Abstract
Context: Swietenia mahagoni L. Jacq. (Meliaceae) is a medium to large evergreen tree native to Southern Florida, Cuba, The Bahamas, Hispaniola, and Jamaica.
Objective: To evaluate the antidiabetic and antioxidant potential of S. mahagoni bark.
Materials and methods: In the present study, the antidiabetic activity of the methanol extract of S. mahagoni (MESM) bark in streptozotocin (STZ; 65 mg/kg body weight)-induced diabetic rats was evaluated. Glibenclamide (0.5 mg/kg; orally) was taken as the reference drug. The blood glucose levels and body weights were measured every 5th day over a period of 15 days. Antioxidant effects were assayed in diabetic rats by estimating thiobarbituric acid reactive substances (TBARS), glutathione (GSH), and catalase (CAT) levels.
Results and discussion: Oral administration of MESM at the doses of 25 and 50 mg/kg b.w. resulted in a significant (p < 0.001) reduction in blood glucose levels in diabetic rats. Body weights were significantly (p < 0.001) reduced in STZ-induced diabetic rats when compared to normal rats, while the extract significantly restored body weight. The present study was further undertaken to evaluate the antioxidant activity of MESM in STZ-induced diabetic rats. Decreased levels of TBARS and increased levels of GSH and CAT activity indicated a reduction in free radical formation in tissues such as the liver and kidney of diabetic rats.
Conclusion: These findings showed the significant hypoglycemic and antioxidant activity of the extract (MESM) in diabetic rats.
Introduction
Oxidative stress is the imbalance between the generation of oxidative chemical species (OCS) and the body defense mechanisms. Environmental pollutants, toxic habits (drug consumption, smoking, and/or alcohol consumption), inadequate nutrition, excess solar radiation, large exposure to toxic substances (i.e. fertilizers and pesticides), drug metabolism (side-effects), and high physical or mental stress are the most common exogenous factors originating OCS in the human organism (CitationAlberto, 2005). Oxidative stress has also been implicated in the pathogenesis of diabetes, liver damage, nephrotoxicity, inflammation, cancer, cardiovascular disorders, and neurological disorders, as well as in the process of aging (CitationMarx, 1987).
Swietenia mahagoni L. Jacq. (Meliaceae), commonly called “mehgoni” in Bengali and “West Indian mahogany” in English, is a medium to large evergreen tree, native to southern Florida, Cuba, The Bahamas, Hispaniola, and Jamaica (CitationMorton, 1987). The seeds reached India in 1795, where it grew so well that fine timber was produced in Calcutta (West Bengal). S. mahagoni is a tall tree, up to 30 m high, with a short, buttressing base, up to 1 m in diameter, many heavy branches, and dense shade. The bark is smooth and gray on young trees, turning to scaly, dark reddish-brown on large trees. The bark is considered an astringent, and is taken orally as a decoction for diarrhea, as a source of vitamins and iron, and as a medicine to treat hemorrhage. The bark decoction is used orally to increase appetite and to restore strength in cases of tuberculosis (CitationAnonymous, 1986). The seeds and bark of this plant are used for the treatment of hypertension, diabetes, malaria, and epilepsy as a folk medicine in Indonesia and India (CitationKadota et al., 1990; CitationPullaiah, 2006). The bark contains tannin, and may serve as an antipyretic, tonic, and astringent (CitationMcFarland, 1944; CitationKhare, 2007). Traditionally the bark decoction of S. mahagoni is used to treat anemia, diarrhea, dysentery, fever, loss of appetite, and toothache. The leaf decoction of S. mahagoni is used against nerve disorders, the seed infusion against chest pain, and a leaf or root poultice against bleeding (CitationMiroslav, 2005).
The methanol extract of S. mahagoni (bark) has been reported for its inhibitory activity against human immunodeficiency virus-1 (HIV-1) protease (CitationWatanabe & Shibuya, 1999; CitationOtake et al., 2006). The local people of East Midnapore (West Bengal) and Balasore (Orissa) traditionally use the aqueous extract of the seeds and bark of S. mahagoni for curing psoriasis, diabetes, and diarrhea, and it is also used as an antiseptic in cuts and wounds (CitationKhare, 2007). The present study attempts to report the antidiabetic and antioxidant properties of S. mahagoni bark in streptozotocin-induced diabetic rats.
Materials and methods
Collection of plant
The stem bark of S. mahagoni was collected in the month of October 2007 from the hill region of Midnapore, West Bengal, India. The plant was authenticated by M. S. Mondal, Botanical Survey of India, Kolkata, India, and a voucher specimen (PMU-3/JU/2007) has been preserved in the Pharmacology Research Laboratory, Jadavpur University, Kolkata for future reference.
Extraction
The bark of S. mahagoni was shade dried and then powdered with a mechanical grinder. The powder (550 g) was defatted with petroleum ether at 60–80°C in a Soxhlet extraction apparatus and then extracted similarly with methanol. The solvents were completely removed under reduced pressure to obtain a dry mass. The yields of the petroleum ether and methanol extracts were found to be 5.20 and 12.00%, w/w, respectively. The extracts were stored in a vacuum desiccator for further use. The preliminary phytochemical screening of the methanol extract of S. mahagoni (MESM) showed the presence of mainly tannins, flavonoids, and triterpenoids (CitationHarborne, 1998).
Animals
Adult male Wistar albino rats weighing 170–200 g were used for the present investigation. They were housed in a clean polypropylene cage and were fed with a standard pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The animals were acclimatized to laboratory conditions for 1 week prior to the experiment. All procedures described were reviewed and approved by the university animal ethics committee (367001/C/CPCACA).
Chemicals
Streptozotocin (STZ), 5,5-dithio bis-2-nitrobenzoic acid (DTNB), glutathione (GSH), (SISCO Research Laboratories, Mumbai, India), thiobarbituric acid, trichloroacetic acid (TCA) (Merck, Mumbai), potassium dichromate, and glacial acetic acid (Ranbaxy, Mumbai) were used for the study, and glibenclamide (Hoechst, India) was used as the reference drug. Daonil® tablets were powdered and made into a suspension in distilled water using 3%, v/v, aqueous Tween 80 as suspending agent.
Acute toxicity study
The extract was administered orally (p.o.) to five groups of eight Swiss albino mice of either sex, (25, 50, 100, 150, 200 mg/kg b.w.) to evaluate the acute toxicity (CitationLorke, 1983). The animals were observed for signs and symptoms of toxicity and mortality over a period of 48 h.
Induction of diabetes
Diabetes mellitus was induced in overnight-fasted adult Wistar male albino rats weighing 170–200 g, by a single intraperitoneal injection of 65 mg/kg b.w. STZ (CitationPellegrino et al., 1998). The STZ was freshly dissolved in citrate buffer (0.01 M, pH 4.5). The injection volume was prepared to contain 65 mg/mL and injected as 0.1 mL/100 g of body weight (CitationMurali et al., 2002). After 5 days, blood glucose levels were measured, and hyperglycemic (>225 mg/dL blood glucose level) animals were taken for the investigation (CitationEwart et al., 1975).
Experimental design and testing of fasting blood glucose level
Thirty male Wistar albino rats (170–200 g) were divided into five groups (n = 6). The first group served as the normal non-diabetic (saline control) group, while diabetes was induced in the other groups (II–V) through STZ injection. Group II served as the diabetic control. After 24 h of STZ, groups III and IV received MESM 25 and 50 mg/kg b.w. (p.o.) once a day for 15 days and group V received the reference drug glibenclamide 0.5 mg/kg, orally once a day for 15 days (CitationOyelola, 2007). Body weight and blood glucose levels were measured at zero time and after 5, 10, and 15 days by using a one-touch glucometer (Accu-Chek®). Twenty-four hours after the last dose, all the animals were sacrificed by cervical dislocation; the liver and kidneys were removed for estimation of various biochemical parameters including lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and glutathione (GSH) and catalase (CAT) activity.
Assay of thiobarbituric acid reactive substances (TBARS)
TBARS were estimated in the liver and kidney by the method of CitationFraga et al. (1981) and are expressed as mmol/100 g of tissue.
Assay of glutathione (GSH)
GSH was determined by the method of CitationEllman (1959) and the GSH activity is expressed as mg/100 g of tissue.
Assay of catalase (CAT)
CAT was assayed according to the method described by CitationSinha (1972) and is expressed as µmol of H2O2 consumed/min/mg protein.
Statistical analysis
All results are expressed as the mean ± SEM (standard error of the mean). The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Dunnett’s test using GraphPad InStat version 3.05 (GraphPad Software, USA).
Results
The acute toxicity study carried out in Swiss albino mice revealed that the LD50 value of MESM was 200 mg/kg b.w.
The body weights of normal and diabetic rats are summarized in . The final body weights were significantly (p < 0.001) decreased in the STZ-control group when compared with the saline control group. The observed data showed an improvement of body weight after treatment with the extract (p < 0.05) with respect to the STZ-control group. The measured blood glucose levels of normal and STZ-induced diabetic rats are shown in . Administration of MESM in STZ-induced diabetic rats, at the doses of 25 and 50 mg/kg b.w., produced a significant reduction in blood glucose levels when compared with the STZ-control group. The effect produced by MESM at the dose of 25 mg/kg b.w. was comparable to that produced by the reference drug, glibenclamide (0.5 mg/kg b.w.).
Table 1. Body weight of normal and methanol extract of S. mahagoni-treated diabetic rats.
Table 2. Fasting blood glucose levels in normal and methanol extract of S. mahagoni-treated diabetic rats.
The level of TBARS and GSH and CAT activities in tissues of experimental diabetic rats are shown in , , and , respectively. There was a significant elevation of lipid peroxide in the liver and kidney with diabetes when compared to the saline control group. It was found that the administration of MESM tended to decrease liver and kidney TBARS levels (p < 0.05, p < 0.001), which is an indication of the inhibition of oxidative damage of hepatic and renal tissues. There was a significant decrease (p < 0.001) in the level of GSH in the STZ-control group when compared with the saline control group. Administration of MESM at doses of 25 and 50 mg/kg b.w. increased the GSH level in the liver and kidney of STZ-induced rats. There was a significant (p < 0.001) reduction in the activity of CAT in the liver and kidney (p < 0.01) during diabetes. Administration of MESM at 25 and 50 mg/kg b.w. increased the CAT activity in the liver (p < 0.001) and kidney to near normal.
Table 3. TBARS levels in tissues of normal and methanol extract of S. mahagoni-treated diabetic rats.
Table 4. Levels of GSH in tissues of normal and methanol extract of S. mahagoni-treated diabetic rats.
Table 5. CAT activitya in tissues of normal and methanol extract of S. mahagoni-treated diabetic rats.
Discussion
STZ is widely used to induce diabetes in experimental animals (CitationJunod et al., 1969; CitationLike & Rossini, 1976). In the present investigation it was observed that administration of the methanol extract of S. mahagoni (MESM) bark at the doses of 25 and 50 mg/kg b.w. to STZ-induced diabetic rats produced strong antihyperglycemic and antioxidant activity. MESM 25 mg/kg b.w. was found to be effective in comparison to MESM 50 mg/kg b.w. MESM showed a significant effect in controlling the loss of body weight that was caused during diabetes in rats.
Preliminary phytochemical analysis of MESM indicated the presence of mainly tannins, and these types of polyphenols are well-known natural antioxidants due to their electron-donating properties, either scavenging the principal propagating radicals or halting the radical chain (CitationBors et al., 1990; CitationDreosti, 2000). Thus, the antioxidant potential of the methanol extract of S. mahagoni bark may be due to the presence of polyphenolic compounds. However, this claim demands further research to isolate the antioxidant principle, since the present study was a preliminary investigation.
Under normal physiological conditions, the human body can compensate for a mild degree of oxidative stress, and remove oxidatively damaged molecules by activating antioxidant enzymes such as catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), etc. (CitationZhu et al., 2004). These antioxidants are able to resist oxidative stress by scavenging free radicals, inhibiting lipid peroxidation, and increasing glutathione and catalase activity (CitationAlberto, 2005).
Lipid peroxidation is a natural phenomenon involved in peroxidative loss at unsaturated lipids, thus bringing about lipid degradation and membrane disorganization. Peroxidized lipid has been considered to play a significant role in the pathogenesis of several diseases, and may be taken as a molecular mechanism of cell injury under pathological conditions (CitationKuaimoto et al., 1981). Lipid peroxidation is usually measured through its catabolite malondialdehyde (MDA) in terms of TBARS as a marker of oxidative stress (CitationJanero, 1990). A marked increase in the concentration of TBARS in STZ-induced diabetic rats indicated enhanced lipid peroxidation leading to tissue injury and failure of the antioxidant defense mechanism to prevent the formation of excess free radicals. MESM showed the ability to prevent STZ-induced increased TBARS levels, suggesting that MESM inhibited lipid peroxidation and improved the pathological condition of diabetes.
Glutathione (GSH) is one of the abundant tripeptide non-enzymatic biological antioxidants present in the liver and kidney. It plays an important role in detoxification and in the protection of cellular constituents against reactive oxygen species (CitationPreet et al., 2005). CitationLoven et al. (1986) suggested that a decrease in hepatic GSH could be the result of decreased synthesis or increased degradation of GSH by oxidative stress in STZ-induced diabetes. Treatment with MESM elevated the reduced level of GSH, thus protecting the liver and kidney from oxidative stress induced by STZ.
Catalase (CAT) is a heme-containing enzyme widely distributed in the peroxisomes or microperoxisomes of all animal tissues. This enzyme catalyzes the decomposition of H2O2 to water and oxygen, and thus protects the cell from oxidative damage by H2O2 or OH− (CitationVenukumar & Latha, 2002). It was found that CAT activity was decreased in the STZ-induced diabetic rats. MESM supplementation in diabetic rats showed normalization of CAT, thus preventing oxidative injury of the liver and kidney.
The present investigation demonstrates that the methanol extract of Swietenia mahagoni bark possesses strong antihyperglycemic and antioxidant activity.
Declaration of interest
One of the authors (S.P.P.) is grateful to the All India Council for Technical Education, New Delhi, India, for financial assistance, and the authorities of Jadavpur University for providing all other facilities.
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