Bangladesh Journal of Pharmacology Research Article Anti-diabetic activity of Allium wallichii on streptozotocin-nicotinamide-induced type-2 diabetes in rat BJP Introduction Type-2 diabetes mellitus is majorly caused due to insulin resistance and accounts for 90% of the total number of cases, which continues to rise daily due to the overburden of associated factors such as aging, rapid urbanization, and obesity (Khan et al., 2020). Some of the common factors responsible for causing diabetes include living a sedentary lifestyle, drinking alcohol, smoking, and inactivity, which may result in a reduction of insulin sensitivity and glucose tolerance, leading to obesity (Chatterjee et al., 2017). Worldwide, ethnobotanical and plant-based traditional medicines research leads to the identification of several bioactive and nutraceutical leads for the prevention and treatment of various diseases including diabetes (Rag- hav et al., 2022). In this context, some of the important high valued traditional ethnomedicinal plants along with their bioactive such as Aegle marmelose (Sabu and Kuttan, 2004), Allium cepa (Akash et al., 2014), Allium sativum (Eidi et al., 2006), Aloe vera (Bunyapraphatsara et al., 1996), Azadirachta indica (Satyanarayana et al., 2015), Gymnema sylvestre (Baskaran et al., 1990), Hibiscus rosa-sinesis (Venkatesh et al., 2008), Mangifera indica (Irondi et al., 2016), Momordica charantia (Joseph and Jini, 2013), Tinospora cordifolia (Sharma et al., 2015) and Trigonella foenum-graecum (Geberemeskel et al., 2019) have been already validated for anti-diabetic activities in recent past. Allium wallichii Kunth (Family: Amaryllidaceae) was documented for medicinal remedies such as dysentery, cholera, blood cholesterol levels, hypertension, intes- tinal pain, liver diseases, high altitude sickness, etc (Rana et al., 2022a). Among phytochemicals such as diosgenin, 1,2 bis (methylthio) ethene, tigogenin, 2,4 dimethyl thiophene, dimethyl disulfide, and trisulfide are some few identified compounds among flavonoids, glycosides, steroids, reducing sugars, organosulfur and terpenoids (Kamal and Sharma, 1984; Kattel and Maga, 1995; Bhandari et al., 2017). The plant has antimicrobial, antioxidant, anti-cancer, and anti-inflammatory activi- ties via in vitro and in vivo studies (Acharya et al., 2011; Abstract The aim of the present study was to investigate the effect of Allium wallichii leaves extract against streptozotocin-nicotinamide-induced type-2 diabetes in rats. The hydroalcohol extract of A. wallichii leaves (200 and 400 mg/kg) was tested for oral glucose tolerance test and in non-insulin-dependent diabetes mellitus-induced elevated serum glucose levels in the rat. Interestingly, single oral administration of A. wallichii caused a significant (p<0.05) reduction in the serum glucose level (mg/dL). On other hand, A. wallichii normalized plasma glucose levels after repeated oral administration for up to 21 days in diabetic rats (p<0.05). The A. wallichii in the dose of 400 mg/kg showed significant effects on streptozotocin-nicotinamide-induced type-2 non-insulin- dependent diabetes mellitus in rats. In conclusion, therapeutic potential of A. wallichii as per claims is validated towards anti-diabetic potential. Article Info Received: 17 October 2022 Accepted: 14 November 2022 Available Online: 19 November 2022 DOI: 10.3329/bjp.v17i4.62273 Cite this article: Rana VS, Sethiya NK, Singhal M. Anti -diabetic activity of Allium wallichii on streptozotocin-nicotinamide-induced type-2 diabetes in rat. Bangladesh J Pharmacol. 2022; 17: 141-146. Anti-diabetic activity of Allium wallichii on streptozotocin- nicotinamide-induced type-2 diabetes in rat Vijay Singh Rana, Neeraj Kumar Sethiya and Manmohan Singhal Faculty of Pharmacy, DIT University, Mussoorie Diversion Road, Dehradun, Uttarakhand 248009, India. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2022; 17: 141-146 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Bhandari et al., 2017; Wang et al., 2016; Iqbal et al., 2018; Sørnes et al., 2021). Since ancient times this plant have a rich traditional and medicinal application. However, only a few activities have been performed in modern medicine. Therefore, the present work has been design- ed to investigate the effect of A. wallichii leaves extract against streptozotocin-nicotinamide-induced type-2 diabetes in rats. Materials and Methods Plant materials Plant samples of A. wallichii were collected from the outskirts of Pitthoragarh (Uttarakhand, India) from August to September 2021 and identified by Dr. M. C. Bharti (Department of Botany, Hemwati Nandan Bahuguna Central Garhwal University, Srinagar, Uttar- akhand, India). Voucher specimens of the plant (No. Herbarium/bot./1070) had been deposited at the Depa- rtment of Botany, Hemwati Nandan Bahuguna Central Garhwal University, Srinagar, Uttarakhand, India. Animals Thirty-six Sprague-Dawley (SD) rats of either sex (Ave- rage weight 200 g and 6 weeks old) were purchased from the National Institute of Biologicals, Noida, India and maintained under standard animal husbandry conditions (temperature 23 ± 2°C, relative humidity 55% ± 10% and 12 hours light-dark cycle). The animals were fed a standard rat pellet diet and had free access to water. Preparation of leaves extract Powdered leaves of A. wallichii were subjected to mace- ration in hydroalcohol (30:70 v/v) for 72 hours. After complete maceration, the filtrate was evaporated under reduced pressure, and the percentage yield, color, and consistency were determined (Bhatt et al., 2010). Acute toxicity studies Six Sprague Dawley rats were fasted overnight without withholding water supply. A single dose of 2 mL of 2000 mg/kg of the test sample was administered via a gavage needle to each of the rats. After dosing, rats were individually observed once every 30 min perio- dically during the first 24 hours, and daily thereafter for 14 days. The animals were observed for any change in fur (fur lost), eyes and mucous, breathing pattern (pan- ting), tremors, convulsion, abnormal defecation (diarr- hea), lethargy, severe pain, distress, and moribund (Perumal et al., 2022). Anti-diabetic activity The A. wallichii extract was screened by the oral glucose tolerance test and streptozotocin-nicotinamide-induced type-II diabetic rat model with slight modification as standard reported methods (Agrawal et al., 2013). Oral glucose tolerance test The oral glucose tolerance test was performed in overnight (18 hours) fasted normal rats. A total of 30 Sprague Dawley rats were divided into five groups of six rats in each group. Group I (positive control) rats received only saline water and the remaining groups 30 min after the administration of the test sample were administered with glucose (2 g/kg) orally. Group II (vehicle control) rats treated with saline water followed by glucose. Group III rats were treated with standard drug (glibenclamide, 1 mg/kg; East West Pharma India) followed by glucose. Group IV and Group V rats were treated with A. wallichii extract at a dose (200 or 400 mg/kg) followed by glucose. Blood samples were collected from the tail vein of all the test animals after a different time interval (30, 60, and 120 min) of glucose administration and the blood glucose levels were esti- mated using a blood glucose monitoring system (BG-03; Morepen Laboratories Limited, India). Streptozotocin-nicotinamide-induced type-2 diabetes After the oral glucose tolerance test, rats were taken to carry out streptozotocin-nicotinamide (National Chemi- cals, India)-induced type-2 diabetes studies for 21 days. In this study, Group I (positive control) rats received normal saline only and the remaining groups 30 min after the administration of the test sample were admini- stered with a single dose of streptozotocin-nicotinamide on day 1. Group II (vehicle control) rats treated with saline water followed by streptozotocin-nicotinamide. Group III rats were treated with the standard drug glibenclamide (1 mg/kg) followed by streptozotocin- nicotinamide. Group IV and Group V rats were treated with A. wallichii extract at a dose (200 or 400 mg/kg) followed by streptozotocin-nicotinamide. Type-2 diabetes was induced by a single intraperitoneal injection of nicotinamide (230 mg/kg) followed by streptozotocin (65 mg/kg; Otto Chemie Pvt. Ltd. India) 15 min afterward on day 1 of the study. Nicotinamide (National Chemicals, India) was dissolved in normal saline and streptozotocin was dissolved in 0.1 M citra- ted buffer (pH 4.5) immediately before use Histological procedures The histological procedure was performed as per the standard reported method (Balamash et al., 2018). In brief, excised pancreas tissues from all the groups were fixed in 10% neutral buffered formalin and processed with an automatic tissue processor. Embedding was done in paraffin wax and sectioned at 5 μm on a rotatory microtome. The sections were stained with hematoxylin and eosin and mounted on distyrene plasticizer xylene. Photomicrographs at magnifications of ×250 were taken from all the groups with the aid of a digital microscope camera. 142 Bangladesh J Pharmacol 2022; 17: 141-146 Statistical analysis All results were reported as mean ± SD. The variation in a set of data has been estimated by performing Turkey multiple comparison post-test to measure one-way ANOVA using non-parametric methods in Graph pad prism. Results Preparation of leaves extract The percentage yield of leaves extract was found to be 11.9% with dark green color. Acute toxicity studies with pharmacological behavioral screening A. wallichii extract (2000 mg/kg) did not show any toxi- city and was found to be non-toxic and safe as evi- denced by observations study of 14 days. Anti-diabetic activity Oral glucose tolerance test A single administration of A. wallichii extract (200 and 400 mg/kg) and glibenclamide (1 mg/kg) significantly reduced serum glucose levels at 30, 60, and 120 min after administration. In the oral glucose tolerance test, the administration of the glucose load (2 g/kg) increas- ed serum glucose levels significantly after 30 min in non -diabetic rats. Glibenclamide (1 mg/kg) and A. wallichii extract (200 and 400 mg/kg) produced a significant increase in the glucose threshold within 60 min, which was then reversed at 120 min after glucose loading. The reductions in serum glucose from basal value (before) to 120 min after glibenclamide (1 mg/kg) and A. walli- chii extract (200 and 400 mg/kg) were shown in Figure 1A. Streptozotocin-nicotinamide-induced type-2 diabetes Positive control rats were found to be stable in their body weight, but diabetic rats (vehicle control) showed a significant reduction in body weight. Streptozotocin followed by nicotinamide caused body weight reduc- tion, which was reversed by both glibenclamide and A. wallichii extract-treated groups after day 7, 14, and 21 of the treatment (data not shown). Similarly, administra- tion of glibenclamide (1 mg/kg) and A. wallichii extract (200 and 400 mg/kg) caused significant reductions in the serum glucose level, which was observed on day 0, 7, 14, and 21 (Figure 1B). Histological findings Figure 2 indicates changes in the quality of the islet cell structure in all experimental groups. The pancreatic tissue sections showed that there was a distortion of the normal histo-architecture of the cells in the islet area of the streptozotocin-nicotinamide-treated group when compared with the control group. Such distortion was reduced and islet cell quality was restored to near normal in the A. wallichii extract-treated groups. Discussion A single administration of A. wallichii extract signifi- cantly reduced serum glucose levels during the course of study suggesting potential application as an anti- hyperglycemic agent in a dose-dependent manner. Moreover, a significant reduction in the consistent serum glucose level was observed during 21 days by A. wallichii extract. Histological evidence, where recovery 10x A B Figure 1: Effect of hydroalcohol extract of A. wallichii on oral glucose tolerance test (A); and anti-diabetic effect in streptozotocin- nicotinamide type-2 diabetic rat (B). Superscript a, b, c, or d denotes p<0.05 when compared with positive control, vehicle control, glibenclamide (1 mg/kg) and extract (200 mg/kg) B lo o d g lu c o s e l e v e l (m g /d L ) 0 50 100 150 200 250 0 100 200 300 400 500 0 7 14 21 Day 30 60 120 Time (min) Positive control Vehicle control Glibenclamide (1 mg/kg) Extract (200 mg/kg) Extract (400 mg/kg) a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c a,b,c a,b,c a,b,c a,b,d a,b a,b a,b a,b a,b a,c a,c a a a a a a a a a a b Bangladesh J Pharmacol 2022; 17: 141-146 143 of pancreas towards streptozotocin-nicotinamide-indu- ced lesion was also observed. Induction of diabetes through streptozotocin was al- ready established to reduce the bodyweight in diabetic animals may be due to disturbance in glucose homeo- stasis associated with endocrine pancreatic cells (Dama- sceno et al., 2014). In this connection, during current study A. wallichii extract was found to reversed the streptozotocin induced body weight reduction. The reversal in body weight reduction by A. wallichii extract may be attributed due to improvement in glucose homeostasis via normalization of pancreatic function- ing as evidence from similar effect produced by other Allium species as reported earlier (Kim et al., 2011). Past evidence shown significant contribution of several Allium species including A. sativum (Eidi et al., 2006), A. saralicum, A. hookeri (Fazelipour et al., 2021), A. cepa (Akash et al., 2014) and many more (Thomson et al., 2015) towards the prevention and treatment of diabetes. Similarly, diallyl trisulfide, which was found to be present in A. wallachii also exhibit anti-diabetic activities as per literature (Tsai et al., 2015). However, A. wallichii still lacking any similar study and not explored for anti- diabetic potential earlier (Rana et al., 2022b). Therefore, in this study, anti-diabetic potential of A. wallichii, on streptozotocin-nicotinamide induced type-2 diabetes in rat was established first time. Moreover, several Allium based dietary components on regular consumption have been found to exhibit multiple health benefits including lowering of blood glucose levels and impro- vement in the performance of pancreas as reviewed on the basis of several studies outcome (Wan et al., 2019). In brief presence of dietary organosulfur compounds in several Allium species already established for preven- tion and treatment of type-2-diabetes by acting as natural hydrogen sulfide donors (Melino et al., 2019). Similarly, A. wallichii (an underutilized dietary vegeta- bles enriched with organosulfur compounds) as per present study able to exhibit blood glucose lowering effect followed by recovering pancreas to normal vascu- lature as evidence from histological finding. Based on findings this study provides supports to various tradi- tional and ethnomedicinal dietary claims on A. wallichii towards health promotion and dietary application. Conclusion A. wallichii extract exhibited significant antihypergly- cemic activities against type-2 diabetic rat. Financial Support Self-funded Ethical Issue The protocols and procedures were approved by the Institutional Animal Experimentation Ethics Committee, Faculty of Pharmacy, DIT University, Dehradun, Uttarakhand, India (Reg. No. DITU/IAEC/21-22/07-09; dated 10th July 2021). The toxicity level of the A. wallichii extract was evaluated according to the guidelines of OECD 420. 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