Sharon Kenechukwu Okonkwo1, Ricardo Reyes-Chilpa2, Manuel Jiménez-Estrada2 and Ezra
Béjar3,4,5. 1Department of Biology, University of California, San Diego 9500 Gilman Drive, La
Jolla, CA 92093. 2Institute of Chemistry, Universidad Nacional Autónoma de México,
Coyoacán, 04510, México, D.F. 3College of Sciences, San Diego State University 5500
Campanile Drive, San Diego, Ca 92182. 4 Plant Bioassay™, 5702 Baltimore Dr # 265, La
Mesa, CA 91942-1667. 5 To whom correspondence should be addressed.

Byrsonima crassifolia (Nanche) is a tropical tree indigenous to México. Its medicinal importance
has been documented historically since the sixteenth century and its uses preserved today in
several communities in México and other Latin American countries. In 1984, an extensive
ethnobotanical survey of medicinal plants conducted by the Mexican Social Security Institute
(IMSS), revealed that B. crassifolia was among the ten most frequently mentioned plants used by
traditional healers for gastrointestinal disorders, especially for the treatment of diarrhea, and
dysentery. A comprehensive program was initiated--including phytochemical and
pharmacological investigation of the plant--to evaluate its potential as a phytomedicinal agent in
the treatment of irritable bowel syndrome. Chemical analysis revealed the presence of 21
chemicals extracted by methanol from the dried leaves and the biological activity of extracts and
pure compounds was studied on a number of bioassays (Bejar, et al 1995). Among the isolated
chemicals from the leaves, b-sitosterol, and betulin (triterpenes), pipecolic acid and proline
(amino acids), catechin, and quercetin (flavonoids) were used as chemical markers for a
comparative analysis of several formulations from the plant.

Since the presence of the chemical markers had not been previously documented in the
traditional preparations (teas) used by traditional healers, our goal in this project was to compare
chemical profiles of the preparations using pure chemical markers isolated from the raw plant as
standards. This information would be of extreme importance to prepare standardized
formulations of traditional remedies for additional bioassay testing and to conduct clinical trials.

Using Thin Layer Chromatography (TLC) and 1H-NMR analysis, we investigated the presence
of chemical markers in teas from bark and leaves compared to leaf and bark methanol extracts
used as positive controls. TLC analysis of both methanol extracts demonstrated the presence of
the reference compounds previously isolated by Bejar et al in 1995. In contrast, bark and leaf
teas afforded only catechin in higher concentrations in the bark than in the leaves. Other chemical
markers were not detected. 1H-NMR analysis of the bark and leaves teas--extracted with ethyl
acetate--displayed characteristic signals for monomers and oligomers of catechin, as well as
sugars.

This report concludes, methanol is a most suitable solvent to isolate triterpenes, aminoacids, and
other flavonoids different to catechin, while hot water, fails to extract these but apparently
concentrate polysaccharides and olygomeric catechins. Because of the difference in chemical
profile, is likely teas will have also a different pharmacological profile than the one reported for
solvent-extracted formulations.

Béjar, E., Amarquaye, A. Che, C.-T., Malone M.H. and Fong, H.H.S. Constituents of
Byrsonima crassifolia and their spasmogenic activity, Int. J. Pharmacog. 1995, 33:1, 25-32.

Supported by a grant from the SDSU-UCSD Minority International Research Training (MIRT)
of the Fogarty International and by Armadillo Pharmaceuticals, Inc. Hakalau HI, 96710