EFFECT OF ETHANOLIC EXTRACTS OF INDIAN MEDICINAL PLANTS ON THE NON-ENZYMATIC ANTIOXIDANT SYSTEM IN STREPTOZOTOCIN INDUCED DIABETIC RATS IN COMPARISON WITH GLIBENCLAMIDEHTML Full Text
EFFECT OF ETHANOLIC EXTRACTS OF INDIAN MEDICINAL PLANTS ON THE NON-ENZYMATIC ANTIOXIDANT SYSTEM IN STREPTOZOTOCIN INDUCED DIABETIC RATS IN COMPARISON WITH GLIBENCLAMIDE
Department of Biotechnology, Periyar University PG Extension Centre, Dharmapuri - 636701, Tamil Nadu, India.
ABSTRACT: Objective: To investigate the effects of ethanolic extracts of leaf and fruit of Trichosanthes dioica and leaf of Clitoria ternatea were studied on the altered non-enzymatic antioxidant system such as reduced glutathione (GSH), Vitamin E, C and A in streptozotocin-induced diabetic Wistar rats. Methods: Male adult Wistar albino rats divided into eleven groups of six rats each were assigned to non-diabetic and diabetic groups (Group I to XI). Diabetes was induced in Albino rats by single intraperitoneal administration of STZ (60 mg/kg body weight), on confirming diabetes after 48 h of injection. Group I and II were kept as non-diabetic and diabetic control. The other diabetic groups (Group III to Group X) were treated with both individual and combined ethanolic extracts of T. dioica and C. ternatea at the doses of 200, and 400 mg/kg of body weight were administrated orally at a single dose per day for 28 consecutive days. Group XI was treated with Glibenclamide (600 μg/kg body weight), a standard oral hypoglycemic drug used as a reference drug for comparison. After completion of experimental period serum, liver and kidney were used for estimating GSH, plasma, and liver for estimating Vitamin E, C and A, and pancreas, liver and kidney were used for histopathological changes in the diabetic rats. Results: A significant increase in GSH, Vitamin E, C and A levels were observed in diabetic rats treated with ethanolic extracts of T. dioica (leaf and fruit) and C. ternatea (leaf) compared to diabetic control rats. Histopathological studies demonstrated the reduction in the pancreas, liver and kidney damage and confirmed the biochemical findings. Conclusion: These results suggest that T. dioica and C. ternatea are beneficial in the control of diabetes by the noticeable antioxidant property.
Trichosanthe dioica, Clitoria ternatea, Non-enzymatic antioxidants, Glibenclamide, Histopathology
INTRODUCTION: Diabetes mellitus persistent hyperglycemia causes increased production of free radicals via, auto-oxidation of glucose. It may lead to the disruption of cellular functions, affects antioxidant reactions catalyzed by reactive oxygen species (ROS) scavenging enzymes 1 and endothelial dysfunction 2, 3.
Scavengers of oxidative stress may have an effect in reducing the increased serum glucose level in diabetes and may alleviate diabetes as well as reduce its secondary complications 4.
Antioxidants are the substances that prevent or slow down the oxidation reactions. All organisms possess the most important antioxidant system includes enzymatic such as superoxide dismutase, catalase, and glutathione peroxidase and the non-enzymatic group includes reduced glutathione, Vitamins A, C and E. They are located in the cell and in the extracellular fluid which is produced either endogenously or derived from dietary sources.
These are responsible for scavenging and to protect the human body against ROS generated in the cells 5. They stop the free radical generation by trapping the free radicals, and thus they inhibit the chain reactions which can lead to the destruction of healthy cells 6. Supplementation of exogenous antioxidants or boosting endogenous antioxidant defenses of the body is a promising way of combating the undesirable effects of ROS induced oxidative damage. Plants have an innate ability to biosynthesize a wide range of some chemical substances especially secondary metabolites and non-enzymatic antioxidants which serve as sources of antioxidants and do scavenging activity 7, 8.
Diabetes mellitus is a multifactorial disease, and therefore, it would require more than a single drug agent to reverse all or the majority of the aspects of the disease. The human body is much better suited to treatment with herbal remedies than with the isolated chemical medicines. Polyherbal therapy which is the use of a combination of various agents from different plant sources for a therapeutic approach in the management of diabetes and has the advantage of producing maximum therapeutic efficacy with minimum side effects 9. In the traditional system of Indian medicinal plant formulations and several cases, combined extracts of plants are used as a drug of choice rather than individual 10.
In the present investigation, the first plant Trichosanthes dioica Roxb (family: Cucurbitaceae) is a dioecious (male and female) vine (creeper) perennial herb distributed in the plains of tropical Asia, Polynesia and Australia 11. It is extensively cultivated as a vegetable crop in the Eastern part of India, particularly in Orissa, West Bengal, Assam, Bihar, Uttar Pradesh Tripura and also in Tamil Nadu 12, 13, 14. It has been used for fever, constipation, diuretics, skin infection, dysentery, diarrhea, convalescents, bronchitis and cancer like conditions. The plant extract has shown a significant reduction in liver enzymes (alanine transaminase and alkaline phosphatase) and serum creatinine 15. The leaves and fruits of the plant have been reported to have hypoglycemic activity 16.
The second plant Clitoria ternatea Linn (family Fabaceae) is a perennial twining herb found in Africa, Australia, America, India, China, Philippines, and Madagascar. In traditional Ayurvedic medicine, it has been used to treat infertility, urinogenital disorder, bronchitis, purgative and diuretic 17, 18, 19, 20. A recent study showed that it has anti-hyperglycemic, anti-hyperlipidemic 21, anti-inflammatory 22 and anti-helminthic 23 activities.
Hence, the objective of the present study is to investigate the above-mentioned plant materials are used individually and in combination to evaluate the anti-diabetic, non-enzymatic antioxidant effectiveness and histological studies in STZ-induced diabetic rats and compared to the effect of standard drug Glibenclamide.
MATERIALS AND METHODS:
Chemicals: Streptozotocin (STZ) was purchased from Siga Chemical Company (USA). Glibenclamide was obtained from Aventis Pharmaceuticals Limited (India). All the chemicals and reagents used in the experiments were of analytical grade obtained from BDH (England and India), E. Merck (Germany), Siga Chemical Company (U.S.A), LOBA - Chemie Indo Austranol Co., (India) whenever necessary the solvents were redistilled before use.
Collection and Authentication of Plant Materials: Fresh unripe fruit and leaf of T. dioica and the leaf of C. ternatea were collected from SKM Herbal Research Centre, Erode, Tamil Nadu, India. With the help of local flora, a voucher specimen (no. VOCB 2307 and VOCB 2453) was retained in Ethnopharmacology Unit, Research Department of Botany, V. O. Chidambaram College, Tuticorin, Tamil Nadu for further reference.
Preparation of Ethanolic Extracts of T. dioica and C. ternatea: Freshly collected leaf and fruit of T. dioica and leaf of C. ternatea were washed with distilled water, and the fruits were cut into small pieces. Both fruits and leaves were dried under shade for two weeks. The shade dried leaves and fruits were coarsely powdered separately. The powdered materials were kept in airtight containers to use. About 500 g of dried coarse powdered samples were weighed and subjected to 1250 ml of ethanol in a Soxhlet extractor for 24 h. All the extracts were filtered through Whatmann no. 41 filter paper separately and the extracts were concentrated in vacuum at 60 ºC using a rotary evaporator. To evaporate the remaining solvent, the extracts were kept in an oven at a temperature of 40-50 ºC for 8h that were used in the present study.
Collection of Experimental Animals: Healthy male adult Albino rats of Wistar strain approximately of the same age, weighing around 160-180 g were procured from Nandha College of Pharmacy. The entire process was approved by the Institutional Animal Ethics Committee (IAEC) which is certified by the Committee for Control and Supervision of Experiments on Animals, (CPCSEA), India (Proposal number: NCP/IAEC/ PHD/01/2007-2008), Nandha College of Pharmacy, Erode, Tamil Nadu, India.
Preparation of Streptozotocin-induced Diabetic Wistar Rats: Diabetes was induced by single dose intraperitoneal administration of streptozotocin at a dose of 60 mg/kg body weight in 0.1 M citrate buffer (pH 4.5) and then injected into the tail of the sixty rats. The injection volume was prepared to contain 1 ml/kg bw 18. After 72 h of STZ administration, the blood glucose content was measured. The animals with blood glucose levels ≥ 250 mg/dl were considered to be diabetic and used for the experiment.
Experimental Design of Animals: In the present investigation, the rats were divided into eleven groups of six rats in each group as follows:
Group I: Normal control rats received normal saline (0.9% sodium chloride).
Group II: STZ-induced diabetic control rats received normal saline.
Group III: Diabetic rats received ethanolic leaf extract of T. dioica (200 mg/kg body weight).
Group IV: Diabetic rats received ethanolic leaf extract of T. dioica (400 mg/kg body weight).
Group V: Diabetic rats received ethanolic fruit extract of T. dioica (200 mg/kg body weight).
Group VI: Diabetic rats received ethanolic fruit extract of T. dioica (400 mg/kg body weight).
Group VII: Diabetic rats received ethanolic leaf extract of C. ternatea (200 mg/kg body weight).
Group VIII: Diabetic rats received ethanolic leaf extract of C.t ernatea (400 mg/kg body weight).
Group IX: Diabetic rats received combined ethanolic extracts of T. dioica leaf (200 mg/kg body weight) and C. ternatea leaf (200 mg/kg body weight).
Group X: Diabetic rats received combined ethanolic extracts of T. dioica fruit (200 mg/kg body weight) and C. ternatea leaf (200 mg/kg body weight).
Group XI: Diabetic rats received standard drug glibenclamide (600 μg/kg body weight) for 28 d orally by using an intragastric catheter tube.
Determination of Blood Glucose Level (Electronic Glucometer Method): The blood collected from the tail vein was used to determine the glucose level. As bleeding starts, the animal was held to the blood glucose test strip and allowed the drop of blood to fall on the strip which reacted with the blood. After a few seconds the blood glucose level was displayed on the screen of the glucometer. The blood glucose was estimated every 7 days in control as well as experimental animals for 28 days.
Collection of Blood and Preparation of Tissue Homogenate: At the end of the treatment, all rats were sacrificed by cervical dislocation. Blood was collected from the experimental animals by direct cardiac puncture. Serum was separated by centrifugation at 2500 rpm for 10 min and stored at –20°C until used for the non-enzymatic antioxidant assays. Liver and kidney of the sacrificed animals were excised immediately and thoroughly washed with cold physiological saline and kept in a deep freezer at –20 °C till used. The homogenate was filtered and then centrifuged at 10,000 rpm for 20 min at 4 °C.
Quantification of Phytochemicals in Ethanolic Extracts of Leaf and Fruit of T. dioica and Leaf of C. ternatea: Total phenolic content was measured by the method 24, flavonoids was estimated by the method 25, tannins and saponins were determined by the method 26, 27, alkaloids was determined according to the method 28, and Vitamin C was measured by the method 29.
Estimation of Non-Enzymatic Antioxidants Activities: The changes in the levels of the most important non-enzymatic antioxidants includes GSH (reduced glutathione) activity was studied by the method 30, Vitamin E, Vitamin C, and Vitamin A were estimated according to the methods 31, 32, 33.
Histopathological Studies: The pancreas, liver, and kidney of the sacrificed rats were dissected, removed and fixed in 10% formalin solution. The fixed specimens were then trimmed, washed and dehydrated in ascending grades of alcohol. These specimens were cleared in xylene, embedded in paraffin, sectioned at 4-6 microns thickness and stained with hematoxylin and eosin then to observe histopathological changes by microscopically.
Statistical Analysis: All the experimental values are expressed as means ± SD for groups of six animals each. Student's t-test performed statistical analyses. The values are statistically significant at three levels, ***p<0.001, **p<0.01, *p<0.05. But NS if p>0.05.
Quantification of Phytochemicals and Vitamin C: The quantitative analysis of phytochemicals in the ethanolic extracts of the investigated plants were given in Table 1.
Among the studied plant samples, C. ternatea leaf extract was found to contain an appreciable amount of flavonoids, total phenolics and tannins whereas T. dioica fruit was found to contain a higher amount of saponins and alkaloids. But T. dioica leaf contained lesser amounts of flavonoids, total phenolics, tannins, saponins, and alkaloids.
Total flavonoid content was expressed as rutin equivalent, total phenolics and tannin content were expressed as gallic acid equivalent and tannic acid equivalent respectively. C. ternatea leaf contained a rich amount of Vitamin C when compared to the other two samples.
TABLE 1: QUANTITATIVE ANALYSIS OF PHYTOCHEMICALS AND VITAMIN C IN ETHANOLIC EXTRACTS OF LEAF AND FRUIT OF T. DIOICA AND LEAF OF C. TERNATEA
|Phytochemicals||T. dioica||C. ternatea
|Flavonoids (mg RE/ g extract)||36.1 ± 2.02||48.2 ± 1.06||74.5 ± 3.03|
|Total phenolics (mg GAE/ g extract)||26.0 ± 0.03||38.1 ± 0.03||98.2 ± 1.02|
|Tannins (mg TAE/ g extract)||38.26 ± 3.81||65.72 ± 7.10||78.75 ± 2.09|
|Saponins (%)||0.38 ± 0.02||0.78 ± 0.01||0.66 ± 0.06|
|Alkaloids (gm/ 100 g)||0.11 ± 0.01||0.21 ± 0.01||0.18 ± 0.01|
|Vitamin C (mg AAE/ g extract)||52.26 ± 0.13||60.03 ± 0.17||118.83 ± 0.47|
Values are mean of three independent analyses of the extract ± standard deviation (n = 3). RE-Rutin equivalent; GAE – Gallic acid equivalent; TAE-Tannic acid equivalent; AAE–Ascorbic acid equivalent
TABLE 2: DETERMINATION OF BLOOD GLUCOSE LEVEL IN NORMAL AND EXPERIMENTAL RATS AT DIFFERENT TIME INTERVALS (DAYS)
|Treatment groups||Dose (mg/kg bw)||Blood glucose level (mg/dl)|
|Days of Treatment|
|I Normal control||Normal saline||81.67±2.91||79.23 ± 2.14||85.92 ± 4.08||80.14 ± 2.84||86.91 ± 2.48|
|II DC||Normal saline||196.85 ± 8.43**||218.56 ± 1.32**||234.68 ± 5.99***||238.63±7.94***||231.57±2.56***|
|III D+TDL||200||178.45 ± 4.81*||167.56 ± 3.87*||143.43 ± 3.95NS||136.95 ± 6.11NS||123.91 ± 4.62a|
|IV D+TDL||400||187.56 ± 3.74**||154.66 ± 2.85*||141.49 ± 9.34NS||132.78 ± 3.71NS||126.75 ± 3.56a|
|V D+TDF||200||194.66 ± 5.86**||143.56 ± 4.87*||130.04 ± 2.91NS||121.42 ± 5.67aa||109.48 ± 2.74a|
|VI D+TDF||400||189.29 ± 4.67*||141.93 ± 2.39NS||125.99 ± 2.58aa||116.83 ± 3.55aa||102.69 ± 2.45aa|
|VIID+CTL||200||191.54 ± 6.45**||164.76 ± 6.34*||153.94 ± 5.93*a||143.54 ± 3.04a||131.87 ± 4.39a|
|VIIID+CTL||400||182.87 ± 2.50*||141.56 ± 2.82NS||121.08 ± 2.94*a||116.87 ± 2.56aa||99.26 ± 2.53aa|
|IX D+ TDL+ CTL||200 +200||198.45 ± 5.23**||151.46 ± 3.78*||132.87 ± 3.87aa||127.59 ± 3.02aa||116.78 ± 2.45a|
|X D+TDF +CTL||200 +200||187.45 ± 2.97*||135.77 ± 3.67NSa||126.76 ± 2.95aa||118.26 ± 2.79aa||102.56 ± 2.95aa|
|XI D+Glibenclamide||0.6||192.67 ± 3.51**||143.56 ± 2.85a||135.68 ± 2.11aa||120.59 ± 3.14aa||108.77 ± 2.05aa|
DC: Diabetic control, D: Diabetic, TDL: T. dioica leaf, TDF: T. dioica fruit, CTL: C. ternatea leaf
Blood Glucose Level: Blood glucose level was determined in various groups of experimental animals at frequent intervals of 7 days for 28 days. The results were illustrated in Table 2. The levels of glucose in blood of STZ-induced diabetic rats (Group II) were found to be significantly (p<0.001; p<0.01) elevated when compared with normal control rats (Group I). Oral administration of individual and combined ethanolic extracts of test samples to the experimental groups (Group III to Group X) showed a significant (p<0.01; p<0.05) reduction in blood glucose level when compared with diabetic control rats (Group II). At the 28th day of treatment, a maximum reduction of blood glucose level was seen in Group VI, VIII, Group X, and XI. On the other hand, low dose (200 mg/kg bw) of T. dioica leaf (Group III) treated group the treatment was less effective in all the different durations.
Effect of Ethanolic Extracts of Leaf and Fruit of T. Dioica and Leaf of C. Ternatea on Serum, Liver and Kidney of Non-Enzymatic Anti-oxidant (GSH) levels in STZ-induced Diabetic Rats: Table 3 represented the concentration of GSH in the serum, liver, and kidney of normal control, diabetic control, and diabetic treated groups of rats.
There was a significant (p<0.05) decrease in the activity of GSH in the serum, liver, and kidney of STZ-induced diabetic rats (Group II) compared to the normal group (Group I). A significant (p<0.05) elevation of serum, hepatic and kidney GSH level were observed in the extracts treated diabetic rats which were dose-dependent. Among these group of animals a significant (p<0.01) increase in the levels of GSH were found in serum of Group VIII and IX, liver of Group VI, VII, and VIII), kidney of Group VIII and standard drug glibenclamide treated Group XI, when compared with diabetic control group (Group II).
TABLE 3: EFFECT OF ETHANOLIC EXTRACTS OF LEAF AND FRUIT OF T. DIOICA AND LEAF OF C. TERNATEA ON REDUCED GLUTATHIONE IN SERUM, LIVER, AND KIDNEY OF CONTROL AND EXPERIMENTAL RATS
|Dose (mg/kg bw)||GSH|
|I Normal control||Normal saline||34.59±1.24||53.21±2.63||21.78±1.34|
|II DC||Normal saline||16.26±1.08*||14.50±2.11*||13.93±1.09*|
|IX D+TDL+CTL||200 + 200||33.99±1.27aa||48.59±2.93a||21.87±1.59a|
|X D+TDF+ CTL||200 + 200||37.56±1.19a||50.91±2.07a||20.01±0.48a|
|XI D + Glibenclamide||0.6||34.27±1.73a||56.33±2.29aa||22.59±1.64a|
DC: Diabetic control, D: Diabetic, TDL: T. dioica leaf, TDF: T. dioica fruit, CTL: C. ternatea leaf. Values are reported as mean ± SD for six animals in each group. *p<0.05, **p<0.01, ***p<0.001 significance between normal control vs. diabetic control and drug-treated groups; ap<0.05, aap<0.01 significance between diabetic control vs. drug-treated groups; NS: Not significant. # mg of glutathione/ dl ## Units/ mg protein
TABLE 4: EFFECT OF ETHANOLIC EXTRACTS OF LEAF AND FRUIT OF T. DIOICA AND LEAF OF C. TERNATEA ON NON-ENZYMIC ANTIOXIDANTS IN PLASMA AND LIVER OF CONTROL AND EXPERIMENTAL RATS
|I Normal control||Normal saline||1.89±0.12||0.87±0.02||1.48±0.02||1.21±0.05||1.56±0.03||1.87±0.04|
|II DC||Normal saline||0.67±0.04*||0.46±0.03*||0.51±0.04**||0.62±0.06*||0.34± 0.04**||0.56±0.05*|
|IV D+TDL||400||1.54±0.06a||0.61±0.08 NS||1.21±0.05a||1.03±0.02 NS||0.98±0.07a||1.23±0.06a|
|VI D+TDF||400||1.68±0.07a||0.72±0.04a||1.36±0.04a||1.19±0.02 NS||1.31±0.04aa||1.59±0.03a|
|X D+TDF+ CTL||200+200||1.96±0.08a||0.91±0.07a||1.42±0.03a||1.30±0.03a||1.49±0.03a||1.67±0.08a|
|XI D + Glibenclamide||0.6||1.83±0.04a||0.87±0.04a||1.39±0.07a||1.48±0.08a||1.21±0.04a||1.55±0.05 NS|
DC: Diabetic control, D: Diabetic, TDL: T. dioica leaf, TDF: T. dioica fruit, CTL: C. ternatea leaf. Values are reported as mean ± SD for six animals in each group. *p<0.05, **p<0.01, ***p<0.001 significance between normal control vs diabetic control and drug-treated groups; ap<0.05, aap<0.01 significance between diabetic control vs drug-treated groups; NS: Not significant. #mg/ dl; ##µg/ mg protein
Effect of Ethanolic Extracts of Leaf and Fruit of T. dioica and leaf of C. ternatea on Plasma and Liver of Non-Enzymatic Antioxidants (Vitamin E, C and A) levels in STZ-induced Diabetic Rats: Free radicals inactivate the enzymic antioxidants, and hence the presence of non- enzymic antioxidant is presumably essential for the removal of these radicals 34. The results of the non-enzymic antioxidants vitamin E (α-tocopherol), vitamin C (ascorbic acid) and vitamin A (total carotenoids) in plasma and liver of normal control and an experimental group of rats were depicted in Table 4.
The levels of these non-enzymic antioxidants in plasma and liver were decreased significantly (p<0.01 and p<0.05) in STZ-induced diabetic rats. On the other hand, treatment of diabetic rats with ethanolic extracts significantly (p˂0.05) increased the non-enzymic antioxidant (vitamin E, C, and A) levels to near normal and was found to be dose-dependent. The plasma level of vitamin E was found to be maximum in diabetic rats treated with combined extracts of T. dioica fruit and C. ternatea leaf (Group X) (1.96 ± 0.08 mg/dl) which is very near to normal control group I (1.89 ± 0.12 mg/dl).
Histopathological Studies of Experimental Rat Pancreas: Histopathological changes were noted in pancreatic tissue. The results were presented in Fig. 1 (a- h).
(a) The pancreas of normal control rats (Group I) showed patches of abundant β-cells. (b) The pancreas of diabetic control rats (Group II) showed a severe reduction in the number of pancreatic β-cells that confirmed the destruction of islets and cells due to the effect of streptozotocin. (c) The pancreas of diabetic rats treated with high dose (400 mg/kg bw) of T. dioica leaf (Group IV) showed partial recovery of pancreatic β-cells.
FIG. 1: HISTOLOGICAL EXAMINATIONS OF PANCREAS OF CONTROL AND EXPERIMENTAL RATS AFTER 28 DAYS OF TREATMENT
(d), (e), (f) and (g) pancreas of diabetic rats after treated with high doses (400 mg/kg bw) of T. dioica fruit (Group VI) and C. ternatea leaf (Group VIII) and combined extracts of T.dioica leaf + C. ternatea leaf (Group IX) and T.dioica fruit + C. ternatea leaf (Group X) showed more or less as T. dioica leaf. (h) The pancreas of diabetic rats after treated with standard drug glibenclamide (Group XI) showed pancreatic β-cells similar to that of the control.
Histopathological Studies of Experimental Rat Liver: Histopathological changes were noted in the liver. The results were depicted in Fig. 2 (a-h).
(a) Liver of normal control rats (Group I) showed the normal architecture of hepatocytes with central lobule. (b) Liver of diabetic control rats (Group II) showed congestion and cellular necrosis. (c) Liver of diabetic rats treated with high dose (400 mg/kg bw) of T. dioica leaf (Group IV) showed the mild congestion. (d) Liver of diabetic rats after treated with high dose (400 mg/kg bw) of T. dioica fruit (Group VI) showed the moderate congestion of hepatocytes with mild necrosis. (e) Liver of diabetic rats after treated with high dose (400 mg/kg bw) of C. ternatea leaf (Group VIII) showed the normal architecture of hepatocytes with mild congestion. (f) Liver of diabetic rats after treated with a combined extract of T. dioica leaf + C. ternatea leaf (Group IX) showed normal architecture with prominent hepatocytes. (g) Liver of diabetic rats after treated with a combined extract of T. dioica fruit + C. ternatea leaf (Group X) showed normal cellular arrangement. (h) Liver of diabetic rats after treated with the standard drug (Group XI) showed normal cell arrangement.
FIG. 2: HISTOLOGICAL EXAMINATIONS OF LIVER OF CONTROL AND EXPERIMENTAL RATS AFTER 28 DAYS OF TREATMENT
Histopathological Examinations of Experimental Rat Kidney: Histopathological changes were noted in the kidney. The results were shown in Fig. 3 (a-h). (a) Kidney of normal control rats (Group I) showed normal alternating areas of convoluted tubules glomeruli and straight tubules. (b) Kidney of diabetic control rats (Group II) showed congestion of convoluted tubules, deranged glomeruli. (c) Kidney of diabetic rats treated with high dose (400 mg/kg bw) of T. dioica leaf (Group IV) showed the mild congestion of tubules and necrosis of cells. (d) Kidney of diabetic rats after treated with high dose (400 mg/kg bw) of T. dioica fruit (Group VI) showed the moderate congestion of tubules and necrosis of cells with clear glomeruli. (e) Kidney of diabetic rats after treated with high dose (400 mg/kg bw) C. ternatea leaf (Group VIII) showed the normal architecture of tubules and glomeruli. (f) Kidney of diabetic rats after treated with a combined extract of T. dioica leaf + C. ternatea leaf (Group IX) showed distinguishable renal corpuscle, glomerulus, and glomerular capsule. (g) Kidney of diabetic rats after treated with a combined extract of T. dioica fruit + C. ternatea leaf (Group X) showed normal convoluted tubules with distinguishable renal corpuscle, glomerulus, and glomerular capsule. (h) Kidney of diabetic rats after treated with the standard drug (Group XI) showed normal cell arrangement.
FIG. 3: HISTOLOGICAL EXAMINATIONS OF LIVER OF CONTROL AND EXPERIMENTAL RATS AFTER 28 DAYS OF TREATMENT
DISCUSSION: Medicinal plants consist of a number of biologically active chemical constituents which are formed during the plant's normal metabolic processes. These chemicals are often referred to as “phytochemicals or secondary metabolites.” They have two categories, i.e., primary and secondary constituents. Primary constituents have chlorophyll, proteins, sugar, and amino acids and secondary constituents contain terpenoids, flavonoids, coumarins, glycosides, phenols, tannins, terpenes, terpenoids and alkaloids 35. The WHO is encouraging, promoting and facilitating the effective use of herbal medicine, only a small percentage (5-15%) of the estimated 400,000 - 500,000 plant species have been scientifically and systematically evaluated for their pharmacological activities. Medicinal plants maintain the health and energy of individuals and treat various diseases, including diabetes without causing toxicity 36.
The phytotherapeutic effects of plant materials are unique to the particular plant species, and its medicinal effects are due to the combination of secondary product present in plant 37. Many conventional drugs have been derived from prototypic molecules in medicinal plants. In the present study, flavonoids, total phenolics, tannins, saponins, alkaloids, and vitamin C were present in considerable quantity in the plant extracts. Flavonoids are a large family of bioactive compounds synthesized by plants. They are the promising alternative for diabetes and its associated complications. It improves and stabilizes the secretion of insulin from pancreatic beta cells, keeping the blood glucose level optimum, reduced aldose reductase activity and compromising continued damage of human islets as well as stabilizing the cellular components is more essential for effective diabetic management. "Insulinomimmetic" activities of flavonoids, which are beneficial and desirable effects for diabetics 38, 39. It acts as protectors for a wide variety of environmental stresses and is responsible for the radical scavenging effect in humans.
Polyphenols are widely distributed in plant foods. They are the most abundant antioxidants in the diet of human beings 40. Dietary polyphenol is associated with lower rates of diabetes and cardiovascular disease 41, 42. Phenolic compounds have strong in-vitro and in-vivo antioxidant activities associated with their ability to scavenge free radicals, break radical chain reactions and chelate metals ions 43. Several plant polyphenols were reported to inhibit α-amylase and sucrase activity and decreasing postprandial blood sugar level 44, 45. Plant tannins are natural polyphenolic compounds of high molecular weight. They have a high antioxidant activity 46; improved body antioxidant status can protect against degenerative diseases. They have been considered to be cardio-protective, anti-carcinogenic, anti-inflammatory and anti-mutagenic activities. It enhances glucose uptake and inhibits adipogenesis. It can improve the pathological oxidative state of a diabetic situation 47. Largest and most prevalent of photochemical groups are the alkaloids, terpenes, and phenolics can often have anti-diabetic effects 48. Saponins reduce the uptake of glucose and cholesterol at the gut and delay glucose transfer from the stomach to the small intestine 49.
The study showed that the ethanolic extracts of test drugs possess a good amount of vitamin C. It also shows antioxidant activity. Phytochemicals working together with nutrients may help to reduce the risk of many diseases, including diabetes mellitus, cancer, heart disease, stroke and high blood pressure 50. A blood glucose level of the diabetic rats treated with individual and combined ethanolic extracts of leaf and fruit of T. dioica and leaf of C. ternatea produced a significant reduction when compared with diabetic control rats. C. ternatea leaf extract indicated a maximum reduction in blood glucose level nearly to the level of normal control rats at 28th day of treatment. This might be due to the antihyperglycemic activity of the plant extracts.
The antidiabetic effect of ethanolic extracts of leaf and fruit of T. dioica and leaf of C. ternatea could be due to the possible presence of the constituents mentioned above in the above said part of the plants used in this particular study, which could act independently or synergistically to enhance the activity of glycolytic and gluconeogenic enzymes. The study revealed that the ethanolic extracts of test drugs possess a good amount of vitamin C. It also exhibits antioxidant activity. Phytochemicals working together with nutrient vitamin C may help to slow the aging process and reduce the risk of many diseases, including diabetes mellitus, high blood pressure, cancer, heart disease and stroke 50.
Non-enzymic antioxidants such as reduced glutathione (GSH), vitamin C, E and A play an excellent role in protecting the cells from oxidative damage 51. Reduced glutathione is a major endogenous non-enzymic antioxidant which counterbalance free radicals mediated damage. It has a multifaceted role in the antioxidant defense system. It is a direct scavenger of free radicals as well as a co-substrate for peroxide detoxification 52.
It is well established that GSH is involved in the protection of normal cells and tissue structure and function by maintaining the redox homeostasis, quenching of free radicals, participation in detoxification of xenobiotic reactions, regulation of immune function and keeps up the cellular levels of the active forms of vitamin C and E by neutralizing the free radicals 53.
The decreased levels of GSH in the serum, liver, and kidney of STZ-induced diabetic rats were due to chronic oxidative stress is seen in diabetic condition. GSH protects the cells against oxidative stress by reacting with peroxides and hydro-peroxides. Decreased activity of GSH is due to decrease GSH formation which requires NADPH and glutathione reductase 54. Maintenance of NADPH/NADP+ ratio plays a crucial role in the regeneration of GSH from GSSG 55. GSH is also used by aldose reductase for the reduction of glucose to sorbitol through the polyol pathway. The competition for NADPH could be responsible for the decreased glutathione levels found in diabetes mellitus 56.
A significant elevation of serum, liver, and kidney GSH level were observed in the extracts treated diabetic rats (Group III-Group X). This indicated that the extracts could either increase the biosynthesis of GSH or reduce the oxidative stress leading to less degradation of GSH or have both effects. Administration of ethanolic extracts of leaf and fruit of T. dioica and leaf of C. ternatea, combined leaf extracts of T. dioica + C. ternatea and fruit extract of T. dioica + leaf extract of C. ternatea to the diabetic rats, maintained the levels of non-enzymic antioxidants to near normal by improving the GSH status in serum, liver and kidney.
A non-enzymic antioxidant like reduced glutathione, ascorbate and α-tocopherol play an excellent role in protecting the cells from oxidative damage 51. All these acts synergistically as cellular antioxidants. The most important antioxidant in the cell membrane is α-tocopherol 57. This molecule is known as chain-breaking antioxidant because its function is to intercept lipid peroxyl radicals (LOOº) and so terminate lipid peroxidation chain reactions. The resultant radical is relatively stable and in normal circumstances, insufficiently reactive to initiate lipid peroxidation itself. This is an essential criterion of a good antioxidant thus protecting cell structures against damage. It helps to build normal and red blood cells as well as working as an antioxidant.
In the present study, the level of vitamin E is lower in diabetic rats which represented an increased utilization of the vitamin due to oxidative stress in diabetes. This Vitamin exists in interconvertible (reduction and oxidized) form. Thus the reduction in the level of antioxidant Vitamin E can be attributed to reduced regeneration from their oxidized form. People with diabetes may also have greater antioxidant requirements because of increased free radical production with hyperglycemia 58, 59.
Elevated levels of Vitamin E in experimental groups documented that it prevented the destructive damage that may occur in diabetes. It also may be effective in reducing glycosylation 60, 61. Ascorbic acid is known to act as an antioxidant both in-vivo and in-vitro. It functions as a free radical scavenger and successfully prevents detectable oxidative damage under all types of oxidative stress. It plays an important role in the detoxification of reactive intermediates produced by cytochrome P450, which detoxify xenobiotics. Reduction in tissue ascorbic acid was observed in STZ-induced 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 62.
The significant increase of Vitamin C in ethanolic extracts and glibenclamide treated groups when compared to diabetic control might be due to the potent antioxidant effect of the plant extracts. It can protect cell membranes and lipoprotein particles from the oxidative damage by regenerating the antioxidant form of vitamin E. Vitamin C and E act synergistically in scavenging a wide variety of ROS 63. The total carotenoids (Vitamin A) have been shown to inhibit tissue lipid peroxidation 64. Beta-carotene and other carotenoids are also believed to provide antioxidant protection to lipid-rich tissues. Research suggests beta-carotene may work synergistically with vitamin E 65.
In the present study, the decreased level of Vitamin A observed in untreated diabetic groups might be due to the liberation of lipid peroxide. It is known to be an important natural antioxidant capable of counteracting oxygen free radicals and exerts a protective effect of antioxidant 66. Increased levels of vitamin A in plant extracts treated rats might be due to decreased levels of lipid peroxides. This can be attributed to the free radical scavenging potential of the plant extracts shown by the in-vitro analysis of the study. The reference drug Glibenclamide treated diabetic group also showed increased levels of non-enzymic antioxidants.
Histopathological Studies in Pancreatic Tissues: One of the major findings of this study is that the histopathological investigation along with the biochemical evaluations demonstrated the possibility of the pancreatic tissue regeneration upon combined extract treatment. The regeneration of the pancreas of the STZ destructed islets is probably because pancreas contains few stable cells which have the capacity of regeneration. In this regard, both the plant extracts may contain progenitors cell which may be mobilized into injured pancreatic tissue. On the other hand, progenitor cells may be participating in this repair mechanism. However, the source and nature of these progenitor cells were not determined in this study.
The findings of histopathological investigations in liver and kidney revealed a normal cellular architecture in the normal control group. Cellular necrosis in liver and congestion of convoluted tubules along with deranged glomeruli in kidney were noticed in the diabetic control group. Combined plant extracts treated diabetic groups presented a pattern similar to normal control group however individual extracts revealed the same pattern with some modifications. In the present findings, combined plant extract more effectively stimulate tissue repair than the other individual plant extract treatment and may be clinically beneficial as an agent to restore or maintain tissues after injury.
CONCLUSION: From the present study, it can be concluded that the combined extract of T. dioica fruit and C. ternatea leaf have potent antidiabetic activity. In this sense, the antihyperglycemic effect may be due to the presence of phytochemicals like flavonoids, phenolics, and tannins in the test plants which are responsible for antioxidant actions and inhibiting the progression of oxidative stress in STZ induced diabetic rats. Hence, these plants can be used in the management of diabetes and diabetic complications.
CONFLICT OF INTEREST: The author declares that there is no conflict of interest.
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How to cite this article:
Kavitha R: Effect of ethanolic extracts of Indian medicinal plants on the non-enzymatic antioxidant system in streptozotocin induced diabetic rats in comparison with Glibenclamide. Int J Pharm Sci & Res 2019; 10(1): 418-30. doi: 10.13040/IJPSR.0975-8232.10(1).418-30.
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