Thin Layer Chromatographic Detection of Steroid and Alkaloid Glycosides in An Ethanolic Extract of Winter Cherry (Physalis alkekengi) Fruits
M. Vessal PhD,* M. Akmali MSc, N. Bambaee-Row MSc
Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz , Iran
Background-The winter cherry (Physalis
alkekengi) fruits have some antiestrogenic
properties.
Objective-To analyze an ethanolic
extract of winter cherry fruits for the presence of
steroid and alkaloid glycosides.
Methods-gel G in -An
ethanolic extract of winter cherry fruits was subjected
to thin layer chromatography on silica n-butanol-glacial
acetic acid-water (100:10:10) and sprayed with specific
reagents for steroids, glycosides and alkaloids.
Results-A major steroid
glycoside, a free sugar and a glycoalkaloid were
identified on the chromatograms.
Conclusion-The antiestrogenic
property of the extract is probably due to the presence
of the steroid glycoside. It is also likely that the
glycoalkaloid found in this extract has antiparasitic
action similar to alkaloid glycosides reported from other
Solanacea species.
Key Words · Steroid glycosides · alkaloid glycosides · Physalis alkekengi
Introduction
The estrogen antagonistic properties of an aqueous extract of Physalis alkekengi fruits on the activities of several estrogen-induced enzymes in the uterus,1,2 liver,3 pituitary gland and hypothalamus4,5 were previously reported from this laboratory. It was suggested that perhaps the aqueous extract of this fruit contained steroid derivatives with polar side chains such as the one present in glycyrrhiza.6 In order to test this hypothesis, the aqueous extract of this fruit was subjected to thin layer chromatography using chloroform-acetone (9:1) and at least two steroid compounds were detected in that extract.3 To further elucidate the nature of the side chain (s) in these, or rather in steroid derivatives with more polar side chains, the aqueous extract was subjected to ethanol extraction and thin layer chromatography in a polar solvent and sprays were used to detect steroid, sugar, and alkaloid-containing compounds on the chromatograms. This preliminary investigation reports the presence of a major component containing a steroid glycoside and two minor components namely a glycoalkaloid and a free carbohydrate.
Materials and Methods
Silica gel G, cholesterol, o- phosphoric acid, ethyl acetate, and n- butanol were obtained from Merck (Darmstadt, Germany). Progestrone and 17- B- estradiol were from Sigma (St. Louis, MO). Tomatine and sodium metaperiodate were secured through Fluka Chemical Company (Switzerland) and vanillin was from B.D.H. (Poole, Dorset, U.K.). All other reagents of high purity were obtained through other commercial sources. Winter cherry fruits (Physalis alkekengi L.: family Solanaceae) were authenticated and extracted with water as described previously.3
The aqueous extract contained water soluble materials from 1.5 g dried fruit powder per ml. The aqueous extract (20 ml) were brought to 85.5% ethanol upon addition of 180 ml of 95% (v/v) ethanol. The suspension was shaken for about 5 min. After centrifugation at 3000 ´ g for 10 min, the supernatant fraction containing ethanol extractable material from 0.15 g fruit powder per ml was directly used for thin layer chromatography of steroids, glycosides and alkaloids.
The ethanolic extract of winter cherry fruits was chromatographed on several 500 m m thick silica gel G thin layer plates (20 ´ 20 cm) along with various steroids (17-ß-estradiol, progestrone and cholesterol), a sugar (glucose) and a steroid-alkaloid- glycoside (tomatine) as standards. The solvent was n-butanol-glacial acetic acid-water (100: 10: 10). Detection of steroid containing spots was accomplished by spraying the plates with a solution of 1g vanillin in 100 ml 50% aqueous solution of o-phosphoric acid followed by heating for 10- 20 min at 120 ° C.7 The steroid containing compounds appeared as violet spots on a white background. Sugar containing components were seen as white spots on a blue background upon spraying the plates first with a 0.1% aqueous solution of sodium metaperiodate and then spraying the half dried chromatograms with a second spray containing 2.8 g benzidine in 80 ml 96% ethanol to which 70 ml water, 30 ml acetone and 1.5 ml 1 N HCl had been added.7 Detection of alkaloids on thin layer plates was performed by use of Dragendorff's reagent.7 Alkaloids appeared as orange spots on a grey background.
Results
Thin layer chromatograms of steroid, carbohydrate, and alkaloid-containing compounds of the ethanolic extract of Physalis alkekengi fruits are shown on Figs. 1, 2, and 3, respectively. As seen on Figs. 1 and 2, a major spot (spot b) with an Rf value of 0.38-0.42 appeared almost in the same position on both the steroid (Fig. 1, spot b) and the carbohydrate (Fig. 2, spot b) chromatograms. This is an indication for the presence of a major steroid glycoside in the ethanolic extract of Physalis alkekengi fruits. Fig. 2 also shows a faster moving spot (spot c) which did not appear in either the steroid or the alkaloid chromatograms. It was therefore identified as a free carbohydrate. Comparison of the three figures also reveals the presence of an alkaloid containing spot with a carbohydrate moiety (compare Fig. 3, spot a and Fig. 2, spot a). Therefore, it appeared that the ethanolic extract of winter cherry fruits also contained a glycoalkaloid. Tomatine (steroid alkaloid glycoside) was run as a standard in all three chromatograms and reacted positively with all three specific sprays employed.
Discussion
Comparing the results of the thin layer chromatography (Figs. 1, 2, 3) indicates that the ethanolic extract of winter cherry fruits prepared in this investigation contains a major steroid glycoside. This steroid glycoside is probably the estrogen antagonistic compound of Physalis which was observed to reduce the activities of estrogen induced enzymes in several estrogen target tissues.1-5 This observation is consistent with the findings of Tamaya et al6 who reported the presence of a steroid glycoside (glycyrrhizin) with mineralocorticoid, glucocorticoid and antiestrogenic activities in the roots of glycyrrhiza. The results also demonstrate the presence of a glycoalkaloid (compare Figs. 2,3) in this ethanolic extract. Presence of glycoalkaloids have been demonstrated in many species of the Solanaceae family.8 Several Solanum steroidal alkaloids and glycoalkaloids have also been demonstrated to inhibit the growth of the red flour beetle, Tribolium castaneum, and tobacco horn worm, Manduca sexta.9 There has also been a report on the inhibitory effect of several Solanum glycoalkaloids on the growth of Trypanosoma cruzi in culture.10 Further purification and structural analyses of the steroid glycoside and the glycoalkaloid of Physalis alkekengi fruits are needed to evaluate the potential of the former as an estrogen antagonist and of the latter as an antiparasitic agent.
References
1 Vessal M, Mehrani HA, Omrani GH. Effects of an aqueous extract of Physalis alkekengi fruit on oestrus cycle, reproduction and uterine creatine kinase-BB isozyme in rats. J Ethnopharmacol 1991;34:69-78.
2 Vessal M, Yazdanian M. Comparison of the effects of an aqueous extract of Physalis alkekengi fruits and/or various doses of _-estradiol on rat estrus cycle, and uterine glucose-6-phosphate dehydrogenase activity. Comp Biochem Physiol 1995;112C:229- 36.
3 Vessal M, Mostafavi-Pour Z, Kooshesh F. Age and sex dependence of the effects of an aqueous extract of Physalis alkekengi fruits on rat hepatic glucose-6-phosphate dehydrogenase activity. Comp Biochem Physiol 1995;111B:675- 80.
4 Vessal M, Rasti M. Estradiol antagonistic effects of winter cherry extract on pituitary and hypothalamic G6PD activities. Iranian J Med Sci 1995;20:152- 8.
5 Vessal M, Rasti M, Kooshesh F. Modulation of the pituitary and basomedial hypothalamic lysyl- aminopeptidase activities by B-estradiol and/or an aqueous extract of Physalis alkekengi fruits. Comp Biochem Physiol 1996;115B:267-71.
6 Tamaya T, Sato S, Okada HH. Possible mechanism of steroid action of the plant herb extracts glycyrrhizin, glycyrrhetinic acid and paeoniflorin: inhibition by plant herb extracts of steroid protein binding in the rabbit. Am J Obstet Gynecol 1986;155:1134-9.
7 Waldi D. Spray reagents for thin layer chromatography In: Thin layer Chromatography (Stahl E, ed.), Berlin: Springer Verlag, 1962;pp:483-502.
8 Chen S, Derrick PJ, Mellon FA, Price KR. Analysis of glycoalkaloids from potato shoots and tomatoes by four sector tandem mass spectrometry with scanning array detection: comparison of positive ion and negative ion methods. Anal Biochem 1994;218:157-69.
9 Wissenberg M, Levy A, Svoboda JA, Ishaaya I. The effects of some Solanum steroidal alkaloids and glycoalkaloids on larvae of red flour beetle, Triboliumcastaneum, and the tobbaco hornworm, Manduca sexta. Phytochemistry 1998;47:203-9.
10 Chataing B, Concepcion JL, Lobaton R, Usubillaga A. Inhibition of Trypanosoma cruzi growth in vitro by Solanum alkaloids: a comparison with ketoconazole. Planta Med 1998; 64: 31-6.