinalis, Nardostachys jatamansi, Withania somnifera and Panax ginseng have been used extensively in various traditional systems of therapy because of  their adaptogenic and psychotropic properties. Inclusion of these well-established CNS affecting plants in the arsenal of 14

O

(83)

modern therapeutics has revived the faith of researchers in the plants. In present review article, amongst 143 plants reported to possess antianxiety activity (Table 1): ( a) only 07 plants have been tested clinically, (b) preliminary antianxiety activity screening on crude extracts has been carried out on 90 plants. Such plants need to be explored with a view to isolate active constituents and their mode of actions,

 (a) 100 mg/kg, p.o. (b) 50 mg/kg, p.o.

100 and 200 mg/kg, p.o. 200 mg/kg, p.o. for seven days

Powder of whole plant

Flavonoidal moiety

(a) Methanol extract (b) Polyphenol fraction

Ethanol extract (95%) of leaves

Fatty acid: trideca7,9,11-trienoic acid(1) isolated from methanol extract of aerial parts

Aqueous extract of stem bark

Aqueous extract of bark

Acorus calamus Linn. (Araceae) Bach/Bacopa monnieri Linn. (Scrophulariaceae) Brahmi

Actaea spicata Linn. (Apiaceae) Baneberry, Grapewort

Adiantum tetraphyllum Humb. & Bonpl. ex Willd. (Adiantaceae) Fourleaf maidenhair

Aethusa cynapium Linn. (Apiaceae) Fool’s Parsley

Albizzia julibrissin Durazz. (Fabaceae) Silktree, Mimosa, Nemunoki

Albizzia lebbeck Benth. (Fabaceae) Siris tree, Albizia

03

04

05

06

07

08

Saponins rich n-butanolic fraction of petroleum ether extract from leaves

2 mg/kg, p.o.

Aqueous extract of flowers

Achillea millefolium Linn. (Compositae) Yarrow, Milfoil

02

25 or 50 mg/ kg, p.o.

20 mg/kg, p.o.

200 mg/kg, p.o.

500 mg TDS for 6 weeks

12 mg/kg, p.o.

50 and 100 mg/kg, orally once daily for 3 days

Ethanol extract of leaves

Abies pindrow Royle (Pinaceae) Talispatra, Silver Fir, Pindrow Fir

01

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Animal/ Human being

EPM

EPM

Male SD rats

Albino Swiss mice

EPM

[1-(3-chlorphenyl)piperazine] induced hypolocomotion test

Male SD rats

Swiss albino mice

OFT, EPM, Acoustic startle response test

EPM

Laca mice

Male Sprague Dawley rats

EPM

Electrophysiological parameters like EEG, ECG

Conflict behaviour

Elevated plus maze (EPM), Open field test (OFT), Elevated zero maze (EZM)

Experimental model/ Assessment of clinical parameters

Laca mice

81 Patients suffering from anxiety disorder

Female Wistar rats

Wistar rats

Table 1: List of various plants reported to possess antianxiety activity.

Inhibition of GABAergic transmission

Interaction with 5-HT1Areceptor

Serotonergic system

—

—

—

—

—

—

—

Mechanism of action

Anxiolytic and nootropic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Improvement in nervousness, restlessness, irritability, poor concentration, sleep and loss of appetite

Anxiolytic

Anxiolytic

Activity

[49]

[48]

[47]

[46]

[45]

[44]

[43]

[42]

[41]

[40]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

15

16 25 and 50 mg/ kg, i.p. 25 and 50 mg/ kg, i.p. 25 and 50 mg/ kg, p.o.

Furanocoumarin – Phellopterin(2) isolated from methanol extract of roots

Riparin III(3) isolated from unripe fruits

Riparin I (4) isolated from unripe fruits

Riparin- III (3) isolated from unripe fruits

Hexane extract of leaves

Palmitone(5) isolated from hexane extract of leaves

Alpinia zerumbet(Pers.) Burtt & RM (Zingiberaceae) Shell flower, Pink porcelain lily

Angelica

Angelica dahurica (Fisch. ex Hoffm.) Benth. (Apiaceae) Dahurian angelica

Aniba riparia (Nees) Mez (Lauraceae) Rosewood

Annona cherimolia Mill. (Annonaceae) Cherimoya, Custard apple

Annona diversifolia Saff. (Annonaceae) Llama, Anona blanca

10

11

12

13

14

15

21 mg/kg, p.o.

Essential oil

0.3, 1, 3, 10 and 30 mg/kg i.p.

6.25, 12.5, 25.0 and 50.0 mg/kg, p.o.

IC50 = 0.36 microM

30.0 mg/kg, p.o.

Essential oil

Inhalation 3.5 mg/L air

1.0,10.0 and 100.0 mg/kg, p.o.

Ethanol extract of aerial parts

Essential oil from leaves

1.56 to 50 mg/ kg, i.p.

Hydro-alcoholic extract (60% ethanol) of leaves

Aloysia polystachya Griseb. (Verbenaceae) Burrito

09

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

Albino mice

Albino mice

Male Swiss mice

Male Swiss mice

Male Swiss mice

In vitro

Male Wistar rats

Male Swiss mice

EPM

Mouse avoidance exploratory behavior, Marble burying test (MBT)

OFT, EPM, HBT

EPM, OFT, HBT

EPM, FST

—

Social interaction in rats (SI), Hole Board Test (HBT)

EPM, LDM

Light/Dark model (LDM) , OFT, EPM

EPM

Swiss albino male mice

Male ICR mice

EPM, Forced Swimming Test (FST)

Experimental model/ Assessment of clinical parameters

Female Sprague Dawley rats

Animal/ Human being

—

GABA/BZD receptor complex

—

—

—

BZD receptors agonist

—

—

—

Other mechanism than BZD-bs modulation at the GABAA receptors

—

Mechanism of action

Anxiolytic

Anxiolytic

Anxiolytic but devoid of sedative activity

Anxiolytic

Anxiolytic, antidepressant

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic without sedative effects

Anxiolytic and antidepressant

Activity

[60]

[59]

[58]

[57]

[56]

[55]

[54]

[53]

[52]

[51]

[50]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 10, 20, 50, 100 and 200 mg/ kg, p.o. 500 mg/kg/ day × 15 days

Fruit juice

Aqueous extract from leaves

Aqueous extract from leaves

Ethyl acetate extract of leaves

Aqueous extract of leaves

Quercetin(7) isolated from methanol extract of aerial parts

Alcoholic extract of peeled roots

L-theanine(8)

Aronia melanocarpa Michx. (Rosaceae) Black chokeberry

Azadirachta indica A. Juss. (Meliaceae) Neem tree

Baphia nitida Lodd. (Fabaceae) African sandalwood, Barwood

Byrsocarpus coccineus Schurn. and Thonn. (Connaraceae) Kimbar mahalba

Calluna vulgaris Linn. (Hull) (Ericaceae) Heather

Calotropis gigantea (L.) Dryand. (Apocynaceae) Giant Milkweed, Crown Flower, Aak

Camellia sinensis (L.) O. Kuntze (Theaceae) Green tea

18

19

20

21

22

23

10 mg/kg, p.o.

250 and 500 mg/kg, p.o.

41µg/mg

200 and 400 mg/kg, p.o.

100-400 mg/ kg, p.o.

5 and 10 ml/ kg, p.o.

Sprague Dawley rats

Albino rats of either sex

—

Albino mice of either sex

Adult albino mice of either sex

Male Charles-Foster albino rats

Wistar rats

Wistar rats

Male BL6/C57J mice

>0.02 mg/kg, p.o.

Kaempferol(6) isolated from hydro-alcoholic extract (70% ethanol) of leaves

17

Male C75 BL/6 mice

30 and 125 mg/kg, p.o.

Ethanol extract of leaves

Apocynum venetum Linn. (Apocynaceae) Dogbane

16

EPM

EPM, Hot plate method, Acetic acid induced writhing, Assessment of locomotor activity, rota rod and PTZinduced convulsions

In vitro

Hexobarbitone induced sleeping time, Y-maze, EPM, HBT

EPM, Y maze

OFT and Morris water maze

EPM, OFT

SI, OFT

EPM

EPM

Increase in dopamine levels but not GABAA receptor interaction

—

Inhibition of MAO-A

—

—

Increase in ascorbic acid level of brain which falls during brain ischemia

—

—

BZD receptor interaction

Involvement of GABAergic system

Anxiolytic

Anxiolytic, anticonvulsant, analgesic and sedative

Anxiolytic

Anxiolytic and sedative

[70]

[69]

[68]

[67]

[66]

[65]

Anxiolytic

Anxiolytic

[64]

[63]

[62]

[61]

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

17

18 795 and 1000 mg/kg, p.o.

10 mg/kg, i.p.

3.2 g/kg/day for 5 days 1 and 1.5 g/kg, i.p.

Essential oil from leaves

Barakol (10)

(a) Aqueous extract of leaves (b) Butanolic fraction of aqueous extract of leaves

Petroleum ether extract of seeds

Oil of seeds

Casimiroa pringlei (S. Watson) Engl. (Rutaceae) Pringle’s Zapote

Cassia siamea Lam. (Fabaceae) Kasod, Siamese cassia

Cecropia glazioui Sneth (Urticaceae) Embauba, Yarumo

Celastrus paniculatus Willd. (Celastraceae) Jyotishmati, Maak kangni

27

28

29

(a) 0.5 and 1.0 g/kg, p.o. (b) 25-100 mg/kg, p.o.

Wistar rats

Albino mice

Male adult Swiss mice

Male wistar rats

Wistar rats

OFT, EPM, Thirsty rat conflict paradigm

Behavioural disinhibition model

EPM

EPM

EPM, OFT, HBT

Spontaneous motor activity, EPM, FST, HBT, MBT

Male and female SpragueDawley rats

40, 80, 160, and 320 mg/ kg, p.o. in mice, or 1.56, 3.12, 6.25,12.5 and 50 mg/kg, i.p. in rats

Hydro-alcoholic (60% ethanol) extract of leaves

26

EPM, OFT

Wistar rats

Casimiroa edulis Llave & Lex. (Rutaceae) White Sapote, Zapote blanco

25

EPM, Vogel conflict test

Male Wistar rats

25 and 35 mg/ kg, i.p.

15, 30 and 60 nmol, intra-BNST bilateral injections

Cannabidiol(9)

EPM, Vogel conflict test

Experimental model/ Assessment of clinical parameters

Male Wistar rats

Animal/ Human being

Aqueous extract of Leaves

15, 30 and 60 nmol, intra-dlPAG (Dorsolateral peri aqueductal gray)

Cannabidiol(9)

Cannabis sativa Linn. (Cannabaceae) Bhang

24

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

Serotonergic mechanism

—

—

—

—

—

—

Facilitates local 5-HT1A receptormediated neurotransmission

Cannabidiol interaction with 5HT1A receptors in dIPAG in brain

Mechanism of action

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and sedative

Anxiolytic, antidepressant and sedative

Anxiolytic

Anxiolytic

Anxiolytic

Activity

[80]

[79]

[78]

[76, 77]

[75]

[74]

[73]

[72]

[71]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 1 g/kg, p.o. 0.5 and 1.0 g/ kg, p.o.

Valepotriate – valtrate(12)

Methanol extract of leaves and pods

50% Ethanol extract from stem barks

Hydro-alcoholic extract (70% ethanol) of leaves

Essential oil from peel (EOP) of leaves

Essential oil from fruits

Essential oil

Methanol extract of roots

Ethyl acetate fraction of ethanol extract of the aerial parts

Essential oil

Centranthus ruber (L.) DC (Valerianaceae) Red valerian

Ceratonia siliqua Linn. (Fabaceae) Carob tree

Cinnamomum cassia Blume. (Lauraceae) Cassia Bark, Chinese cinnamon

Cissus sicyoides Linn. (Vitaceae) Possum grape vine, Princess vine

Citrus aurantium Linn. (Rutaceae) Bitter Orange

Citrus sinesis Linn. (Rutaceae) Sweet Orange, Blood Orange

Clitoria ternatea Linn. (Papilionaceae) Butterfly pea

Convulvulus pluricaulis Choisy. (Convolvulaceae) Shankhpuspi

Copaifera reticulata Ducke (Leguminosae) Brazilian copaiba

32

33

34

35

36

37

38

39

100, 400 and 800 mg/kg, i.p.

100 mg/kg, p.o.

100-400 mg/ kg, p.o.

100, 200 and 400 µl

300, 600 and 1000 mg/kg, i.p.

750 mg/kg, p.o.

Pods - 12.17 ng and Leaves - 18.7 ng diazepam equivalent

5 mg/kg, p.o.

Wistar rats

Sprague-Dawley rats and Swiss albino mice

Male Swiss albino mice and Wistar rats

Wistar male rats

Male Swiss mice

Male Swiss mice

Male and female Swiss albino mice

Male ICR mice

In vitro

Wistar rats

EPM

EPM, OFT and rotarod performance

EPM, LDM

EPM, LDM

LDM, MBT

EPM, OFT

EPM, HBT, MBT, Sodium Pentobarbital-induced sleeping time, PTZ-induced convulsion

EPM

—

Inhibition of orientation reflexes and motor activity

EPM, OFT, SI, locomotor activity, punished drinking, novel cage test

Male Sprague-Dawley (SD) rats

(a) 500 mg/kg, p.o. (b) 3047 mg/ kg, p.o. (c) 111 mg/kg, p.o. (d) 3 mg/kg, p.o.

(a) Marketed formulations (b) Methanol extract (c) Ethyl acetate extract (d) Asiaticoside(11)

31

Significantly attenuated the peak of acoustic startle response amplitude

Double-blind, placebocontrolled study in 20 subjects

12 g/day, p.o.

Powdered drug

Centella asiatica (L.) Urb. (Umbelliferae) Gotu Kola

30

—

—

—

—

—

—

—

Regulation of 5-HT1A and GABA receptor system

BZD receptor interaction

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic, anticonvulsant

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

—

—

Anxiolytic

—

[92]

[91]

[90]

[89]

[88]

[87]

[86]

[85]

[84]

[83]

[82]

[81]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

19

20 LDM

EPM, OFT, Pentobarbital sleeping time, Rotarod test

Wistar rats

Razi male mice

50 mg/kg, i.p.

(a) 56, 80, 320 and 560 mg/ kg, i.p. (b) 50, 200 and 600 mg/ kg, i.p. (c) 0.05, 0.15 and 0.35 ml/ kg, i.p.

20 mg/kg, i.p.

200 mg/kg, i.p.

0.5 and 1.0 g/ kg, i.p.

Crocin(13) isolated from aqueous extract of red dried stigmas

(a) Aqueous extract of stigmas (b) Crocin(13) (c) Safranal (14)

Proanthocyanidin(15) rich fraction isolated from aqueous extract of bark

Methyl eugenol(16) from essential oil

Curcumin(17)

Citral (18) or tea abafado

Essential oil

Hydro-alcoholic extract (70% ethanol) of stems

Hydro-alcoholic extract (50% ethanol) of leaves

Croton celtidifolius Baill. (Euphorbiaceae) Sangue-de-adave

Croton zehntneri Pax & Hoffman (Euphorbiaceae) Canela de Cunha

Curcuma longa Linn. (Zingiberaceae) Curcuma, Turmeric

Cymbopogon citratus (DC.) Stapf (Poaceae) Lemongrass, Ginger grass

Davilla rugosa Poiret (Dilleniaceae) Cipo-Caboclo, Fire vine

Drymaria cordata (L.) Willd. ex Roem. & Schult. (Caryophyllaceae) Tropical chickweed

42

43

44

45

46

47

100 mg/kg, p.o.

15 mg/kg, p.o.

1, 3 and 10 µl/100 g, p.o.

3 mg/kg, i.p.

Swiss albino mice

Male Wistar rats

Swiss male mice

Male albino Swiss mice

Swiss albino mice

Male Wistar rats

Wistar rats

EPM, LDM, OFT, HBT

EPM, OFT

EPM, LDM

OFT, Rota-rod test, Spontaneous motor activity, Barbiturate sleeping-time, Transcorneal electroshock, PTZ-induced convulsions, Punished response test

EPM, OFT, LDM, SI

OFT, SI, EPM, HBT, FST

EPM

EPM

Crocus sativus Linn. (Liliaceae) Saffron, Autumn crocus

Male albino mice

41

100 mg/kg, p.o.

Aqueous extract of seeds

Experimental model/ Assessment of clinical parameters

Coriandrum sativum Linn. (Umbelliferae) Coriander, Dhaniya

Animal/ Human being

40

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

—

—

—

—

Involvement of inducible NOS

—

—

—

—

—

Mechanism of action

Anxiolytic

Anxiolytic

Anxiolytic

Central Nervous depressant

Anxiolytic

Antidepressant and mild anxiolytic

Anxiolytic

Anxiolytic (At lower dose), hypnotic (At higher dose)

Anxiolytic

Anxiolytic

Activity

[102]

[101]

[100]

[99]

[98]

[97]

[96]

[95]

[94]

[93]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 Male TO mice

50 mg/kg, i.p.

Acute (200 mg/kg, p.o.) chronic (50 mg/kg, p.o. for 7 days)

Hydro-ethanol extract (80%) of the plant flowers

(a) Aqueous, hydroalcoholic extracts (b) Hydrolyzed fraction obtained from whole plant

Hydro-alcoholic extract (70% ethanol) from the inflorescence

Water : Alcohol (7:3) extract of inflorescence

Erythrinian alkaloids i.e (+)-α– hydroxyersotrine(19), erythravine(20) and (+)-11-α–hydroxy erythravine(21) isolated from hydro-alcoholic extract of flowers

Eclipta alba Linn. (Asteraceae) Bhringaraj, False daisy

Erythrina mulungu Mart. (Papilionaceae) Mulungu, Corticeira

51

52

Male Swiss Mice

3 and 10 mg/ kg, p.o.

EPM, LDM

ETM

Male Wistar rats

Acute study 200 and 400 mg/kg, p.o. and chronic study for 21 days, 50 and 200 mg/kg, p.o.

Locomotor activity, EPM, HBT, Cold restraint induced gastric ulcer and white blood cell count in the milk induced leukocytosis challenge Elevated T maze (ETM), LDM, Cat odor test

Wistar rats

EPM

EPM

EPM, SI, shock induced social avoidance test, OFT

EPM, Spontaneous motor activity, Ketamine-induced sleep time

Male Wistar rats

(a) 150 and 300 mg/kg, p.o. (b) 30 mg/kg, p.o.

Male NMRI albino mice

5, 10, 30, 62.5, 80 and 125 mg/kg, i.p.

Aqueous extract of flowers

Echium amoenum Fisch. Et Mey. (Boraginaceae) Viper’s bugloss, Red feathers

50

Male Wistar rats

3-7 mg/kg, p.o.

(a) E. purpurea root extract (ethanol 4% v/v; Echinacoside 4%) (b) E. purpurea herb extract (ethanol 60% m/m; total phenols 4%) (c) E. angustifolia root extract (ethanol 85% v/v; Echinacoside 4%) (d) E. purpurea root extract (ethanol 70% v/v)

Echinacea purpurea (L.) Moench. (Asteraceae) Cone flower

49

Swiss albino mice

25, 50, 100, 200 and 400 mg/kg, p.o.

Essential oil

Ducrosia anethifolia Boiss. (Apiaceae) Hazza, Hazzaz

48

—

—

—

—

—

—

—

Anxiolytic

Anxiolytic

Anxiolytic

Nootropic, sedative, anxiolytic and antistress

Anxiolytic

Anxiolytic

Only extract (d) showed anxiolytic activity

Anxiolytic but not sedative

[111]

[110]

[108, 109]

[107]

[106]

[105]

[104]

[103]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

21

22 Acute study - 200 and 400 mg/kg, p.o., and chronic study - 50 and 200 mg/kg, p.o. 50 and 100 mg/kg, p.o. for 23-26 days

Water : Alcohol (7:3) extract of stem bark

Hydro-ethanol extract of stem bark

Aqueous extract of whole plant

(a) Methanol extract (b) adenosine(24) isolated from pulp or flesh

Hydro-alcoholic (50% ethanol) extract of leaves

Erythrina velutina Willd. (Fabaceae) Bico-De-Papagaio

Eschscholzia californica Cham. (Papaveraceae) California poppy, Gold poppy

Euphorbia hirta Linn. (Euphorbiaceae) Asthma weed

Euphoria longana Lamarck (Sapindaceae) Longan Arillus

Euphorbia nerrifolia Linn. (Euphorbiaceae) Indian spurge tree, Oleander spurge

54

55

56

57

58

100 to 300 mg/kg, i.p.

70% ethanol extract of aerial parts

400 mg/kg, p.o.

(a) 2 g/kg, s.c. (b) 30 mg/kg, s.c.

12.5 and 25 mg/kg, i.p.

25 mg/kg, i.p.

Hydro-alcoholic extract (60% ethanol) of aerial parts

3 and 10 mg/ kg, p.o.

Alkaloids – Erysodine(22) and erysothrine(23) isolated from hydro-alcoholic extract of flowers

Erythrina suberosa Roxb. (Fabaceae) Coral tree

3-10 mg/kg, p.o. CE (50, 100, 200 and 400 mg/kg, p.o.)

Crude extract (CE), Erythrinian alkaloids: (+)-α– hydroxyersotrine(19), erythravine(20) and (+)-11-α–hydroxy erythravine(21) isolated from hydro-alcoholic extract of flowers

53

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

Swiss albino mice

Male ddY mice

Swiss albino mice

Male Wistar rats

EPM

Vogel type anti-conflict method

Stair case test, LDM

CCl4 induced neuropathic pain, hot plate and carrageenan induced pain

LDM

EPM

Adult male Swiss albino mice Male Swiss mice

ETM

EPM, LDM

T-maze, Locomotor activity test

Experimental model/ Assessment of clinical parameters

Male Wistar rats

Male albino mice

Male Swiss mice

Animal/ Human being

—

—

—

—

BZD receptor interaction

—

—

—

—

Mechanism of action

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and antineuropathic pain

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Activity

[119]

[118]

[117]

[116]

[115]

[114]

[110]

[113]

[112]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 Swiss albino mice ICR-CD1 male mice

15 mg/kg, i.p.

150 mg/kg, p.o. 5C, 9C and 30C dilutions

Galphimine B(25), galphimine A(26) and galphimine rich fractions (GRFs) obtained from methanol extract of aerial parts

Methanol extract of aerial parts

Capsules containing 310 mg of aqueous extract of aerial parts

Kamishoyosan

(a) Aqueous extract of rhizomes (b) Phenolic constituents: 4-hydroxyl-benzyl alcohol, and benzaldehyde and its phenolic constituents

Methanol extract of roots and rhizomes

Centesimal dilutions of hydro-alcoholic extract of plant as in homeopathic system

Galphimia glauca Cav. (Malpighiaceae) Calderona amarilla

Gardenia jasminoides Ellis (Rubiaceae) Cape jasmine

Gastrodia elata Blume (Orchidaceae) Tian ma (China); Gastrodia Tuber(English name)

Gelsemium sempervirens (L.) Ait. (Loganiaceae) Carolina yellow Jasmine

62

63

64

(a) 400 mg/kg, p.o. (b) 50 and 100 mg/kg, i.p.

Male ICR mice

LDM, OFT

EPM

EPM

SI

HAMA scale, the clinical global impression scale and patient global evaluation

A controlled randomized double blind clinical trial

310 mg twice daily for 4 weeks Male ddY mice

EPM, LDM, FST

ICR albino mice

125, 250, 500, 1000 and 2000 mg/kg, p.o.

50-200 mg/kg, p.o.

EPM

EPM, OFT and rotarod performance

EPM, OFT, Foot shock induced flighting behaviour

Male ICR mice

Sprague-Dawley rats and Swiss albino mice

61

100 mg/kg, p.o.

Ethyl acetate fraction of ethanol extract of the aerial parts

Evolvulus alsinoides Linn. (Convolvulaceae) Shankhpushpi

Albino mice

60

0.3 g/kg, p.o. for 5 days twice daily

Chloroform, n-butyl alcohol and water fractions obtained from methanol extract of roots

Eurycoma longifolia Jack (Simaroubaceae) Tongkat ali, Penawar bias

59

—

—

Interaction with 5-HT(1A) receptor

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

—

—

Anxiolytic and antidepressant

Anxiolytic

Anxiolytic, neuromuscular coordination and antioxidant

Anxiolytic

—

—

—

—

[127]

[126]

[125]

[124]

[123]

[122]

[121]

[91]

[120]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

23

24 Charles Foster rats

0.6 mg/kg, p.o.

0.5 and 1.0 g/ kg, p.o. for 7 days; 1 and 2 mg/kg, p.o. for five days

Ginkgolic acid(27) conjugates (GAC) isolated from chloroform: methanol extract (2:1) of the leaves

G. biloba extract (GBE), standardized to contain 24% ginkgoflavoglycosides and 6% ginkgo-terpenoid lactones or ginkgolide A(28)

Hydro-alcoholic extract of roots and rhizomes

Ethanol extract of aerial parts

Ethanol extract of fruits

Aqueous, hydroalcoholic, and ethanol extract of calyxes of plant

Isoquinoline alkaloid: Montanine(29) isolated from ethanol extract of bulbs

Glycyrrhiza glabra Linn. (Leguminosae) Licorice, Mulethi

Hedyosmum brasiliense Mart. (Chloranthaceae) Cha de bugre

Heteropterys glabra Hook. & Arn. (Malpighiacae) Redwing

Hibiscus sabdariffa Linn. (Malvaceae) Jamaica sorrel, Red sorrel

Hippeastrum vittatum (L’Herit) Herbert (Amaryllidaceae) Amaryllis

66

67

68

69

70

Anxiolytic and sedative (1-10 mg/kg, i.p.), anticonvulsant (30 and 60, mg/kg, i.p.)

300 mg/kg, p.o.

350 mg/kg, p.o.

100 mg/kg, i.p.

10-300 mg/kg, i.p.

Wistar AF rats

8-16 mg/kg, i.p.

Ginkgo biloba extract (EGb-761)

Swiss albino mice

Wistar rats

DBA/2J mice

Male Swiss albino mice

Swiss albino mice

Male ddY mice

In vitro using rat brain mitocondrial extract

5 and 10 mg equivalent

Aqueous and ethanol extracts of leaves

Ginkgo biloba Linn. (Ginkgoaceae) Ginkgo, Maidenhair tree

Animal/ Human being

65

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

EPM, Sodium pentobarbitalinduced sleep, PTZ-provoked convulsions, FST

EPM, ketamine- induced sleep

Sleep wakefulness cycle, electroencephalogram (EEG) and visual evoked potentials (VEP)

EPM, OFT, Barbiturate-induced sleeping time test

EPM, foot shock induced aggression

EPM

EPM, OFT, novelty-induced feeding latency and SI

SI

—

Experimental model/ Assessment of clinical parameters

—

—

—

—

—

Other mechanism but not through GABA/ BZD/ Cl- channel receptor interaction

—

GABA/ BZD/ Cl- channel receptor interaction

Inhibition of monoamine oxidase (MAO A and B)

Mechanism of action

Anxiolytic, mild sedative and anticonvulsant but not antidepressant

Anxiolytic and sedative (at multiple doses)

Anxiolytic and sedative

Anxiolytic and sedative

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Activity

[136]

[135]

[134]

[133]

[132]

[131]

[130]

[129]

[128]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 150 and 300 mg/kg, p.o. 200-400 mg/ kg, p.o.

H. perforatum extract LI 160

Hydro-alcoholic extract of whole plant

Ethanol extract of leaves

380 mg/kg/ day chronic administration

H. perforatum extract LI 160

Kielmeyera coriacea Mart. (Clusiaceae) Páu santo

300 mg/kg, p.o

H. perforatum extract LI 160

73

300 mg/kg, p.o. for 21 days

H. perforatum extract LI 160

120 mg/kg/ day, p.o.

40 mg/kg, s.c.

100 or 200 mg/kg, p.o. OD for 3 days

Hydro-alcoholic extract of whole plant

Vitexin(32), isoorientin(33) and orientin(34) from methanol extract of Stems

5 mg/kg, p.o.

Lyophilized aqueous extract

Jatropha ciliata M. Arg. (Euphorbiaceae) Huanarpo

2778 and 1852 mg/kg, p.o.

—

Standardized extract of the whole plant, containing 0.54% total hypericins [0.11% hypericin(30) and 0.43% pseudohypericin(31)]and 0.09% protoforms

72

H. perforatum extract LI60

Hypericum perforatum Linn. (Guttiferae) St John’s wort

71

Male Wistar rats

Male ddY mice

Male Laca mice

Swiss albino mice

C57BL/6J Mice

Male albino Swiss mice

EPM

Vogel type Anticonflict effect in mice

Mirrored chamber, EPM, EZM

MBT, FST

OFT, LDM, FST

ETM

Mouse defense test battery

EPM, OFT, EZM, novelty-induced suppressed feeding latency, SI

Wistar rats

Male albino Swiss mice

EPM

OFT, LDM

—

Male albino Swiss mice

Male Sprague–Dawley rats

In vitro

—

—

—

—

—

—

—

Affect monoamines concentration in rats’ brain

—

Inhibitory influence on glutamatergic transmission mediated by NMDA receptors

β receptor activation

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and antidepressant

Anxiolytic and antidepressant

Anxiolytic

[147]

[146]

[145]

[144]

[143]

[142]

[141]

[140]

Anxiolytic

Anxiolytic

[139]

[138]

Anxiolytic

Anxiolytic

[137]

Anxiolytic

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

25

26

(a) Hydro-alcoholic extract (70% ethanol) (b) 5,7-dimethoxyflavone (1), 5,7-dimethoxy-6methylflavone (2), 5-hydroxy-7-methoxy-6methylflavone (3) and 5-hydroxy-7-methoxy6,8-dimethylflavone (4)

Three chemotypes of essential oil (EO1, EO2, EO3) from leaves

Daphnoretin(35) isolated from hydro-alcoholic extract (60% ethanol) of whole plant

Ethanol extract of leaves

Leptospermum scoparium J.R. et G. Forst. (Myrtaceae) Manuka or Tea tree

Lippia alba (Mill.) N.E. Brown (Verbenaceae) Cidreira, Bushy matgrass

Loeselia mexicana Brand (Polemoniaceae) Mexican false calico, Espinosilla

Magnolia dealbata Zucc. (Magnoliaceae) Eloxochiti

75

76

77

78

100 and 300 mg/kg, p.o.

1.8, 3.7, 7.5 and 15.0 mg/ kg, i.p.

EO1 and EO3 (100 mg/kg, i.p.) and EO2 (25 mg/kg, i.p.)

(a) 250 mg/kg, p.o. (b) IC50values of 2.1 microM (1), 45 microM (2), 3.3 microM (3) and 40 microM (4)

—

I ml/100 g, i.p.

Lavender oil

Lavender odour

Inhalation 0.1-1.0 ml

Essential oil from leaves

Lavandula angustifolia Miller (Lamiaceae) English Lavender

74

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

Male Swiss albino mice

Male ICR mice

Male Swiss Mice

(a) Rats (b) In vitro radio receptor assay with [3H] Flunitrazepam

Mature male and female gerbils

Male ICR Mice

Adult male SpragueDawley albino rats

Animal/ Human being

EPM, HBT, exploratory rearings, Sodium pentobarbital-induced hypnosis, PTZ-induced seizures

OFT, EPM

EPM, OFT and rotarod

Locomotion study

EPM

Galler type conflict test

Open field behavior test

Experimental model/ Assessment of clinical parameters

—

—

—

Interaction with GABAA/BZD receptor

—

—

Lavender oil potentiates the responses of GABA receptors at low concentrations and inhibits responses of GABA receptors at high concentrations in vitro

Mechanism of action

Anxiolytic, sedative and anticonvulsant

Anxiolytic

Anxiolytic and myorelaxant

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Activity

[156]

[155]

[154]

[152, 153]

[151]

[150]

[148, 149]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders



Cyracos : hydro-alcoholic (30% ethanol) extract of aerial parts

Methanol, ethyl acetate and alkaloid rich fraction of stem bark

(a) Methanol extract of fruits (b) Butanolic fraction

Decoction from bark of the roots

Neferine(39) isolated from methanol extract of embryos of the seeds

Hydro-alcoholic extract (80% ethanol) of aerial parts

Methanol extract of leaves

Mitragyna parvifolia Roxb. (Rubiaceae) Kaim, Gulikadam

Morinda citrifolia Linn. (Rubiaceae) Noni, Indian mulberry

Nauclea latifolia J.E.Smith (Rubiaceae) Negro peach, African peach

Nelumbo nucifera Gaertner (Nymphyaeaceae) Sacred water lilly, Pink lotus

Nepeta persica Boiss. (Lamiaceae) Catmint

Ocimum gratissimum Linn. (Lamiaceae) Vana Tulsi

82

83

84

85

86

87

200 and 400 mg/kg, p.o.

50 mg/kg, i.p.

100 mg/kg, i.p.

80 and 160 mg/kg, i.p.

(a) IC50 – 22.8 µg/ml (b) IC50 – 27.2 µg/ml

200 and 400 mg/kg, p.o.

120, 240 and 360 mg/kg, p.o. for 15 days

30 mM

Apigenin(38)

Melissa officinalis Linn. (Lamiaceae) Lemon balm, Common balm

3 mg/kg, i.p.

Apigenin(38) isolated from aqueous extract of branchlets with flowers

81

0.2, 0.5 and 1.0 mg/kg, p.o.

Obovatol (37)isolated from leaves

Marticaria chamomila Linn. or Matricaria recutita (Asteraceae) German chamomile, Amerale

0.2 mg/kg, seven days, p.o.

Honokiol (36)

80

0.2-1 mg/kg, p.o. for seven days

Honokiol (36)

Magnolia Obavata Thunb. (Magnoliaceae) Japanese bigleaf magnolia

79

Swiss albino mice

Male NMRI mice

Male ICR mice

Adult Swiss male mice

In vitro

Swiss albino mice

C57Bl/6Jico mice

In vitro, Rats

Male C Fl mice

ICR male mice

Male mice of the ddY strain

Male mice of the ddY strain

—

OFT, PTZ-induced seizure test

EPM

EPM

EPM, Diazepam-induced sleep, MES-, Strychnine-, PTZ-induced convulsions test, OFT

EPM, MBT

EPM

—

EPM, HBT, Locomotor activity test, Horizontal-wire test, Seizure testing

EPM, HBT

EPM

EPM

—

—

—

—

GABAA agonist

Interaction with GABA receptors

Inhibits GABA-T (transaminase) activity and increase GABA level in brain

Interaction with GABAA/BZD receptor

Interaction with GABAA/BZD receptor

GABA-BZDreceptors / Cl- channel activation

—

—

Anxiolytic and anticonvulsant

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and mild sedative at 10 times dose

Anxiolytic

Anxiolytic

Anxiolytic

[170]

[169]

[168]

[167]

[166]

[165]

[164]

[161163]

[160]

[159]

[158]

[157]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

27

28 500 mg/ capsule twice daily after meal

100 mg/kg, p.o.

Aqueous extract of whole plant

Ethanol extract of seeds

Ginseng extract G-115

Aqueous extract of white and red roots powder

Pachyrhizus erosus Linn. (Leguminosae) Bangkwang, Jicama

Panax ginseng C.A.Meyer (Araliaceae) Chinese, Japanese, Korean ginseng, Ninjin

Panax quinquefolius Linn. (Araliaceae) American ginseng

89

90

91

EPM

Male ICR mice

(a) 50 and 100 mg/kg, p.o. (b) 5, 10 and 25 mg/kg, p.o. once daily for 3 days

(a) Ginseng aqueous extract (b) Ginsenosides Rg3(41) and Rh2(42) from roots

EPM, LDM, HBT

EPM

Male ICR albino mice

(a) 300, 600 and 1200 mg/ kg, p.o (b) 50, 100, and 200 mg/ kg, p.o (c) 2.5, 5 and 10 mg/kg, i.p

(a) Ginseng root powder (b) Crude saponin ginseng fraction (c) Ginsenoside Rb1(40)

Male Swiss albino mice

EPM

Albino mice

RG (100 mg/ kg, p.o.) and SG (25 and 50 mg/kg, p.o.)

Butanol fractions of roots of red (RG) and sun ginseng (SG)

50 and 100 mg/kg, p.o.

EPM, OFT, conflict behavior in thirsty rats, foot shock induced fighting in paired mice

Male Wistar strain albino rats and albino mice

20 and 50 mg/ kg, p.o. twice daily for 5 days

Saponins

Vogel conflict procedure

Staircase test, EPM, aggressive behavior, Pentobarbitone induced sleeping time, locomotor activity, rotorod test

Hamilton’s brief psychiatric rating scale (BPRS)

35 male and female human beings

Swiss albino mice

EPM, Passive avoidance paradigm, Scopolamine and diazepam induced amnesia

Experimental model/ Assessment of clinical parameters

Swiss Mice

Animal/ Human being

Wistar rats

150 mg/kg, p.o.

200 mg/kg, p.o.

Aqueous extract of whole plant

Ocimum sanctum Linn. (Lamiaceae) Tulsi, Holy basil

88

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

—

Interaction with GABA/BZD receptors

—

—

Decrease MAO activity in brain

—

—

—

—

Mechanism of action

[179]

[178]

Anxiolytic

Anxiolytic

[177]

[176]

[175]

[174]

[173]

[172]

[171]

Ref.

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Sedative, antianxiety muscle relaxant and antiaggressive activity

Anxiolytic

Anxiolytic and nootropic effects

Activity

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 EPM EPM, MBT

Adult female Wistar rats Adult male Swiss mice

Adult male Wistar rats

Wistar rats Adult male Swiss mice

50 and 100 mg/kg, i.p. 1 mg/kg, i.p.

50, 100 and 150 mg/kg, i.p. (a) 230 mg/kg, p.o. (b) 100 mg/kg, p.o. (c) 30 mg/kg, p.o.

400 and 800 mg/ kg, p.o. 50, 100 and 150 mg/kg, i.p. 100 and 300 mg/kg, p.o.

Aqueous extract of leaves

Chrysin (43)

Hydro-alcoholic extract (40% ethanol) of leaves

(a) Aqueous extract (b) Total flavonoid fraction (c) Luteolin-7-O-(2rhamnosyl glucoside) (44) from total flavonoid fraction of aqueous extract of leaves

Spray dried powder of aqueous extract of leaves

Aqueous extract

Aqueous extract of mature fruits and its butanolic fraction

Passiflora coerulea Linn. (Passifloraceae) Blue Passion flower

Passiflora edulis Sims (Passifloraceae) Bat-Leaved Passion flower

Passiflora incarnata Linn. (Passifloraceae) Passion flower, Maypop

94

95

96

Swiss Albino mice Swiss Albino mice

125 mg/kg, p.o. 100 mg/kg, p.o. 10 mg/kg, p.o.

Methanol extract of aerial parts

Homoeopathic formulations

Benzoflavone nucleus as basic moiety compound from methanol extract

Swiss Albino mice

45 drops/day for 4 weeks

Aqueous extract (PassipayTM, Iran Darouk)

A double blind randomized trial on 36 patient with GAD

Male CF1 mice

Wistar rats

Adult male Wistar rats

400 and 800 mg/ kg, p.o.

Spray dried powder of aqueous extract of leaves

EPM

EPM

EPM

HAMA scores

LDM, Ethyl ether–induced hypnosis, PTZ-induced convulsions

EPM

EPM

EPM, HBT

EPM

EPM

EPM

Adult female Wistar rats

50,100 or 150 mg/kg, i.p.

Hydro-alcoholic extract (40% ethanol) of leaves

EPM

Passiflora alata Dryander (Passifloraceae) Winged-stem Passion flower

Male albino-Swiss mice

93

HE (300 and 600 mg/kg, p.o.) ME (100 and 300 mg/kg, p.o.)

Hydro-ethanol (HE) and Methanol (ME) Extract from leaves

Passiflora actinia Hooker (Passifloraceae) Wild bell apple, maracujá-do-mato

92

—

—

—

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and sedative but not anticonvulsant

—

—

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

—

—

—

—

Interaction with BZD receptors

—

—

—

GABAA receptor interaction

[195]

[194]

[190193]

[189]

[188]

[183, 184]

[182]

[187]

[181]

[185, 186]

[183, 184]

[182]

[181]

[180]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

29

30 250 and 500 mg/kg, p.o.

10 mg/kg, p.o.

100 and 200 mg/kg, i.p. and p.o. 300 and 900 mg/kg, p.o.

Rosmarinic acid (45)and caffeic acid(46) isolated from hydro-alcoholic extract of leaves

Hexane, hydro-alcoholic, and precipitated hydro-alcoholic extract (50%) of roots

Whole plant extract

Perilla frutescens (L.) Britton (Lamiaceae) Purple Perilla, Wild red basil

Petiveria alliacea Linn. (Phytolaccaceae) Guinea hen weed

98

99

Male albino Swiss mice

Female Swiss mice

Albino mice

Adult male Wistar rats and Swiss mice

EPM

Male C57BL/6J mice

(a) 150 and 300 mg/kg, p.o. (b) 2.1 and 4.2 mg/kg, p.o. (c) 0.17 and 0.34 mg/kg, p.o.

(a) Hydro-alcoholic extract (50% ethanol) of aerial parts (b) Butanol fraction (c) Chloroform extract

Aqueous and ethanol extract of leaves

EPM

Male BL6/C57 J mice

375 mg/kg, p.o.

Hydro-ethanol extract (50% ethanol) of aerial parts

EPM, OFT

EPM, OFT

FST

EPM, OFT, HBT

Numerical rating scale, Trieger dot test and the digit-symbol substitution test

A double blind placebocontrolled study on 60 patients with anxiety

500 mg, p.o.

EPM

Tablet containing 1.01 mg benzoflavone (BZF)

Male Sprague-Dawley rats

Experimental model/ Assessment of clinical parameters

2 mg/kg, i.p.

Animal/ Human being

Methanol extract of aerial parts and Chrysin

Passiflora quadrangularis Linn. (Passifloraceae) Giant granadilla

Dose

97

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

—

—

Modulation of the α1A- adrenoceptormediated signal transductions and also attenuates the down regulation of BDNF transcription

—

—

Interaction with GABA receptors

—

Interaction with GABA/BZDreceptors

Mechanism of action

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Activity

[205]

[204]

[203]

[202]

[201]

[199, 200]

[198]

[196, 197]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders



Piper methysticum Forst. (Piperaceae) Kava, Kawa

Piper solmsianum C. DC. (Piperaceae) Pariparoba

100

101

Anxiolytic

Anxiolytic

—

—

Baroreflex control of heart rate (BRC) and respiratory sinus arrhythmia (RSA) HAMA Scale, Hospital Anxiety and Depression Scale (HADS), Self- Assessment of Resilience and Anxiety (SARA)

Patients suffering with GAD

280 mg/day for 4 weeks 50 mg/day for 4 weeks

120-240 mg/ kg, p.o. 400 mg/day

Cockerels (Gallus gallus; strain W36)

125 mg/kg and 88 mg/kg, i.p. 50 mg/day for 4 weeks

50-300 mg/ day for 4 weeks

Kava extract standardized to 30% kavalactones

Kava-Kava special extract WS 1490

Hydro-alcoholic extract of roots

Kava Kava LI150 extract

Samples containing 12.8-100% total kavalactones, and fractions containing kavalactones 1-6(47-52) in varying concentration (0.1-67.5%)

Ethanol extract of the aerial parts

Kava-Kava special extract WS 1490

Kava-Kava special extract WS 1490

Emulsion of the essential oil from aerial parts

5 or 10% v/v

Anxiolytic

—

The total score of the Anxiety Status Inventory (ASI) observer rating scale, structured well-being self-rating scale (Bf-S) and CGI HAMA Scale, subjective well- being scale (Bf-s), Erlanger Anxiety, Tension, Aggression Scale (EAAS), CGI, The Brief Personality Structure Scale and The Adjective Checklist

A randomized double-blind placebo-controlled clinical trial on 141 patients suffering from neurotic anxiety A randomized double-blind placebo-controlled clinical trial on 230 patients suffering from neurotic anxiety

EPM

Anxiolytic

—

Mirrored chamber avoidance assay and EPM

Swiss albino mice

Swiss male mice

Anxiolytic

—

Chick social separation procedure

i.p. injections of different concentrations

—

Anxiolytic

Anxiolytic

Anxiolytic

—

HAMA Scale and Boerner Anxiety Scale (BoEAS), CGI, a sleep questionnaire (sf-13), and a quality of life questionnaire

A randomized double-blind placebo-controlled clinical trial on 129 patients suffering from GAD

—

Anxiolytic

—

EPM

Wistar rats

A randomized double-blind placebo-controlled clinical trial on 37 patient with DSM-IV GAD

Anxiolytic

—

CGI scale, AMDP – module, HAMA, Hamilton depression scale, Beck anxiety inventory, Speilberger trait anxiety inventory

Controlled clinical trial on a 37-year-old female outpatient with GAD, SP and SAD

3 tablets daily equivalent to 135 mg kava pyrones daily for 12 weeks

Kava extract LI 150

Anxiolytic

—

HAMA, somatic and psychic anxiety, Clinical Global Impression (CGI), Self-Report Symptom Inventory-90 Items revised, and Adjective Mood Scale

25-week multicenter randomized placebocontrolled double-blind trial on 121 outpatients suffering from anxiety of non-psychotic origin

50 mg, p.o.

WS 1490 extract

[217]

[215, 216]

[214]

[213]

[212]

[211]

[210]

[209]

[208]

[207]

[206]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

31

32 Male adult Swiss mice

Male adult Swiss mice

HE, fractions (250, 500 and 1000 mg/kg), p.o., Dihydrostyryl2-pyrones and styryl-2pyrones (0.3 fmol–25 pmol, i.c.v.) 0.3 fmol–25 pmol, i.c.v.

40, 80 and 160 mg/kg, p.o. 10, 25 and 50 mg/kg i.p. or p.o.

Three dihydrostyryl-2pyrones I-III (54-56) and four styryl-2-pyrones I-IV (57-60) isolated from ethyl acetate fraction of hydro-ethanol (HE) extract of whole plant

Dihydrostyryl-2pyrones(54-56) and styryl-2- pyrones(57-60) isolated from ethyl acetate fraction of hydro-ethanol (HE) extract of whole plant

Polygala saponins

α and β amyrin (61-62) pentacyclic triterpenes isolated from stem bark resin

Chlorogenic acid(63) isolated from fruits

Methanol extract of aerial parts

Polygala sabulosa A.W. Bennett (Polygalaceae) Timutu-pinheirinho

Polygala tenuifolia Willd. (Polygalaceae) Yuan Zhi

Protium heptaphyllum (Aubl.) March. (Burseraceae) Brasil resintree

Prunus domestica Linn. (Pleuronectidae) Mirabelle, Plum, Alu bukhara

Pulsatilla nigricans Stoerck (Ranunculaceae) Pasqueflower, Windflower, Meadow anemone

104

105

106

107

200 mg/kg, p.o.

20 mg/kg, i.p.

Laca mice

Swiss albino male mice

Male Swiss mice

Male adult Swiss mice

Swiss male mice

103

50 and 100 mg/kg, i.p

Piplartine (53) amide alkaloid isolated from roots

Piper tuberculatum Jacq. (Piperaceae) Pimenta darta and Pimenta Longa

Animal/ Human being

102

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

EPM

EPM, LDM, free exploratory test

EPM, OFT

EPM, OFT, HBT

EPM

EPM, Pentobarbital-and ethyl ether-induced hypnosis, PTZ-induced convulsions, Rota-rod test

EPM, OFT

Experimental model/ Assessment of clinical parameters

—

—

BZD receptor interaction

—

BZD receptor interaction

—

—

Mechanism of action

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Hypnotic, anticonvulsant and anxiolytic

Anxiolytic

Activity

[224]

[223]

[222]

[221]

[220]

[219]

[218]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 125, 250, 500, 1000 and 2000 mg/kg, p.o. 12.5 mg/kg, i.p.

Hexane extract of leaves

Ethanol extract of leaves

Ethanol extract of aerial parts

Aqueous extract of flowers

A diterpenoid CMP I

Salvinorin-A(64)

Hydro-alcoholic (60% ethanol) extract of leaves and flower

60% ethanol extract of leaves

Diterpene quinine – Miltirone(65) isolated from ethereal extract of roots

Hydro-alcoholic extract (80% ethanol) of aerial parts

Rollinia mucosa (Jacq.) Baill. (Annonaceae) Wild sugar apple

Rubus brasiliensis Martius (Rosaceae) Amora branca

Ruta chalepensis Linn. (Rutaceae) Fringed rue, herb-of-grace

Salix aegyptiaca Linn. (Salicaceae) Egyptian muskwillow

Salvia cinnabarina M.Martens & Galeotti (Lamiaceae) Sage, Wild Kus

Salvia divinorum Epling & Játiva (Lamiaceae) Diviner’s sage

Salvia elegans Vahl. (Lamiaceae) Scarlet pineapple

Salvia miltiorrhiza Bge. (Lamiaceae) Red sage

Salvia reuterana Boiss. (Lamiaceae) Sage

110

111

112

113

114

115

116

117

118

100 mg/ kg, i.p.

10-60 mg/kg, p.o.

0.001-1000 µg/kg, s.c.

10 mg/kg, p.o.

100 mg/kg, i.p.

300 mg/kg, p.o.

150 mg/kg, per gavage

1.62 to 6.25 mg/kg, p.o.

15 mg/kg, p.o.

Hydro-alcoholic extract (contains 3% rosavin and 1% salidroside)

Rhodiola rosea Linn. (Rhizophoraceae) Arctic root, Golden root, Roseroot

109

100, 250, and 500 mg/kg, p.o.

Ethanol extract of seeds

Punica granatum Linn. (Punicaceae) Pomegranate, Granada

108

Male Syrian mice

Albino mice

Sprague Dawley rats

Male ICR mice

Adult male Sprague-Dawley rats

Albino mice

Male NMRI mice

Male Swiss albino mice

Male Wistar rats and Swiss mice

Albino mice

Male CD1 mice

Young and old male Swiss albino mice

EPM, Spontaneous locomotor activity

Four plate test

EPM, FST

EPM, LDM, OFT

EPM, FST, Spontaneous motor activity in mice, Tail suspension test

EPM, FST

EPM

PTZ-induced seizures, sodium pentobarbital-induced hypnosis, exploratory activity, anxiety by unfamiliar environment and nociception

EPM

Avoidance exploratory behavior paradigm

LDM

EPM, Pentobarbital-induced sleeping time, FST, tail flick and hot plate test

—

BZD receptor interaction

—

—

k-opioid and endocannabinoid systems

—

—

—

Interaction with GABAA receptor

GABA/BZD receptors interaction

—

—

Anxiolytic

Anxiolytic

Psychotropic

Anxiolytic

Anxiolytic, antidepressant

Anxiolytic

Anxiolytic

Anxiolytic, anticonvulsant, sedative, antinociceptive

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic, antidepressant and antinoceceptive

[237]

[236]

[235]

[234]

[233]

[232]

[231]

[230]

[228, 229]

[227]

[226]

[225]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

33

34 EPM

Radio receptor BZD-S assay

Radio receptor BZD-S assay

Vogel shock conflict test

Male ICR mice

In vitro, Forebrains of Sprague-Dawley rats

In vitro

Male ICR mice

7.5, 15 and 30 mg/kg, p.o.

IC50 values 0.008 to 100 µM 6.05 mM

Baicalein (10 mg/kg, i.p.) and baicalin (20 mg/kg, i.p.)

Essential oil

A monoflavonoid Wogonin(66), isolated from dichloromethane extract of roots

Only 2’-OH flavones isolated from dichloromethane, water extracts of roots

(a) 5,7,2’-trihydroxy-6,8dimethoxy flavones (b) 5,7-dihydroxy-6methoxyflavone isolated from dichloromethane extract of roots

Flavonoid baicalin(67) and its aglycone baicalein(68)

Scutellaria baicalensis Georgi (Labiatae) Huangqin

Scutellaria lateriflora Linn. (Labiatae) Blue skullcap, Hoodwort

Securidaca longepedunculata Fresen (Polygalaceae) Violet tree

121

122

123

100-400 mg/ kg, p.o.

Albino mice of either sex

EPM, Y-maze, Strychnine- and Picrotoxin-induced seizure, Hexobarbitone- induced sleep test, Exploratory activity

EPM, SI

Adult male SpragueDawley rats

(a) 40 mg/g, p.o. (b) 33 mg/g, p.o. (c) 1.6 mg/g, p.o. (d) 31 mg/g, p.o.

(a) Flavonoid baicalin(67) in ethanol extract of roots (b) baicalein(68) in ethanol extract of roots (c) Ethanol extract of roots (d) glutamine in water extract of roots

Aqueous roots extract

EPM

Male Sprague-Dawley rats

Behavioural responses

100 mg/ml, orally

Anxiety in a woman in labour

Aqueous extract of roots

Inhalation

EPM, Y-maze, HBT, Actophotometer, MBT

Saussurea lappa C.B. Clarke (Asteraceae) Kuth, Kustha

Albino mice

120

200 and 400 mg/kg, p.o.

Methanol extract of seeds and fruits

Experimental model/ Assessment of clinical parameters

Sapindus mukorossi Gaertn. (Sapindaceae) Soapberry,Ritha

Animal/ Human being

119

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

—

Interaction with GABAA / BZD receptor

Interaction with GABAA / BZD receptor

Interaction with BZD binding site of GABAA receptors

(a) Interaction with GABAA / BZD receptor (agonist) (b) Interaction with GABAA / BZD receptor (selective antagonist)

Interaction with GABAA / BZD receptor

Interaction with GABA/ BZD receptor

—

GABAergic transmission

Mechanism of action

Anxiolytic, anticonvulsant, sedative

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Activity

[246]

[245]

[245]

[244]

[242, 243]

[241]

[240]

[239]

[238]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 EPM

Albino Wistar mice and rats of either sex

Male TO mice

Male Syrian mice

100 mg/kg, p.o.

100 mg/kg, i.p.

PF (25 and 50 mg/kg, i.p.) EF (25 and 50 mg/kg, i.p.) AF (50 mg/kg, i.p.)

Hydro-alcoholic Extract (50% ethanol) from fully grown flowering herb

Ethanol extract of leaves

Hydro-alcoholic extract (80% ethanol) and essential oil of aerial parts

Petroleuum ether (PF), ethyl acetate (EF) and water (AF) fractions of hydro-alcoholic extract (80% ethanol) of aerial parts

Spondias mombin Linn. (Anacardiaceae) Hog plum, Jobo, Yellow mombin

Stachys lavandulifolia Vahl. (Lamiaceae) Lavendelblaetrige and Wood betony

Theobroma cacao Linn. (Sterculiaceae) Cacao, Chocolate tree, Kakao

127

128

129

Mass or cake

EPM

Swiss albino male mice

PE (10 mg/kg, i.p.), EE (10 mg/kg, i.p.), WE (30 mg/kg, i.p.)

Petroleum ether extract (PE), 90% ethanol extract (EE), Water extract (WE) of flowers

100 mg/100g, o.s.

12.5 to 100 mg/kg, i.p.

Male Wistar strain rats

Albino wistar rats and Swiss mice

ETM

Muricidal action of rats, Porsolt’s FST

EPM

EPM, OFT, Foot shock induced aggression

EPM

Sphaeranthus indicus Linn. (Asteraceae) Mundi

Adult male Swiss mice

126

30-300 mg/kg, p.o.

Hydro-alcoholic (50% ethanol) and dichloromethane extract from aerial parts

EPM, MES-, PTZ-, Strychnine-, Lithium-pilocarpine-and electrically induced seizures, Pentobarbital-induced sleep, Amphetamine antagonism

Sonchus oleraceus Linn. (Asteraceae) Sow thistle, Milky tassel

Male Sprague-Dawley rats and male mice (NIN strain)

125

100 and 200 mg/kg, p.o.

Benzene:ethyl acetate (BE) fraction of the acetone soluble part of petroleum ether extract of leaves

Sesbania grandiflora (L.) Poir. (Fabaceae) Agati

124

Conditional fear relating behaviour, but did not affect the concentration of brain monoamines such as norepinephrine, serotonin and dopamine

—

—

GABAA receptor

—

—

—

Increase in the brain content of GABA and 5-HT

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and antidepressant

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and anticonvulsant

[254]

[253]

[252]

[251]

[250]

[249]

[248]

[247]

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

35

36 Male Swiss albino mice

Laca mice Laca mice

10-300 mg/kg, p.o.

25 mg/kg, p.o. 50 mg/kg, p.o. 2 mg/kg, p.o.

Aqueous extract of inflorescences [Quercetin(7) and kaempferol(6) may be active constituents]

Hydro-alcoholic extract (70% ethanol) of Inflorescences and butanol fraction

Isofavones (MF11RCE)

Methanol extract of aerial parts

Homoeopathic formulations

Apigenin(38) isolated from methanol extract of aerial parts

Aqueous extract of roots

Anthocyanin fraction from 96% ethanol extract of berries

Hydro-alcoholic (70% ethanol) extract of roots

Tilia tomentosa Moench (Malvaceae) Silver Lime

Trifolium pratense Linn. (Fabaceae) Red clover

Turnera aphrodisiaca Ward (Turneraceae) Damiana

Uncaria rhynchophylla (Miq.) Jacks (Rubiaceae) Cat’s Claw herb

Vaccinium ashei Reade (Ericaceae) Blueberry

Valeriana edulisssp. Procera Nutt. ex Torr. (Valerianaceae) Tobacco root

131

132

133

134

135

136

100, 300 and 1000 mg/kg, p.o.

0.6–1.0 and 2.6–3.2 mg/ kg/day, p.o.

200 mg/kg/ day p.o for 7 days

80 mg

Male ICR mice

Adult male Swiss mice (aged 3 months)

Male SD rats and male ICR mice

Laca mice

Women with postmenopausal anxiety

Swiss albino Mice

Albino ICR mice

25-100 mg/kg, p.o.

Methanol extract from bracts and flowers [Tiliroside(70) main constituent]

Dose equivalent to 1 g of plant material

Male Swiss albino mice

1 to 10 mg/kg, i.p.

β-sitosterol(69) isolated from hexane extract of inflorescences

Tilia americana Linn.var. mexicana (Malvaceae) American basswood, American linden

130

Animal/ Human being

Dose

Extract/Fraction/Isolate

Biological source

S. No.

Table 1: Continued

PTZ-induced seizures, Exploratory rearing, Rotarod

EPM, OFT, Inhibitory avoidance

EPM, HBT

EPM

EPM

EPM

HADS, Zung’s self- rating depression scale

EPM, HBT

EPM, HBT, sodium pentobarbital (SP)-induced hypnosis potentiation, ambulatory Activity

EPM

EPM, HBT, sodium pentobarbital-induced hypnosis and ambulatory activity

Experimental model/ Assessment of clinical parameters

—

—

Serotonergic nervous system

—

—

—

—

Anxiolytic, anticonvulsant and myorelaxant

Memoryenhancing, anxiolytic and locomotion increasing effects

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic and antidepressant

Anxiolytic

Anxiolytic and sedative

—

Interaction with BZD receptors

Anxiolytic

Anxiolytic and Sedative at higher dose (30 mg/kg, i.p.)

Activity

—

—

Mechanism of action

[266]

[265]

[264]

[263]

[262]

[261]

[260]

[259]

[258]

[257]

[255, 256]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 4 and 7 mg/kg, i.p. 0.2 g/kg, p.o. 3 mg/kg, i.p.

20 and 50 mg/ kg, p.o. for 5 days 50, 200 and 500 mg/kg

0.5 g/kg, p.o. 320 mg/kg, p.o. thyroid tablet for nine days 2.0 mg/kg, p.o.

Flavonoid linarin(72)

Dichloromethane extract of roots

Valerenic acid(73) isolated from hydroalcoholic extract of roots

Ethanol extract of roots

Glycowithanolides isolated from the roots

Aqueous extract of roots

Benzene fraction of acetone soluble part of petroleum ether extract of dried rhizomes

Ethanol extract of seeds

Alcoholic extract of seeds

Vitex negundu Linn. (Verbenaceae) Five-leaved chaste tree

Withania somnifera (Linn.) Dunal (Solanaceae) Ashwagandha

Zingiber officinale Linn. (Zingiberaceae) Ginger

Ziziphus jujuba Miller (Rhamnaceae) Desi Ber

139

140

141

142

Sanjoinine-A(74) from alkaloidal fraction of seeds

Female hooded rats

Hesperidin (4 mg/kg, i.p.), 6-methylapigenin (1 mg/kg, i.p.)

6-methylapigenin (70) and hesperidin(71), isolated from roots and rhizomes

15 and 30 mg/ kg, i.p.

100 and 200 mg/kg, p.o.

Male Wistar rats

83.1 mg per day

Valepotriates

EPM, OFT, HBT

Yin deficiency mice

Male ICR mice

EPM

EPM

SI, Novelty-induced suppressed feeding latency, EPM

EPM, LDM

EPM

EPM

HBT

EPM , HBT

Psychic factor of HAMA

Behavioural studies in locomotion test and FST

EPM, OFT

Black/white test, EPM and ambulatory behaviour test EPM, LDM

Male ICR mice

Male Sprague-Dawley rats

Wistar rats

Wistar rats

Swiss albino mice

Albino mice

Adult male Wistar rats

36 patients with GAD DSM III-R

Female albino mice

Valdan drops o.s. daily for 15 days

Hydro-alcoholic extract called valdan drops

Valeriana officinalis Linn. (Valerianaceae) Valerian

Male albino Swiss mice

138

10 mg/kg, p.o.

Valepotriate fraction

Valeriana glechomifolia Meyer (Valerianaceae) Valerian

137

Increase the GABA and expression of GABAA GABAergic transmission

—

—

—

—

—

GABA(A) –ergic system

—

—

Interaction with GABAA / BZD receptor

Significant reduction in the psychic factor of HAMA

—

—

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

Anxiolytic

[282]

[281]

[280]

[279]

[278]

[277]

[276]

[274, 275]

[273]

[272]

[270, 271]

[269]

Anxiolytic

Anxiolytic

[268]

[267]

Anxiolytic

Anxiolytic

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

37

38

Monoterpene alcohol – Isopulegone(82)

Indole alkaloid alstonine(83)

Aswal

Iridol containing compounds iridoids

04

05

06

Inhalation 100 µl

Neroli essential oil

03

Inhalation 1 ml

Lemon oil

10 mg/kg, p.o.

100- 250 mg/l, i.p.

1 mg/kg, i.p.

25, 50 mg/kg, i.p.

12.5, 25, 50 mg/ kg, p.o.

Inhalation (1.0, 2.5 or 5.0% w/w)

Rose oil

Monoterpenic phenol – Carvacrol(81) from essential oil fraction of Oregano and Thyme

1 ml/100 g, i.p.

Rose oil

Male rats and patients with arterial hypertension I - II degree, accompanied by psycho-emotional disturbances

Guinea pigs and Long-Evans rats

Male Swiss mice

Male Swiss mice

Male Swiss mice

Gerbils

ICR strain mice

Adult Wistar male rats

Male ICR mice

Male ICR mice

1 ml/100 g, i.p.

Essential oils:

(a) Lavender oil – 2-phenethyl alcohol, citronellal(75) (b) Rose oil – 1,8 cineole(76), menthone(77), pulegone(78), methyl alcohol, caryophyllene(79) (c) Peppermint - menthol(80)

Male ICR mice

1600 mg/kg, i.p.

Lavender oil

Male ICR mice

Animal/Human beings

200–1600 mg/kg, i.p.

Dose

Oils of rose, ylang-ylang, and Chamomile extracted from flowers of Rosa sp., Cananga odorata, and Anthenis nobilis (or Matricaria chamomilla), respectively. orange oil extracted from the rind of Citrus sp..

Formulation/Extract/Fraction/Isolate

02

01

S. No.

Table 2: List of various anxiolytic formulations and compounds.

Behavioural parameters

Extracellular and whole cell patch clamp recordings on CA1 pyramidal neurons

LDM

OFT, EPM, HBT, Tail suspension and FST

EPM

Locomotor activity, FST

EPM, FST, OFT

EPM

Geller conflict test and Vogel’s conflict test

(a) Anticonflict (b) Increased ambulatory effect (c) Increased ambulatory effect

Geller type conflict test

Geller conflict test and Vogel’s conflict test

Experimental model/Clinical studies parameters

Hetero-geneous with calcium antagonism

—

—

GABAergic transmission

—

5-HTnergic pathway and the suppression of DA activity related to enhanced 5-HTnergic neurons

—

—

[294]

[293]

Anxiolytic and antiepileptic Anxiolytic

[292]

[291]

[290]

Anxiolytic

Anxiolytic and antidepressant

Anxiolytic

[289]

[288]

Anxiolytic and antidepressant

Anxiolytic

[287]

[286]

[285]

Anxiolytic

Anxiolytic

Anxiolytic

Dopamine receptor involvement

[284]

[283]

Only rose oil exhibited anxiolytic activity

Anxiolytic

Ref.

Activity

—

Other mechanism but not through GABA/BZD

Mechanism of action

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

 600 mg

Standardized product containing Melissa officinalis and Valeriana officinalis

Zingicomb, a preparation consisting of Z. officinale and G. biloba extracts

Polyherbal formulation

Suanzaorentang, Chinese medicine (Semen Ziziphi Spinosae, Rhizoma Chuanxiong, Poria, Rhizoma Anemarrhenae, Radix et Rhizoma Glycyrrhizae)

Sho-ju-sen (SK), a Japanese herbal medicine, contains a water extract of Sasa kurinensis Makino et Sibata (Kumazasa; Poaceae) leaves (SS), ethanol extract of Pinus densiflora Siebold et Zucearini (Japanese red pine; Pinaceae) (PN) and Panax ginseng C.A. Meyer (Ginseng; Araliaceae) (PX) in the ratio of 8:1:1

Kami-Shoya-San (TJ-24) is one of the traditional Chinese herbal medicine: Bupleurum scorzoneraefollium Willd. (Bupleuri Radix; Bupleuraceae), Paeonia lactiflora Pallas (Paeoniae Radix; Paeonaceae), Atractylodes lancera (Thunb.) DC. (Actractylodis Lanceae Rhizoma; Compositae), Archangelica officinalis Hoffm. (Angelicae Radix; Umbelliferae), Poria cocos (Schw.) Wolf (Hoelen; Polyporaceae), Gardenia jasminoides Ellis (Gardeniae Fructus; Rubiaceae), Paeonia suffruticosa Andr. (Moutan Cortex; Paeonaceae), G. glabra (Glycyrrhizae Radix; Leguminosae), Z. officinale (Zingiberis Rhizoma; Zingiberaceae), Mentha arvensis Malinvaud (Menthae Herba; Labiatae)

08

09

10

11

12

13

Human beings

Male mice of the ddY strain

Albino mice

5g

SK (10% solutions for 7 days)

25-100 mg/kg, p.o.

SI

EPM

EPM and FST —

One-trial step-through avoidance task

Male Wistar rats

Male Swiss mice

Defined Intensity Stressor Simulation (DISS) and Cognitive performance

Two self-rated scale and one observer rated scale

Double blind, placebo controlled, randomized cross over, 24 healthy volunteers

Clinical trial (20 patients with situationally induced anxiety)

50, 100, 300 mg/ kg, p.o.

0.5, 1, 10 or 100 mg/kg, intragastrically

150 mg for a week

Kavosporal forte (Standardized extract of Kava)

07

5α-reductase inhibitor, involvement of neurosteroid synthesis followed by GABA receptor stimulation

—

Decrease serotonergic activity

—

—

—

—

[301]

[302]

Anxiolytic

[299, 300]

Anxiolytic

Anxiolytic

[298]

[297]

[296]

[295]

Antianxiety and antidepressant

Anxiolytic

Ameliorated the negative effects of the DISS on ratings of anxiety

Amelioration of anxiety arises in connection with mammary biopsy

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

39

40

Dietary products: Dietary soy phytoestrogens

(a) Aq. Extract of Melissa officinalis (b) Aq. extracts of Centella asiatica and Valeriana officinalis (c) Aq. extracts of Maricaria recutita and Humulus lupulus

(a) Aqueous extract of the Rutaceae family (b) hydro-alcoholic extract of Alchemilla erythropoda Juz. (Ladies Mantle; Rosaceae)

Botanical extracts

Formulation/Extract/Fraction/Isolate

Phytoestrogenrich Phyto-600 diet

(a) IC50 – 0.35 mg/ml (b) 1 mg/ml (c) 0.11-0.65 mg/ ml

Homoeopathic complex comprising Aconite, Avena sativa, Passiflora incarnata, Scutellaria laterifolia, Stramonium and Valeriana Tablet, granule, capsules or oral liquid of methanol extract of Rumex madaio Lozenges containing Citrus pectin, trisodium citrate, raw sugar, water, glucose fructose syrup, and mixture of lavender oil, extracts of Melissa, hop and oat Phenolic compounds having phenolic molecule covalently linked an oxygen containing group, a nitogen or another oxygen containg group and C1-C4 alkoxy group obtained from monocotyledon plants like corn

09

12

10 11

05 06 07 08

02 03 04

L-tryptophan, linseed oil, thyme oil, aqueous extracts of St-John’s wort, Arenaria blossom, Valerian, Elecampane Theanine, green tea, red ginseng, Sasamorpha purpurascens extracts Water, aq.-alcoholic and CO2 extract of Forget-me-not (Myosotis) Tablet, pill or granule comprising Bupleuri Radix, Rhizoma Anemarrhenae, saponin component isolated from Semen Ziziphi spinosae Extracts of plants containing betulinic acid and its derivatives Cassia tora aq. Extract Rosamarinic acid isolated from Perilla extract Hydro-alcoholic extract of Piper methysticum leaves

Composition of formulation

Long–Evans males and females rats

In vitro

Chick

Animal/Human beings

01

S. No.

Dose (a) 28 and 56 mg/ kg, i.p. (b) 12.5 and 25 mg/kg, i.p.

Table 3: List of anxiolytic patented formulations.

15

14

S. No.

Table 2: Continued

Activity

—

(a) inhibit GABA transaminase (b) stimulate glutamic acid decarboxylase (c) inhibit glutamic acid decarboxylase

—

Mechanism of action

Anxiolytic

Anxiolytic

Anxiolytic

Activity

Antianxiety

Antianxiety Antianxiety

Management of stress including sleep disturbances, aggresiveness, instability of temper and state of anxiety Management of anxiety and stress Anxiolytic, nootropic, anticonvulsant and cerebroprotective activity Treatment of acute anxiety (Panic anxiety) and chronic anxiety (Generalized anxiety) Anxiolytic Anxiolytic Antianxiety and antidepressant Anxiolytic, anticonvulsant, muscle relaxant, analgesic, sleep inducing, antiinflammatory and neuroprotective Antianxiety

EPM

Chick social separation-stress procedure

Experimental model/Clinical studies parameters

[317]

[315] [316]

[314]

[310] [311] [312] [313]

[307] [308] [309]

[306]

Ref.

[305]

[304]

[303]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders



Clinical reports

Phytoconstituents, Pharmacological reports and Mechanism of action

Phytochemical reports, Pharmacological reports

Pharmacological reports

07

—

Piper methysticum, Bacopa monniera, Kava

08

09

10

Foods

Brazilian plants (39-anxiolytic and 28- hypnotic)

Herbal drugs / Herbal constituents

Flavonoids, essential oils, phenolic acids, alkaloids

—

Nervine, anxiolytic

—

Hawthorn and California Poppy, Immature Oat Seed, Passionflower, Lemonbalm, Vervain, Lavender and Linden

Pharmacological reports and Clinical reports

Anxiolytic

—

Kava, Skullcap, Lemon balm (Melissa officinalis), Valeriana officinalis, Passiflora, Dietary supplements

Pharmacological reports

06

Anxiolytic

—

Ginkgo biloba, Hypericum perforatum, Valeriana officinalis, Panax ginseng

Pharmacological reports, Mode of action

05

—

Piper methysticum, Ginkgo biloba, Galphimia glauca, Matricaria recutita, Passiflora incarnata, Valeriana officinalis

Clinical reports

04

—

(a) Ephedra species, Paullinia species, Catha edulis (b) Cannabis sativa, Tabernanthe iboga, Psychotria viridis, Banisteriopsis (c) Passiflora incarnata, Valeriana, Piper methysticum

Pharmacological reports

03

—

—

Anxiolytic and hypnotic

Anxiolytic and antidepressant

—

—

—

—

Interaction with receptors of CNS (γ-aminobutyric acid, glutamate, dopamine, muscarinic and adenosine receptors)

—

—

—

—

Mode of action reported

Psychiatric disorders

Anxiolytic

Anxiolytic, Generalized anxiety disorders

(a) Adaptogen (b) Hallucinogenic (c) Analgesic and anxiolyitc

Psychoactive

—

Catha edulis, Cola species, Datura species, Pausinystalia yohimbe, Tabernanthe iboga

Traditional uses, chemical constituents, pharmacological reports

02

Anti-anxiety and antidepressant

Therapeutic activity reported

—

Phytoconstituents reported

Valeriana officinalis, Melissa officinalis, Passiflora incarnata, Humulus lupulus, Lavendula officinalis, Piper methysticum, Tilia platyphyllos, Leonurus cordiaca, Hypericum perforatum

Plant drugs

Pharmacological reports

Information available

01

S. No.

Table 4: List of review articles published on anxiolytic plants, and their constituents and formulations.

[328]

[327]

[326]

[325]

[324]

[323]

[322]

[321]

[320]

[319]

[318]

Ref.

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

41

42

Phytochemical reports, Pharmacological reports

Pharmacological reports

Pharmacological reports, Mode of action

Pharmacological reports, Mode of action

Clinical reports

Pharmacological reports

Pharmacological reports, Clinical reports

Efficacy, Safety Profile, Pharmacological reports

Safety profile

Clinical reports

Pharmacological reports, Side effects, Mode of action

12

13

14

15

16

17

18

19

20

21

Information available

11

S. No.

Table 4: Continued

Kava lactones

Kava

Kava

Kava

Hypericum perforatum

(a) Kava kava roots (b) Ginkgo extract

Natural remedies such as St John’s Wart, Kava Kava, Passion flower, Inositol, Valerian root, Melatonin, Omega-3-fatty acids, s-adenosyl-Lmethionine

Ethanol extracts of 31 traditional plants

Methanol extracts of traditional plants

Eighty five herbal drugs

Brazilian plants

Plant drugs

—

—

—

—

—

(a) Analgesic, anesthetic (b) Anxiolytic Side effects- Skin rash and kava dermopathy

Anxiolytic

Anxiolytic

Anxiolytic

Antidepressant, anxiolytic, nootropic, sedative, analgesic, anticonvulsant, antischizophrenic, alcohol, nicotine and caffeine deaddiction

(a) Anxiolytic (b) Nootropic

Anxiolytic

—

—

Anxiolytic and antiepileptic

Psychotherapeutic activity

(a) Non-opiate pathway (b) GABA receptor binding

—

—

[342]

[338341]

[337]

[336]

[335]

—

—

[334]

[333]

—

—

GABAA – BZD receptor, Inhibition of GABA transaminase

[332]

[331]

In vitro radioligand receptor binding and enzyme assays such as acetylcholine esterase, choline acetyl transferase, monoamine oxidase A and B. Selectively on GABAA, NMDA and MAO receptors

[330]

—

[329]

—

(a) Analgesic, antipyretic, antianxiety, hypnotic (b) Hallucinogen, stimulant (c) Antipyretic, antianxiety (d) Hallucinogen (e) Antianxiety (f) Hypnotic Anxiolytic, antidepressant, neuroleptic, antidementia

Ref.

Mode of action reported

Therapeutic activity reported

—

—

—

(a) Flavonoids (b) Alkaloids (c) Essential oil (d) Lignans (e) Tannins (f) Triterpene and saponins

Phytoconstituents reported

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders



Phytochemical reports, Pharmacological reports

Pharmacological reports

Phytoconstituents, Pharmacological reports

Phytoconstituents, Pharmacological reports

Phytoconstituents, Pharmacological reports

Phytoconstituents, Pharmacological reports, Mode of action

Phytoconstituents, Pharmacological reports

Phytoconstituents, Pharmacological reports, Mode of action

Phytoconstituents, Pharmacological reports, Mode of action

22

23

24

25

26

27

28

29

30

Zizyphus jujuba

—

—

—

—

—

—

—

Matricaria recutita

—

Anxiolytic

Wogonin (Flavonoid)

Anxiolytic

BZD binding site of GABAA and modulation of receptor activity

[351]

[350]

— Sedative, anxiolytic, antinociceptive, anticonvulsant, hallucinogenic

Terpenoids: Monoterpenoids (linalool, α-thujone, borneol, valepotriates); Sesquiterpenoids (valerenic acid, artemisinin); Diterpenoids (ginkgolides, forskolin, salvinorine A); Triterpenoids (ginsenosides); Meroterpenoids (cannabinoids)

[349]

—

Antinociceptive, memory enhancing, anxiolytic

Food proteins – δ-opioid peptides, gluten, exorphins, rubiscolins

[348]

Anxiolytic

[347]

[346]

[345]

[344]

[343]

Flavonoids

BZD site on GABAA

—

Sedative, hypnotic, anxiolytic, antidepressant, antipsychotic, anticonvulsant

Cannabinoids, δ9tetrahydrocannabinol, cannabidiol Flavonoids – chrysin, apigenin and semisynthetic derivatives of flavone

—

Narcotic-anxiolytic, hallucinogenic

Alkaloids in Sceletium and Mesembryanthamaceae

—

—

Antioxidant, antimicrobial, antiplatelet, antiinflammatory, antimutagenic, antispasmodic, anxiolytic, cholesterol lowering Anxiolytic, sedative, hypnotic, aphrodisiac, anticancer, hypotensive, antiinflammatory

—

Flavonoids, phenolic compounds, essential oil

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

43

Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders

(c) chemical constituents responsible for antianxiety activity have been reported in 53 plants and (d) possible mechanism of action has been reported in 41 plants. Seven formulations of plant drugs, 02 well known classes of phytoconstitunts and 03 pure constituents present in various plants, and nutraceuticals reported to possess antianxiety activity in battery of experimental models of anxiety have been compiled in present work (Table 2). Twelve anxiolytic formulations containing plants have been patented (Table 3). A survey of literature revealed that 30 review articles have been published on anxiolytic plant formulations and specific plant covering broad aspects as phytochemistry, pharmacology, clinical studies, toxicology and safety profiles (Table 4). This review article would of immense help to natural product researchers to select traditionally used and clinically potential plants for their future research work.

AKNOWLEDGEMENT Authors are grateful to Mr Rakesh Chawla, Lecturer, S.D. College of Pharmacy, Barnala for providing necessary full research articles for compilation of this review.

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311. Rathod MR, Shethia BD, Pandya JB, Dodiya PJ, Palit G, Chatterjee M, et al. Process for the preparation of herbal extract of Cassia tora leaves for treating anxiety disorders. PCT Int Appl, WO 2010109318 A1 20100930, 35p; 2010. 312. Takeda H,Tsuji M, Hayashi M, Inazu M,Yamada T, Miyamoto J, et al. Antidepressant/ antianxiety agents containing rosmarinic acid. Japanese Patent JP 2002275061 A20020925, 7 p; 2002. 313. Bueter B. Plant extract.PCT Int. Appl. WO 2002007743 A2 20020131; 2002. 314. Farrington D, Farrington T. An anti-anxiety homeopathic complex. PCT Int Appl, WO 2009047004 A1 20090416, 77p; 2009. 315. Cheng Y, Mei R, Chen X, Zhao J. Method for manufacturing antianxiety agent containing extract of Rumex madaio as active ingredients. Chinese Patent CN 101972310 A 20110216, 10p; 2011. 316. Finzelberg GmbH & Co. KG, Germany. Lozenges containing plant extracts and essential oils as anxiolytics and aroma-therapeutic agents. German Patent DE 202004013132 U1 20050310, 3 p; 2005. 317. Shelby NJ, Godfrey MT, Rosenfeld MJ. Methods for inducing anti-anxiety and calming effects in animals and humans. U.S. Patent US 20050250772 A1 20051110, 34 p; 2005. 318. Nowak G. Herbal medicines with anti-anxiety and antidepressant activity. Herb Pol. 2009; 55(1):84-97. 319. Stafford GI, Jager, AK, van Staden J. African psychoactive plants. African Nat Plant Prod. 2009 Dec 20; 1021:323-46. 320. Carlini EA. Plants and the central nervous system. Pharmacol Biochem Behav. 2003 Jun; 75(3):501-12. 321. Faustino TT, de Almeida RB, Andreatini R. Medicinal plants for the treatment of generalized anxiety disorder: a review of controlled clinical studies. Rev Bras Psiquiatr. 2010 Dec; 32(4):429-36. 322. Ozarowski M, Mikolajczak PL, Kujawski R, Bobkiewicz-Kozlowska T, Mrozikiewicz PM. The influence of biologically active compounds of medicinal plants on the central nervous system receptors-basis of potential interaction with synthetic drugs mechanisms. Herb Pol. 2008; 54(3):113-36. 323. Weeks BS. Formulations of dietary supplements and herbal extracts for relaxation and anxiolytic action: Relarian. Int Med J Exp Clin Res. 2009 Nov; 15(11):RA256‑62. 324. Abascal K, Yarnell E. Nervine herbs for treating anxiety. Altern Complement Ther. 2004 Dec; 309-15. 325. Ernst E. Herbal remedies for anxiety - a systematic review of controlled clinical trials. Phytomedicine. 2006 Feb; 13(3):205-8. 326. Sousa FC, Melo CTV, Cito MCO, Felix FHC, Vasconcelos SMM, Fonteles MMF, et  al. Medicinal plants and their bioactive constituents: a scientific review of bioactivity and potential benefits in the anxiety disorders in animal models. Rev Bras Farmacogn. 2008; 18(4):642-54. 327. Rodrigues E, Tabach R, Galduroz JCF, Negri G. Plants with possible anxiolytic and/or hypnotic effects indicated by three Brazilian cultures -Indians, AfroBrazilians, and river-dwellers. Stud Nat Prod Chem. 2008; 35:549-95. 328. Gould EM, Parkar S, Crawford K, Forbes D, Skinner MA, Scheepens A. Plant based ‘mood foods’- targeting anxiety. Curr Top Nutraceutical Res. 2008; 6(1):29‑40. 329. Rodrigues E, Mendes FR, Negri G. Plants indicated by Brazilian Indians for disturbances of the Central Nervous System: a bibliographical survey. Curr Med Chem Cent Nerv Syst Agents. 2006; 6(3):211-44. 330. Zhang ZJ. Therapeutic effects of herbal extracts and constituents in animal models of psychiatric disorders. Life Sci. 2004 Aug 20; 75(14):1659-99. 331. Misra R. Modern drug development from traditional medicinal plants using radioligand receptor-binding assays. Med Res Rev. 1998 Nov; 18(6):383-402. 332. Awad R, Ahmed F, Bourbonnais-Spear N, Mullally M, Ta CA, Tang A, et al. Ethnopharmacology of Q’eqchi’ Maya antiepileptic and anxiolytic plants: Effects on the GABAergic system. J Ethnopharmacol. 2009 Sep 7; 125(2):257-64. 333. Kinrys G, Coleman E, Rothstein E. Natural remedies for anxiety disorders: potential use and clinical applications. Depress Anxiety. 2009; 26(3):259-65. 334. Schilcher H. Psychopharmaceuticals of plants origin. A new classification according to indication groups. Dtsch Apoth-Ztg. 1995; 135(20):17-20, 25-8. 335. Can OD, Ozturk Y, Ozkay UD. Farmakoloji AD, Eczacilik F. A natural antidepressant: Hypericum perforatum L.: review. Turkiye Klinikleri Tip Bilimleri Dergisi. 2009; 29(3):708-15. 336. Sarris J, LaPorte E, Schweitzer I. Kava: a comprehensive review of efficacy, safety, and psychopharmacology. Aust N Z J Psychiatry. 2011 Jan; 45(1):27-35. 337. Stevinson C, Huntley A, Ernst E. A systematic review of the safety of kava extract in the treatment of anxiety. Drug saf. 2002; 25(4):251-61. 338. Cauffield JS, Forbes HJ. Dietary supplements used in the treatment of depression,  anxiety and sleep disorders. Lippincott’s Prim Care Pract. 1999 May‑Jun; 3(3):290-304.

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339. Pittler MH, Ernst E. Efficacy of kava extract for treating anxiety: systematic review and meta-analysis. J Clin Psychopharmacol. 2000 Feb; 20(1):84-9. 340. Pittler MH, Edzard E. Kava extract for treating anxiety. Cochrane Database Syst Rev. 2001; CD003383. 341. Singh YN, Singh NN. Therapeutic potential of kava in the treatment of anxiety disorders. CNS Drugs. 2002; 16(11):731-43. 342. Anonymous. Piper methysticum (kava kava). Altern Med Rev. 1998; 3(6):458-60. 343. McKay DL, Blumberg JB. A review of the bioactivity and potential health benefits of chamomile tea (Matricaria recutita L.). Phytother Res. 2006 Jul; 20(7):519-30. 344. Mahajan RT, Chopda MZ. Phyto-pharmacology of Ziziphus jujuba Mill - a plant review. Phcog Rev. 2009; 3(6):320-9. Available from: http://www.fitoica.com/ Biblioteca/Revistas/Pharmacognosy%20review/Pharmacognosy/2009/N2/10.pdf 345. Smith MT, Crouch NR, Gericke N, Hirst M. Psychoactive constituents of the genus Sceletium N.E.Br. and other Mesembryanthemaceae: a review. J Ethnopharmacol. 1996 Mar; 50(3):119-30.

346. Ashton CH, Moore PB, Gallagher P, Young AH. Cannabinoids in bipolar affective  disorder: A review and discussion of their therapeutic potential. J Psychopharmacol. 2005 May; 19(3):293-300. 347. Paladini AC, Marder M, Viola H, Wolfman C, Wasowski C, Medina JH. Flavonoids and the central nervous system: from forgotten factors to potent anxiolytic compounds. J Pharm Pharmacol. 1999 May; 51(5):519-26. 348. Jager AK, Saaby L. Flavonoids and the CNS.Molecules. 2011 Feb 10; 16(2):1471‑85. 349. Yoshikawa M, Takahashi M, Yang S. Delta opioid peptides derived from plant proteins. Curr Pharm Des. 2003; 9(16):1325-30. 350. Passos CS, Arbo MD, Rates SMK, von Poser GL. Terpenoids with activity in the central nervous system (CNS). Rev Bras Farmacogn. 2009; 19(1A):140-9. 351. Tai MC,Tsang SY, Chang LYF, Xue H.Therapeutic potential of wogonin: a naturally occurring flavonoid. CNS Drug Rev. 2005 Summer; 11(2):141-50.

ABBREVIATIONS ADAA American Psychiatric Association APA Anxiety Disorders Association of America ASI Anxiety Status Inventory BRC Baroreflex control of heart rate BZD Benzodiazepines BoEAS Boerner Anxiety Scale CNS Central nervous system CGI Clinical Global Impression dlPAG Dorsolateral peri aqueductal ECG Electrocardiogram EEG Electroencephalographic EPM Elevated plus maze EZM Elevated zero maze EAAS Erlanger Anxiety, Tension, Aggression Scale FRA Federal Regulatory Authorities FST Forced swimming test GABA Gamma-amino butyric acid GAD Generalized anxiety disorder GRAD Global Research on Anxiety and Depression HAMA Hamilton Anxiety Scale



HBT HADS 5-HT1A i.p. LDM MBT MAO NOS OCD OFT PD PTZ p.o. PTSD SARA SI SAD SP s.c. TDS

Hole board test Hospital Anxiety and Depression Scale 5-hydroxytryptamine 1A Intraperitoneally Light / Dark model Marble burying test Monoamine oxidase Nitric oxide synthase Obsessive–compulsive disorder Open field test Panic disorder Pentylenetetrazole Per oral Post-traumatic stress disorder Self Assessment of Resilience and Anxiety Social interaction Social phobia or Social anxiety disorder Specific phobia Subcutaneously Thrice daily

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Pharmacognosy Communications

www.phcogcommn.org

Volume 1 | Issue 1 | Jul-Sep 2011

Review Article Problems of Reproducibility and Efficacy of Bioassays Using Crude Extracts, with reference to Aloe vera I. E. Cocka,b* a Biomolecular and Physical Sciences, Nathan Campus, Griffith University, 170 Kessels Road, Nathan, Queensland 4111, Australia. bEnvironmental Futures Centre, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111,Australia

ABSTRACT: Aloe vera has a long history of medicinal usage and its biological activities have been well documented in a variety of bioassays. However, isolated Aloe vera leaf components generally do not display the same bioactivities, or have lower efficacies than crude juice/extracts. It is likely that several components work in a synergistic manner in the crude mixture, resulting in increased bioactivities. Furthermore, different laboratories often report varying bioactivities using the same extraction procedure on plant material from the same species. Individual Aloe vera cultivars may have widely varying levels of the bioactive phytochemicals. Due to the structure and chemical nature of many of the Aloe vera phytochemicals, it is likely that many of its reported medicinal properties are due to anti-oxidant or pro-oxidant effects. The anti-oxidant/prooxidant activities of many of Aloe vera’s phytochemicals is dependent not only on their individual levels, but also on the ratios of various components, and on their individual redox states. Therefore, discrepancies between bioactivity studies are likely when using different crude mixtures. The potential differences between these crude mixtures need to be taken into account when analysing the reproducibility and efficacy of bioassays of crude extracts. KEY WORDS: Aloe barbadensis Miller, Aloe vera, anti-oxidant, pro-oxidant, medicinal plant, crude extracts.

Introduction Plants have a long history of usage as medicinal agents and were the main source of medicines prior to the advances of modern medicine. In many developing countries, herbal medicinal systems remain important in the treatment of many ailments. Ayuvedic medicine is still commonly practiced within India with an estimated 85% of Indians still using crude plant preparations for the treatment of a wide variety of diseases and ailments.[1] Traditional Chinese medicine (TCM) and African medicinal systems also account for a major portion of health care in these regions. Even in countries where allopathic/Western medicine is dominant, much is also owed to plant medicinal systems. Furthermore, many users are returning to herbal medicinal systems due to the perception that natural medicines are often safer than allopathic drugs, as well as seeking treatments to diseases for which modern medicine does not yet have solutions. Many of the prescription drugs currently marketed for a wide variety of ailments were originally isolated from plants or are

*Correspondence: Tel.: +61 7 37357637; fax: +61 7 37355282. E-mail: [email protected] (I. E. Cock). DOI: 10.5530/pc.2011.1.3

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semi-synthetic analogues of phytochemicals. It has been estimated that approximately 25% of all prescription drugs currently in use are of plant origin.[2,3] Furthermore, approximately 75% of new anticancer drugs marketed between 1981 and 2006 were derived from plant compounds.[3] Traditionally, plant based medicines have been used as crude formulations such as infusions, tinctures and extracts, essential oils, powders, poultices and other herbal preparations. The current trend is to isolate and characterise the individual phytochemical components with the aim of producing an analogue of increased bioactivity/bioavailability. Such studies have given rise to many useful drugs such as quinine (from Cinchona spp.) and digoxin (from Digitalis spp.) as well as the anticancer drugs vincristine and vinblastine (from Vinca rosea). However, the bioactivities seen for crude extracts are often much enhanced, or even totally different to those seen for the individual components.[4,5] Crude plant extracts may contain hundreds, or even thousands of different chemical constituents that interact in complex ways. Often it is not known how an extract works, even when its therapeutic benefit is well established. The study of crude extracts is itself fraught with difficulties. Plants grown under varied conditions will often produce different phytochemical profiles, or at least different quantities of the individual components.[6,7] Similarly, different cultivars within a

(c) Copyright 2011 EManuscript Publishing Services, India

Cock: Problems of Reproducibility and Efficacy of Bioassays

species may also produce different levels of other bioactive components or other constituents which enhance/counteract their medicinal activities.[8] Therefore, the bioactivity of crude extracts may be reliant on the conditions in which the plant grows, the season, and the individual plant itself. Other contributing factors may even include induced chemical defences against predators or pathogens. The extraction procedure, treatment and handling of crude plant extracts may also affect the condition and therefore the bioactivity/efficacy of the phytochemical components. Most plant extracts contain a complex mixture of terpenes, phenolic compounds and alkaloids, many of which can undergo oxidation/reduction processes.[6] The alteration of the redox state may change the behaviour of phytochemicals. Indeed, the maintenance of cellular redox state has been associated with the treatment and prevention of many diseases and ailments including atherosclerosis, inflammatory injury and cancer,[9,10] cardiovascular disease[11] and neurological degenerative disorders.[12] Redox control is also linked with diabetes/anti-diabetic bioactivities[13] and has been associated with the reduction of obesity.[14] Antioxidants can directly scavenge free radicals, protecting cells against oxidative stress related damage to proteins, lipids and nucleic acids.[15] The following discussion will examine some problems associated with reproducibility and efficacy of using crude extracts in bioassays, with reference to the well characterised medicinal plant, Aloe vera.

Variability in bioactivity and efficacy of crude Aloe vera extracts Aloe barbadensis Miller (commonly known as Aloe vera) has a long history of usage as a food, cosmetic and as a medicinal agent. Amongst its noted therapeutic activities, Aloe vera has been reported to have anti-bacterial,[16,17] anti-fungal,[16] antiviral,[18,19] immune-stimulatory,[20] anti-inflammatory[21,22] and

anti-diabetes[23] bioactivities. However, many studies examining the therapeutic potential of Aloe extracts report conflicting results, showing either a lack of therapeutic bioactivity for some Aloe species,[24] or even toxicity associated with some Aloe vera preparations.[25-27] It is well known that plant age is an important determinant of Aloe vera bioactivities. With respect to anti-oxidant potential, the bioactivity has been shown to fluctuate within a given cultivar in relation to the age of the plant, with highest anti-oxidant levels reported for 3 year old plants.[28] This is complicated further as the relative levels of a plants anti-oxidant phytochemicals also fluctuates seasonally.[29] Furthermore, the phytochemical profiles of individual plants will change, dependent on a variety of other environmental and growth conditions.[6,7] Plants may produce a wide variety of secondary metabolites which have no apparent role in primary plant growth or development processes. These molecules are often unique to plants from a single species and increase during times of high stress such as drought, fire and bacterial infection.[6] Therefore, whilst Aloe vera plant growth may be optimal during times of good growth conditions, it is likely that the level of useful bioactive phytochemicals will be elevated in conditions which stress the plant. Many of these secondary metabolites may exhibit anti-microbial, antioxidant, cytotoxic and other medicinally useful properties.[6]

A. barbadensis phytochemistry Anthraquinones

Many bioactive phytochemical components have been isolated from Aloe vera leaves and their bioactivities extensively examined. In particular, the anthraquinones, anthrones and chromones have been particularly well studied and have been shown to be effective at counteracting various disease states.[21,30] The anthraquinones aloe emodin (Figure 1a) and aloin (Figure 1b)

Figure 1: Chemical structures for the anthraquinones (a) aloe emodin and (b) aloin, the chromone (c) aloesin, (d) anthrone, (e) cinnamic acid and (f) β-sitosterol.



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Cock: Problems of Reproducibility and Efficacy of Bioassays

are thought to exert their reported therapeutic potentials via an anti-oxidant mechanism. For example, aloe emodin has high inhibitory free radical scavenging activity and has been shown to act as an anti-oxidant by inhibiting lipid peroxidation.[31]

acid derivatives also have concentration dependent anti-oxidant/ pro-oxidant activities. Cinnamic acid derivatives behave as antioxidants at lower concentrations, but convert to pro-oxidants at concentrations above 5 µM.[44]

Interestingly, aloe emodin and aloin have been shown to be capable of behaving as either an anti-oxidant or as a pro-oxidant, with their action being dependent upon their concentration.[32] Aloe emodin exerts anti-oxidant behaviour at lower concentrations, yet acts as a pro-oxidant at high concentrations. In contrast, aloin has an anti-oxidant effect at higher concentrations, yet a pro-oxidant effect at low concentrations. Thus, the variable effects reported for crude Aloe vera extracts in various studies may be due to differing level of aloe emodin and/or aloin present in the extract.

In contrast, Yen et al. (2000) demonstrated that the chemical structure of anthrone (Figure 1d) predisposes it to function as an electron acceptor (electrophile), hence as a strong anti-oxidant, independent of its concentration within an extract.[31] It therefore remains possible that Aloe vera extracts with high concentrations of anthrone may maintain anti-oxidant potential, even under conditions which would otherwise predispose the extract to function as a pro-oxidant. For example, Aloe vera extracts containing high aloe emodin and low aloin concentrations (both of which favour pro-oxidant bioactivity) may still function as an anti-oxidant if high enough levels of anthrone are present to maintain the redox state of these anthraquinones. Conversely, low levels of anthrone may predispose an extract to display pro-oxidant activities. It is therefore likely that the redox character of an extract is not only dependent on the levels of the different phytochemicals present, but also on the ratios of several important components within the mixture.

Other phenolic Aloe vera constituents

Similar pro-oxidant effects have been reported for other antioxidant phytochemicals including flavonoids,[33] tannins[34] and curcumin.[35] Previous studies have shown that transition metal ions, such as copper or iron, can enhance the conversion of the anti-oxidant to the pro-oxidant state.[36,37] The pro-oxidant/antioxidant effect of plant extracts is due to a balance between the free radical scavenging activities and reducing power of their phytochemical components. This can be explained using the anti-oxidant vitamin ascorbic acid as an example. Although ascorbic acid has well characterised anti-oxidant bioactivities, it is also known to act as a pro-oxidant at high concentrations.[38] This is due to the greater reducing power of ascorbic acid compared to its free radical scavenging activity. In the presence of transition metal ions, ascorbic acid will function as a reducing agent, reducing the metal ions. In the process, it is converted to a pro-oxidant. Therefore, high dietary intake of ascorbic acid in individuals with high iron levels (e.g. premature infants) may result in unexpected negative health effects due to the induction of oxidative damage to susceptible biomolecules.[39-41] The anti-oxidant activity of aloesin (Figure 1c) and other chromones has also been extensively described.[42,43] In contrast, a literature search did not reveal any studies examining the potential pro-oxidant activity of these compounds. One study reported several chromones to have higher reducing power than ascorbic acid.[42] The relatively high reducing power of ascorbic acid is believed to be responsible for its ability to function as a pro-oxidant.[38] It is therefore possible that aloesin and other Aloe vera chromones may have a similar anti-oxidant/pro-oxidant profile to ascorbic acid (i.e. anti-oxidant activity at lower concentrations and pro-oxidant activity at higher concentrations). However, it must be emphasised that this possibility is based on the reported higher reducing power of the chromones compared to their free radical scavenging activity[42] and has not been adequately tested. Cinnamic acid (Figure 1e) and its derivatives are phenolic molecules which are present in many fruits, vegetables and whole grains, as well as in Aloe vera leaves. Studies indicate that cinnamic 54

Aloe vera leaves also contains a number of other medicinally important phytochemicals including β-sitosterol (Figure 1f) and β-sitosterol glucosides. These phytosterols have been shown to promote arterial endothelial cell proliferation.[45] They also promote the expression of proteins involved in angiogenesis and thus have potential applications in the management of chronic wounds. Recently, β-sitosterol has also been trialled for the treatment of breast cancer[46] and diabetes,[47] although the efficacy is still under investigation. It appears that these therapeutic bioactivities may be due, at least in part, to their redox state of the molecule. A recent study has indicated that β-sitosterol treatment results in glutathione reduction as well as maintaining the anti-oxidant enzymes superoxide dismutase and glutathione peroxidise in a reduced state.[48] This bioactivity in turn is related to the redox state of the sterol. Interactions between the various components within the crude extracts may also play a role in converting otherwise anti-oxidant molecules into pro-oxidants in the extract or vice versa. Non-phenolic components

Other phytochemical components of Aloe vera leaf extracts include acemannan (Figure 2), a long chain polymer of β (1→4) linked galactomannan saccharides.[49,50] Acemannan has been reported to accelerate wound healing,[51-54] activate macrophages[55,56] and have synergistic anti-viral activity in combination with azidothymidine and acyclovir.[19] It has been reported that acemannan also has anti-oxidant properties and that these properties may be responsible for its therapeutic activities.[57] Furthermore, the anti-oxidant potential of Aloe vera polysaccharides is dependent upon the concentration of the molecule and the degree of acetylation of the monomeric units.‌[58] High polysaccharide concentrations (>8 mg mL-1) were found

Cock: Problems of Reproducibility and Efficacy of Bioassays

Figure 2: The structure of acemannan (a major polysaccharide component of Aloe vera leaves) consists of a polymer of β (1→4) linked galactomannan sugars.

to be necessary for Aloe vera polysaccharides to display antioxidant activity. The same study also showed that increased acetylation enhances the anti-oxidant activity of Aloe vera polysaccharides.[58] However, the polysaccharide components within Aloe vera leaves are not constant. Instead, the composition and concentration of the polysaccharides fluctuate with changes in the growing environment and conditions. Aloe vera leaves also contain inorganic minerals in variable concentrations (e.g. calcium, magnesium, zinc, iron and copper). As previously discussed, the redox state of many Aloe vera phytochemicals is affected by the presence of metal ions, converting otherwise anti-oxidant components into pro-oxidants. Thus, Aloes growing in soil containing elevated levels of metallic ions would be expected to have higher concentrations of metal ions, and thus tend towards pro-oxidant rather than anti-oxidant bioactivities. Other molecules (such as vitamins, amino acids and proteins) may also have an effect on the redox state of the phytochemical components.

Medicinal effects of Aloe vera requiring multiple phytochemicals The multitude of phytochemicals present in an Aloe vera crude extract not only affect each others redox state and ability to function as an anti-oxidant/pro-oxidant, but several phytochemicals may also be required for different aspects of the same therapeutic effect. Some of the medicinal properties associated with plant extracts require the concerted action of several bioactivities. The following discussion examines several therapeutic properties of Aloe vera extracts that require the synergistic action of several bioactivities, each of which may be reliant on multiple phytochemicals. This is by no means a complete examination of the therapeutic properties of Aloe vera extracts, but instead serves to illustrate the difficulties of assigning a therapeutic effect to a single component. Similarly, it further illustrates the 

problems with reproducibility when analysing crude extracts by bioassay due to differences in the levels of specific phytochemicals, their redox state, and their ratio to other components. Anti-inflammatory Activity

Inflammation is a complex response by the body to injury. It typically follows a variety of insults including burns, wounds, bites and stings etc. It is characterised by a wide variety of symptoms[59] including: • Swelling. Injury may result in increased capillary permeability which allows leukocyte migration and fluid accumulation in the damaged tissue. This accumulation results in the swelling characteristic of inflammation. • Redness and heat are caused by vasodilation, reducing blood pressure and increasing circulation. • Pain is a complex reaction resulting from the release of short peptides and prostaglandins. These inflammatory processes require the cellular release of several classes of molecules. Vasoactive substances (e.g. bradykinin, prostaglandins and vasoactive amines) are required to dilate blood vessels, opening junctions between cells to allow leukocytes to pass through capillaries. Any compound capable of blocking these vasoactive substances would potentially have a therapeutic effect on the symptoms of inflammation. β-sitosterol is the most abundant phytosterol in Aloe vera extracts. β-sitosterol stimulates smooth muscle cells to release of prostacyclin (PGI2).[60] However, β-sitosterol treatment blocks the release of PGI2 and prostaglandin E2 (PGE2) from macrophages.[60] Thus, β-sitosterol treatment would be expected to affect vasodilation and, therefore, have a therapeutic effect on inflammation. The Aloe vera leaf chromone aloesin, and its derivatives, inhibit cyclooxygenase-2 and thromboxane A2 synthesis through their anti-oxidant activities.‌[61,62] Thus, Aloe vera chromones produce anti-inflammatory effects. In contrast, anthraquinones have been shown to stimulate PGE2 release[63] and would, therefore, be expected to promote pro-inflammatory activity. 55

Cock: Problems of Reproducibility and Efficacy of Bioassays

The peptidase bradykinase has been isolated from Aloe vera leaves and has been shown to break down the vasoactive peptide bradykinin.[64,65] As bradykinin treatment results in vasodilation, hydrolysing this protein would result in decreased vasodilation and, therefore, inhibit leukocyte passage and fluid leakage from the capillaries into the surrounding tissue. Aloe vera leaf bradykinase would, therefore, be expected to contribute to the therapeutic effects on the symptoms of inflammation. Chemotactic factors, including several proteins and peptides, are required to increase cell motility, especially the motility of leukocytes during inflammation. Blocking these chemotactic factors, or blocking their effects, prevents inflammatory swelling. Several compounds in Aloe vera extracts have been shown to be capable of blocking chemotaxis. Anthraquinones suppress cytolytic T-lymphocytes in favour of suppressor cells.[66,67] Furthermore, anthraquinones decrease cytokine production and IL-2 mRNA expression in activated T lymphocytes,[68] thereby decreasing chemotaxis. More recent studies have demonstrated that the anthraquinone emodin decreases plasma levels of the cytokines IL-2 and TNF-α, whilst increasing IL-10 (which itself down-regulates IL-2 and TNF-α cytokine activity).[69] None of these studies, however, examined the relationship of the redox state of the anthraquinones with these effects. Furthermore, these studies have not rigorously examined the effects of a range of doses of these phytochemicals. In contrast, Aloe vera polysaccharides (including acemannan) have a stimulatory effect on chemotaxis. Acemannan exposure stimulates cytokine production and activates lymphocytes.[70,71] Specifically, pure acemannan isolated from Aloe vera leaves has been shown to stimulate macrophages to release IL-1, IL-6, interferon, GM-CSF and TNF-α in vitro.[72] Similarly, Aloe vera lectins stimulate cytokine production. Aloctin A, the best characterised of the Aloe lectins, has been shown to stimulate the production of IL-2[73] and to enhance the production and activation of macrophages.[73] Therefore, Aloe vera extracts contain both chemotactic stimulatory and inhibitory compounds. The chemotactic effect of Aloe vera extracts would, therefore, be dependent on the levels and ratios of the factors affecting chemotaxis as well as their redox state. Aloe vera extracts contain multiple active phytochemicals. It  is  likely that several of these may be required to address different aspects of the inflammatory process. Failure to consider this is likely to be responsible for past ambiguities about the efficacy of Aloe extracts in relation to its anti-inflammatory activity. Antiseptic activity

The interruption of the external epidermal barrier by a wound, burn or other such event allows microbes to enter and infect the wound. The invasion of microorganisms may cause or intensify inflammation (described in section 4.1.) and may hinder wound healing (described in section 4.3) and/or cause disease. 56

Aloe vera leaf extracts have been previously shown to display good anti-bacterial[16,74,75] and anti-fungal bioactivities.[16,76] Early anti-bacterial studies of Aloe vera extracts have provided confounding and even contradictory results. Some of these studies indicate that the bioactive agent(s) are anthraquinones,[77,78] whilst other studies found Aloe vera anthraquinones to be inactive as anti-bacterial agents.[79] Numerous subsequent studies have demonstrated the anti-bacterial activity of isolated anthraquinones from Aloes[80,81] and various other plant species.[82-84] Whilst the mechanism of anti-bacterial activity is still subject to investigation, it has been suggested that aloe emodin and aloesin function by inducing bacterial membrane disruption.[80] This study also determined that the form of aloe emodin and aloesin tested also affects their anti-bacterial activity. It was demonstrated that anthraquinone loaded liposomes had strong anti-bacterial activity, whilst the purified free anthraquinones did not. It is, therefore, possible that some of the observed differences in the anti-bacterial activities of anthraquinones and Aloe vera extracts may be due to the form of anthraquinones that the bacteria were tested against. Whilst this study showed that anti-bacterial activity is dependent on the form of anthraquinone tested, the effect of concentration was not extensively examined. MIC values were determined by testing across a range of concentrations, although only relatively low concentrations were tested. It is possible that higher concentrations may have a very different effect, analogous to the concentration effects already described for anthraquinone anti-oxidant/pro-oxidant activity. Other Aloe vera components have also been implicated in the antibacterial activity of leaf extracts. A recent study tested anthraquinone free leaf extracts and isolated components.[85] This study showed that cinnamic acid, coumaric acid, ascorbic acid and pyrocatechol purified from Aloe vera gel all display good anti-bacterial activity, especially towards Gram-positive bacteria. It was postulated that the phenolic anti-bacterial agents functioned by disrupting bacterial cell membranes, as well as by denaturing bacterial proteins. Furthermore, cinnamic acid is known to block bacterial glucose uptake and ATP production,[86] therefore, inhibiting bacterial growth. Coumaric acid has been shown to inhibit bacterial enzymatic activity.[87] A number of other phenolic components were also found to have low to moderate anti-bacterial activity. In addition to direct inhibitory effects on bacteria, Aloe vera components may also function by selectively modulating the cells of the immune system (described in detail in section 4.4). Furthermore, acemannan also inhibits bacteria adhering to epithelial cells and establishing an infection.[88] It is likely that the anti-bacterial activity of Aloe vera extracts in vivo is due to the synergistic effects of multiple bioactive components, functioning through several mechanisms. Anti-fungal activity has received less attention, although some studies have demonstrated the ability of Aloe vera extracts to

Cock: Problems of Reproducibility and Efficacy of Bioassays

inhibit fungal growth.[16,76,89] Anthraquinones, especially aloe emodin and aloesin, were implicated in this anti-fungal activity,[76] however, the identity of anti-fungal components and their mechanisms of action have not been extensively examined. Similarly, the anti-viral activity of Aloe vera leaf extracts has been demonstrated,[18,90] although detailed purification, identification and mechanistic studies are required. Wound Healing

Whilst anti-inflammatory and anti-microbial bioactivities are complex processes requiring the synergistic action of several bioactivities, wound healing is more so. Wound healing, a relatively well studied therapeutic property of Aloe vera, is the result of several bioactivities including: • Inflammation, which has summarised in section 4.1. • Antiseptic bioactivity, which has summarised in section 4.2. • Cell growth and proliferation • Matrix remodelling The growth of endothelial, epithelial and fibroblast cells are critical in wound healing. As a first step in wound healing, a fibrin clot is formed as a temporary repair. This step is vital as it helps avoid microbial infection which may retard the healing process. The wound is subsequently invaded by a variety of cell types, some of which stimulate an inflammatory response, and others which are directly involved in the repair mechanism. [91] The effects of Aloe vera extract components on inflammation processes and chemotaxis have already been summarised in  section 4.1. Wound repair itself occurs in three phases: the migration of epithelial cells and fibroblasts to the wound site, proliferation of cells and cellular maturation. It is likely that the wound healing effect of Aloe vera extracts involves the synergistic action of multiple components on several pathways. Aloe vera anthraquinones reportedly possess contradictory effects on cell growth and proliferation. For instance, Aloe emodin has been shown to stimulate a 2.5 fold increase in rat hepatocyte DNA synthesis with a corresponding increase in cell growth.‌[92] Additionally, aloe emodin has been shown to protect hepatocytes from apoptosis.[69] In contrast, other studies have shown aloe emodin to induce apoptosis in pro-myeloleukemic HL-60 cells[93] and human lung squamous cell carcinoma,[94,95] and to inhibit human neuroectodermal tumour growth.[96] Some studies have postulated that the pro-apoptotic effect of aloe emodin is due to an induction of caspase 3 activity, together with a decrease in the levels of the anti-apoptotic protein Mcl‑1.‌[93] Another study has implicated caspase 8 mediated cleavage in the apoptotic activity of emodin.[97] Studies into the pro-apoptotic mechanism of aloe emodin are ongoing. Similarly, anthrones have also been shown to induce cell death. In a recent study, an anthrone from the Ethiopian medicinal plant Kniphofia foliosa was shown to  induce  rapid death in mouse and human cancer cells via necrosis.‌[98] 

Other Aloe vera phenolic compounds have also been implicated in the wound healing effects of Aloe vera extracts. β -sitosterol and β-sitosterol glucosides promote endothelial cell proliferation and angiogenesis,[45] although their activity appears to be dependent on its redox state.[48] The reduced sterol has anti-oxidant activity and stimulates wound healing processes, whilst oxidised sterols are pro-oxidants and induce cell death. β -sitosterol and β-sitosterol glucosides, therefore, have potential applications in wound management in their reduced state. The Aloe vera chromone aloesin has also been reported to stimulate cellular proliferation.‌[51,61,99] It is possible that the proliferative effect of aloesin is due to its high anti-oxidant activity.[42,43] In contrast, cinnamic acid has been shown to down-regulate expression of cell proliferation and anti-apoptotic gene products, although the affects of both high and low concentrations were not examined.‌[100,101] The redox environment affects cellular signal transduction, DNA and RNA synthesis, protein synthesis, enzyme activation, regulation of the cell cycle, ligand binding, DNA binding and nuclear translocation, and therefore ultimately cell proliferation/ death.[102,103] Transcription factors are active in their reduced form and their translocation to the nucleus is also redox dependent.‌[104] A reducing environment favours cellular proliferation whilst an oxidising environment results in an increase in reactive oxygen species, initiating cell death.[105,106] Therefore, extract conditions favouring anti-oxidant activity (e.g. low aloe emodin, high aloin, low cinnamic acid, low ascorbic acid, low transition metal and high anthrone concentrations) would be expected to favour cellular proliferation whilst conditions favouring pro-oxidant activity (e.g. high aloe emodin, low aloin, high cinnamic acid, high ascorbic acid, high transition metal and low anthrone concentrations) would favour cell death. The non-phenolic components, particularly acemannan, have also been shown to have a role in wound healing. For example, the stimulation of gingival fibroblast proliferation has been demonstrated when treating oral wounds with high doses of acemannan.[107] This stimulatory effect was found to be due to an induction in expression of the growth factors KGF-1, VEGF and an increase in collagen expression. This study only examined the effects of relatively high concentrations of acemannan, in the range that would correlate to anti-oxidant activity. As lower concentrations may correlate to pro-oxidant activities, it is possible that the induction of fibroblast proliferation may not be seen at these concentrations. Indeed, as lower concentrations of acemannan correspond to pro-oxidant effects, it is possible that at lower concentrations, cell death may be induced. The concentration dependent redox effect of acemannan may also contribute to the discrepancies seen between proliferative studies of Aloe vera extracts. As well as requiring cellular growth and proliferation, wound healing also requires matrix remodelling. Aloe vera gel extracts have been shown to stimulate and speed up the production of hyaluronic acid and dermatan sulphate.[52] Activities of the enzymes 57

Cock: Problems of Reproducibility and Efficacy of Bioassays

β-glucuronidase and N-acetyl glucosaminidase are increased during wound healing, resulting in increased carbohydrate turnover at the site of the wound. Other studies also demonstrated that wounded diabetic rats treated with Aloe vera gel show increased collagen formation[53] and cross linking.[54] It is evident that a synergistic action is required by several Aloe vera extract components on multiple wound healing associated bioactivities. The reported discrepancies between different studies may be due to differences in concentrations, ratios and redox states of these components. Immunomodulation

Manipulation of the immune system has therapeutic potential in the treatment of a variety of diseases. Aloe vera leaf extracts have been reported to have both good immuno-stimulatory[20] and immune-suppressive activities (as reviewed in Boudreay and Beland[108]); however, rigorous scientific examination of these effects is limited. Much of the studies into the immune-modulatory potential of Aloe vera extracts have focused on the immunestimulatory effects, particularly of the polysaccharide components. Whilst numerous Aloe vera polysaccharide components have been shown to have immune-modulatory effects,[109-111] acemannan has been particularly well studied. The immune-modulatory effects of acemannan are thought to be due to activation of macrophage cells and antigen processing. The activated macrophages secrete cytokines including IL-1, IL-6, interferon, GM-CSF and TNF-α in vitro.[72] The release of these cytokines is itself associated with further pathology through the induction of inflammation. Acemannan also enhances macrophage sensitivity to IFN-γ, inducing apoptosis.[20] Neither acemannan nor IFN-γ was capable of inducing apoptosis alone. Instead, a synergistic effect is required and this effect appears to function through the inhibition of the expression of Bcl-2 proteins.[20] Studies have also highlighted the immune-modulatory properties of the smaller phenolic components of Aloe vera leaves. Aloe emodin and other anthraquinone derivatives have been shown to have an immune-suppressive effect by blocking lymphocyte proliferation.[66,67] Emodin also reduced IL-1, IL-2 and IL-2 receptor expression.[66] It was suggested that emodin suppresses both macrophages and lymphocytes. Further studies have identified 37 other anthraquinones with the ability to block cytolytic T lymphocyte induction and the ability to prevent antibody production.[67] The effect of concentration and the ratio between anthraquinones were not tested in these studies. It has been postulated that Aloe vera extracts may exert immunemodulatory effects through their functioning as anti-oxidants, inhibiting/stimulating the production of free radicals.[28] Treating streptozotocin induced diabetic[112] or gamma-irradiated rats[113] with Aloe vera leaf extracts reduces lipid peroxidation and the formation of hydroperoxides whilst increasing the levels of anti-oxidant enzymes (e.g. reduced glutathione, glutathione peroxidise, glutathione-S-transferase, catalase, superoxide dismutase) in the liver, lungs and kidney. Similarly, Aloe vera gel has been shown to inhibit ROS production in colorectal mucosa 58

cells.[114] Interestingly, this study found the Aloe gel extract lacks this activity at either higher or lower concentrations, indicating a concentration dependence similar to that reported for the redox effects of Aloe vera components.[32] It is, therefore, possible that the variable immune-modulatory effects reported for Aloe vera extracts in different studies may be due to the concentrations, ratios and redox states of several important compounds in the tested extracts, with extract conditions favouring anti-oxidant bioactivity resulting in immune-stimulation. Conversely, conditions favouring pro-oxidant activity would be expected to result in immune-suppression, although this has not been extensively tested. Anti-Diabetic activity

Diabetes mellitus refers to a group of metabolic disorders that result in increased blood glucose concentrations, either because the pancreas does not produce enough functional insulin (type 1 diabetes), or because cells do not respond to the insulin which is produced (type 2 diabetes). The causes of diabetes mellitus include the auto-immune destruction of pancreatic cells,[115] viral infections,[116] genetic and environmental factors,[117] insulin or insulin receptor gene mutations[118] and altered pancreatic prostaglandin metabolism.[119] Diabetes has significant health effects, impacting on the quality of life and life expectancy of those suffering with it. A number of studies have indicated the beneficial effects of Aloe vera extracts in diabetic patients.[23,120] Administration of Aloe vera extracts to streptozotocin-induced diabetic rats resulted in a decrease in blood glucose and a corresponding increase in liver glycogen.[120] The maintenance of glucose homeostasis by Aloe vera extracts in diabetic rats was shown to involve a number of mechanisms. Aloe vera extract treatment altered the activities of multiple enzymes: glycogen phosphorylase activity was decreased and glycogen synthetase increased, resulting in increased hepatic glycogen stores.[120] Hexokinase activity and mRNA levels were decreased in diabetic rats,[121] yet treatment with Aloe vera extract returned these parameters towards normal levels.[120] Similarly, increased lactate dehydrogenase, glucose-6-phosphatase and fructose-1, 6-bisphosphatase activities were seen in diabetic rats.[122] Aloe vera extract treatment significantly restored these enzyme activities.[120] Glycosylation of blood proteins including haemoglobin, albumin and lipoproteins is also characteristic of diabetes mellitus.[123] Under the hyperglycaemic conditions of diabetes mellitus, blood glucose interacts with specific amino acids on the proteins surface, forming glycosylated protein products which may undergo a series of further chemical modifications resulting in the production of advanced glycation end products (AGE).[124] The binding of AGEs to their receptors results in altered cell signalling which in turn results in free radical production.[125] Indeed, diabetes mellitus has been shown experimentally to be associated with an increase in free radical formation and an associated decrease in anti-oxidant potential.[126,127] Studies have directly linked oxidative stress with

Cock: Problems of Reproducibility and Efficacy of Bioassays

the impaired maintenance of glucose homeostasis and the enhanced lipid peroxidation seen in diabetes mellitus.[127] Furthermore, increased total anti-oxidant levels have been measured in the blood and saliva of diabetic patients, further supporting the proposed role of oxidative stress in diabetes mellitus.[128] Oxidative stress induction has also been suggested to be the common link between the diverse medical complications (including cardiovascular disease, renal and neural degeneration, impaired vision and erectile dysfunction) seen in diabetes mellitus.‌[129,130] Therefore, treatment with anti-oxidants would be expected to counteract many of these complications. Aloe vera has a number of compounds (both phenolics and non-phenolic compounds) that can act as anti-oxidants (as described in section 3. - A. barbadensis phytochemistry). As many of these compounds can potentially behave as either anti-oxidant or pro-oxidant dependant on their concentration, redox state and ratio between compounds, it is not surprising that studies using Aloe vera crude extracts to treat diabetes mellitus have had mixed success. Anti- Cancer activity

The growth and development of healthy cells depends on fine regulation of growth promoting and inhibiting pathways. Protooncogenes and tumour suppressor genes are responsible for encoding proteins that regulate cell division/cell cycle, as well as for the repair of damaged DNA and cell programmed death by apoptosis. Mutations within these genes have been implicated in the onset of cancer.[131] Such mutations result in cells that no longer require external signals to proliferate. Furthermore, these cells fail to recognise signals that restrict cell division, resulting in uncontrolled cell growth. In tumour genesis, multiple genes may be altered and transmitted to daughter cells, which subsequently escape normal growth restraints and form a tumour, which may be benign or malignant. The induction of oxidative stress has been linked with several types of cancer.[132,133] Chromosome instability is also a common feature of many of the cancers that have been linked with oxidative stress, suggesting that increased oxidative stress may contribute to development of genetic instability. Oxidative stress leading to genetic instability may result in the emergence of new tumour phenotypes. In such populations, a decrease in apoptosis but an increase in tumour growth and subsequent tumour progression is observable. Currently used anti-cancer agents (e.g. doxorubicin, daunorubicin, mitomycin C, etoposide, cisplatin, arsenic trioxide, ionising radiation, photodynamic therapy) depend exclusively, or in part, on the production of ROS for cytotoxicity. Sensitivity of tumour cells to oxidative stress and/or apoptosis may affect treatment success.[134,135] Studies indicated that WEHI7.2 mouse thymoma cells over expressing catalase (CAT38) or thioredoxin (THX) were resistant to glucocorticoid-induced apoptosis in vitro.[136,137] This suggested that glucocorticoid-induced apoptosis occurred by a ROS dependant/independent mechanism. It was observed 

that average tumour weights increased in severe combined immune-deficient (SCID) mouse tumour xenografts from cells over expressing catalase or thioredoxin.[137] Tumours from both transfectants contained fewer apoptotic cells but mitotic cell numbers were similar. This suggested that anti-oxidant over expression resulted in increased tumour size due to a decrease in apoptosis. The cell proliferation/apoptosis inducing abilities of Aloe vera extracts and isolated components have been described in Section  4.3. Briefly, ROS based tumour therapy may induce regression in apoptosis/oxidatant sensitive tumour cells. Thus, if Aloe vera components were present in concentrations and ratios consistent with pro-oxidant activity, the extract would induce apoptosis and, therefore, would have anti-cancer activity. If the levels of components were consistent with a reducing environment, anti-oxidant activity would result and the extract would not have anti-cancer activity. Conversely, should the protocol be repeated on a tumour with apoptotic resistant/ oxidant resistant cells, the converse would apply and tumour progression would be likely.

Conclusions The problems associated with reproducibility and efficacy of bioassays using Aloe vera juice and/or crude extracts illustrates some of the difficulties encountered in natural products research. Individual extract batches may vary widely with regards to individual phytochemical profiles, ratios between various components, and the redox state of these components. These variances may have profound effects on the reported bioactivities and are likely to account for the reported discrepancies between different studies bioassaying crude mixtures. Despite these difficulties, the use of crude extracts is often necessary as the individual components often do not show the same bioactivities, or have different efficacy to crude extracts. This is true for Aloe vera. Aloe vera juice, or Aloe vera crude extracts, often display higher efficacy than the purified components. It is likely that the biological activity of Aloe vera is a synergistic and perhaps additive action of the different classes of compounds found within the plant, rather than a single constituent or just a few compounds. Furthermore, these compounds are required in the correct levels/ ratios/redox states for bioactivity to be observed.

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Pharmacognosy Communications

www.phcogcommn.org

Volume 1 | Issue 1 | Jul-Sep 2011

Research Article Cassane-type diterpenoids from the genus Caesalpinia R. A. Dickson1*, T. C. Fleischer1, P. J. Houghton2 Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical, KNUST Kumasi, Ghana. 2Pharmacognosy Research Laboratories, Pharmaceutical Sciences Research Division, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK

1

ABSTRACT: Medicinal plants belonging to the Caesalpinia (Ceasalpiniaceae) genus are widely distributed in most tropical countries and have been frequently employed in folkloric medicine worldwide in the treatment of various ailments including skin diseases, malaria, cancer, infections, erectile dysfunction, pain and wounds. Interest in this genus has increased considerably over the years and the biological properties of different phytoconstituents, such as the cassane-type diterpenoid isolates, have been studied. Over the past 60 years, a number of cassane-type diterpenoids have been isolated from species of this genus and some of them possess interesting biological activities. Recently, three novel cassane-type diterpenoids, benthaminin 1, 2 and 3, which demonstrate antimicrobial and antioxidant properties, have been isolated from Caesalpinia benthamiana growing in Ghana. This review seeks among other things to collate all these isolated compounds, recognising their diversity and commenting on their relevance as bioactive compounds. KEY WORDS: Cassane-type diterpenoids, Caesalpinia, Ceasalpiniaceae, Biological activity.

INTRODUCTION Caesalpinia is the name of a genus of enormous size and of ancient origin. It is named after the Italian naturalist, Andreas Caesalpino, of Arezzo (1519-1603). He was also a botanical collector, systematist and philosopher, chief physician to Pope Clement VIII, and a professor of medicine and botany in Pisa and Rome.[1] Caesalpinia consists of about 200 species, consisting of shrubs, tall climbers, small and tall trees, mostly armed with spines and curves, hooked, sharp thorns and rarely unarmed. Their leaves are bipinnate, lacy, and attractive, while the leaflets are few to many, opposite, rarely alternate, small or large, herbaceous or leathery. The flowers are yellow, red, or variegated, showy, handsome, medium to large and are multiflowered. Finally, the pods are variable, often prickly, flat, straight or beaked.[2,3]

THE CAESALPINACEAE FAMILY The leguminous trees fall under the sub-families Caesalpinaceae, Fabiaceae and Mimosaceae. The trees of Caesalpinaceae are by far the most scenic, exhibiting many-coloured splendour. The leguminosae family is currently divided into three subfamilies and 36 tribes. Subfamily Caesalpinioideae comprises of four tribes and 2,250 species, subfamily Mimosoideae four tribes and 3,270 species, and subfamily Papilionoideae 28 tribes and 13,800 species.[2,3] Polhill and Vidal,[4] divided the Caesalpinieae *Correspondence: [email protected]; +233 204620000 DOI: 10.5530/pc.2011.1.4

into 8 informal generic groups: the Gleditsia group (2 genera), the Acrocarpus group (monogeneric), the Sclerolobium group (3 genera), the Peltophorum group (13 genera), the Caesalpinia group (16 genera), the Poeppigia and Pterogyne groups (both monogeneric) and the Dimorphandria group (10 genera). These authors stated that the tribe is ‘a remarkable mixture of relics and complexes of relatively recent speciation, providing many pitfalls for formal systematics and biogeographical interpretations.‌[4] Since 1980, several studies have cast new light on intergeneric relationships within the Caesalpinieae, necessitating the restructuring of some of the nine informal generic groups.[5,6] Without doubt, the genus with the greatest taxonomic and nomenclatural complexity within the Caesalpinieae is the genus Caesalpinia, which in its broadest sense comprises about 140 species and contains 25 generic names in synonymy.[4]

GEOGRAPHICAL DISTRIBUTION OF THE GENUS CAESALPINIA Members of Caesalpinia are widely distributed throughout the tropics and subtropics, primarily in America and Asia, and extending to Australia, Polynesia Madagascar and Africa[7,8] (Table 1). In Africa it is widespread in the western and southern areas with C. benthamiana being the most common. About 25  species are found in the Caribbean, 10 in Cuba and the Bahamas, 2-3 species in Mexico and a few extending to Central America.[4] C. pulcherrima is found in the Philippines and the Caribbean where it is an ornamental plant. It is red with yellow margins, and this variety is the national flower of Barbados, known as ‘Pride of Barbados’.[9]

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Dickson, et. al.: Review on C. major

Caesalpinia is also found in Colombia, Ecuador, Peru, Paraguay and Argentina and is very popular in Brazil. Indeed, the name ‘Brazil’, had its origin in the Portuguese word ‘Bresil’ or ‘brazil’ which means bright red and resembling glowing coals, and was used to describe the colour of Caesalpinia wood abundant in this area.[10] Nine species are widespread in Asia with about two confined to China.[11] C. major is widely distributed in Southeast Asia.[12] The genus Caesalpinia is also popular in Thailand and Indonesia[13] and C. minax, is found in China.[14] Apart from their medicinal importance, Caesalpinia species may also serve as garden ornamentals and hedge plants. The beauty of C. pulcherrima, whose showy red flowers are borne in long spikes, is reflected in its common names: pride of Barbados, paradise flower, Spanish carnation, peacock’s crest, flower tree, and others. This semi-drought-resistant species flowers in favourable habitats when only 8 months old.[10]

ETHNOPHARMACOLOGICAL USES The roots of C. benthamiana are considered to be an effective dysentery remedy in Ghana.[15] The powdered roots are mixed with shea butter or palm kennel oil to treat skin diseases and wounds.[15] An infusion of the dried root is consumed or used in bathing for general malaise in Senegal.[16] In the Philippines and the Caribbean, decoctions of the leaves, bark and roots of C. pulcherrima are used traditionally to treat liver disorders, ulcers of the mouth and throat. It also reduces fevers, acts as an abortificient, and alleviates fungal infections. The fruit is also used to check bleeding and prevent diarrhoea and dysentery.[17] The flowers have also been used to combat oxidative stress by eliminating free radicals from the system.[18] Within southeast Asia, C. major has traditionally been implemented as a tonic, anthelmintic, and for rheumatism and back-ache.[19] In Thailand, the seeds of this plant are used as an expectorant and antitussive agent.[19] C. minax finds use in Chinese folk medicine in the treatment of common colds, fever and dysentery.[20] Also in folk medicine in Kagoshima in Japan, C. decapetala is used in the treatment of neuralgia. A decoction from the pods of C spinosa is used in eye washes in the Callera district of the Czech

Republic.[21] In addition, plant part decoctions are employed in folk medicine to treat intermittent fever and as an abortifacient, emmenagogue, and as a general tonic. Bonducin, an amorphous, white bitter glycoside, is abundant in the seed cotyledons of C. bonduc Roxb., C. bonducella Flem., and C. crista L.[22,23] It is sometimes referred to as “poor man’s. quinine” because it is used as a substitute for quinine in the treatment of intermittent fever. The seeds of C. bonducella are grey, round, smooth and stony. The buoyancy of the seed accounts, in part, for this species being widely dispersed tropically by ocean currents. They are used as talismans and beads, and also by children as marbles. They yield oils for cosmetics and use in medical preparations. It is a shrubby tree of Argentina and Chile, exuding a golden yellow gum that contains approximately 80% arabin. It is completely soluble in water and is an acceptable substitute for gum Arabic.[24] C. echinata Lam. is the national tree of Brazil. The name ‘Brazil’, had its origin in the Portuguese words ‘Bresil’ or ‘brazil’ which means bright red, resembling glowing coals and were used to describe the colour of caesalpinia wood abundant in this area.

BIOLOGICAL ASPECTS OF THE GENUS CAESALPINIA The genus Caesalpinia (Ceasalpiniaceae) has been associated with a number of biological activities. Plants in this group have been employed globally in folkloric medicine in the treatment of numerous diseases. Various investigations have been carried out on plants belonging to the genus Caesalpinia in order to validate the folkloric uses of these plants to determine its antimicrobial, antimalarial and anticancer properties, among others. Antimicrobial Activity

Extracts and compounds from the seeds have been screened against pathogenic organisms that include viruses, bacteria and fungi. Cassane furanoditerpenes, designated as caesalmin C-G, were evaluated for their effects on the proliferation of the Para 3 virus. The tetracyclic furanoditerpenoid isolates showed significant activity against the Para 3 virus, with IC50 values ranging between 7.8 and 14.8 µg/mL. However, caesalmin G, which is the only furanoditerpenoid lactone, is highly toxic, with a therapeutic index (TI) value of 3.0.[25] It is noteworthy that the

Table 1: Distribution of Caesalpinia species Selected species C. benthamiana (Baill.) Herend. & Zarucchi C. brevifolia Baill. C. coriaria (Jacq.) Willd. C. crista L. C. decapetala (Roth) Alst. C. japonica S. & Z. C. percherrima (L.) Sw. C. sappan L. C. spinosa (Mol.) Ktze.

64

Geographical location

References

W. Africa

Irvine (1961), Burkill (1994)

France Philippines Hawaii, USA Zimbabwe S. Africa Japan Philippines Hawaii, USA S. Africa

Naudin (1894) Banados & Fernandez (1954) Allen & Allen (1936b) Corby (1974) Grobbelaar & Clarke (1974) Asai (1944) Banados & Fernandez (1954) Allen & Allen (1936b) Grobbelaar & Clarke (1974)

Dickson, et. al.: Review on C. major

therapeutic index (TI) value of caesalmin C is almost the same as that of ribavirin (an inhibitor of DNA and RNA viruses), which serves as a positive control in the bioassay. It can be concluded that the anti-Para3 virus activity of tetracyclic furanoditerpenoids is better than that of the furanditerpenoid lactone.[25] Since the major components of the seed of C. minax possess such potent activity, it may be feasible to develop a new antiviral agent from this medicinal plant. Furthermore, macrocaesalmin, a cassane furanoditerpenoid lactone from the seeds of C. minax, was evaluated for antiviral activities against RSV, Para-3 and influenza Type A viruses according to an established protocol which showed inhibitory activity against the RSV (IC50 = 24.2µg/mL, TC 50 = 138.3µg/‌mL and SI = 5.7) in cell culture, and the corresponding values for the positive control (ribarivin) were 3.4, 60.6 and 17.8µg/mL, respectively. The antiviral activity of the compound was less than the positive control; however, the selectivity index for natural products was considered significant (SI > 4). However, it is inactive against the para-3 virus (IC 50 = 51.9µg/mL, TC 50 = 137.5µg/mL and SI = 2.6) with the corresponding values for the positive control being 2.7 µg/mL, 62.5µg/mL and 23.1µg/‌mL, respectively. Similarly, macrocaesalmin was inactive against the influenza Type A virus. Respiratory viral infections have long been recognized as important contributors to morbidity and mortality in young children and older adults, and the search for natural products as antiviral agents against respiratory viruses has attracted considerable attention in recent years. The isolation of macrocaesalmin and the evaluation of its efficacy on three major respiratory pathogens thus provide useful clues in the search for antiviral drugs against RSV infection.[26] Four new cassane-type furanoditerpenoids possessed antimicrobial activities against several bacteria including S. aureus, E. coli, P. aeruginosa and B. subtilis and fungi (C. albicans and T. mentagrophytes have been isolated from the air-dried leaves of C. pulcherrima. A cassane-type diterpene ester, pulcherralpin, isolated from the stems of this plant has potential fertility regulating and antitumor activities.[27] Two antitubercular cassane furanoditerpenoids, namely 6 β-benzoyl-7 β-hydroxyvouacapen-5 α-ol and 6 β cinnamoyl7β‑hydroxyvouacapen-5 α-ol, have been isolated from the root of Caesalpinia pulcherrima. It was observed that 6β-cinnamoyl7β-hydroxyvouacapen-5 α-ol possessed stronger antitubercular activity demonstrating a minimum inhibitory concentration (MIC) of 6.25 µg/mL, while the benzoyl analogue 6 β-benzoyl-7 β-hydroxyvouacapen-5 α-ol was less active (MIC 25 µg/mL). Both compounds exhibited moderate cytotoxic activity towards KB (human oral carcinonoid cancer), BC (human breast cancer) and NCl-H187 (small cell lung cancer) cell lines.[28] According to Mexican folklore, plants belonging to the genus Caesalpinia have found use in the treatment of kidney ache, cystitis, urethritis, prostate inflammation, fever, tooth-ache and 

abdominal cramps.[29] In the Philippines, decoctions of the leaves, bark and roots are used to manage liver bleeding and prevent diarrhoea and dysentery, whilst the flowers are utilized to combat oxidative stress.[30] These plants are used in the treatment of common cold, fever and dysentery in China.[31] In South Sulawesi of Indonesia, the seed kernel of the plant has been traditionally used as an anthelminthic and antimalarial.[32] The seeds of plants belonging to this genus are used as expectorant and antitussive agents in the herbal medicine practice of Thailand.[33] Their usefulness in the treatment of rheumatism and back-ache and as a tonic has also been reported in Indonesia.[34,35] Antiviral and anticancer activities from these plants have also been reported.‌[36,37] In Caribbean folk medicine, plants of this genus have been employed extensively.[38] Medicinal plants belonging to this group have been used in traditional medicine in the management of diseases in African countries including Senegal, Nigeria, Sudan and Liberia.[39] Ghana is not an exception in this regard as plants belonging to the genus are extensively employed in folkloric practice in the treatment of various ailments such as skin diseases and wounds, gonorrhoea, sleeping sickness and constipation.[40] The taxonomy of the family Ceasalpiniaceae has been the subject of much debate. It was previously referred to as Fabaceae, and prior to this was known as Leguminosae. Antimalarial Activity

Forty four cassane-and norcassane-type diterpenes isolated from Caesalpinia crista of Myammar and Indonesia were evaluated for their antimalarial activity against the malaria parasite Plasmodium falciparum (FCR – 3/A2 clone in vitro). Most of the tested diterpenes displayed antimalarial activity, and norcaesalpinin E showed the most potent activity with an IC50 value of 0.090µM, a greater potency than the clinically used drug chloroquine (IC50, 0.29µM).[41] Ten new cassane diterpenes including caesalpinins H-P and norcaesalpinin F were tested for their inhibitory activities on the growth of Plasmodium falciparum (FCR – 3/A2 in vitro. All displayed activity in a dose dependent manner. Among the newly isolated compounds, caesalpinin K and norcaesalpinin F showed the most potent inhibitory activity with an IC50 value of 120 and 140nM respectively, which is lower than the value reported for the well-characterizedantimalarial drug, chloroquine (IC50, 282 – 291nM).[42] Three new cassane furanoditerpenoids (1-3) exhibiting antimalarial activity against the multidrug-resistant K1 strain of Plasmodium falciparum have been isolated from kernels of Caesalpinia bonduc. Anticancer Activity

Caesaldekarin J possesses inhibitory activity against glutathione S-transferase, an enzyme that has been implicated in resistances during treatment of cancer and parasitic infections, and can be isolated from the ethanolic extract of Caesalpinia bonduc bark.[43] Two new cassane butenolides, caesalpinolide A (1) and B (2), epimeric at the hemiketal position, were isolated from the marine 65

Dickson, et. al.: Review on C. major

creeper Caesalpinia bonduc. They exhibited inhibitory effects on MCF-7 breast cancer cell lines, with IC50 values of 12.8 and 6.1 (μM), respectively, and also inhibited endometrial and cervical cancer cell lines.[44] Similarly, Phanginin I, a cassane-type diterpenoid isolated from the seeds of Caesalpinia sappan exhibited cytotoxic effects against KB cell line with IC50 value of 4.4μg/‌ml.‌[45] Basic Molecular Skeleton of Cassane-type Diterpenoids

Plants belonging to the genus Caesalpinia have proven to be a rich source of cassane-type diterpenoids.[46,47-51] These cassane diterpenoids are characterized by a molecular skeleton constructed from the fusion of three cyclohexane rings A, B and C and a furan ring (1) .Ring C may sometimes be aromatic as in compounds 24, 38, 59-62. The existence of an exocyclic methylene group at position 14 is a characteristic of some of these cassane-type diterpenoid compounds (see 62, 76, 81, 83, 88). Generally, these diterpenoids give a red colour with Ehrlich reagent, suggesting the presence of a furan ring in their molecular structure. However, not all cassane-type diterpenes have this furan ring (e.g 5, 6) and therefore will not respond to this test. Cassane-type diterpenoids from the genus Caesalpinia

The isolation of the cassane-type diterpenoids may have begun in the mid 1950’s from other sources other than the genus Caesalpinia. However, Jiang et al (2001), isolated pulcherrimin A (3) and ε-caesalpin (4) from Caesalpinia pulcherrima.[52] In 1992, the roots of Caesalpinia decapetala yielded caesaljapin (9), a cassane diterpenoid.[53] Caesaldekarin A (15), C (16), D (16i) and E (16ii) were also isolated from the roots of Caesalpinia major.[54]

plant, two cassane diterpenes neocaesalpin A (18) and B (19) were isolated.[55] From the roots of the same plant in the following year, caesaldekarin F (20) and G (21), were isolated.[56] In the same year, and again from the roots of this plant, seven cassane diterpenoids that included caesaldekarin A (15), H, I, J, K and L (22-26) and demethylcaesaldekarin C (27) were isolated.[57] Additional diterpenoid lactone compounds, caesalmins A, B, C, D, E, F and G (28-34) were isolated from the seeds of C. minax.‌[58] Four novel diterpenoid compounds (35-38), possessing both antibacterial and antifungal activities, have been isolated from the leaves of C. pulcherrima.[59] A novel diterpenoid named macrocaesalmin (39), together with caesalmin B, D and H possessing antiviral and anticancer activities were isolated from the seeds of C. minax.[31,60] This was followed in the following year by the isolation of cassane diterpenoid compounds (+)-vouacapenic and (+)- vouacapenate (2, 7) from Vouacapoua americana belonging to the Leguminosae.[61] Novel norcassane-type diterpenes norcaesalpinin A, B and C (44-48), have also been obtained from the seed kernels of C. crista.‌[61] In the following year, five new cassane-type diterpenes, caesalpinins MA-ME 1-5 (49-53) and three new norcassane-type diterpenes, norcaesalpinins MA-MC (54-56) together with known cassane-type diterpenes ( 29, 30, 32 and 51) were isolated from C. crista.[62] Nine new cassane-type diterpenes taepeenin A-I

O

Caesalpinin 1 (17), a cassane furanoditerpene, has been isolated from Caesalpinia bonducella roots.[48] From the seeds of the same

H CH3

H HO

16

O

15 11

12

1 2

4

HOOC

O

13

O

14 10

3

O

OH

9

17

8 7

5 6

Figure 1: Carbon skeleton of cassane-type diterpenoids

Figure 2: Pulcherrimin A

O

O OAc AcO H OH Figure 3: e-caesalpin

66

H

HOOC

H

H

OH

MeOOC Figure 4: Caesaljapin

H

O

Dickson, et. al.: Review on C. major

O

O

H

H

OH OAc

OH

MeOOC

Figure 5: Caesaldekarin A

Figure 6: Caesaldekarin C

O

O

OAc

O

H

AcO

OH

H

OAc

O

O

HO

OH

OH

OAc

Figure 7: Caesalpinin 1

Figure 8: Neocaesalpin A

O OAc

HO

AcO

O

O

H

H

H

H

H

OH

MeOOC

OH

Figure 10: Caesaldekarin F

Figure 9: Neocaesalpin B

OH O

O H

H

H MeOOC Figure 11: Caesaldekarin G



OH

H AcO

OH CH2

Figure 12: Caesaldekarin H

67

Dickson, et. al.: Review on C. major

O

O

H H OH

OH CH2OH

OH

MeOOC

Figure 13: Caesaldekarin I

Figure 14: Caesaldekarin J

O H

HO

OH

OH

H

HOCH 2

Figure 15: Caesaldekarin A

Figure 16: Caesaldekarin B

O

O OH

H

H

OH

HOOC

OH

Figure 17: Demethylcaesaldekarin C

H OAc

Figure 18: Caesalmin A

O OAc

O OAc

H H

H OH H

O

H H

H

OH OAc

Figure 19: Caesalmin B

OAc

Figure 20: Caesalmin C

O OAc

H

O OAc

OH

H OH OAc 68

O

H

H

H

Figure 21: Caesalmin D

OH

H

H MeOOC

H

O

H H

OAc

OH OAc Figure 22: Caesalmin E

OH OAc

Dickson, et. al.: Review on C. major

O

O

OH

OAc

H

H H

OMe OAc

OH OAc

OH

Figure 23: Caesalmin F

H

H H OAc

Figure 24: Caesaldekarin G

O

O

H

H

H

H

OH O

OH O

OH

O

O

Figure 25: Isovouacapenol A

Figure 26: Isovouacapenol B

O

O

H H

H

OH

OH O

OH O

O

O

Figure 27: Isovouacapenol C

Figure 28: Isovouacapenol D

O O

H H O

Figure 29: Macrocaesalmin



O OAc

H O

H H

O

H OAc

OH

H OH

Figure 30: Caesalmin H

69

Dickson, et. al.: Review on C. major

O OH

O OAc

H O

H

H

AcO

O

H

OH H OAc

OH

Figure 31: Bonducellpin D

Figure 32: Norcaesalpinin A

OH

O

O

OAc

OAc

H

O OH

OH

Figure 33: Norcaesalpinin B

Figure 34: Norcaesalpinin C

O

O AcO

AcO

H

H CH3

H AcO

COOCH3 H

H OH

OH

Figure 35: Caesalpinin MA

Figure 36: Caesalpinin MB

O

O

AcO

AcO

AcO

Me

Me AcO

OH

OH

OAc

Figure 37: Caesalpinin MC

Figure 38: Caesalpinin MD 4

OH

H O

H AcO

H

AcO

Figure 39: Caesalpinin ME

70

H

O OH

OAc Figure 40: Norcaesalpinin MA

O

Dickson, et. al.: Review on C. major

OH

O

O

AcO

AcO

H O

H

OAc

OH

OH

OAc

Figure 41: Norcaesalpinin MB

Figure 42: Norcaesalpinin MC

O

O

H

MeOOC

H

HOOC

Figure 43: Taepeenin A

Figure 44: Taepeenin B

O

O

H

MeOOC

MeOOC

OH

Figure 45: Taepeenin C

H COOMe

Figure 46: Taepeenin D

O

O

MeOOC

O

H CHO

H COOMe

Figure 47: Taepeenin E

Figure 48: Taepeenin F

O

H

H OH

H

H H Figure 49: Taepeenin G



MeOOC

H CHO

Figure 50: Taepeenin H

71

Dickson, et. al.: Review on C. major

O

O H

H H H CH2OH

MeOOC

O

H H MeOOC

Figure 51: Taepeenin I

Figure 52: Nortaepeenin A

O

O

H

OAc O

H H OH MeOOC

AcO

Figure 53: Nortaepeenin B

OH

Figure 54: Caesaldekarin e

O

O OAc

OAc AcO

AcO

AcO

HO

OH

Figure 55: 2-Acetoxycaesaldekarin e

OH

Figure 56: 2-Acetoxy-3-deacetocaesaldekarin e

O

O

OAc

OAc H H

OH

OH

OAc Figure 57: 6-Acetoxy-3-deacetoxycaesaldekarin e

Figure 58: 14 (17)-Dehydrocaesalmin F

O O

O OAc

H COOMe

H OH

OH

72

COOMe H

H H

OAc Figure 59: Bonducellpin B

H

OH

OH H

Figure 60: Bonducellpin C

Dickson, et. al.: Review on C. major

O OAc

O

H

O

COOMe

H

H

H

H

OAc

OH

OAc

OH

H

OAc

Figure 61: 7-Acetotoxybonducellpin C

Figure 62: 1-Deacetoxy-1-oxocaesalmin C

O

O O

O

H

H H

H OAc

OH OH

OH

Figure 63: S-Caesalpin

OAc OAc

Figure 64: 1-Deacetylcaesalmin C

O

O

OAc

OAc

H

H

OAc

OH

AcO

O

O

H

O

OH OAc

OAc

Figure 65: Caesalpinin C

H

Figure 66: Caesalpinin D

O

O O

OAc

H

AcO

O

H AcO

H

OH

Figure 67: Caesalpinin E

OAc

OH OAc Figure 68: Caesalpinin F

O OH

O O

H

H

O H OH Figure 69: Caesaldekarin H



O H

O OH

OAc

O

OAc

Figure 70: Caesaldekarin I

73

Dickson, et. al.: Review on C. major

O O

O

H

OAc

H

OH

H

OAc

OH

H

H

COOMe

OH

OAc Figure 72: Caesalpinin K

Figure 71: Caesalpinin J

O OAc

O

H

OAc

COOMe

H

H

H

OAc

OH

CHO

H

H OH

OH

OH Figure 74: Caesalpinin N

Figure 73: Caesalpinin M

O

O

OAc

OAc O

H

AcO

O OH

H OH

OH

Figure 75: Caesalpinin O

Figure 76: Caesalpinin P

O

O OAc

OAc

OAc

OH

OH

OAc

H

H

H

H

OAc

Figure 77: Caesalpinin MF

Figure 78: Caesalpinin MG

O OAc

O

COOH

H

H

OH

OH

OH

OAc 74

H

H

H

H

Figure 79: Caesalpinin MH

COOMe

H

COOMe

H

Figure 80: Caesalpinin MI

OH

Dickson, et. al.: Review on C. major

O

O

O

OAc

H

H

H

H OH Figure 81: Caesalpinin MJ

OAc

OH

OAc

OAc Figure 82: Caesalpinin MO

O O

O

H

O

O

H

OAc

OH OAc Figure 83: Norcaesalpinin MD

H

AcO

O

H AcO

OH

Figure 84: Norcaesalpin D

O

O

O

O

H

O

H

Figure 85: Norcaesalpin E

O

H

OH

OH

OH

OH

H

OAc Figure 86: Norcaesalpin F

O

O

H H

MeOOC

H

MeOOC

Figure 87: Benthaminin 1

H

Figure 88: Benthaminin 2

O H H H MeOOC Figure 89: Benthaminin 3



(57‑65) and two new norcassane-type diterpenes nortaepeenin A-B (66-67) were also isolated from the stems and roots of C.  crista.[63] From the seed kernels of the same plant, known cassane and norcassane-type diterpenes including compounds 29, 30, 32, 34, 46-49, 54, 68-78 and new cassane-type diterpenes, namely caesalpinins C-K (65-68), M-P (73-76), caesalpinins MFMJ (77-81), MO (82) and norcaesalpins MD (83), D-F (84-86) possessing antimalarial activity, have been isolated. Norcaesalpinin E (85), displayed the most potent antimalarial activity.[62] 75

Dickson, et. al.: Review on C. major

CONCLUSION Cassane-type diterpenoids continue to be isolated from medicinal plants. Three novel cassane-type diterpenoids- benthaminin 1 (87), 2 (88) and 3 (89) possessing antimicrobial and antioxidant properties have been isolated from Caesalpinia benthamiana.[51] Similarly, two novel cassane-type diterpenoids designated magnicaesalpin and neocaesalpin O together with three known ones named caesalmin D and E and neocaesalpin L have been isolated from the seeds of Caesalpinia magnifoliolata.[52] A number of these cassane-type furanoditerpenoids have been found to manifest various biological activities including antibacterial, antifungal,[29] anti-inflammatory, anti-analgesic,[52,53] antiviral and anticancer,[31] antimalarial[54] and antituberculosis activities.[36] Thus, these cassane diterpenoids are of interest due to their structural diversity and their broad spectrum of biological activities. Further studies should be performed to indicate which of these isolated bioactive chemical constituents may serve as lead compounds in the synthesis of biomolecules to tackle the numerous global health challenges due to the emerging and ongoing drug resistance associated with long term use of conventional medicines used in the treatment and management of infectious diseases.

16. 17. 18. 19.

20. 21.

22. 23. 24. 25. 26.

27. 28. 29.

ACKNOWLEDGEMENTS R. A. Dickson is grateful to the Commonwealth Scholarship Commission, UK and the Kwame Nkrumah University of Science and Technology, Ghana for sponsorship.

30. 31. 32.

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6. 7. 8. 9. 10. 11. 12. 13.

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76

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Ogawa K, Aoki I, Sashida Y. Caesaljapin, a cassane diterpenoid from Caesalpinia decapetala var. japonica. Phytochemistry 1992; 31:2897-2898. 54. Peter S, Tinto WF, Mclean S, Reynolds WF, Yu M. Cassane diterpenes from Caesalpinia bonducella. Phytochemistry. 1998; 47:1153-1155. 55. Peter SR, Tinto. WF. Bonducellpins A-D, new cassane furanoditerpenes of Caesalpinia bonduc. J. Nat. Prod. 1997; 60:1219-1221. 56. Peter SR, Tinto WF, Mclean S, Reynolds WF, Yut M. Cassane Diterpens from Caesalpinia Bonducella, Phytochemistry. 1998; 47:(6) 1153-1155. 57. Lyder, DL, Peter SR, Tinto WF, Bissada SM, McLean S, Reynolds, WF. Minor  cassane diterpenoids of Caesalpinia bonducella. J. Nat. Prod. 1998; 61:1462‑1465. 58. Li DM, Ma L, Liu GM, Hu LH. Cassane diterpene-lactones from the seed  of   Caesalpinia minax Hance. Chemistry and Biodiversity. 2006; 3(11): 1260‑1265. 59. Ragasa CY, Hofilena JG, Rideout JA. New furanoid diterpenes from Caesalpinia pulcherrima. J. Nat. Prod. 2002; 65:1107-1110. 60. Jiang RW, Paul PHB, Shuang-Cheng MA, Ye WC, Chan SP, Thomas CWM, Zhen‑Dan H, Wang H, Siu-Pang C, Eng-Choon OV, Hong-Xi X, Mak CW. Molecular structures and antiviral activities of naturally occurring and modified cassane furanoditerpenoids and friedelane triterpenoids from Caesalpinia minax. Bioorganic and Medicinal Chemistry. 2002; 10:2161-2170. 61. Banaskota AH, Attamimi F, Usia TZ Linn YT, Kaluani SK. Kadota S. Novel norcassane-type diterpene from the seed kernels of Caesalpinia crista. Tetrahedron Letters. 2003; 44:6879-6882. 53.

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Pharmacognosy Communications

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Volume 1 | Issue 1 | Jul-Sep 2011

Research Article Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves Dinesh kumar B1, Analava Mitra2*, Manjunatha M2 Department of Pharmaceutics, PSG College of Pharmacy, Coimbatore-641 004, India. 2School of Medical Science and Technology, Indian Institute of Technology, Kharagpur -721302, West Bengal, India

1

ABSTRACT: Introduction: Azadirachta indica (Meliaceae) leaves are used traditionally in the Indian Ayurvedic medicinal system to treat diabetes. The aim of the present study is to investigate the effect of azadirachtolide (tetranortriterpenoid from  Azadirachta indica leaves) on blood glucose and serum lipid profiles on streptozotocin-induced diabetic rats. Methods:  Streptozotocin-induced diabetic rats were used for the study. Azadirachtolide (at a dose 50 and 100 mg/kg) was administrated intra-peritoneally in diabetic rats once a week for 30 days. Biochemical parameters notably fasting blood sugar, total cholesterol, triglycerides, low-density lipoprotein, very low-density lipoprotein and high-density lipoprotein were determined. The in vitro alpha amylase and alpha glucosidase inhibitory effects of azadirachtolide were measured and IC50 values were determined. Results: Azadirachtolide exhibited significant (P < 0.05) anti-diabetic as well as hypolipidemic effects by lowering FBS, TC, TG, LDL, and VLDL levels; but also with elevation of HDL level in diabetic rats. Azadirachtolide showed appreciable alpha amylase (IC50 value of 55.80 ± 1.7 µg/ml) and alpha glucosidase inhibitory effects (IC50 value of 47.85 ± 1.4 µg/‌ml) compared with acarbose (IC50 value of 83.33 ± 1.8 µg/ml). Conclusion: The present study indicated that azadirachtolide possesses anti-hyperglycemic and anti-lipidemic effects. Thus, results suggested azadirachtolide has a beneficial effect in the management of diabetes associated with abnormal lipid profile and related cardiovascular complications. KEYWORDS: Azadirachta indica, Azadirachtolide, Anti-diabetic, Hypolipidemic

Introduction Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion or insulin action, or both.[1] Broad research on diabetes has resulted in the development of a number of oral hypoglycemic agents including biguanides, sulphonylureas and thiozolidinediones which are available commercially for the management of diabetes. However, these drugs also produce nondesirable side effects.[2] Hence, there is a need to develop alternative anti-diabetes medicines. The herbal medicines are widely used for the treatment of disease because of their effectiveness, safety, affordability and acceptability. [3] Medicinal plants including their phyto-compounds have been used in the Indian traditional systems of medicine for treatment of diabetic populace all around the world with less known scientific basis of their functioning.[4-7] Hence, phyto-products from medicinal plants need to be investigated by scientific methods for their anti-diabetic activity. Various medicinal effects have been reported for anti-inflammatory, anti-arthritic, antipyretic, *Correspondence: +913222-282220/282657; Fax: +913222-282221; Email: [email protected], [email protected] DOI: 10.5530/pc.2011.1.5

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antifungal, anti-bacterial, diuretic, immunomodulatory and anti‑tumor properties. Phyto-compounds such as azadirachtins, nimocinol, isomeldenin, 2, 3′-dehydrosalanol gedunin, nimbin, nimolicinol from Azadirachta indica have been reported in the leaves.[8] Tetranortriterpenoids has been reported for anticancer, antiviral, anti-allergic and anti-inflammatory activities.[9-12] There is no report on azadirachtolide (tetranortriterpenoid from Azadirachta indica leaves) for antidiabetic and hypolipidemic activities. Therefore, the effect of azadirachtolide (tetranortriterpenoid from Azadirachta indica leaves) on blood glucose and serum lipid profiles on streptozotocin-induced diabetic rats was investigated. Further, in vitro alpha amylase and alpha glucosidase an inhibitory effect of azadirachtolide was evaluated.

Materials and Methods Chemicals and reagents

Streptozotocin, starch azure, porcine pancreatic amylase, alpha glucosidase from yeast Saccharomyces cerevisiae, para-nitrophenyl gluco-pyanoside and Tris-HCl buffer were procured from Sigma Chemicals, USA. Dimethyl sulfoxide, acetic acid, calcium chloride, ethanol, chloroform, petroleum ether, potassium bromide,

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Kumar, et. al.: Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves

deuterated chloroform, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, Whatmann filter paper and sodium carbonate were purchased from Merck, India. Thin layer chromatography plates were obtained from Merck (silica gel 60 F254 grade, Germany). Diagnostic kits and reagents for fasting blood sugar, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein and very low density lipoprotein were obtained from Merck, India. Acarbose was gifted by Zota Pharmaceutical Pvt. Ltd., Chennai. Glibenclamide (Aventis Pharma- Mumbai) was procured from local medical market.

twin-trough glass chamber previously saturated with mobile phase vapor for 20 min. After developing the plate, it was dried at 105ºC for 15 min and then it was scanned using Scanner 3 (CAMAG, Switzerland) at 254nm using WinCATS 4 software. IR spectrum was recorded using a Thermo Nicolet Nexus 870 FT‑IR Spectrophotometer using potassium bromide pellets. Mass spectrum was recorded on Electro-Spray Ionization Mass Spectroscopy (Waters, UK). NMR spectra were recorded in CDCl3 in a Bruker 400 MHZ spectrometer using Topspin software.

Plant materials

In vitro alpha amylase inhibitory assay

Azadirachta indica leaves (Rutaceae) were collected from the locality of IIT Kharagpur campus, West Bengal, India in the month of September and October 2007. The leaves were inspected to be  healthy and botanically identified and authenticated by Dr. M. Senthilkumar, Plant Biotechnologist, Prathyusha Institute of Technology and Management, Chennai. The herbarium Azadirachta indica leaves was deposited in the Prathyusha Institute of Technology and Management (PITAM) against voucher no. PITAM/ CH/00015/ 2007. Azadirachta indica leaves after collection were dried at room temperature (27-30ºC) for 25‑30 days. After complete drying (inspection), the dried materials were ground into fine powder using a domestic electric grinder (Product: GX 21, Bajaj appliances, Mumbai, India) and used for extraction. Extraction and isolation

Dried plant powder of Azadirachta indica leaves (500 g) was extracted with ethanol (1 L) at room temperature. Then extract was filtered (Whatmann filter paper, 110mm, Cat. no 1001 110). The filtrate was evaporated by rotary evaporation (Buchi Rotavapor R-210) to get a dark greenish solid residue. These greenish solid residues (15 g) was successively extracted with petroleum ether (3.5 g) and chloroform (5.2 g) and subjected for in vitro alpha amylase inhibitory activity. The chloroform fraction showed appreciable alpha amylase inhibitory compared to petroleum ether fraction. The chloroform fraction was subsequently subjected to column chromatography using gradient elution using acetone and chloroform as solvents (10% acetone in chloroform for 15 mins, 20% acetone in chloroform for 15  mins and 30% acetone in chloroform for 15 mins). The fractions obtained with 20% acetone in chloroform afforded compound-I (10 mg). These fractions were subjected to preparative TLC with mobile phase hexane: ethyl acetate (8.5:1.5) for isolation of compound-I. Compound-I was identified as azadirachtolide by comparing its FTIR, ESI-MS and NMR with previously published literature (Ragasa et al., 1997). General experimental procedure

HPTLC (CAMAG, Switzerland) analyzes was performed using silica gel 60 F254 TLC plate. All collected fractions were spotted (10 µl) on a silica gel 60 F254 (Merck, Darmstadt, Germany) TLC plate. The plate was air dried and then developed using the solvent system hexane: ethyl acetate (8.5:1.5) in a CAMAG

The assay was carried out following the standard protocol with slight modifications.[13] Starch azure (2 mg) was suspended in a tube containing 0.2ml of 0.5 M Tris-Hcl buffer (pH 6.9) containing 0.01 M calcium chloride (substrate). The tube was boiled for 5 min and then pre-incubated at 37º C for 5 min. Azadirachtolide was dissolved in 0.1% of dimethyl sulfoxide in order to obtain concentrations of 10, 20, 40, 60, 80 and 100 µg/ml. Then 0.2 ml of azadirachtolide of a particular concentration was put in the tube containing the substrate solution. 0.1 ml of porcine pancreatic amylase in Tris-Hcl buffer (2units/ml) was added to the tube containing the azadirachtolide and substrate solution. The reaction was carried out at 37 ºC for 10 min. The reaction was stopped by adding 0.5 ml of 50% acetic acid in each tube. The reaction mixture was then centrifuged (Eppendorf -5804 R) at 3000 rpm for 5 min at 4ºC. The absorbance of resulting supernatant was measured at 595 nm (Perkin Elmer Lambda 25 UV-VIS). The concentration of the azadirachtolide required to inhibit 50% of alpha amylase activity under the conditions was defined as the IC50 value. The experiments were repeated thrice with the same protocol. The alpha amylase inhibitory activity was calculated as follows: Alpha amylase inhibitory activity = (Ac+) – (Ac–) – (As–Ab) × 100 (Ac+) – (Ac–) Where, Ac+, Ac–, As, Ab are defined as the absorbance of 100% enzyme activity (solvent with enzyme alone), 0% enzyme activity (solvent without enzyme), a test sample (with enzyme) and a blank (a test sample without enzyme) respectively. In vitro alpha glucosidase inhibitory assay

The assay was performed using a standard protocol.[14] Alpha glucosidase (2U/ml) was premixed with 20 µl of azadirachtolide at various concentrations (10, 20, 40, 60, 80 and 100 µg/ml) and incubated for 5 min at 37ºC. 1mM para-nitrophenyl glucopyanoside (20 µl) in 50mM of phosphate buffer (pH 6.8) was added to initiate the reaction. The mixture was further incubated at 37ºC for 20 min. The reaction was terminated by addition of 50 µl of 1 M sodium carbonate and the final volume was made up to 150 µl. Alpha glucosidase activity was determined spectrophotometrically at 405nm on a Biorad microplate reader 79

Kumar, et. al.: Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves

by measuring the quantity of para-nitrophenol released from pNPG. The assay was performed in triplicate. The concentration of azadirachtolide required to inhibit 50% of alpha glucosidase activity under the conditions was defined as the IC50 value. The experiments were repeated thrice with same protocol. Animal studies

Adult male Wistar Rats (weighing 150-200 g) were used for this investigation. The animals were acclimatized to the laboratory conditions for a period of 2 weeks prior to the experiment. They were maintained at an ambient temperature (25 ± 2 ºC) and relative humidity (40-60%), with 12/12 h of light/dark cycle. The animals were maintained on balance diet and water ad libitum. Institutional Animal Ethical Committee (IAEC) approved the study and all the experiments were carried out by following the guidelines of CPCSEA, India. Induction of diabetes and blood sample collection

A freshly prepared solution of streptozotocin (45mg/kg) in 0.1M citrate buffer pH 4.5 was injected intra-peritoneally in overnight fasted rats. After 3 days, blood was collected from the tail vein of overnight fasting rats under the supervision of a veterinary surgeon using aseptic conditions. The FBS level of blood was checked regularly up to the stable hyperglycemia stage, usually one week after streptozotocin injection. Animals with marked hyperglycemia (FBS 250 mg/dl) were selected for the study.[15] Experimental design

Group I - Normal control Group II - Diabetic control Group III - Diabetic +50 mg/kg (i.p.) azadirachtolide Group IV – Diabetic +100 mg/kg (i.p.) azadirachtolide Group V - Diabetic + 0.5 mg/kg (i.p.) glibenclamide The experiment was carried on five groups (I, II, III, IV and V) of six rats each. Group-I served as normal control. Group-II served as diabetic control. Group III-diabetic + 50 mg/kg (i.p.) of azadirachtolide. Group IV-diabetic + 100 mg/kg (i.p.) of azadirachtolide. Group V-diabetic + 0.5 mg/kg (i.p.) of glibenclamide and served as positive control. The azadirachtolide was suspended in 0.3% w/v sodium carboxy methyl cellulose (Sodium CMC) as a vehicle and injected intra-peritoneally into rats once a week for one month with a dose of 50 mg/kg and 100 mg/kg body weight. The blood samples were collected from each rat by retro-orbital vein-puncture. Biochemical parameters were estimated at the beginning and after 30 days of experiment.

Statistical analysis

All values were expressed mean ± standard deviation. Statistical analysis of in vivo results were performed by one-way analysis of variance (ANOVA) followed by Student’s t-test. P < 0.05 was considered statistically significant. In vitro inhibitory assay statistical difference and linear regression analysis were performed using Graphpad prism 5 statistical software.

Results Azadirachtolide (10 mg) was isolated from 500 g of dried leaves of Azadirachta indica (Figure 1). HPTLC analyzes indicated that F2 contained azadirachtolide and the retention factor (Rf) values of azadirachtolide was found to be 0.31 (Figure 2). The F2 fractions were subjected to preparative TLC with the solvent system hexane: ethyl acetate (8.5:1.5) to get the compound-1 (azadirachtolide). FTIR (KBr disc) is shown in Figure 3: peak at 3444 cm-1 indicated presence of OH group, peak at 2925 cm-1, 2854 cm-1 was due to presence of C-H, peak at 1370 cm-1 showed C-H bending, peak at 1736 cm-1 indicated presence of ester carbonyl group, peak at 1666 cm-1 showed presence of C-O group and peak at 1458 cm-1 indicated presence of CH-CH bending (Figure 3). ESI-mass spectroscopy showed the presence of a molecular weight peak of azadirachtolide at 593. ESI-MS (m/z, % intensity): m/z 593 [M-H]-. Proton NMR (CDCl3 solvent) showed senecioyloxy subtitutent δ 1.88 (3H), δ 2.20 (3H), δ 5.70 (1H). an acetate δ 1.97 (3H). Four additional methyl singlet δ 0.8, δ 1.25, δ 1.28, δ 1.30, two olefinic hydrogen δ 5.57, δ 5.71, methylene hydrogen bonded to oxygenated carbons δ 4.15 (1H), δ 3.81 (1H), δ 3.68 (1H), δ 3.59 (1H) and methine hydrogen bonded to oxygenated carbons δ 4.12 (1H), δ 4.15 (1H), δ 5.30 (1H), δ 5.47 (1H). Azadirachtolide showed appreciable alpha amylase (IC50 value of 55.80 ± 1.7µg/ml) and alpha glucosidase inhibitory effects (IC50 value of 47.85 ± 1.4µg/ml) as compared with acarbose (IC50 value of 83.33 ± 1.8µg/ml) (Figure 4). The body weight was slightly increased in normal control rats compared to initial body weight whereas streptozotocin-induced diabetic rats showed loss of body weight (172.6 ± 2.05 g) after 30 days as compared with initially weight of diabetic rats (186.6 ± 1.24 g). However, body weight of diabetic rats was restored by treating with

Biochemical parameters

Biochemical parameters notably fasting blood sugar (FBS), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) levels and highdensity lipoprotein (HDL) level in blood serum were measured spectrophotometrically (Semi-Autoanalyzer, Microlab 300, Merck) as per the manufacturers instructions using diagnostic kits and reagents obtained from Merck, India. 80

Figure 1: Structure of azadirachtolide.

Kumar, et. al.: Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves

Figure 2: HPTLC peaks of collected column fractions CE-Crude extract (Pink peak), F1-10% acetone in chloroform (Violet peak), F2-20% acetone in chloroform (Green peak), F3-30% acetone in chloroform (Orange peak).

Figure 3: FTIR Spectrum of azadirachtolide.

glibenclamide (0.5 mg/kg) and azadirachtolide (at a dose of 50 mg/kg and 100 mg/kg, i.p.) for 30 days (Table 1). Streptozotocin treatment resulted in elevation of fasting blood glucose, triglycerides, total cholesterol, low density lipoproteins, very low density lipoproteins and a reduction in high densitylipoprotein levels as compared to the normal control rats (Table 2). Intra-peritoneal administration of azadirachtolide (at a dose of 50 mg/kg and 100 mg/kg, once a week for 30 days) exhibited significant (P < 0.05) reduction in fasting blood sugar levels (204.0 ± 2.94 and 198.3 ± 2.86 mg/dl in diabetic rats. Diabetic 

rats treated with azadirachtolide (at a dose of 50 mg/kg and 100 mg/‌kg, i.p.) once a week for 30 days on being compared with diabetic rats exhibited significant (P < 0.05) reduction in fasting blood sugar levels (204.0 ± 2.94 and 198.3 ± 2.86 mg/dl respectively). The standard glibenclamide (0.5 mg/kg, i.p.) also showed anti-diabetic activity with reduction of fasting blood sugar level (215.0 ± 2.18 mg/dl) on 30 days as compared to the diabetic control. There was a significant (P < 0.05) reduction in triglycerides, total cholesterol, low density lipoprotein and very low density lipoprotein levels of diabetic rats treated with azadirachtolide (50 and 100 mg/‌kg, i.p.) on being compared with diabetic control. Also, there was a significant (P < 0.05) elevation of HDL level in azadirachtolide (50 and 100 mg/kg, i.p.) treated diabetic rats. 81

Kumar, et. al.: Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves

Figure 4: Alpha amylase and alpha glucosidase inhibitory effects of azadirachtolide.

Table 1: Body weights of streptozotocin-induced diabetic rats after treatment with azadirachtolide. Group

Initial body weight

Final body weight

Normal control Diabetic control 50 mg/kg of azadirachtolide 100 mg/kg of azadirachtolide 0.5 mg/kg of glibenclamide

191.0 ± 0.81 186.6 ± 1.24 183.0 ± 1.60 182.0 ± 1.63 180.3 ± 0.47

200.0 ± 0.63 172.6 ± 2.05 178.3 ± 1.69* 177.6 ± 1.69* 176.3 ± 1.24*

*(P