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Vol 3 | Issue 2 | Apr – Jun 2024                                                                                     Indian J Pharm Drug Studies | 70  

Original Article  

In-vitro Anti-Diabetic activity of leaves of Cicer arietinum 

Supriya Mhamane1, Priyanka Adat2 

From, 1Sahyadri College of Pharmacy, Methwade, Sangola, Maharashtra, 2Assistant Professor, Pharmaceutical Chemistry, 

Sahyadri College of Pharmacy, Methwade, Sangola, Maharashtra, India. 

ABSTRACT 

Chickpea, (Cicer arietinum), also known as garbanzo bean or Bengal gram, yearly plant of the pea family Fabaceae, widely grown 

for its beneficial seeds. Chickpeas are cultivated in more than 50 countries. Largest areas under chickpeas are India, Pakistan, 

Ethiopia, Turkey and Mexico. This study incorporates the Cicer arietinum leaves anti diabetic activity. It has traditional medicinal 

uses like anti-diabetic and it is also used in illness to optimize the taste. This work envisaged the anti-diabetic activity of leaves of 

Cicer arietinum by using UV-Visible spectrophotometer at 540nm. The maximum peak of inhibition of alpha-amylase is observed at 

10µg/ml.  

Key words: Cicer arietinum, Alpha amylase, Enzyme inhibition, 3, 5-dinitro salicylic acid. 

iversity in the medicinal plants is the real wealth of 

India. Since time immemorial many different types of 

medicinal herbs and their formulation have been used. 

Therapy using medicinal plants is more esteemed than using 

synthetic chemicals shown by practical experience and several 

modern researches works. Medicinal plants growing wild or 

cultivated are rich in the world, which forms a enormous 

natural and economical health which must be protected, 

increased for the development of economy, wealth of nation 

and health of people [1]. Rich flora of medicinal plants in 

developing countries is potential source of new biologically 

active substances [2]. Regarding modern research enterprise, 

drug development from plants must necessarily suggest a 

multi-disciplinary approach. As sources of many potent drugs, 

plants are used medicinally worldwide [3].  

Diabetes mellitus is not a single disease but is a group of 

metabolic disorders affecting a huge number of populations in 

the world. It is mainly characterized by chronic hyperglycemia, 

resulting from defects in insulin secretion or insulin action [4]. 

Even though the cases of diabetes are increasing day by day, 

except insulin and oral hypoglycemic drugs no other way of 

treatment has been successfully developed so far. Amylase and 

glucosidase are mainly used for evaluating the anti-diabetic 

activity of a particular drug [5]. 

Access this article online 

Received – 24th Jan 2024 

Initial Review – 02nd Feb 2024 

Accepted – 08th Feb 2024 Quick Response Code 

MATERIALS AND METHODS  

Method of Extraction: 50g of leaf powder has to be subjected 

to soxhlet extraction process using alcohol as the solvent. Make 

sure that all the powder should be wet and the solvent passes 

from powder and settled to the bottom. Due to heating to the 

apparatus at 550C the solvent system again goes at top of 

powder and by passing powder it again settle at bottom in RBF 

(Round Bottom Flask) along with the chemical constituents 

which are dissolve in the solvent system. Heat this apparatus 

upto the solvent becomes colorless. After that stop heating and 

keep for 15min for cooling. After cooling, collect all the extract 

in a clean beaker, then transfer some extract in a petri plate for 

air dry [6].  

In vitro anti-diabetic activity: When blood glucose (sugar) is 

very high, then it is said to be diabetes (hyperglycemia). The 

main source of energy in our body is blood glucose, which is 

obtained from the food that we eat. Insulin is a hormone 

secreted from beta cells of Islets of Langerhans in pancreas. 

With the help of insulin blood glucose enters in the cells and 

utilized as a source of energy [7]. α-amylase and α-glucosidase 

are carbohydrate hydrolyzing enzymes. α-amylase converts 

polysaccharides like starch, glycogen to disaccharides by 

breaking 1, 4-glycosidic linkage by hydrolysis and digest the 

carbohydrate. α-glucosidase converts this disaccharides to 

monosaccharides, which are responsible for postprandial 

hyperglycemia [8]. The agents / drugs that lower the 

abnormally high glucose (sugar) level in the blood [9]. 

__________________________________________________ 

Correspondence to: Supriya Mhamane, Korwali, Tal. Mohol, 

Dist. Solapur, Maharashtra, India. Email: 

supriyamhamane99@gmail.com. Tel.: +91 8080244609. 

D 

mailto:supriyamhamane99@gmail.com


Mhamane & Adat                                                                                         Cicer arietinum Leaf Anti-Diabetic Activity 

Vol 3 | Issue 2 | Apr – Jun 2024                                                                                     Indian J Pharm Drug Studies | 71  

α-Amylase inhibitory activity 

Chemicals used in the assay: α-amylase, potato starch, 

sodium acetate trihydrate, glacial acetic acid, sodium 

hydroxide, 3,5-dinitro salicylic acid, sodium potassium 

tartarate, ethanol[10,11]. 

Preparation of reagents 

1. α-amylase solution: Weigh 27.5mg α-amylase. Take 

250ml beaker and pour 100ml distilled water and weighed 

27.5mg α-amylase. Stir it for 2 min [10,11]. 

2. Sodium acetate buffer: Weigh 2.72gm sodium acetate 

trihydrate and add it in 80 ml of distilled water. Adjust the 

pH at 4.8 with glacial acetic acid. Make up the volume at 

100 ml with distilled water [12]. 

3. Starch solution (0.1% w/v): Weigh 0.1gm potato starch 

and add it in 100ml sodium acetate buffer. Stir to mix 

properly [10,11,15]. 

4. 2N NaOH: Weigh 2gm NaOH and add it in 25ml distilled 

water and stir it [13]. 

5. 3, 5-dinitro salicylic acid reagent: Weigh 1gm 3, 5-dinitro 

salicylic acid and add it in 50ml distilled water. Weigh 

30gm sodium potassium tartarate and add it in above 

solution by continue stirring. Add 20ml 2N NaOH and 

dilute this solution to 100ml with distilled water [10,11]. 

Table 1: Preparation of reagent for In-vitro anti-diabetic 

activity of leaves of Cicer sarietinum. 

Reagent Quantity Required 

α-amylase solution 0.5ml 

Sodium acetate buffer [Q.S] 

Starch solution (0.1% w/v) 0.5ml 

2N NaOH [Q.S.] 

3, 5-dinitro salicylic acid reagent 1ml 

Preparation of test solution: The stock solution of leaf extract 

was prepared of 100µg/ml by using ethanol and 70% aqueous 

ethanol as a solvent. For this, 0.001gm (1mg) semidried drug 

extract were added in 10ml of solvent. From this stock 

solution, 5 different concentrations of 2, 4, 6, 8 and 10µg/ml 

were prepared by pipetting 0.2, 0.4, 0.6, 0.8 and 1ml of stock 

solution and dilute it up to 10ml [14].  

Procedure: In this assay, 0.5ml of test solution of Cicer 

arietinum leaf extract of different concentration such as 

2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml and 10µg/ml prepared in 

ethanol and hydro-alcohol were taken in different test tubes. 

Then 0.5ml of α-amylase enzyme solution and 0.5ml of starch 

solution were added and allow standing for 3 minutes for 

reaction among the drug extract, enzyme and starch. Then add 

1ml of 3, 5-dinitro salicylic acid to stop the reaction. At the 

same time same procedure follow for control, instead of 0.5ml 

test solution add 0.5ml distilled water. Then measure the 

absorbance at 540nm using UV- Visible spectrophotometer [14]. 

 
Figure 1: Dilution of Anti-diabetic Activity 

Calculation: Inhibition of α-amylase were calculated by using 

following formula: 

% Inhibition= × 10 

RESULTS 

In-vitro Anti-diabetic Activity  

Table 2: In-vitro anti-diabetic activity of Cicer arietinum for 

ethanolic and hydro-alcoholic extract   

Conc. 

(µg/ml) 
Percentage 

Inhibition 

(Acarbose) 

(%) 

Percentage 

Inhibition 

(Ethanolic) 

(%) 

Percentage 

Inhibition 

(Hydro-alc.) 

(%) 

2 66.78 12.89 1 

4 69.31 29.92 3.84 

6 82.78 38.13 6.47 

8 84.12 54.02 27.88 

10 99.86 99.64 80.1 

               

Graph 1: In-vitro anti-diabetic activity of Cicer arietinum 

for ethanolic and hydro-alcoholic extract. 

DISCUSSION 

Cicer arietinum plant shows presence of various plant 

constituents like alkaloids, glycosides, steroids, carbohydrates, 



Mhamane & Adat                                                                                         Cicer arietinum Leaf Anti-Diabetic Activity 

Vol 3 | Issue 2 | Apr – Jun 2024                                                                                     Indian J Pharm Drug Studies | 72  

amino acids, inorganic elements. These constituents exhibit 

anti-diabetic activity [16]. Alpha amylase converts 

polysaccharide into glucose and maltose. That’s why blood 

glucose level increases and causes the postprandial 

hyperglycemia. By inhibiting the alpha amylase enzyme we 

can prevent breakdown of carbohydrate into glucose and 

maltose [17,18]. Alpha amylase inhibitory assay is firstly 

applied to study the anti-diabetic activity of the leaves of Cicer 

arietinum. Demonstration of the alpha amylase inhibition by 

ethanolic and hydro-alcoholic extract was done by this study. 

This study shows percentage inhibition of alpha amylase. 

Ethanolic extract shows high percentage inhibition at 10µg/ml 

i.e. 99.64%. The percentage inhibition increases as 

concentration increases as shown in table and graph. Hydro-

alcoholic extract shows high percentage inhibition at 10µg/ml 

i.e. 80.10%. The percentage inhibition increases as 

concentration increases as shown in table and graph [19,20]. 

CONCLUSION  

These studies conclude that, the ethanolic and hydro-alcoholic 

extract of leaves of Cicer arietinum shows anti-diabetic activity 

by alpha amylase inhibition assay. But the ethanolic extract has 

high percentage inhibition as compared with hydro-alcoholic 

extract. Ethanolic extract at 10µg/ml shows high percentage 

inhibition i.e. 99.64%. The percentage inhibition increases as 

concentration increases from 2µg/ml to 10µg/ml as shown in 

table and graph. 

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How to cite this article: Supriya Mhamane, Priyanka Adat. 

In-vitro anti-diabetic activity of leaves of Cicer arietinum. 

Indian J Pharm Drug Studies. 2024; 3(2):70-72. 

Funding: None;                 Conflicts of Interest: None Stated 

 

 

http://dx.doi.org/10.52700/pjbb
https://www.niddk.nih.gov/health-information/diabetes/overview/%20what-is-diabetes#:~:text=Diabetes%20is%20a%20disease% 20that,to%20be%20used%20for%20energy
https://www.niddk.nih.gov/health-information/diabetes/overview/%20what-is-diabetes#:~:text=Diabetes%20is%20a%20disease% 20that,to%20be%20used%20for%20energy
https://www.niddk.nih.gov/health-information/diabetes/overview/%20what-is-diabetes#:~:text=Diabetes%20is%20a%20disease% 20that,to%20be%20used%20for%20energy
https://www.britannica.com/science/diuretic
https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/BI501%20Advanced%20Biochemistry%20and%20Immunology.pdf
https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/BI501%20Advanced%20Biochemistry%20and%20Immunology.pdf
https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/BI501%20Advanced%20Biochemistry%20and%20Immunology.pdf
https://www.virusys.com/rsticketspro/9-sop-106-preparation-of-2n-naoh/article
https://www.virusys.com/rsticketspro/9-sop-106-preparation-of-2n-naoh/article
https://doi.org/10.21203/rs.3.rs-42363/v1

