Phytochemical and Biological Investigation of Aloe Grandidentata Salm-Dyck

The crude alcoholic extract of the leaves of Aloe grandidentata Salm.-Deck showed significant antimicrobial activity (200 mg/ml), potent anti-inflammatory and chronic antihyperglycemic (100 mg/kg b.wt.) compared to standard positive drugs. Phytochemical studies of the potent extract revealed the isolation and characterization of seven compounds; two new compounds; 1,1',8,8'-tetrahydroxy -3'acetyl -3-methyl -5,5'bianthracene -9, 9',10,10'-tetraone (2) and 1,6,8-trihydroxy–7–methoxy–3methyl anthraquinone (3), five known compounds, β-sitosterol (1), emodin (4), chrysophanol (5), physicon (6) and β-sitosterol-3-O-β–D-glucoside (7). This is the first report of the isolation of emodin and β-sitosterol-3-O-β–D-glucoside from genus Aloe and physicon from family Liliaceae. All structures of the isolated compounds were determined using several spectroscopic techniques; UV, IR, MS, NMR (1H NMR and 13C NMR) and by comparison with literature data. *Corresponding author: Taghreed Abdou Ibrahim Shehata, Associate Professor of Pharmacognosy, College of Pharmacy, King Saud University, Saudi Arabia, Tel: 00966556639972; Fax: 00966112062183; E-mail: tshehata@ksu.edu.sa Received October 27, 2013; Accepted December 18, 2013; Published December 21, 2013 Citation: Ibrahim TA, Fiki NME, Shehata IA, Sleem AA, Shoukry MM (2013) Phytochemical and Biological Investigation of Aloe Grandidentata Salm-Dyck. Med chem 4: 298-302. doi:10.4172/2161-0444.1000155 Copyright: © 2013 Ibrahim TA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Aloe (Liliaceae) is a large genus of 400 species native to Africa, Madagascar, and Arabia [1]. Aloe has a wide range of medicinal application such as laxative effect, wound healing effect, reduces blood sugar in diabetes, soothes burns, eases intestinal problems, reduces arthritic swelling, ulcer curative effect, stimulates immunes response against cancer etc. [2]. Studied pharmacological effects of Aloe as in vitro or in animals include antimicrobial [3], anti-inflammatory and anti-arthritic activity [4,5] and hypoglycemic effects [6][7][8]. Several constituents were isolated from different Aloe species; sterols, lignin, saponins, anthrones, their dimmers, chromones, flavones, C-gylycosides of anthrone and chromones [9] and glycoproteins and polysaccharides [9,10]. Aloe grandidentata is a green fleshy plant reaches up to 30 cm height, flourishes in Egypt and flowers in January till June; the subterranean part consists of rhizome and adventitious roots [11]. Nothing was found about chemical constituents and biological activity of A. grandidentata, so the present study was planned to investigate both chemical constituents and biological effect of the plant. yield fraction B (petroleum ether, 18.6 g), fraction C (chloroform, 9.1 g), fraction D (ethyl acetate, 4.3 g) and fraction E (n-butanol 6.1 g).
Fraction D (4 g) was similarly, subjected to silica gel column chromatography, subfraction 42 (187 mg, ethyl acetate: CH 3 OH 9:1) was rechromatographed on LH20 column to afford 15 mg of β-sitosterol-3-O-β-D-glucoside (7). Compounds 1, 4-7 were detected through comparison of TLC behavior with authentic standard, melting point and mixed melting point. In addition to comparing their spectroscopic data with that reported in literatures. In addition, compounds 2,3 were investigated through their spectroscopic data.

1,6,8-trihydroxy-7-methoxy-3-methyl anthraquinone (compound 3)
MP: 236-238ºC. Antimicrobial activity: The antimicrobial activity was tested using agar disc diffusion method [12]. A suspension of the tested microorganism (0.1 mL of 108 cells per mL) was spread on solid media plates. Aliquots of 15 μg of the alcohol extract (fraction A) dissolved in dimethyl sulfoxide (DMSO, Merck, Germany) were applied on sterile paper discs (6 mm diameter). Ciprofloxacin and nystatin were used as standard antibacterial and antifungal agents, respectively, as positive controls, and DMSO without the extracts was used as a negative control. The discs were deposited on the surface of inoculated agar plates. These plates were held at 4°C for 2 h, followed by incubation at 37°C for 24 h for bacteria, or at 30°C for 48 h for yeasts. The diameters of the inhibitory zones were measured in millimeters. All tests were performed in triplicate.

Anti-inflammatory activity:
This effect was determined according to the method described by Winter et al. [13]. Eighteen male albino rats, weighing (130-150g) were randomly divided into three groups, each of six animals, first group; received 1ml of saline serving as control, second group; received 100 mg/kg b. wt. of the ethanol extract (A), third group; received 20 mg/kg b. wt. of the reference drug Indomethacin. One hour later after drug administration, all the animals received a sub plantar injection of 0.1ml of 1% carrageenan solution in saline in the right hind paw and 0.1ml saline in the left hind paw. Four hours after drug administration, the rats were sacrificed; both hind paws excised and weighed separately. The percentage of oedema (inflammation) was calculated according to the following equation: Antihyperglycemic activity: The alcohol extract of A. grandidentata was tested for its anti-hyperglycemic activity over long period (2 months). The blood glucose level was monitored after 4 and 8 weeks from zero time. Thirty male albino rats of the Sprague Dawely strain (130 -140 g) were injected intraperitoneal with alloxan (150 mg/ kg body weight) to induce diabetes mellitus [14]. Animals were divided into 3 groups; Hyperglycemia was assessed after 72 hours by measuring blood glucose [15] and after 1 and 2 months intervals from treatment. First group; diabetic rats that served as positive control, second groups; diabetic rats that received 100 mg/kg b. wt. of the ethanol extract (A), third group; diabetic rats that received 150 mg/kg b. wt. of Metformin as reference standard drug. At the end of each study period, blood samples were collected from the retro orbital venous plexus through the eye canthus of anaesthetized rats after an overnight fast. Serum was isolated by centrifugation and the blood glucose level was measured [15]. Blood glucose level was measured at zero G 0 and after treatment G t . Percentage of change in blood glucose level was calculated from the following equation: At the end of the experiments, all dead animals were getting rid by frozen till incineration.
The structure ( Figure 1B In addition the λ MeOH max at 260, 390 and 440 nm besides the high molecular weight supported that compound 2 was a new polyhydroxybianthraquinoid isolated for the first time. On the basis of the available spectral evidences the structure was established to be 1,1',8,8'-tetrahydroxy-3'-acetyl -3-methyl -5, 5bianthracene -9,9',10,10'-tetraone. Compound 3 ( Figure 1B) was obtained as orange crystals with m.p. 236-238°C, soluble in chloroform and gives positive tests for anthraquinone. A molecular formula of C 16 H 12 O 6 was determined for compound 3 on the basis of the observed molecular ion peak at m/z 300. The 1 H -NMR spectra of compound 3 displayed a signal for CH 3 appeared at 2.51 (3H, s, CH 3 -3), the hydroxyl group appeared at 4.45 (1H, brs, H-6) this elucidation is comparable to that of emodin but it differs by the presence of a sharp signal appeared at 3.80 (3H,s) denoting the presence of methoxy (OCH 3 ) group typical for aromatic methoxy group at position 7. Those signals were beside the aromatic proton signal which appeared at 6.83 (1H, s,H-2). The meta coupling protons is confirmed by the presence of a signal at 7.72 (1H, d, J = 3, H-4) and a signal at δ 7.42 (1H, s , H-5). In addition to a characteristic methoxy group signal at δ68.17 and methyl at 22.29, the 13 C NMR spectra of compound 3 displayed 14 carbon signals. The chemical shifts of these 14 resonance signals suggested the presence of two aromatic rings and two doubly conjugated carbons (δ c 180.06, 192.88). Accordingly, the structure of this new compound (3) was determined as 1,6,8-trihydroxy-7-methoxy-3-methylanthraquinone.
The results of antimicrobial activity of the alcoholic extract of A. grandidentata (200 mg/ml) are shown in Table 1. It showed mild antibacterial activities against G +ve bacteria, G -ve bacteria and fungi with the strongest effect being detected against Pseudomonas aeruginosa. These results were in agreement with observations previously reported for several Aloe species [3]. The antimicrobial activities of the alcoholic extract of A. grandidentata might be attributed to its contents of anthraquinones [21] or β-sitosterol [22].
The results of experiments examining the effects of the alcoholic extract of A. grandidentata leaf on carrageenan-induced paw edema are presented in Table 2.
The assay revealed that oral administration of 100 mg/kg b. wt. alcoholic extract reduced paw edema by 59.7%. The results showed  that the alcoholic exerted 92.01% potency as that standard antiinflammatory drug indomethacin. These results were in agreement with findings previously reported for several Aloe species [4,5,13].
The observed data (Table 3) revealed that A. grandidentata Salm. -Dyck at dose 100 mg /kg, reduced the blood glucose level by (45.03%) and (56.24%) after treatment for 4 weeks and 8 weeks respectively. This study represented the first report; for A. grandidentata Salm. -Dyck to have a potent antidiabetic effect equivalent to 95.26% and 86.83% after 4 weeks and 8 weeks respectively comparing to the standard antidiabetic drug Metformin. These results were in agreement with findings previously reported for Aloe vera [6][7][8].
The hypoglycemic effect of alcoholic extract of A. grandidentata may be attributed to its content of chrysophanol [27].
Potency calculated as compared to the standard anti-inflammatory drug Indomethacin * Significantly different from control group at p <0.01 % of change is calculated as regard to the control group. S.E.=standard error