Abstract
Pongamia pinnata (L.) Pierre (Fabaceae), popularly known as “Karanj” or “Karanja” in Hindi, and Indian beech in English, is a medium-sized glabrous tree. In the present study we report the antihyperglycemic activity of stem bark of alcohol extract of Pongamia pinnata (PPSBAE). Based on acute oral toxicity data, PPSBAE showed no mortality in normal mice up to 5,000 mg/kg. PPSBAE was administered as three doses (i.e., 100, 200, and 400 mg/kg) to diabetic mice, and the serum glucose level and body weight were measured. The onset of serum glucose reduction was observed at 2 h (130.32 mg/dl), peak at 4 h (151.79 mg/dl) and sustained at 6 h, but waned at 24 h. In the subacute study, maximum reduction (305.72 mg/dl) in serum glucose was observed at a dose of 400 mg/kg on day 28. An oral glucose tolerance test (OGTT) was carried out after administration PPSBAE (200 mg/kg) in non-diabetic mice previously loaded with 2.5 g/kg, per oral (p.o.) of glucose. The PPSBAE (200 mg/kg) showed increased glucose threshold in non-diabetic mice. These results suggest that the PPSBAE possesses antihyperglycemic activity.
Introduction
There is an increasing trend towards using herbal drugs reported to have antidiabetic activity. Many herbs and plant products are used widely even when their biologically active compounds are unknown, because of their effectiveness, fewer side effects, and relatively low cost (CitationBailey & Day, 1989).Pongamia pinnata (L.) Pierre (Fabaceae) synonyme Pongamia glabra Vent., popularly known as “Karanj” or “Karanja” in Hindi, and Indian beech in English, is a medium-sized glabrous tree, found throughout India (CitationAiman, 1970; CitationKrishnamurthi, 1969). In the Ayurvedic literature, different parts of this plant have been recommended as a remedy for bronchitis, whooping cough, rheumatic joints, and to quench dipsia in diabetes (CitationKirtikar & Basu, 1987). The bark is used internally for bleeding piles, beriberi (CitationKhare, 2004), diabetes (CitationAiman, 1970) and as an antimicrobial (CitationKoysomboon et al., 2006; CitationAlam, 2004). Punitha et al. Antihyperglycemic and antilipidperoxidase activities of aqueous (CitationPunitha et al. 2006) as well as ethanol extract (CitationPunitha & Manoharan, 2006) of Pongamia pinnata flowers in alloxan-induced diabetic rats have been reported. However, the antihyperglycemic activity of the stem bark has not been reported. The present investigation studied the antihyperglycemic activity of Pongmia pinnata stem bark.
Materials and Methods
Collection and authentication of plant material
Pongamia pinnata bark was collected during April and May 2006 from the hill area of Bhandara, Bhandara District, Maharashtra State, India. The plant was identified and authenticated by Dr. A. M. Mujumdar, Department of Botany, Agharkar Research Institute, Pune, India and a voucher specimen was deposited at that Institute (voucher specimen sample no. AHMA-23892).
Drugs and chemicals
Glyburide (Ranbaxy Pharma Ltd., India), alloxan monohydrate (Spectrochem, India), glucose estimation kit (Accurex Biomedical Pvt. Ltd., India) and d-glucose (S.D. Fine-Chem. Ltd, India), gum acacia (Research-Lab, India) were purchased from respective vendors.
Preparation of alcohol extract of Pongamia pinnata
The stem bark of Pongmia pinnata was shade-dried and powdered (Mesh size-16) in a grinder. The powdered bark (500 g) was macerated in 99.9% alcohol (2.5 L ethanol) at room temperature for 7 days and filtered. The filtrate was dried on a tray dryer at 40°C (yield – 12.5% w/w). The semi-solid extract was suspended with 2% gum acacia in distilled water to prepare the drug solution of concentration of 100 mg/ml and used for pharmacological studies.
Preliminary phytochemical screening
The preliminary phytochemical analysis was carried out for the alkaloid (Mayer's, Hager's, Dragendorff's, Wagner's test), flavonoids (Shinoda test), tannins, phenolic compounds and saponins (CitationKokate, 1991).
Experimental animals
Swiss albino mice (25–30 g) of either sex were purchased from National Toxicology Centre, Pune, India. They were maintained at a temperature of 25° ± 1°C and relative humidity of 45 to 55% under 12 h light: 12 h dark cycle. The animals had free access to food pellets (Chakan Oil Mills, Pune, India) and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Pune.
Acute oral toxicity studies
Healthy adult albino mice of either sex were subjected to acute toxicity studies as per guidelines (AOT No. 425) suggested by the Organization for Economic Co-operation and Development (CitationOECD, 2001). The mice were observed continuously for 2 h for behavioral, neurological and autonomic profiles and for any lethality or death for the next 48 h.
Induction of experimental diabetes
Diabetes was induced in mice by a single intravenous injection of aqueous alloxan monohydrate (80 mg/kg, intravenous) solution. After 48 h, the animals showing serum glucose level above 300 mg/dl (diabetic) were selected for the study. All the animals had free access to tap water and pellet diet.
Collection of blood and determination of serum glucose level
Blood samples from the experimental mice were collected by retro-orbital plexus technique using heparinized capillary glass tubes. The blood samples collected were analyzed for glucose levels by glucose oxidase peroxidase (GOD/POD) method as described earlier (CitationAbdel-Barry et al., 1997) and serum glucose levels were expressed in mg/dl.
Effect of PPSBAE on serum glucose in alloxan-induced diabetic mice
Diabetic Swiss albino mice of either sex were divided into five groups (n = 6): group I-vehicle (gum acacia 2%, 10 ml/kg), group II-glyburide (10 mg/kg), group III-PPSBAE (100 mg/kg), group IV-PPSBAE (200 mg/kg), Group V-PPSBAE (400 mg/kg). PPSBAE was given orally.
Acute study involved estimation of serum glucose level at 0, 1, 2, 4, 6, and 24 h after PPSBAE administration.
The subacute study involved repeated administration of PPSBAE for 28 days (once a day) at prefixed times and serum glucose levels were estimated in samples withdrawn at 4 h after PPSBAE administration on day 7, 14, 21, and 28. The data were represented as mean serum glucose level and standard error of mean (SEM).
Effect of PPSBAE on body weight in diabetic mice
During the study period of 28 days the mice were weighed daily and their body weights were recorded. From these data, mean change in body weight and SEM were calculated and tabulated.
Effect of PPSBAE on Oral Glucose Tolerance Test (OGTT)
The non-diabetic mice were fasted overnight before commencing the experiment. The mice divided into three groups (n = 6): group I-vehicle (gum acacia 2%, 10 ml/kg), group II-glyburide (10 mg/kg) and group III-PPSBAE (200 mg/kg). PPSBAE and glyburide was given orally. The mice of all the groups were loaded with d-glucose (2.5 g/kg, p.o.) solution after half an hour of drug administration (CitationBadole et al., 2006; CitationLatha & Pari, 2003). Blood samples were withdrawn by the retro-orbital technique before drug administration and at 30, 60, and 120 min after glucose loading. The serum glucose was estimated immediately in the samples.
Statistical analysis
Data were expressed as mean ± SEM and statistical analysis was carried out by one-way ANOVA with post hoc Dunnett's test performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, www.graphpad.com. P value was considered significant when P < 0.05.
Results
The preliminary phytochemical analysis of PPSBAE showed the presence of alkaloids, flavonoids, tannins, and phenolic compounds.
Acute toxicity studies revealed that the extract was safe up to a dose level of 5,000 mg/kg of body weight. No lethality or any toxic reactions were found up to the end of the study period.
Single administration of PPSBAE (100, 200, and 400 mg/kg) as well as glyburide (10 mg/kg) significantly reduced serum glucose levels at 2, 4 h after PPSBAE administration. The reduction in serum glucose from the basal value (before) at 4 h after 200 and 400 mg/kg of PPSBAE was 151.79 and 229.27 mg/dl, respectively. The antihyperglycemic effect was sustained at 6 h but waned at 24 h. The onset of the antihyperglycemic effect of glyburide was at 2 h, the peak effect was at 6 h ().
Table 1. Effect of PPSBAE on serum glucose level in alloxan-induced diabetic mice (acute study).
In the subacute study, repeated administration (once a day for 28 days) of the PPSBAE as well as glyburide caused significant (P < 0.01) reduction in the serum glucose level as compared to vehicle treated group. On day 28th reduction in serum glucose by the doses of 100, 200, and 400 mg/kg of PPSBAE were 210.55, 276.20, and 304.42 mg/dl, respectively. Glyburide treated animals showed maximum reduction (243.59 mg/dl) on day 28 ().
Table 2. Effect of PPSBAE on serum glucose level in alloxan-induced diabetic mice (subacute study).
Body weight of vehicle-treated diabetic mice decreased during the study period. PPSBAE (100, 200, and 400 mg/kg) and glyburide (10 mg/kg) prevented a decrease in body weight of diabetic mice. On the other hand, mice gained body weight, which indicated a beneficial effect of PPSBAE ().
Table 3. Effect of PPSBAE on body weight in alloxan-induced diabetic mice.
In the oral glucose tolerance test, PPSBAE (200 mg/kg) produced a significant (P < 0.01) increase in glucose threshold, 120 min post-glucose loading in non-diabetic mice ().
Table 4. Effect of PPSBAE on oral glucose tolerance test in non-diabetic mice.
Discussion
Alloxan, a β -cytotoxin, induces “chemical diabetes” in a wide variety of animal species including mice by damaging the insulin secreting β -cells of the pancreas. It causes time and concentration-dependent degenerative lesions of the pancreatic β -cells (CitationJorns et al. 1997; CitationLenzen & Panten, 1988) Induction of diabetes by alloxan (intravenously) is reflected by glycosuria, hyperglycemia, polyphagia, polydipsia, and body weight loss compared with normal animals (CitationSzkudelski, 2001).
In the present study, significant reduction in serum glucose level was seen at 2 h and peak reduction occurred at 4 h after treatment with PPSBAE in acute study as well as subacute administration. The PPSBAE showed short onset and short duration of antihyperglycemic action.
Subacute treatment for 28 days with the PPSBAE in the tested doses brought about improvement in body weights of alloxan treated diabetic mice indicating its beneficial effect in preventing loss of body weight in diabetic mice (CitationXie et al., 2003; CitationSwanston-Flatt et al., 1989). The ability of PPSBAE to protect against body weight loss seems to be due to its ability to reduce hyperglycemia.
In the present study, PPSBAE significantly enhanced glucose utilization in an oral glucose tolerance test in non-diabetic mice. From the data obtained in the oral glucose tolerance test, it is clear that serum glucose level reached a peak and returned to base level after 2 h in normal mice. In contrast, in vehicle-treated mice, serum glucose level remained high even after 2 h. The administration of PPSBAE effectively prevented the increase in serum glucose level without causing a hypoglycemic state. The effect may be due to restoration of the delayed insulin response. In this context, other medicinal plants, such as Pleurotus pulmonarius (CitationBadole et al., 2006), Cassia auriculata (CitationLatha & Pari, 2003) have been reported to possess similar effects.
A relationship between oxidative stress and diabetes mellitus has been well demonstrated in many experimental animal models (CitationPunitha et al., 2006; CitationPunitha & Manoharan, 2006; CitationBartosikova et al., 2003).
Free radical formations are involved in the pathogenesis of diabetes and the development of diabetic complications (CitationGiugliano et al., 1995; CitationColman et al., 1989). The oxidative stress is significantly increased in diabetes because prolonged exposure to hyperglycemia increases the generation of free radicals and reduces capacity of antioxidation defense systems (CitationColman et al., 1989).
Flavonoids are potent antioxidants and are known to modulate the activities of various enzyme systems due to their interaction with various biomolecules (CitationDevipriya & Shyamaladevi, 1999; CitationCatapano, 1997). CitationKameswararao et al. (1997) reported that flavonoids, alkaloids, tannins, and phenolics have bioactive antidiabetic principles. CitationChakravarthy et al. (1980) reported that flavonoids regenerate the damaged β -cells in the alloxan diabetic rats.
Preliminary phytochemical analysis of the stem bark of Pongamia pinnata showed presence of alkaloids, glycosides, phenolic compounds, flavonoids, furanoflavones, chromenoflavones, flavonodiaketones, pongaflavonol, pyranochalcones, and flavonoid glycoside (CitationKrishna & Grampurohit, 2006; CitationYin et al., 2006a, Citation2006b, Citation2005; CitationCarcache-Blanco et al., 2003; CitationAsalkar et al., 2000; CitationTanaka et al., 1992; CitationPathak et al., 1983; CitationTalapatra et al., 1982). The antihyperglycemic activity of PPSBAE may be due to the presence of several bioactive antidiabetic principles. It is thus apparent that the PPSBAE possesses antihyperglycemic effect.
Conclusions
Administration of PPSBAE significantly reduced high blood glucose level and prevented loss in body weight in alloxan-induced diabetic mice. PPSBAE also increased glucose threshold in normal mice. Our results indicate that PPSBAE possesses antihyperglycemic activity and also prevents further loss of body weight in diabetic animals. Identification of active compound(s) with significant antihyperglycemic activities from the stem bark of Pongmia pinnata may provide an opportunity to develop a novel class of anti-diabetic agents.
Acknowledgements
The authors would like acknowledge Dr. S. S. Kadam, Vice-Chancellor and Dr. K.R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, India, for providing necessary facilities to carry out the study. We are also thankful to the All India Council of Technical and Education (AICTE), India for financial support by awarding National Doctoral Fellowship for the research work.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- JA Abdel-Barry, IA Abdel-Hassan, and MH Al-Hakiem. (1997). Hypoglycaemic and antihyperglycaemic effects of Trigonella foenum graecum leaf in normal and alloxan induced diabetic rats. J Ethnopharmacol 58:149–155.
- R Aiman. (1970). Recent research on indigenous antidiabetic medicinal plants-an overall assessment. Ind J Physiol Pharmacol 14:65–76.
- S Alam. (2004). Synthesis and studies of antimicrobial activity of lanceolatin B. Acta Chim Slov 51:447–452.
- LV Asalkar, KK Kakkar, and OJ Chakre. (2000): Second Supplement to Glossary of Indian Medicinal Plants with Active Principles. Part I (A-K) (1965–81). New Delhi, National Institute of Science Communication Publishing, pp. 265–266.
- SL Badole, SN Shah, NM Patel, PA Thakurdesai, and SL Bodhankar. (2006). Hypoglycemic activity of aqueous extract of Pleurotus pulmonarius in alloxan induced diabetic mice. Pharm Biol 44:421–425.
- L Bartosikova, J Necas, V Suchy, R Kubinova, D Vesela, L Benes, T Bartosik, J Illek, J Salpachata, J Klusakova, L Bartokova, V Strnadova, P Frana, and J Franova. (2003). Monitoring of antioxidative effect of morine in alloxan induced diabetes in labolatory rat. Acta Vet Brno 72:191–200.
- CJ Bailey, and C Day. (1989). Traditional treatment for diabetes. Diab Care 12:533–564.
- AL Catapano. (1997). Antioxidant effect of flavonoids. Angiol 48:39–44.
- EJ Carcache-Blanco, YH Kang, EJ Park, BN Kardono, LBS Su, S Riswan, HHS Fong, JM Pezzuto, and AD Kinghorn. (2003). Constituents of the stem bark of Pongamia pinnata with the potential to induce quinone reductase. J Nat Prod 66:1197–1202.
- PG Colman, LI Wang, and KJ Lafferty. (1989): Molecular biology and autoimmunity of type I diabetes mellitus. In: B Drazini, S Melmed, and D LePoith. eds., Molecular and Cellular Biology of Diabetes Mellitus. Insulin Secretion. New York, Alan R. Liss Inc., pp. 125–137.
- BK Chakravarthy, S Gupta, SS Gambir, and KD Gode. (1980). Pancreatic β cell generation-A novel antidiabetic mechanism of Pterocarpus marsupium Rox. Ind J Pharmacol 12:123–127.
- S Devipriya, and CS Shyamaladevi. (1999). Protective effect of quercetin in cisplatin induced cell injury in the rat kidney. Ind J Pharmacol 31:422–426.
- D Giugliano, A Ceriello, and G Paolisso. (1995). Diabetes mellitus, hypertension and cardiovascular disease: Which role for oxidative stress. Metabol 44:363–368.
- A Jorns, R Munday, M Tiedge, and S Lenzen. (1997). Comparative toxicity of alloxan, N-alkylalloxan and ninhydrin to isolated pancreatic islets in vitro. J Endocrinol 155:283–293.
- B Kameswararao, R Giri, MM Kesavulu, and C Apparao. (1997). Herbal medicines: In the treatment of diabetes mellitus. Manphar Vaidya Patrika 1:33–35.
- CP Khare. (2004): Encyclopedia of Indian Medicinal Plants. Springer-Verlag editor. New York, pp 378–379.
- A Krishnamurthi. (1969): The Wealth of India VIII. New Delhi, Publication and Information Directorate, CSIR.
- KR Kirtikar, and BD Basu. (1987): Indian Medicinal Plants vol. 1, 2nd edition, E Blatter, JF Caius, and KS Mhaskar. eds., published by Basu LM, Allahabad, India., pp 830–832.
- CK Kokate. (1991): Practical Pharmacognosy. 3rd eds., published by Jain MK, Vallabh prakashan, New Delhi., pp 107–113.
- S Koysomboon, IV Altena, S Kato, and Chantrapromma. (2006). Antimicrobacterial flavonoids from Derris indica. Phytochemistry 67:1034–1040.
- VN Krishna, and ND Grampurohit. (2006). Studies on the alkloids of stembark of Pongamia pinnata Linn. Ind Drugs 43:383–387.
- M Latha, and L Pari. (2003). Antihyperglycaemic effect of Cassia auriculata in experimental diabetes and its effects on key metabolic enzymes involved in carbohydrated metabolism. Clin Exp Pharmacol Physiol 30:38–43.
- S Lenzen, and U Panten. (1988). Alloxan: History and mechanism of action. Diabetol 31:337–342.
- OECD (Organisation for Economic Co-operation and Development) (2001): Guidance Document on Acute Oral Toxicity Testing, Paris, Environment Directorate, OECD, pp. 1–24.
- VP Pathak, TR Saini, and RN Khanna. (1983). Glabrachalcone-A chromenochalcone from Pongamia glabra seeds. Phytochemistry 22:1303–1304.
- R Punitha, and S Manoharan. (2006). Antihyperglycemic and antilipidperoxidative effects of Pongamia pinnata (Linn.) Pierre flowers in alloxan induced diabetic rats. J Ethnopharmacol 105:39–46.
- R Punitha, K Vasudevan, and S Manoharan. (2006). Effect of Pongamia pinnata flowers on blood glucose and oxidative stress in alloxan induced diabetic rats. Ind J Pharmacol 38:62–63.
- SK Swanston-Flatt, C Day, PR Flatt, BJ Gould, and CJ Bailey. (1989). Glycaemic effects of traditional European plant treatments for diabetes: Studies in normal and streptozotocin diabetic mice. Diab Res 10:69–73.
- T Szkudelski. (2001). The mechanism of alloxan and streptozotocin action in β cells of the rat pancreas. Physiol Res 50:536–546.
- SK Talapatra, AK Malik, and B Talapatra. (1982). Isopongaglabol and 6-methoxyiso-pongaglabol, two new hydroxyfuranoflavones from Pongamia glabra. Phytochemistry 21:761–766.
- T Tanaka, M Iinuma, Y Fujii, K Yuki, and M Mizuno. (1992). Flavonoids in root bark of Pongamia pinnata. Phytochemistry 31:993–998.
- JT Xie, A Wang, S Mehendale, J Wu, HH Aung, L Dey, S Qiu, and CS Yuan. (2003). Anti-diabetic effects of Gymnema yunnanense extract. Pharmacol Res 47:323–329.
- H Yin, S Zhang, and J Wu. (2005). Prenylated flavonoids from Pongamia pinnata. Z Naturforsch 60b:356–358.
- H Yin, S Zhang, J Wu, H Nan, L Long, J Yang, and Q Li. (2006a). Pongaflavanol-A prenylated flavonoid from Pongamia pinnata with a modified ring A. Molecules 11:786–791.
- H Yin, S Zhang, J Wu, and H Nan. (2006b). Dihydropyranoflavones from Pongamia pinnata.. J Braz Chem Soc 17:1432–1435.