|Year : 2014 | Volume
| Issue : 1 | Page : 76-81
Neuroprotective effect of a triterpenoid saponin isolated from Momordica cymbalaria Fenzl in diabetic peripheral neuropathy
Raju B Koneri, Suman Samaddar, SM Simi, Srinivas T Rao
Department of Pharmacology, Karnataka College of Pharmacy, Bangalore, Karnataka, India
|Date of Submission||01-Sep-2012|
|Date of Decision||22-Jun-2013|
|Date of Acceptance||08-Oct-2013|
|Date of Web Publication||16-Jan-2014|
Raju B Koneri
Department of Pharmacology, Karnataka College of Pharmacy, Bangalore, Karnataka
Source of Support: None, Conflict of Interest: None
Objectives: To investigate the neuroprotective potential of a saponin isolated from the roots of Momordica cymbalaria against peripheral neuropathy in streptozotocin-induced diabetic rats.
Materials and Methods: A steroidal saponin (SMC) was isolated from M. cymbalaria Fenzl and purified by preparative high-performance liquid chromatography. Diabetes was induced in male Wister rats by injecting streptozotocin 45 mg/kg. Diabetic rats were divided into six groups for neuroprotective effect-three each for preventive and curative groups. Neuropathic analgesia was assessed by tail-flick and hot-plate methods. Dorsal root ganglion (DRG) neurons and sciatic nerves were isolated, and histopathological analysis was performed. Antioxidant activity (superoxide dismutase, catalase, and inhibition of lipid peroxidation) of the saponin was also carried out on the isolated DRG neurons and sciatic nerves to assess total oxidative stress.
Results: In both preventive and curative protocols, rats administered with SMC showed significant decrease in tail immersion latency time and increase in pain sensitivity when compared to diabetic control group. There was improvement in the myelination and degenerative changes of the nerve fiber in both the groups, and an obvious delay in the progression of neuropathy was evident. SMC treatment showed significant decrease in superoxide dismutase, catalase activity, and lipid peroxidation in the nerves.
Conclusions: The steroidal saponin of M. cymbalaria (SMC) possesses potential neuroprotective effect in diabetic peripheral neuropathy with respect to neuropathic analgesia, improvement in neuronal degenerative changes, and significant antioxidant activity.
Keywords: Antioxidant, diabetic peripheral neuropathy, neuroprotection, saponin of Momordica cymbalaria
|How to cite this article:|
Koneri RB, Samaddar S, Simi S M, Rao ST. Neuroprotective effect of a triterpenoid saponin isolated from Momordica cymbalaria Fenzl in diabetic peripheral neuropathy. Indian J Pharmacol 2014;46:76-81
|How to cite this URL:|
Koneri RB, Samaddar S, Simi S M, Rao ST. Neuroprotective effect of a triterpenoid saponin isolated from Momordica cymbalaria Fenzl in diabetic peripheral neuropathy. Indian J Pharmacol [serial online] 2014 [cited 2020 Aug 4];46:76-81. Available from: http://www.ijp-online.com/text.asp?2014/46/1/76/125179
| » Introduction|| |
Neuropathy, a common complication of diabetes mellitus, is related to duration and severity of hyperglycemia.  Usually, more than 50% patients with duration of diabetes of 25 years or more are affected, making it as one of the most common diseases of the nervous system.  Diabetic neuropathy may manifest as third nerve palsy, mononeuropathy, diabetic amyotrophy, autonomic neuropathy, and thoracoabdominal neuropathy. Diabetic neuropathy affects all peripheral nerves including pain fibers, motor neurons, and the autonomic nervous system.
Ethno-botanical information reports around 800 plants that may possess antidiabetic activity when assessed using the presently available experimental techniques.  Momordica cymbalaria Fenzl (MC) (Cucurbitaceae) is a species found in Karnataka and Andhra Pradesh, India. Its tuber is traditionally used as an abortifacient and also for the treatment of diabetes mellitus. Its fruit powder and extract were reported to have antidiabetic activity in experimental type 1 diabetic models. ,, We have previously reported the antidiabetic activity of saponins of M. cymbalaria (SMC) possibly due to reversal of the atrophy of the pancreatic islets of β-cells, resulting in increased insulin secretion and hepatic glycogen levels which may attenuate hyperinsulinemia. The alpha-adrenergic blocking effect may also contribute to their insulin secretion and sensitizing effects. 
Antidiabetic plants may exhibit additional neuroprotective, effect but such a study has not been reported with M. cymbalaria. Hence, this study is an attempt to elucidate the effect of the phytochemicals isolated from the roots of M. cymbalaria on peripheral diabetic neuropathy.
| » Materials and Methods|| |
Drugs and Chemicals
Solvents for extraction were obtained from Merck, India. Regular insulin was obtained from Biocon, Bangalore; streptozotocin from Sigma-Aldrich, USA; and trichloroacetic acid (TCA) and thiobarbituric acid (TBA) from Himedia Labs, Mumbai, India. Kits for estimation of serum glucose, glycated hemoglobin, cholesterol, triglycerides, and high-density lipoprotein (HDL) were sourced from Prism Diagnostics Pvt. Ltd, Mumbai, India.
The fresh roots of M. cymbalaria Fenzl were collected from Gadag district of Karnataka and authenticated by the Department of Botany, Bangalore University, Bangalore, India. The roots of M. cymbalaria were isolated, chopped into small pieces, dried under shade at room temperature for 7 days, and were powdered.
Extraction and Isolation
The root powder (100 g) was extracted with 1000 mL methanol in a reflux condenser for three cycles of 7 h each till the volume reduced to half. Extract was filtered through Whattman filter paper no. 1 and evaporated to dryness. Five grams of methanolic extract was subjected to saponification in a 500-mL conical flask and was hydrolyzed with 100 mL of 0.5 N potassium hydroxide for 1-2 h. After hydrolysis, free fatty acids were separated. Unsaponified fraction of this free fatty acid is then extracted with 100 mL diethyl ether and allowed to stand for a few minutes. Upper layer was collected to get unsaponified fatty acids and lower water layer to get free fatty acids. Upper layer (organic layer) was extracted with diethyl ether for complete separation of sterols. This ethereal layer was completely evaporated at room temperature and mixed and filtered through the fat-free filter paper to remove the solid particles. The separator was rotated carefully for few minutes, without violent shaking and allowed the liquids to separate. Ether and 1% aqueous KOH were added and ether layer was separated. The aqueous layer was extracted twice with ether. Finally, the ethereal layers were combined and washed with distilled water and checked for alkalinity with phenolphthalein solution.
The ethereal solution was transferred to a weighed flask. The ether was distilled off and 6 mL acetone was added and shaken. By the aid of gentle current of air, the solvent was completely removed from the flask, which was held obliquely and rotated almost entirely immersed in boiling water. The residue was dried to a constant weight at a temperature not above 80°C to get the unsaponified matter. Chemical tests were conducted to confirm the presence of triterpenoids.
Male Wistar rats (200-250 g), procured from Indian Institute of Science, Bangalore, were maintained at room temperature of 25 ± 2°C with 12 h light-dark cycle. The animals were provided with normal pellet chow and water ad libitum, except during experimentation. The study protocols were duly approved by the Institutional Animal Ethics Committee (IAEC) of Karnataka College of Pharmacy, Bangalore. Studies were performed in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. The oral acute toxicity study was performed using the up and down procedure as per Office of Prevention, Pesticides and Toxic Substances (OPPTS) guidelines (OPPTS 870.1100).
Induction of Experimental Diabetes
After overnight fasting, diabetes was induced in the animals by intravenous injection of streptozotocin dissolved in 0.1 M sodium citrate buffer pH 4.5 at a dose of 45 mg/kg. The animals were allowed to drink 5% w/v glucose solution overnight to overcome the drug-induced hypoglycemia. On the third day, animals with blood glucose level greater than 250 mg/dL were treated as diabetic rats and were used for the further experiments. 
The diabetic animals were divided into six groups (three for preventive and three for curative) containing 10 rats each.
Group 1: Normal control (sodium citrate p.o. for 30 days).
Group 2: Diabetic control-streptozotocin (45 mg/kg i.v.) observed for 30 days
Group 3: Diabetic rats treated with insulin (4 U/kg, s.c. for 30 days [day 3-day 33])
Group 4: Diabetic rats treated with saponins of M. cymbalaria (SMC) (100 mg/kg, p.o. for 30 days [day 3-day 33]).
Group 5: Diabetic control-streptozotocin (45 mg/kg i.v.) observed for 86 days
Group 6: Diabetic rats treated with insulin (4 U/kg, s.c. for 30 days [day 56-day 86])
Group 7: Diabetic rats treated with SMC (100 mg/kg, p.o. for 30 days [day 56-day 86]).
After 2 h of insulin and SMC treatment on the last day in both preventive and curative groups, the animals were subjected to Eddy's hot-plate test and tail-flick method to assess the developed neuropathy. Blood was collected retro-orbitally for the estimation of serum glucose, glycosylated hemoglobin, triglycerides, total cholesterol, low-density lipoprotein, and HDL using respective assay kits (Prism diagnostics Pvt. Ltd, Mumbai, India) using a semi-automatic biochemical analyzer.
Analgesic Activity by Tail-flick Method
The tail of each rat was submerged in 29°C water for 30 min before beginning the test. Thereafter, the whole tail of each rat was submerged in 49°C water and the time taken for the rat to show a characteristic tail-flick response was recorded. The test was repeated three times for each rat and the average of the three measurements was recorded as the withdrawal latency. ,
Analgesic Activity by Hot-plate Method
Each rat was placed on the hot plate maintained at 55°C-56°C and the time taken for the response to occur (either licking of paw or jumping) was recorded. A cutoff time of 10 s was kept to avoid damage to the paw of the animal. Prior to any treatment, the rats were allowed to acclimatize to the test procedure and apparatus and baseline values were obtained. ,,
Isolation of Sciatic Nerve and Dorsal Root Ganglion Neurons
Sciatic nerve was isolated as per the procedure described by Mizisin A.  The animals were killed and the skin of the lateral surface of the left thigh was incised. A cut was made directly through the biceps femorus to expose and isolate the sciatic nerve. Isolation of dorsal root ganglion (DRG) neurons was done as per the procedure described by Kamiya H et al.  Briefly, the spinal column of the animal was removed and opened from the dorsal side to reveal the spinal cord. The spinal cord was removed under dissecting microscope and the DRGs dissected out by grasping each with micro forceps and cutting the root on either side with micro scissors.
One DRG and sciatic nerve were preserved for histopathological analysis in 10% v/v formalin solution. The remaining DRG and sciatic nerve were placed in 10% w/v KCl solution, homogenized, and centrifuged at 5000 rpm for 10 min. The supernatant was separated and estimated for superoxide dismutase (SOD), catalase levels, and markers of lipid peroxidation.
Assessment of Diabetes-induced Oxidative Stress in Nerve
Estimation of SOD
To 2.78 mL sodium carbonate buffer (0.05 mM, pH 10.2), 100 μL of 1 mM EDTA and 20 μL tissue supernatant were added and incubated at 30°C for 45 min. The reaction was initiated by adding 100 μL of adrenaline. The change in the absorbance was recorded at 480 nm for 3 min. Sucrose was used as a blank.  The values were expressed as units/milligram protein.
Estimation of Catalase
To 1.9 mL phosphate buffered saline (pH 7.0), 100 μL of supernatant was added. To this 1 mL of H 2 O 2 was added and the change in the absorbance was recorded at 240 nm for 3 min.  The values were expressed as units/milligram protein.
Based on the Liu and Ng  method, the presence of markers of lipid peroxidation was determined. 0.5 mL supernatant, 0.1 mL of 10 mM FeSO4, and 0.1 mL of 0.1 mM ascorbic acid were incubated at 37°C for 1 h. The reaction was stopped by addition of 0.75 mL of 28% (w/v) TCA and 0.5 mL of 1% (w/v) TBA, successively. The mixture was then heated at 100°C for 45 min. After centrifugation, all precipitated proteins were removed and the color of the malondialdehyde (MDA)-TBA complex in the supernatant was detected at 532 nm. The values of MDA are expressed as nmol/mg of protein.
The results are expressed as mean ± standard error of mean and one-way analysis of variance (ANOVA) followed by Dunnett's test was used to determine statistical significance.
| » Results|| |
Acute Toxicity Test
Mortality in the acute toxicity test of SMC was seen in the limit test at the dose of 5000 mg/kg. Mortality was not seen in the main test up to a dose of 1750 mg/kg and hence 100 mg/kg was selected for the study.
STZ Induced Diabetic Neuropathy: Tail-flick
and Hot-plate Method
Diabetic control rats treated in both preventive (group 2) and curative groups (group 5) showed a statistically significant (P < 0.001) increase in the tail-flick latency time and a statistically significant (P < 0.001) decrease in the response time with hot-plate method when compared with vehicle control (group 1). The groups receiving insulin and SMC in both preventive and curative groups (groups III, IV, VI, and VII) showed a statistically significant (P < 0.001) increase in the tail-flick latency time and a statistically significant (P < 0.001) decrease in the response time with hot-plate method when compared with the respective diabetic control groups [Table 1].
|Table 1: Effect of saponin of Momordica cymbalaria (SMC) on tail-flick latency and hot-plate response time and in streptozotocin (STZ)-treated rats|
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Diabetic control rats in preventive group (group 2) after 30 days and in curative group (group 5) after 86 days showed a significant (P < 0.001) increase in the levels of serum glucose, triglycerides, cholesterol, and glycosylated hemoglobin and decrease in HDL when compared with normal control (group 1).
Rats administered with insulin and SMC (groups 4, 5, 6, and 7) showed a significant (P < 0.001) decrease in the levels of serum glucose, triglycerides, cholesterol, and glycosylated hemoglobin and increase in HDL when compared with their respective diabetic control rats [Table 2].
|Table 2: Effect of saponin of Momordica cymbalaria (SMC) on biochemical parameters and glycosylated haemoglobin (HbA1C) in streptozotocininduced diabetic rats|
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Morphology of DRG Neurons and Sciatic Nerves
Histopathological analysis of DRG neurons and sciatic nerves showed degenerative changes in terms of neuronal cell bodies, nerve fiber, neuronal cell soma, and loss of myelination in the diabetic control animals in both preventive and curative groups [Figure 1].
|Figure 1: Histopathological pictograms revealing effects of saponin of Momordica cymbalaria on DRG neurons and sciatic nerves of streptozotocininduced diabetic rats in both preventive and curative groups (H&E 100×), n = 10|
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Treatment with SMC showed improvement in the myelination and degenerative changes of the nerve fibers in both groups. Even though a complete restoration of neuronal integrity was not achieved, a delay in the progression of diabetic neuropathy is obvious under the action of the SMC. Preventive group animals showed better improvement when compared to the curative groups. Insulin-treated groups show loss of encapsulation of neuronal cell bodies and disrupted bundles of nerve fibres and neuronal cell soma. Most of these neuronal cell bodies appear disintegrated.
Assessment of diabetes-induced oxidative stress in nerve
Diabetic control rats treated in preventive (group 2) and curative groups (group 5) showed a significant (P < 0.001) increase in SOD, catalase activity, and lipid peroxidation in DRG neurons when compared with vehicle control (group 1).
Rats administered with insulin and SMC (groups 4, 5, 6, and 7) showed a significant (P < 0.001) decrease in super oxide dismutase, catalase activity, and lipid peroxidation in DRG neurons when compared with their respective diabetic control groups [Table 3].
|Table 3: Effect of saponin of Momordica cymbalaria on oxidative stress in streptozotocin-induced diabetic rats|
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| » Discussion|| |
Crude saponins from M. cymbalaria have been demonstrated to have antidiabetic and antihyperlipidemic activities. Momordica charantia is reported to contain saponins such as charantin, momordicine, insulin-like steroidal saponin, and triterpenes saponins, etc., which are responsible for its antidiabetic activity. 
Increase in glycosylation of proteins including hemoglobin is seen with uncontrolled or poorly controlled diabetes. The glycation of myelin protein may contribute to the impairment of nerve conduction. These advanced glycation end products are also present in peripheral nerves which could interfere with axonal transports.  There is loss of pain perception in diabetes probably due to nerve damage and induction of peripheral neuropathy. , Thermal hypoalgesia has been reported in diabetic rats using the tail-flick test or the hot-plate test. , In this study, streptozotocin-induced diabetic control rats showed significant hypoalgesia in the hot-plate and tail-flick method. Hypoalgesia was more significant in curative than preventive group, indicating long-term diabetes causes more hypoalgesia. Previous studies also have shown similar hypoalgesia.  Treatment with insulin and SMC showed a significant decrease in the tail immersion latency and increase in pain sensitivity when compared with diabetic control group, suggesting protective effect against neuropathy.
An increase in the percentage of glycosylated hemoglobin was observed in both preventive and curative diabetic control groups. Treatment with insulin and SMC prevented this elevation in both preventive and curative diabetic rats groups. This could be due to the result of improved glycemic control produced by steroidal saponins of M. cymbalaria. Previous studies also have shown similar results. 
Histopathological analysis of DRG neurons and sciatic nerves showed degenerative changes in terms of neuronal cell bodies, nerve fiber, neuronal cell soma, and loss of myelination in the diabetic control animals which is similar to observations in other studies.  Treatment with SMC showed improvement in the myelination and degenerative changes of the nerve fiber in both the groups. Even though a complete restoration of neuronal integrity is not evident, a delay in the progression of diabetic neuropathy is obvious under the action of the SMC.
The generation of oxygen-derived free radicals in diabetes is the leading cause of the development of diabetic neurological complications such as neuropathic pain and depression. , The free radicals so produced may react with polyunsaturated fatty acids in cell membranes leading to lipid peroxidation, which in turn again results in production of free radicals.  Lipid peroxidation-mediated tissue damage has been observed in the development of both types I and type II diabetes. Lipid peroxidation effects are greater in nerve root and DRG because the blood-nerve and perineurial barriers are lower at these sites. In this study, diabetic control rats showed a significant increase in lipid peroxidation in DRG and sciatic nerve in both preventive and curative groups. Lipid peroxidation was significantly reduced in both groups upon treatment with insulin and SMC.
SOD has been implicated as one of the most important enzymes in the antioxidant defense system which catalyses the dismutation of superoxide radicals to produce H 2 O 2 and molecular oxygen.  The observed decrease in SOD activity could result from inactivation by H 2 O 2 or glycation of enzymes. The superoxide anion has been known to inactivate catalase, which is involved in the detoxification of hydrogen peroxide.  Thus, increase in SOD activity may indirectly play an important role in the activity of catalase. Diabetic control rats showed a significant decrease in SOD and catalase activity in the DRG neurons and sciatic nerves in both preventive and curative groups. On treatment with insulin and SMC, there was improvement in both groups.
In conclusion, the steroidal saponin of M. cymbalaria revealed significant preventive and curative effects on diabetic neuropathy pertaining to improvement in myelination and restoration of neuronal integrity, thereby delaying the progression of neuropathy. The neuronal antioxidant activity may facilitate neuroprotective action. Further studies are warranted to prove its efficacy in humans.
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[Table 1], [Table 2], [Table 3]
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