Evaluation of Acute toxicity, In-vitro , In-vivo Antidiabetic Potential of the Flavonoid Fraction of the plant Chenopodium album L.

Evaluation of Acute toxicity, In-vitro , In-vivo Antidiabetic Potential of the Flavonoid Fraction of the plant phytochemicals bioactive constituents various therapeutic activities anticancer antioxidant, antinociceptive activity, etc. 13,14 . The methanolic roots extract was earlier reported for the treatment of diabetes when evaluated using streptozotocin (STZ)-induced diabetes in Wistar rats 15, and the aqueous extract of the aerial part of the C. album was showed considerable α-amylase inhibition assay of 98.72% at 3mg/ml of concentration 16 . However, the aerial parts' Bioactivity-guided fractionation was not evaluated for antidiabetic potential and acute toxicity study. This study pertains to investigate the potential fraction against the high-fat diet and STZ induced diabetic rat model and in-silico prediction of the possible mechanism of the bioactive fraction. ABSTRACT Background: The Chenopodium album L . commonly recognized as Bathua , is widely distributed globally and contains various phytoconstituents that help treat several diseases. However, until now, aerial parts' antidiabetic potential and the plant's acute toxicity at fraction level have never been established. Objectives: To investigate the acute toxicity, the in-vitro , in-vivo antidiabetic potential of the plant at fraction level. Materials and Methods : The aerial parts of the plant were fractionated into different fractions, i.e., flavonoid fraction (CAFF), tannin fraction (CATF), alkaloid fraction (CAAF), saponin fraction (CASF), and were analyzed for in-vitro alpha-amylase inhibition assay. The CAFF, CATF, and CAAF were selected based on in-vitro alpha-amylase inhibition assay results and were further screened for its acute toxicity and in vivo antidiabetic activity using a high-fat diet and streptozotocin-induced diabetes model. The CAFF was characterized by LC-MS, and a molecular docking study was carried out. Results: The in-vitro alpha-amylase inhibition assay revealed that CAFF was found to be more potent than standard Acarbose having IC 50 values 122.18 ± 1.15 and 812.83± 1.07 µg/ml, respectively. The CAFF fraction was found to possess potent antidiabetic activity in a dose-dependent manner in both in vitro and in vivo diabetic models and did not produce any sign of severe toxicity. Furthermore, the bioactive CAFF fraction was characterized by LC-MS, showed the presence of quercetin 3-O-(2’’,6’’-di-O-rhamnosyl) glucoside (QRG) or quercetin 3-O-(2’’,6’’-di-Orhamnosyl) galactoside (QRGa) and quercetin 3-O-rutinoside (rutin) (QR). It is predicted from the molecular docking study that the CAFF fraction primarily acts as an alpha-amylase inhibitor. Conclusion: The CAFF fraction was found to poses dose-dependent potent antidiabetic activity and did not produce any sign of severe toxicity and primarily act as an alpha-amylase inhibitor.


INTRODUCTION
Diabetes is one of the foremost causes of death across the globe 1 . In 2017, around 425 million adults in the age group of 20-79 years were reported to be affected by diabetes, and it is estimated to rise to 629 million by 2045 2 . Type 2 diabetes is a metabolic disorder characterized by hyperglycemia [3][4][5] and also associated with the risk of cardiovascular disease and obesity 6,7 . Clinical evidence showed that improving lifestyle by maintaining healthy body weight and modest physical exercise can avoid Type 2 diabetes mellitus. Moreover, in a short time, lifestyle modification fails to exert its effects on diabetes, and it is tough to maintain the modified lifestyle 8,9 . Nowadays, combined therapy of several oral hypoglycemic agents exhibits an efficient treatment for glycemic control in the management of diabetes. However, the available combinational therapies possess numerous side effects 10 . The inclusion of phytoconstituents in combination therapy provides more efficient treatment with significantly decreased side effects in the management of diabetes 11 . Phytochemicals are considered less toxic compared with their synthetic counterparts 12 . Therefore, it is the need of the hour to explore phytochemicals for drug intervention that can help prevent and treat Type 2 diabetes mellitus with fewer side effects.
The Chenopodium album L. commonly known as Bathua in Hindi, is classified into the Chenopodiaceae family, widely grown globally, and contains various bioactive constituents that possess various therapeutic activities like anticancer hepatoprotective, antioxidant, antinociceptive activity, etc. 13,14 . The methanolic roots extract was earlier reported for the treatment of diabetes when evaluated using streptozotocin (STZ)-induced diabetes in Wistar rats 15, and the aqueous extract of the aerial part of the C. album was showed considerable α-amylase inhibition assay of 98.72% at 3mg/ml of concentration 16 . However, the aerial parts' Bioactivity-guided fractionation was not evaluated for antidiabetic potential and acute toxicity study. This study pertains to investigate the potential fraction against the high-fat diet and STZ induced diabetic rat model and in-silico prediction of the possible mechanism of the bioactive fraction.

Processing of the plant material
The aerial part (3000 g) of C. album was collected on maturity, adequately cleaned, and was further air-dried at room temperature to prevent microbial growth, and after drying, the aerial parts were powdered using a mechanical grinder and preserved till further use in an airtight container.

Fractionation of total tannins and flavonoids
The powdered aerial parts were defatted using petroleum ether (40-60 °C), followed by successive extraction with chloroform and ethyl acetate using microwave-assisted extraction. The dried ethyl acetate extract, which was then dissolved in the aqueous phase and 10% NaCl solution, was added to the aqueous solution, centrifuge the solution to precipitate tannins (CATF), and further, the supernatant liquid was partitioned with ethyl acetate. The ethyl acetate layer was evaporated to dryness under reduced pressure to achieve the total flavonoids (CAFF) 17 .

Fractionation of total alkaloids
The aerial part of the plant was mixed with NH 4 OH (25%) to form a slurry, further extracted with ethyl acetate using microwave-assisted extraction. The dried ethyl acetate extract, dissolved in water acidified with sulfuric acid to maintain pH 3-4, was further extracted with petroleum ether (40-60 °C) followed by diethyl ether eliminate the lipophilic, acidic, and neutral impurities. The solution was basified to pH 9-10 with NH 4 OH. The resulting solution was further extracted with chloroform and washed with distilled water, concentrated to dryness under reduced pressure to obtain total alkaloids (CAAF) 18 .

Fractionation of total saponin
Defatting of the powdered aerial part was done as mentioned earlier, followed by successive extraction using chloroform and methanol as a solvent by microwave-assisted extraction. The dried methanolic extract was suspended in water, partitioning was done using diethyl ether and saturated n -butanol to isolate the total saponin (CASF) 19 .

In-vitro alpha-amylase inhibitory activity assay
The alpha-amylase inhibitory assay was carried out using the iodinestarch method. Acarbose was used as a standard. The assay depends on developing an iodine and starch complex, i.e., blue, and exhibits maximum absorbance at 580 nm. The positive control solution was prepared using alpha-amylase enzyme and starch in the absence of an inhibitor to achieving 100% enzymatic activity with minimum absorbance value. Whereas the negative control solution contains the starch that converts into the dark green colored complex after the addition of iodine solution having maximum absorbance due to the absence of inhibitor and alpha-amylase and possess no enzymatic activity. However, the test solution absorbance and color intensity should lie in the middle of positive and negative control absorbance 20,21 .

Preparation of stock sample solution
The stock solution 1000 ppm concentration was prepared for each fraction (CAFF, CATF, CAAF, and CASF) by dissolving 10 mg in 10 ml methanol.

Preparation of sample solution
The various sample concentrations were prepared by withdrawing 0.5, 1, 1.5, 2, and 2.5 ml of the stock solution into 10 ml of different volumetric flasks. Volume was prepared using methanol and labeled the sample solutions 50, 100,150, 200, and 250 µg/ml. % Inhibition and enzyme activity were calculated using the formula mentioned below: Enzyme activity = (Abs. of negative control -Abs. of positive control)-Abs. of test sample/ (Abs. of negative control-Abs. of positive control) *100 % Inhibition= 100-enzyme activity.
Inhibition concentration (IC 50 ): The drug concentration where 50% enzyme inhibition takes place.

Experimental animals
Female albino mice were procured from the central animal facility from the Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, for acute toxicity study. Sprague-Dawley (SD) rats of either sex procured from National Institutes of Pharmaceutical Education and Research, Mohali, for in-vivo antidiabetic study. The animals were kept in standard polypropylene cages and maintained under controlled conditions, i.e., room temperature (22±2 °C) and relative humidity (55±5%) with 12:12 h light and dark cycle, and the experiments were conducted in the light cycle. All the rats and mice were supplied commercially accessible rat regular pellet diet (NPD) and water ad libitum before the dietary management. The animal experiments were conducted after approval from the Institutional Animal Ethics Committee of Lovely Professional University (Approval No. LPU/ IAEC/2019/53), and experiments were conducted as per the guidelines of CPSCEA (Govt. of India). For the acute toxicity, the female mice were selected as per the OECD test guidelines 425, and for the antidiabetic activity, SD rats were selected for HFD and STZ induced diabetic rat model as per the reported literature 24,25.

Acute toxicity assay
The acute toxicity assay was carried out as per the OECD Test Guidelines 425(Up and Down Procedure). The fractions (CAFF, CATF, and CAAF) were selected based on the in-vitro alpha-amylase assay results. In this study, non-pregnant female albino mice with an age group of 8-10 weeks and 28 ± 4 g weight were selected. The mice fasted for 3-4 h before dosing but had access to water ad libitum and a single dose of 2000 mg/ kg; p.o of the fractions (CAFF, CATF, and CAAF) were administrated according to the bodyweight of the single mice from each test group. The animals were closely monitored initially for 30 min, then for four h for any sign of toxicity. The food was restored after 1-2 h of dosing. After the drug-treated mouse's survival, all the remaining four mice in each group were administered with the same dose. A similar protocol was carried out for all the vehicle control group mice by administrating 1% CMC in the same volume as the treated group. After the single-dose administration of all the fractions, the groups were closely monitored for any toxic effect, and behavioral parameters were also recorded for the first 30 min, 4 hrs, and 24 hrs and after that at regular intervals 14 days. The bodyweight of mice was measured at regular intervals. At the end of the protocol, the mice excised by cervical dislocation under general anesthesia and organ weight of heart, liver, and kidney were measured. Blood samples were withdrawn by cardiac puncture and sent to the pathology laboratory to estimate biochemical and hematological parameters. The isolated organs, i.e., heart, liver, and kidney, were preserved in 10% formalin solution for histopathology evaluation 22,23 .

Biochemical analysis
All the samples were sent to the pathology lab (National Laboratories, Phagwara, Punjab) to analyze blood glucose, triglyceride, cholesterol, HDL, LDL, VLDL, and creatinine urea, bilirubin, AST, ALT, alkaline phosphate, total protein, globulins, and albumin.

Histopathological study
The isolated vital organs (heart, liver, and kidney) of mice were fixed in 10% formalin after sacrificing, after processing fixed in paraffin wax. Paraffin sections (5mm) were stained with eosin and hematoxylin. The slides were kept beneath the light microscope, and magnified tissue structure images were captured for analysis.

Diabetes induction and in vivo experimental design
To evaluate and identify the potential novel antidiabetic agents from medicinal plants for the cure of type 2 diabetes mellitus, various fractions (CAFF, CATF, and CAAF) of C. album were selected using Sprague-Dawley rats (Both Male and Female) for the development of high-fat diet (HFD) feeding and administering a low dose (35 mg/kg) of Streptozotocin (STZ) 22,23 . The rats were categorized into two different dietary regimens, i.e., NPD or HFD composition 26 (58% fat, 25% protein, and 17% carbohydrate, as a percentage of total kcal) ad libitum, respectively, for the initial period of 2 weeks. The NPD and HFD rats were further divided into NPD, HFD + STZ, HFD + STZ + Test compound CAFF, CATF, and CAAF having six animals in each group. The ingredients of HFD are according to 27 . After two weeks of dietary manipulation, all the rats from the HFD-fed group were injected with STZ low dose (35 mg/kg; i.p.). After that, body weight and biochemical estimations were conducted on the 7 th day. The test compounds CAFF, CATF, and CAAF, were fed orally at two different concentrations, i.e., 250mg/kg and 500 mg/kg continuous for seven days. Therefore, the higher dose (500 mg/kg) was selected as per the previously reported literature of the plant 15 and rationalized the study; the lower dose was selected, i.e., 250 mg/kg. The blood samples were collected from rat's retro-orbital plexus under light anesthesia using capillary tubes to analyze the plasma glucose level, total cholesterol level, and triglycerides level. Histopathology of the pancreas was conducted after sacrificing 50% of the animals at the end of the protocol. The non-fasting rats with a plasma glucose level of ≥300 mg dl −1 were considered diabetic and chosen for further experimental studies. Animal feed water intake was also measured. The rats were allowed to continue with the feed as per the protocol.

Histopathological study
The rats' isolated pancreas was fixed in 10% formalin after sacrificing and processing, fixed in paraffin wax. 5mm paraffin sections stained with eosin and hematoxylin. The slides were kept beneath the light microscope, and magnified tissue structure images were captured for analysis.

Statistical analysis
The results were expressed as Mean ± SD, and the statistical significance among the groups was analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. P ≤ 0.05 was considered statistically significant.

Liquid chromatography-mass spectrometry (LC-MS) analysis of CAFF
The bioactive CAFF fraction obtained from the plant's aerial parts was characterized through LC-MS Spectra for the bioactive molecules responsible for the activity. The LC-MS data components present in the CAFF fraction correlated the mass fragments with reported literature data 28,29 .

Molecular docking
The molecular modeling software, Autodock-vina 30,31, was used to study molecular docking. PDB or proteins 3WY2, 4GQR, and 3SZ1 for alpha-glucosidase, alpha-amylase, and PPAR-gamma proteins were selected and extracted from the protein data bank. The structures of these molecules were drawn by ChemDraw and changed to 3D. Further minimizations of energy were carried out using the MM2 Interface program on ChemBio3D Ultra 12.0, and molecules were saved in pdb format (Cambridge Soft). For the identification of the most active molecule, initially, the removal of internal ligand was done and docking was carried out in the same manner as an actual ligand with set RMASD of 1.5A o .

In-vitro Alpha-amylase inhibition assay
All the fractions of the plant were evaluated by alpha-amylases assay. The CAFF showed potent alpha-amylase inhibitory activity when compared with standard Acarbose having IC 50 values 122.18 ± 1.15 and 812.83 ± 1.07 respectively, whereas the CATF, CAAF found to be less active with that of the standard in terms of % inhibition., The CASF was found to be least active in comparison to the other fractions. The results are mentioned in Table 1.

Acute toxicity study
The acute toxicity was conducted as per OECD Guidelines. CAFF, CATF, and CAAF at a concentration of 2000 mg/kg were evaluated. No mortality was observed in any of the treated and vehicle control groups. All the animals were observed at a regular interval, and observations were noted during the study phase, i.e., 14 days.

Behavioral pattern and body weight
The itching was observed in all the treated groups in the first 30 min, and after that, sleepy and drowsing effects were observed in CAFF and CATF fraction during the first 4 hrs. The itching might be due to some mild toxicity caused by the fractions. During the acute toxicity study, it was observed that there is a slight increase in body weight in the treated and vehicle control group reported in Figure 1.

Organ to body weight index
There was no significant difference observed in organ to body weight index for both treated and vehicle control groups. There were no lesions  Values are expressed as Mean ± SD; CAFF-Chenopodium album flavonoid fraction; CATF-Chenopodium album tannin fraction; CAAF-Chenopodium album alkaloid fraction; CASF-Chenopodium album saponin fraction. observed at the organ level (heart, liver, and kidney) in any of the groups at a 2000 mg/kg concentration, p.o. The organ to body weight index was recorded and reported in Figure 2.

Biochemical analysis
In CAFF fraction, there is an increase in the total cholesterol, HDL, LDL, VLDL, triglycerides, urea, creatinine, protein, SGOT, SGPT levels when compared with vehicle control. Furthermore, in the CATF fraction, the levels of the total cholesterol, LDL, Triglycerides, urea, protein, SGOT, ALP were also increased, whereas in the CAAF fraction, there is an increase in total cholesterol, LDL, urea, SGOT, and ALP levels were recorded. The biochemical results indicate that all the fractions at 2000 mg/kg produce mild toxicity symptoms in albino mice without producing any severe toxicity at the organ level. The data are represented in Table 2.  Table 3.

Histopathology analysis
All the fractions CAFF, CATF, and CAAF of the plant did not show any severe sign of toxicity at the organ level, suggesting that all the fractions are safe at a concentration of 2000 mg/ kg, p.o. The histopathological research findings are summarized for the heart (Figure 3), Kidney (Figure 4), and liver ( Figure 5). The results indicate that all the plant fractions produce only mild toxicity at the heart, liver, and kidney, but the CAAF fraction produces mild to moderate liver toxicity. These results are also supported by the organ to body weight index, biochemical and hematology findings.

Biochemical analysis
The plasma glucose, total cholesterol, and total triglycerides were compared on the 22 nd and 29 th day reported in Table 4. After one week of STZ injection, i.e., on the 22 nd day, there is a highly significant increase in glucose, cholesterol, and triglyceride level in all the HFD+STZ treated groups that confirm the type 2 diabetic condition. After one week of treatment, i.e., 29 th day, there is a highly significant decrease in the glucose, cholesterol, and triglyceride level in a CAFF fraction at a dose of 500 mg/kg, p.o. when compared with the experimental group and found to be more potent than the standard Acarbose. Furthermore, the CATF fraction also significantly reduces all the biochemical levels when compared with the experimental group. There is no significant difference observed with the CAAF fraction treated group than the experimental group ( Figure 6).

Histopathology analysis
After a single dose of STZ administration, there is the development of type-2 diabetes and leads to necrosis of β-cell, which can be observed in the experimental group. After a week of administration of the CAFF fraction, the organs were isolated on the 29 th day. The findings represented that the islet of pancreatic cells retains their standard structure, and mild necrosis was observed that supports the CAFF fraction's potential antidiabetic effect, whereas, in the CATF fraction, there is less recovery of islet cell. However, no recovery was observed in the CAAF treated group than the experimental control group (Figure 7).        Table 5.

Molecular docking
Based on LC-MS's prediction, major constituents were studied insilico for their antidiabetic activity choosing α-glucosidase, α-amylase, and PPAR gamma receptors. Among the phytochemicals, QRGa is supposed to be most active with -6.5 kcal/mol, -9.3 kcal/mol, and -7.0 kcal/mol α-glucosidase, α-amylase, and PPAR gamma, respectively in comparison to standard Acarbose as mentioned in Table 6. A detailed interaction for QRGa was studied for α-glucosidase, α-amylase, and   Values are presented as Mean ± SD, N= 5, Statistical analysis was performed using one way ANOVA followed by Turkey's multiple comparison test, # represents P<0.050 vs. Vehicle Control; VC-Vehicle control; CAFF-Chenopodium album flavonoid fraction; CATF Chenopodium album tannin fraction; CAAF-Chenopodium album alkaloid fraction.  Values are presented as Mean± SD, n=6. Statistical analysis was performed using one way ANOVA followed by Turkey's multiple comparison test, # represents p< 0.05 vs. Vehicle Control, * represents p< 0.05 vs. Experimental Control.

Group Number
PPAR gamma, respectively. QRGa showed four hydrogen bonding to ASN301 at the binding site of alpha-glucosidase (Figure 8a), while electrostatic interactions with alpha-amylase (Figure 8b), QRGa showed one hydrogen bond with PPAR-gamma protein (Figure 8c). The RMSD set for molecular docking was 1.5A o .

DISCUSSION
Type 2 Diabetes mellitus is the most common type of diabetes that arises from insulin production defects or reduced peripheral tissue response towards insulin 32,33 . The complications associated with Type 2 diabetes are nephropathy, retinopathy, neuropathy, and cardiovascular disease 34 . The phytochemical treatment in type 2 diabetes has fewer adverse effects and might be considered a better replacement of the existing oral therapy 35,36 . The inclusion of phytoconstituents in combination therapy provides more efficient treatment with significantly decreased side effects in diabetes management 37,38 . The plant's available literature represents that the methanol extract was influential in the treatment of diabetes; however, the study was conducted at pilot scale only, and no reports were available for the responsible specialized metabolites or compounds responsible for its therapeutic activity. Additionally,   toxicity studies were also not conducted to establish the safety profile of the plant 15 . Therefore, our research mainly focuses on exploring the safety profile, in-vitro and in-vivo antidiabetic potential of the plant at the fraction level, identifies the responsible phytoconstituents, and predicts its plausible mechanism of action.
The plant's aerial parts were fractionated into different fractions and screened for in-vitro alpha-amylase assay. The CAFF fraction showed potent alpha-amylase inhibitor activity compared to standard Acarbose with IC50 values 122.18 ± 1.15 and 812.83± 1.07µg/mL, respectively. Other fractions (CATF and CAAF) showed less potent activity. However, the CASF fraction did not produce any significant in-vitro alpha-amylase inhibition activity. Therefore, it is predicated from an alpha-amylase assay that the CAFF fraction plays a vital role in starch and glycogen metabolism, i.e., by decreasing the blood glucose level, thereby reducing the conversion rate of starch to monosaccharides. The fractions (CAFF, CATF, and CAAF) were analyzed for their safety profile based on the alpha-amylase activity. These fractions were screened for acute toxicity at a concentration of 2000 mg/kg, p.o. in albino mice as per OECD guideline 425. The results represent itching for the first 30 min in all the fractions, and afterward, sleepy and drowsing effects were observed in CAFF and CATF fraction. No significant difference was observed in organ body weight index; lesions were not observed in any isolated organs. However, the initial behavioural changes were observed during the first 4 hrs., probably indicating mild toxicity symptoms.
Furthermore, the biochemical parameters represent a significant increase in the lipid profile, i.e., total cholesterol, LDL, VLDL, triglycerides levels; urea in kidney function test; protein, albumin, SGOT/AST levels in liver function test. The increased levels of biochemical parameters in all the fractions indicate the mild toxicity symptoms in CAFF and CATF fractions whereas mild to moderate toxicity symptoms was observed in CAAF fraction.. The hematology parameters also represent the significant increase in the platelets and various blood cells. A significant difference was observed in WBC only in the CAAF fraction in comparison to the control group. These findings indicate the mild toxicity of the fractions. The biochemical results were supported by histopathology results, representing that the fractions (CAFF and CATF) produce mild granular degeneration in the heart, liver, and kidney, whereas the CAAF exhibits mild to moderate granular degeneration in all the organs. Therefore the acute toxicity study indicates that all the fractions have an LD 50 more than 2000 mg/ kg. Despite this, it is recommended to explore the mechanistic insights for the mild toxicity by performing sub-acute and chronic toxicity of the bioactive fractions with detailed mechanistic studies.  The fractions (CAFF, CATF, and CAAF) were evaluated for their invivo antidiabetic potential in HFD+ STZ induced Type 2 diabetes. The result represents that after one week of CAFF administration at a 500 mg/kg dose, p.o. There is a highly significant decrease in the glucose, cholesterol, and triglyceride level compared with the experimental group, which supports the potential role of CAFF as it was found to be more potent than the standard Acarbose that acts as a positive control. The histopathology findings of the CAFF fraction indicate that the islet of pancreatic cells retains their standard structure, and mild necrosis was observed that supports the potential anti-diabetic effect of the CAFF fraction, whereas, in the CATF fraction, moderate necrosis was observed. However, no recovery is observed in CAAF treated group when compared to the experimental control group. The in-vivo and in-vitro antidiabetic study represent that the CAFF fraction exhibit potential antidiabetic activity among all the fractions.
The highly active fraction CAFF fraction was further explored by LCMS, and three principal components were identified, i.e., QR, QRG, and QRGa, which are probably responsible for the therapeutic activity of the fraction, reported in Table 5. In literature, these flavonoids QR, QRG, and QRGa are reported for antidiabetic and antioxidant activity [39][40][41][42][43][44] and mainly act by interfering in the carbohydrate absorption in the form of glucose and also by assimilation of glucose via insulin release. This represents that due to these flavonoids' presence, the CAFF fraction showed an antidiabetic effect [39][40][41][42][43][44] .
Thus, to predict the CAFF fraction mechanism, the molecular docking study was carried out against various targets. A detailed interaction for QRGa was studied for alpha-glucosidase, alpha-amylase, and PPAR gamma, respectively. The molecular docking results represent that the QRGa was most active with -6.5 kcal/mol, -9.3 kcal/mol, and -7.0 kcal/ mol alpha-glucosidase alpha-amylase, and PPAR gamma, respectively, and it is predicated that CAFF fraction mainly acts as an α-amylase inhibitor.

SUMMARY AND CONCLUSION
The current study results represent that CAFF fraction was found to possess potent antidiabetic activity dose-dependently in both in vitro and in vivo diabetic models. In contrast, the CATF was less active, and the CAAF fraction did not produce any therapeutic activity. Furthermore, all the fractions did not produce any sign of severe toxicity. Only mild toxicity was observed at a dose of 2000 mg/kg, p. o. In conclusion, the CAFF fraction enhanced the overall diabetic situation by acting as an alpha-amylase inhibitor and further exploring the formulations.

Neeraj Choudhary
He has been working as an Associate Professor in Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, PCTE Group of Institutes, Ludhiana. My research areas of interest are natural products and herbal drug technology that also extends into identifying, isolation, characterization of phytocostituents from the medicinal plants.

DR. ASHISH SUTTEE
Dr. Ashish Suttee is currently working as an Associate Professor at the School of Pharmaceutical Sciences, Lovely Professional University, Punjab, India. His primary research interest is exploration, standardization and screening of medicinal plants (in vitro, in vivo and molecular docking ) as potential therapeutic interventions for the treatment of diabetes and liver diseases. He had been authored more than 30+ research and review articles including book chapters. He is a member of APTI, IPA, and IPCA.