Sunday, July 31, 2011

Nilavembu-Good for Diabetics -An evidence base

Ravikumar. R * et al /International Journal Of Pharmacy & Technology
IJPT | Dec-2010 | Vol. 2 | Issue No.4 | 1016-1027
Page 1016
ISSN: 0975-766X
Available Online through Research Article
www.ijptonline.com
ANTIDIABETIC AND ANTIOXIDANT EFFICACY OF ANDROGRAPHIS PANICULATA IN
ALLOXANIZED ALBINO RATS
Ravikumar. R*, Krishnamoorthy. P, Kalidoss. A
Post Graduate and Research Department of Biotechnology.
JJ College of Arts and Science, Pudukkottai, Tamil Nadu, India- 622 422.
Email: ravimicrobiotech@gmail.com
Received on 12-10-2010 Accepted on 29-10-2010
Abstract: Antidiabetic potential and antioxidant activity of ethanol extract of Andrographis paniculata leaves were
assessed in alloxan-induced diabetic rats. Results revealed that, diabetic rats showed increase in blood glucose and
decrease in plasma insulin (p<0.01) levels after 48 hrs of alloxan administration. The oral administration of ethanol
extract at a dose of 100 and 200 mg/kg of body weight, for 30 days exhibited a significant reduction in the blood
glucose level compared to the standard drug-treated rats and diabetic control. The level of serum triglycerides,
phospholipids and total cholesterol were significantly increased in diabetic rats. The concentration of urine,
glucose, glycosylated Haemoglobin was significantly increased, whereas haemoglobin, total protein, albumin and
liver glycogen were significantly decreased in diabetic animals. Administration of A. paniculata leaf extract
decreased the diabetic complication. The marker enzymes of liver toxicity such as serum alanine transaminase
(ALT), serum aspartate transaminase (AST), serum acid phosphatase (ACP) and serum alkaline phosphatase (ALP)
were elevated significantly in diabetic control. The liver glycogen levels also increased significantly in alloxaninduced
diabetic rats. Catalase and vitamin C levels were increased Andrographis paniculata fed rats.
Phytotherapy with the A. paniculata ethanol extract showed significant restoration in enzymatic and non enzymatic
activities in diabetic animals. The phyto-treatment showed more efficient antihyperglycemic effect than the
standard drug glibenclamide.
Keywords: Andrographis paniculata, alloxan, antidaiabetic, antioxidant, blood-glucose, insulin
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Introduction
Diabetes is a serious metabolic disorder with micro and macro vascular complications that results in
significant morbidity and mortality. The increasing number of ageing population, consumption of calories rich diet,
obesity and sedentary life style have led to increase the number of diabetes world wide. The current treatment,
although provide a good glycemic control but do a little in preventing complications (Vats et al., 2004). There is an
increased demand to use natural products with antidiabetic activity due to the side effects associated with the use of
insulin and oral hypoglycemic agents (Holman et al., 1991). The World Health Organization (WHO) (1980) has
also recommended the evaluation of the effectiveness of plants in condition where we lack safe modern drugs
(Upathaya et al., 1991). The pharmaceutical drugs are either too expensive or have undesirable side effects.
Treatment with sulphonylureas and biguanides are also associated with side effects (Rang et al., 1991).
Andrographis paniculata (Burm.f) Nees is used extensively in the Indian traditional system of medicine. In the
present investigation antidiabetic efficacy of the plant extract of Andrographis paniculata on alloxan induced
diabetic rats.
Study area:
Materials and Methods
Adult Wistar rats weighing 160-200 g of either sex were maintained in large polypropylene cages in a wellventilated
room temperature with natural day-night cycle and fed with balanced rodent pellet and water ad libitum
throughout the experimental period. They were quarantined for 1 week prior to the experiments to acclimatize them
to laboratory conditions. The study protocol was approved by the IAEC (Institutional Animal Ethics Committee of
CPCSEA, New Delhi, Govt. of India). Fresh whole plants of Andrographis paniculata were collected from
Sivapuram Pudukkottai District, during the months of September-December 2006 and identified by Dr.P.
Jayaraman (Director), Plant Anatomy Reasearch centre, Chennai. The leaves were collected and dried in shade for
15 days and made to coarse powder and extracted with ethanol by the method of Harborne, 1973. The extract was
preserved in an air tight container, in a refrigerator and used to evaluate the antidiabetic and antioxidant efficacy.
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Alloxan was purchased from Loba chemie. All the other chemicals used in the experiment were analytical
grade. A total of 36 rats (30 diabetic surviving rats, 6 normal rats) were experimented. The rats were divided into 6
groups of 6 rats each. Rats were orally fed with two doses of ethanol extracts (100 and 200 mg/kg body weight per
day) and glibenclamide (600μg/kg). Experimental diabetes was induced by using alloxan monohydrate following
the method of Trivadi et al., 2004.
Blood samples were drawn at weekly intervals till the end of study (i.e., 3 weeks). At the end of 3 weeks, all
the rats were killed by decapitation under pentobarbitone sodium (60 mg/kg) anesthesia. Blood was collected in
tubes containing potassium oxalate and sodium fluoride solution for the estimation of blood glucose and plasma
was separated for assay of insulin. Liver and kidney were dissected out, washed in ice cold saline, patted dry and
weighed. The following biochemical analyses were carried out: Urine Sugar was detected by reagent based Uristix
from Bayer Diagnostics. Total protein, Blood Glucose (Sasaki et al., 1972), estimation of haemoglobin (Dacie and
Lewis, 1977), glycosylated haemoglobin (Bannon, 1982), liver glycogen (Morales et al., 1973), Cholesterol (Zlatkis
et al., 1953), Triglycerides (Foster and Dunn, 1973), Estimation of phospholipids (Rouser et al., 1970), estimation
of Protein (Lowry et al., 1951), estimation of serum albumin, (Reinhold, 1953), Determination of serum aspartete
transaminase (Mohun and Cook, 1957), determination of serum alanine transaminase (Mohun and Cook, 1957),
alkaline Phosphatase (King, 1965), acid phosphatase (Gutman and Gutman, 1940), estimation of urea (Natelson,
1957) assay of catalase (Sinha, 1972) and Vit C (Omaye et al., 1979). Statistical analysis was performed using the
SPSS 11.5 software package. Group mean values were compared with Duncan’s Multiple Range Test and analyzed
with one way Analysis of Variance (ANOVA).
Results
Urine sugar was significantly increased in diabetic rats when compare to the control rats,after treatment of
Andrographis paniculata urine sugar level is significantly decreased when compare to the diabetic control rats. A
significant increase in blood glucose and significant reduction in body weight were observed in diabetic rats,
compared to control rats. Oral administration of Andrographis paniculata (100 and 200 mg/kg body weight) for 21
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days shows significant reduction in blood glucose compared to untreated diabetic rats. Blood glucose levels were
found to be increased significantly by % in diabetic control, whereas the rats treated with 100 mg/kg of
Andrographis paniculata showed a decrease at % and 200 mg/kg at %. The decrease in blood glucose levels
was significantly better than the diabetic rats treated with glibenclamide.Haemoglobin levels were found to be
decreased significantly in alloxanized diabetic rats. Liver glycogen,total protein, and albumin were significantly
decreased in diabetic rats when compare to the normal control rats.Administration of Andrographis paniculata
extract significant changes in liver glycogen, total protein and albumin.There was a significant reduction in
heamoglobin and serum protein while glycosylated hemoglobin and blood urea significantly increased in diabetic
rats when compared with control rats. Oral administration of Andrographis paniculata (100 and 200 mg/kg b.wt)
significantly restored the value to near normal. Control animals administered with Andrographis paniculata 200
mg/kg body weight did not show any significant change in any of the parameters studied. A significant increase in
the total cholesterol, triglycerides,urea and phospholipids were found in the liver and kidney of the diabetic rats
compared to the tissue of normal control rats. SGOT, SGPT, ACP and ALP were significantly increased in diabetic
rats when compared to the normal control rats. Catalase and vitamin C levels were significantly decreased in the
serum of the diabetic rats, compared to the normal control rats. Analyzed data are expressed as means with their
standard deviations.
Table-1: Antidiabetic efficacy of Andrographis paniculata.
Experimental
Group
Urine
sugar
Blood glucose
(mg/dl)
Haemoglobin
(g/dl)
Glycosylated
haemoglobin
(mg/Hb)
Liver glycogen
(Mg/g tissue)
Total protein
(g/dl)
Albumin
(g/dl)
Urea
(mg/dl)
Normal control Nil 96.0±3.23a 14.0±0.54c 1.61±0.03a 3.9±0.16d 6.92±0.45d 4.13±0.24d 28.4±2.10a
Diabetic control +++ 224.10±6.67c 9.93±0.42a 3.2±0.14c 2.4±0.08b 4.93±0.26a 3.1±0.26a 39.2±2.72d
Diabetic +APE
(100) mg/kg
+ 134.21±5.38ab 11.5±0.32b 2.21±0.08bc 2.9±0.06a 5.26±0.51b 3.8±0.32bc 35.2±3.1c
Diabetic +APE
(200mg/kg)
+ 124.34±4.9b 13.21±0.52bc 1.91±0.07a 3.4±0.14cd 6.12±0.28cd 3.91±0.36c 29.6±3.2b
Diabetic +
glibenglamide
+ 126.32±3.8b 13.19±0.63bc 2.1±0.08b 3.1±0.12c 5.93±0.35c 3.73±0.29b 31.2±2.9b
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Values are expressed as Mean ± Standard Deviation (SD); Level of significance=0.05; APE= Andrographis
paniculata extract
Table-2: Effect of Andrographis paniculata leaf extract on total cholesterol, triglyceroids and
phospholipids.
Experimental
Group
Total
cholesterol
(mg/dl)
Triglycerides
(mg/dl)
Phospolipids
(mg/dl)
Plasma Liver Kidney Plasma Liver Kidney Plasma Liver Kidney
Normal control 4.25±
0.22a
105.16±
5.69a
5.25±
0.27a
5.05±
0.25a
72.13±
3.71a
41.50±
2.06a
94.00±
4.48a
82.00±
4.10a
148.62±
7.49a
Diabetic control 9.50±
0.45d
230.12±
11.25d
9.98±
0.49d
11.01±
0.56d
124.62 ±
6.23d
73.07±
3.80d
158.01±
8.26d
152.01±
6.32d
188.38±
9.57d
Diabetic+APE
(100mg/kg)
5.12±
0.23c
154.13±
6.82c
7.23±
0.52c
8.01±
0.43c
110.52±
5.83c
65.91±
3.01c
112.21±
5.44c
113.52±
5.2c
172.31±
7.21c
Diabetic+APE
(200mg/kg)
4.51±
0.33b
125.42±
5.42b
5.75±
0.30a
7.32±
0.31b
83.84±
4.64b
52.0±
2.71b
107.43±
4.23b
96.36±
5.28b
161.90±
8.10bc
Diabetic +
glibenglamide
5.25±
0.26b
130.12±
6.19bc
6.32±
0.29b
7.90±
0.38b
92.71±
4.48bc
56.50±
2.96bc
108.21±
3.39bc
100.52±
5.87b
154.00±
7.74b
Values are expressed as Mean ± Standard Deviation (SD); Level of significance=0.05;
APE= Andrographis paniculata extract
Table-3: Antioxidant efficacy of Andrographis paniculata leaves.
Experimental
Group
SGOT
(IU/L)
SGPT
(U/L)
Acid
phospatase
(IU/L)
Alkaline
phospatase
(IU/L)
Catalase
(μ moles of
H2O2
utlized/min
/mg/protein)
Vitamin C
(mg/dL-1)
Normal control 78.54±3.88a 67.04±3.43a 5.67±0.29a 9.56±0.48a 24.70±1.18 12.50±0.62
Diabetic control 147.50 ±6.76d 95.78±4.96d 8.50±0.48d 23.85± 1.41d 14.10±0.82 7.50±0.53
Diabetic +APE
(100 mg/kg)
110.50±7.21c 82.13±3.56c 7.21±0.52c 14.81±0.51c 18.82±1.06 8.93±0.62
Diabetic +APE
(200mg/kg)
Diabetic +
glibenglamide
97.70±5.21bc
90.03±4.53b
78.54±4.23b
82.37±4.13c
6.02±0.33b
7.08±0.41bc
11.68±0.65b
12.75±0.72bc
21.06± 1.01
17.87±0.93
11.02±0.61
10.21±0.67
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Values are expressed as Mean ± Standard Deviation (SD); Level of significance=0.05;
APE= Andrographis paniculata extract
Discussion
Blood sugar increased in alloxan-injected animals, since alloxan causes a massive reduction in insulin release,
by the destruction of the beta-cells of the Islets of Langerhans and inducing hyperglycemia (Goldner and Gomori,
1943). Oral administration of (100 and 200 mg/kg Andrographis paniculata body wt.) resulted in a significant
reduction in the blood glucose. Protein synthesis is decreased in all tissues due to absolute or relative deficiency of
insulin in alloxan-induced diabetic rats (Jorda et al.,1982). Present result protein level in diabetic rats significantly
decreased when compares to the control rats. . A significant elevation in serum creatinine and urea levels indicate an
impaired renal function of diabetic animals.( Shinde and Goyal,2003). In the present study urea level was
significantly increased in diabetic rats.
The present study also indicates that Andrographis paniculata can inhibit alloxan renal toxicity as evident from
the blood urea level. In the present study, the glycosylated hemoglobin level was high showing that the diabetic
animals had high blood glucose level. The values decreased significantly in Andrographis paniculata administered
animals showing the influence of the leaf extracts on sugar reduction. Insulin influences the intracellular utilization
of glucose in a number of ways. Increase in glycogen in liver can be brought about by an increase in glycogenesis
and/or a decrease in glycogenolysis. So the Andrographis paniculata extract could have stimulated glycogenesis
and/or inhibited glycogenolysis in the diabetic rat liver. Insulin and sulfonylurea drugs (e.g., glibenclamide) cause
hypoglycemia when taken in excessive doses and overt hypoglycemia is the most worrisome effect of these drugs
(Chakrabarti and Rajagopalan, 2002). The significant increase in the level of triglycerides in liver and kidney of
diabetic control rats may be due to the lack of insulin. Since under normal condition, insulin activates the enzyme
lipoprotein lipase and hydrolysis triglycerides (Frayn, 1993). Andrographis paniculata reduces triglycerides,
cholesterol and phospolipids in tissues of alloxan-induced diabetic rats. Further more, the present study reveals,
higher levels of triglycerides, cholesterol and phospholipids in diabetic rats (Khosla, 1995).The decrease in protein
and albumin may be due to microproteinuria and albuminuria, which are important clinical markers of diabetic
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nephropathy (Mauer et al., 1981) and/or may be due to increased protein catabolism (Almdal and Vilstrup, 1988).
The results of the present study demonstrated that the treatment of diabetic rats with the aqueous extract of
Andrographis paniculata caused a noticeable elevation in the plasma total protein and albumin levels as compared
with their normal levels. Such improvement of serum protein and albumin was previously observed after the oral
administration of Balanites aegyptiaca to experimentally diabetic rats (Mansour and Newairy, 2000). A significant
elevation in serum urea levels indicates an impaired renal function of diabetic animals (Shinde and Goyal, 2003).
Thus, it would appear that the Andrographis paniculata leaves supplement lowered the plasma urea levels by
enhancing the renal function that is generally impaired in diabetic rats. These results are similar to other previous
studies on the mesocarp extract of B. aegyptiaca (Saeed et al., 1995) and herbal formulation D-400 (Dubey et al.,
1994). The serum AST and ALT levels increased as a result of metabolic changes in the liver, such as
administration of toxin, cirrhosis of the liver, hepatitis and liver cancer including diabetes (Chalasani et al., 2004).
Similarly in the present study, it was observed that the levels of serum AST and ALT in alloxan induced diabetic
rats were elevated. It may be due to leaking of enzymes from the tissues and migrating into the circulation by the
adverse effect of alloxan (Stanely, 1999). AST and ALT were used as markers to assess the extent of liver damage
in streptozotocin induced diabetic rats (Hye et al., 2005). In this study, the administration of ethanol extract to
alloxan-induced diabetic rats reduces AST and ALT levels.
The serum AST and ALT levels increase as a result of metabolic changes in the liver, such as
administration of toxin, cirrhosis of the liver, hepatitis and liver cancer including diabetes.44 Similarly in the
present study, it was observed that the levels of serum AST and ALT in alloxan induced diabetic rats were elevated.
It may be due to leaking out of enzymes from the tissues and migrating into the circulation by the adverse effect of
alloxan.45 AST and ALT were used as markers to assess the extent of liver damage in streptozotocin induced
diabetic rats.46 In this study, the administration of water soluble fraction of ethanol extract to alloxan-induced
diabetic rats reduces AST and ALT levels efficiently than aqueous extract treated rats. In addition to the assessment
of AST and ALT levels during diabetes, the measurement of enzymatic activities of phosphatases such as acid
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phosphatase (ACP) and alkaline phosphatase (ALP) is of clinical and toxicological importance as changes in their
activities are indicative of tissue damage by toxicants.47 In our study, serum ACP and ALP increased considerably
in alloxan induced diabetic rats. Elevated level of these enzymes in diabetes may be due to extensive damage to
liver in the experimental animals by alloxan. Treatment with water soluble fraction of ethanol extract in alloxaninduced
diabetic rats produces a more significant decline in these levels than the aqueous extract treated rats. From
the present observation, it was evident that water soluble fraction of ethanol extract protects the adverse effects of
lipid peroxide mediated tissue damage in alloxan induced diabetic rats.
In addition to the assessment of AST and ALT levels during diabetes, the measurement of enzymatic
activities of phosphatases such as acid phosphatase (ACP) and alkaline phosphatase (ALP) is of clinical and
toxicological importance as changes in their activities are indicative of tissue damage by toxicants (Som Nath
Singh, et al., 2001). In Present study, serum ACP and ALP increased considerably in alloxan induced diabetic rats.
Elevated level of these enzymes in diabetes may be due to extensive damage to liver in the experimental animals by
alloxan.
Catalase causes reduction of H2O2 whereas GPx reduces H2O2 and lipid peroxides (Shull and Marsh, 1991).
Several studies have demonstrated lowered non-enzymatic antioxidant levels and enzymatic antioxidant activities in
streptozotocin induced diabetic rats (Ananthan et al., 2004; Venkateswaran, 2002). In the present study A.
paniculata leaf extract is found to increase serum concentrations of catalase (CAT). Vitamin C, a major extra
cellular non-enzymatic antioxidant, has crucial role in scavenging several reactive oxygen species. It functions as a
free-radical scavenger and successfully prevents detectable oxidative damage under all types of oxidative stress.
Reduction in tissue ascorbic acid was observed in STZ-diabetic rats. The decrease could have been due to increased
utilization of ascorbic acid as an antioxidant defense against increased reactive oxygen species or to a decrease in
the GSH level, since GSH is required for the recycling of ascorbic acid (Hunt, 1996). The present study reveals that
considerable changes in all parameters after the treatment of A. paniculata leaf extract.
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Conclusion: Therefore, A. Paniculata an indigenous herb shows potential antioxidant and antidiabetic properties,
which could be due to the presence of potent antihyperglycaemic factors. Further study is underway in our
laboratory to isolate the active principles.
Acknowledgement: Authors are thankful to the management of J.J College of Arts and Science, Pudukkottai,
Tamil Nadu, India for providing the general basic laboratory facilities.
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Corresponding Auhtor:
Ravikumar. R*
Email: ravimicrobiotech@gmail.com

3 comments:

Dr.G.சிவராமன் said...

Hi Sathya,
good one.
One suggestion, when you post please make sure the text, table alignment are right..that will give pleasant reading experience. Thanks

Kannan Siddha said...

dear sathya useful information. thank u

Kannan Siddha said...

hi sathya very useful information thank u