Comparative evaluation of anti-diabetic action and pancreatic histopathology of rats treated with two alkaloidal plant extracts

This study aimed to understand Strychnosnuxvomica and Holarrhena pubescens Stem bark extract action towards M3 receptor in controlling blood glucose levels. Strychnos nux vomica  and Holarrhena pubescens are both alkaloidal drugs can help in controlling Hyperglycemic level. This will be useful in the formulation of a new herbal drug molecule for treating diabetes. Chloroform and ethanolic extracts of selected alkaloidal plants were extracted using the soxhlet apparatus and obtained quotes were tested for acute toxicity studies and carried out anti-diabetic action on Wister albino rats for 21 days. Results obtained from Blood glucose levels and histopathological study of test groups are compared with blood glucose levels of standard group, and highly significant action was identified by the chloroform extract of Strychnos nux vomica and Holarrhena pubescens group. Moderate anti-diabetic action was observed remaining two groups of ethanolic extracts. Strychnos nux vomica and Holarrhena pubescens ethanolic extract groups are acting on M3 receptors and controlling Hyperglycemic levels.

Strychnos nux vomica, Holarrhena pubescens, M3 Receptor action, Controlling glucose action A This study aimed to understand Strychnosnuxvomica and Holarrhena pubescens Stem bark extract action towards M 3 receptor in controlling blood glucose levels. Strychnos nux vomica and Holarrhena pubescens are both alkaloidal drugs can help in controlling Hyperglycemic level. This will be useful in the formulation of a new herbal drug molecule for treating diabetes. Chloroform and ethanolic extracts of selected alkaloidal plants were extracted using the soxhlet apparatus and obtained quotes were tested for acute toxicity studies and carried out anti-diabetic action on Wister albino rats for 21 days. Results obtained from Blood glucose levels and histopathological study of test groups are compared with blood glucose levels of standard group, and highly signi icant action was identi ied by the chloroform extract of Strychnos nux vomica and Holarrhena pubescens group. Moderate anti-diabetic action was observed remaining two groups of ethanolic extracts. Strychnos nux vomica and Holarrhena pubescens ethanolic extract groups are acting on M3 receptors and controlling Hyperglycemic levels.
Holarrhena pubescens Wall.exG. Don is a medicinal plant broadly found in India, particularly in Himalayan ranges. Uses of this plant are mentioned in the classical Ayurvedic literature, and it has many folklore claims. Holarrhena pubescens is categorised as lactiferous, small deciduous tree, which grows to a height of 13 m, a width of 1.1 m and girth with 3-7 m. It is having leaf size of 15 to13 cm length and 4 to 12 cm width; leaves are having the shape of obtuse, rounded or acute apex; its veins are around 10-14 in number, having opposite venation pattern; it is ovate, leaves have 1.5 cm size petioles, and its veins are 3-6 cm in diameter. Flowers are odourless and has a white colour and are in a terminal corymbose cyme. Corolla puberulous outside; 8-13 mm long tube, slightly in lated near the base over stamens (Gopal and Chauhan, 2006).

Weston et al. work is showing that acetylcholine
Muscuranic three receptors (M3) on the pancreas are one of the reasons for the dysregulation of insulin. Stimulation of the M3 receptor located on the pancreas is involved in regulating the cholinergic pathway which is stimulated by glucose leads to insulin release from the beta cells of islets of langerhans (Singh et al., 2009). Receptors upon stimulation result in the release of insulin, chloroform extract of Strychnos nux vomica and Holarrhenapubescens Stem bark extracts have shown M3 receptors' stimulation on the pancreas has caused the insulin release and controls the excess glucose levels in blood (Roca et al., 2004). This was further supported by our study based on alloxan-induced pancreatic damage model in Wister albino rats by biological parameters evaluation and histopathology of pancreatic cells.

Selected Plants
Strychnous nux-vomica stem bark was processed from the Nirmal district, Telangana, India, during October 2018. The plant materials were authenticated by Dr.Prasanna P.V scientist F and HOD at India's botanical survey, Deccan regional centre, Hyderabad, Telangana, India. Voucher specimen No: BSI/DRC/18-19/Tech./ 589, has been deposited at the Botanical Survey of India (BSI).
The stem bark of Holarrhena pubescens wall ex G.Don was collected from Nirmal district, Telangana, India, during November 2016. Dr.Rasingam L did authentication of plant material, and scientist In-charge at the BSI, Deccan regional centre, Hyderabad, Telangana, India. Voucher specimen No: BSI/DRC/16-17/Tech./681, deposited at BSI

Dose ixation studies
Swiss albino mice of 20-25g were chosen for determining acute toxicity studies. Animals were categorised into control and test groups; each group contains six animals. 5% normal saline was given to the control group, and the test group was treated with chloroform and ethanolic extract of S.Nuxvomica stem bark extracts and Holarrhenapubescens stem bark extracts respectively. Experimental animals were screened for 4 hours to 48 hours for behavioural and mortality changes; there, by LD50 value is calculated (Yeh et al., 2003).
Graph 1: Graphical representation of Blood glucose in various drug treatment groups

Animals selection for anti-diabetic action
Wister albino rats of either sex of 150-250 g weight were taken for the study. The approval process

Induction of diabetes
Induction of Diabetes in experimental animals was carried by administering alloxan at a dose of 110 mg/kg b.w i.p, 24 hours fasting process was monitored for the experimental animals before the  (Matkovic et al., 1996). Seventy-two hours after administration of alloxan blood samples were analysed for blood glucose levels. Animals which were exhibiting more than 200mg/dl of blood glucose levels were considered as diabetic and further used in the evaluation of antidiabetic action of strychnos nux vomica stem bark extracts and H.pubescens stem bark extracts simultaneously.

Process of Experiment
Animals which are having more 200mg/dl of blood glucose levels were categorised into seven groups consists of 6 animals in each group. Drug protocol treatment was given in Table 1.

Process of drug administration
5% gum acacia was used as a suspending agent for mixing of selected stem bark extracts with distilled water which were administered to selected experimental animals using an oral feeding needle.

Screening of biochemical parameters
Anti-diabetic property of animals was determined by collecting the blood samples at 0, 7, 14 and 21 days from retro-orbital plexus of rats using microcapillary tubes. Collected blood samples were centrifuged at 10,000rpm for 10 minutes, and glucose levels were estimated using Blood glucose-PAP (glucose oxidase/phenol/4-amino-antipyrine) method by using Elitech Glucose S.L.Kit in an autoanalyser. The blood glucose level was determined as mg/dl (Cheng, 2005) and the values of blood glucose levels were given in Table 2. Obtained results were graphically represented in Graph 1.

Statistical analysis
Obtained results were subjected to One-way Analysis of variance (ANOVA) for comparison, the values p<0.05 was considered as signi icant, in Toxic group that is diabetic control group ***P<0.001 compared to the normal control and test groups results are considered as signi icant at *P<0.05, **P<0.01, ***P<0.001 compared to standard groups results.
The result obtained from the studies was subjected to a one-way analysis of variance (ANOVA) for comparison among different groups. The minimum value of p<005 was considered as signi icant. The diabetic control group results were signi icant at ***P<0.001 compared to the normal control group, the results of the test group are considered signi icant at *P<0.05, ** P<0.01, *** P<0.001 compared to Diabetic control group.

Histology Examination
At the end of 21 days, all rats were sacri iced using chloroform, and the pancreatic tissues were collected from the animals and rapidly ixed in 10% normal saline for 48h. Selected tissue samples were then processed routinely by paraf in embedding and stained with hematoxylin and eosin. The slides were evaluated using a light microscope and photographed using an Olympus digital camera (Nikon 2). Histopathology images of rat's pancreas in various groups were given in Figures 1, 2, 3, 4, 5, 6 and 7. From the histopathological studies results, the Control group pancreas histology reveals normal cell structure of islets and normal nucleus appearance [ Figure 1]. Diabetic control group histological architecture cell damage, fewer islets of Langerhans, and degranulation of cells and necrosis [ Figure 2]. The standard group has shown an increased number of islets of Langerhans, and cell regaining function was observed [ Figure 3]. The chloroform extract of SN has shown highly signi icant action by increasing the number and decreased the necrotic changes than the diabetic control group [ Figure 4]. Ethanolic extract of SN has shown moderated signi icant activity in islets regaining and nucleus structure [ Figure 5]. HP's chloroform extract has shown the rearrangement of beta cells and ibrotic growth, but the number of islets has been increased [Figure 7]. Ethanolic extract of HP has demonstrated the least signi icant action by less necrosis, degeneration, ibrosis and vacuolisation process (Weston-Green et al., 2013) [ Figure 6]. Alloxan's diabetogenic action is due to oxidative stress, and histol-ogy changes were best described by (Coskun et al., 2005).

CONCLUSION
The present comparative studies on alkaloidal plants of Strychnos nux vomica and Holarrhena pubescens chloroform and ethanolic extracts were showing signi icant protection against alloxaninduced pancreatic cell damage. Further investigations need to be done to isolate the chemical constituents of selected plants; chloroform extracts need to be carried to determine the mechanism behind the hypoglycaemic activity. It was found that the selected alkaloidal stem bark especially chloroform extracts of both the selected plants were exhibiting signi icant anti-diabetic action by stimulating M3 receptors on the pancreas which causes insulin release and control the excess glucose in the blood.