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Vol 2 | Issue 4 | Oct – Dec 2023                                                                          Indian J Pharm Drug Studies | 179  

Original Article 

In-vitro anti-inflammatory 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 

Cicer arietinum also known as Bengal gram. This plant belongs to the family Fabaceae. It is cultivated in India in Madhya 

Pradesh, Uttar Pradesh, Rajasthan, and Maharashtra. This study shows that Cicer arietinum leaves anti-inflammatory activity. 

It is used to decrease inflammation. This work estimates the anti-inflammatory activity of leaves of Cicer arietinum by using a 

UV-Visible spectrophotometer at 660nm. The maximum peak of inhibition of protein denaturation by using ethanolic and 

hydro-alcoholic extract is observed at 500µg/ml. By comparing both the extract ethanolic extract shows maximum percentage 

inhibition.  

Keywords: Cicer arietinum, Diclofenac sodium, Protein denaturation, Inflammation, Bovine serum albumin. 

atural products are compared to pure chemicals, 

they are less toxic. Therapeutic activity is seen in 

plant or plant extract, but may not be in isolated 

pure components. One component may decrease the 

toxicity and increases the usefulness of another. The herbs 

have a wide range of applications in the medicinal field. 

From ancient times herbs are used as medicines. Herbs are 

used in the treatment of diseases.  ROS like superoxide 

(O2
●-) and hydroxyl (OH●), hydroperoxyl (OOH●), peroxy 

(ROO●) and alkoxy (RO●) radicals, and non-free radicals, 

e.g., hydrogen peroxide (H2O2) and hypochlorous acid 

(HOCl), which are constantly produced in the human body 

during cell metabolism. Others are reactive nitrogen 

species (RNS) consisting of nitric oxide (NO●), 

peroxynitrite (ONOO●), and nitrogen dioxide (NO2). 

Signal transduction, gene expression, and activation of 

receptors can be regulated by free radicals [1].  

Excessive free radicals if not eliminated immediately then 

they can also be toxic to living cells which can lead to 

oxidative damage to functional macromolecules like DNA, 

proteins, and lipids. As a result, it is critical to look for 

endogenous antioxidant substances that act as direct 

scavengers of free radicals or as metal ion chelating agents 

Access this article online 

Received – 21st July 2023 

Initial Review – 26th July 2023 

Accepted – 1st August 2023 

 

 
 

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that catalyze the generation of radical species, which could 

delay the progression of many chronic diseases or reduce 

chronic inflammation. Antioxidant plant components can 

decrease the formation of free radicals and also decreases 

diseases by oxidative stress [2].  

The phenolics and flavonoids of plant extract involve 

in the antioxidant activities of plants and act as anti-

inflammatory agents. The formation of proinflammatory 

molecules like TNF-α and nitric oxide (NO) can regulate 

inflammation. Free radicals react with inflammatory 

molecules leading to cell death and tissue damage [3]. 

Chickpea (Cicer arietinum L.) is high in protein (17-23%), 

as well as carbs (70%), lipids (4-10%), vitamins (B group), 

and minerals such as potassium, phosphorus, magnesium, 

and calcium. Chickpea proteins have excellent 

bioavailability, and peptides with hypolipidemic, 

antioxidant, and antibacterial action, among others, have 

been found due to their amino acid composition after 

hydrolysis. However, no anti-inflammatory bioactive 

peptides extracted from enzymatically degraded chickpea 

proteins have been reported [3].  

MATERIALS AND METHODS 

Extraction method 

Soxhlet apparatus is used for the extraction of 50gm of 

_______________________________________ 

Correspondence to: Supriya Mhamane, Korwali, Tal. 

Mohol, Dist. Solapur, Maharashtra, India. Email: 

supriyamhamane99@gmail.com Tel.: +91 8080244609 

N 

mailto:supriyamhamane99@gmail.com


Mhamane S & Adat P                                         In vitro anti-inflammatory activity of leaves of Cicer arietinum 

Vol 2 | Issue 4 | Oct – Dec 2023                                                                          Indian J Pharm Drug Studies | 180  

powdered drug and 200 ml of the solvent system. Wet the 

powder by using a solvent system and take three cycles of 

solvent on heating mantle at 550c and continuous water 

supply to condenser for cooling of solvent. Due to this it 

again comes in powdered drug and again goes in the round 

bottom flask and continues the cycle up to solvent 

becomes colorless [4]. 

In-vitro anti-inflammatory activity 

Inflammation: The body’s response (defense) to injury or 

infection is called inflammation. It shows redness, pain, 

swelling, and heat-like symptoms [5]. 

Treatment of inflammation 

1. Acute inflammation: Rest, ice, and good wound care. 

2. Chronic inflammation: Vitamin supplements 

(Vitamins A, C, and D), zinc [5]. 

Bovine serum albumin is used to search for anti-

inflammatory drugs at an early stage without the use of 

animals. It combines with water, salt, fatty acid, vitamins, 

and hormones and transfers it to tissues and cells. 

Chickpea is the preferred protein source during 

inflammation [6]. 

Inhibition of protein denaturation 

Chemicals used for the assay: Potassium dihydrogen 

phosphate, disodium hydrogen phosphate, sodium 

chloride, HCl, bovine serum albumin (BSA), diclofenac 

sodium, methanol, ethanol, 70% ethanol [1]. 

Reagent preparation 

 1) Phosphate buffer 

For 200ml buffer, weigh 0.136gm potassium dihydrogen 

phosphate, 0.176gm disodium hydrogen phosphate, and 

0.7gm sodium chloride. Mix all the chemicals in a 250ml 

beaker and add 100ml distilled water. Maintain the pH at 

6.3 with the help of HCl. Make up the volume up to 200ml 

[1,9]. 

2) Bovine Serum Albumin (BSA) 

Weigh 5gm BSA and add in a 100 ml buffer [1]. 

Standard preparation: Diclofenac sodium is used as a 

standard. For the preparation of the stock solution, dissolve 

100mg diclofenac sodium in 100ml methanol to produce 

1000µg/ml. From this stock solution, 3 different 

concentrations of 100, 200, and 500µg/ml were prepared 

by pipetting 1, 2, and 5ml of stock solution and diluting it 

up to 10ml [1]. 

Test solution preparation: Weigh 100mg semisolid drug 

extract and dissolve it in 100ml solvents (Ethanol and 70% 

Hydroethanol) to produce 1000µg/ml. From this stock 

solution, 3 different concentrations of 100, 200, and 

500µg/ml were prepared by pipetting 1, 2, and 5ml of 

stock solution and diluting it up to 10ml [1,7]. 

Procedure: In different 7 test tubes, add 0.9ml BSA. In 

the first 3 test tubes add 0.1ml standard i.e. diclofenac 

sodium of different concentrations (100µg/ml, 200µg/ml, 

and 500µg/ml), in the next 3 add 0.1ml test solution of 

Cicer arietinum leaves extract of different concentration 

(100µg/ml, 200µg/ml and 500µg/ml), in remaining 1 test 

tube add 0.1ml distilled water (Figure 1). Then the test 

tubes were incubated at room temperature for 5 minutes 

and then add 2.5ml phosphate buffer in each test tube. 

Measure the absorbance at 660nm by using a UV Visible 

spectrophotometer [1,9].   

 
Figure 1 - Dilution for Anti-inflammatory Activity 

Calculation: Percentage inhibition was calculated by 

using the following formula: 

% Inhibition =    × 10 

Results and Discussion: Inflammation is a physiological 

response to bacterial infection or damaged vascularized 

living tissue. Although this tissue's response is normally to 

protect itself, when it becomes uncontrollable, recurring, 

or chronic, it can be linked to disorders such as asthma, 

obesity, and rheumatoid arthritis, among others. The most 

common treatments for inflammation are steroidal and 

non-steroidal medicines; however, their usage is limited 

due to their numerous adverse effects; thus, interest is 

growing in the development of alternative therapies 

derived from natural sources that do not pose health 

hazards [10]. Cicer arietinum plant shows anti-



Mhamane S & Adat P                                         In vitro anti-inflammatory activity of leaves of Cicer arietinum 

Vol 2 | Issue 4 | Oct – Dec 2023                                                                          Indian J Pharm Drug Studies | 181  

inflammatory activity due to the presence of alkaloids, 

glycosides, steroids, carbohydrates, amino acids, and 

inorganic elements. 

Due to injury to the body, the body’s defense system gets 

activated and releases some mediators which can lead to 

an increase in blood flow which causes an increase in the 

size of the blood vessel and then swelling of that body part 

i.e. inflammation. To overcome the inflammation, anti-

inflammatory agents are used. Inhibition of protein 

denaturation activity is first performed to study the anti-

inflammatory activity of the leaves of Cicer arietinum. 

The determination of inhibition of protein denaturation 

activity by ethanolic and hydro-alcoholic extract was 

shown by this study. This study gives the percentage 

inhibition. The ethanolic extract shows high percentage 

inhibition at 500µg/ml i.e. 66.67%. The percentage 

inhibition increases as concentration increases, which is 

shown in the table and graph. The hydro-alcoholic extract 

shows high percentage inhibition at 500µg/ml i.e. 36.11%. 

The percentage inhibition increases as the concentration 

increases, which is shown in the table and graph. These 

values are compared with the standard (Table 1, Figure 2) 

[8].  

Table 1 - In-vitro anti-inflammatory activity of Cicer arietinum 

Conc. 

(µg/ml) 

Percentage Inhibition (Diclofenac 

sodium) (%) 

Percentage Inhibition 

(Ethanolic) (%) 

Percentage Inhibition (Hydro-

alc.) (%) 

100 38.89 25 22.22 

200 63.89 41.67 30.56 

500 77.78 66.67 36.11 

 

Figure 2 - In-vitro anti-inflammatory activity of Cicer arietinum for ethanolic and hydro-alcoholic extract. 

CONCLUSION 

Based on this study, it is concluded that the ethanolic and 

hydro-alcoholic extract of leaves of Cicer arietinum shows 

anti-inflammatory activity by inhibition of protein 

denaturation activity further studies were suggested to 

isolate the active principles responsible for the activity. 

The ethanolic extract shows a high percentage of 

inhibition as compared with the hydro-alcoholic extract. 

Ethanolic extract at 500µg/ml shows high percentage 

inhibition i.e. 66.67%. The percentage inhibition increases 

as the concentration increases from 100µg/ml to 500µg/ml 

and it is compared with the standard. 

REFERENCES 

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Mhamane S & Adat P                                         In vitro anti-inflammatory activity of leaves of Cicer arietinum 

Vol 2 | Issue 4 | Oct – Dec 2023                                                                          Indian J Pharm Drug Studies | 182  

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How to cite this article: Supriya Mhamane, Priyanka 

Adat. In-vitro anti-inflammatory activity of leaves of 

Cicer arietinum. Indian J Pharm Drug Studies. 2023; 

2(4):179-182. 

Funding: None          Conflict of Interest: None Stated  

 

https://my.clevelandclinic.org/health/symptoms/21660-inflammation
https://my.clevelandclinic.org/health/symptoms/21660-inflammation
https://www.jeffersonhealth.org/your-health/living-well/the-best-foods-for-an-anti-inflammatory-diet
https://www.jeffersonhealth.org/your-health/living-well/the-best-foods-for-an-anti-inflammatory-diet

