Journal club 3 3 dec

Researcher, Drug Specialist um MGIMS, SEVAGRAM, WARDHA
26. Jan 2021

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Journal club 3 3 dec

  1. Dr Ashishkumar Baheti JR 1 ,MD Pharmacology MGIMS , Sevagram JOURNAL CLUB 3 3/12/2019 1
  2. Antidiabetic activity of hydro-alcoholic stem bark extract of Callicarpa arborea Roxb. with antioxidant potential in diabetic rats Julfikar Ali Junejo, Mithun Rudrapal, Lalit Mohan Nainwal, Kamaruz Zaman Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam, India Biomedicine & Pharmacotherapy Vol.95 , 2017 2
  3. 1. Introduction • Diabetes mellitus (or Diabetes) is a metabolic disease characterized by hyperglycemia with abnormal carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action/both. • Oxidative stress (OS) is believed to be the underlying cause of cellular injury, tissue damage or organ dysfunctions commonly associated with diabetic complications. • Callicarpa arborea Roxb., known as beautyberry (English), is a shrub or small evergreen tree, which belongs to family Verbenaceae. 3
  4. Ethnobotanical study indicates that paste of the stem bark and juice of C. arborea has been used by the tribals of Mizoram state of Northeast India to cure cuts and wounds. The leaves infusion has also been used for the management of diabetes. Moreover, since a biochemical relationship exists between diabetic hyperglycemia and cellular oxidative stress, the antidiabetic activity evaluation of the hydro-alcoholic extract (HAE) of C. arborea stem bark was carried out along with the antioxidant activity study. 4
  5. 2. Materials and Methods 2.1 Chemicals All chemicals and reagents, Commercial reagent kits were procured from Rankem, Mumbai and Himedia Laboratories Ltd., Mumbai, and SPAN Diagnostics Ltd., Surat (India). 2.2 Plant material Stem bark of Callicarpa arborea Roxb. were collected from forest areas of Dibrugarh district, Assam during the month of December 2014. The plant species was identified and authenticated. 5
  6. 2.3 Preparation of hydro-alcoholic extract (HAE) 100 g of powdered stem barks was used for the preparation of extract. Powdered stem barks were extracted using sufficient quantity (1000 ml) of ethanol:water mixture (7:3) by cold maceration for 24 h. 2.4 Test Animals Healthy Wistar male albino rats (240–260 g) 2.5 Phytochemical screening Preliminary phytochemical screening of the HAE was carried out for detection of the presence of various phytoconstitutents. 6
  7. 2.6 Estimation of phenolic and flavonoid contents The total phenolic content of the HAE was evaluated following the Folin-Ciocalteu colorimetric method The total flavonoid content was estimated using the aluminum chloride colorimetric method 2.7 In vitro antioxidant activity The in vitro antioxidant activity of HAE was carried out by the following three assay methods in accordance with previously reported procedures with minor modifications. Lipid peroxidation scavenging activity, Hydroxyl radical scavenging activity and Superoxide radical scavenging activity. 7
  8. 2.8 Acute oral toxicity study Over-night fasted rats were randomly divided into six groups of six animals each. Rats of different groups were administered with increasing doses (250, 500, 1000, 2000 and 2000 mg/kg b.w.) of the HAE. One group was maintained as normal control and was given vehicle alone. The animals were observed individually for first one hour for any gross behavioral changes if any, and then periodically for the next 24 h, and then at every 24 h for any signs of acute toxicity over a period of 14 days. 8
  9. 2.9 Oral glucose tolerane (OGT) test Glucose 2 g/kg was fed orally 30 min after the administration of HAE and metformin hydrochloride. Blood was withdrawn from the tail vein at 0, 30, 60, 90 and 120 min Group 1 (n=6) Normal control vehicle (0.5% CMC w/v in NS). Group 2 (n=6) Test group HAE at 250 mg/kg b.w. Group 3 (n=6) Test group HAE at 500 mg/kg b.w. Group 4 (n=6) Treatment group metformin hydrochloride (5 mg/kg b.w.). 9
  10. 2.10 Hypoglycemic activity in streptozotocin-induced diabetic rats Diabetes was induced in overnight fasted animals by a single intraperitoneal (i.p) injection of streptozotocin (STZ, 55 mg/ kg b.w. in normal saline). Blood was collected from the tail vein each time for the determination of glucose levels on 0, 7, 14 and 21 day. Group 1 (n=6) Normal control vehicle (0.5% CMC w/v in NS). Group 2 (n=6) Diabetic control vehicle (0.5% CMC w/v in NS). Group 3 (n=6) Test group 1 HAE at 250 mg/kg b.w. Group 4 (n=6) Test group 2 HAE at 500 mg/kg b.w. Group 5 (n=6) Standard group metformin hydrochloride (5 mg/kg b.w.). 10
  11. 2.11 Liver and kidney function tests Blood was collected , The serum was separated by centrifugation (2000 rpm, 10 min) for estimation of various biochemical parameters. Serum insulin levels were measured by the microplate ELISA method using a commercial kit (SPAN Diagnostics Ltd.). Serum lipid profile was estimated using commercially available kits (SPAN Diagnostics kit). Serum was used to estimate glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP), total protein (TPR) and creatinine (CRTN). 11
  12. 2.12 In vivo antioxidant activity On 21st day, all the groups of animals were anaesthetized using diethyl ether, liver was dissected out, washed with normal saline and one part was preserved in 10% formalin for histopathological studies. The other part of liver was homogenized by ice chilled Tris- HCl buffer and used for activities/levels of superoxide dismutase (SOD), catalase, reduced Glutathione (GSH), Glutathione peroxidase (GPx), and malondialdehyde (MDA). 12
  13. 2.13 Histopathological studies At the end of 21th day of treatment, the animals were fasted for 12 h, anaesthetized using diethyl ether and sacrificed by cervical dislocation. Pancreas and liver were instantly dissected out, excised and rinsed in ice-cold saline solution. Tissues were processed and after fixation, tissues were dehydrated in ethanol (70–95%), cleared in xylene, and embedded in paraffin, solid transverse sections of 4–5 mm thickness were obtained by using a rotary microtome. The section were stained with haematoxin-eosin and histopathological observations were carried out under a light microscope. 2.14 Statistical analysis one-way ANOVA followed by Student’s t-test. 13
  14. 3. Results 3.1 Phytochemical screening The percent yield of crude dried extract was found to be 68.78%, w/w per dry weight of powdered stem barks. The results of preliminary phytochemical screening revealed the presence of alkaloids, glycosides, saponins, tannins, flavonoids and phenolic compounds. 3.2 Acute toxicity study No sign and symptoms of acute toxicity and mortality up to 2000 mg/kg body weight dose were observed during the whole experimental period. For further studies, the doses were fixed as 250 and 500 mg/kg body weight. 14
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  16. 3.3 In vitro antioxidant activity Fig. 3.3.1. Superoxide radical scavenging activity. Values are mean ± SEM of three replicate experiments. HAE, quercetin and gallic acid exhibited 78.47 ± 0.20%, 82.12 ± 0.44% and 85.33 ± 0.34% of scavenging effect respectively at the highest tested concentration of 250 mg/ml. 16
  17. Fig.3.3.2. Hydroxyl radical scavenging activity. Values are mean ± SEM of three replicate experiments. The percent inhibition of hydroxyl radicals were 83.45 ± 0.18%, 87.37 ± 0.30% and 86.14 ± 0.49 for HAE, quercetin and gallic acid respectively at the concentration of 250 mg/ml. 17
  18. Fig. 3. Lipid peroxidation scavenging activity. Values are mean ± SEM of three replicate experiments. %Scavenging activity of HAE is statistically significant at p < 0.05, compared to quercetin & gallic acid (standards). 64.11 ± 0.27%, while quercetin and gallic acid inhibited by 74.27 ± 0.65%, and 71.09 ± 0.34%, respectively at 250 mg/ml 18
  19. Fig. 4. OGT test. Values are mean ± SEM of three replicate experiments. Activities of HAE and metformin are statistically significant at p < 0.05, compared to normal control. In OGT, HAE (250 and 500 mg/kg) showed significant (p < 0.05) reduction of glucose load (plasma glucose level) as compared to normal control group. 19
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  22. 3.8. Effect of HAE on lipid profile in diabetic rats In diabetic rats, the levels of triglycerides (TG), total cholesterol (TC), and low density lipoprotein (LDL) were significantly increased and high density lipoprotein (HDL) level was significantly decreased. In HAE (250 and 500 mg/kg) treated groups the TG, TC and LDL levels activities were significantly (p < 0.05) reduced and the HDL level was significantly (p < 0.05) increased as compared to diabetic control rats. 22
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  24. Increased levels of MDA, an indicator of LPO, in diabetic rats were significantly (p < 0.05) reduced after treatment with HAE (250 & 500 mg/kg) as compared to the normal rats. 24
  25. Fig. 5. Histology of pancreas of experimental rats after treatment with HAE, 500 mg/kg. (A) Normal control, (B) Diabetic control, (C) Diabetic treated with HAE (500 mg/kg), (D) Diabetic treated with metformin. 25
  26. Fig. 6. Histology of liver of experimental rats after treatment with HAE 500 mg/kg. (A) Normal control, (B) Diabetic control, (C) Diabetic treated with HAE (500 mg/kg), (D) Diabetic treated with metformin. 26
  27. 4. Discussion  The intraperitoneal administration of STZ damages partially the insulin secreting b-cells of the pancreas by breaking DNA strands leading to decreased endogenous insulin release which ultimately results in diabetes mellitus.  Administration of HAE to diabetic rats showed a significant reduction in the levels of blood glucose and an increase in the levels of serum insulin and it was further supported by histopathological observations.  The liver glycogen content was markedly reduced in diabetic animals, which was in proportion to insulin deficiency.  Diabetic rats treated with HAE increased significantly the liver glycogen content as compared to the diabetic control, which could be due to increased insulin secretion. 27
  28.  Under normal circumstances insulin activates enzyme lipoprotein lipase and hydrolyses triglycerides.  Diabetic rats treated with HAE significantly improved serum TG and TC.  The significant control of the levels of serum lipids in the HAE treated diabetic rats might be attributed to improvements in insulin levels.  Significant lowering of LDL cholesterol and raise in HDL cholesterol were observed in treated diabetic rats. 28
  29.  Diabetic rats showed significantly increased level of CRTN.  The significant reduction in the level of CRTN in HAE treated diabetic rats indicated that the HAE prevented the progression of renal damage in diabetic rats.  In diabetes mellitus, high glucose level can inactivate antioxidant enzymes SOD, CAT, GSH and GPx by glycating these proteins thus producing induced oxidative stress, which in turn, causes lipid peroxidation.  Furthermore, malonaldehyde (MDA) is one of the end products in LPO process. 29
  30.  LPO in the tissue homogenate was determined by measuring the amounts of MDA produced primarily.  SOD, CAT, GSH and GPx activities were increased to normal indicating the efficacy of HAE in attenuating the oxidative stress (OS) and eventual inhibition of LPO in diabetic liver.  Decrease in MDA level indicated reduced rate of LPO in HAE treated diabetes.  In this study, a marked increase in the concentration of TBARS and MDA were observed in STZ induced diabetic rats indicating the LPO of tissues under OS.  Since the HAE significantly decreased TBARS levels as well as MDA in liver of diabetic rats indicating strong lipid peroxidation scavenging activity of the HAE as antioxidant agent. 30
  31.  Superoxide directly initiates LPO and plays an important role in the formation of other ROS like hydroxyl radicals, which induce oxidative damage in lipids, proteins and DNA.  Hydroxyl radical is directly involved in the LPO process. Hydroxyl radicals are more potent than superoxides, and HAE could effectively scavenge these radicals together with the inhibition of LPO, where in it scavenged active oxygen species by preventing the propagation of free radical chain reaction as antioxidant.  It is now assumed that the antioxidant activity is responsible for the antidiabetic action of the HAE, and phenolic compounds and flavonoids present in the HAE may be involved in reducing underlying cellular OS and eventual hypoglycemic reactions. 31
  32. 5. Conclusion  The present study concludes that HAE showed potent hypoglycemic activity in diabetic rats compared to normal rats with significant improvement in body weight, levels of serum insulin, liver glycogen, serum lipids, liver and kidney biochemical markers and liver antioxidant enzymes levels of experimental animals.  The antioxidant activity of C. arborea stem bark reported herein signifies the potential of this plant species as herbal antioxidant with possible role in the prevention of oxidative stress-induced diabetes and associated disease complications. The authors declare that there are no conflicts of interest. 32
  33. Criticism 1)Title – Clear, brief Study design was not mentioned 2) Abstract - Provided an accurate summary of the background, key methods, principal findings and conclusions of the study Introduction 3) Background- Included sufficient scientific background 4) Objectives - Yes, objectives were Clearly described. 33
  34. Methods 5) Ethical statement – Yes, animal ethics committee approval was taken 6) Study design - The number of experimental and control groups was mentioned for each experiment; sampling method was mentioned 7) Experimental procedures – were explained in detail, method of euthanasisa also mentioned. 8) Experimental animals - Provided details of the animals used, including species, strain, sex, developmental stage, weight, source of animal 34
  35. 9) Housing and husbandary – type of facility, type of cage, number of cage companions was mentioned. 10) Sample size - the number of animals in each group was mentioned in each experiment. Doesn’t mention How sample size was calculated? 11) Allocating animals to experimental groups – full details of how animals were allocated, treated and assessed in different experimental groups given. 12)Experimental outcomes -Yes, they were Clearly defined 13) Statistical methods – Yes, details of the statistical method used for each analysis was mentioned 35
  36. Results 14) Baseline data- Yes, it was mentioned. 15) Numbers analysed- the mean of all the animals 6/6 present in each group was used in analysis 16) Outcomes and estimation – Yes, it was mentioned. P < 0.05 was considered statistically significant, all the outcomes were mentioned graphically and in tabular form. 17) Adverse events- No, there were no adverse events 36
  37. Discussion 18) Interpretation/ scientific implications- Yes, interpretations of the study were mentioned 19) Generalisability/ translation- Yes, the findings of the study were relevant to human biology 20 ) Funding – NO, source of funding was not mentioned. 37
  38. THANK YOU 38
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  40. FIG. 3 3.3.3. Lipid peroxidation scavenging activity LP induced by Fe2+/ascorbate in rat liver homogenate was found to be inhibited by the HAE in a concentration dependant manner and a considerable amount of lipid peroxidation inhibitory effect was observed by 64.11 ± 0.27%, while quercetin and gallic acid inhibited by 74.27 ± 0.65%, and 71.09 ± 0.34%, respectively at 250 mg/ml (Fig. 3). Test results were considered statistically significant when compared to standard drugs (p < 0.05). 40
  41. FIG. 5 3.11. Histopathological observations 3.11.1. Effect of HAE on pancreatic section in normal and diabetic rats Histopathological studies of pancreas (Fig. 5) of STZ-treated diabetic rats exhibited reduction in the dimensions of islets, damaged b-cell population and extensive necrotic changes followed by fibrosis and atrophy (B). HAE (500 mg/kg) and metformin treated rats restored the necrotic and fibrotic changes and also increased the number and increased the size of the islets (C). In normal control group normal acini and normal cellular in the islets of langerhans in the pancreas were observed (A). 41
  42. FIG. 6 3.11.2 Effect of HAE on liver section in normal and diabetic rats Photomicrographs of liver (Fig. 6) showed normal hepatic cells with well preserved cytoplasm, nucleus, nucleolus and central vein (A). In case of diabetic rats, the normal lobular structure was preserved. The central vein was prominent and prominently congested. Focal areas of hemorrhage were also seen. Vacuoliza- tion and fatty change were evident. The portal tracts appeared normal (B). In diabetic treated group (HAE 500 mg/kg), the hepatocytes portal tracts and central veins appeared normal (C). HAE treated group is comparable with metformin treated group (D) and changes in treated groups are significant (p < 0.05) compared to normal control group. 42

Hinweis der Redaktion

  1. Hindi- kumhar, nepali – guren,
  2. like drowsiness, restless- ness, writhing, convulsions and symptoms of toxicity and mortality The acute toxicity study was done as per OECD guideline-425.
  3. Carboxymethylcellulose (CMC) solution (0.5% w/v in normal saline) was used as vehicle.
  4. The animals confirmed as diabetic (after 72 h of STZ injection) by the elevated plasma glucose levels (200– 300 mg/dl) was used for the experiment. Carboxymethylcellulose (CMC)
  5. The total phenolic content of the HAE, calculated from the calibration curve of gallic acid (R2 = 0.986), was 52.17 ± 2.48 GE/g, and the total flavonoid content (R2 = 0.988), calculated from the calibration curve of quercetin was 39.10 ± 2.15 QE/g.
  6. 3.6. Effect of HAE on blood glucose levels in diabetic rats  STZ-treated diabetic rats exhibited significant increase in the levels of blood glucose in comparison to normal rats. After treatment with HAE the blood glucose levels were significantly (p < 0.05) reduced compared to the diabetic control rats at both the doses 250 & 500 mg/kg Table 2.
  7. The activity of HAE was found less than that of metformin (5 mg/kg) treated group. In diabetic rats the body weight, insulin level and glycogen content were significantly decreased. After 21 days of treatment with HAE at 250 and 500 mg/kg the body weight was significantly (p < 0.05) increased, insulin level and glycogen content were also significantly (p < 0.05) increased as compared to diabetic rats
  8. Metormin (5 mg/kg) treated rats also showed significant effects on blood levels of SGOT, SGPT, ALKP, TPR and CRTN in diabetic rats. 3.9. Effect of HAE on SGOT, SGPT, ALKP, TPR and CRTN in diabetic rats   There was a significant increase in activities of SGOT, SGPT and ALKP in diabetic rats. After treatment with HAE (250 & 500 mg/kg) the activities of SGOT, SGPT and ALKP activities were significantly (p < 0.05) reduced as compared to diabetic control rats. A significant decrease in serum total protein (TPR) level and a significant increase in creatinine (CRTN) level were observed in diabetic rats. After treatment with HAE at 250 and 500 mg/kg doses for 21 days the TPR level was significantly increased (p < 0.05) and CRTN level was significantly (p < 0.05) decreased compared to diabetic control rats.
  9. Metformin (5 mg/kg) also showed significant (p < 0.05) reduction of these enzymes. 3.10. Effect of HAE on liver enzymes and MDA   In treated diabetic rats, the activities of SOD, CAT, GSH and GPx were significantly increased. There was a significant (p < 0.05) reduction in the activities of these antioxidant enzymes in diabetic rats as compared to normal rats.
  10. which clearly revealed that diabetic treated (HAE) animals showed increase in the number of islets, lesser degree of shrinkage and restoration of necrosis of b-cells of pancreas. The decrease in liver glycogen content in diabetes is due to the lack of insulin which ultimately results in the inactivation of glycogen synthase enzyme.The significant increase in the glycogen levels of the HAE treated diabetic animals might be because of the reactivation of glycogen synthase system.
  11. In insulin deficient diabetics, the plasma free fatty acid concentration is elevated as a result of increased free fatty acid outflow from fat depots, where the balance of the free fatty acid esterification–triglyceride lipolysis cycle is displaced in favour of lipolysis.
  12. SOD, CAT, GSH and GPx are enzymatic antioxidants that play a vital role in preventing oxidative damage to cells. SOD reduces the superoxide radical into hydrogen peroxide (H2O2). The other enzymatic antioxidant CAT catalyzes the reduction of hydrogen peroxides into water molecule and protects the tissues against reactive hydroxyl radicals. When cell has increased levels of SOD without a proportional increase in GPx, cells face a peroxide overload challenge. Attenuating – reducing
  13. STZ produces oxygen radicals in the body, which cause pancreatic injury and could be responsible for increased blood sugar level in animals. Moreover, abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to the development of insulin resistance.
  14. The changes in pancreas morphology in metformin treated group (D) are similar to HAE treated rats, where changes are significant (p < 0.05) compared to normal control group.