Abstract: Background and Objective: Salvia species have been used since ancient times for more than 60 different ailments. Salvia splendens shows a wide availability in Jharkh and region. According to existing literature, its hypoglycemic activity has already been established in diabetic-rodent model. Therefore, an effort has been made to further explore the efficacy of the active fraction of the plant against diabetes and diabetic neuropathy. Materials and Methods: The plant material was collected, shade dried and extracted with methanol. Ethyl acetate fraction of the methanolic extract (SSEA) was subjected to various tests for neuropathic pain assessment (Tail flick, Cold allodynia, R and all Selitto), biochemical tests (fasting blood glucose level, lipid peroxidation, reduced glutathione, catalase levels in brain), Motor Nerve Conduction Velocity (MNCV) determination and histopathological evaluation of the sciatic nerve. Results and Conclusion: There was a significant decrease in blood glucose level in animals treated with SSEA. At a dose of 400 mg kg-1, SSEA showed reduction of MDA and increase in SH and catalase levels in brain. The MNCV in diabetic rats (STZ-NA induced) treated with SSEA 200 and 400 mg kg-1 showed somewhat elevation in nerve conduction velocity as compared to that of diabetic rats. Transverse section of sciatic nerve showed changes due to diabetic nephropathy such as axonal swelling, derangement of nerve fibres which were reverted by 400 mg kg-1 of SSEA.
INTRODUCTION
Diabetes Mellitus (DM) is defined as a group of metabolic disorders characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins1. It is a chronic medical condition that is expected to affect over 300 million people worldwide by 20252.
Diabetic neuropathy is a common complication of diabetes. It usually progresses gradually and involves small and large sensory fibres. The symptoms include, loss of ability to sense pain, loss of temperature sensation and then developing a neuropathic pain, following a "glove and stocking" distribution, beginning in the lower limbs, first affecting the toes and then progressing upwards3. Relatively common conditions which may be associated with diabetic neuropathy, include third nerve palsy, mononeuropathy, mononeuropathy multiplex, diabetic amyotrophy, polyneuropathy, autonomic neuropathy and thoracoabdominal neuropathy. These conditions are thought to result from diabetic microvascular injury involving small blood vessels that supply nerves (Vasa nervorum) in addition to macrovascular conditions that can culminate in diabetic neuropathy4,5. The primary cause of diabetic neuropathy is thought to be hyperglycaemia6. Peripheral nerve involvement is highly frequent in diabetes mellitus and it has been documented that one third of diabetic patients have peripheral neuropathy7. The risk factors involved in the development of diabetic neuropathy include, advanced age, alcohol intake, dyslipidemia, genetic factors, hypertension, increased height, poor glycaemic control and tobacco abuse8.
Pharmacological agents with proven efficacy for painful diabetic neuropathy include the tricyclic antidepressants, the selective serotonin and noradrenaline reuptake inhibitors, anticonvulsants, opiates, membrane stabilizers, the antioxidant alpha-lipoic acid and topical agents, including capsaicin9. The wide variety of medical therapies used to treat diabetic neuropathy attests to the lack of an ideal treatment. Moreover, their use is often associated with problems such as development of adverse side effects, insufficient efficacy and cost–effectiveness. Therefore, clinicians looked for other drugs to increase the overall analgesic effect without causing unacceptable side effects10. Salvia splendens belonging to family, Lamiaceae has been scientifically proven to possess antidiabetic, anticoagulant, anti-inflammatory, antioxidant, antimicrobial, antitumor and mosquito larvicidal properties. The presence of significant antioxidant property in methanolic extract due to terpenoid and anthocyanin11 and antihyperglycaemic activity in both the aqueous and the methanolic extract from the aerial parts of S. splendens have already been reported12. Preliminary phytochemical screening showed that the ethyl acetate fraction of the methanolic extract of S. splendens contained more amounts of terpenoids, flavonoids, phenols and tannins. Quantitative estimations revealed high phenolic and flavonoidal contents in the same fraction. So, the present study was aimed to evaluate the potential of ethyl acetate fraction from the aerial parts of the plant to alleviate Streptozotocin-Nicotinamide induced diabetic neuropathy in rats.
MATERIALS and METHODS
Plant material and extraction: The aerial parts of Salvia splendens were collected from the campus of B.I.T., Mesra, Ranchi in the month of April-May, 2013. The plant was taxonomically identified by the Botanical Survey of India (BSI), Howrah.
The dried and powdered plant material was subjected to extraction in a soxhlet apparatus using methanol. The obtained extract was fractioned using n-hexane, chloroform and ethyl acetate and evaporated using rotary evaporator. The dried extract was subjected to preliminary phytochemical investigation using standard methods13,14 and the ethyl acetate fraction was chosen for further experiments.
Chemicals and reagents: Tris HCl buffer, Thiobarbituric acid (TBA), Trichloroacetic acid (TCA), 5, 5-dithiobis-2-nitrobenzoic acid (DTNB) reagent were obtained from Sigma Aldrich, USA. All other chemicals were of analytical grade and obtained from Merck, USA.
Animals: Wistar albino rats of either sex having a body weight of 180-250 g were used in this study. Animals were procured from CPCSEA approved Institutional animal house of Birla Institute of Technology, Mesra. All animals were kept in polyacrylic cages and maintained under standard housing conditions (24-27°C and humidity 60-65% with 12:12 light: dark cycles). Food was provided in the form of dry pellets (FDA) and water ad libitum. The animals were allowed to get acclimatized to the laboratory conditions for 7 days before the commencement of the experiment.
Induction of diabetes and development of diabetic neuropathic model Streptozotocin-Nicotinamide induced type-2 diabetes rat model: Streptozotocin was dissolved in citrate buffer (pH 4.5) and nicotinamide was dissolved in normal physiological saline. Non-insulin dependent diabetes mellitus was induced in overnight fasted rats by a single intraperitoneal injection of 60 mg kg-1 streptozotocin, 15 min after the i.p administration of 120 mg kg-1 of nicotinamide. The streptozotocin-nicotinamide injected animals were then given 5% w/v glucose solution for 5-6 h following the injection to prevent initial drug-induced hypoglycaemic mortality. Blood glucose was measured using glucometer after 72 h and then on day 7.
The animals with blood glucose concentration more than 200 mg dL-1 were used in the study. Nociceptive measurement was carried out to check the development of neuropathy in animal models. The electrophysiological and biochemical experiments were carried out on the 35th day of the experiment15.
Preparation of the dose: The SSEA (S. splendens ethyl acetate fraction) was dissolved in 5% Tween 80 to prepare two doses (200 and 400 mg kg-1) and given at a dose of 0.1 mL/100 g body weight.
Treatment protocol: Type-2 diabetes was induced by intraperitoneal administration of streptozotocin and nicotinamide. The extract was given for 35 days orally in two different doses. The animals were divided into the following groups with 5 rats in each group.
| Group 1: | Served as normal control and the animals were kept untreated |
| Group 2: | STZ-NA induced animals which served as the diabetic neuropathic animals |
| Group 3: | STZ-NA treated diabetic neuropathic animals received SSEA extract at a dose of 200 mg kg-1 b.wt. by oral route, daily |
| Group 4: | STZ-NA treated diabetic neuropathic animals received SSEA dose of 400 mg kg-1 b.wt. by oral route, daily |
Tail flick method: Thermal sensitivity was assessed for all the rats of various groups by tail immersion test. The lower 5 cm portion of the tail was marked and immersed in warm water bath maintained at a temperature of 52°C±0.5. A cut-off time of 12 sec was maintained in all the case. Shortening of the tail withdrawal time indicates hyperalgesia due to central mechanism16.
Cold allodynia test: Cold allodynia was assessed after 2 h of assessment of hyperalgesia by measuring tail withdrawal latency. Ice cold water (10°C±0.5) was taken in a beaker. The paws of animals were gently submerged in ice cold water and the withdrawal time was observed. Cut off time of 15 sec was taken. Shortening of the tail withdrawal was reported as allodynia17.
Randall Selitto test: Mechanical nociceptive threshold is an index of mechano-hyperalgesia which was be assessed by Randall Selitto method. Pressure was applied to the centre of the dorsal surface of right hind paw increasing at a linear rate. Cutoff of 250 g was maintained in each case to avoid tissue injury. When the animal displayed pain by withdrawal of the paw, the applied paw pressure was registered by the analgesia- meter and expressed in mass units (grams). Five tests separated by at least 15 min were performed for each animal18.
Fasting blood glucose measurement: Blood from the tail vein of the animals was withdrawn and the blood glucose level was measured using a calibrated glucometer (model no. B 1801 Contour TS, Bayer) from 7th day to 35th day of the experiment at intervals of 7 days.
Animal sacrifice and organ collection: After the experimental period of 35 days, the animals from each experimental group were starved for 12 h and sacrificed by cervical dislocation and whole brains were immediately dissected out and washed in ice-cold saline to remove the blood. The brains were weighed and 10% tissue homogenate was prepared with 0.025 M Tris HCl buffer, pH 7.5. After centrifugation at 2000 rpm for 10 min, the clear supernatant was used to estimate enzyme activities19.
Estimation of lipid peroxidation: The method estimates Malonaldehyde (MDA), a product of lipid peroxidation. One molecule of MDA reacts with two molecules of thiobarbituric acid (TBA) under mild acidic condition to form pink colored chromogen and intensity was measured colorimetrically at 535 nm.
Supernatant was mixed with Trichloroacetic acid TCA (20%) and centrifuged at 2000 rpm for 15 min and supernatant was collected and mixed with TBA (0.067% in 1 M Tris hydrochloride, pH 7) and absorbance was measured at 535 nm.
In 2 mL of tissue supernatant, 2 mL TCA (20%) was added, centrifuged in a cooling centrifuge and supernatant was separated. To 2 mL of this supernatant, 2 mL of TBA was added, kept in boiling water bath for 10 min and cooled. Blank was similarly prepared using 1.5 mL distilled water, 1.5 mL TCA, 3mL of second supernatant and 3 mL TBA. The absorbance of the test against blank was measured at 535 nm using spectrophotometer20.
Reduced glutathione (GSH) estimation: Glutathione present in RBC consists of some sulfhydryl groups. 5, 5 dithiobis 2-nitrobenzoic acid (DTNB), a disulphide compound gets easily attacked by these sulfhydryl groups and forms a yellow colored anion which is measured colorimetrically at 412 nm.
To 1 mL of tissue supernatant, 1 mL of TCA (10%) was added, cooled for 10 min and centrifuged at 2000 rpm. To 0.5 mL of the supernatant so obtained, 4 mL of DTNB (60 mg of DTNB was mixed in 100 mL of 1% sodium citrate solution) and 1.5 mL phosphate buffer (0.2 M, pH 8) were added. Blank was similarly prepared substituting 1 mL distilled water for the tissue supernatant. The samples were mixed well and kept at room temperature for 5 min. The absorbance is taken against blank using spectrophotometer at 412 nm21.
Catalase estimation: In the Ultra Violet range H2O2 shows a continued increase in absorbance with decreasing wavelength. The decomposition of H2O2 can be followed directly by the decrease in absorbance at 240 nm. The difference in the absorbance per unit time is a measure of the catalase activity.
Phosphate buffer (50 mmol/L, pH 7.0) preparation.
| • | The 6.81 g KH2PO4 was dissolved in distilled water up to 1000 mL |
| • | The 8.9 g Na2HPO4 was dissolved in distilled water up to 1000 mL |
Reagents a and b were mixed in the proportion of 1:1.5 prior to experiment.
To 0.1 mL of brain homogenate 1.9 mL of phosphate buffer (0.05 M, pH 7) and 1 mL of freshly prepared 30 mM H2O2 were added and change in absorbance was measured at 240 nm for 3 min intervals of 30 sec. One unit of catalase activity was defined as change in absorbance of 0.01 units per minute22.
Motor Nerve Conduction Velocity (MNCV): The MNCV was done in the sciatic posterior tibial conducting system in a temperature controlled environment. The left sciatic nerve was first exposed through surgical procedure and signal was recorded by 4 channel MP 35 Biopac Systems incorporation, USA. BSL STM, attached to channel 2 of MP 35 for providing a stimulus of 8 V for 0.2 msec. The response of the stimulus as recorded from the channel 1 with self designed EMG electrodes.
The electrodes were placed on the foot (paw) and stimulus was given to the sciatic nerve.
Differential EMG was recorded to analyze the nerve conduction. The time taken by the impulse to reach from one electrode to other was recorded; further the distance between the impulse transmitting electrodes was measured23,24.
Histopathological evaluation of sciatic nerve: Sciatic nerve was isolated after sacrifice and the samples of sciatic nerve were kept in the fixative solution (10% formalin) and cut into 4 μm sections using microtome. Staining was done by using hematoxylin and eosin. Nerve sections were analyzed under microscope at 100X for axonal derangement and other defects25.
RESULTS
Tail flick: Animals of the diabetic group (STZ-NA) exhibited marked decrease in the pain threshold from a noxious stimulus as compared to the normal control animals (p<0.05). However treatment of STZ-NA induced diabetic animals with Salvia splendens (200 and 400 mg kg-1) significantly increased the pain threshold in both the treatment groups (Table 1, Fig. 1).
Cold allodynia: Animals of the diabetic group (STZ-NA) exhibited significant decrease in pain threshold in response to non-noxious stimuli as compared to normal rats from 14th day of induction of STZ.
Treatment of STZ-NA induced diabetic group with S. splendens (200 and 400 mg kg-1) significantly decreased the pain threshold in diabetic group (Table 2, Fig. 2).
Randall Selitto test: Paw-withdrawal threshold in the Randall-Sellito test was reduced in the STZ-NA induced diabetic rats as compared to controls. Treatment of STZ-NA induced diabetic group with Salvia splendens (200 and 400 mg kg-1) partially corrected this variation at the end of study (Table 3).
Blood glucose level: Effect of SSEA at dose levels of 200 and 400 mg kg-1 b.wt. was observed on the blood glucose levels of STZ-NA diabetic group as shown in Table 4. The blood glucose levels were markedly raised in diabetic control as compared to normal, on the 7th, 14th, 21st, 28th and 35th day of the experiment. The treatment groups showed significant reduction in blood glucose level on 7th, 14th, 21st, 28th and 35th day with respect to diabetic control (STZ-NA induced). The above study revealed that on daily treatment with ethyl acetate fraction of S. splendens, STZ-NA induced diabetic group showed marked improvement in blood glucose levels which proved its efficacy in attenuating diabetes (Table 4).
Estimation of reduced glutathione in brain: The antioxidant enzyme activity i.e. estimation of reduced glutathione is shown in Table 5. From this study, it was evident that glutathione (μg mg-1 of protein) was found to significantly decrease (p<0.05) in the diabetic control group as compared to the normal control group.
On treatment of STZ-NA induced diabetic group with ethyl acetate fraction of S. splendens at doses of 200 and 400 mg kg-1, it was found that the GSH level significantly increased (p<0.05) in the treatment groups compared to diabetic control group.
Estimation of malonaldehyde of brain: The antioxidant enzyme activity i.e., estimation of malonaldehyde is shown in Table 5. From this study it was evident that malonaldehyde (nmoles/mg of protein) was found to be significantly elevated (p<0.05) in the diabetic control rats as compared to the normal control group. On treatment of STZ-NA induced diabetic group with ethyl acetate fraction S. splendens at doses of 400 mg kg-1 it was found that the malonaldehyde (nmoles/mg of protein) level significantly decreased (p<0.05) showing reversal of peroxidation but there was no significant change in the malonaldehyde level in group treated with 200 mg kg-1 of extract when compared to the diabetic group.
Estimation of catalase activity: The antioxidant enzyme activity i.e. estimation of catalase activity is shown in Table 5. From this study it was evident that catalase level (U mL-1 tissue) was found to significantly decrease (p<0.05) in the diabetic control rats as compared to the normal control. Treatment of STZ-NA induced diabetic group with S. splendens at a dose of 400 mg kg-1 showed significant change compared to diabetic control group (STZ-NA induced).
Motor nerve conduction studies: Six weeks after induction of diabetic neuropathy, the sciatic motor nerve conduction of STZ-NA (12.3 m sec-1) was compared with that in the nondiabetic control group (23 m sec-1) which was significantly slower by approximately 46.52%. However, the MNCV in diabetic rats (STZ-NA induced) treated with SSEA 200 and 400 mg kg-1 showed somewhat elevation in nerve conduction velocity as compared to that of diabetic rats. However further studies are to be conducted with larger number of animals in order to get statistically validated result (Table 6, Fig. 3a-d).
Histopathological studies: Normal control animals showed normal arrangement of fibres and there was no axonal swelling observed in this group. The diabetic group showed derangement of nerve fibers with significant axonal swelling. Animals treated with SSEA 400 mg kg-1 significantly attenuated diabetes induced axonal degeneration of fibres and axonal swelling was reverted back which was evident from the architecture of sciatic nerve but those treated with 200 mg kg-1 of dose, did not show the histological arrangement similar to the control group thus inferring that the changes were not reverted back to normal (Fig. 4).
DISCUSSION
S. splendens Sellow ex Roem. and Schult. (Lamiaceae) has been used by tribals of Chotanagapur, Bihar and Chhatisgarh as medicine and in culinary purposes. The methanolic extract of the plant has already been reported to have antioxidant26 and antidiabetic12 activities. Therefore, active fraction from the methanolic extract of the plant was identified based on preliminary phytochemical estimation.
Streptozotocin is most commonly used to induce diabetes in experimental animals and administration of STZ-NA caused diabetic neuropathy27,28 probably due to partial destruction of β-cells which leads to a decrease in blood insulin and an increase in blood glucose and decreased utilization by tissues in these animal models. STZ-NA induced diabetic model was found to be a suitable animal model for short term and long term studies.
According to previous studies, it has been well established that oxidative stress leads to nerve deficits in diabetic rats29. Some studies indicated that reduction in neural blood flow and endoneurial hypoxia may cause functional and morphological abnormalities of nervous system30. In addition, increased free radicals due to hyperglycemia may affect central and peripheral nervous system31.
To assess thermal and mechanical sensory responses, behavioural tests have been performed. In thermal test, the tail withdrawal latency (flicking response) or signs of struggle were observed and recorded. In cold allodynia, shortening of tail withdrawal time indicated allodynia. The hyperalgesia effect due to tail withdrawal effect is attributed to central mechanism. Another diabetes-related phenomenon, mechanical hyperalgesia, was revealed by assessment of paw-withdrawal thresholds by the Randall-Sellilo test.
In the present study, there has been marked hyperalgesia in untreated group followed by neuropathic pain at the end of 35 days. The diabetic group developed significant thermal and cold hyperalgesia (p<0.05). Several researchers have reported hyperalgesia in diabetic rats32,33. Previous preclinical studies have demonstrated an association between elevated TNF-α levels and altered pain behavior34,35. TNF-α concentration in spinal cord dorsal horn are elevated in several neuropathological disorders associated with hyperalgesia36,37. The levels of TNF-α and TNF-α receptor 1 in dorsal root ganglia and spinal cord dorsal horn increase after peripheral nerve injury and in other neuropathic pain models.
During the study, it was observed that treatment of STZ-NA induced diabetic rats with SSEA at a dose of 200 and 400 mg kg-1 exhibited significant reduction (p<0.05) in nociception due to thermal and cold induced algesia. When checked for mechanical algesia, SSEA showed significant increase in mechanical threshold.
The result of present study showed a significant decrease in the blood glucose (p<0.05) in the treatment group animals compared to the diabetic control group. This result showed that SSEA was a potent antihyperglycemic agent and was thus decreasing the parameters involved in the progression of diabetic neuropathy.
Free radical oxidative stress has also been well reported in STZ-diabetic rats38. Lipid peroxidation and glucose oxidation leads to increased level of toxic oxidants in diabetic condition39. Reduction in endogenous antioxidant enzymes is also reported in STZ induced diabetes39. In the present study, there was an increase in malondialdehyde levels in brain of diabetic group animals and decrease in the level of GSH and catalase enzyme in the diabetes induced model (p<0.05). Decreased GSH in diabetic group rats was supported by earlier studies suggesting that the decrease in GSH is a sensitive and a potentially relevant indicator of neurotoxicity and plays a role in development of diabetic complications in brain40.
Treatment of STZ-NA induced rats with SSEA at a dose of 400 mg kg-1 showed significant reduction of MDA and increased level of GSH and catalase in the brain (p<0.05), diabetic group treated at lower dose (200 mg kg-1) exhibited insignificant result. This may be due to anti-oxidant activity of Salvia splendens due to the presence of higher amount of flavonoid, anthocyanin and phenolic compounds.
There is a decrease in the motor nerve conduction velocity in the diabetic animal as compared the normal control rat. Diabetic neuropathy is characterized by neuroanatomical changes and by decreased nerve conduction velocity. A relationship has been demonstrated between structural lesions and slowed nerve conduction velocity in chronically diabetic animals41,42. The reduced nerve conduction velocity has been associated with a diminished activity of Na, K-ATPase43. Decrease in the endoneurial blood flow leads to the degeneration of myelin sheath causing decrease in the nerve conduction velocity44.
Transverse section of sciatic nerve shows axonal swelling and derangement of nerve fibers. These findings are consistent with previously published reports45. The SSEA at a dose of 400 mg kg-1 reverted the histological changes that occurred due to diabetic neuropathy but SSEA at a dose of 200 mg kg-1 did not show significant changes in the histopathology of neuropathy. Therefore, the present study supports the potential use of S. splendens ethyl acetate extract in treatment of diabetic complications. Results of the study demonstrate the protective effect of S. splendens extract in diabetic neuropathy which may be due to the improved glycaemic control and antioxidant system. After completion of the study protocol, it was found that the ethyl acetate fraction of aerial parts of extract were efficacious in attenuating the progression of diabetic neuropathy but further studies need to be performed to validate the data which can alter due to several factors.
ACKNOWLEDGMENTS
The authors would like to acknowledge the Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India, for providing the necessary facilities to carry out the study.