Oriental Pharmacy and Experimental Medicine 2010 10(2), 90-102
Antidiabetic activity of Argyreia speciosa (sweet) (Burm.f.)Boj. in normogl- ycemic and Streptozotocin-induced diabetic rats
PV Habbu1,*, KM Mahadevan2, VH Kulkarni3, Marietta P3, Pratap V3, BS Thippeswamy3 and VP Veerapur4
1Post graduate department. Division of Pharmacognosy, S.E.T’s College of Pharmacy, Dharwad-580 002, India;
2Post graduate department in chemical sciences and Research, Kuvempu University Shankarghatta, Shimoga, India; 3Post graduate department. Division of Pharmacology, S.E.T’s College of Pharmacy, Dharwad-580 002, India; 4Department Pharmaceutical Chemistry, S.E.T’s College of Pharmacy, Dharwad-580 002, India
Received for publication June 11, 2009; accepted May 10, 2010
SUMMARY
Effect of ethanol (ASE) and water (ASW) extracts of Argyreia speciosa on blood glucose and lipid profile was investigated in normoglycemic and Streptozotocin (STZ)-induced diabetic animals. In oral glucose and sucrose tolerance test, treatment with ASE and ASW (100 and 200 mg/kg) and Glidenclamide (10 mg/kg) significantly improved the glucose and sucrose tolerance in normal animals. In addition, respective treatment for fifteen-day resulted in significant percentage reduction in serum glucose (SG) ie., 30.39% (lower dose of ASE) and 33.21% (higher dose of ASW). In standardized STZ (50 mg/kg, iv)-induced diabetic rats, a single dose of ASE and ASW treatment exhibited reduction in SG levels at different time intervals compared to basal levels.
Administration of both the doses of ASE and ASW for fifteen-day days exhibited greater percentage reduction in glycemia (24.6%, 24.7%, 23.9% and 21.9% respectively) and also ameliorated restored to near normal value of all tested lipid parameters. Further, treatment also exhibited significantly improved glucose tolerance over the period of 120 min compared to diabetic control group. Eventhough treatment failed to increase serum insulin levels significantly but peripheral utilization of insulin was increased as evident by insulin tolerance test. Taken together, present study supports the traditional usage of title plant in the treatment of diabetes mellitus.
Key words: Argyreia speciosa; Antidiabetic; Antihyperlipidemic; Rasayana; Vruddhadaruka.
INTRODUCTION
Diabetes is a disorder in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by insulin. This results primarily in
elevated fasting and postprandial blood glucose levels. If this imbalanced homeostasis does not return to normalcy and continues for a protracted period of time, it leads to hyperglycemia that in due course turns into a syndrome called Diabetes Mellitus (Lernmark, 1998). The global prevalence of diabetes is estimated to be 2.8% in 2000 and 4.4% in 2030. The total number of Diabetes is projected to rise from 171 million in 2000 to 366 million in 2030. Medication often has an important
*Correspondence: Postgraduate department of Phar- macognosy, S.E.T’s College of Pharmacy, Dharwad- 580 002, Karnataka, India. Tel: +918362448540; Fax:
+918362467190; E-mail: prasannahabbu@ymail.com, prasherbs@yahoo.com
DOI 10.3742/OPEM.2010.10.2.090
role to play particularly for the control of blood glucose and lipids profiles (Wildet al., 2004).
External insulin preparations can be used to treat type 1 diabetes mellitus as well as in uncontrolled type 2 diabetes mellitus (T2DM) conditions. The oral hypoglycemic agents such as Sulfonylureas (Tolbutamide, Glibenclamide), Biguanides (Phen- formin, Metformin), Thiazolidinediones (Troglitazone, Rosiglitazone), α-Glucosidsase inhibiotrs (Acarbose, Miglitol) have been extensively used in T2DM.
But none of them are ideal in correcting blood glucose levels round the clock, with single dose administration. In large doses, these agents cause hypoglycemia and in smaller doses, unchecked hyperglycemia and both these extremes is equally life threatening. Moreover, it has been clearly established that insulin and sulfonylureas, even with excellent glycemic control do not normalise diabetes dyslipidemia or improve hypertension (Position statement, Int. Diab. Federation, 2005).
Parallel to this, recent developments in understanding the pathophysiology of the disease process have opened up several new avenues to identify and develop novel therapies to combat the diabetic plague. In the Indigenous system of medicine like Ayurveda, many herbal medicines have been recommended for the treatment of diabetes or madhumeha and some of them experimentally evaluated (Roa et al., 2004).
Concurrently, phytochemicals identified from traditional medicinal plants are presenting an exciting opportunity for the development of new types of therapeutics. In recent years, there has been renewed interest in the treatment against different diseases using herbal products as they are generally non-toxic and WHO has also recommended the evaluation of the effectiveness of plants in condition where we lack safe modern drugs. This has accelerated the global effort to harness and harvest those medicinal plants that bear substantial amount of potential phytochemicals showing multiple beneficial effects in combating diabetes and diabetes-related complications.
Therefore, as the disease is progressing unabated, there is an urgent need of identifying indigenous natural resources in order to procure them, and study in detail, their potential on different newly identified targets in order to develop them as new therapeutics (Ashoket al., 2001).
Argyreia speciosa (Burm.f) Boj. (Convulvulaceae) is a rasayana plant, commonly known as Vrudhadaruka in Indian system of medicine. Roots of A. speciosa are used traditionally as aphrodisiac, rejuvenating, intellect promoting, brain tonic, in the treatment of infected wounds, bronchitis, syphilis and pulmonary tuberculosis (Warrier, 1994; Sharma, 2004). The plant has been screened for anti- inflammatory (Gokhale et al., 2002), immunomo- dulatory (Gokhale et al., 2003), and hepatoprotective (Habbu et al., 2008), antimicrobial and antitubercular (Habbu et al., 2009) and nootropic (Habbu et al., 2009) activities. Phytochemical investigations on the roots of A. speciosa have resulted in the isolation of a range of biologically active substances like Flavonoid sulphates (Petra et al., 1999), Stigmasteryl p-hydroxycinnamate and a Coumarin (Srivastava and Shukla, 1998) and many phenolic compounds. We reported the isolation, characterization, antimicrobial antituberculosis efficacy of flavanoid suphates and other fractions of A. speciosa (Habbu et al., 2009).
The paucity of scientific information regarding the effects of Argyreia speciosa root extracts on serum glucose and lipid levels, the present study is planned to investigate the hypoglycemic and hypolipidemic activity in normoglycemic and diabetic animals.
MATERIALS AND METHODS Drugs and chemicals
Streptozotocin (STZ) purchased from HI-Media, Mumbai. Insulin injection biphasic isophane, Ph.
Eur. Human mixtard manufactured by Torrent Pharmaceuticals, Ltd., Standard drug Glidenclamide (GLB) 5 mg (Daonil) obtained from Avents Pharma,
Ltd., Goa. Acarbose (glucobay) Bayer Pharma, Pvt Ltd. Standard kits for Glucose, Triglycerides, Cholesterol, and HDL-Cholesterol were obtained from ERBA Mannheim, Manufactured by Transasia Biomedical Ltd, Baddi, India. Serum insulin was estimated by radioimmunoassay method using the kit from Bhabha Atomic Research Centre, Mumbai, India.
Plant material
Roots of Argyreia speciosa were collected from hilly areas (900 m) surrounding Dharwad, Karnataka province, India and authentication of the plant was done by Dr. G.R. Hedge, Department of Botany, Karnataka University, India. A herbarium specimen of the plant was kept in department of Pharmacognosy (SETCPD/Ph.cog/herb/33/2006), SET’s college of Pharmacy, Dharwad, Karnataka, India. The collected material was washed with running water. The roots were chopped in to small pieces and dried under shade. Dried roots were coarsely powdered and used for fractionation.
Preparation of plant extract
Coarse plant material was cleaned by passing the powder material through 120 mesh sieve to remove any fine dust or powder, and coarse powder was used for extraction. Dried powder of root was exhaustively extracted with 95% ethanol (ASE) in a Soxhlet apparatus. Further water extract (ASW) was prepared by maceration using chloroform water. Both the fractions were concentrated by rotary flash evaporator under reduced pressure and controlled temperature, followed by freeze drying and stored in a desiccator.
Animals
Young adult male Wistar rats weighing 150 - 200 g were obtained from inbred animal house Venkateshwara enterprises, Bangalore, Karnataka.
The animals were housed in polypropylene cages in standard environmental conditions, 12 h light and 12 h dark cycle at 25 ± 2oC. Before and during
the experiments, the rats were fed with standard laboratory pellet diet and water ad libitum obtained from Venkateshwara enterprises, Bangalore. Animals were acclimatized to the laboratory condition for at least 15 days prior to the experiment and were maintained in a well ventilated animal house. The experimental protocol was approved by the Institutional animal Ethical Committee (IAEC) animals and the care of the laboratory was taken as per the CPCSEA regulation.
Effect of ASE and ASW in normoglycemic rats Oral glucose tolerance test (OGTT)
The experimental rats were divided into six groups of five rats each. Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o.);
Group 2: NC rats treated with ASE (100 mg/kg, p.o.); Group 3: NC rats treated with ASE (200 mg/kg, p.o.); Group 4: NC rats treated with ASW (100 mg/kg, p.o.); Group 5: NC rats treated with ASW (200 mg/kg, p.o.); Group 6: NC rats treated with GLB (10 mg/kg, p.o.)
Different doses of ASE/ASW, GLB and vehicle were given orally to normoglycemic rats fasted for 18 h. After thirty minutes, glucose (2 g/kg in distilled water) was administered orally. Blood samples were collected from the retro-orbital plexus at 0 min (i.e. immediately after glucose load), 30, 60 and 120 min after glucose administration.
Serum glucose (SG) was estimated by the enzymatic glucose oxidase method using diagnostic reagent kit. The results are expressed as integrated area under curve for glucose (AUCglucose) calculated by trapezoid rule.
AUCglucose=
Multiple-dose fifteen-day study
The same groups of animals were further treated with respective doses of ASE, ASW and GLB for fifteen consecutive days. Whereas, GLB (0.5 mg/
kg, p.o./day) was administered for fifteen days.
Percentage reduction in glycemia was calculated C1+C2
( )
---2 ×(t2–t1)
with respect to the initial (0 day) level according to: percentage reduction in glycemia = [(Gi-Gt) / Gi] × 100; Where Gi is initial glycemia and Gt is the glycemia value at 7, 10 and 15 days (Yanardag et al., 1998).
Oral sucrose tolerance test (OSTT)
The experimental rats were divided into six groups of five rats each. Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o.);
Group 2: NC rats treated with ASE (100 mg/kg, p.o.); Group 3: NC rats treated with ASE (200 mg/kg, p.o.); Group 4: NC rats treated with ASW (100 mg/kg, p.o.); Group 5: NC rats treated with ASW (200 mg/kg, p.o.); Group 6: NC rats treated with Acarbose (7 mg/kg, p.o.)
Different doses of ASE/ASW/Acarbose and vehicle were given orally to normal rats fasted for 18 h. Thirty minutes later, sucrose (2 g/kg in distilled water) was administered orally. Blood samples were collected from the retro-orbital plexus at 0 min (i.e. immediately after sucrose load), 30, 60 and 120 min post sucrose administration (Li et al., 2004). SG was estimated by the enzymatic glucose oxidase method using diagnostic reagent kit. The results are expressed as integrated area under curve for glucose (AUCglucose) calculated by trapezoid rule using formula as mentioned above.
Effect of ASE and ASW in STZ-induced diabetic rats Induction of diabetes mellitus
Diabetic condition was induced in male Wistar rats by single intravenous injection of STZ (50 mg/kg) [Chosen optimum dose: In house data]
after overnight fasting for 12 h (Siddique et al., 1987). Rats showing SG level > 200 mg/dl seven days after STZ administration were considered diabetic and included in the study. Diabetic rats were randomized into different groups based on their SG levels.
Single-dose one-day study
The experimental rats were divided into seven
groups of five rats each. Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o.);
Group 2: Diabetic control (DC) received 0.5%
tragacanth (10 ml/kg, p.o.); Group 3: DC rats treated with ASE (100 mg/kg, p.o.); Group 4: DC rats treated with ASE (200 mg/kg, p.o.); Group 5:
DC rats treated with ASW (100 mg/kg, p.o.);
Group 6: DC rats treated with ASW (200 mg/kg, p.o.); Group 7: DC rats treated with GLB (10 mg/
kg, p.o.)
Blood samples were collected at 0, 2 and 4 h after extracts/GLB administration. SG was estimated by the enzymatic glucose oxidase method. Blood glucose was estimated by the enzymatic glucose oxidase method using a commercial glucometer (Accu-chek® Active, Roche diagnostic, Mannheim, Germany). Percentage reduction in glycemia was calculated with respect to the initial (0 h) level according to: Percentage reduction in glycemia = [(Gi-Gt)/Gi] × 100; Where Gi is initial glycemia and Gt is glycemia at 2 and 4 h (Yanardag et al., 1998).
Multiple-dose fifteen-day study
The above groups of animals were further treated with respective doses of ASE, ASW and GLB for fifteen consecutive days in order to evaluate the chronic effect of extracts/GLB treatment on hyperglycemia. Whereas, GLB (0.5 mg/kg, p.o./
day) was administered for fifteen days. Percent reduction in glycemia was calculated with respect to the initial (0 day) level by above mentioned formula.
OGTT
On 10th day, glucose tolerance of various groups was estimated by a simple OGTT. Glucose (2 g/kg) was administered to 12 h fasted rats and blood samples were collected from the retro-orbital plexus at 0 (before glucose load), 30, 60 and 120 min after glucose administration. SG was estimated by the enzymatic glucose oxidase method. The results were expressed as integrated area under curve for glucose (AUCglucose), which was calculated by
trapezoid rule. Also serum insulin was estimated 0 (before glucose load), 30 and 60 min after glucose administration. Serum insulin (SI) was estimated by radioimmunoassay method using the kit from Bhabha Atomic Research Centre, Mumbai, India.
The results were expressed as integrated area under curve for insulin (AUCinsulin), which was calculated by trapezoid rule.
Insulin tolerance test (ITT)
Insulin tolerance test is a measure of the extent of peripheral utilization of glucose. On 13th day, insulin (2 U/kg, i.v) was administered to six h-fasted rats. Blood samples were collected from the retro-orbital plexus at 0 (just before insulin load), 10, 20 and 30 min after insulin injection. SG was estimated by the enzymatic glucose oxidase method. The results were expressed as integrated area under curve for glucose (AUCglucose), which was calculated by trapezoid rule.
Estimation of biochemical parameters
At the end of the treatment schedule, blood samples were collected from retro-orbital plexus.
Serum was separated and analysed spectrophoto- metrically for triglyceride (STG), total cholesterol (STC), HDL-cholesterol (HDL-c), using diagnostic reagent kit ERBA diagnostics Mannheim GMBH, Germany. Homeostatic model assessment (HOMA) as a measure of insulin resistance was calculated by the following formula (Rajasekar et al., 2005).
HOMA =
VLDL-cholesterol (VLDL-c) and LDL-cholesterol (LDL-c) in serum were calculated as per Friedewald’s equation (Friedewald et al., 1972)17.
LDL-c = Total cholesterol - - HDL-c
The markers of dyslipidemia such as TC/HDL-c
and LDL-c/HDL-c ratios were also calculated.
Glucose uptake by isolated hemi-diaphragm of diabetic rats
Isolation of diaphragm
After collection of blood for biochemical estimations, experimental rats were killed by cervical dislocation.
The diaphragms were dissected out quickly with minimal trauma and divided into two equal halves. The hemi-diaphragms were then rinsed in cold Tyrode solution (without glucose) to remove any blood clots (Chattopadhyay et al., 1992). Two diaphragms of five animals (10 diaphragms) in each group were used for the study.
Treatment protocol
Seven sets of ten graduated test tubes each, were grouped as, Group 1 : 2 ml of Tyrode solution with 2 g % glucose [Normal control]; Group 2 : 2 ml of Tyrode solution with 2 g % glucose [Diabetic control]; Group 3 : 2 ml of Tyrode solution with 2 g % glucose + ASE (50 µg/ml); Group 4 : 2 ml of Tyrode solution with 2 g % glucose + ASE (100 µg/ml); Group 5 : 2 ml of Tyrode solution with 2 g % glucose + ASW (50 µg/ml); Group 6 : 2 ml of Tyrode solution with 2 g % glucose + ASW (100 µg/ml); Group 7 : 2 ml of Tyrode solution with 2 g % glucose + insulin (Nova Nordisk) 0.62 ml of 0.4 U/ml solution [Insulin treated group]
The volumes of all graduated test tubes were made up to 4 ml with distilled water and estimated the initial glucose concentration by commercially available glucose kit based on glucose-oxidase method. The hemi-diaphragms were placed in test tubes and incubated for 30 min at 37oC in bubbled with oxygen with continuous shaking.
Glucose uptake per gram of tissue was calculated as the difference between the initial and final glucose content in the incubated medium (Swanston- Flatt et al., 1991; Ghosh et al, 2004).
Statistical analysis
The data were expressed as mean ± S.E.M for six insulin µU/ml × glucose mmol/L
22.5
VLDL c–
( )= Triglyceride 5
Triglyceride 5
rats in each group. Statistical comparisons were performed by one-way ANOVA followed by Tukey’s post-test.
RESULTS OGTT
Administration of glucose (2 g/kg) produces significant change in SG level of normal rats.
Treatment with lower dose of ASE (100 mg/kg) and ASW (100 mg/kg) and GLB (10 mg/kg) significantly (P < 0.01; P < 0.001) improved the glucose tolerance (Fig. 1. A and B), whereas, treatment
with higher dose of ASE and ASW (200 mg/kg) did not significantly reduced the AUCglucose compared to normal control group.
Multiple-dose fifteen-day study
Treatment with ASE (100 and 200 mg/kg) and ASW (100 and 200 mg/kg) once daily for fifteen days showed hypoglycemic activity (P < 0.05; P <
0.01;P < 0.001) ASE (100 mg/kg) showed significant (P < 0.05; P < 0.01) percentage reduction (15.14%
and 30.39%) in SG levels at 7th and 15th day, compared to basal values (0 day) and activity is better than ASE (200 mg/kg). Whereas, higher dose of ASW (200 mg/kg) exhibited higher activity than ASW (100 mg/kg) at all tested days (Fig. 2).
OSTT
Animals subjected to Sucrose (2 g/kg) load produces significant change in SG level of normal rats. Treatment with different dose of ASE (100 and 200 mg/kg), ASW (100 and 200 mg/kg) and Acarbose (7 mg/kg) exhibited significant (P < 0.05;
Fig. 1. Effect of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on glucose tolerance in fasted normal rats. A) SG levels were measured prior to, and after p.o. administration of glucose alone (2 gm/kg body weight), or in combination with ASE, ASW or GLB. B) Area under curve for glucose (AUCglucose) values for 0 - 120 min post glucose load.
Data represent the mean ± S.E.M., for 5 rats. aP < 0.05;
bP < 0.01 ; cP < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).
Fig. 2. Effect of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on SG levels in Normoglycemic rats.
Bar graph represents the percentage reduction in glycemia with respect to the initial (0 day) level. Each value represents mean ± S.E.M., n = 5. bP < 0.01, compared to normal control of the same time interval.
One-way ANOVA followed by Tukey’s post-test.
P < 0.01) reduction in SG level over the period of 120 min compared to normal control group (Fig.
3A). Further, treatment of ASE /ASW/Acarbose significantly (P < 0.05; P < 0.001) improved the Sucrose tolerance (Fig. 3B) suggested that the components of extracts may inhibit a-glucosidase.
Moreover, treatment of lower dose of ASE and ASW were better than higher dose to improve sucrose tolerance in normal rats.
Effect of ASE and ASW in STZ-induced diabetic rats Single-dose one-day study
A single dose of ASE (100 and 200 mg/kg) and ASW (100 and 200 mg/kg) treatment exhibited reduction in SG levels at different time intervals
compared to basal levels (0 h). However, administration of GLB showed significant (P < 0.05; P < 0.001) reduction is SG levels with maximum reduction (50.94%) at 4 h post GLB treatment compared to their basal levels, whereas, ASE treated animals showed dose dependent percentage reduction in SG levels compared to their basal levels (Fig. 4).
Multiple-dose fifteen-day study
Repeated administration of ASE (100 and 200 mg/kg) and ASW (100 and 200 mg/kg) for 15 days, showed significantly (P < 0.05; P < 0.01) reduced levels of SG compared to respective basal values (0 day). On 15th day, tested doses of ASE and ASW showed significantly (P < 0.001) greater percentage reduction in glycemia (24.6% : 24.7%
and 23.9% : 21.9% respectively) compared to diabetic control (Fig. 5).
OGTT
On 10th day, oral administration of glucose (2 g/kg) did not produced significant change in SG level of normal control rats and AUC for the 120 min Fig. 3. Effect of ethanol (ASE) and water extract
(ASW) of Argyreia speciosa on sucrose tolerance in fasted normal rats. A) SG levels were measured prior to, and after p.o. administration of sucrose alone (2 g/
kg body weight), or in combination with ASE, ASW or Acarbose. B) Area under curve for glucose (AUCglucose) values for 0 - 120 min post sucrose load.
Data represent the mean ± S.E.M., for 5 rats. aP < 0.05;
bP < 0.01; cP < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).
Fig. 4. Effect of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on SG levels in STZ-induced rats [Single-dose one-day study]. Bar graph represents the percentage reduction in glycemia with respect to the initial (0 h) level. Each value represents mean ± S.E.M., n=5. aP < 0.05; bP < 0.01; cP < 0.001 compared to diabetic control of the same time interval. One-way ANOVA followed by Tukey’s post-test.
interval was not altered. The diabetic rats exhibited significant elevation in fasting SG (at time zero) and showed significant impairment in glucose tolerance to exogenously administered glucose compared to Normal rats (Fig. 6A). Treatment with different dose of ASE ASW (100 and 200 mg/kg), and GLB (10 mg/kg) significantly (P <
0.05; P < 0.01) improve the glucose tolerance (Fig.
6B). Further, treatment of ASE and ASW exhibited significant (P < 0.05; P < 0.01) reduction in SG level over the period of 120 min compared to diabetic control group.
Administration of glucose (2 g/kg) stimulated the release of higher levels of insulin in normal control rats, whereas glucose load was ineffective in stimulating the release of insulin in diabetic rats, suggesting that these diabetic rats resembled severe diabetic (type I) condition in which a maximum pancreatic damage occurred, whereas, treatment of ASE and ASW to diabetic rats enhanced the glucose stimulated insulin release from pancreatic β-cells but response was not significant
(Fig. 7A). Integrated areas under the insulin curve over 60 min (AUCinsulin) of diabetic group was significantly lower (P < 0.001) compared to normal control. Treatment with GLB produced a significantly (P < 0.05) increased AUCinsulin compared to diabetic control, whereas, administration of different doses of ASE and ASW fail to increase significantly (Fig. 7B).
Effect on blood glucose, serum insulin and HOMA On 10th day, diabetic rats exhibited significant (P <
0.001) hyperglycemia (354.42 ± 15.95; SG levels rose to between 296 to 406 mg/dl) and hypoinsulinemia (8.25 ± 1.1) as compared to normal control rats.
Fig. 5. Effect of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on SG levels in STZ- induced diabetic rats [Multiple-dose fifteen-day study]. Bar graph represents the percentage reduction in glycaemia with respect to the initial (0 day) level.
Each value represents Mean ± S.E.M., n = 5. aP < 0.05;
bP < 0.01, cP < 0.001 compared to diabetic control of the same time interval. One-way ANOVA followed by Tukey’s post-test.
Fig. 6. Effect of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on glucose tolerance in fasted diabetic rats. A) SG levels were measured prior to, and after p.o. administration of glucose alone (2 gm/kg body weight), or in combination with ASE, ASW or GLB. B) Area under curve for glucose (AUCglucose) values for 0 - 120 min post glucose load.
Data represent the mean ± S.E.M., for 5 rats. aP < 0.05;
bP < 0.01; cP < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).
The degree of insulin resistance as calculated by HOMA values were similar in both diabetic and
normal control rats suggested that, the diabetic rats were not under insulin resistance condition (i.e. peripheral utilization of glucose was not compromised).
Oral administration of ASW (100 mg/kg) to diabetic rats, significantly (P < 0.05) decreased SG levels, whereas all other tested doses of ASE and ASW fail to produce significant effect. Moreover, tested doses of ASE and ASW did not significantly increase SI levels. However, HOMA values were near to normal levels (Table 1).
ITT
On 13th day, SG levels were measured following insulin challenge (2 U/kg, i.v). Surprisingly, in contrast to established reports, severe (type I) diabetic rats subjected to insulin challenge did not exhibit a marked fall in SG levels suggested that, these diabetic rats were not able utilize the exogenously administered insulin to reduce the SG levels. This contrary observation may be due to the marginal loss of insulin sensitivity in diabetic rats, even though these diabetic rats were in type I diabetic condition (as evident by lower insulin levels after glucose challenge and HOMA values).
Moreover, the blood glucose levels and AUCglucose
in diabetic rats treated with ASE and ASW were significantly (P < 0.01; P < 0.001) lower at 10, 20 min compared to the glucose levels at the corresponding time points in the diabetic rats receiving the vehicle (Fig. 8A and B).
Fig. 7. Effect of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on serum insulin levels. A) Incremental AUC insulin values for 0 - 60 min. B) post glucose (2 gm/kg body weight) challenge performed on tenth day of treatment with ethanol (ASE) and water extract (ASW) of Argyreia speciosa. Data represent the mean ± S.E.M., for 5 rats. aP < 0.05; bP <
0.01; cP < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).
Table 1. Effect of ten days treatment of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on SG, SI and HOMA levels in STZ-induced diabetic rats
Treatment groups SG (mg/dl) SI (µU/ml) HOMA
Normal Control 91.43 ± 3.70 30.0 ± 2.20 6.67 ± 0.52
Diabetic Control 345.54 ± 25.95 8.25 ± 1.10 6.83 ± 0.44 ASE (100 mg/kg) 256.67 ± 23.52 12.50 ± 2.40 7.5 ± 0.76
ASE (200 mg/kg) 237.38 ± 39.2 13.25 ± 2.30 7.1 ± 0.4
ASW (100 mg/kg) 211.10 ± 25.3a 11.30 ± 1.90 5.60 ± 0.2
ASW (200 mg/kg) 235.60 ± 47.10 11.30 ± 2.4 6.0 ± 0.4
GLB (0.5 mg/kg) 280.30 ± 19.25 11.75 ± 0.9 8.0 ± 0.31
Each value represent mean ± S.E.M; n = 5; aP < 0.05 compared to Diabetic control. One way ANOVA followed by Tukey’s post test.
Estimation of lipid parameter
Diabetic rats showed significantly (P < 0.001) increased levels of STG, STC, VLDL-c and LDL-c levels, whereas HDL-c was decreased in diabetic rats compared to normal rats (Table 2). The markers of dyslipidemia such as TC/HDL-c and LDL-c/HDL-c ratios were significantly elevated in the diabetic group. Oral administration of different doses of ASE and ASW for fifteen-days exhibited significant reduction (P < 0.001) in all tested lipid parameters and restoring them to near-normal values (Table 2).
Glucose uptake by isolated hemi-diaphragm of diabetic rats
Normal rats showed 2.154 mg glucose uptake per gram of diaphragm tissue, whereas fifteen-day post STZ (50 mg/kg, i.v.) treated rats showed significant (P < 0.01) reduction in glucose uptake (0.328 mg/g of tissue). This observation suggested that these STZ administered rats were under insulin resistance condition (even though these STZ rats exhibited type I diabetic condition as evident by lower insulin levels post glucose challenge and HOMA values). Furthermore, this observation (reduction in glucose uptake by diaphragm of diabetic rats) was in line with data of insulin tolerance test.
Treatment of insulin (62 µU) caused significant (P < 0.001) stimulation of glucose uptake leading Fig. 8. Effect of ethanol (ASE) and water extract
(ASW) of Argyreia speciosa on insulin tolerance in six h fasted diabetic rats. A) SG levels were measured prior to, and after p.o. administration of insulin alone (2U/
kg body weight), or in combination with ASE, ASW or GLB. B) Area under curve for glucose (AUCglucose) values for 0 - 30 min post insulin injection. Data represent the mean ± S.E.M., for 5 rats. aP < 0.05; bP <
0.001; cP < 0.001 as compared with diabetic rats (one way ANOVA followed by Tukey’s post-test).
Table 2. Effect of ethanol (ASE) and water extract (ASW) of Argyreia speciosa on Lipid Profile in STZ-induced diabetic rats (multiple dose fifteen-days study)
Serum parameter Normal control
Diabetic control
ASE 100mg/kg
ASE 200mg/kg
ASW 100mg/kg
ASW 200mg/kg
GLB 0.5 mg/kg STG(mg/dl) 84.9 ± 3.13 131.77 ± 7.5 84.9 ± 3.13c 84.04 ± 5.5c 62.2 ± 13.0c 88.9 ± 4.3c 91.9 ± 6.7c STC(mg/dl) 68.41 ± 3.9 126.62 ± 4.7 68.41 ± 3.13c 68.68 ± 3.8c 59.9 ± 8.9c 70.5 ± 3.2c 82.9 ± 6.9c HDL-c(mg/dl) 34.1 ± 2.0 14.52 ± 1.8 30.03 ± 3.24 28.75 ± 3.8 19.3 ± 1.6 25.2 ± 3.2 26.6 ± 1.9 VLDL-c(mg/dl) 12.8 ± 0.7 26.35 ± 1.5 16.98 ± 0.63c 16.81 ± 1.1c 12.4 ± 2.6c 17.8 ± 0.9c 18.4 ± 1.3c LDL-c(mg/dl) 16.3 ± 3.3 85.75 ± 3.4 21.4 ± 5.46c 23.12 ± 6.5c 28.1 ± 5.8c 27.6 ± 5.6c 37.9 ± 8.5c TC/HDL-c ratio 1.9 ± 0.1 9.17 ± 1.2 2.35 ± 0.24c 2.57 ± 0.5c 3.1 ± 0.4c 2.9 ± 0.5c 3.2 ± 0.4c LDL-c/HDL- c ratio 0.5 ± 0.1 6.26 ± 0.9 0.77 ± 0.21c 0.96 ± 0.4c 1.5 ± 0.3c 1.2 ± 0.4c 1.5 ± 0.4c Each value represent mean ± S.E.M. n = 5; cP < 0.001 compared to Diabetic control. One way ANOVA followed by Tukey’s post test
to ~5 fold increase compared to diabetic control values whereas, treatment of different doses of ASE (50 and 100 µg/ml) and ASW (50 and 100 µg/ml) failed to stimulate glucose uptake.
DISCUSSION
Diabetes mellitus is a metabolic disease as old as mankind and is characterized by hyperglycemia associated with impairment in insulin secretion/
action along with altered carbohydrate, protein and lipid metabolism. The function of insulin is to maintain normal blood glucose levels either by suppression of glucose output from liver or by the stimulation of glucose uptake and its metabolism.
Insufficient release of insulin or loss of insulin action at target tissues causes abnormal glucose and lipid metabolism. This results in elevated glucose levels in blood, the hallmark of diabetes.
Type-1 diabetes results from autoimmune destruction of pancreatic b-cells resulting in insulin deficiency.
Before the introduction of insulin in 1922 the treatment of diabetes mellitus relied heavily on dietary measures which included the use of traditional plant therapies. Many traditional plant treatments for diabetes exist (Gray et al., 1997, 1999). However, few have received scientific or medical scrutiny and the World Health Organization has recommended that traditional plant treatments for diabetes warrant further evaluation (WHO, 1980). Insulin therapy affords effective glycemic control, yet its drawbacks such as ineffectiveness on oral administration, short shelf life, need for constant refrigeration and hypoglycemia on excess dosage limits its usage (Kasiviswanath et al., 2005). Therefore efforts continue to find insulin substitutes from synthetic or plant sources.
STZ-induced experimental diabetes is a valuable model for induction of type 1 diabetes.
Further it is generally accepted that severe diabetes (SD) is similar to type 1 and mild diabetes (MD) is similar to type 2 diabetes (Matti et al., 2005;
Sharma et al., 2006). To our knowledge, this is the
first detailed study to investigate the effect of ethanol (ASE) and aqueous (ASW) fractions of Argyreia speciosa root on the glucose levels, lipid profile in STZ-induced diabetic rats.
The severity and insulin resistance (reduced peripheral utilization of glucose) condition in diabetic rats induced by STZ (50 mg/kg, i.v) was confirmed by lower insulin levels post glucose- challenge, HOMA (measure of insulin resistance) values and lack of glucose uptake by isolated hemi-diaphragm of diabetic rats. Both ASE and ASW produced hypoglycemia and improved glucose tolerance in normal rats in spite of counter regulatory factors avoiding reduction in blood glucose levels. Therefore hypoglycemic activity of ASE and ASW could be mediated by stimulation of surviving β-cells to release more insulin and may be through extra-pancreatic mechanisms. Furthermore, treatment of ASE and ASW significantly improve the sucrose tolerance in normal rats, suggested that the components of extracts may inhibit a-glucosidase, a membrane bound enzyme located on the epithelium of the small intestine, catalyzing the cleavage of disaccharides to form glucose. Therefore, inhibitors can retard the uptake of dietary carbohydrates and suppress post-prandial hyperglycemia. STZ is well known for its selective pancreatic islet β-cell cytotoxicity and has been extensively used to induce DM in animals. It interferes with cellular metabolic oxidative mechanisms.
The present data suggested that ASE and ASW significantly reduced hyperglycemia in both single- dose one-day and multiple-dose fifteen-day diabetic studies. The efficacy of the ASE is better than ASW. This could be mediated by improving the glycemic control mechanisms (extra-pancreatic) and increasing insulin secretion from remnant pancreatic b-cells in diabetic rats.
DM is often linked with abnormal lipid metabolism. The impairment of insulin secretion results in enhanced metabolism of lipids from the adipose tissue to the plasma (Ananthan et al.,
2004). It has been demonstrated that insulin deficiency in diabetes leads to a variety of disruptive changes in metabolic and regulatory processes, which in turn lead to accumulation of lipids (Goldberg, 1981). It has also been shown that insulin significantly normalizes lipid levels in diabetic rats (Pathak et al., 1981). The extract supplementation also results in significant attenuation in STG, STC, VLDL-c, and LDL-c. These effects may be due to low activity of cholesterol biosynthesis enzymes or low levels of lipolysis. Increased TC/
HDL-c and LDL-c/HDL-c ratios are well known markers of dyslipidemia in STZ-induced diabetic rats (Rajkumar et al., 2005). ASE and ASW administration reinstated dyslipidemic markers to near-normal values.
The phytochemical examination of ethanol (ASE) and water (ASW) extract of Argyreia speciosa revealed the presence of alkaloids, carbohydrate, flavanoids, tannins and phenolics.Several authors have reported flavanoids, phenolics and steroidal glycoside as bioactive antidiabetic principles (Shani et al., 1974; Punitha et al., 2006). In addition, we reported antimicrobial, adaptogenic potentiality of flavanoid sulphates and their aglycones (quercetin and kaempferol) from the title plant (Habbu et al., 2009, 2010). The observed antidiabetic and hypolipidaemic activity of title plant may be attributed to the presence of these bioactive principles and their synergistic properties. Therefore we conclude that the ethanol (ASE) and water (ASW) extract of Argyreia speciosa root has endowed with anti-diabetic (single-dose one-day study and multi- dose fifteen-day study), anti-hyperlipidaemic activity in standardized STZ-induced diabetic rats, justifying its use in the traditional system of medicine.
ACKNOWLEDGEMENTS
Authors are thankful to President, Soniya education trust and Principal SET’s College of Pharmacy, Dharwad for providing necessary facilities and Dr. G R Hegde, Karnatak University, Dharwad,
India for identification and authentication of plant material.
REFERENCES
Ananthan R, Latha M, Pari L, Baskar C, Narmatha V.
(2004) Modulatory effects of Gymnema montanum leaf extract on alloxan induced oxidative stress in wistar rats. Nutrition 20, 280-285.
Ashok K, Tiwari J, Madhusudana Rao. (2001) Diabetes mellitus and multiple therapeutic approaches of phytochemicals. Present status and future prospects Pharmacology Division and Natural Product Laboratory, Organic-I Division, Indian Institute of Chemical Technology, Hyderabad: India.
Chattopadhyay RR, Sarkar SK, Ganguly S, Banerjee RN, Basu TK. (1992) Effect of extract of leaves of Vinca rosea Linn. on glucose utilization and glycogen deposition by isolated rat hemidiaphragm. Ind. J.
Physiol. Pharmacol. 36, 137-138.
Friedewald WT, Levy RI, Fredrickson DS. (1972) Estimation of low-density lipoprotein cholesterol in plasma, without use of the preparative centrifuge.
Clin. Chem. 18, 499.
Ghosh R, Sharatchandra KH, Rita S, Thokchom IS.
(2004) Hypoglycemic activity of Ficus hispida (bark) in normal and diabetic rats. Ind. J. Pharmacol. 36, 222-225.
Gokhale AB, Damre AS & Saraf MN. (2003) Investigations in to the Immunomodulatory activity of Argyreia speciosa. J. Ethnopharmacol. 84, 109-114.
Gokhale AB, Damre AS, Kulkarni KR & Saraf MN.
(2002) Preliminary evaluation of anti-inflammatory and anti-arthritic activity of S. lappa, A. speciosa and A. aspera. Phytomedicine 9, 433-437.
Goldberg RB. (1981) Lipid disorders in diabetes.
Diabetes Care 4, 561-572.
Gray AM, Flatt PR. (1997) Nature’s own pharmacy:
the diabetes perspective. Proc. Nut. Soc. 56, 507-517.
Gray AM, Flatt PR. (1999) Insulin-secreting activity of the traditional antidiabetic plant Viscum album (mistletoe). J. Endocrinol. 160, 409-414.
Habbu PV, Shastry R., Mahadevan KM, Joshi HK &
Das SK. (2008) Hepatoprotective and antioxidant effects of Argyreia speciosa in rats, Afr.J .Trad. Compl.
Altern. Med. 5, 158-164.
Habbu PV, Shastry RA, Mahadevan KM & Sneha C.
(2009) Antiamnesic activity of Argyreia speciosa in mice. Int. J. Green Pharm. (In Press).
Habbu PV, Shastry RA, Mahadevan KM, Manjunath H. (2009) Antimicrobial activity of flavanoids sulphates and other fractions of Argyreia speciosa (Burm.f.) Boj. Ind. J. Exp. Bio. 47, 121-127.
Habbu PV, Mahadevan KM, Kulkarni PV, Daultsingh C, Veerapur VP, Shastry RA (2010) Adaptogenic and in vitro antioxidant activity of Argyreia speciosa (Burm.f) Boj. In acute and chronic stress paradigms in rodents. Ind. J. Exp. Bio. 48, 53-60.
International Diabetes Federation. (2005) Position Statement. Brussels.
Kasiviswanath R, Ramesh A, Kumar KE. (2005), Hypoglycemic and antihyperglycemic effect of Gmelina asiatica Linn. in normal and Alloxan- induced diabetic rats. Biol. Pharm. Bull. 28, 729-732.
Lernmark A. Ott J. (1998) Sometimes it’s hot.
Sometimes its not. Nature Genet. 19, 213-218.
Li Y, Wen S, Kota BP, Peng G, Qian Li G, Yamahara J.
et al. (2004) Punica granatum flower extract, a potent a - glucosidase inhibitor, improves postprandial hyperglycemia in Zucker diabetic fatty rats. J.
Ethnopharmacol. 99, 239-244.
Matti R, Das UK, Ghosh D. (2005) Attenuation of hyperglycemia and hyperlipidemia in streptozotocin- induced diabetic rats by aqueous extract of seed of Tamarindus indica. Biol. Pharm. Bull. 28, 1172-1176.
Pathak RM, Ansari S, Mahnood A. (1981) Changes in chemical composition of intestinal brush border membrane in alloxan induced chronic diabetes. Ind.
J. Exp. Biol. 9, 503-505.
Petra M, Britta T, Macki K & Eckart E. (1999) Flavanoid sulphates from Convolvulaceae. Phytochemistry 50, 267.
Punitha R, Manoharan S. (2006) Antihyperglycemic and antilipidperoxidative effects of Pongamia pinnata (Linn.) Pierre flowers in alloxan induced diabetic rats. J. Ethanopharmacol. 105, 39-46.
Rajasekar P, Kaviarasan S, Anuradha, CV. (2005) L- carnitine administration prevents oxidative stress in high fructose-fed insulin resistant rats. Diabetologica Croatica 34, 21-28.
Rajkumar M, Uttam KD, Debidas G. (2005) Attenuation of hyperglycemia and hyperlipidemia in streptozotocin-
induced diabetic rats by aqueous extract of seed of Tamarindus indica. Biol. Pharm. Bull. 28, 1172-1176.
Roa KN, Kirishna MB, Srinivas N. (2004) Effect of chronic administration of Boerhaavia diffusa Linn.
Leaf extract on experimental diabetes in rats. Trop. J.
Pharm. Res. 3, 305-309.
Shani J, Goldschmied A, Joseph B, Ahronoson Z, Sulman FG. (1974) Hypoglycemic effect of Trigonella foenum graecum and Lupinus terminis (Leguminosae) seed and their major alkaloids in alloxan diabetic and normal rats. Arch. Inter. Pharmacodyna. Ther.
210, 27-31.
Sharma PC, Yelne MB, Dennis TJ. (2004) Database on medicinal plants used in Ayurveda. pp 550, Central Council for Research in Ayurveda and Siddha New Delhi.
Sharma SB, Nasir A, Prabhu KM, Murthy PS, Dev G.
(2003) Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxan-induced diabetic rabbits. J. Ethanopharmacol.
85, 201-206.
Siddique O, Sun Y, Lin JC, Chein YW. (1987) Facilitated transdermal transport of insulin. J. Pharmacol. Sci.
76, 341-345.
Srivastava A, Shukla Y. (1998) Aryl esters and a Coumarin from Argyreia speciosa, Ind. J. Chem. 37B, 192-194.
Swanston-Flatt SK, Flatt PR, Day C, Bailey CJ. (1991) Traditional dietary adjuncts for the treatment of diabetes mellitus. Proc. Nut. Soc. 50, 641-651.
Warrier PK, Nambiar VP, Ramankutty C. (1994) Indian medicinal plants-a compendium of 500 species. pp191. Orient Longman New Delhi.
Wild S, Roglic G, Green A, Sicree R, King H. (2004) Global prevalence of diabetes. Diabetes Care 27, 1047-1053.
World Health Organization. (1980) Second Report of the WHO Expert Committee on Diabetes Mellitus.
pp.66 Technical Report Series. 646, Geneva.
Yanardag R, Colak H. (1998) Effect of chard (Beta vulgaris L. var. cicla) on blood glucose levels in normal and alloxan-induced diabetic rabbits. Pharm.
Pharmacol. Commun. 4, 309-311.
