|Year : 2014 | Volume
| Issue : 4 | Page : 442-446
Dolichos biflorus Linn. ameliorates diabetic complications in streptozotocin induced diabetic rats
Yogesh Saxena1, Brijesh Purwar1, Harsh Meena2, Parth Sarthi3
1 Department of Physiology, Himalayan Institute of Medical Sciences, SRH University, Dehradun, Uttarakhand, India
2 Department of Pathology, Himalayan Institute of Medical Sciences, SRH University, Dehradun, Uttarakhand, India
3 Department of Physiology, Integral Institute of Medical Sciences and Research, Integral University, Lucknow, Uttar Pradesh, India
|Date of Web Publication||18-Jun-2015|
Asso. Prof., Department of Physiology, Himalayan Institute of Medical Sciences, SRH University, Dehradun - 248 140, Uttarakhand
Source of Support: SRH University, Dehradun, Conflict of Interest: None
| Abstract|| |
Background: Horsegram (Dolichos biflorus Linn.) is a known antilithiatic, hypolipedemic and has free radical scavenging activity and increased production of reactive oxygen species play a role in pathophysiological mechanisms that trigger diabetic complications. Aim: To see the effect of daily oral feeding of D.biflorous on nephropathy and retinopathy in streptozotocin (STZ) induced-diabetic rats. Materials and Methods: A total of 24 healthy rats were randomly grouped into controls, diabetic and diabetic on Dolichos. Diabetes was induced by a single dose of STZ (55 mg/kg) and animals were given prepared food and water ad libitum. Dolichos was orally given at 300 mg/kg/day to rats in diabetic on Dolichos group for next 30 days. Fasting blood glucose levels was monitored at beginning and at the end of the experiment while assessment of serum creatinine levels and histopathological study of kidney and retina was carried only at the end of the experiment. Statistical differences between groups were analyzed using analysis of variance followed by, Bonferroni test as posthoc test. Results: Results indicated improvement in serum creatinine levels and reduced glomerular sclerosing and Bowman's space with interstitial alterations and significantly reduced renal hypertrophy in diabetic rat son Dolichos diabetic rats (P < 0.001). Retinal layers showed inconsistent improvement in the width of the neuronal layers and decreased vacuolization of plexiform layers and retinal vessel density. Conclusion: D. biflorus at doses of 300 mg/kg/day for 30 days resulted in gradual but significant decreased diabetic nephropathy.
Keywords: Diabetic nephropathy, diabetic retinopathy, Dolichos biflorus, experimental diabetes
|How to cite this article:|
Saxena Y, Purwar B, Meena H, Sarthi P. Dolichos biflorus Linn. ameliorates diabetic complications in streptozotocin induced diabetic rats. AYU 2014;35:442-6
|How to cite this URL:|
Saxena Y, Purwar B, Meena H, Sarthi P. Dolichos biflorus Linn. ameliorates diabetic complications in streptozotocin induced diabetic rats. AYU [serial online] 2014 [cited 2020 Feb 18];35:442-6. Available from: http://www.ayujournal.org/text.asp?2014/35/4/442/159022
| Introduction|| |
Diabetes mellitusis a global epidemic with ~20% of the diabetics residing in South-East Asia  and is associated with vascular and non-vascular complications. [2,3] Progressive complications like retinopathy leading to blindness [4,5] and nephropathy leading to renal failure [5,6] enhances the outlook of this metabolic disorder. Obvious shared features include their dependence on duration, accumulation of metabolites independent of insulin involving vessels and basement membrane of tissues.
Putative pathogenic mechanisms involves the increased production of reactive oxygen species [7,8] and impaired sorbitol pathway  leading to deposition of advanced glycosylated products and hemodynamic changes. 
Although insulin therapy is effective in prevention of complications, it has its demerits of daily parental administration, variability of effectiveness and high incurred cost.
Dolichos biflorous Linn. known as "Kulthi0" is widely grown and consumed in hills of Uttrakhand. It is a common ingredient of several of Ayurvedic preparations for bowel disorders and renal stones.  Evidence suggest that it has antilithotic,  antihepatotoxic,  hypolipedemic,  sugar lowering effect [15,16] and has potential antioxidant and free radical scavenging property. 
In the above context study was conducted to assess the effect of D. biflorous on diabetic complications of retinopathy and nephropathyin experimental diabetic rat model.
| Materials and Methods|| |
The interventional study was conducted at the Department of Physiology of HIHT University during a period of 2 months. Clearance for this study was taken from the Institutional Animal Ethics Committee (IAEC) of the university (No. HIHTPHARMA/I-1/2010/1954; dt. 18.02.10). Adult Wistar stain rats weighting 120-150 g (30-45 days old) were used for experimental diabetes models.
Streptozotocin (STZ) was used to produce experimental diabetes. It was supplied by Sigma Chemicals Co., St. Louis, MO, USA. A single dose of freshly prepared buffered solution of STZ (Inj. streptozotocin was prepared by dissolving it in 3 ml of 0.1 Mcold sodium citrate buffer of pH 4.5) was used to produce experimental diabetes.
D. biflorus was procured as seed powder from Gem Energy Industry Ltd, Chennai (US patent no 5916567; authorized dealer) in the amount of 1 Kg.
Healthy adult Wistar rats weighing 120-150 g (30-45 days old) were procured from central animal house of the university. The animals were individually housed in labeled plastic cages (43 cm χ 29 cm χ 15 cm) under standard laboratory conditions. The room temperature was maintained at 25°C ± 3°C and animals were allowed freshly prepared food and water ad libitum. They were subjected to the regular cycle of the day and night and observed for gain in weight for 3-4 days.
A total of 24 rats were studied, which were randomly grouped under control, diabetic rats and diabetic rats on Dolichos. Each group comprised of 8 rats.
- Group I: Control rats
- Group II: Diabetic rats
- Group III: Diabetic rats on D. biflorus (n = 8).
Method of induction of diabetes (Group II and III)
Every time six healthy rats (STZ 50 mg vial sufficient for only 6 rats) were fasted and their fasting blood glucose (FBS) was assessed. Following anesthesia (Ether) each rat were weighed and single dose of freshly prepared STZ (at 55 mg/kg) was given in the tail vein using all aseptic precautions. Those animals whose FBS level exceeded 250 mg/dl at 72 h after injection were considered as diabetic. 
Control animals were injected with the equivalent amount of cold citrate buffer (pH 4.5). All the doses were administered at a volume not exceeding 1 ml/100 g body weight of rats. The site was cleaned and smeared with antibiotic ointment to prevent infection.
Following the procedure all the studied rats were fed freshly prepared feed and had access to water ad libitum. Rats of Group III were given measured Dolichos seed powder at of 300 mg/kg/day in suspension by intra gastric feeding tube (Ramson no. 6) for 30 days. At the 30 th day (0 day is the day of onset of diabetes), the rats were measured for weight, FBS, Serum creatinine and sacrificed for harvesting of tissues for histology.
Bio chemical analysis
- Measurement of Glucose: For the determination of blood glucose using Gluco-check (model: Abbot`s Glucometer XCE115-3098), whole blood was taken from the tail vein of all rats 
- Plasma creatinine: Blood samples for the measurement of blood chemistry were drawn into pre-chilled Ethylene di-amine tetra acetic acid (EDTA-containing tubes and immediately placed on ice. All tubes were centrifuged within several minutes of collection and the plasma samples were assayed for creatinine using RA-50 semi auto-analyzer.
For the study and comparison of pancreatic islets of Langerhans, retina and kidney, rats from all the groups were sacrificed by cervical dislocation and dissected for retrieval of pancreatic tissue, enucleation of eyeballs and resection of both the kidneys. The samples were fixed in 10% formalin, stained with H and E and photographed by microscope with × 20 and × 40 magnifications. In diabetic rats tissue of pancreatic Langerhans and the beta cells degenerated irreversibly.  The kidneys were observed for histological changes like glomerular thickening with decreased bowmen space, tubular vacuolization and moderate interstitial fibrosis [Figure 1]a-c. H and E sections of the retina were observed for decreased neuronal layer with thickening of basement membrane, blood vessels invasion and vacuolization of plexiform layers [Figure 2]a-c.
|Figure 1: Normal rat: Kidney at ×20 normal Bowman`s space with normal architecture and no evident sclerosis seen: Normal tissue. (b) Diabetic rat ×40: Giant retinal vessels occupying the ganglion layer and inner plexiform layer. Lumen filled with red blood cells; Lucentare as seen in inner plexiform. (c) Diabetic rat on Dolichos: Kidney at ×20: Bowman`s space are not decreased with minimal disruption of architecture; protein deposits are minimal with less tubular obliteration; minimal infilteration|
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|Figure 2: (a) Normal rat: All layer of retina are distinct and regularly arranged. (b) Diabetic rat ×40: Giant retinal vessels occupying the ganglion layer and inner plexiform layer. Lumen filled with red blood cells; Lucentare as seen in inner plexiform. (c) Diabetic rats on Dolichos: ×40: Less disruption of layers including photo receptors layer; few blood vessels in ganglion cell layer; little vacuolization in plexiform layer|
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The data were expressed as mean ± standard error of the mean statistical differences between groups were analyzed using analysis of variance (ANOVA) followed by, Bonferroni test as posthoc test. The difference was considered to be statistically significant at P < 0.05. Statistical software IBM SPSS (Version -17) was used for analysis.
| Results|| |
Rats that had received STZ became diabetic at a frequency of 75% (12/16 × 100). Weight and basal FBS levels of all the groups were not significantly different from each other. Diabetes was associated with reduced gain in body weight when compared with the control rats and the reduction was statistically significant (P = 0.008, 0.02). However, the reduction was less in diabetic rats on Dolichos. The data related to the body weight are shown in [Table 1].
No statistically significant difference was observed between groups in the mean FBS values prior to administration of STZ suggesting that all animals were non-diabetics. Repeated measure ANOVA for the differences in the mean values of the FBS between the groups suggested that FBS values of diabetic rats were higher at both 3 rd and 30 th day of development of diabetes and the difference was statistically significant (P < 0.001) in comparison to controls. Group III showed statistically significant difference to control at 3 rd day only (P < 0.001). Although higher value of FBS was observed on 30 th day in group III but it was not statistically significant when compared with the controls [Table 2].
No significant difference in creatinine mean values was observed between the groups. However there was a definite increase in the plasma creatinine levels in diabetic rats. The diabetic rats showed significant renal hypertrophy (ratio of renal weight/rat weight) which decreased in diabetic rats on Dolichos (0.85 ± 0.1; P < 0.001) [Table 3].
|Table 3: Biochemical analysis in blood and renal hypertrophy (kidney weight/rat weight)|
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The kidney specimens of the diabetic group showed markedly severe destruction in glomerular and tubule-interstitial lesions such as glomerular sclerosis, atrophy, interstitial expansion and interstitial cellular infiltration [Figure 1]b as compared with those of the control group [Figure 1]a. In the diabetic rats on D. biflorus, general morphology of glomerulus and tubule-interstitial lesions were much improved and showed quite normal appearance [Figure 1]c.
Retinal histology shows infiltration of blood vessels in the layers of retina (ganglion layer) and disruption of the plexiform layer with vacuolization and thickening of the basement membrane [Figure 2]b when compared with the normal rats [Figure 2]a. The retinal layers of the diabetic rats on Dolichos showed lesser development of the features of diabetes following 30 days of Dolichos only in five cases; however, rest of the three diabetic rats did show early changes of diabetic retinopathy (DR).
| Discussion|| |
Plants are part and parcel of human society from the dawn of civilization and extensive use of D. biflorus as food for both animals and human beings is well-known. The seeds are consumed by humans after cooking or frying. They are eaten whole or after grinding into a meal, unlike other seeds, which are consumed after splitting.
The present study provided evidence those 30 days of D. Biflorus at 300 mg/kg/day may provide benefit in the diabetic nephropathy, but is less likely to prevent the development of DR. Dolichos treated diabetic rats had significantly decreased bowman's space, less sclerosis and inflammatory cells when compared with diabetic rats. In diabetes mellitus, increased blood glucose, lipids, oxidized low-density lipoprotein and oxygen free radicals can induce glomerulo-sclerosis and chronic tubule-interstitial damage in the kidneys leading to DN. [3,20] A progressive decline in the glomerular filtration rate due to loss of functioning nephrons alters the physiological functioning of diabetic kidney.  Elevated serum creatinine seen in diabetic rats levels was not elevated in Dolichos treated diabetic rats. Decreased blood glucose levels in these Dolichos treated rats, shows that the effect is dependent on the serum glucose levels, which were decreased to near normal in them. The markedly reduced total body weight with elevated kidney weight (renal hypertrophy) of the diabetic group when compared with control group was significantly decreased with amelioration of sclerosis of glomerulus and decreased interstitial infiltration during 30 days of the supplementation of Dolichos seed powder at 300 mg/kg in group of diabetic rats on Dolichos.
Microvascular changes in DR might be related to hyperglycemia-induced intramural pericyte death and thickening of the basement membrane and may lead to disruption of the blood-retinal barrier.  In present study, retinal vessel density is significantly increased, with thickened basement membrane and vacuolization of plexiform layer seen in diabetic animals as compared to normal rats. Intracellular hyperglycemias has been linked to an overproduction of extracellular matrix; hyperglycemia may also decrease production of trophic factors for endothelial and neuronal cells and the intracellular oxidative stress in endothelial cells plays a key role in endothelial dysfunction.  Dolichos treated rats showed variable response to the Dolichos treatment for 30 days. Some of the diabetic rats showed decreased thinness of retinal layers with less disruption of ganglionic and plexiform layers when treated with D. biflorus for 30 days while others did show changes in DR even after Dolichos intervention.
In diabetes, there is also increased production of free radicals  that can initiate per-oxidation of lipids, which in turn stimulates glycation of protein and causes long-term complications.  Researchers have shown antioxidant potential of methanolic extract of D.biflorus, thus it may reduce long-term complication produced in diabetes.  Dolichos is known for its antioxidant and free radical scavenging effect [17,26] and may therefore decrease the oxidative damage of the membrane lipids in renal tissues. Earlier studies have shown its hypolipidemic effect,  which may also contribute to the lesser degree of damage in the renal tissue.
Fibers of the Dolichos make a sheath in the intestine to reduce the absorption of carbohydrates and natural insulin into the blood stream. This enables energy to be sustained for a period of at least 3-4 h. Decreased levels of sugar in blood were observed by the authors in their earlier work.  Both good control of sugar and presence of antioxidant effect could have decreased the onset of nephropathy, which was observed in this study. In agreement to these findings, researcher reported that antioxidants and good control of diabetes leads to improved renal functions.  These findings suggest that Dolichos may improve the disturbed metabolism associated with diabetes.
Furthermore, effect of the Dolichos powder remains for a longer time when compared with the conventional available medicines as after the intake of normal conventional medicine, the left over insulin in the pancreas is absorbed very quickly in the blood stream when compared with the Dolichos powder and hence the frequency of the intake of the powder will be less.
| Conclusion|| |
D. biflorus at doses of 300 mg/kg/day for 30 days resulted in gradual but significant decreased diabetic nephropathy and fasting blood sugar. It is therefore suggested that the seed of Dolichos may be used as a supplement to the conventional anti-diabetic therapy; however, further studies are required to look for its long-term effect and its possible mechanism of action.
| References|| |
Chan JC, So W, Ma RC, Tong PC, Wong R, Yang X. the complexity of vascular and non-vascular complications of diabetes: The Hong Kong diabetes registry. Curr Cardiovasc Risk Rep 2011;5:230-9.
Kumar A. Diabetic blindness in India: The emerging scenario. Indian J Ophthalmol 1998;46:65-6.
Rahi JS, Sripathi S, Gilbert CE, Foster A. Childhood blindness in India: Causes in 1318 blind school students in nine states. Eye (Lond) 1995;9:545-50.
Nakai S, Shinzato T, Nagura Y, Masakane I, Kitaoka T, Shinoda T, et al
. An overview of regular dialysis treatment in Japan as of 31 Dec 2003. Ther Apher Dial 2005;9:431-58.
Riordan EP. Eye. In: Tierney LM, Mcphee SJ, Papadakis MA, editors. Current Medical Diagnosis and Treatment. 42 nd
ed., Vol. 46. New York: Lange Medical Books/McGraw - Hill; 2003. p. 1777-8.
Lane PH, Steffes MW, Fioretto P, Mauer SM. Renal interstitial expansion in insulin-dependent diabetes mellitus. Kidney Int 1993;43:661-7.
Jedziniak JA, Chylack LT Jr, Cheng HM, Gillis MK, Kalustian AA, Tung WH. The sorbitol pathway in the human lens: Aldose reductase and polyol dehydrogenase. Invest Ophthalmol Vis Sci 1981;20:314-26.
Marjani A. Plasma lipid peroxidation zinc and erythrocyte Cu-Zn superoxide dismutase enzyme activity in patients with type 2 diabetes mellitus in Gorgan City (South East of the Caspian Sea). Internet J Endocrinol 2005;2:1647-8.
Nobécourt E, Jacqueminet S, Hansel B, Chantepie S, Grimaldi A, Chapman MJ, et al
. Defective antioxidative activity of small dense HDL3 particles in type 2 diabetes: Relationship to elevated oxidative stress and hyperglycaemia. Diabetologia 2005;48:529-38.
Ayurnepal Nepal, Medicinal-Plant. Available from: http://www
.ayurnepal.com/medicinal-plants/142-dolichos-biflorus-Linn.html. [Last cited on 2013 Aug 20].
Garimella TS, Jolly CI, Narayanan S. In vitro
studies on antilithiatic activity of seeds of Dolichos biflorus
Linn.and rhizomes of Bergenia ligulata
Wall. Phytother Res 2001;15:351-5.
Laskar S, Bhattarcharyya UK, Sinhababu A, Basak BK. Antihepatotoxic activity of kulthi (Dolichos biflorus
) seed in rats. Fitoterapia 1998;69:401-2.
Muthu AK, Sethupathy S, Manavalan R, Karar PK. Hypolipidemic effect of methanolic extract of Dolichos biflorus
Linn. in high fat diet fed rats. Indian J Exp Biol 2005;43:522-5.
Pant MC, Uddin I, Bhardwaj UR, Tewari RD. Blood sugar and total cholesterol lowering effect of Glycine soja
(Sieb and Zucc.), Mucuna pruriens
(D.C.) and Dolichos biflorus
(Linn.) seed diets in normal fasting albino rats. Indian J Med Res 1968;56:1808-12.
Parthsarthi, Purwar B, Saxena Y.Effect of Dolichos biflorus
on blood sugar and lipids in diabetic rats. Indian J Physiol Pharmacol 2013;57:63-71.
Hazra B, Sarkar R, Mandal S, Biswas S, Mandal N. Studies on antioxidant and antiradical activities of Dolichos biflorus
seed extract. Afr J Biotechnol 2009;8:3927-33.
Levi JA, Wiernik PH, Diggs CH. Combination chemotherapy of advanced previously treated Hodgkin′s disease with streptozotocin, CCNU, adriamycin and bleomycin. Med Pediatr Oncol 1977;3:33-40.
Wong KK. Reduction by streptozocin of blood glucose utilization during the appearance of the streptozocin induced early hyperglycaemia in fasting rats. Biochem Mol BiolInt 1996;39:191-5.
Yoshida M, Kimura H, Kyuki K, Ito M. Effect of combined vitamin E and insulin administration on renal damage in diabetic rats fed a high cholesterol diet. Biol Pharm Bull 2005;28:2080-6.
Yamabe N, Yokozawa T, Oya T, Kim M. Therapeutic potential of (-)-epigallocatechin 3-O-gallate on renal damage in diabetic nephropathy model rats. J Pharmacol Exp Ther 2006;319:228-36.
Edwards PJ, Barnard L, Leonard VK, Yi JS, Moloney KP, Kongnakorn T, et al
. Understanding users with diabetic retinopathy: Factors that affect performance in a menu selection task. Behav Inf Technol2005;24:175-86.
Chrissobolis S, Miller AA, Drummond GR, Kemp-Harper BK, Sobey CG. Oxidative stress and endothelial dysfunction in cerebro-vascular disease. Front Biosci (Landmark Ed) 2011;16:1733-45.
Erciyas F, Taneli F, Arslan B, Uslu Y. Glycemic control, oxidative stress, and lipid profile in children with type 1 diabetes mellitus. Arch Med Res 2004;35:134-40.
Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 1991;40:405-12.
Muthu AK, Sethupathy S, Manavalan R, Karar PK. Antioxidant potential of methanolic extract of Dolichos biflorus
Linn in high fat diet fed rabbits. Indian J Pharmacol 2006;38:131-2.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]