Phytochemistry 57 (2001) 427–435 www.elsevier.com/locate/phytochem
Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts Rohan Kermanshaia,1, Brian E. McCarryb, Jack Rosenfeldc, Peter S. Summersa, Elizabeth A. Weretilnyka, George J. Sorgera,* a Department of Biology McMaster University, Hamilton, Ontario, Canada L8S 4K1 Department of Chemistry, McMaster University, Hamilton, Ontario, Canada, L8S 4K1 c Department of Pathology, McMaster University, Hamilton, Ontario, Canada, L8S 4K1
b
Received 3 February 2000; received in revised form 22 December 2000
Abstract Papaya (Carica papaya) seeds were extracted in an aqueous buffer or in organic solvents, fractionated by chromatography on silica and aliquots tested for anthelmintic activity by viability assays using Caenorhabditis elegans. For all preparations and fractions tested, anthelmintic activity and benzyl isothiocyanate content correlated positively. Aqueous extracts prepared from heattreated seeds had no anthelmintic activity or benzyl isothiocyanate content although both appeared when these extracts were incubated with a myrosinase-containing fraction prepared from papaya seeds. A 10 h incubation of crude seed extracts at room temperature led to a decrease in anthelmintic activity and fractionated samples showed a lower benzyl isothiocyanate content relative to non-incubated controls. Benzyl thiocyanate, benzyl cyanide, and benzonitrile were not detected in any preparations and cyanogenic glucosides, which were present, could not account for the anthelmintic activity detected. Thus, our results are best explained if benzyl isothiocyanate is the predominant or sole anthelmintic agent in papaya seed extracts regardless of how seeds are extracted. # 2001 Published by Elsevier Science Ltd. All rights reserved. Keywords: Carica papaya; Caricaceae; Anthelmintic; Benzyl isothiocyanate; Caenorhabditis elegans; Myrosinase; Nematode
1. Introduction Papaya seeds have been used for centuries as a vermifuge in India (Lal et al., 1976), Central and South America (Roig y Mesa,1974) and throughout the world (Werner, 1992). Clinical trials with humans have led to seemingly contradictory results with Robinson (1958) claiming that papaya seeds are effective and Fernando (1959) claiming they are not. However, laboratory studies have confirmed that various preparations of papaya seeds can kill helminths effectively in vitro and in infected animals (Krishnakumari and Majumder, 1960; Dar et al., 1965; Lal et al.,1976). The number and identity of anthelmintic compounds present in papaya seeds has not yet been established. * Corresponding author. Tel.: +1-905-525-9140, ext. 24376; fax: +1-905-522-6066. E-mail address:
[email protected] (G.J. Sorger). 1 Permanent address: Department of Biology, Faculty of Sciences, Esfahan University, Esfahan, Iran.
Previous work has shown that seeds ground and extracted with either water or organic solvents, including alcohol, all produce crude extracts with anthelmintic activity and contain bioactive compounds such as benzyl isothiocyanate (BITC) (Dar et al., 1965; Ettlinger and Hodgkins, 1956; Krishnakumari and Majumder, 1960; Tang, 1971; Tang et al., 1972). In some cases, diethyl ether could be used to concentrate the bioactive principle(s) from water soluble seed extracts but, while the material that partitioned to the diethyl ether or organic solvent layer was shown to have anthelmintic activity and contain BITC, fractions that were produced by the initial extraction of the seeds with water or which partitioned to the water layer were apparently never tested for anthelmintic activity (Ettlinger and Hodgkins, 1956; Tang, 1971, 1973). Steam distillates using watersoluble extracts of papaya seeds also yielded BITC and the activity of this fraction against helminths was demonstrated (quoted in Ettlinger and Hodgkins, 1956). Since BITC is volatile and relatively insoluble in water, distillation of this compound from an aqueous solution
0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00077-2
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at a relatively low temperature is not surprising. However, the steam distillation carried out by these researchers would not have identified any other putative bioactive compounds that may have been heat-labile. Given the variety of solvents and extraction conditions that can produce extracts with bioactive properties against helminths, there is little compelling evidence that BITC is the only anthelmintic principle that can be extracted from papaya seeds. The anthelmintic effect of papaya seeds has been variously ascribed to carpaine (an alkaloid) and carpasemine (later identified as benzyl thiourea by Panse and Paranjpe, 1943), and BITC (Krishnakumari and Majumder, 1960; Tang, 1971). Benzyl thiourea is reported to be an artifact that arose during purification of the bioactive principle due to a reaction between ammonia and BITC (Ettlinger and Hodgkins, 1956) and it was present in preparations from seeds at a concentration of one-tenth that of BITC. Subsequently, Dar et al. (1965) tested BITC and benzyl thiourea individually for bioactivity and showed BITC to be about 20 times more toxic to Ascaris lumbricoides than benzyl thiourea. Since this study only tested compounds already known to be present in papaya seed extracts it falls short of proving that BITC is the only active anthelmintic principle in these seeds. In addition to anthelmintic properties, papaya seed extracts also contain antimicrobial activity (Ettlinger and Hodgkins, 1956; Emeruna, 1982) due, at least in part, to BITC (Das et al., 1954). Again, however, it is not clear whether all the antibiotic activities are due to one or more compounds present in these seed preparations. In papaya seeds, BITC is formed from benzyl glucosinolate (Gmelin and Kjær, 1970; Tang, 1973; Bennett et al., 1997), the major or perhaps only glucosinolate present. Glucosinolates in the seeds of many plants are metabolized to yield isothiocyanates by the action of enzymes commonly called myrosinases (thio-glucoside hydrolases, EC 3.2.3.1) (Ettlinger and Hodgkins, 1956; Palmieri et al., 1982), enzymes that are brought into contact with their substrate(s) upon damage to seeds in which they are found. The myrosinase and glucosinolates are in different compartments of the papaya seed (the endosperm and sarcotesta of the seed, respectively), although a small fraction of substrate and enzyme reside together in the embryo (Tang, 1973). It follows that papaya seeds must be crushed or otherwise damaged to produce substantial amounts of the antibiotic BITC. Different myrosinase/glucosinolate combinations give rise to different products, with some producing a combination of thiocyanates and isothiocyanates, others a mix of nitriles, thiocyanates and isothiocyanates and still others isothiocyanates alone (Saarivirta and Virtanen, 1963; Virtanen, 1965). It remains to be determined whether the myrosinase/benzyl glucosinolate combination of papaya seed gives rise to all the possible products listed above or only BITC.
More recently, Bennett et al. (1997) found that papaya leaves and stems contain cyanogenic glucosides. This finding was unanticipated because plants that contain glucosinolates do not, as a rule, contain cyanogenic glucosides (Bennett et al., 1997; Conn, 1980). However, cyanogenic glucosides offer a potential source of highly toxic cyanide if they are also present in seeds. The prospect of cyanide contributing to the antibiotic properties of papaya seed preparations is difficult to address since seeds were not tested for these glucosides in the earlier study (Bennett et al., 1997) and so we felt it necessary to test for the presence of cyanogenic glucosides in papaya seeds. In this study we elected to use C. elegans as an endpoint to measure anthelmintic activity. While C. elegans is a free living nematode and not a parasite, it is considered to be a good nematode model with important genetic similarities to parasitic nematodes (Blaxter, 1998). Furthermore, parasitic nematodes have yet to be propagated outside of their hosts. Our findings show that anthelmintic activity is present or recovered from either water-soluble (‘‘aqueous’’) or non-polar organic solvent (‘‘nonaqueous’’) extracts prepared from papaya seeds and that BITC is present in both extracts. We propose that BITC is the major and probably only source of anthelmintic activity present in papaya seeds and that it is produced in preparations of these seeds primarily, and probably exclusively, as the result of the action of resident myrosinase(s).
2. Results Commercial BITC (98% pure) was toxic to C. elegans, as expected (Table 1). Extracts of fresh papaya seeds prepared with water were also toxic to C. elegans and were shown to contain BITC (Table 1). A volume of 10–20 ml of these aqueous extracts, when freshly prepared, representing the contents of about 1.2–2.4 mg of seed, was sufficient to kill C. elegans in our 0.5 ml assay. BITC was also found in oil concentrates prepared from seeds that were Soxhlet extracted with pentane and between 0.04 and 0.1 ml of this oil, representing the contents of 4–10 mg of seed, killed the nematodes in our assay (Table 1). If one considers the measure of toxicity as the LC90 or the concentration of the compound needed to kill >90% of the nematodes within 4–5 h, this value is roughly equivalent regardless of the source of BITC (Table 1). Exposures for periods exceeding 12 h were generally lethal to all the nematodes in the assay when the concentration of BITC was at the LC90, irrespective of the source of BITC. Fig. 1 shows the log of the concentration of BITC in commercial BITC and in the aqueous and oil extracts of papaya seeds plotted against the log of the minimum volume of these preparations needed to kill 90% of the nematodes. A highly significant constant
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R. Kermanshai et al. / Phytochemistry 57 (2001) 427–435 Table 1 BITC and papaya seed extracts decrease the viability of Caenorhabditis elegansa Preparation
BITC standardb Seed extract a b c d
Dose to kill>90% nematodes
Crude aqueous Crude oil
BITC
Volume (ml)
Seed numberc
Concentration (M)
LC90d (mM)
0.001–0.002 10–20 0.04–0.1
– 0.03–0.06 0.1–0.25
7.46 0.0006 0.196
15.30 12–24 16.39
Representative results shown, experiment was repeated three times. Commercially available, 98% pure. One seed weighs approximately 40 mg after the mucilaginous coat has been removed. Concentration of BITC in the 0.5 ml nematode viability assay required to kill >90% of the C. elegans in 4–5 h.
relationship is found with an r2 of 0.989, an outcome that is best explained if BITC is the active anthelmintic principle in each of these preparations. When the seed residue, after having been thoroughly extracted with pentane to remove BITC, is re-extracted with water, the resulting extract is still a potent anthelmintic (100 ml of this extract kills >90% of C. elegans). This could be explained if one or more of the active principles present is soluble in water and not in pentane, or if a water-soluble precursor of the active principle is converted to the active principle, that is only then soluble in pentane. Since benzyl glucosinolate is water soluble, is known to be present in papaya seeds (Tang, 1973; Bennett et al., 1997) and is a precursor of BITC that, in turn, is soluble in pentane, the latter hypothesis is plausible and could explain why both extractions yield BITC (Table 1). Myrosinase, an enzyme that catalyzes the conversion of glucosinolates to isothiocyanates (Tang, 1973; Palmieri et al., 1982), was present in water extracts of ground papaya seeds and was found to elute in the void volume of a
Sephadex G-50 column, as expected, because of the high molecular weight of myrosinases (Botti et al., 1995). Anthelmintic activity eluted much later from the column and was absent from the void volume (results not shown). The results in Table 2 show that aqueous extracts of seeds ground after heat-treatment of the seed exhibit no apparent nematode killing activity relative to extracts of untreated seeds at the volumes tested. Also, subjecting the extract of heat-treated seeds to a cyclocondensation reaction with benzene dithiol (Zhang et al., 1992) produced an absorption spectrum with no symmetrical peak at 365 nm thus giving no evidence for the presence of BITC (Fig. 2A). However, when extracts from these heat-treated seeds were incubated with an aliquot of the void volume obtained from a Sephadex G-50 column loaded with an extract prepared from fresh seeds, the nematode killing potential of the mixture increased markedly (Table 2). When this preparation was subjected to the cyclocondensation reaction described above, the absorbance spectrum produced (Fig. 2B) is typical of that published for pure isothiocyanates processed in the
Fig. 1. Regression plot showing the relationship between BITC concentration of various preparations and dose required to kill >90% of Caenorhabditis elegans in a nematode killing assay.
Fig. 2. Absorption spectra of an extract of heat-treated seed before (A) and after (B) incubation of the extract with a VoG50 fraction containing myrosinase activity. For panel B, the sample was incubated for 30 min and diluted eight fold with 10 mM potassium phosphate (pH 6) buffer before measurement.
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same fashion (Zhang et al., 1992). No nematode killing activity was found in samples where the fresh seed extract obtained from the void volume of a Sephadex G-50 column was boiled for 10 mm prior to incubation with the heat-treated seed extract (Table 2). In a comparable experiment the LC90 of BITC for the sample subjected to activation conditions was determined to be 20 mM. This LC90 is similar to that listed for all the preparations used in Table 1, again suggesting that BITC is the active nematode killing principle here. An oil fraction of papaya seed, prepared by Soxhlet extraction with pentane, was applied to a silica column and compounds present were separated by elution from the column with increasingly polar organic solvents. The BITC content and the C. elegans killing activity eluted in the same fraction (Table 3). In preliminary experiments, 40–60% of the nematode killing activity Table 2 Loss of toxicity in aqueous papaya seed extracts due to heat treatment can be restored upon incubation with a protein fraction prepared from fresh papaya seedsa Incubation mixture
Dosage tested in nematode viability test
Main componentb
Additionc
Volume (ml)
Nematodes killed (%)
Buffer Extract of heat-treated seed in buffer
VoG50d Buffere VoG50
100 100 50 100 100
0 0 100 100 0
Boiled VoG50 a
Representative results shown, experiment was repeated five times. 0.5 ml Volume with buffer comprised of 10 mM potassium phosphate (pH 6.0) and 1 mM ascorbic acid. c Volume of addition was 0.2 ml. d VoG50 is the void volume fraction from a Sephadex G-50 column (see Experimental). No nematode killing activity was detected in this fraction. e 10 mM potassium phosphate (pH 6.0) and 1 mM ascorbic acid. b
Table 3 BITC content and toxicity of silica column fractions of papaya seed extractsb Solvent for elution of fraction
Crude oil 100% Hexane 85% Hexane/15% dichloromethane 100% Dichloromethane 100% Methanol a
eluted in a 90% hexane (H)/10% dichloromethane (DCM) mixture with the balance of the killing activity eluting in a 75% H/25% DCM mixture. The 90% H/ 10% DCM fraction was examined by GC and found to contain a single prominent peak that upon subsequent examination by mass spectroscopy and infrared spectroscopy, was found to correspond to BITC (analysed by the Center for Mass Spectrometry, McMaster University). The 75% H/25% DCM fraction was examined by TLC, and upon exposure to iodine vapour, was found to contain a number of spots, so no further tests were performed with this fraction (results not shown). In subsequent fractionations, all the BITC and all the anthelmintic activity co-eluted from the column in a mixture of 85% H/15% DCM (Table 3). Despite the presence of other compounds as shown by TLC and a recovery of BITC that was less than 100% of the crude, this fraction yields an LC90 estimate which is similar to that determined for the crude oil from which it was produced and one that lies within the range of values quoted in Table 1. We noted that the nematode killing power of aqueous extracts of papaya seeds incubated at room temperature for 10 h decreased to 30–50% of a freshly prepared extract (Table 4). Aqueous extracts of papaya seeds were either extracted immediately with diethyl ether or following a 10 h incubation at room temperature. Both diethyl ether extracts were then fractionated on a silica column and the C. elegans killing activity and BITC content of each solvent fraction were determined. For both the unincubated and incubated samples, the nematode killing activity and BITC co-eluted in the 85% H/15% DCM fraction (Table 4). In the case of the sample that had first been incubated for 10 h, the BITC content of the 85% H/15% DCM fraction was only half that found in the same fraction of the unincubated Table 4 Incubating crude seed extracts of papaya for 10 h reduces their BITC content and toxicity to nematodes Seed extract incubationa
Fraction
BITC (mM)
Minimum volume required for 100% nematode kill (ml)c
None
Crude 85% Hexane/15% dichloromethane
n.d.b 504
30–50 16
10 h
Crude 85% Hexane/15% dichloromethane
n.d. 240
BITC Concentration (mM)a
LC90 (mM)
196 0 130
39 0 29
0 0
0 0
The volume of each fraction was reduced by evaporation and, where necessary, volumes were made equivalent by the addition of hexane. b Representative data shown. The experiment was completed twice with identical results.
100 50
a Incubation at room temperature prior to chromatographic separation of crude extract on silica and the subsequent testing for BITC content and toxicity to nematodes. b n.d. Means not determined. c Volumes were corrected for differences in resuspension volume of the chromatographic fractions (see Experimental for silica chromatography and GC quantification of BITC).
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sample (Table 4). In separate experiments aliquots of the aqueous extract were incubated for up to 9 h at room temperature before extraction with diethyl ether. The diethyl ether layer was then assayed for anthelmintic activity and BITC content. After six hours of incubation, only 75% of the anthelmintic activity and 70% of BITC remained relative to those measured for the nonincubated sample. After nine hours of incubation, these values decreased to 50 and 39% for anthelmintic activity and BITC level, respectively. Thus, the same conclusion was reached, C. elegans killing activity and BITC content decreased with the length of incubation. In an effort to identify other putative anthelmintic compounds in papaya seed extracts we attempted to obtain carpaine from the papaya seeds following the procedure of Govindachari et al. (1965) but all our attempts were unsuccessful. Seed extracts of Lepidium sativum have been found by colorimetric methods to contain BC (benzyl cyanide) and BTC (benzyl thiocyanate) as well as BITC, their proportions varying with extraction conditions (Virtanen, 1962; Saarivirta and Virtanen, 1963, 1965; Gil and MacLeod, 1980). Preparations from Tropaeolum and Sinapsis alba seeds, on the other hand, contained only BITC (Saarivirta and Virtanen, 1963). Papaya seeds have not been examined specifically for their content of BTC, BC and benzonitrile (BN). An aqueous preparation of seeds was extracted twice with diethyl ether, the diethyl ether layer was evaporated in vacuuo and the resulting oil resuspended in hexane. This sample, which contained all the anthelmintic activity from the extracted seeds, was applied to a silica column and the anthelmintic activity containing fraction (85% H/15% DCM), was subjected to GC under conditions that separate BITC, BTC and BC (Fig. 3, inset). BITC was found as was an unknown compound tentatively identified as butylated hydroxytoluene (BHT is introduced as the stabilizing additive for the diethyl ether) but no BTC or BC was detected in this preparation (Fig. 3). By an in vitro assay, BITC was more potent as an anthelmintic than BTC and far more potent than BC (Table 5). Taken together, these observations indicate that BTC and BC do not appear to contribute significantly towards the anthelmintic power of papaya seed extracts. A commercial source of benzonitrile (BN) was also examined for its toxicity towards C. elegans and was found to have a very high LC90 of >19 mM (Table 5). Also, BN has a retention time of 2.98 min when subjected to the same GC elution conditions as used to separate BITC, BTC and BC (data not shown) and no such peak was noted on the chromatograph shown in Fig. 3. Therefore, insufficient BN is present in the biologically active fraction for BN to be considered responsible for killing C. elegans. Bennett et al. (1997) reported that leaves and stems of papaya seedlings contained cyanogenic glucosides with the highest concentration found in leaves (extracts
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Fig. 3. GC analysis of papaya seed preparations with anthelmintic activity shows that BITC is present but BC and BTC are absent. An aqueous papaya seed extract was extracted with diethyl ether, concentrated and dissolved in hexane and this was fractionated on a silica column. The 85% H/15% DCM fraction containing anthehnintic activity was evaporated to dryness and dissolved in CS2 for GC analysis (see Experimental). This experiment was performed twice with similar results. The inset shows a GC separation of commercially available BC, BTC and BITC.
Table 5 Toxicity of various compounds to Caenorhabditis elegans Compounda
LC90 (mM)
BITC Benzyl thiocyanate Benzyl cyanide BN KCN
15–45 200 5000 >19,000 >100
a Commerically available compounds were dissolved in DMSO. DMSO controls had no negative effect on nematodes.
showing 8 mM cyanide) but seeds were not tested. We found that the C. elegans LC90 for KCN exceeded 100 mM in our killing assay (Table 5) and while cyanide at this concentration caused nematodes to move more slowly relative to the assay without cyanide, they recovered completely within 5 h. The cyanide content of aqueous extracts of papaya seeds was always less than 28 mM even after the extracts had been incubated with b-glucosidase at room temperature for two hours. When these extracts are used in a nematode killing assay, the final concentration of cyanide present in the assay at which 90% of the nematodes are killed is less than 5 mM, a concentration well below the 100 mM KCN found to be non-lethal to nematodes. Processing a papaya seed sample with KCN added to a final concentration of 20 mM yielded a sample whose concentration was equivalent to that of the original sample plus
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the added KCN, demonstrating that cyanide was not disappearing from the extract during the extraction procedure. BITC added to extracts and to the cyanide assay mixture did not give rise to cyanide, hence there was no complication from endogenous glucosinolates or BITC. It follows from the above that the cyanide/cyanogenic glucoside contents of the papaya seed extracts do not contribute to their anthelmintic power.
3. Discussion 3.1. BITC is the active compound in papaya seed preparations Our results show that the anthelmintic activity of papaya seed extracts prepared using water or pentane co-purifies with BITC during column chromatography. Further, re-extraction of aqueous extracts with diethyl ether leads to a complete recovery of both bioactivity and BITC in the diethyl ether fraction. We noted that changes in nematode killing activity of preparations were paralleled by changes in their BITC content. For example, the increase in anthelmintic activity obtained by incubating extracts of heat-treated seeds with a soluble protein fraction containing myrosinase activity is accompanied by an increase in BITC concentration. Also, when extracts are left for 10 h at room temperature, there is a decrease in killing activity of the extract that coincides with a decrease in BITC concentration. Aqueous extracts of papaya seeds do not appear to contain enough cyanogenic glucosides to be toxic to nematodes nor is there any detectable BTC, BC or BN in the fraction containing the anthelmintic activity. Taken together with the previous literature, all these results indicate that the main and perhaps the only effective anthelmintic in the papaya seed preparations we investigated is BITC. This conclusion supports the earlier proposal by Dar et al. (1965), but provides a more thorough investigation of possible candidate bioactive compounds naturally occurring in the papaya seed extract. 3.2. Toxicity of papaya seed preparations According to our measurements it takes the equivalent of 1.2–2.4 mg of seed to kill >90% of the C. elegans in our 500 ml assay within 4–5 h and completely rid the assay mixture of live nematodes within 12 h. Given these in vitro viability results and assuming that the volume of the small intestine of an adult human is approximately 1.3 l (Masoro, 1973), then a dose of 3.1– 6.2 g of papaya seeds would be equivalent to that which is effective against C. elegans in our assay. However, this dosage is likely to be an underestimate given that it does not consider factors that would reduce the intestinal BITC concentration over time including the movement
of contents through the gut [with the residence time of a bolus being very short for the stomach and 3–7 h for the small intestine, (Osterwald, 1990; Fleisher et al., 1999)], and the likelihood that BITC is absorbed by the intestinal epithelium. Our calculated dose approaches the published daily dose recommended for the medicinal use of papaya seeds in Cuba (1–1.5 g three times a day, Roig y Mesa, 1974). If one gives the equivalent of 3.1– 6.2 g of seeds to a 55 kg human, this amounts to a dose of 56–112 mg/kg. Despite the speculative nature of this dosage, this value was shown to be non-lethal to mice and rats (Chinoy et al., 1994) and a 5–10-fold lower dose was shown to be effective against Ascaris in children (Robinson, 1958). A consideration relevant to the medicinal use of papaya seeds, is that BITC must be released from benzyl glucosinolate by a papaya myrosinase. It follows that factors that could inhibit myrosinase or inactivate BITC could compromise the concentration of BITC in the small intestine. One such factor might be exposure to acid (pH 2) in the stomach (Magee, 1973). We tested the effect of acid exposure on the potency of papaya seed extracts by deliberately acidifying aqueous seed extracts to pH 2.6 with HCl and then neutralized the extract to pH 6.5 with 2 M Na2CO3 either immediately or after one hour. Comparison of the in vitro anthelmintic activity of these acidified and neutralized extracts relative to that of an untreated control seed extract showed that all the preparations were of equivalent potency. An additional consideration that was not evaluated in this study is that there may be variability among papayas with respect to myrosinase or glucosinolate content. Such variation or indeed any factor that could affect BITC content of seed extracts would, in turn, lead to differences in nematode killing potency between preparations. The BITC content of papaya seeds could be lethal to some beneficial as well as harmful intestinal microorganisms (Emeruna, 1982). Thus while treatment with papaya seeds might eliminate intestinal bacteria, this treatment would not lead to an infection of the intestine by eukaryotic parasites, such as Candida, since they would also be killed by the BITC (Stoll and Seebeek, 1948). In addition to nematode killing activity, BITC appears to elicit several biological effects. BITC inhibits chemically induced carcinogenesis in animal models and cell cultures (reviewed in Wattenberg, 1977; Zhang and Talalay, 1994) but is potentially toxic at therapeutic dosages since it is reported to be goitrogenic, carcinogenic and mutagenic (Yamaguchi, 1980; Fenwick et al., 1983). Thus a concern in any therapeutic application of BITC must address how different are the therapeutic and toxic/lethal doses of the vermifuge. Fenwick et al. (1983) analysed data that suggests that BITC is goitrogenic and proposed that the goitrogenic effect is probably more associated with iodine deficiency
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than to a direct effect of BITC alone. If such is the case, any adverse effects of BITC might be averted by an iodine supplement. Lohiya et al. (1994) showed that papaya seed extracts administered to male rats for 30–60 days (at a dose equivalent to 67–333 mg/kg/day) can result in temporary but completely reversible sterility. This daily dose is comparable to our speculative dosage (see above), but the comparatively short duration for therapy to kill nematodes in humans should pose no threat in this regard. Yamaguchi (1980) found that BITC at 50 mM (roughly equivalent to 7.5 mg of papaya seed/l) doubled the spontaneous reversion rate of Salmonella typhimurium strain TA100 and that activation by rat liver extract S-9 had no effect on this reversion rate. TA100 is much more sensitive than the wild type strain to mutagens and a petri dish assay is very different from the inside of a mammalian intestine, but the 7.5 mg seed/l is only slightly greater than that used to kill C. elegans in our assay (2.4–4.8 mg of seed/l) and could be, therefore, a source of concern. In contrast, at much higher concentrations in experimental animals, BITC seems to act as an anticarcinogen that seems to be due to the antagonistic effect of BITC on the enzymatic activation of mutagens (Wattenberg, 1977; Zhang and Talalay, 1994; Talalay and Zhang, 1996; Fahey et al. 1997). Isothiocyanates, formed from their respective glucosinolates, have been found in a number of different plants (notably in members of the Cruciferae) where they have been shown to have anticarcinogenic properties (Wattenberg, 1977; Zhang and Talalay, 1994; Talalay and Zhang, 1996). With respect to cyanide/cyanogenic glucosides in papaya seeds, a fatal dose of KCN for humans, according to Merck (1989), is between 1.85 and 2.22 mM and the fatal blood level of CN is reported as 115 mM (Henry, 1979). These levels are obviously well above those found by us in the seeds, let alone the levels expected after dilution by the digestive tract, the bloodstream and inter- plus intrastitial water. It would appear from the foregoing discussion and from many generations of traditional use of papaya seeds as an anthelmintic that this work, based upon chemical and biological analysis of papaya seed extracts, supports existing practices and provides insight into how they may be improved.
4. Experimental 4.1. Strains and materials Caenorhabditis elegans strain N2 was obtained from Dr. J. Culotti, Hospital for Sick Children, Toronto, ON, Canada. BITC, 1,2 benzenedithiol, BTC, BC, BN,
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N-chlorosuccinimide, succinimide, pyridine and barbituric acid were from Aldrich. Papaya (Carica papaya) was purchased locally. 4.2. Growth and maintenance of C. elegans and toxicity assay C. elegans was maintained on solid NGM medium (Sulston and Hodgkin, 1988). Between 20 and 50 nematodes were placed in 500 m1 of S medium (Sulston and Hodgkin, 1988) in microtitre wells (Falcon No 3047, Becton Dickinson) containing 30 ml of a 5 times concentrated overnight culture of E. coli WP2 (uvrA trp malB, Witkin, 1975) resuspended in S medium. Aliquots (5–100 ml) of aqueous extracts (in water or 10 mM potassium phosphate, pH 6) were added directly to the wells while concentrated oils, fractions in non-aqueous solvents and commercial BITC were diluted with dimethyl sulfoxide (DMSO) prior to being added to the wells as 10 ml volumes. Dilute extracts prepared in non-aqueous solvents were concentrated first by evaporation under N2, the residue resuspended with 1 ml of hexane, dried with air and the final residue was dissolved in 10 ml of DMSO which was then added to the test wells. Controls with the equivalent volumes of DMSO alone were negative. Nematode survival was measured 4–5 h after addition of the extracts and again 12–13 h later. 4.3. Preparation of seed extracts Seeds harvested from papaya fruit had their mucilaginous coating removed prior to extract preparation. ‘‘Aqueous extracts’’ were prepared by grinding seeds with a mortar and pestle using distilled water (1 g seeds to 10 ml water), at room temperature and removing the debris by centrifugation at 16,000 g at 4 C for 30 min. ‘‘Oils’’ were prepared by grinding seeds in a mortar and pestle, with no added solvent and then adding the ground material to a thimble and extracting by refluxing 10 times in 8 volumes of pentane, in a Soxhlet extractor. The final extract in pentane was concentrated in vacuuo at 38 C. A typical extraction of 14 g of seeds yielded approximately 0.9 ml of crude oil following Soxhlet extraction. 4.4. Fractionation of extracts Aqueous extracts (10 ml), prepared as described above, were extracted with 10 ml of diethyl ether and the aqueous phase was recovered and re-extracted two more times with 10 ml of diethyl ether each time. The three diethyl ether fractions were pooled and evaporated in vacuuo. The resulting residue was dissolved in 1 ml of hexane and 0.45 ml was loaded onto a 10 g silica (Silica gel, 63–200 mm mesh, BDH) column (231.3 cm). Oil prepared from seeds (described above) was also
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fractionated by a comparable silica column. In this case, oil (0.45 ml) was loaded without further preparation onto the matrix. For fractionation of either aqueous or oil extracts, samples were eluted from the column at a rate of 0.7 ml min 1 with successive 100 ml volumes of 100% hexane, 85% hexane/15% dichloromethane, 100% dichloromethane and 100% methanol. The resulting eluates were evaporated in vacuuo at 42 C and the residues were made to a final volume of 500 ml with hexane and stored at 18 C. Aliquots (10–100 ml) of each fraction were evaporated to dryness, the residue dissolved in 10 ml DMSO which was then used in the nematode killing bioassay to titre the potency of the fraction. 4.5. Activation of heated seed extracts with myrosinase Seeds (with their mucilaginous coats removed) were either left unheated or were heat-treated by immersing a tube containing seeds in boiling water for 10–20 mm or by autoclaving the seeds at 18 psi for 20 mm. Aqueous extracts were then prepared from the unheated and heat-treated seeds by grinding 1 g seeds with 10 ml of 10 mM potassium phosphate (pH 6) buffer. One milliliter of extract from unheated seeds was loaded onto a Sephadex G-50 column (10 ml bed volume). The fraction eluting in the 3.5 ml void volume (VoG50) contained the myrosinase activity (1.25 mmol sinigrin disappearing min 1 ml 1, using the assay of Palmieri et al., 1982). Heat-treated seed extracts (0.5 ml) were incubated with 0.2 ml of the above VoG50 fraction for 30 mm at room temperature and then placed on ice. lAscorbic acid was included at a final concentration of 1 mM in the activation incubations. Aliquots of the activated extract were removed and used to measure killing of C. elegans as described above (5–100 ml per assay) and measure BITC concentration (10–100 ml for colorimetric determinations or 1–5 ml of 10–50-fold diluted preparation for GC measurements). 4.6. Measurement and detection of compounds TLC was used as an initial method for determining purity of extracts. Samples were spotted onto silica Gel-G plates, developed in methanol–n-butanol–conc. HCl–H2O (10:10:1:1; v/v/v/v), and plates were then exposed to iodine vapours to visualize organic compounds Colorimetric measurement of BITC was made following a procedure modified from that described by Zhang et al. (1992). Two aliquots of a sample to be measured were each made to a final volume of 500 ml with 10 mM Tris–HCl (pH 7.5). To one aliquot, 500 ml of 13 mM benzene dithiol in methanol was added while to the second aliquot 500 ml of methanol (control) was added. A blank reaction with buffer and benzene dithiol
was also prepared. All of the reactions were incubated at 65 C for 60 min in a microfuge tube and then microfuged for 5 min. Absorbance of the supernatant was taken at 365 nm with a Uvikon 930 spectrophotometer and compared to those of a standard curve prepared using commercial BITC. For BITC determinations, measurement using GC was found to have greater specificity. GC was performed on a Hewlett Packard 5790A equipped with a flame ionization detector. The column was a Megabore DBI column with a 30 m length, 0.52 mm inner diameter and 0.24 mm film thickness. The carrier gas was H2 maintained at a flow rate of 12 ml min 1. Injector port and flame ionization detector were set at 250 and 300 C, respectively. Hydrogen and compressed air were delivered to the detector at flow rates of 120 and 300 ml min 1, respectively. Typical injection size was 1–3 ml of sample dissolved in CS2 or diethyl ether. At injection, the oven temperature was 65 C and was maintained at that temperature for 4 min and then increased at a rate of 5 C per min to a final temperature of 160 C. The output signals from the detector were recorded and integrated with a Shimadzu CR3A Chromatopac Integrator. Calibration using a dilution series of standards with known concentrations was analysed prior to each set of sample measurements. Concentrations were determined by comparing the responses from samples with those of the standards for injections of the same volume. For GC analysis, papaya oil samples were diluted directly in CS2 or diethyl ether. Aqueous samples were extracted into diethyl ether and either injected directly into the GC or evaporated under N2 gas, the residue dissolved in CS2 and then injected as above. Measurement of cyanogenic glucosides and cyanide followed the procedure of Halkier and Møller (1989). Papaya seeds were ground with 50 mM Mes-NaOH (pH 6.5) and extracts incubated at room temperature with almond b-glucosidase at 1 mg ml 1 (Sigma G-0395) for 2 h. Reaction mixtures were adjusted to 0.83 M NaOH and incubated for 1 h to liberate free CN. A 50 ml aliquot of glacial acetic acid was then added to each reaction mixture and the whole mixture was assayed for CN content (Halkier and Møller, 1989). The absorbance of the coloured complex formed was read at 585 and 650 mm and the difference in absorbance values were used to quantify CN levels by comparison to a standard curve prepared with KCN.
Acknowledgements We gratefully acknowledge K. Green, McMaster Regional Centre for Mass Spectrometry for mass spectrometry analysis. This research was supported in part by a research grant to E.A.W. by the Natural Sciences and Engineering Research Council of Canada.
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