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Original Article
Assessment of antidiabetic potential of Cissampelos pareira leaf extract in streptozotocinenicotinamide induced diabetic mice Kuldeep Singh Yadav a, Narayan Prasad Yadav a,*, Karuna Shanker b, Shiny C. Thomas a, Saurabh Srivastav a, Shruti Srivastava a, Vineet Kumar Rai a, Nidhi Mishra a, Priyam Sinha a a
Herbal Medicinal Products Department, CSIR-Central Institute of Medicinal and Aromatic Plants, P. O. CIMAP, Lucknow 226 015, India b Analytical Chemistry Department, CSIR-Central Institute of Medicinal and Aromatic Plants, P. O. CIMAP, Lucknow 226 015, India
article info
abstract
Article history:
Objective: To validate the traditional use of Cissampelos pareira as an antidiabetic agent in
Received 8 May 2013
streptozotocinenicotinamide induced diabetic male mice.
Accepted 19 June 2013
Methods: Antidiabetic effect of aqueous extract of C. pareira leaves (CPAE) was evaluated at
Available online 22 August 2013
250 mg/kg and 500 mg/kg body weight, p.o. doses in male albino mice over the period of 14 days. Random blood glucose level and body weight were observed periodically. Liver
Keywords:
glycogen level, organ coefficient and other biochemical parameters were also determined
Biochemical parameters
after completion of the study.
Cissampelos pareira Linn.
Result: Significant ( p < 0.001) changes were observed in random blood glucose levels of
Hypoglycemic
mice after 14 days of treatment period at 500 mg/kg CPAE treatment. Aspartate trans-
Liver glycogen
aminase, alanine transaminase, alkaline phosphatase, total bilirubin, triglycerides and creatinine levels were significantly ( p < 0.05) decreased while liver tissue glycogen and serum protein levels were significantly ( p < 0.05) increased after CPAE administration. No significant changes were observed in body weight and organ coefficient. Conclusions: The present study concluded that the aqueous extract of C. pareira at 500 mg/kg body weight was capable in reducing diabetic attritions so it might be a valuable candidate for diabetes treatment. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Cissampelos pareira Linn. (Menispermaceae), is a climbing shrub found throughout tropical and subtropical parts of India, East Africa and America. Locally, it is known as
“Laghupatha” and prescribed as a medicine for various human ailments in “Ayurveda”. Roots and aerial parts of C. pareira have been reported to contain several alkaloids, such as hayatin, hayatidin, hayatinin, cissampeline, cissampareine, warifterine, tetradrine,
* Corresponding author. Tel.: þ91 522 2718657. E-mail address:
[email protected] (N.P. Yadav). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.06.027
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Table 1 e Effect of aqueous extract of C. pareira on oral glucose tolerance test. Groups
Glucose level (mg/dl) Fasting
Glucose control Metformin CPAE-250 CPAE-500
87.80 85.20 78.60 82.40
7.06 5.60 2.48 1.86
15 min 299.60 240.40 244.80 216.00
30 min 216.60 156.80 187.80 157.40
22.58 8.54d 7.84 11.85e
60 min
20.22 8.71d 6.65 8.03d
179.80 110.40 145.40 117.00
19.67 7.33e 7.22 6.23e
90 min 144.40 87.80 101.20 88.20
12.31 8.06e 11.44d 3.76e
120 min 121.20 62.20 90.40 76.00
6.48 3.06f 4.50e 3.27f
Values are expressed as mean SEM, n ¼ 5 in each group; dp < 0.05, ep < 0.01, fp < 0.001 compared to glucose fed normal mice.
pareirubrines A and B, sepeerine, bebeerine, cissampeloflavone, quercitol, sterol, saponins, essential oil and quaternary ammonium bases.1,2 Plant has been documented to possess antioxidant,3 hepatoprotective,4 antifertility,5 antinociceptive, antiarthritic,6 anti-inflammatory,7 antimalarial,8 antidiarrheal,9 immunomodulatory,10 cardioprotective activity,11 and effective in agerelated cognitive decline.12 Based on diversified pharmacological properties and traditional use of this plant, the aim of the present study was to assess the antidiabetic potential of C. pareira leaf extract in streptozotocinenicotinamide (STZeNIC) induced hyperglycemia in mice.
2.
Materials and methods
2.1.
Plant material
The leaves of C. pareira Linn. were collected locally and authenticated from CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow. Voucher specimen (CIMAP-13663) has been deposited in the herbarium of the Institute.
2.2. Preparation of aqueous extract of leaves of C. pareira (CPAE) Shade dried leaves were powdered and macerated with distilled water with occasional shaking and the mixture was filtered after 48 h. The filtrate was concentrated to dryness using rotary evaporator (yield 9.1% w/w).
2.3.
Experimental animals
Swiss albino male mice, weighing between 24 3 g were selected for this study. The animals were acclimatized for one week. The animals were fed with standard rodent pellet diet and water ad libitum. The experimental protocols were duly approved by Institutional Animal Ethical Committee (IAEC) according to CPCSEA (Government of India) guidelines (Reg. No. 400/01/AB/CPCSEA, AH-2012-08).
2.4.
Oral glucose tolerance test (OGTT)
Swiss albino male mice were fasted approximately for 18 h before commencing the experiment and divided into four groups of 5 animals each (n ¼ 5). Group-I was kept as glucose control and vehicle (distilled water) was administered at a dose of 10 ml/kg body weight and group-II was used as positive control with metformin administration at dose of 200 mg/ kg. Group-III and IV were treated as test groups and CPAE was given at dose of 250 and 500 mg/kg respectively. In addition, mice of all groups were administered glucose solution at the dose of 2 g/kg after 30 min of the administration of their respective doses. All the treatments were given orally. Blood was withdrawn from tail-vein just prior to the respective dose administration (fasting glucose level) and at 15, 30, 60, 90, and 120 min after glucose loading. Blood glucose level was measured using glucometer.13,14
2.5.
Induction of diabetes in mice
In another set of experiment, mice with overnight fasting were treated with streptozotocin (STZ; 200 mg/kg) dissolved in
Table 2 e Effect of C. pareira on random blood glucose level. Days
Glucose level (mg/dl) Normal control
Day Day Day Day Day
1 4 7 10 15
109.00 98.80 101.60 120.60 103.00
10.61 8.60 2.48 12.42 5.56
Diabetic control 358.71 341.86 408.57 369.86 427.71
c
4.75 6.13c 21.48c 9.63c 23.52c
Metformin 334.00 266.71 242.86 209.71 216.71
8.44 20.03e 20.64e 15.90f 13.59f
CPAE-250 342.57 309.29 339.14 350.43 308.71
19.17 11.04 19.63 21.34 11.97f
CPAE-500 321.43 277.14 325.14 297.43 296.57
10.16 8.39e 14.87d 12.59d 11.80f
Values are expressed as mean SEM, n ¼ 5 in each group; cp < 0.001 compared to normal control group; dp < 0.05, ep < 0.01, fp < 0.001 compared to diabetic control group.
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Table 3 e Effect of C. pareira on body weight and organ coefficient. Groups
Body weight (g) Day 0
Normal control Diabetic control Metformin CPAE-250 CPAE-500
22.57 0.87 24.22 1.34 24.66 1.8 24.61 0.79 22.88 1.39
Organ coefficient (mg/g)
Day 15 23.72 17.31 20.74 18.67 19.92
0.82 1.29b 1.71 0.74 1.43
Liver
Spleen
41.00 2.89 66.49 3.13b 59.87 5.30 56.85 4.99 50.83 4.27
23.02 9.50 31.25 9.43 28.60 7.80 29.74 10.28 28.11 7.90
Kidney 41.72 47.06 41.84 42.24 41.50
2.15 10.97 9.40 10.29 9.34
Values are expressed as mean SEM, n ¼ 5 in each group; bp < 0.01 compared to normal control group. Organ coefficient ¼ [organ weight (mg)/ body weight (g)] 100.
0.1 M citrate buffer, i.p., just after 15 min of nicotinamide (NIC; 110 mg/kg) injection except in vehicle control group which was injected similarly with vehicle only i.e. normal saline and citrate buffer. All the animals received 5% glucose solution for 12 h to avoid hypoglycemic shock. Hyperglycemia was confirmed after 3 days and steady state of hyperglycemia was reached after 10 days. Blood glucose level was determined using glucometer and the mice having serum glucose 300 mg/dl were selected for the investigation.14
2.6.
Evaluation of antidiabetic activity
The diabetic animals were randomly allocated into four groups of five animal each (n ¼ 5). Group-A served as normal control (non-diabetic), group-B as diabetic control (diabetic) and group-C was positive control (diabetic þ metformin200 mg/kg). The animals of group D (diabetic þ CPAE-250 mg/ kg) and group-E (diabetic þ CPAE-500 mg/kg) served as test control. The respective doses were administered once orally to all animals for 14 days. Blood glucose level was measured on day 1, 4, 7, 10 and 15 randomly. After 24 h of last dose administration, blood samples were collected by heart puncture under deep ether anesthesia and animals were sacrificed by cervical dislocation. Liver, kidney and spleen were excised, washed in ice cold 0.1 M phosphate buffer saline, soaked on tissue paper and weighed. Serum was used to perform biochemical analysis namely alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin (TBIL), total protein (TP), serum creatinine, urea, triglycerides (TG), high density lipid (HDL) and low density lipid (LDL) using commercially available kits and 10% liver tissue homogenate was prepared to estimate glycogen content.13
2.7.
Characterization of CPAE through HPLC
The chromatographic separation was achieved using a Phenomenex C-18 (4.6 250 mm, 5 mm) at 35 C. The mobile phase was 0.5% AcOH in water (solvent A) and acetonitrile containing 0.5% AcOH (solvent B). The step gradient elution was started with 5% B with a flow rate of 1.0 ml/min. The percentage of B was increased to 15% at 10 min, 85% at 45 min. At 50 min the percentage of B was changed to 95% and at 55 min this was reduced to 15%. Finally, initial conditions were reverted at 60 min. The injection volume was 20 ml. The data acquisition was performed in the range of 190e400 nm to monitor chromatographically separated peaks. For HPLC fingerprint 254 nm was selected considering optimum signal response.
2.8.
Statistical analysis
The results are expressed as mean SEM. Statistical analysis was done using analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison test using GraphPad PRISM version 4.01 (GraphPad software, USA). The value of p < 0.05 was considered statistically significant.
3.
Results
The oral glucose tolerance test (Table 1) revealed that treatment with CPAE at dose of 500 mg/kg significantly ( p < 0.05) suppressed elevated blood glucose level at all checked time points. After 14 days treatment, significant ( p < 0.001) recovery from hyperglycemic condition ( p < 0.001) to normal level was observed in both CPAE doses; 250 and 500 mg/kg (Table 2).
Table 4 e Effect of C. pareira on liver tissue glycogen and serum liver parameters in STZ-NIC intoxicated albino mice. Groups
Normal control Diabetic control Metformin CPAE-250 CPAE-500
Liver tissue glycogen (mg/100 mg) 483.90 237.52 402.60 345.53 394.78
28.75 19.59c 24.91e 4.47d 8.22e
Serum biochemical liver parameters ALP (IU/L) 112.8 189.3 125.4 158.1 142.8
5.6 9.5c 7.9f 10.5 10.2d
AST (IU/L) 84.89 208.24 112.43 179.52 147.87
8.65 10.51c 8.22f 6.92d 9.41f
ALT (IU/L) 41.5 188.4 86.2 153.4 112.1
5.4 9.2c 9.8f 9.4 7.1f
TBIL (mg/dl) 0.31 2.43 0.68 1.47 0.86
0.08 0.10c 0.07f 0.07f 0.08f
TP (g/dl) 6.41 4.21 5.02 5.88 5.97
0.99 0.75 1.02 0.33 0.41
Values are expressed as mean SEM, n ¼ 5 in each group; cp < 0.001 compared to normal control group; dp < 0.05, ep < 0.01, fp < 0.001 compared to diabetic control group.
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Body weight of diabetic control group was decreased significantly ( p < 0.05). No significant change was observed in body weight of test groups after CPAE treatment for 14 days. Furthermore, no significant changes were noticed in organ coefficient of any experimental group except liver coefficient of diabetic control mice which was significantly increased ( p < 0.01) as compared to normal control mice (Table 3). Significant ( p < 0.001) elevation in liver enzyme levels namely ALP, AST, ALT and TBIL was observed in serum of diabetic control as compared to normal control mice. CPAE at both doses recovered liver enzyme levels significantly ( p < 0.05) towards normal level while total bilirubin levels were decreased significantly ( p < 0.001) (Table 4). Plasma HDL levels were significantly ( p < 0.05) reduced in diabetic control mice when compared with normal control and CPAE (500 mg/kg) significantly recovered ( p < 0.01) HDL levels towards normalization. Plasma TG levels were also significantly ( p < 0.001) increased in diabetic control compared to normal control mice and CPAE (500 mg/kg) exhibited significant recovery ( p < 0.05) towards normal level. Plasma LDL levels did not show any significant change in diabetic as well as treatment groups (Fig. 1a). STZeNIC induced diabetic mice showed significant reduction in liver tissue glycogen levels ( p < 0.001) as compare to normal control group while CPAE treatment at both doses significantly ( p < 0.05) recovered the liver tissue glycogen to 43.84% and 63.83% respectively (Table 4). CPAE 250 and 500 mg/kg body weight treatment also reduced serum creatinine levels significantly ( p < 0.01) but serum urea levels were significantly ( p < 0.01) reduced by CPAE at dose of 500 mg/kg only (Fig. 1b).
Fig. 1 e Effect of C. pareira on serum lipid and kidney parameters in STZeNIC treated albino mice. Values are expressed as mean ± SEM, n [ 5 in each group; a p < 0.05, bp < 0.01, cp < 0.001 compared to normal control group; dp < 0.05, ep < 0.01, fp < 0.001 compared to diabetic control group; Value shown in parenthesis is percent recovery calculated as [(diabetic controlLtreatment control)/(diabetic controlLnormal control)] 3 100.
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In order to obtain reproducible chromatographic fingerprint of CPAE for quality control, the method validation of HPLC-PDA fingerprint analysis was performed on the basis of the retention time and the peak area. The experiment was conducted to examine the classification and concentration of phytochemicals in three categories according to their polarity. The possible separated chemical flux under experimental condition, which have chromophoric group have been shown in the chromatogram. A typical chromatograms of aqueous extract of C. pareira Linn. (CPAE) is shown in Fig. 2. It could be concluded that most of the reverse-phase separated compounds were of medium polar nature, presumably belongs to chalconeeflavones by characteristic UV spectra. The possibility of any alkaloids was ruled out by negative dragendorff test of eluent of this region.
4.
Discussion
The fundamental basis of hyperglycemia in diabetes mellitus is over-production (excessive hepatic glycogenolysis and gluconeogenesis) and decreased utilization of glucose by the tissues leading to persistent hyperglycemia which might be responsible for most diabetic complications. Lowering blood glucose to near-normal levels should be aimed to treat all diabetic patients.15 CPAE has capacity to reduce blood glucose level significantly in glucose fed hyperglycemic normal mice during OGTT. This effect may occur due to reduction in intestinal glucose absorption or induction of glycogenic process along with reduction in glycogenolysis and glyconeogenesis.16 Streptozotocin (STZ) causes selectively necrotize pancreatic b-cells. Metformin (a biguanide) is often used as a standard antidiabetic drug in STZ-induced experimental diabetes.17 The results demonstrated that CPAE significantly reduced the blood glucose level which is associated with the effectiveness of C. pareira for controlling hyperglycemia. The extra cellular glucose in the presence of insulin converts into glycogen in the liver cells and the enzymes glycogen synthase and glycogen phosphorylase are responsible for glycogen metabolism. Our results demonstrated that there was significant loss in liver tissue glycogen level in diabetic animals. Treatment with CPAE significantly increased liver
Fig. 2 e HPLC chromatogram of C. pareira leaves extract (detail chromatographic conditions are mentioned in the experimental section).
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glycogen which might be associated with stimulation of glycogenesis and/or inhibition of glycogenolysis in the liver of diabetic mice. Hypertriglyceridemia is most common abnormality in diabetes.15 A significant increased state of triglycerides was observed in toxin treated animals. In diabetic state, LDL carries cholesterol to its depositing site (i.e. peripheral tissues) whereas HDL helps in excreting cholesterol, means it transports cholesterol from peripheral tissues to the liver. Hence increase in LDL level is atheromatic. Our study demonstrated that CPAE treatment significantly increased HDL level while LDL level was unaffected in all experimental groups. The ALP, AST, ALT, TBIL, and TP are considered as sensitive indicator of liver injury.18 Rise in serum level of AST, ALT, ALP and total bilirubin have been attributed to the damaged structural integrity of the liver. The significant decrease in liver enzymes namely AST, ALT, ALP and total bilirubin levels were noticed after oral administration of CPAE as compared to diabetic animals. It implies the normal functioning and protective effect of liver and supports hepatoprotective claim of C. pareira.4 The present study demonstrated increase in glucose metabolism and decrease in the gluconeogenesis as evidenced by increase in liver glycogen, serum lipids and creatinine levels. This affirms that other active ingredient(s) may impart for the in-vivo antihyperglycemic effect. This study unveils that the decrease in blood glucose level may be attributed to the stimulation of glucose uptake by peripheral tissues and/ or decrease in the gluconeogenesis. Hence, the antihyperglycemic effect may be probably due to an extrapancreatic mechanism and/or the regeneration of pancreatic b-cells.
Conflicts of interest All authors have none to declare.
Acknowledgment The authors are highly thankful to Director, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India for providing research facilities for the completion of the present investigation. Authors are also thankful to JPR solutions for their partial funding to publish this research work.
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