Mutation Research, 224 (1989) 209-212
Elsevier MUTGEN 01459
Mutagens from roasted seeds of Moringa oleifera I r e n e M. V i l l a s e n o r 1, C l a r a Y. L i m - S y l i a n c o i a n d F a b i a n D a y r i t 2 I Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101 (Philippines) and 2 Department of Chemistry, Ateneo de Manila University, Loyola Heights, Quezon City (Philippines) (Received 22 November 1988) (Revision received 7 March 1989) (Accepted 13 March 1989)
Keywords: Moringa oleifera; Mutagenic compounds
Summary A number of biosynthetically and chemically related compounds were isolated from the roasted seeds of
Moringa oleifera. The micronucleus test, an in vivo method, using albino mice as the test system, was used for monitoring the mutagenicity of the isolated compounds. Structure-activity correlation studies showed that 4(a-L-rhanmosyloxy)phenylacetonitrile, 4-hydroxyphenylacetonitrile, and 4-hydroxyphenyl-acetamide exhibited mutagenic activity.
Moringa oleifera is commonly known as either the horseradish tree, drumstick tree, radish tree, or West Indian ben. The seeds, when roasted, are used externally for the treatment of gout and acute rheumatism (Quisumbing, 1978). However, reports exist in the literature documenting the mutagenicity (Cabalfin et al., 1982) and clastogenicity (Lim-Sylianco et al., 1985) of the roasted seeds after metabolic activation. The mutagens present in the roasted seeds of Moringa oleifera have recently been isolated and their structures elucidated (Villasenor, 1988). Solvent partitioning was the procedure adopted for isolating the mutagens from the ethanolic extract of the roasted seeds. The extracts were purified through successive column chromatographic runs using silica gel 60G. The structures of the
Correspondence: Dr. I.M. Villasenor, Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101 (Philippines).
mutagens were elucidated by spectral methods, including Fourier transform infrared, electron ionization mass spectrometry, ~H- and 13C-nuclear magnetic resonance and 2-dimensional correlation spectroscopy. In this study, the structure-activity correlations of the mutagens were examined using the micronucleus test (Schmid, 1975). A probable mutagenic mechanism is proposed based on the results of the bioassay. Materials and methods
Chemicals 4(c~-L-Rhamnosyloxy)benzylisothiocyanate, 4 (a-L-rhamnosyloxy)phenylacetonitrile, 4-hydroxyphenylacetonitrile, 4-hydroxyphenylacetamide, and 4-hydroxyphenylacetic acid were isolated from the roasted seeds of Moringa oleifera Lam. The reagents used for the micronucleus test were fetal calf serum (Gibco), Giemsa stain (Merck), May-Grunwald stain (Sigma) and dimethyl sulfoxide (DMSO) (Fluka) as solvent.
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
210 TABLE 1 STRUCTURE-ACTIVITY CORRELATION STUDIES OF THE COMPOUNDS ISOLATED FROM ROASTED SEEDS OF Moringa oleifera USING THE MICRONUCLEUSTEST Compound
Number of MN-PCE/1000 PCE (mean + SD)
4( a-L-Rhamnosyloxy)benzylisothiocyanate 4( et-l~-Rhamnosyloxy)phenylacetonitrile 4-Hydroxyphenylacetonitrile 4-Hydroxyphenylacetamide
8.8 43 43 80 40 47
2.67 + 0.42 11.38 + 0.60 9.93_+0.68 11.27 + 1.36 8.07_+0.93 3.53 _+0.81 2.52+0.62 8.40 + 0.57
4-Hydroxyphenylaceticacid DMSO (8.0 ml/kg) Tetracycline
a Five mice per concentration; 3 slides per mouse.
Micronucleus test Swiss Webster albino mice, 7-12 weeks old, were used. Five mice were used per dosage of the test compounds and 5 mice each for the spontaneous, positive, and solvent controls. The required weights of the test compounds were dissolved in DMSO. Two doses, 24 h apart, were administered intraperitoneally. Six hours after the second administration, the mice were killed by dislocation of the neck. Both femora were removed by cutting through the pelvis and tibia. After removal of the muscle tissues, the distal epiphyseal portion was torn off. The proximal end of the femur was shortened carefully with scissors until a small opening to the marrow canal was seen. About 0.2 ml of fetal calf serum was introduced into a 1-ml syringe and then the hypodermic needle was inserted a few mm into the proximal end of the marrow. The femur was then submerged completely in 2 ml serum in a 5-ml test tube and the marrow was aspirated. After flushing and aspirating several times, the test tube was centrifuged at 1000 rpm for 5 rain and the supernatant was decanted. The cells in the sediment were mixed carefully using a Pasteur pipette and aspirator and were then smeared on glass slides. The 3 slides prepared for each mouse were left overnight to dry. The following procedure was used in staining the slides: 3 min in undiluted M a y - G r u n w a l d stain; 2 rain in 50% M a y - G r u n w a l d stain solution diluted with distilled water; and 10 rain in 15% aqueous Giemsa stain solution.
The number of micronucleated polychromatic erythrocytes (MN-PCE) per 1000 polychromatic erythrocytes (PCE) was counted using a highpowered microscope.
Results Based on the results of the micronucleus test (Table 1), the number of M N - P C E for 4(Ot-Lrhamnosyloxy)phenylacetonitrile (11.38), its aglycone (9.93), and 4-hydroxyphenylacetamide (8.07) indicates that they are mutagenic to mice at a dosage of 40 m g / k g body weight. A dosage of approximately 96 m g / k g of the aglycone caused the death of mice while increasing the dosage of the amide to 80 m g / k g caused a corresponding increase in the number of MN-PCE. 4(Ct-L-Rhamnosyloxy)benzylisothiocyanate was toxic even at low dosages of 50 mg and 22 m g / k g . The mice died after the first intraperitoneal administration of the compound. At the non-toxic dose of 8.8 m g / k g , 4(a-t-rhamnosyloxy)benzylisothiocyanate did not induce micronucleus formation in PCE.
Discussion The micronucleus test has the advantage of being an in vivo cytogenetic method. In addition to the detection of chromosomal damage, the micronucleus test is also highly suitable for detecting chromosome loss due to the partial impairment of the spindle apparatus (Heddle et al., 1983). Micro-
211 CH z - C==N
(~ HO - - ~
C Hz"C= ~ -
"C= NH -P H '
I 4. CHz'-C=NH2~-. ~ H O
CH:,- C \ NHz
C--H ~ ~HO J OH
C--H I OH
Fig. 1. Proposed metabolicactivation pathwayfor 4(a-L-rhamnosyloxy)phenylacetonitrile,its aglycone,and 4-hydroxyphenylacetamide.
nuclei originate from chromatin which lags in anaphase. 4( ct-t-Rhamnosyloxy)benzylisothiocyanate was identified as an active antimicrobial agent from the seeds of Moringa oleifera (Eilert et al., 1981). It is formed by the enzymatic hydrolysis of the parent glucosinolate (Badgett, 1964). Thermal degradation of the same glucosinolate produces 4(aL-rhamnosyloxy)phenylacetonitrile and it is well documented that nitriles arise from the thermal degradation of glucosinolates (MacLeod et al., 1981; Gil and MacLeod, 1980).
In the present study, structure-activity correlation suggests that 4-hydroxyphenylacetonitrile is the proximate mutagenic agent in roasted seeds of Moringa oleifera since significant increases in MN-PCE were observed. The deoxy-sugar moiety of 4(et-L-rhamnosyloxy)phenylacetonitrile is readily cleaved by a glycosylase while 4-hydroxyphenylacetamide is readily converted to 4-hydroxyphenylacetonitrile by protonation and subsequent dehydration. 4-Hydroxyphenylacetonitrile is readily converted to 4-hydroxybenzaldehyde by ct-hydroxylation and subsequent release of cyanide
(CN-). Studies (Kaplita and Smith, 1986; Freeman and Hayes, 1987) showed that saturated nitriles undergo hepatic biotransformation in mice and rats to release C N - via a cytochrome P-450dependent oxidation of the a-carbon yielding a cyanohydrin intermediate (Fig. 1). The mutagenicity of several aldehydes is well documented. 4-Hydroxyphenylacetic acid did not show positive mutagenic activity. This is reasonable based on an experiment by Neudecker and Henschler where in the presence of cyanamide, an inhibitor of aldehyde dehydrogenase (ALDH), there was an increase in the activity of acrolein, a known mutagenic aldehyde. ALDH converts aldehydes to their corresponding acids, thus inhibition of ALDH will lead to an increase in the concentration of the highly mutagenic aldehydes.
Acknowledgements This work was supported by the Department of Science and Technology (DOST), the Natural Science Research Institute (NSRI), and the Office of Research Coordination (ORC). Thanks are due to the U.P.-AdMU-DLSU Ph.D. Consortium for the fellowship of I.M.V. and to Ms. Angela Alcantar of the Department of Chemistry, Ateneo de Manila University.
References Badgett, B.L. (1964) Part one. The mustard oil glucoside from Moringa oleifera seed. Part two. Ascorbic acid analogue with deoxy side chain, Diss. Abs., XXV, 1556-1557.
Cabalfin, E.A., C.Y. Lim-Sylianco and J.A. Concha (1982) Mutagenicity studies on forty one Philippine medicinal plants, Asian J. Pharm., IV, 9-17. Eilert, U,, B. Wolters and A. Nahrstedt (1981) The antibiotic principle of seeds of Moringa oleifera and Moringa stenopetala, Planta Med., 42, 55-61. Freeman, J., and E.P. Hayes (1987) Metabolism of acetonitrile to cyanide by isolated rat hepatocyte, Fund. Appl. Toxicol., 8, 263-271. Gil, V., and A.J. MacLeod (1980) Degradation of glucosinolares of Nasturtium officinale seeds, Phytochemistry, 19, 165:7-1660. Heddle, J.A., M. Hite, B. Kirkhart, K. Mavournin, J.T. MacGregor, G.W. Newell and M.F. Salamone (1983) The introduction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency GeneTox Program, Mutation Res., 123, 61-118. Kaplita, P.V., and R.P. Smith (1986) Pathways for the bioactivation of aliphatic nitriles to free CN in mice, Toxicol. Appl. Pharmacol., 84, 533-540. Lim-Sylianco, C.Y., I. Panizares and A.P. Jocano (1985) Clastogenic effects on bone marrow erythrocytes of some medicinal plants, Phil. J. Sci., 114, 39 52. MacLeod, A.J., S.S. Panesar and V. Gil (1981) Thermal degradation of glucosinolates, Phytochemistry, 20, 977-980. Neudecker, T., and D. Henschler (1985) Allylisothiocyanate is mutagenic in Salmonella typhimurium, Mutation Res., 156, 33 37. Quisumbing, E. (1978) Medicinal Plants of the Philippines, Katha Publishing Co., Inc., Quezon City. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Villasenor, I.M. (1988) Isolation and Structure Elucidation of the Mutagenic Components of Roasted Seeds of Moringa oleifera Lam., Ph.D. Dissertation, University of the Philippines.