Irene M. Villaseñor

Mutagens from roasted seeds of moringa oleifera

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(alpha-L-rhamnosyloxy)phenylacetonitrile, 4-hydroxyphenylacetontrile, and 4-hydroxyphenyl-acetamide exhibited mutagenic activity.

Anticarcinogenicity potential of spinasterol isolated from squash flowers

Spinasterol, an antimutagen, was isolated from squash flowers by solvent partitioning and repeated vacuum liquid chromatography. Spinasterol was then tested for its anticarcinogenic potential by using the mouse skin tumor assay. There was a 90% skin tumor incidence for the positive control group (DMBA + croton oil + acetone). At a concentration of 15.0 microg/0.2 ml acetone, spinasterol decreased the incidence of skin tumors by 55.6% and decreased the number of tumors by 65.0% when applied immediately after croton oil. Hence, spinasterol showed antitumorigenic potential. It is not a co-carcinogen nor a co-tumor promoter as there was no increase in the incidence of skin tumors after spinasterol application. Teratogenesis Carcinog. Mutagen. 20:99-105, 2000.

A new antimutagen from Mentha cordifolia Opiz

The CHCl(3) extract of Mentha cordifolia Opiz. showed antimutagenicity against tetracycline. An antimutagen was purified by solvent partitioning and repeated normal phase-vacuum liquid chromatography (NP-VLC) using a micronucleus test-guided isolation and purification. Spectral analyses showed that the isolated antimutagen is possibly 6,7-bis-(2,2-dimethoxyethene)-2,11-dimethoxy-2Z,4E,8E,10Z-dodecatetraendioic acid. It inhibited the mutagenicity of tetracycline by 68.7% at a dosage of 0.01 mg per 20 g mouse. Statistical analysis using Kruskal-Wallis one-way analysis of variance (ANOVA) by ranks showed that its variance differs from that of the solvent control group (tetracycline+dimethylsulfoxide (DMSO)) at alpha=0.001. Moreover, the isolated antimutagen did not exhibit mutagenic activity at the same dosage. Statistical analysis showed that it is not mutagenic at 0.001 level of significance because its variance differs from that of tetracycline.

Clastogenicity of red pepper (Capsicum frutescens L.) extracts.

Extracts from the fruits of Capsicum frutescens L. were tested for their clastogenicity using the mouse-bone-marrow micronucleus (mouse-MN) assay. Results of the mouse-MN, an in vivo method, indicated that the isolate CF-1 is clastogenic at the maximum tolerated dose of 1.22 mg/kg mouse. Statistical analysis using the Wilcoxon two-sample test showed that the null hypothesis, mu tetracycline = muCF-1, is acceptable at 0.05 and 0.01 degrees of significance. Hence, the clastogenicity of CF-1 is statistically similar to that of tetracycline, a known clastogen, at the 5% and 1% levels of significance.

Antigenotoxic spinasterol from Cucurbita maxima flowers

The antigenotoxic constituent of squash flowers was isolated by solvent partitioning and repeated vacuum liquid chromatography. The micronucleus test, an in vivo method, was used to monitor the antigenotoxicity of the various fractions during the isolation process. Isolate SQFwB2D from the chloroform extract of squash flowers is the most antigenotoxic isolate. It decreased the mutagenicity of tetracycline by 64.7% at a dosage of 100 mg/kg mouse. Statistical analysis using Kruskall Wallis one-way analysis of variance by Ranks showed that SQFw2D is different from the control group (tetracycline + corn oil) at alpha = 0.001. GC-MSD of isolate SQFwB2D shows 2 peaks at Rt = 19.860 (SQFwB2D-1) and 20.242 min (SQFwB2D-2) with relative peak heights of 16:1, respectively. Spectral analyses show that SQFwB2D-1 is 24 alpha-ethyl-5 alpha-cholesta-7,trans-22-dien-3 beta-ol or spinasterol.

Cytotoxic C-Methylated Chalcones from Syzygium samarangense.

Abstract The flavonoids 2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), 2′,4′-dihydroxy-6′-methoxy-3′-methyldihydrochalcone (4) and 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethyldihydrochalcone (5), isolated from Syzygium samarangense. (Blume) Merr. & L.M. Perry (Myrtaceae), were subjected to cytotoxicity testing using the dimethylthiazoldiphenyl tetrazolium (MTT) assay. The cell lines used were the Chinese hamster ovarian (CHO-AA8) and the human mammary adenocarcinoma, (MCF-7 and SKBR-3). Among the test compounds, 2 exhibited significant differential cytotoxicity against the MCF-7 cell line with an IC50 of 0.0015 ± 0.0001 nM. It was also cytotoxic against the SKBR-3 cell line with an IC50 of 0.0128 ± 0.0006 nM. Doxorubicin, the positive control, had an IC50 of 2.60 ± 0.28 × 10−4 nM against the MCF-7 cell line and an IC50 of 2.76 ± 0.52 × 10−5 nM against the SKBR-3 cell line. When tested in a mechanism-based yeast bioassay for detecting DNA-damaging agents using genetically engineered Saccharomyces cerevisiae. RS322Y (RAD52) mutant strain and (LF15/11) (RAD+) wild-type strain, 2 showed significant selective cytotoxicity against the RAD52 yeast mutant strain. It had an IC12 of 0.1482 nM, as compared with the positive control, streptonigrin, which had an IC12 of 0.0134 nM. Hence, 2 is a cytotoxic natural product with potential anticancer application.

Prolyl endopeptidase inhibitors from Syzygium samarangense (Blume) Merr. & L. M. Perry.

Compounds isolated from the hexane extract of the leaves of Syzygium samarangense (Blume) Merr. & L. M. Perry were tested for inhibitory activity against the following serine proteases: trypsin, thrombin and prolyl endopeptidase. The compounds were identified as an intractable mixture of α-carotene and β-carotene (1), lupeol (2), betulin (3), epi-betulinic acid (4), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (5), 2′-hydroxy-4′,6′-dimethoxy-3′- methylchalcone (6), 2′,4′-dihydroxy-6′-methoxy-3′,5v-dimethylchalcone (7), 2′,4′-dihydroxy- 6′-methoxy-3′-methyldihydrochalcone (8) and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (9). Hydrogenation of compounds 5, 6 and 7 yielded compound 8, 2′-hydroxy-4′,6′-dimethoxy- 3′-methyldihydrochalcone (10) and 2′,4′-dihydroxy-6′-methoxy-3′,5-dimethyldihydrochalcone (11), respectively. The hydrogenated products of compounds 6 and 7 were also tested for enzyme inhibitory activity. In addition, β-sitosterol (12) and β-ᴅ-sitosterylglucoside (13) were also isolated. This is the first report of the isolation of compounds 1-6, 8 and 13 from this plant. Compounds 3-8 and 10 exhibited significant and selective inhibition against prolyl endopeptidase among three serine proteases. This is the first report of this kind of activity for all these compounds.

Bioactivity studies on the alkaloid extracts from seeds of Leucaena leucocephala

The chloroform soluble (CS) and ethyl acetate (ES) soluble alkaloidal extracts from the seeds of Leucaena leucocephala Lam. (de Witt) were tested for their analgesic, anthelminthic, mutagenic, antimutagenic and pharmacological activities. The CS alkaloidal extract is analgesic at a dose of 5 mg/20 g mouse as it showed a comparable result to mefenamic acid using the acetic acid‐induced writhing method. An in vitro test for anthelminthic activity using Ascaris suum showed that the CS alkaloidal extract gave a comparable result to mebendazole (Antiox) at a concentration of 5 mg/mL. Results of the micronucleus test, an in vivo method, showed that both the CS and ES alkaloidal extracts were not mutagenic but instead exhibited antimutagenic activities at 0.5 mg/20 g mouse. The CS‐ and ES‐soluble alkaloidal extracts reduced the number of micronucleated polychromatic erythrocytes induced by tetracycline, a known mutagen, by 67.0% and 71.2%, respectively. Preliminary pharmacological screening showed that both extracts caused a relative depression in the central nervous system as evidenced by a decrease in respiratory rate and depth and a decrease in motor activity.

Menthalactone, a new analgesic from Mentha cordifolia Opiz. leaves.

Menthalactone, a new long-chain alkene with a bicyclic lactone moiety, was isolated as an analgesic constituent from the leaves of Mentha cordifolia Opiz. At a dosage of 100 mg/ kg mouse, it decreased the number of squirms induced by acetic acid by 67.3%. Statistical analysis using Kruskall Wallis one-way analysis of variance by ranks showed that menthalactone is different from the solvent control at α = 0.01 and approximates the analgesic activity of mefenamic acid at 0.001 level of significance.

Spasmolytic Flavonoids from Syzygium samarangense (Blume) Merr. & L.M. Perry

The hexane extract of Syzygium samarangense (Ss.Hex) dose-dependently (10D1000 μg/ ml) relaxed the spontaneously contracting isolated rabbit jejunum. Four rare C-methylated flavonoids with a chalcone and a flavanone skeleton were isolated from Ss.Hex and were subsequently tested for spasmolytic activity. All flavonoids, identified as 2′-hydroxy-4′,6′- dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (4), showed dose-dependent spasmolytic activity in the rabbit jejunum with IC50 values of 148.3 ± 69.4, 77.2 ± 43.5, 142.4 ± 58.6 and 178.5 ± 37.5 μg/ml (mean ± SEM), respectively. The dihydrochalcone derivative of compound 1, 2′-hydroxy-4′,6′-dimethoxy-3′- methyldihydrochalcone (5), when tested for spasmolytic activity, did not significantly relax the smooth muscle relative to the other compounds. Verapamil, a standard spasmolytic, has an IC50 value of 0.16 ± 0.04 μg/ml. This is the first report of the relaxant activity of chalcones, specifically of compounds 1-3.