Brijesh Ranjan

[Downloaded from http://www.phcog.com on Wednesday, July 12, 2017, IP:-] Pharmacogn. Mag. ORIGINAL ARTICLE A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products www.phcog.com | www.phcog.net Evaluation of the Antidiabetic Properties of S‑1708 Mulberry Variety Brijesh Ranjan, Randhir Kumar1, Neeraj Verma2, Swati Mittal2, Pranab Lal Pakrasi1, R. Venkatesh Kumar Department of Applied Animal Sciences, Sericulture Laboratory, Babasaheb Bhimrao Ambedkar University, Lucknow, 1Department of Zoology, Embryo Physiology Laboratory, Banaras Hindu University, 2Department of Zoology, Skin Physiology Laboratory, Banaras Hindu University, Varanasi, Uttar Pradesh, India Submitted:- Revised:- Published:- ABSTRACT SUMMARY Background: Diabetes is a metabolic disease prevalent worldwide in all age group of people. The source of diabetes is due to an oxidation process that can produce free radicals. An increase in oxidative free radicals in the body is reported to be one of the several causes of diabetes. The best remedy to combat oxidative stress is the use of antioxidants, which inhibit and scavenge free radicals. Aim: This study has been undertaken to evaluate the antioxidant activity and antidiabetic effect of mulberry leaf extract in diabetic mice. Materials and Methods: Antioxidant activity of mulberry leaves was determined by 2,2‑diphenyl‑1‑picryl‑hydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) assay. Antidiabetic assay of mulberry leaf extract was analyzed by oral administration of leaf extract up to 3 weeks in diabetic mice induced by streptozotocin. Results: In vitro antioxidant activity in both DPPH and FRAP assays showed significantly (P < 0.05) higher inhibition of free radicals than that with ascorbic acid. Diabetic mice fed with mulberry leaf extract showed increment (+25.88%) in body weight and a significant reduction in blood glucose concentration (−71.58%). Further, glucose‑6‑phosphate dehydrogenase enzyme activity was significantly (P < 0.05) increased, whereas activities of other enzymes particularly catalase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase were decreased in diabetic mice after oral administration of mulberry leaf extracts. Histology of liver revealed regeneration of hepatocytes, central vein, and nucleus. Conclusion: This study demonstrated that S‑1708 mulberry variety has a potential therapeutic value in diabetes and related complications. Key words: 2,2‑diphenyl‑1‑picryl‑hydrazyl, antioxidant, ferric reducing/antioxidant power, mulberry, streptozotocin • Diabetes mellitus is a grave metabolic deviations and responsible for many complications affecting various organs in the human body. In spite of the known antidiabetic medicine available in the market, diabetes and the associated impediments sustained to be a major medical crisis. Medicinal plants have been proven to be useful in diabetes due to their rich therapeutic value. In the current study, S-1708 mulberry variety not only authenticated the earlier results obtained from other medicinal plants but also turn out to be known as a potential source for treating diabetes by demonstrating tremendous ant- diabetic properties. INTRODUCTION Natural antioxidant compounds from plant origin are medicinally helpful for human health. Antioxidant compounds including ascorbic acid, carotenoids, flavonoids, and tannins are well known to play an important role in the prevention of several chronic diseases.[1] The capacity between the production and neutralization of reactive oxygen species (ROS) by antioxidants is very reasonable, and if this balance tends toward the overproduction of ROS, the cells start to suffer the penalties of oxidative stress.[2] Oxidative stress is linked with numerous pathologic conditions such as cardiovascular and neurodegenerative diseases. Dietary antioxidants including Vitamins E, C, and carotenoids are well known to be effective in the prevention of oxidative stress‑related diseases.[3] Mulberry is rich in alkaloids, polyphenols, flavonoids, and anthocyanin which have been suggested to be accountable for health benefits.[4] Mulberry leaves contain a variety of essential micronutrients, for example, ascorbic acid, carotene, Vitamin B, Vitamin D and flavonol glycosides.[5] Traditionally, mulberry leaves have been used as medicinal agents to nourish the blood, benefit the and treat weakness, fatigue, anemia, and premature graying of hair. It is also used to treat urinary incontinence, tinnitus, dizziness, and constipation in the aged patient. Mulberry leaves S280 Abbreviations used: S-1708, DPPH, FRAP. Correspondence: Dr. R. Venkatesh Kumar, Department of Applied Animal Sciences, Sericulture Laboratory, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow ‑ 226 025, Uttar Pradesh, India. E‑mail:-DOI: 10.4103/pm.pm_490_16 Access this article online Website: www.phcog.com Quick Response Code: have other pharmacological properties such as analgesic, antiasthmatic, anti‑rheumatic, antitussive, and astringent.[6] The phytochemicals of mulberry vegetative parts are getting more attention nowadays due to their numerous applications in served industries such as food, pharmaceutical, nutraceuticals, and cosmetics.[7] Further, mulberry leaves have been used to treat hyperglycemia, inflammation, cough, hypertension, cancer, and fever.[8] Mulberry acts as a growing resource of combating stress‑related diseases. Hence, this study was aimed to determine the antioxidant and antidiabetic properties of mulberry (variety‑S‑1708) in streptozotocin (STZ)‑induced diabetic mice. This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact:-Cite this article as: Ranjan B, Kumar R, Verma N, Mittal S, Pakrasi PL, Kumar RV. Evaluation of the antidiabetic properties of S-1708 mulberry variety. Phcog Mag 2017;13:S280-8. © 2017 Pharmacognosy Magazine | Published by Wolters Kluwer - Medknow [Downloaded from http://www.phcog.com on Wednesday, July 12, 2017, IP:-] BRIJESH RANJAN, et al.: Antidiabetic Properties of Mulberry MATERIALS AND METHODS Chemicals 2,2‑diphenyl‑1‑picryl‑hydrazyl (DPPH), 2,4,6‑Tripridyl‑s‑triazine (TPTZ), were purchased from Sigma‑Aldrich, USA. supplied by Mumbai, India. Ascorbic acid, sodium acetate buffer (pH‑3.6), glacial acidic acid, sodium acetate, were procured from Loba Chemie Pvt. Ltd., Mumbai, India. Glucose‑6‑phoshtate were dehydrogenase (G6PDH), β‑nicotinamide adenine dinucleotide phosphate (β‑NADP) were purchased from Sisco Research Laboratory Pvt., Mumbai, India. Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were obtained from ARKRAY Healthcare Pvt. Ltd., India. STZ was bought from HiMedia, Mumbai, India. All other chemicals used were of analytical grade. Collection of sample A sample of mulberry leaves (variety‑S‑1708) was collected from the Central Sericultural Germplasm Resources Centre (CSGRC), Hosur, Tamil Nadu, India. The plant species was identified by Dr. P. Sharawati, an eminent scientist in the mulberry division of the CSGRC. The identification of the variety of mulberry was based on the different morphological characters present in different varieties of mulberry. A catalog, having all aspect of different characters of mulberry is also provided by CSGRC to scientists to make the identification easy and precise. The variety was further confirmed by another eminent scientist Dr. M. M. Borpuzari of CSGRC Hosur, Tamil Nadu, India, which is also serving as repository center for the identification of mulberry Germplasm. Preparation of plant extracts Leaves were washed with distilled water, left to dry naturally at room temperature and powdered with a grinder. The powdered leaves (50 mg) was extracted in both methanol and ethanol solvents, separately for 48 h. The extracts were filtered using Buchner funnel and Whatman No. 1 filter paper and freshly prepared extracts were used for the analysis of antioxidant activity through DPPH and ferric reducing/antioxidant power (FRAP) assay. The powder leaf material (100 mg) was dissolved in 10 ml of 20% ethanolic solvent for 2 h,[9] an extract was filtered and used for in vivo study of diabetic mice. Hence, composition of extracts was not analyzed due to the limitation of the facility. Determination of free radical scavenging activity using 2,2‑diphenyl‑1‑picryl‑hydrazyl method The free radical scavenging activity of mulberry leaf extracts was measured by the spectrophotometric method for the assay of hydrogen donating free radical.[10] Different concentrations (50, 100, 200, 300, 400 µl/ml) of mulberry leaf extracts and ascorbic acid as standard were prepared in both methanol and ethanol solvents separately. Thereafter, 3 ml of 0.004% DPPH reagent was added. The reaction mixture was mixed thoroughly and left for incubation at room temperature in dark. The absorbance was measured at 517 nm using a spectrophotometer and antioxidant activity was expressed as percentage inhibition. Percentage inhibition I % = (Ablank − Asample/Ablank) × 100. Where Ablank is the absorbance of the control (without test material) and Asample is the absorbance of the test material. The assay was carried out in triplicate. Determination of ferric reducing/antioxidant power assay FRAP assay was carried out with minor modifications following Szeto et al. 2002.[11] The FRAP reagent was prepared from 300 mM acetate buffer (pH 3.6), 20 mM ferric chloride and 10 mM TPTZ in 40 mMHCl. All three solutions were mixed together in the ratio of 10:1:1 (v/v/v). The absorbance was measured at 595 nm using a spectrophotometer. The results were expressed in µmole/Fe [II] mg. The assay was carried out in triplicate. Ethics clearance All the experiments were conducted in the Department of Zoology, Banaras Hindu University, in accordance with the Institutional practice and within the framework of experiment of experimental animals (scientific procedure) Act of 2007, of the Committee for the Purpose of Supervision and Control of Experiments on Animals, Government of India. Test animals and induction of diabetes Male Park strain mice 4–6 week old (25 ± 5 g) were used for the antidiabetic test. The mice were housed in an individual cage in an air‑habituated room with 12 h light/dark cycle at the temperature of 25°C ± 20°C with free access to food and water. All mice were acclimatized to the laboratory conditions for 7 days before the experiment. Inductions of diabetes in mine were carried out with minor modification of Hua et al.[12] Diabetes in 18 mice (overnight fasted) was induced by intraperitoneal injection of 1% STZ prepared in 0.1 M citrate buffer (pH 4.5) at a single dose of 125 mg/kg body weight. After 72 h, fasting blood glucose (FBG) levels of the mice was examined. Mice with FBG values >226 mg/dl were considered hyperglycemic. For further experiments, mice were divided into five groups according to their FBG and weight (six animals in each group) as following: • Group I: Control • Group II: Diabetic (FGB > 226 mg/dl) • Group III: Diabetic treated with Insulin (4 U/dl) • Group IV: Control treated with S‑1708 mulberry leaf extract • Group V: Diabetic treated with S‑1708 mulberry leaf extract. Feeding schedule Standard feed of laboratory diet was purchased from Paramount Techno Chem, Varanasi, Uttar Pradesh, India. Groups (IV and V) of experimental mice were orally fed by mulberry leaf extract (4 U/dl) two times (7 am and 7 pm) in 24 h.[13] Group III mice were given insulin at the same dose and time.[14] Measurement of body weight and blood glucose level The effect of mulberry leaf extract in different groups of diabetic mice was measured by changes in body weight of mice and FBG level at 7, 14, and 21 days. Hepatic enzyme assay After 21 days of the experiment, mice of all groups were anesthetized under diethyl ether; liver was excised, washed with phosphate buffer saline (PBS) at pH‑7.4 and homogenized with cold PBS containing protease inhibitors. Homogenate was then centrifuged at 10,000 × g for 15 min, and supernatant was collected and stored at −80°C. The supernatant was used for the assay of catalase, glucose‑6‑phasphate dehydrogenase, SGOT, and SGPT. Catalase (EC 1.11.1.6) The activity of catalase was assayed by the method of Beers and Sizer.[15] The assay system contained 1.9 ml sodium phosphate buffer Pharmacognosy Magazine, Volume 13, Issue 50, April-June 2017 (Supplement 2) S281 [Downloaded from http://www.phcog.com on Wednesday, July 12, 2017, IP:-] BRIJESH RANJAN, et al.: Antidiabetic Properties of Mulberry (0.05 M, pH 7.0), 0.1 ml liver supernatant and 1.0 ml H2O2 (0.059 M in buffer). The change in absorbance was read at 240 nm for 3 min at 30 s intervals against blank containing 0.1 ml distilled water instead of enzyme source. The specific activity was calculated by a molar absorbance index for H2O2 as 43.6 and expressed as moles of H2O2 decomposed/min/mg protein. Glucose‑6‑phosphate dehydrogenase (EC1.1.1.49) The enzyme activity was measured as per Worthington enzyme manual.[16] The assay system contained 2.7 ml of 0.055 M Tris–HCl buffer (pH 7.8 with 0.0033 M MgCl2), 0.1 ml liver supernatant, 100 µl ml of 0.006 mM NADP+ and 0.1 ml glucose‑6‑phosphate (0.1 M). The change in absorbance was recorded at 340 nm for 5 min against blank containing 0.1 ml of distilled water instead of enzyme source. The specific activity was expressed as micromoles of NADP+ reduced/min/mg protein using extinction coefficient for NADPH as 6.22 cm2/μmol. in Table 2. Mulberry leaf revealed lower IC50 value of methanolic and ethanolic solvents were 196.12 mg/ml and 143.56 mg/ml as compared to ascorbic acid 271.73 mg/ml and 218.319 mg/ml, respectively. Antioxidant reducing activity of ferric reducing/antioxidant power FRAP assay was used to determine the antioxidant activity of mulberry leaf (Variety S‑1708) was in the methanolic and ethanolic solvents. The reducing activity of mulberry leaf and ascorbic acid are summarized in Table 3 and ethanolic extracts was found to be 4107.22 ± 97.6 µM/Fe(II) mg and 3223.7 ± 85.1 µM/Fe(II) mg, respectively, at the concentration of 400 µg/ml, whereas ascorbic acid recorded in methanolic solvent 3540.60 µM/Fe(II) mg and ethanolic 1698.7 µM/Fe(II) mg at the same concentration. In vivo streptozotocin drug exposures Change in body weight of different group of mice The activities of SGOT and SGPT in liver tissue of each group of mice were assayed by commercial span kit obtained from ARKRAY Healthcare Pvt., Ltd., India.[17‑24] In the time growth of diabetic study, during in vivo exposure of mice to STZ, the body weight decreased significantly 23.99% from control Group I (25.7 ± 1.53 g) to experimental Group II (19.7 ± 3.06 g), after 21 days. Administration of mulberry leaf extracts to diabetic mice, however, results in significant gain in body weight 25.88% form (Group II 19.7 ± 3.06 g to Group IV 24.8 ± 2.21 g). Experimental changes in other groups of mice are expressed in Table 4. Histopathological study of liver tissue Change in glucose concentration of different group of mice To evaluate the histopathological alterations, after 21 days treatment of mulberry leaf extract, mice of each group were anesthetized with diethyl ether. Liver was excised and fixed in aqueous Bouin’s fluid, following Bancroft and Gamble.[25] The fixed tissues were then dehydrated in an ethanol series of ascending concentration, cleared in cedar wood oil and embedded in paraffin wax (melting point 58°C–60°C) (E‑Merck, Mumbai, India). Serial sections were cut at a thickness of 6 µm using a Leica Rotary Microtome (Model RM 2125RT; Leica Microsystems, Bensheim, Germany). The sections were mounted on ethanol cleaned glass slides and were kept in an oven at 37°C overnight to dry. Sections were deparaffinized and were stained with Ehrlich’s hematoxylin and eosin (Ehrlich 1886).[26] The stained sections were dehydrated in an ascending ethanol series, cleared in xylene and mounted in distrenedibutylphthalate xylene. Glucose concentration levels in the STZ induced diabetic mice showed a significant (P < 0.05) (+322.39%) increment after induction of STZ at 21 days (Group I 134 mg/dl to Group II, 566 mg/dl. Diabetic mice treated with mulberry leaf extracts, however, showed a significant (P < 0.05) decline in the concentration of glucose level up to a value of- mg/dl (71.58%), (which was almost equivalent to those of normal control group) as compared to STZ diabetic mice. Consequentially, diabetic treated with mulberry leaf had been significant decline more a percentage (−71.58%) as compared to insulin diabetic mice (−70.37%). Glucose concentration changes in different groups of mice are summarized in Table 5. Serum glutamic oxaloacetic transaminase and serum glutamic pyruvate transaminase Statistical analysis Data were analyzed by applying one‑way analysis of variance followed by Dunnett’s post hoc test and results were analyzed as mean ± standard deviation. Levels of significance were tested at the level of P < 0.05 by using IBM SPSS (version 20, Armonk, New York, USA) package. RESULTS In vitro antioxidant activity assay Antioxidant scavenging activity of 2,2‑diphenyl‑1‑picryl‑hydrazyl In this study, mulberry leaf (variety S‑1708) exhibited higher scavenging activity of DPPH as free radicals compared to the ascorbic acid used as a standard. The concentration dependent percent inhibition of mulberry leaf and ascorbic acid are summarized in Table 1. Maximum activity is observed in both methanol (71.58% ±1.71%) and ethanol (82.06% ±0.4%) solvents of the mulberry leaf at 400 µg/ml concentration. In contrast to mulberry leaf, standard compounds of ascorbic acid revealed less activity in both methanol (62.37% ±0.6%) and ethanol (72.89% ±0.9%) solvents at the same concentration. Further, IC50 value of methanolic and ethanolic extracts of mulberry leaf and ascorbic acid are calculated and summarized S282 Table 1: 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of mulberry leaf extract and ascorbic acid Concentration (µl) Ascorbic acid percentage inhibition Mulberry variety S-1708 percentage inhibition Methanol Ethanol Methanol Ethanol 50 13.70±0.2 12.46±1.2 16.29±-±0.5*-±1.2 26.55±2.6 32.46±1.24* 36.05±1.2*-±0.3 43.41±1.8 48.80±0.30* 54.09±0.6*-±0.4 59.29±2.2 61.29±1.85* 72.07±1.3*-±0.6 72.89±0.9 71.58±1.71* 82.06±0.4 Value are expressed are mean±SD (n=3); mean bearing similar superscript. *(50-400 µl) in same column do not differ significantly from each other based on one-way ANOVA analysis followed by S-N-K post hoc multiple range test. Level of significant was tested at the level of P<0.05. Ascorbic acid treated as control and compared with the S-1708 mulberry variety. ANOVA: Analysis of variance; SD: Standard deviation Table 2: IC50 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of mulberry leaf extract and ascorbic acid Solvent Methanol Ethanol Ascorbic acid (mg/ml- Mulberry variety S-1708 (mg/ml- Pharmacognosy Magazine, Volume 13, Issue 50, April-June 2017 (Supplement 2) [Downloaded from http://www.phcog.com on Wednesday, July 12, 2017, IP:-] BRIJESH RANJAN, et al.: Antidiabetic Properties of Mulberry Change in enzyme activity of different groups of mice In STZ‑induced diabetic mice the activity of catalase was significantly increased (P