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Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 33  

Research Article 

In vitro pharmacological investigation of fruit of Acacia nilotica   

Pradnya Shinde1, Shraddha Kadav2, Ashwini Kamble3, Ashpak Tamboli4  

From, 1,2,3Sahyadri College of Pharmacy, Methwade, Sangola, Maharashtra, 4Associate Professor, HOD, Pharmaceutical chemistry, 

Sahyadri College of Pharmacy, Methwade, Sangola, Maharashtra.  

Correspondence to: Pradnya Shinde, Vidyanagar, Akluj road, Malshiras, Solapur, Maharashtra, India Email: 

pradnyashinde6859@gmail.com  

ABSTRACT 

The famous medium-sized Acacia nilotica tree, often known locally as "Babul" or "Kikar," is widely distributed throughout tropical and 

subtropical regions. It has a wide variety of possible medical applications and antioxidant action. This study's goal was to ascertain the 

fruits of Acacia nilotica's Invitro anti-inflammatory and antioxidant activities. Acacia nilotica hydroalcoholic extract's Invitro anti-

inflammatory and anti-oxidant activities were assessed. The significant activity of Inhibition of Protein denaturation shown a maximum 

of 76% inhibition at 500 µg/ml concentration. From the hydroalcoholic extract 400 g/ml showed a maximum of 99.85% of DPPH radical 

scavenging activity. 

Key words: Protein denaturation, Acacia nilotica, DPPH, free radical scavenging 

ndia's variety of medicinal plants is its true source of 

wealth. Since the beginning of time, numerous varieties of 

medicinal herbs and their preparation have been used. 

Developing nations have a diverse range of medicinal plants that 

could be used to create novel biologically active compounds. 

Drug development from plants must inevitably entail a 

multidisciplinary strategy in terms of a contemporary research 

project. Mineral, plant, and animal products were the primary 

sources of medicines for a very long time [1].  

There is evidence that almost all ancient civilizations used 

herbs for the treatment of illnesses and for reviving bodily 

systems. Herbal medicines are typically made from a single 

extract, a fraction of that extract, or a combination of fractions 

and extracts from various plants. These substances must be 

carefully standardized for safety, efficacy, and cost-

effectiveness [2–4]. Along with other areas of human activity, 

the study of medicine and related sciences has advanced 

quickly. The body's antioxidant defence mechanisms are only 

effective when the number of free radicals is within the normal 

physiological range; otherwise, oxidative stress, which can lead 

to tissue damage and consequent illnesses [5, 6].  

MATERIAL AND METHODS  

Method of Extraction   

Weighed 45 gm of powdered drug of fruits of Acacia nilotica 

and it was extracted in soxhlet apparatus by using 

hydroalcoholic (viz. water: ethanol) solvent. Soxhlet apparatus 

filled with powder of Acacia nilotica fruit then added the solvent 

system into it and started heating at 55 0 C after completing one 

cycle of the assembly. Stopped heating when solvent became 

colourless then extract was collected and air dried.  

Invitro Anti-Inflammatory Activity  

Inflammation is the body's natural defence mechanism and is 

characterized by pain, swelling, heat generation, and loss of 

function in the wounded area. The location and severity of the 

damage will determine how much function the afflicted part 

loses. The body's reaction to an accidental cut is comparable to 

the reaction to other types of tissue injury brought on by burns 

from heat, radiation, bacterial, or viral attack since inflammation 

is the body's generalised defence mechanism. Inflammation is a 

complicated process that is linked to pain and includes things 

like increased protein denaturation, increased vascular 

penetrability, and layer change. Prostaglandin, kinins, and 

histamine are chemical mediators released by injured bodily 

tissue (cells) during inflammation. Vasodilation and capillary 

permeability are increased as a result. This causes the blood flow 

to the injured location to increase. Various in vitro and in vivo 

models are employed to research anti-inflammatory efficacy. 

Acacia nilotica invitro model is assessed for the current study of 

anti-inflammatory activities.  

Invitro Protein Denaturation Inhibition Bioassay  

Chemicals used for the activity: BSA (Bovine Serum 

Albumin), NaCl, Disodium hydrogen phosphate, Potassium 

dihydrogen phosphate, Methanol, Standard diclofenac sodium.  

I 



Shinde et al                                                                    In vitro pharmacological investigation of fruit of Acacia nilotica 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 34  

Preparation of reagent % Bovine serum Albumin: Dissolve 

5 gm. of BSA in phosphate buffer saline of pH 6.3  

Phosphate buffer saline of pH 6.3: Adjust the pH of the 

solution to 6.3 by adding HCL after dissolving 0.68 grammes of 

potassium dihydrogen phosphate, 0.895 grammes of disodium 

hydrogen phosphate, and 3.50 grammes of sodium chloride.  

Preparation of standard solution: The standard stock solution 

of diclofenac sodium in methanol was generated at a 

concentration of 10,000 mg/ml, and three different 

concentrations of 100, 200, and 500 mg/ml were prepared from 

this stock solution.  

Test solution preparation: Using methanol as the solvent, 

stock solutions of various fruit extracts containing 10,000 g/ml 

were created. Three distinct concentrations of 100, 200, and 500 

g/ml were made from this stock solution.  

Procedure: The procedure for the current study's assay of 

protein denaturation inhibition is provided below. In this assay, 

0.1 ml of test solution of Acacia nilotica fruit extract of different 

concentrations (100 g/ml, 200 g/ml, and 500 g/ml produced in 

methanol) and 0.9 ml of bovine serum albumin (5 percent 

aqueous solution) were taken in different test tubes. In place of 

the test solution for the control, 0.1 ml of distilled water was 

employed. For 0.1 ml of diclofenac as is customary. Instead of 

the test solution, sodium was used (different concentrations of 

methanol were made, namely 100, 200, and 500 g/ml). The 

mixture was incubated at 37 °C for 5 min, then test tubes were 

heated at 55 °C for 3 min, and finally allowed to cool. Each test 

tube received 2.5 ml of phosphate buffer saline with a pH of 6.3 

after being cooled. At 660 nm, the absorbance was 

spectrophotometrically quantified [14].  

Standardization: By employing standardised diclofenac 

sodium, the Invitro serum albumin denaturation inhibition 

bioassay was standardised. 

 

Fig. no. 1:  Images Of Anti – inflammatory Activity 

Calculation: The inhibition to the protein denaturation was 

measured as  

            Abs of control – Abs of treated 

% Inhibition=   ----------------------------------------   X 100 Abs 

of control  

Invitro Anti-Oxidant Activity  

Oxidation: The chemical reaction known as oxidation involves 

the transfer of an electron or hydrogen atom from a material to 

an oxidising agent. Free radicals are produced as a result of the 

oxidation reaction.  

Antioxidants: By preventing the start of oxidative chain 

reactions, antioxidants are substances that prevent or delay the 

oxidation of other molecules. The reducing agents are frequently 

antioxidants.  

DPPH Radical Scavenging Activity  

Principle: Due to its unpaired electron, the persistent free 

radical known as DPPH (1, 1-diphenyl-2-picrylhydrazyl) has a 

rich purple color. When an antioxidant provides the DPPH with 

an electron, the DPPH's deep violet color decolorizes to a pale 

yellow non-radical form. When a solution of DPPH is mixed 

with a solution of a substrate that can give off a hydrogen atom, 

at that point this gives rise to the reduced form with the loss of 

this violet shading. This change in colour of DPPH and the 

subsequent drop in absorbance are observed spectrophotometric 

ally at a maximum of 517nm.  

Chemicals used for the activity: DPPH (2, 2-diphenyl-1-

picrylhydrazyl), Ascorbic acid, Methanol.  

Preparation of reagents:   

1. DPPH solution: Dissolve 0.025 mg of DPPH in 1000 ml of 

methanol. Prepare fresh solution while using.  

2. Standard solution (Ascorbic acid solution): Dissolve 100 mg 

of ascorbic acid in 100 ml of methanol.  

3. Test solution (Extract solution): dissolve 50 mg of test 

sample in 50 ml of methanol.  

Procedure: Using a methanolic solution of DPPH, the ability of 

Acacia nilotica fruit extracts to scavenge free radicals was 

evaluated. A free radical is DPPH. Antioxidants convert DPPH 

to the 517 nm-measured 2, 2diphenyl-1-picrylhydrazine. The 

common antioxidant utilised is ascorbic acid. The assay is 

carried out utilising the brand Williams et al. method. The 

reaction combination used to assess this activity contains 3.9 ml 

of methanolic DPPH solution and 0.1 ml of various extract 

solutions (containing 25, 50, 100, 150, 200, and 400 g/ml). 

Instead of extract, 0.1 ml of ascorbic acid was utilised for the 

standard. Moreover, 0.1 ml of methanol was employed for the 

blank preparation. This combination was incubated at room 

temperature for 30 minutes in the dark. 517 nm absorbance 

measurement. All extracts’ percentage DPPH radical 

scavenging activity was evaluated and compared to the 

reference value [14].  



Shinde et al                                                                    In vitro pharmacological investigation of fruit of Acacia nilotica 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 35  

 
Fig. no. 2: Images of Antioxidant Activity  

Standardization: Ascorbic acid was used to standardise the 

Invitro antioxidant DPPH free radical scavenging activity.  

Calculation: The percent inhibition was measured as  

       Abs of control – Abs of treated 

% Inhibition=   ----------------------------------------   X 100  

                                     Abs of control  

RESULT  

Invitro Anti-inflammatory Activity:  

Invitro Protein Denaturation inhibition bioassay: Bioassay for 

In Vitro Suppression of Protein Denaturation In vitro percentage 

(percent) inhibition of protein denaturation caused by heat of 

standard Diclofenac sodium in table no.1   

Invitro Antioxidant Activity: Invitro Antioxidant Activity: 

DPPH free radical scavenging activity  

Table No. 1: Heat induced protein Denaturation inhibition 

of standard Diclofenac sodium  

Standard 

Drug  

Concentration 

in μg/ml  

Absorbance  (%) 

inhibition  

Diclofenac 

sodium  

100  0.115  38.50  

200  0.076  59.35  

500  0.043  77.00  

Table No. 2: Heat induced protein denaturation inhibition 

of different extracts of Acacia nilotica Fruits.  

Plant 

Extract  

Concentration 

in μg/ml  

Absorbance   (%) 

inhibitin  

Control   -  0.024  -  

Hydroalc 

oholic 

extract  

100  0.014  41.66  

200  0.011  54.16  

500  0.006  76  

Table No. 3: Percentage of DPPH free radical scavenging 

activity of fruit extract of Acacia nilotica.  

Solution   Con. in μg/ml  (%) inhibition  

Plant extract   25  80.6  

 50  81.67  

 100  85.67  

 150  99.21  

 200  99.35  

 400  99.85  

Standard solution  -  99.87  

 

Graph No. 1: Percentage inhibition of protein denaturation 

at different concentration of Fruit extract of Acacia nilotica 

 

Graph No. 2: DPPH radical scavenging activity of Extract 

of Acacia nilotica fruits  

DISCUSSION   

Acacia nilotica contain number of phytochemicals such as 

Alkaloids, Flavonoids, Phenols and terpenes which are 

responsible for Anti-inflammatory and antioxidant activity. 

Inflammation include events such as expansion of vascular 

permeability and increment of protein denaturation. 

Inflammation leads to tissue injury which causes release of 

chemical mediator such as prostaglandins, kinins and histamine. 

These mediators leads to vasodilation which causes increase 

blood flow at the site of injury. Antiinflammatory compounds 

gives inhibitory effect against inflammation by inhibiting 

release of chemical mediators    

The protein denaturation inhibition assay is being used for 

the first time in this work to evaluate the antiinflammatory 

41.66 

54.16  

76 

0 
10 
20 
30 
40  
50 
60 
70 
80 

100 200 500 

Conc. of Extracts 

Percentage (%) inhibition 

Percentage (%) inhibition 

  

0 
20 
40 
60  
80 

100  
120 

25 50 100 150 200 400 - 

Plant extract Standard solutio 

1 2 

Percentage (%) inhibition 

Percentage (%) inhibition  



Shinde et al                                                                    In vitro pharmacological investigation of fruit of Acacia nilotica 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 36  

efficacy of the fruit extracts of the ethnomedical herb Acacia 

nilotica. Heat causes denaturation of proteins. Evaluation of 

hydro-alcoholic extract's inhibition of protein denaturation. 

According to the results of the current investigation, percentage 

inhibition rises as concentration goes from 100 g/ml to 500 g/ml. 

In comparison to the other fruit extracts of Acacia nilotica, these 

all exhibit the highest percentage inhibition of 76 percent at 500 

g/ml concentration as shown in table no. 2. Following that, the 

% inhibition of standard Diclofenac sodium is compared to that 

of the hydro-alcoholic extract. At a concentration of 500 g/ml, 

diclofenac sodium exhibits a 77 percent inhibition in percentage 

as shown in table no. 1. ROS include free radicals which is 

produced by oxidation process. But the excessive generation of 

free radicals leads to oxidative stress and causes various 

diseases. Antioxidant compound reduces the generation of free 

radicals and prevent the disease caused by oxidative stress. The 

Phenolics and Flavonoids leads to prevention of ROS formation 

by inhibiting oxidative enzyme.  

The DPPH free radical scavenging assay is being used for 

the first time in this work to evaluate the anti-oxidant activity of 

the fruit extract of the ethnomedicinal herb Acacia nilotica. The 

hydro-alcoholic and aqueous concentration's activity to 

scavenge DPPH free radicals has come to an end. In comparison 

to the other fruit concentrates of Acacia nilotica, the 400 g/ml 

concentration of the Acacia nilotica fruit extract exhibits 

considerable DPPH free radical scavenging activity of 99.85 

percent as shown in table no. 3. The common antioxidant 

utilised is ascorbic acid nociceptive action it blocks the pain 

sensation caused by inflammation.  

Protective extent of Acacia nilotica: Acacia nilotca provide 

effect against peripheral as well as CNS inflammation. It shows 

antinociceptive action that it blocks the pain sensation caused by 

inflammation. It act as antiinflammatory by inhibiting formation 

of pain mediators i.e. prostaglandins and kinins at peripheral 

target sites. Acacia nilotica shows the CNS depressant activity 

which helps to treat pain attacks, anxiety. It increases the 

activity of GABA receptor which shows calming effect which 

used to treat anxiety, pain sensation and insomnia.   

CONCLUSION  

In the current investigation, the hydroalcoholic extract's protein 

denaturation inhibition assay method is used to assess and 

confirm the invitro anti-inflammatory effect. These analyses 

lead to the conclusion that, when compared to the industry 

standard, diclofenac sodium, the hydroalcoholic extract 

significantly inhibits protein denaturation by 76% at a 

concentration of 500 g/ml. DPPH radical scavenging activity is 

another method of confirming the in vitro antioxidant activity of 

Acacia nilotica fruit. According to the results of this test, the 

hydroalcoholic extract exhibits 99.85 percent radical 

scavenging activity at a concentration of 400 g/ml.  

For this test, the standard antioxidant is ascorbic acid. From 

all of the research, it has been determined that the fruit extract 

of Acacia nilotica may have antioxidant and anti-inflammatory 

properties.  

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How to cite this article: Pradnya Shinde, Shraddha Kadav, 

Ashwini Kamble, Ashpak Tamboli. In vitro 

pharmacological investigation of fruit of Acacia nilotica. 

Indian J Pharm Drug Studies. 2023; 2(1) 33-36. 

Funding: None                  Conflict of Interest: None Stated 

 


