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Barringtonia acutangula improves the biochemical parameters in diabetic rats
Chinese Journal of Natural Medicines 2014, 12(2): 01260130
Chinese Journal of Natural Medicines
Barringtonia acutangula improves the biochemical parameters in diabetic rats Marslin Gregory1, 2*, Vinoth Kumar Megraj Khandelwal3, Revina Ann Mary4, V. K. Kalaichelvan1, V. Palanivel1, 5* 1
Department of Pharmacology, Faculty of Pharmacy, Annamalai University, Chidambaram, India; Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), Department of Biology, University of Minho, Portugal; 3 Consorzio Mario Negri Sud, Department of Translational Pharmacology, Santa Maria Imbaro, Italy; 4 Department of Nursing Science, A.J. College of Nursing, Chennai, Tamilnadu, India; 5 Padmavathi College of Pharmacy and Research Institute, Periyanahalli, Dharmapuri, Tamilnadu, India 2
Available online 20 Feb. 2014
[ABSTRACT] AIM: This study was aimed at evaluating the anti-diabetic activity of the ethanol and aqueous extracts of the leaf material of Barringtonia acutangula in a diabetic animal model. METHODS: The # #_ ^" ^} #_ kg1 body weight) of the leaves of B. acutangula were assessed for antidiabetic activity in a streptozotocin (STZ)-induced diabetes animal model following 21 days of treatment. Glibenclamide (0.6 mgkg1 p.o.) was used as a positive control. The hematological parameters, such as blood glucose level, urea, creatinin, cholesterol, HDL-C, and LDL-C levels were examined. RESULTS: An acute toxicity study (5 000 mgkg1, p.o.) did not produce any symptoms of toxicity. Significant reductions in blood glucose level, and serum total cholesterol and triglyceride levels were noted in animals treated with the extract. The high density lipoprotein-cholesterol (HDLC) level was found to increase as compared with the diabetic control group. CONCLUSION: These results suggest that the leaf aqueous and ethanolic extracts of B. acutangula have anti-diabetic effects. The aqueous extract of B. acutangula produced a similar effect when compared with the ethanol extract. It is proposed that consumption of B. acutangula in some form like tea may help the management of diabetes. [KEY WORDS] Barringtonia acutangula; Ethanolic extract; Aqueous extract; Anti-diabetic activity
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)02-0126-05
Introduction Diabetes mellitus is a metabolic disorder affecting carbohydrate, fat, and protein metabolism. Diabetes mellitus has been recognized as one of the most important health problems all over the world. Over the past ten years, the number of diabetics has risen by 5% per annum (p.a.) to approximately 250 million, and has been projected to become one of [Received on] 25-Nov.-2012 [Research funding] This project was supported by University Grant Commission (UGC), New Delhi, India (No. 32/135/2006 (SR)). [ Corresponding authors] Marslin Gregory & Palanivel: E-mail: [email protected], [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
the world’s major killers within the next 25 years [1]. More than 70% of the population in developing countries relies on traditional medicine for their primary health care needs [2], and traditional medicines are being explored for various activities [3-4]. Plants have always been an exemplary source of drugs, and many of the currently available drugs have been derived directly or indirectly from plants [5]. With the ever growing interest in natural medicines, many plants have been screened and reported to be useful in treating and managing diabetes [6]. Barringtonia acutangula (L.) Gaertn. (Lecythidaceae) is an important traditional medicinal plant distributed throughout India, mostly near the low lying areas, and has been used by indigenous groups for a wide range of medicinal purposes [7]. It has several vernacular names, including Indian oak, Indian putat, and hillol. In south India it is called samudra pazham. A literature survey revealed that various parts of B.
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Marslin Gregory, et al. / Chin J Nat Med, 2014, 12(2): 126130
acutangula have been used as a folk medicine for curing various ailments including hemiplegia, pain in the joints, eye diseases, stomach disorders, anthelmintic, diarrhea, cough, dyspnea, leprosy, intermittent fever, splenic disorders, and poisoning [8-9]. Many of the traditionally ascribed activities of medicinal plants were later confirmed and accepted following intensive research. A good example is the antinociceptive, antidiarrheal, and neuropharmacological activity of B. acutangula recently reported [10]. Moreover, Babre and co-workers studied the hydroalcoholic root extract of B. acutangula on streptozotocin-induced diabetic rats. They reported that administration of the root extract of B. acutangula produces a significant reduction, followed by a steady decrease of the blood glucose level, and concluded that the antidiabetic activity may be due to the presence of saponins, tannins, phenols, and alkaloids [11]. In the present investigation, the anti-diabetic effects of the ethanolic and aqueous extracts of the leaves of B. acutangula were studied.
used to study the acute toxicity of the B. acutangula extracts. Animals were fasted overnight and orally administered with the ethanolic extract (50, 500, 2 000 and 5 1). The same conditions were maintained for toxicity study of aqueous extract. A group of animals which received an equal volume of PBS served as the control. Changes in the animal behavior were noted before and after administration for 24 h. Treated animals were further observed for up to 14 days for any signs of toxicity [14]. Evaluation of anti-diabetic activity Graphical expression of experimental work
Materials and methods Plant material The plant material of B. acutangula was collected from Pondycherry Botanical Garden, (Plot no 21), and authenticated by Mr. Sukumar, Agriculture Officer, Pondycherry Botanical Garden, India. The leaves were dried under the shade, powdered, and stored in an airtight container. For ethanol extract, dried leaf powder (250 g) was loosely packed in the thimble of Soxhlet apparatus and extracted with ethanol for 18 h at 55 ºC. The extract was air dried at 2530 ºC for 4 h. For oral administration, the extract was dissolved in 10 ml phosphate buffered saline (PBS) at different concentrations [12]. The aqueous extract was prepared according to the traditional method: the leaf powder (50 g) was dissolved in boiling distilled water (100 mL). The hot infusion was then left to reach room temperature (ca. 15 min) and filtered. The filtrate was used as such for biological tests on animals. Phytochemical screening The ethanol and aqueous extracts of the root bark of B. acutangula were evaluated for the presence of flavonoids, tannins, alkaloids, saponins, glycosides, and sterols/ triterpenes using the methods described by Brain et al [13]. Animals ¥ ® ^ ^ # ? # #_ in the Central Animal House, Raja Muthaiya Medical College, in polypropylene cages under standard conditions. During the whole experimental period, animals were fed a balanced diet (rat chow) and water ad libitum. Experiments were carried out in accordance with the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) Guidelines, and the study was approved by the Institutional Animal Ethical Committee (IAEC/CPCSEA/ 03/002/11). Acute toxicity study Five groups (n = ` # }
Induction of diabetes in rats The animals were fasted for 24 hours, and diabetes was induced by a single injection of STZ (60 mgkg1). The blood glucose level was checked before and 72 h after STZ injection to confirm the development of diabetes in animals. The # ^ `_ ^ ` # }
mL1) were selected for further studies [15]. Experimental design All of the diabetic animals were randomly divided into seven groups with six animals in each group and treated once a day for 21 days as follows: Group I (normal healthy control) received vehicle PBS. Group II served as a diabetic control and received only STZ (60 mgkg1). Groups III and IV diabetic rats received B. acutangula ethanolic extract at 250 and 500 mgkg1 bw, respectively. Groups V and VI diabetic rats received B. acutangula aqueous extract at 250 and 500 mgkg1 bw, respectively. Group VII diabetic rats received glibenclamide (GLB) at 10 mgkg1 bw. Blood sample collection After 21 days of treatment the animals were deprived of food overnight, and the blood was collected from the retro orbital plexus in tubes containing EDTA for the estimation of blood glucose level, creatinine, urea, cholesterol, HDL-C, and LDL-C levels in serum. Body weights were determined. Statistical Analysis The results are expressed as x ± s. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test. P < 0.05 was considered as significant.
Results
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Diabetes mellitus is a chronic metabolic disorder which is
Marslin Gregory, et al. / Chin J Nat Med, 2014, 12(2): 126130
characterized by hyperglycemia resulting from deficiency in the production of insulin by the pancreas, or reduced insulin activity [16]. The etiology of type 1 diabetes is the absolute deficiency of insulin secretion, while that of type 2 is a combination of insulin resistance with inadequate compensatory insulin-secretory response [17] The anti-diabetic activities of the ethanolic and aqueous extracts of B. acutangula are reported. Acute toxicity studies revealed the non-toxic, safe nature of both the aqueous and ethanolic extracts of the leaves of B. acutangula. No mortality was observed in the extract-treated mice, and the behavior (tremors, convulsions, diarrhea, salivation, and paralysis) of the treated mice also appeared normal in most of the animals. Neither lethality nor any toxic reaction was found at any dose selected until the end of the study. Even at the dose of 5 000 1, there were no symptoms of toxicity or mortality up to 14 days. According to Lorke et al any substance that is not toxic at 5 1
is considered relatively safe [18]. The preliminary phytochemical screening of the extracts of B. acutangula revealed the presence of carbohydrates, phytosterols, terpenoids, flavonoids, glycosides, tannins, and saponins. Similar results were reported previously for the same plant [19]. Administration of a single intraperitoneal injection of 60
1 bw of freshly-prepared STZ produced diabetes in rats after a week. The effect of different doses of both aqueous and ethanolic extracts of the leaves of B. acutangula on fasting blood glucose levels in normal and STZ induced diabetic rats are summarized in Table 1. The results showed that in the diabetic control group, the blood glucose, urea, and creatinine levels were higher than those of the normal control group (Fig. 1, 2). In particular, the greatest decrease of glycemia level was observed in rats treated with 1 bw of B. acutangula, and the positive control. The low levels of blood glucose suggest that B. acutangula might be considered for further anti-diabetic studies.
Table 1 Anti-diabetic activity of B. acutangula ethanolic and aqueous extracts in STZ-induced diabetic rats ( x ± s) Treatment
Dose /(mgkg1)
Blood glucose/} _L1)
Creatinine/} _L1)
Urea/} _L1)
Normal
88.16 ± 2.2
1.13 ± 0.02
32.2 ± 1.2
Diabetic control (STZ)
60
235.13 ± 5.2
1.89 ± 0.09
69.5 ± 3.4
***
***
32.5 ± 2.3***
Control (Glibenclamide)
0.6
97.16 ± 3.5
Ethanolic extract of B.
250
121.16 ± 1.4***
1.36 ± 0.02***
34.5 ± 0.7***
acutangula
500
***
97.14 ± 1.6
***
1.27 ± 0.04
32.4 ± 1.8***
Aqueous extract of B.
250
120.52 ± 1.6***
1.38 ± 0.02***
35.3 ± 0.7***
500
***
***
31.8 ± 1.2***
acutangula
96.24 ± 1.4
1.28 ± 0.03
1.29 ± 0.06
***
P < 0.001 vs diabetic control group
Fig. 1 Effect of aqueous and ethanolic extracts on blood glucose and urea in STZ-induced diabetic rats. The results show significant reduction in blood glucose level of treated groups compared with diabetic control. When compared with the standard group, almost similar results were obtained
Total lipids, triglycerides, and total cholesterol of normal and experimental animals were examined (Table 2). There was a significant elevation in all of the biochemical parameters in the diabetic animals as compared with the non-diabetic control group, although HDL-C was exceptional (Fig. 3). The effect of administration of B. acutangula aqueous and etha# " ^ #_ 1 bw and glibenclamide decreased glycemia, total lipids, triglycerides, and total
Fig. 2 Effect of B. acutangula extract on creatinine level in STZ induced diabetic rats
cholesterol, although they were higher than normal controls. The results listed in Table 3 show the changes of body weight among the groups of diabetic and normal rats. A normal body weight gain was observed in Groups I, III, IV, V, and VI, whereas a significant decrease in body weight was observed with the diabetic control group (Fig. 4). Finally, the overall comparative study between the aqueous and ethanolic extracts of B. acutangula shows that both extracts produce the
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Marslin Gregory, et al. / Chin J Nat Med, 2014, 12(2): 126130
Table 2 Effect of extracts on lipid profile in STZ induced diabetic rats ( x ± s, n = 6) Treatment
Dose/} 1)
Normal
Diabetic control (STZ)
62.12 ± 2.5
60
Standard (Glibenclamide)
Hematological estimation Cholesterol/} _L1) Triglycerides/} _L1)
0.6
96.16 ± 2.6
102.23 ± 3.1
HDL-C/} _L1) 45.15 ± 2.0
LDL-C/} _L1) 125.16 ± 2.1
183.18 ± 4.4
28.18 ± 2.1
141.13 ± 1.9
65.33 ± 1.4***
122.26 ± 5.4***
36.25 ± 1.9***
129.16 ± 0.7***
Ethanolic extract of B.
250
66.25 ± 0.8***
118.18 ± 5.1***
39.18 ± 1.6***
121.18 ± 3.7***
acutangula
500
64.24 ±1.3***
110.21 ± 1.8***
41.12 ± 1.5***
118.12 ± 1.5***
***
***
119.12 ± 1.8***
***
117.17 ± 1.2***
Aqueous extract of B. acutangula P <0.01 vs diabetic control group
250 500
***
67.26 ± 0.5
116.23 ± 4.4
***
***
65.32 ± 1.6
109.26 ± 1.5
43.16 ± 2.0
44.21 ± 1.7
***
anti-diabetic activity in dose dependent manner, and both the extract exhibits almost similar activity. Recent hypoglycemic studies in animals revealed that various phytochemical constituents, such as polysaccharides, flavonoids, terpinoids, tannins, saponins, and alkaloids, are active compounds for hypoglycemic activity [20]. Any one of these active compounds may be well-solubilized in both extracts, and that could be the reason both the aqueous and ethanol extracts showed almost similar anti-diabetic effects. Fig. 3 Effect of extract on lipid profile in STZ induced diabetic rats. Results express the significant reduction in cholesterol, triglycerides, LDL-C, and HDL-C levels compared with diabetic control
Discussion The present study was designed to evaluate the anti- diabetic potential of B. acutangula aqueous and ethanolic extracts in STZ-induced diabetic rats. The administration of
Table 3 Effect of extracts on body weight in STZ induced diabetic rats ( x ± s, n = 6) Treatment
Dose/} 1)
Normal Diabetic control (STZ) Standard (Glibenclamide)
60 0.6 250 500 250 500
Ethanolic extract of B. acutangula Aqueous extract of B. acutangula ***
Body weight/g Initial day 210.24 ± 2.6 215.23 ± 3.2 212.12 ± 3.4 208.16 ± 2.3 214.14 ± 3.1 210.16 ± 2.9 215.1.7 ± 3.1
Day 21 228.12 ± 3.2 202.14 ± 3.6 231.23 ± 2.8*** 224.25 ± 3.1*** 229.27 ± 2.5*** 223.21 ± 2.3*** 230.16 ± 2.6***
P <0.01 vs diabetic control group
Fig. 4 Effects of extract on body weight in STZ-induced diabetic rats before and after treatment. The results show the reduction in the body weight of the diabetic animals
streptozotocin in animals can induce necrosis of beta-cells of the islets of Langerhans, thus causing hypoinsulinemia and hyperglycemia [21]. In this study, it was found that 21-day treatment with different concentrations of B. acutangula aqueous and ethanolic extracts significantly reduced the fasting blood glucose level in diabetic rats. The results also showed that the blood glucose level was lowered in the drug-treated groups. Diabetes is associated with profound alterations in the plasma lipid and lipoprotein profiles. In uncontrolled type 2 diabetes mellitus, there is an increase in cholesterol, triglycerides, urea, creatinine, and LDL-C and cholesterol with decrease in HDL-C which contributes to the coronary artery disease [22]. Treatment with both aqueous and ethanol extracts resulted in the elevation of HDL-Cholesterol levels, when compared to the diabetic control group. The maximum in-
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Marslin Gregory, et al. / Chin J Nat Med, 2014, 12(2): 126130
crease in HDL-Cholesterol value (44.21) was obtained in the animals treated with 500 mgkg1 aqueous leaf extract. This value is doubled when compared with the diabetic control and is more similar to the normal animals. The increased HDL-cholesterol and decrease in LDL-C levels may be beneficial due to the negative correlation between HDL-C levels and cardiovascular diseases. This could be due to the presence of a hypolipidemic agent more in the aqueous leaf extract B. acutangula.
[8]
[9]
[10]
[11]
Conclusion From this study, it is clear that B. acutangula leaf aqueous and ethanolic extracts have significant anti-diabetic activity in animal models. It is suggested that the anti-diabetic activity is probably due to the presence of flavonoids. The extract is non-toxic even at relatively high concentrations (5 000 mgkg1 b.w). Similar results were obtained for animals treated with the aqueous extract. Hence it is proposed that consumption of B. acutangula leaf in some form, like a tea, would control the blood glucose level in diabetic patients. Further studies are being carried out to explore the mechanism underlying the biological activities of the compounds present in the extract.
[12]
[13]
[14]
[15]
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Abdulrashid U, Qamar A, Bala M, et al. Anti-hyperglycemic activity of the leaves of Tetracera scandens Linn. Merr (Dilleniaceae) in alloxan induced diabetic rats [J]. J Ethnopharmacol, 2010, 131 (1): 140-145. Antwi S, Martey K, Donkor LK, et al. Anti-diarrhoeal activity of Blighia sapida (Sapindaceae) in rats and mice [J]. J Pharmacol Toxicol, 2009, 4 (3): 117-115. Vinoth KM, Kh #_ ##_¯² ¥et al. Effect of Hemidesmus indicus R. Br. on the ischemia- reperfusion injury in the isolated rat heart [J]. Pharm Biol, 2010, 48 (6): 611-614. Vinoth KM, Khandelwal, Balaraman R, et al. Hemidesmus indicus and Hibiscus rosa sinensis affect ischemia reperfusion injury in isolated rat hearts [J]. Evid Based Complemen Alternat Med, 2011, 1 (1): 1-8. Bopp A, De Bona KS, Belle LB, et al. Syzygium cumini inhibits adenosine deaminase activity and reduces glucose levels in hyperglycemic patients [J]. Fundament Clin Pharmacol, 2009, 23 (4): 501-507. Vinoth KM Khandelwal, Koneri R, Balaraman, et al. Biological activities of some Indian medicinal plants [J]. J Adv Pharm Ed Res, 2011, 1(1): 12-44. Mishra S, Sahoo S, Routl KK, et al. Hepatoprotective effect of Barringtonia acutangula Linn. leaves on carbon tetrachloride-induced acute liver damage in rats [J]. Indian J Nat Prod Resourc, 2011, 2: 515-519.
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Rahman MM, Polfreman MD, MacGeachan J, et al. Antimicrobial activities of Barringtonia acutangula [J]. Phytother Res, 2005, 19 (6): 443-445. Padmavathi D, Susheela L, Bharathi RV. Pharmacognostical evaluation of Barringtonia acutangula leaf [J]. Int J Ayurveda Res, 2011, 2 (1): 37-41. Imam MZ, Sultana S, Akter S. Antinociceptive antidiarrheal and neuropharmacological activities of Barringtonia acutangula [J]. Pharm Biol, 2012, 50 (9): 1078-1084. Babre NP, Debnath S, Manjunath YS, et al. Antioxidant potential of hydroalcoholic extract of Barringtonia acutangula Linn. root on streptozotocin-induced diabetic rats [J]. Int J Pharm Sci Nanotechnol, 2010, 2 (4): 201-203. Gregory M, Vithalrao KP, Franklin G, et al. Antiulcer (ulcer-preventive) activity of Ficus arnottiana Miq. (Moraceae) leaf methanolic extract [J]. Am J Pharmacol Toxicol, 2009, 4 (3): 89-93. Brain KR, Turner TD. The Practical Evaluation of Phytopharmaceuticals [M]. Bristol: Wright-Scientechnica, 1957: 10-30. Gregory M, Divya B, Revina AM, et al. Anti-ulcer activity of Ficus religiosa leaf ethanolic extract [J]. Asian Pac J Trop Biomed, 2013, 3 (7): 554-556. Kumar S, Kumar V, Prakash OM. Antidiabetic and hypolipidemic activities of Kigelia pinnata flowers extract in streptozotocin induced diabetic rats [J]. Asian Pac J Trop Biomed, 2012, 2 (7): 543-546. Zheng XK, Zhang L, Wang WE, et al. Anti-diabetic activity and potential mechanism of total flavonoids of Selaginella tamariscina (Beauv.) Spring in rats induced by high fat diet and low dose STZ [J]. J Ethnopharmacol, 2011, 137 (1): 662-668. Patel DK, Kumar R, Laloo D, et al. Diabetes mellitus: An overview on its pharmacological aspects and reported medicinal plants having antidiabetic activity [J]. Asian Pac J Trop Biomed, 2012, 2(5):411-420. Lorke D. A new approach to practical acute toxicity testing [J]. Arch Toxicol, 1983, 54: 275-287. Rashmi K, Bhasker Shenoy K, Hegde K, et al. Hepatoprotective effect of Barringtonia acutangula (L.) Gaertn. leaf extracts against CCl 4 -induced hepatic damage [J]. J Pharm Res, 2011, 4 (2): 540-542. Patil K. Evaluation of hypoglycemic activity of Barringtonia acutangula fruit extracts in streptozotocin induced hyperglycemic Wistar rats [J]. J Cell Tissue Res, 2011, 11: 2573-2578 Shu XS, Lv JH, Tao J, et al. Antihyperglycemic effects of total flavonoids from Polygonatum odoratum in STZ and alloxan-induced diabetic rats [J]. J Ethnopharmacol, 2009, 124 (3): 539-543. Arvind K, Pradeepa R, Deepa R, et al. Diabetes and coronary artery diseases [J]. Indian J Med Res, 2002, 116: 163-176.
Cite this article as: Marslin Gregory, Vinoth Kumar Megraj Khandelwal, Revina Ann Mary, V. K. Kalaichelvan, V. Palanivel. Barringtonia acutangula improves the biochemical parameters in diabetic rats [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 126-130
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Chinese Journal of Natural Medicines
Barringtonia acutangula improves the biochemical parameters in diabetic rats Marslin Gregory1, 2*, Vinoth Kumar Megraj Khandelwal3, Revina Ann Mary4, V. K. Kalaichelvan1, V. Palanivel1, 5* 1
Department of Pharmacology, Faculty of Pharmacy, Annamalai University, Chidambaram, India; Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), Department of Biology, University of Minho, Portugal; 3 Consorzio Mario Negri Sud, Department of Translational Pharmacology, Santa Maria Imbaro, Italy; 4 Department of Nursing Science, A.J. College of Nursing, Chennai, Tamilnadu, India; 5 Padmavathi College of Pharmacy and Research Institute, Periyanahalli, Dharmapuri, Tamilnadu, India 2
Available online 20 Feb. 2014
[ABSTRACT] AIM: This study was aimed at evaluating the anti-diabetic activity of the ethanol and aqueous extracts of the leaf material of Barringtonia acutangula in a diabetic animal model. METHODS: The # #_ ^" ^} #_ kg1 body weight) of the leaves of B. acutangula were assessed for antidiabetic activity in a streptozotocin (STZ)-induced diabetes animal model following 21 days of treatment. Glibenclamide (0.6 mgkg1 p.o.) was used as a positive control. The hematological parameters, such as blood glucose level, urea, creatinin, cholesterol, HDL-C, and LDL-C levels were examined. RESULTS: An acute toxicity study (5 000 mgkg1, p.o.) did not produce any symptoms of toxicity. Significant reductions in blood glucose level, and serum total cholesterol and triglyceride levels were noted in animals treated with the extract. The high density lipoprotein-cholesterol (HDLC) level was found to increase as compared with the diabetic control group. CONCLUSION: These results suggest that the leaf aqueous and ethanolic extracts of B. acutangula have anti-diabetic effects. The aqueous extract of B. acutangula produced a similar effect when compared with the ethanol extract. It is proposed that consumption of B. acutangula in some form like tea may help the management of diabetes. [KEY WORDS] Barringtonia acutangula; Ethanolic extract; Aqueous extract; Anti-diabetic activity
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)02-0126-05
Introduction Diabetes mellitus is a metabolic disorder affecting carbohydrate, fat, and protein metabolism. Diabetes mellitus has been recognized as one of the most important health problems all over the world. Over the past ten years, the number of diabetics has risen by 5% per annum (p.a.) to approximately 250 million, and has been projected to become one of [Received on] 25-Nov.-2012 [Research funding] This project was supported by University Grant Commission (UGC), New Delhi, India (No. 32/135/2006 (SR)). [ Corresponding authors] Marslin Gregory & Palanivel: E-mail: [email protected], [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
the world’s major killers within the next 25 years [1]. More than 70% of the population in developing countries relies on traditional medicine for their primary health care needs [2], and traditional medicines are being explored for various activities [3-4]. Plants have always been an exemplary source of drugs, and many of the currently available drugs have been derived directly or indirectly from plants [5]. With the ever growing interest in natural medicines, many plants have been screened and reported to be useful in treating and managing diabetes [6]. Barringtonia acutangula (L.) Gaertn. (Lecythidaceae) is an important traditional medicinal plant distributed throughout India, mostly near the low lying areas, and has been used by indigenous groups for a wide range of medicinal purposes [7]. It has several vernacular names, including Indian oak, Indian putat, and hillol. In south India it is called samudra pazham. A literature survey revealed that various parts of B.
– 126 –
Marslin Gregory, et al. / Chin J Nat Med, 2014, 12(2): 126130
acutangula have been used as a folk medicine for curing various ailments including hemiplegia, pain in the joints, eye diseases, stomach disorders, anthelmintic, diarrhea, cough, dyspnea, leprosy, intermittent fever, splenic disorders, and poisoning [8-9]. Many of the traditionally ascribed activities of medicinal plants were later confirmed and accepted following intensive research. A good example is the antinociceptive, antidiarrheal, and neuropharmacological activity of B. acutangula recently reported [10]. Moreover, Babre and co-workers studied the hydroalcoholic root extract of B. acutangula on streptozotocin-induced diabetic rats. They reported that administration of the root extract of B. acutangula produces a significant reduction, followed by a steady decrease of the blood glucose level, and concluded that the antidiabetic activity may be due to the presence of saponins, tannins, phenols, and alkaloids [11]. In the present investigation, the anti-diabetic effects of the ethanolic and aqueous extracts of the leaves of B. acutangula were studied.
used to study the acute toxicity of the B. acutangula extracts. Animals were fasted overnight and orally administered with the ethanolic extract (50, 500, 2 000 and 5 1). The same conditions were maintained for toxicity study of aqueous extract. A group of animals which received an equal volume of PBS served as the control. Changes in the animal behavior were noted before and after administration for 24 h. Treated animals were further observed for up to 14 days for any signs of toxicity [14]. Evaluation of anti-diabetic activity Graphical expression of experimental work
Materials and methods Plant material The plant material of B. acutangula was collected from Pondycherry Botanical Garden, (Plot no 21), and authenticated by Mr. Sukumar, Agriculture Officer, Pondycherry Botanical Garden, India. The leaves were dried under the shade, powdered, and stored in an airtight container. For ethanol extract, dried leaf powder (250 g) was loosely packed in the thimble of Soxhlet apparatus and extracted with ethanol for 18 h at 55 ºC. The extract was air dried at 2530 ºC for 4 h. For oral administration, the extract was dissolved in 10 ml phosphate buffered saline (PBS) at different concentrations [12]. The aqueous extract was prepared according to the traditional method: the leaf powder (50 g) was dissolved in boiling distilled water (100 mL). The hot infusion was then left to reach room temperature (ca. 15 min) and filtered. The filtrate was used as such for biological tests on animals. Phytochemical screening The ethanol and aqueous extracts of the root bark of B. acutangula were evaluated for the presence of flavonoids, tannins, alkaloids, saponins, glycosides, and sterols/ triterpenes using the methods described by Brain et al [13]. Animals ¥ ® ^ ^ # ? # #_ in the Central Animal House, Raja Muthaiya Medical College, in polypropylene cages under standard conditions. During the whole experimental period, animals were fed a balanced diet (rat chow) and water ad libitum. Experiments were carried out in accordance with the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) Guidelines, and the study was approved by the Institutional Animal Ethical Committee (IAEC/CPCSEA/ 03/002/11). Acute toxicity study Five groups (n = ` # }
Induction of diabetes in rats The animals were fasted for 24 hours, and diabetes was induced by a single injection of STZ (60 mgkg1). The blood glucose level was checked before and 72 h after STZ injection to confirm the development of diabetes in animals. The # ^ `_ ^ ` # }
mL1) were selected for further studies [15]. Experimental design All of the diabetic animals were randomly divided into seven groups with six animals in each group and treated once a day for 21 days as follows: Group I (normal healthy control) received vehicle PBS. Group II served as a diabetic control and received only STZ (60 mgkg1). Groups III and IV diabetic rats received B. acutangula ethanolic extract at 250 and 500 mgkg1 bw, respectively. Groups V and VI diabetic rats received B. acutangula aqueous extract at 250 and 500 mgkg1 bw, respectively. Group VII diabetic rats received glibenclamide (GLB) at 10 mgkg1 bw. Blood sample collection After 21 days of treatment the animals were deprived of food overnight, and the blood was collected from the retro orbital plexus in tubes containing EDTA for the estimation of blood glucose level, creatinine, urea, cholesterol, HDL-C, and LDL-C levels in serum. Body weights were determined. Statistical Analysis The results are expressed as x ± s. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test. P < 0.05 was considered as significant.
Results
– 127 –
Diabetes mellitus is a chronic metabolic disorder which is
Marslin Gregory, et al. / Chin J Nat Med, 2014, 12(2): 126130
characterized by hyperglycemia resulting from deficiency in the production of insulin by the pancreas, or reduced insulin activity [16]. The etiology of type 1 diabetes is the absolute deficiency of insulin secretion, while that of type 2 is a combination of insulin resistance with inadequate compensatory insulin-secretory response [17] The anti-diabetic activities of the ethanolic and aqueous extracts of B. acutangula are reported. Acute toxicity studies revealed the non-toxic, safe nature of both the aqueous and ethanolic extracts of the leaves of B. acutangula. No mortality was observed in the extract-treated mice, and the behavior (tremors, convulsions, diarrhea, salivation, and paralysis) of the treated mice also appeared normal in most of the animals. Neither lethality nor any toxic reaction was found at any dose selected until the end of the study. Even at the dose of 5 000 1, there were no symptoms of toxicity or mortality up to 14 days. According to Lorke et al any substance that is not toxic at 5 1
is considered relatively safe [18]. The preliminary phytochemical screening of the extracts of B. acutangula revealed the presence of carbohydrates, phytosterols, terpenoids, flavonoids, glycosides, tannins, and saponins. Similar results were reported previously for the same plant [19]. Administration of a single intraperitoneal injection of 60
1 bw of freshly-prepared STZ produced diabetes in rats after a week. The effect of different doses of both aqueous and ethanolic extracts of the leaves of B. acutangula on fasting blood glucose levels in normal and STZ induced diabetic rats are summarized in Table 1. The results showed that in the diabetic control group, the blood glucose, urea, and creatinine levels were higher than those of the normal control group (Fig. 1, 2). In particular, the greatest decrease of glycemia level was observed in rats treated with 1 bw of B. acutangula, and the positive control. The low levels of blood glucose suggest that B. acutangula might be considered for further anti-diabetic studies.
Table 1 Anti-diabetic activity of B. acutangula ethanolic and aqueous extracts in STZ-induced diabetic rats ( x ± s) Treatment
Dose /(mgkg1)
Blood glucose/} _L1)
Creatinine/} _L1)
Urea/} _L1)
Normal
88.16 ± 2.2
1.13 ± 0.02
32.2 ± 1.2
Diabetic control (STZ)
60
235.13 ± 5.2
1.89 ± 0.09
69.5 ± 3.4
***
***
32.5 ± 2.3***
Control (Glibenclamide)
0.6
97.16 ± 3.5
Ethanolic extract of B.
250
121.16 ± 1.4***
1.36 ± 0.02***
34.5 ± 0.7***
acutangula
500
***
97.14 ± 1.6
***
1.27 ± 0.04
32.4 ± 1.8***
Aqueous extract of B.
250
120.52 ± 1.6***
1.38 ± 0.02***
35.3 ± 0.7***
500
***
***
31.8 ± 1.2***
acutangula
96.24 ± 1.4
1.28 ± 0.03
1.29 ± 0.06
***
P < 0.001 vs diabetic control group
Fig. 1 Effect of aqueous and ethanolic extracts on blood glucose and urea in STZ-induced diabetic rats. The results show significant reduction in blood glucose level of treated groups compared with diabetic control. When compared with the standard group, almost similar results were obtained
Total lipids, triglycerides, and total cholesterol of normal and experimental animals were examined (Table 2). There was a significant elevation in all of the biochemical parameters in the diabetic animals as compared with the non-diabetic control group, although HDL-C was exceptional (Fig. 3). The effect of administration of B. acutangula aqueous and etha# " ^ #_ 1 bw and glibenclamide decreased glycemia, total lipids, triglycerides, and total
Fig. 2 Effect of B. acutangula extract on creatinine level in STZ induced diabetic rats
cholesterol, although they were higher than normal controls. The results listed in Table 3 show the changes of body weight among the groups of diabetic and normal rats. A normal body weight gain was observed in Groups I, III, IV, V, and VI, whereas a significant decrease in body weight was observed with the diabetic control group (Fig. 4). Finally, the overall comparative study between the aqueous and ethanolic extracts of B. acutangula shows that both extracts produce the
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Table 2 Effect of extracts on lipid profile in STZ induced diabetic rats ( x ± s, n = 6) Treatment
Dose/} 1)
Normal
Diabetic control (STZ)
62.12 ± 2.5
60
Standard (Glibenclamide)
Hematological estimation Cholesterol/} _L1) Triglycerides/} _L1)
0.6
96.16 ± 2.6
102.23 ± 3.1
HDL-C/} _L1) 45.15 ± 2.0
LDL-C/} _L1) 125.16 ± 2.1
183.18 ± 4.4
28.18 ± 2.1
141.13 ± 1.9
65.33 ± 1.4***
122.26 ± 5.4***
36.25 ± 1.9***
129.16 ± 0.7***
Ethanolic extract of B.
250
66.25 ± 0.8***
118.18 ± 5.1***
39.18 ± 1.6***
121.18 ± 3.7***
acutangula
500
64.24 ±1.3***
110.21 ± 1.8***
41.12 ± 1.5***
118.12 ± 1.5***
***
***
119.12 ± 1.8***
***
117.17 ± 1.2***
Aqueous extract of B. acutangula P <0.01 vs diabetic control group
250 500
***
67.26 ± 0.5
116.23 ± 4.4
***
***
65.32 ± 1.6
109.26 ± 1.5
43.16 ± 2.0
44.21 ± 1.7
***
anti-diabetic activity in dose dependent manner, and both the extract exhibits almost similar activity. Recent hypoglycemic studies in animals revealed that various phytochemical constituents, such as polysaccharides, flavonoids, terpinoids, tannins, saponins, and alkaloids, are active compounds for hypoglycemic activity [20]. Any one of these active compounds may be well-solubilized in both extracts, and that could be the reason both the aqueous and ethanol extracts showed almost similar anti-diabetic effects. Fig. 3 Effect of extract on lipid profile in STZ induced diabetic rats. Results express the significant reduction in cholesterol, triglycerides, LDL-C, and HDL-C levels compared with diabetic control
Discussion The present study was designed to evaluate the anti- diabetic potential of B. acutangula aqueous and ethanolic extracts in STZ-induced diabetic rats. The administration of
Table 3 Effect of extracts on body weight in STZ induced diabetic rats ( x ± s, n = 6) Treatment
Dose/} 1)
Normal Diabetic control (STZ) Standard (Glibenclamide)
60 0.6 250 500 250 500
Ethanolic extract of B. acutangula Aqueous extract of B. acutangula ***
Body weight/g Initial day 210.24 ± 2.6 215.23 ± 3.2 212.12 ± 3.4 208.16 ± 2.3 214.14 ± 3.1 210.16 ± 2.9 215.1.7 ± 3.1
Day 21 228.12 ± 3.2 202.14 ± 3.6 231.23 ± 2.8*** 224.25 ± 3.1*** 229.27 ± 2.5*** 223.21 ± 2.3*** 230.16 ± 2.6***
P <0.01 vs diabetic control group
Fig. 4 Effects of extract on body weight in STZ-induced diabetic rats before and after treatment. The results show the reduction in the body weight of the diabetic animals
streptozotocin in animals can induce necrosis of beta-cells of the islets of Langerhans, thus causing hypoinsulinemia and hyperglycemia [21]. In this study, it was found that 21-day treatment with different concentrations of B. acutangula aqueous and ethanolic extracts significantly reduced the fasting blood glucose level in diabetic rats. The results also showed that the blood glucose level was lowered in the drug-treated groups. Diabetes is associated with profound alterations in the plasma lipid and lipoprotein profiles. In uncontrolled type 2 diabetes mellitus, there is an increase in cholesterol, triglycerides, urea, creatinine, and LDL-C and cholesterol with decrease in HDL-C which contributes to the coronary artery disease [22]. Treatment with both aqueous and ethanol extracts resulted in the elevation of HDL-Cholesterol levels, when compared to the diabetic control group. The maximum in-
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crease in HDL-Cholesterol value (44.21) was obtained in the animals treated with 500 mgkg1 aqueous leaf extract. This value is doubled when compared with the diabetic control and is more similar to the normal animals. The increased HDL-cholesterol and decrease in LDL-C levels may be beneficial due to the negative correlation between HDL-C levels and cardiovascular diseases. This could be due to the presence of a hypolipidemic agent more in the aqueous leaf extract B. acutangula.
[8]
[9]
[10]
[11]
Conclusion From this study, it is clear that B. acutangula leaf aqueous and ethanolic extracts have significant anti-diabetic activity in animal models. It is suggested that the anti-diabetic activity is probably due to the presence of flavonoids. The extract is non-toxic even at relatively high concentrations (5 000 mgkg1 b.w). Similar results were obtained for animals treated with the aqueous extract. Hence it is proposed that consumption of B. acutangula leaf in some form, like a tea, would control the blood glucose level in diabetic patients. Further studies are being carried out to explore the mechanism underlying the biological activities of the compounds present in the extract.
[12]
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Cite this article as: Marslin Gregory, Vinoth Kumar Megraj Khandelwal, Revina Ann Mary, V. K. Kalaichelvan, V. Palanivel. Barringtonia acutangula improves the biochemical parameters in diabetic rats [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 126-130
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