Research Article
Phytochemical Investigation of Biologically Active Fractions of Astragalus spinosus Roots Grown in Egypt
Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
Introduction
Astragalus L. (Fabaceae) is one of the largest and most widely distributed genera of about 2000 species, mainly in the North Temperate regions and Tropical African mountains (Evans, 1998 and Boulous, 1999). The genus Astragalus has 37 species indigenous to Egypt. Astragalus spinosus Vahl. is a perennial spiny herb growing in Egypt (Hutchinson, 1973 and Tackholm, 1974). Some Astragalus species are used as forage for livestock and wild animals particularly in Asia, others are used as foods, cosmetics, source of vegetables (Rizk, 1986). A number of Astragalus species are used in Chinese traditional medicine as antiperspirant, antihypertensive, antidiabetic, diuretic and tonic (Konoshima, 1996; Hikino et al., 1976). They also have hepatoprotective, antioxidative, immunostimulant and antiviral properties, whereas others have shown toxic activity (Hartwell, 1970) and have been used for the treatment of leukemia and uterine cancer. In many cases the cytotoxic principles can pass to humans through milk and meat (Rios and Waterman, 1997).
From the aerial parts of A. spinosus, five astragalosides were previously isolated namely, astragaloside I, isoastragaloside I, astragaloside II, astragalosideIV and cycloastragenol-6-0-glucoside (EL-Sebakhy et al., 1990 and Abdallah et al., 1993). Recently, adsurgenic acid was isolated for the first time from the ether extract of the same plant (Darwish, 2002).
Carbon tetrachloride (CCl4) is commonly used as a typical toxicant reflecting the various aspects of toxicity. Doses of CCl4 in the range of several ml kg-1 body weight produce liver necrosis within one or two hours (Edwards et al., 1993). Also, it was known to cause damage to lungs, kidneys, adrenal and central nervous system in humans and experimental animals (Rechnagel, 1989; Zhao and O Brien, 1996; Wong et al., 1998).
The study was planned to asses the ability of the fractionated plant extracts, column fractions, and isolated glycosides of the dried roots of Astragalus spinosus to prevent or reduce the CCl4-induced mortality, hepato-renal dysfunctions and cardiac toxicity.
Materials and Methods
The research work was conducted at Faculty of Pharmacy, Faculty of Science, Alexandria University, Alexandria, Egypt during 1999 to 2002.
Plant material: Fresh flowering and fruiting plant of A. spinosus Vahl. were obtained from area around Alexandria-Cairo Desert Highway at Kilo/65. The plant was identified by Late Prof. V. Tackholm, Faculty of Science, Cairo University. Voucher specimen has been deposited at the herbarium of Faculty of Science, University of Alexandria, Egypt.
NMR spectra were recorded on Bruker AMX 500 (1H NMR 500 MHZ, 13C NMR 125MHz), chemical shifts are expressed in δ values with TMS as internal standard. Mass spectra were carried out on Varian MAT 311 A, FABMS using NBA as matrix. MPLC: Gilson pump, silica gel 60 (15-40 μm Merck). Column chromatography using silica gel 60 (Merck).
TLC was performed on precoated silica gel 60 F254 plates (Merck). Solvent system ethyl acetate-glacial acetic-water (5:1:1) and dichloromethane-methanol (9:1), (8.5:1.5) and (8:2). Detection was carried out by spraying with anisaldehyde-sulphuric and heating at 120 °C.
Extraction and isolation: The air-dried powdered roots of A. spinosus (1 Kg) were extracted with 90% ethanol. The alcoholic extract was concentrated to 200ml, added slowly with continuous stirring to water (1L), left for 5 hours at room temp. and filtered. The filtrate was partitioned into ether, dichloromethane, ethyl acetate and n-butanol, successively. Ether fraction was previously investigated and afforded a new acid analogue (adsurgenic acid) (Darwish, 2002).
Ethyl acetate soluble fraction was fractionated on silica gel column, eluted with dichloromethane-methanol increasing percentage of methanol, fraction A and fraction B eluted with 20% methanol were examined biologically.
The two fractions were biologically active:
Fraction A was subjected to medium pressure liquid chromatography (MPLC) using silica gel 60 (15-40 um, Merck) flow rate 5ml/min, MPLC: column 1x 55cm eluted with dichloromethane-methanol, Omin: 0% methanol, 40 min: 1% methanol, 60 min: 5% methanol, 120 min: 5% methanol isocratic, 180 min :12% methanol, 200min: 20% methanol. Fractions eluted at 120 min. were a mixture of two saponin glycosides; isoastragaloside I (compound I) and trigonoside I (compound 2) (Fig. 1) which were identified by comparison of their spectral data (NMR, EIMS, FABMS) with the reported data.
Fraction B was subjected to silica gel CC using, dichloromethane-methanol as eluant. Fractions eluted with 30% methanol-dichloromethane were collected, concentrated and rechromatographed on another silica gel column. Fractions eluted with methanol- dichloromethane (45%) afforded cycloastragenol 6-0-glycoside (compound 3) (Fig. 1) which was identified by comparison of their spectral data (NMR, EIMS, FABMS) with the reported data together with co-TLC with reference sample (Fig. 1).
Fig. 1: | Structure of glycosides and Adsurgenic acid |
Compound 1 (Isoastragaloside I): FABMS (positive ion) m/z 891 (M++ Na) 868 (M+) for molecular formula C45H72O16, EIMS m/z 43 (100% rel. abund) for the acetyl group. 1HNMR spectrum showed signals at δ2.30 and 1.94 for the two acetyl groups of the sugar together with two signals at δ170, 171 in the 13CNMR spectrum.
Compound 2 (trigonosides I): FABMS m/z 645 (M++ Na), 662 (M+) corresponding to molecular formula C35H58O9. 1H and 13CNMR data for compounds I and II were identical with the reported data.
Compound 3 (cycloastragenol 6-0-glycoside): FABMS (positive ion) m/z 675 (M++ Na), 652 (M+) corresponding to molecular formula C36H60O10 , 1HNMR (500MHz, DMSO):δ 0.18 (1H, d, J = 4.6Hz, H-19), 0.46 (1H, d, J= 4.6 Hz, H-19),1.09 (2x 3H,S M-21 and M-29), 0.9 (3H, S, M-27), 1.0 (3H, S, M-28), 1.11 (3H, S, M-26), 1.18(3H, S, M-18), 1.2 (3H, S, H-30) 4.88 (1H, d, J= 8.8 Hz, H-1 glucose).13CNMR (125 MHZ, DMSO), 104.47(C-1``), 80.91 (C-6), 78.6 (C-3``), 77.65 (C-3), 77.13(C-5``), 74.94 (C-2``),, 73.17 (C-16), 72.61 (C-4``), 70.9(C-25), 62.20 (C-6``), 28.36(C-29), 28.15 (C-27), 27.58 (C-26), 26.9(C-21), 20.17(C-18), 19.88 (C-28) and 15.69(C-30).
Bioassay studies
Animals and treatments: Adult female Bufo regularis (Egyptian toads), weighing about 25±4.3 g, were used throughout the studies. The animals were housed in groups of 20 in glass cages covered with a wire galvanized meshes. The bottom of these glass cages were covered with a wet sponge. Food (earth worms) was available ad libitum. In order to minimize possible nutritional effects, both experimental and control animals were fasted for 18 h before sacrifice.
Carbon tetrachloride: The CCl4 (Analar) was used as the toxicant agent. It was diluted with corn oil to deliver the proper dosage in a volume of 10 ml kg-1 b.wt. All doses were given as a single subcutaneous injection.
Experiment I: Animals were divided into groups of 20 and were administered CCl4 at various doses. Mortalities were recorded at 24 h following CCl4 injection. 24 h LD100 was estimated. It was found 5.93ml Kg-1. b.wt.
Experiment II: The dried alcoholic extract of A. spinosus roots was dissolved in methanol to prepare the desired dose in a volume of 1 ml kg-1 b.wt. Preliminary experiments were carried out to determine the most effective concentration of the alcoholic extract that can reduce the mortality-induced by CCl4. It was found to be 60mg kg-1 b.wt. when administered subcutaneously. The animals were divided into 5 groups, 20 animals in each group (Table 1). Mortalities were recorded after 24 h of CCl4 injection.
Experiment III: The fractions of the alcoholic extract (dichloromethane, ethyl acetate, butanol) and the aqueous fraction left after fractionation of the alcoholic extract with the organic solvents were dissolved in methanol to prepare the desired doses in a volume of 1 ml kg-1 b.wt. Preliminary experiments proved that the s.c. injection of 50 mg of each fraction / Kg b.wt. was the most effective concentration in reducing CCl4-induced lethality. The animals were divided into 10 groups 20 animals in each group (Table 2). Moralities were recorded after 24 h following CCl4 injection. Ethyl acetate proved to be the most potent one in reducing the CCl4-induced mortality.
Experiment IV: The ethyl acetate fraction was subjected to chromatographic separation on silica gel column (as previously mentioned) to yield fractions A and B. Preliminary experiments proved that the s.c. injection of 10 mg Kg-1 b.wt. of each fraction was the most effective concentration in reducing CCl4-induced lethality. The animals were divided into 6 groups, 20 animals in each group (Table 3). Mortalities were recorded after 24 h following CCl4 dosing.
Experiment V: The isolated glycosides (I+ II) and III as well as Adsurgenic acid were used to evaluate their effectiveness in preventing both the CCl4-induced mortality and hepato- renal perturbations. This experiment includes both lethality and biochemical studies.
Lethality studies: Preliminary experiments indicated that the s.c. administration of 2 mg Kg-1 b.wt. of each of (compound I, II) and compound III as well as the adsurgenic acid was the most effective concentration in protecting against CCl4-induced lethality. The animals were divided into 8 groups 20 animals each for lethality studies (Table 4). Mortalities were recorded after 24 h following CCl4 injection.
Biochemical studies: Animals were divided into 8 groups as those done for the lethality studies, while animal specimens were collected after 16 h only. Animals were anaesthetized with ether and blood withdrawn by cardiac puncture. Blood was collected in heparinized tubes for cellular determinations and non-heparinized tubes for serum preparation.
Serum was prepared by centrifugation of the blood at 8000 r.p.m. for 15 minutes. The activities of serum transaminases i.e., aspartate aminotransferase (AST or GOT) and alanine aminotransferase (ALT or GPT) and creatin kinase (CK), in addition to urea and creatinine concentrations in the serum were estimated by using BM/Hitachi system 7,7 Automatic Analyzer.
Statistical analysis: Measured values are presented as the arithmetic mean of seven experiments ± standard error (S.E.). Results were analyzed using students t-test. Differences were considered significant at p < 0.05. The test of significance of differences were calculated between:
a | Means of the control group and means of either of these treated groups: CCl4, compound III, adsugenic acid, CCl4 + (compound I and II), CCl4 + adsurgenic acid were referred by letter a. |
b | Means of CCl4 group and either of these experimental groups: CCl4+ compound (I + II) CCl4 + compound III, CCl4 + and adsurgenic acid were referred by letter b. |
Results
The simultaneous administration of the alcoholic extract of A. spinosus root (60 mg Kg-1 b.wt. s.c.) and CCl4 (5.93 ml kg-1 b.wt. s.c., which represents the 24h LD100) reduced the percentage of mortality that were induced by the latter to 65% (Table 1). The results indicate that the injection of the fractionated extracts ethyl acetate, butanol, aqueous and dichloromethane (50 mg kg-1. b. wt. s. c.), decreased the percentages of CCl4- induced mortality to 50, 55, 60 and 75%, respectively (Table 2).
Thus, the ethyl acetate extract (50 mg kg-1. b. wt. s. c.) gave the best protective effect against CCl4 (24 h LD100) - induced lethality. So, it was used in the subsequent experiments. The data (Table 3) indicate that A is slightly more effective than B. The results (Table 4) shows that the simultaneous administration of compound I and II, compound III and adsurgenic acid decreased the percentage of CCl4 - induced lethality to 45, 35 and 55%, respectively.
The results (Table 5) shows that CCl4 administration resulted in marked increases in liver transaminases (AST and ALT) and CK activities.
The simultaneous administration of compound I+ II, compound III and adsurgenic acid with CCl4 offered partial protective effects against the increases in the activities of AST and ALT that were induced by the latter, since, there were a significant differences between the values of these groups, the control and CCl4 groups. The co-administration of either compound III or adsurgenic acid provides partial protective effects against the CCl4 -induced elevations in the CK activities.
Table 1: | Effect of Alcoholic extract (60 mg kg-1 b.wt.) on the mortality of Bufo regularis |
*: These groups served as negative controls **: Number of animal used in each studied group n=20 |
Table 2: | Effect of the fractionated extracts (50 mg kg-1 b.wt.) on the mortality of Bufo regularis |
*: These groups served as negative controls **: Number of animal used in each studied group n=20 |
Table 3: | Effect of ethyl acetate column fractions (10 mg kg-1 b.wt.,) on mortality of Bufo regularis |
*: These groups served as negative controls **: Number of animal used in each studied group n=20 |
Table 4: | Effect of (isoastragaloside I and trigonoside I), cycloastragenol-6-0-glycoside and adsurgenic acid. (2 mg kg-1 b.wt.) on mortality of Bufo regulari |
*: These groups served as negative controls **: Number of animal used in each studied group n=20 |
On the other hand compound I, II did not provide any protective effects against these elevations in CK activities.
CCl4 administration induced marked increases in urea and creatinine levels in the serum. The data (Table 6) also indicate that administration of either compound I, II and compound III following CCl4 injection succeeded partially in preventing these disturbances. On the other hand, administrated dose of Adsurgenic acid increased the CCl4 B induced elevations in serum urea and creatinine concentrations.
It is important to note that the injection of cycloastragenol-6-0-glycoside (compound III), isoastragaloside I and trigonoside I compound (I and II) alone did not induce any alterations during either the bioassay or the biochemical studies, while the administration of adsurgenic acid alone caused severe toxic effects on urea and creatinine levels.
Discussion
The investigation indicated that the 24 h LD100 of CCl4 for adult female Bufo regularis is 5.93 ml kg-1 b.wt.
The data (Table 5) revealed that CCl4 induced liver dysfunction as indicated by increases in the activities of AST and ALT. A number of reports indicated that CCl4 can result in similar biochemical changes in both man (Ruprah et al., 1985) and experimental animals including gerbils (Cal and Mehendale, 1990), Marino ewes (Vajdovich et al., 1995), rats (Lin et al., 1998) and mice (Wong et al., 1998).
It also caused impairment in the renal function as indicated by elevations in serum urea and creatinine concentrations (Table 6). Ruprah et al. (1985) observed that renal damage, normally manifest within 48h, occurring up to 2 weeks after CCl4 exposure. McGregor and Lang (1996) mentioned that the kidney is one of the primary targets in CCl4 toxicity, moreover, Sanzgiri et al. (1997) detected high concentrations of CCl4 in the kidney of Male Sprague-Dawley rats subjected to equivalent oral and inhalation CCl4 exposures. The present results showed that CCl4 induced significant increase in creatin kinase activity. Creatin kinase activity increases in myocardial infarction and muscular dystrophy (Julian et al., 1998).
In this study, results indicated that alcoholic extract of A. spinosus roots was effective in reducing CCl4-induced lethality (Table 1). Results also depicted that the ethyl acetate extract was the most effective fraction in reducing CCl4- induced mortality. The efficacy of the three other fractionated extracts was as follows: butanol > aqueous > dichloromethane (Table 2). In addition, the data indicated that the ethyl acetate column fractions A and B were effective in reducing CCl4- induced lethality (Table 3). In this investigation the results of the biochemical studies were co- inside with that of the bioassay studies (Table 5 and 6).
Table 5: | Effect of isoastragaloside I and trigonoside I (compound I and II) cycloastragenol-6-0-glycoside (compound III) and adsurgenic acid (2 mg kg-1 b.wt.) on liver transaminases (AST, ALT) and creatine kinase (CK) activities |
a,b= statistically significant (p < 0.005) when compared with values of the control group or CCl4 injected group , respectively |
Table 6: | Effect of isoastragaloside I and trigonoside I (compound I and II), cycloastragenol-6-0-glycoside (compound III) and adsurgenic acid on serum urea and serum creatinine concentration |
a,b= statistically significant (p < 0.005) when compared with values of the control group or CCl4 injected group, respectively |
The bioassay studies of the isolated glycosides indicated that they were useful in decreasing CCl4 induced mortality as compared with adsurgenic acid. The order of their protective effect was as follows: cycloastragenol-6-0- glycoside > (isoastragaloside I and trigonoside I) > adsurgenic acid. The s.c. injection of cycloastragenol-6-0-glycoside succeeded partially in modulating the hepato, renal and cardiac toxicities as compared with the control values, while (isoastragaloside I and trigonoside I) offered partial protection in functions of liver and kidney only, but it failed in improving the CCl4 induced elevation in CK activity. On the other hand adsurgenic acid caused renal toxic effects. This suggest that cycloastragenol-6-0-glycoside is promising in modulating the disturbances in liver, kidney and heart functions that were induced by CCl4 or any other toxicants.
The present research work indicate that the co-administration of the cycloastragenol-6-0-glycoside or of (isoastragaloside I and trigonoside I) along with CCl4 is more effective and advantageous in protecting against lethality and toxicities of the latter, than when it is given with adsurgenic acid.
Acknowledgments
The author is deeply grateful to Dr. Karima Wershana (Associate Prof. Zoology Department, Faculty of Science, University of Alexandria, Egypt) for running all the bioassay and biochemical studies and also to Prof. Ahlam Y. Daabees, Prof. of Comparative Animal Physiology, (Zoology Department, Faculty of Science, University of Alexandria, Egypt) for the valuable assistance. The author thanks Prof. R.Bauer (institute fur Pharmazeutiche Biologie der Heinrich-Heine Universilät, Düsseldorf, Germany) for NMR and Mass spectra.