http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.970286
ORIGINAL ARTICLE
The regulatory effects of Cissus quadrangularis on some enzymes involved in carbohydrate metabolism in streptozotocin-induced diabetic rats R. K. Lekshmi, M. S. Sreekutty, and S. Mini
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Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum, Kerala, India
Abstract
Keywords
Context: Diabetes mellitus (DM) is probably the single most important metabolic disease and is widely recognized as one of the leading causes of death and disability. Cissus quadrangularis Linn. (Vitaceae) is a medicinal food and is reported to possess hypoglycemic activity. Objective: The present study evaluates the effect of ethyl acetate fraction of C. quadrangularis stem (CQSF) on carbohydrate metabolism in hepatic tissues of experimental diabetic rats. The phytochemical compounds present in the CQSF extract were identified by gas chromatography–mass spectrometry (GC–MS) analysis. Materials and methods: Diabetic animals were treated with CQSF (50, 100, and 200 mg/kg body weight) for 45 d. Several indices such as blood glucose, glycated hemoglobin (HbA1c), insulin, liver function tests, hepatic glycogen content, and the activities of carbohydrate-metabolizing enzymes were assayed after 45 d of extract treatment. Results: A pronounced effect was observed with extract doses 100 and 200 mg/kg body weight. CQSF at a dose of 100 mg/kg body weight significantly decreased the altered levels of blood glucose by about 56%. CQSF also modulated the activities of carbohydrate-metabolizing enzymes by significantly increasing the activity of hexokinase (1.9-fold) and pyruvate kinase (2.2-fold) and significantly reducing the activity of glucose-6-phosphatase (41.23%), fructose1,6-diphosphatase (29.43%), and glycogen phosphorylase (35.07%). GC–MS analysis revealed the presence of 10 chemical constituents, and N-methyl-1-adamantane acetamide was found to be the prevailing compound in the extract. Discussion and conclusion: The current study suggests the antidiabetic potential of CQSF, mediated through the regulation of carbohydrate metabolic enzyme activities.
Diabetes mellitus, ethyl acetate fraction, GC–MS analysis, hypoglycemic activity
Introduction Diabetes mellitus (DM) is a group of syndromes characterized by hyperglycemia, altered metabolism of lipids, carbohydrates, and proteins (Davis, 2006). It is regarded as a noncurable but a controllable disease. DM has a complex etiology with interacting genetic factors and lifestyle factors including adiposity, physical activity, and diet (Brito et al., 2009). Apart from the currently available therapeutic options for diabetes, which have limitations of their own, many herbal medicines have been recommended for the treatment of diabetes (Mukherjee et al., 2006). Plant-derived functional foods are in great demand in developing countries as an alternative approach to treat diabetes (Fadzelly et al., 2006). Cissus quadrangularis Linn. (Vitaceae) (CQ) is a perennial tendril climber with a wide range of applications as a food and
Correspondence: Dr. S. Mini, Associate Professor, Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, Kerala, India. Tel: +9495276631. E-mail: minisaraswathy@ gmail.com
History Received 9 November 2013 Revised 20 August 2014 Accepted 22 September 2014 Published online 10 April 2015
traditional medicine. The stems and leaves of CQ have been consumed as vegetables and studies indicated that it possesses hypoglycemic and free radical scavenging activities (Chaudhari et al., 2013; Jainu et al., 2006). Several studies suggest that CQ extracts are beneficial in reducing body weight as well as lowering blood glucose, cholesterol, and triglyceride levels to the normal range (Stohs & Ray, 2013). Cissus quadrangularis is used by common folk in India to hasten the fracture healing process (Deka et al., 1994). Different parts of the plant have been used orally or topically as a medicine for skin infections, constipation, piles, anemia, asthma, irregular menstruation, burns, and wounds (Kirtikar et al., 2000). A phytochemical review of CQ shows the presence of phytosterols, flavonoids, tannins, and triterpenoids and these active components may be responsible for its pharmacological activities (Mehta et al., 2001). Previous studies in our lab revealed that the ethyl acetate fraction from the methanol extract of the CQ stem possesses in vitro antioxidant and anti-glycation activities (Lekshmi & Mini, 2013). However, the regulatory mechanism contributing to the antidiabetic effect of CQ was not elucidated well and
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reports were scanty. Therefore, the present study was designed to investigate the potential role of C. quadrangularis on glucose utilization pathways in experimental DM.
Materials and methods Chemicals Biochemicals were purchased from Sigma Aldrich (St. Louis, MO), Merck Chemical Company (Darmstadt, Germany), and Sisco Research Laboratories (Mumbai, India).
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Plant material Cissus quadrangularis stems were collected from the local areas of Thiruvananthapuram, in March–April 2012 and identified by Dr. Valsala Devi, Department of Botany, University of Kerala (Voucher no: KUBH 5805). The stems of CQ were shade-dried, powdered, and extracted with methanol using a Soxhlet extractor. The crude extract was filtered through Whatman No. 1 filter paper and concentrated in vacuum at 40 C in a rotary evaporator (Heidolph, Schwabach, Germany). The crude methanol extract (ME) was suspended in water and defatted with petroleum ether (PE) and then the remaining aqueous phase was partitioned with ethyl acetate (EA) to yield an ethyl acetate fraction (CQSF) which was concentrated and used for the experimental study. Animals Male Sprague–Dawley rats (200–220 g) bred in our department animal house were used for the study. Animals were housed in polypropylene cages and maintained under standard conditions [12 h light and 12 h dark cycle (25 ± 10 C)]. All the animal care was taken as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), and the experimental protocol was approved by Institutional Animals Ethics Committee (IAEC). Induction of experimental diabetes Rats were rendered diabetic by a single intraperitoneal injection of freshly prepared streptozotocin (STZ) at a dose of 40 mg/kg body weight in 0.1 M citrate buffer (pH 4.5) (Ramesh & Pugalendi, 2006). Diabetes was identified in rats by moderate polydypsia and marked polyuria. After 48 h of STZ induction, blood glucose levels were estimated and rats with a blood glucose ranging between 200 and 400 mg/dL were considered diabetic and used for the experiments. Experimental design Rats were divided into five groups comprising six rats in each group. Group I was normal control rats; group II consisted of STZ-induced diabetic rats; groups III, IV, and V consisted of STZ-induced diabetic rats treated with CQSF at doses 50, 100, and 200 mg/kg body weight, respectively. The daily intragastric treatments using fresh suspension of CQSF were continued for 45 d. After the experimental period, animals were fasted overnight and sacrificed under chloroform anesthesia. Blood and liver were collected immediately for various biochemical estimations.
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Biochemical parameters The fasting serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT) were measured by using commercially available kits (Agappe Diagnostics, Ernakulam, India). Glycated hemoglobin (HbA1c) was estimated by kit based on the ion exchange method (Nathan et al., 1984). Insulin was estimated by the ELISA (rat insulin ELISA kit) method (Grassi & Pradelles, 1991). Activity of glycolytic enzymes was assayed: hexokinase was estimated by the method of Crane and Sols (1953); pyruvate kinase was estimated by the method of Bucher and Pfleiderer (1955). Hepatic glycogen content was estimated by the method of Carroll et al. (1956). Gluconeogenic enzyme activities in the liver were assayed using the following procedures: glucose-6-phosphatase was estimated by the method described by Koide and Oda (1959), fructose1,6-diphosphatase was estimated by the method of Pontremoli (1966), and the activity of glycogen phosphorylase was assayed by the procedure described by Singh et al. (1961). Histopathological analysis Histology of liver was studied using hematoxylin and eosin (H & E) staining. The liver tissues were collected in a 10% formalin solution and immediately processed by the paraffin technique. Thin sections (5 mm) were cut and stained with H & E and photographed under a light microscope for observation of structural abnormality. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the extract was performed using Agilent CP 3000 GC/Saturn 2200 MS (Agilent, Palo Alto, CA) equipped with ECD, PFPD, and MS detectors. For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant flow rate of 1 mL/min. The inlet temperature was set at 250 C. The oven temperature was maintained at 100 C for 1.5 min and gradually increased up to 270 C at the rate of 5 C per min. The diluted samples of 1 mL were injected and the scan range was 40–600. Statistical analysis All the results were expressed as mean ± SD for six animals in each group and experiments were performed in triplicate. The statistical analysis was carried out by one-way analysis of variance (ANOVA) using the SPSS (version 17) statistical analysis program (SPSS Inc., Chicago, IL). Statistical significance was considered at p50.05.
Results Serum glucose, HbA1c, and insulin There was a significant elevation in fasting blood glucose in the diabetic group (344.18 ± 33.02 mg/dL) when compared with other groups. Oral administrations of CQSF significantly (p50.05) decreased blood glucose levels of diabetic rats in groups III (213.53 ± 20.49 mg/dL), IV (152.1 ± 14.59 mg/dL),
Antidiabetic activity of Cissus quadrangularis stem
DOI: 10.3109/13880209.2014.970286
and V (156 ± 14.97 mg/dL) (Figure 1). The data presented in Figure 2 indicated the effect of CQSF extract on HbA1c. There was a significant elevation in HbA1c levels in the diabetic group (10.92 ± 1.05%) when compared with other groups. The extract treatment significantly (p50.05) reduced HbA1c levels by about 35%. The plasma insulin level decreased significantly in the diabetic group (0.68 ± 0.07 ng/ mL) when compared with other groups and it was improved by CQSF administration (Figure 3). However, there were no statistically significant differences among the fasting blood glucose, HbA1c, and plasma insulin levels of diabetic rats of the groups IV and V, which indicated that 100 mg/kg body weight is the optimum dose for obtaining good glycemic control.
Liver toxicity markers were assayed to assess hepatic injury. The activities of ALT, AST, ALP, and GGT were significantly
Serum Glucose 400
Before treatment Aer treatment
a
350
mg/dL
300
b
250 200
c
c
150 100 50 0 I
II
III
IV
V
Groups Figure 1. Fasting blood glucose levels. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,c statistically significant as compared with the diabetic group. Similar alphabets indicate no significant difference between groups.
altered in the DM group, indicating damage to hepatocytes. Treatment with CQSF significantly (p50.05) lowered these enzyme activities (Table 1). There was no statistically significant difference in most of the liver toxicity markers between the groups treated with the extract at doses 50, 100, and 200 mg/kg body weight, which indicated that the hepatocellular damage is notably prevented by treatment with even the lowest dose of CQSF. Hepatic glycogen content and carbohydrate-metabolizing enzymes Hepatic glycogen content in diabetic rats (2.07 ± 0.19 mg/g wet tissue) was found to be significantly reduced (p50.05) compared with the normal control (6.88 ± 0.63 mg/g wet tissue). Treatment with CQSF enhanced the glycogen storage efficiency of liver of diabetic rats compared with diabetic control animals (Figure 4). STZ administration significantly (p50.05) elevated the activity of glycogen phosphorylase in diabetic control rats (83.46 ± 8.01 mmol of Pi liberated/min/mg protein) as compared with the normal animals (52.07 ± 5 mmol of Pi liberated/min/mg protein). Altered activity of the enzyme is reverted to near normal levels by extract administration (61.26 ± 5.88 mmol of Pi liberated/min/mg protein) in diabetic rats (Table 2). In the case of glycogen phosphorylase activity, there was no statistical significance between the extract-treated groups (III, IV, and V). On one hand, the activities of hexokinase and pyruvate kinase were significantly diminished (p50.05) in STZ-induced diabetic rats as compared with normal control animals. However, CQSF treatment significantly increased (p50.05) the activities of hexokinase (1.9-fold) and pyruvate kinase (2.2-fold) in liver tissues of diabetic rats (Table 2). On the other hand, there was an increase in the activities of gluconeogenic enzymes like glucose-6-phosphatase and fructose-1,6-diphosphatase in diabetic rats as compared with the normal rats. Supplementation of CQSF showed restoration of glucose-6-phosphatase (41.23%) and fructose-1, 6-diphosphatase (29.43%) to the basal levels as compared with control rats (Table 2). In most of the carbohydratemetabolizing enzyme activities, there was no statistical
HbA1c Plasma Insulin Levels
a
12
b
10
c
8
c
6 4
ng/mL
% glycated hemoglobin
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Toxicity markers
3
5 4.5 4 3.5 3
c
2.5 2 1.5
a
1 0.5 0
2 0 I
II
III
IV
V
Groups
Figure 2. Glycated hemoglobin levels. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,c statistically significant as compared with the diabetic group. Similar alphabets indicate no significant difference between groups.
I
II
c
b
III
IV
V
Groups
Figure 3. Plasma insulin levels. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with the diabetic group. Similar alphabets indicate no significant difference between groups.
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Table 1. Activities of toxicity markers in liver (IU/L). Parameters
Group I
ALP GGT ALT AST
Group II
114.32 ± 10.96 18.78 ± 1.80 40.83 ± 3.93 66.36 ± 6.36
Group III
170.94 ± 16.41 35.41 ± 3.40a 65.4 ± 5.71a 95.28 ± 9.15a
a
Group IV b
145.52 ± 13.96 27.41 ± 2.64b 56.93 ± 5.46b 79.97 ± 7.67b
Group V c
128.46 ± 12.33 24.48 ± 2.36b 51.04 ± 4.90b 73.17 ± 7.02b
127.48 ± 12.24c 25.57 ± 2.47b 51.83 ± 4.98b 76.57 ± 7.35b
Values are expressed as mean ± SD of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with the diabetic group. Similar alphabets in the same row indicate no significant difference between groups.
Hepatic Glycogen Content
mg/g wet tissue
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8
6
c 4
c
b a
2
0 I
II
III
IV
V
Groups Figure 4. Hepatic glycogen content. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with diabetic group. Similar alphabets indicate no significant difference between groups.
significance between the groups IV and V, indicating that 100 mg/kg body weight is the optimum dose for better hypoglycemic activity. Histopathological analysis of liver The histopathological analysis of liver of the normal control group showed normal architecture (Figure 5A), while the diabetic rats (Figure 5B) showed marked hepatocellular damage and these changes were restored to near normal by the treatment with CQSF (Figure 5C–E). GC–MS analysis Ten compounds were identified in the CQSF extract by GC-MS analysis. To the best of our knowledge, the identified compounds have not been reported previously in this plant. The active principles with their retention time (RT), molecular formula, molecular weight (MW), and peak area % are presented in Table 3.
Discussion The prevention and care of diabetes have tended to diet-based intervention considering safety and economy (Jilin et al., 2011). Cissus quadrangularis is a medicinal food used by the folk healers as a hypoglycemic agent. Hence the present study aimed to evaluate the anti-diabetic potential of ethyl acetate fraction of C. quadrangularis stem on STZ-induced DM. In our study, STZ was selected for induction of diabetes in
rats. STZ is well known for its selective pancreatic b-cell cytotoxicity and has been extensively used to induce DM in animals (Raju & Balaraman, 2008) and it is less toxic and allows a consistent maintenance of DM. The experimentally induced diabetic rats showed severe hyperglycemia interrelated with a decrease in endogenous insulin secretion and release. Rats treated with CQSF extract showed a significant decrease in the level of blood glucose and an increase in the level of serum insulin. These results indicated that CQSF produced anti-hyperglycemic activity and the glucose-lowering activity of CQSF may be attributed to pancreatic-enhancement of insulin secretion. Increment in plasma insulin by CQSF is probably due to the presence of adamantine derivatives which possess insulin secretagogue activity. It may also be due to the regeneration of pancreatic b cells which are destroyed by STZ (Eidi et al., 2006). The level of HbAlc is monitored as a reliable index of glycemic control in diabetes. Elevated HbAlc was observed in the diabetic group which indicates poor glycemic control. Uncontrolled and long-term diabetes was often accompanied with high glycosylated hemoglobin which is responsible for the development of late diabetic complications namely vascular dysfunction, neuropathy, and diabetic nephropathy. In case of diabetic rats treated with the CQSF extract, the HbAlc levels were brought down from elevated level to almost normal. STZ administration was associated with hepatocellular damage. The increased activities of marker enzymes like AST, ALT, ALP, and GGT in serum are suggestive of liver injury, which might be mainly due to the leakage of these enzymes from the liver cytosol into the blood stream (Kasetti et al., 2010). Treatment with CQSF notably prevented the elevation of these enzymes to an extent. Thus CQSF prevents liver cell damage and preserves cell integrity possibly leading to the survival of the functionally active cells, as evidenced by histopathological analysis. According to previous reports, DM was presented with alterations in glucose homeostasis that contribute to persistent hyperglycemia and liver plays a major role in the regulation of glucose metabolism (Ohaeri, 2001). The activity of enzymes like hexokinase, pyruvate kinase, glucose-6-phosphatase, and fructose-1,6-diphosphatase was markedly altered, resulting in hyperglycemia, which leads to the pathogenesis of diabetic complications (Bhavapriya & Govidasamy, 2000). The altered the activity of hexokinase and pyruvate kinase, key enzymes in the catabolism of glucose, diminishing the metabolism of glucose and ATP production in diabetic conditions. The reduction in the activities of these enzymes in the liver tissues of diabetic
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Table 2. Activities of carbohydrate metabolizing enzymes. Parameters
Group I d
Hexokinase Pyruvatekinasee Glucose-6-phosphatasef Fructose 1,6-diphosphatasef Glycogen phosphorylaseg
16.56 ± 1.59 33.09 ± 3.18 19.61 ± 1.70 69.70 ± 6.36 52.07 ± 5.00
Group II
Group III a
7.95 ± 0.77 12.57 ± 1.14a 66.36 ± 6.72a 133.20 ± 13.47a 83.46 ± 8.01a
Group IV b
10.26 ± 0.99 25.40 ± 2.43b 44.93 ± 4.30b 118.40 ± 11.97b 62.79 ± 6.02b
Group V c
15.26 ± 1.53 29.08 ± 2.81c 39.86 ± 4.04b 94.12 ± 9.52c 61.26 ± 5.88b
14.85 ± 1.29c 27.80 ± 2.54b,c 39.81 ± 4.01b 101.50 ± 10.85c 61.80 ± 5.93b
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Values are expressed as mean ± SD of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with the diabetic group. Similar alphabets in the same row indicate no significant difference between groups. dmg glucose phosphorylated/min/mg protein; eunits/mg protein; fmmol of Pi liberated/h/mg protein, gmmol of Pi liberated/min/mg protein.
Figure 5. Histopathology of liver. (A) Normal control – normal hepatocytes showing normal architecture. (B) Diabetic control – extensive hepatocellular damage in the form of inflammation, sinusoidal dilation, fatty changes, and extensive vacuolization with disappearance of nuclei. (C)–(E) Diabetic + CQSF shows only mild inflammation, and there is restoration of normal tissue morphology.
rats is an indication of reduced glycolysis and amplified gluconeogenesis signifying that these two pathways are distorted in diabetes. In agreement with the above reports, the activities of hexokinase and pyruvate kinase were significantly decreased in the STZ-induced DM group. Administration of CQSF to diabetic rats significantly elevated these enzyme activities in liver. The activities of regulatory enzymes in gluconeogenesis, like glucose-6-phosphatase and fructose-1,6-diphosphatase, are elevated in DM (Grover et al., 2000) and increased activities of these enzymes in STZ-induced diabetic rats may be due to insulin insufficiency (Baquer et al., 1998). Glucose-6-phosphatase and fructose-1,6-diphosphatase are dephosphorylating enzymes which impair hepatic glucose utilization. Our results showed that the activities of glucose-6-phosphatase and fructose-1,6-diphosphatase were significantly decreased by the administration of CQSF. Glycogen is the primary intracellular storage form of glucose and its quantity in various tissues is a direct manifestation of insulin activity as insulin supports intracellular glycogen deposition (Pederson et al., 2005). The reduced glycogen store in diabetic rats has been attributed to the loss of glycogen synthase-activating system and/or the increased
activity of glycogen phosphorylase (Golden et al., 1979). In the present study, there was a decrease in the hepatic glycogen content of diabetic rats which suggests the increased glucose output during insulin deficiency. Here diabetic animals showed increased glycogen phosphorylase activity when compared with normal control animals. Treatment with CQSF restored the levels of glycogen, probably by means of decreasing the activity of glycogen phosphorylase. Histological studies were carried out over the liver for the five groups. STZ-induced diabetic rats showed marked hepatocellular damage in the form of inflammation, sinusoidal dilation, fatty changes, and extensive vacuolization with the disappearance of nuclei. Administration of CQSF significantly improved the histological architecture of liver in diabetic rats. Consonant with the histological findings, serum ALT, AST, ALP, and GGT levels were substantially higher in DM rats, but it was restored to near normal levels after the treatment with CQSF. Thus the histopathological analysis of liver showed the protective effect of CQSF in experimental diabetes. The GC–MS analysis of CQSF showed the presence of 10 constituents which were characterized and identified for the first time in this work. The identified compounds
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Table 3. Chemical composition of CQSF extract. RT
Name
9.296 11.538 14.455 17.215 26.201 28.378 38.107 41.651 46.997 52.207
Ethanone, 1-(2-hydroxy-5-methylphenyl)Niacinamide Phenol, 2,4-bis(1,1-dimethylethyl)Diethyl 2,5-pyridinedicarboxylate Cyclohexene, 1,5,5-trimethyl-6-methylene N-Methyl-1-adamantaneacetamide 13-Docosen-1-ol, (Z)N-Nitrosohemanthidine lactone Fluorometholone 5-Benzofuranpropanol, 2-(3,4-dimethoxyph
include phenolic derivatives, fatty alcohols, acetophenones, carbonyl compounds, and aliphatic hydrocarbons and were having medicinal properties such as antioxidant (Yoon, 2006), anti-inflammatory (Constantino et al., 1993), anticancer (Piedrafita & Ortiz, 2006), and hypoglycemic activities (Singab et al., 2005). One of the major compounds present in CQSF was N-methyl-1-adamantane acetamide. Adamantane derivatives are reported to be insulin secretagogues in nature (Garrino & Henquin, 1987). The antidiabetic activity of CQSF may be attributed to the cumulative effect of these major compounds present in it.
Conclusion Our study clearly indicated that CQSF has potential antihyperglycemic activity in STZ-induced diabetic rats. Pronounced activity was observed at the doses of about 100 and 200 mg/kg. The ameliorative effect of CQSF against severe hyperglycemia may be due to the enhancement in the peripheral utilization of glucose, correcting the impaired liver glycolysis and limiting gluconeogenic formation. The major chemical compounds revealed by GC–MS analysis are believed to be responsible for the anti-hyperglycemic activity. Thus, the dietary supplementation of C. quadrangularis stem may be helpful for the management of DM and prevention of diabetic complications.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Molecular formula
Molecular weight
Peak area %
C9H10O2 C6H6N2O C14H22O C11H13NO4 C10H16 C13H21NO C22H44O C17H16N2O6 C22H29FO4 C13H17NO2
150.18 122.12 206.32 223.23 136.23 207.31 324.58 344.32 376.46 219.28
0.14 0.86 22.82 3.35 2.2 37.57 1.31 0.5 0.3 13.14
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Raju K, Balaraman R. (2008). Antidiabetic mechanisms of saponins of Momordica cymbalaria. Pharmacogn Mag 4:197–206. Ramesh B, Pugalendi KV. (2006). Antihyperglycemic effect of umbelliferone in streptozotocin-diabetic rats. J Med Food 9:562–6. Singab AN, El-Beshbishy HA, Yonekawa M, et al. (2005). Hypoglycaemic effect of Egyptian Morus alba root bark extract: Effect on diabetes and lipid peroxidation of streptozotocin-induced diabetic rats. J Ethanopharmacol 100:333–8. Singh VN, Venkatasubramanian TA, Viswanathan R. (1961). The glycolytic enzymes of guinea pig lung in experimental bagassosis. Biochem J 78:728–32. Stohs SJ, Ray SD. (2013). A review and evaluation of the efficacy and safety of Cissus quadrangularis extracts. Phytother Res 27: 1107–14. Yoon MA, Jeong TS, Park DS, et al. (2006). Antioxidant effects of quinoline alkaloids and 2,4-di-tert-butylphenol isolated from Scolopendra subspinipes. Biol Pharm Bull 29:735–9.
ORIGINAL ARTICLE
The regulatory effects of Cissus quadrangularis on some enzymes involved in carbohydrate metabolism in streptozotocin-induced diabetic rats R. K. Lekshmi, M. S. Sreekutty, and S. Mini
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Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum, Kerala, India
Abstract
Keywords
Context: Diabetes mellitus (DM) is probably the single most important metabolic disease and is widely recognized as one of the leading causes of death and disability. Cissus quadrangularis Linn. (Vitaceae) is a medicinal food and is reported to possess hypoglycemic activity. Objective: The present study evaluates the effect of ethyl acetate fraction of C. quadrangularis stem (CQSF) on carbohydrate metabolism in hepatic tissues of experimental diabetic rats. The phytochemical compounds present in the CQSF extract were identified by gas chromatography–mass spectrometry (GC–MS) analysis. Materials and methods: Diabetic animals were treated with CQSF (50, 100, and 200 mg/kg body weight) for 45 d. Several indices such as blood glucose, glycated hemoglobin (HbA1c), insulin, liver function tests, hepatic glycogen content, and the activities of carbohydrate-metabolizing enzymes were assayed after 45 d of extract treatment. Results: A pronounced effect was observed with extract doses 100 and 200 mg/kg body weight. CQSF at a dose of 100 mg/kg body weight significantly decreased the altered levels of blood glucose by about 56%. CQSF also modulated the activities of carbohydrate-metabolizing enzymes by significantly increasing the activity of hexokinase (1.9-fold) and pyruvate kinase (2.2-fold) and significantly reducing the activity of glucose-6-phosphatase (41.23%), fructose1,6-diphosphatase (29.43%), and glycogen phosphorylase (35.07%). GC–MS analysis revealed the presence of 10 chemical constituents, and N-methyl-1-adamantane acetamide was found to be the prevailing compound in the extract. Discussion and conclusion: The current study suggests the antidiabetic potential of CQSF, mediated through the regulation of carbohydrate metabolic enzyme activities.
Diabetes mellitus, ethyl acetate fraction, GC–MS analysis, hypoglycemic activity
Introduction Diabetes mellitus (DM) is a group of syndromes characterized by hyperglycemia, altered metabolism of lipids, carbohydrates, and proteins (Davis, 2006). It is regarded as a noncurable but a controllable disease. DM has a complex etiology with interacting genetic factors and lifestyle factors including adiposity, physical activity, and diet (Brito et al., 2009). Apart from the currently available therapeutic options for diabetes, which have limitations of their own, many herbal medicines have been recommended for the treatment of diabetes (Mukherjee et al., 2006). Plant-derived functional foods are in great demand in developing countries as an alternative approach to treat diabetes (Fadzelly et al., 2006). Cissus quadrangularis Linn. (Vitaceae) (CQ) is a perennial tendril climber with a wide range of applications as a food and
Correspondence: Dr. S. Mini, Associate Professor, Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, Kerala, India. Tel: +9495276631. E-mail: minisaraswathy@ gmail.com
History Received 9 November 2013 Revised 20 August 2014 Accepted 22 September 2014 Published online 10 April 2015
traditional medicine. The stems and leaves of CQ have been consumed as vegetables and studies indicated that it possesses hypoglycemic and free radical scavenging activities (Chaudhari et al., 2013; Jainu et al., 2006). Several studies suggest that CQ extracts are beneficial in reducing body weight as well as lowering blood glucose, cholesterol, and triglyceride levels to the normal range (Stohs & Ray, 2013). Cissus quadrangularis is used by common folk in India to hasten the fracture healing process (Deka et al., 1994). Different parts of the plant have been used orally or topically as a medicine for skin infections, constipation, piles, anemia, asthma, irregular menstruation, burns, and wounds (Kirtikar et al., 2000). A phytochemical review of CQ shows the presence of phytosterols, flavonoids, tannins, and triterpenoids and these active components may be responsible for its pharmacological activities (Mehta et al., 2001). Previous studies in our lab revealed that the ethyl acetate fraction from the methanol extract of the CQ stem possesses in vitro antioxidant and anti-glycation activities (Lekshmi & Mini, 2013). However, the regulatory mechanism contributing to the antidiabetic effect of CQ was not elucidated well and
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reports were scanty. Therefore, the present study was designed to investigate the potential role of C. quadrangularis on glucose utilization pathways in experimental DM.
Materials and methods Chemicals Biochemicals were purchased from Sigma Aldrich (St. Louis, MO), Merck Chemical Company (Darmstadt, Germany), and Sisco Research Laboratories (Mumbai, India).
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Plant material Cissus quadrangularis stems were collected from the local areas of Thiruvananthapuram, in March–April 2012 and identified by Dr. Valsala Devi, Department of Botany, University of Kerala (Voucher no: KUBH 5805). The stems of CQ were shade-dried, powdered, and extracted with methanol using a Soxhlet extractor. The crude extract was filtered through Whatman No. 1 filter paper and concentrated in vacuum at 40 C in a rotary evaporator (Heidolph, Schwabach, Germany). The crude methanol extract (ME) was suspended in water and defatted with petroleum ether (PE) and then the remaining aqueous phase was partitioned with ethyl acetate (EA) to yield an ethyl acetate fraction (CQSF) which was concentrated and used for the experimental study. Animals Male Sprague–Dawley rats (200–220 g) bred in our department animal house were used for the study. Animals were housed in polypropylene cages and maintained under standard conditions [12 h light and 12 h dark cycle (25 ± 10 C)]. All the animal care was taken as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), and the experimental protocol was approved by Institutional Animals Ethics Committee (IAEC). Induction of experimental diabetes Rats were rendered diabetic by a single intraperitoneal injection of freshly prepared streptozotocin (STZ) at a dose of 40 mg/kg body weight in 0.1 M citrate buffer (pH 4.5) (Ramesh & Pugalendi, 2006). Diabetes was identified in rats by moderate polydypsia and marked polyuria. After 48 h of STZ induction, blood glucose levels were estimated and rats with a blood glucose ranging between 200 and 400 mg/dL were considered diabetic and used for the experiments. Experimental design Rats were divided into five groups comprising six rats in each group. Group I was normal control rats; group II consisted of STZ-induced diabetic rats; groups III, IV, and V consisted of STZ-induced diabetic rats treated with CQSF at doses 50, 100, and 200 mg/kg body weight, respectively. The daily intragastric treatments using fresh suspension of CQSF were continued for 45 d. After the experimental period, animals were fasted overnight and sacrificed under chloroform anesthesia. Blood and liver were collected immediately for various biochemical estimations.
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Biochemical parameters The fasting serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT) were measured by using commercially available kits (Agappe Diagnostics, Ernakulam, India). Glycated hemoglobin (HbA1c) was estimated by kit based on the ion exchange method (Nathan et al., 1984). Insulin was estimated by the ELISA (rat insulin ELISA kit) method (Grassi & Pradelles, 1991). Activity of glycolytic enzymes was assayed: hexokinase was estimated by the method of Crane and Sols (1953); pyruvate kinase was estimated by the method of Bucher and Pfleiderer (1955). Hepatic glycogen content was estimated by the method of Carroll et al. (1956). Gluconeogenic enzyme activities in the liver were assayed using the following procedures: glucose-6-phosphatase was estimated by the method described by Koide and Oda (1959), fructose1,6-diphosphatase was estimated by the method of Pontremoli (1966), and the activity of glycogen phosphorylase was assayed by the procedure described by Singh et al. (1961). Histopathological analysis Histology of liver was studied using hematoxylin and eosin (H & E) staining. The liver tissues were collected in a 10% formalin solution and immediately processed by the paraffin technique. Thin sections (5 mm) were cut and stained with H & E and photographed under a light microscope for observation of structural abnormality. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the extract was performed using Agilent CP 3000 GC/Saturn 2200 MS (Agilent, Palo Alto, CA) equipped with ECD, PFPD, and MS detectors. For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant flow rate of 1 mL/min. The inlet temperature was set at 250 C. The oven temperature was maintained at 100 C for 1.5 min and gradually increased up to 270 C at the rate of 5 C per min. The diluted samples of 1 mL were injected and the scan range was 40–600. Statistical analysis All the results were expressed as mean ± SD for six animals in each group and experiments were performed in triplicate. The statistical analysis was carried out by one-way analysis of variance (ANOVA) using the SPSS (version 17) statistical analysis program (SPSS Inc., Chicago, IL). Statistical significance was considered at p50.05.
Results Serum glucose, HbA1c, and insulin There was a significant elevation in fasting blood glucose in the diabetic group (344.18 ± 33.02 mg/dL) when compared with other groups. Oral administrations of CQSF significantly (p50.05) decreased blood glucose levels of diabetic rats in groups III (213.53 ± 20.49 mg/dL), IV (152.1 ± 14.59 mg/dL),
Antidiabetic activity of Cissus quadrangularis stem
DOI: 10.3109/13880209.2014.970286
and V (156 ± 14.97 mg/dL) (Figure 1). The data presented in Figure 2 indicated the effect of CQSF extract on HbA1c. There was a significant elevation in HbA1c levels in the diabetic group (10.92 ± 1.05%) when compared with other groups. The extract treatment significantly (p50.05) reduced HbA1c levels by about 35%. The plasma insulin level decreased significantly in the diabetic group (0.68 ± 0.07 ng/ mL) when compared with other groups and it was improved by CQSF administration (Figure 3). However, there were no statistically significant differences among the fasting blood glucose, HbA1c, and plasma insulin levels of diabetic rats of the groups IV and V, which indicated that 100 mg/kg body weight is the optimum dose for obtaining good glycemic control.
Liver toxicity markers were assayed to assess hepatic injury. The activities of ALT, AST, ALP, and GGT were significantly
Serum Glucose 400
Before treatment Aer treatment
a
350
mg/dL
300
b
250 200
c
c
150 100 50 0 I
II
III
IV
V
Groups Figure 1. Fasting blood glucose levels. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,c statistically significant as compared with the diabetic group. Similar alphabets indicate no significant difference between groups.
altered in the DM group, indicating damage to hepatocytes. Treatment with CQSF significantly (p50.05) lowered these enzyme activities (Table 1). There was no statistically significant difference in most of the liver toxicity markers between the groups treated with the extract at doses 50, 100, and 200 mg/kg body weight, which indicated that the hepatocellular damage is notably prevented by treatment with even the lowest dose of CQSF. Hepatic glycogen content and carbohydrate-metabolizing enzymes Hepatic glycogen content in diabetic rats (2.07 ± 0.19 mg/g wet tissue) was found to be significantly reduced (p50.05) compared with the normal control (6.88 ± 0.63 mg/g wet tissue). Treatment with CQSF enhanced the glycogen storage efficiency of liver of diabetic rats compared with diabetic control animals (Figure 4). STZ administration significantly (p50.05) elevated the activity of glycogen phosphorylase in diabetic control rats (83.46 ± 8.01 mmol of Pi liberated/min/mg protein) as compared with the normal animals (52.07 ± 5 mmol of Pi liberated/min/mg protein). Altered activity of the enzyme is reverted to near normal levels by extract administration (61.26 ± 5.88 mmol of Pi liberated/min/mg protein) in diabetic rats (Table 2). In the case of glycogen phosphorylase activity, there was no statistical significance between the extract-treated groups (III, IV, and V). On one hand, the activities of hexokinase and pyruvate kinase were significantly diminished (p50.05) in STZ-induced diabetic rats as compared with normal control animals. However, CQSF treatment significantly increased (p50.05) the activities of hexokinase (1.9-fold) and pyruvate kinase (2.2-fold) in liver tissues of diabetic rats (Table 2). On the other hand, there was an increase in the activities of gluconeogenic enzymes like glucose-6-phosphatase and fructose-1,6-diphosphatase in diabetic rats as compared with the normal rats. Supplementation of CQSF showed restoration of glucose-6-phosphatase (41.23%) and fructose-1, 6-diphosphatase (29.43%) to the basal levels as compared with control rats (Table 2). In most of the carbohydratemetabolizing enzyme activities, there was no statistical
HbA1c Plasma Insulin Levels
a
12
b
10
c
8
c
6 4
ng/mL
% glycated hemoglobin
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Toxicity markers
3
5 4.5 4 3.5 3
c
2.5 2 1.5
a
1 0.5 0
2 0 I
II
III
IV
V
Groups
Figure 2. Glycated hemoglobin levels. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,c statistically significant as compared with the diabetic group. Similar alphabets indicate no significant difference between groups.
I
II
c
b
III
IV
V
Groups
Figure 3. Plasma insulin levels. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with the diabetic group. Similar alphabets indicate no significant difference between groups.
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Table 1. Activities of toxicity markers in liver (IU/L). Parameters
Group I
ALP GGT ALT AST
Group II
114.32 ± 10.96 18.78 ± 1.80 40.83 ± 3.93 66.36 ± 6.36
Group III
170.94 ± 16.41 35.41 ± 3.40a 65.4 ± 5.71a 95.28 ± 9.15a
a
Group IV b
145.52 ± 13.96 27.41 ± 2.64b 56.93 ± 5.46b 79.97 ± 7.67b
Group V c
128.46 ± 12.33 24.48 ± 2.36b 51.04 ± 4.90b 73.17 ± 7.02b
127.48 ± 12.24c 25.57 ± 2.47b 51.83 ± 4.98b 76.57 ± 7.35b
Values are expressed as mean ± SD of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with the diabetic group. Similar alphabets in the same row indicate no significant difference between groups.
Hepatic Glycogen Content
mg/g wet tissue
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8
6
c 4
c
b a
2
0 I
II
III
IV
V
Groups Figure 4. Hepatic glycogen content. Values are expressed as mean ± SEM of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with diabetic group. Similar alphabets indicate no significant difference between groups.
significance between the groups IV and V, indicating that 100 mg/kg body weight is the optimum dose for better hypoglycemic activity. Histopathological analysis of liver The histopathological analysis of liver of the normal control group showed normal architecture (Figure 5A), while the diabetic rats (Figure 5B) showed marked hepatocellular damage and these changes were restored to near normal by the treatment with CQSF (Figure 5C–E). GC–MS analysis Ten compounds were identified in the CQSF extract by GC-MS analysis. To the best of our knowledge, the identified compounds have not been reported previously in this plant. The active principles with their retention time (RT), molecular formula, molecular weight (MW), and peak area % are presented in Table 3.
Discussion The prevention and care of diabetes have tended to diet-based intervention considering safety and economy (Jilin et al., 2011). Cissus quadrangularis is a medicinal food used by the folk healers as a hypoglycemic agent. Hence the present study aimed to evaluate the anti-diabetic potential of ethyl acetate fraction of C. quadrangularis stem on STZ-induced DM. In our study, STZ was selected for induction of diabetes in
rats. STZ is well known for its selective pancreatic b-cell cytotoxicity and has been extensively used to induce DM in animals (Raju & Balaraman, 2008) and it is less toxic and allows a consistent maintenance of DM. The experimentally induced diabetic rats showed severe hyperglycemia interrelated with a decrease in endogenous insulin secretion and release. Rats treated with CQSF extract showed a significant decrease in the level of blood glucose and an increase in the level of serum insulin. These results indicated that CQSF produced anti-hyperglycemic activity and the glucose-lowering activity of CQSF may be attributed to pancreatic-enhancement of insulin secretion. Increment in plasma insulin by CQSF is probably due to the presence of adamantine derivatives which possess insulin secretagogue activity. It may also be due to the regeneration of pancreatic b cells which are destroyed by STZ (Eidi et al., 2006). The level of HbAlc is monitored as a reliable index of glycemic control in diabetes. Elevated HbAlc was observed in the diabetic group which indicates poor glycemic control. Uncontrolled and long-term diabetes was often accompanied with high glycosylated hemoglobin which is responsible for the development of late diabetic complications namely vascular dysfunction, neuropathy, and diabetic nephropathy. In case of diabetic rats treated with the CQSF extract, the HbAlc levels were brought down from elevated level to almost normal. STZ administration was associated with hepatocellular damage. The increased activities of marker enzymes like AST, ALT, ALP, and GGT in serum are suggestive of liver injury, which might be mainly due to the leakage of these enzymes from the liver cytosol into the blood stream (Kasetti et al., 2010). Treatment with CQSF notably prevented the elevation of these enzymes to an extent. Thus CQSF prevents liver cell damage and preserves cell integrity possibly leading to the survival of the functionally active cells, as evidenced by histopathological analysis. According to previous reports, DM was presented with alterations in glucose homeostasis that contribute to persistent hyperglycemia and liver plays a major role in the regulation of glucose metabolism (Ohaeri, 2001). The activity of enzymes like hexokinase, pyruvate kinase, glucose-6-phosphatase, and fructose-1,6-diphosphatase was markedly altered, resulting in hyperglycemia, which leads to the pathogenesis of diabetic complications (Bhavapriya & Govidasamy, 2000). The altered the activity of hexokinase and pyruvate kinase, key enzymes in the catabolism of glucose, diminishing the metabolism of glucose and ATP production in diabetic conditions. The reduction in the activities of these enzymes in the liver tissues of diabetic
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Table 2. Activities of carbohydrate metabolizing enzymes. Parameters
Group I d
Hexokinase Pyruvatekinasee Glucose-6-phosphatasef Fructose 1,6-diphosphatasef Glycogen phosphorylaseg
16.56 ± 1.59 33.09 ± 3.18 19.61 ± 1.70 69.70 ± 6.36 52.07 ± 5.00
Group II
Group III a
7.95 ± 0.77 12.57 ± 1.14a 66.36 ± 6.72a 133.20 ± 13.47a 83.46 ± 8.01a
Group IV b
10.26 ± 0.99 25.40 ± 2.43b 44.93 ± 4.30b 118.40 ± 11.97b 62.79 ± 6.02b
Group V c
15.26 ± 1.53 29.08 ± 2.81c 39.86 ± 4.04b 94.12 ± 9.52c 61.26 ± 5.88b
14.85 ± 1.29c 27.80 ± 2.54b,c 39.81 ± 4.01b 101.50 ± 10.85c 61.80 ± 5.93b
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Values are expressed as mean ± SD of six rats in each group; significance accepted at p50.05; astatistically significant as compared with the normal group; b,cstatistically significant as compared with the diabetic group. Similar alphabets in the same row indicate no significant difference between groups. dmg glucose phosphorylated/min/mg protein; eunits/mg protein; fmmol of Pi liberated/h/mg protein, gmmol of Pi liberated/min/mg protein.
Figure 5. Histopathology of liver. (A) Normal control – normal hepatocytes showing normal architecture. (B) Diabetic control – extensive hepatocellular damage in the form of inflammation, sinusoidal dilation, fatty changes, and extensive vacuolization with disappearance of nuclei. (C)–(E) Diabetic + CQSF shows only mild inflammation, and there is restoration of normal tissue morphology.
rats is an indication of reduced glycolysis and amplified gluconeogenesis signifying that these two pathways are distorted in diabetes. In agreement with the above reports, the activities of hexokinase and pyruvate kinase were significantly decreased in the STZ-induced DM group. Administration of CQSF to diabetic rats significantly elevated these enzyme activities in liver. The activities of regulatory enzymes in gluconeogenesis, like glucose-6-phosphatase and fructose-1,6-diphosphatase, are elevated in DM (Grover et al., 2000) and increased activities of these enzymes in STZ-induced diabetic rats may be due to insulin insufficiency (Baquer et al., 1998). Glucose-6-phosphatase and fructose-1,6-diphosphatase are dephosphorylating enzymes which impair hepatic glucose utilization. Our results showed that the activities of glucose-6-phosphatase and fructose-1,6-diphosphatase were significantly decreased by the administration of CQSF. Glycogen is the primary intracellular storage form of glucose and its quantity in various tissues is a direct manifestation of insulin activity as insulin supports intracellular glycogen deposition (Pederson et al., 2005). The reduced glycogen store in diabetic rats has been attributed to the loss of glycogen synthase-activating system and/or the increased
activity of glycogen phosphorylase (Golden et al., 1979). In the present study, there was a decrease in the hepatic glycogen content of diabetic rats which suggests the increased glucose output during insulin deficiency. Here diabetic animals showed increased glycogen phosphorylase activity when compared with normal control animals. Treatment with CQSF restored the levels of glycogen, probably by means of decreasing the activity of glycogen phosphorylase. Histological studies were carried out over the liver for the five groups. STZ-induced diabetic rats showed marked hepatocellular damage in the form of inflammation, sinusoidal dilation, fatty changes, and extensive vacuolization with the disappearance of nuclei. Administration of CQSF significantly improved the histological architecture of liver in diabetic rats. Consonant with the histological findings, serum ALT, AST, ALP, and GGT levels were substantially higher in DM rats, but it was restored to near normal levels after the treatment with CQSF. Thus the histopathological analysis of liver showed the protective effect of CQSF in experimental diabetes. The GC–MS analysis of CQSF showed the presence of 10 constituents which were characterized and identified for the first time in this work. The identified compounds
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Table 3. Chemical composition of CQSF extract. RT
Name
9.296 11.538 14.455 17.215 26.201 28.378 38.107 41.651 46.997 52.207
Ethanone, 1-(2-hydroxy-5-methylphenyl)Niacinamide Phenol, 2,4-bis(1,1-dimethylethyl)Diethyl 2,5-pyridinedicarboxylate Cyclohexene, 1,5,5-trimethyl-6-methylene N-Methyl-1-adamantaneacetamide 13-Docosen-1-ol, (Z)N-Nitrosohemanthidine lactone Fluorometholone 5-Benzofuranpropanol, 2-(3,4-dimethoxyph
include phenolic derivatives, fatty alcohols, acetophenones, carbonyl compounds, and aliphatic hydrocarbons and were having medicinal properties such as antioxidant (Yoon, 2006), anti-inflammatory (Constantino et al., 1993), anticancer (Piedrafita & Ortiz, 2006), and hypoglycemic activities (Singab et al., 2005). One of the major compounds present in CQSF was N-methyl-1-adamantane acetamide. Adamantane derivatives are reported to be insulin secretagogues in nature (Garrino & Henquin, 1987). The antidiabetic activity of CQSF may be attributed to the cumulative effect of these major compounds present in it.
Conclusion Our study clearly indicated that CQSF has potential antihyperglycemic activity in STZ-induced diabetic rats. Pronounced activity was observed at the doses of about 100 and 200 mg/kg. The ameliorative effect of CQSF against severe hyperglycemia may be due to the enhancement in the peripheral utilization of glucose, correcting the impaired liver glycolysis and limiting gluconeogenic formation. The major chemical compounds revealed by GC–MS analysis are believed to be responsible for the anti-hyperglycemic activity. Thus, the dietary supplementation of C. quadrangularis stem may be helpful for the management of DM and prevention of diabetic complications.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Molecular formula
Molecular weight
Peak area %
C9H10O2 C6H6N2O C14H22O C11H13NO4 C10H16 C13H21NO C22H44O C17H16N2O6 C22H29FO4 C13H17NO2
150.18 122.12 206.32 223.23 136.23 207.31 324.58 344.32 376.46 219.28
0.14 0.86 22.82 3.35 2.2 37.57 1.31 0.5 0.3 13.14
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DOI: 10.3109/13880209.2014.970286
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Antidiabetic activity of Cissus quadrangularis stem
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