Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 
Users Online: 307 | Home Print this page Email this page Small font size Default font size Increase font size

 Table of Contents  
Year : 2012  |  Volume : 31  |  Issue : 3  |  Page : 132-136

Antioxidant potential and total phenolic content of methanolic bark extract of Madhuca indica (koenig) Gmelin

1 Department of Pharmacy, Bharat Institute of Technology, Partapur, By-Pass road, Meerut, India
2 Department of Pharmacy, Jodhpur National University, Jodhpur, Rajasthan, India

Date of Web Publication4-Nov-2012

Correspondence Address:
Anu Chaudhary
Bharat Institute of Technology, Meerut - 250 103
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0257-7941.103197

Rights and Permissions

This study was carried out to investigate the antioxidant and free radical scavenging activity of methanolic extract of Madhuca indica bark in varios systems. DPPH radical, superoxide anion radical, nitric oxide radical, hydroxyl radical, lipid peroxidation, and total phenolic content assays were carried out to evaluate the antioxidant potential of the extract. The percentage inhibition of 40 mg/ml concentration of MMI in DPPH radical scavenging model was found as 74.1%. The scavenging of nitric oxide by the plant extract was concentration dependent and IC 50 value of rutin was found to be 161.7 μg/ml. MMI elicited significant and concentration-dependent superoxide radical scavenging effect with MMI as well as standard curcumin, which exhibited IC 50 values of 38.1 and 5.84 μg/ ml, respectively. MMI demonstrated significant scavenging activity of OH - radical generated from Fe 2+ -ascorbate-EDTA-H 2 O 2 in a concentration-dependent manner. The extract showed a significant dose-dependent free radical scavenging activity in all the models. The extract showed the presence of high phenolic content corresponding to 98.48 μg equivalent of gallic acid and the antioxidant activity could be attributed to this.

Keywords: Antioxidant activity, DPPH, Madhuca indica, nitric oxide radical

How to cite this article:
Chaudhary A, Bhandari A, Pandurangan A. Antioxidant potential and total phenolic content of methanolic bark extract of Madhuca indica (koenig) Gmelin. Ancient Sci Life 2012;31:132-6

How to cite this URL:
Chaudhary A, Bhandari A, Pandurangan A. Antioxidant potential and total phenolic content of methanolic bark extract of Madhuca indica (koenig) Gmelin. Ancient Sci Life [serial online] 2012 [cited 2023 Mar 30];31:132-6. Available from: https://www.ancientscienceoflife.org/text.asp?2012/31/3/132/103197

  Introduction Top

It has been established that oxidative stress is among the major causative factors in induction of many chronic and degenerative diseases including atherosclerosis, diabetes mellitus, cancer, Parkinson's disease and immune dysfunction and is involved in aging. [1],[2],[3] Antioxidants, both exogenous and endogenous, whether synthetic or natural, can be effective in prevention of the free radical formation by scavenging or promotion of their decomposition and suppression of such disorders. [1],[4] There is growing interest towards natural antioxidants from herbal sources. [5],[6],[7]

Madhuca indica Syn. Madhuca latifolia (Sapotaceae), commonly known as "mahua" in India, is an important economic plant growing throughout the subtropical region of the Indo-Pak subcontinent. [8] Different parts of this plant are used as stimulants, demulcents, emollients, heating, and astringents. [9] The bark is a good remedy for itching, swellings, fractures, and snake bites, as well as for diabetes mellitus. [10] Mahua oil is used for the treatment of skin diseases, rheumatism, headache, and as a laxative. Fruits are astringent and largely used as a lotion for chronic ulcers, in acute and chronic tonsillitis, and in pharyngitis. The constituents reported from M. Indica include fatty acids, [11],[12] sapogenins, [13] sugars, [10] triterpenoids, steroids, [14],[15],[16] saponins, [17],[18] flavonoids, and glycosides. [15],[16],[19]

Keeping this in view, this study has been undertaken to investigate antioxidant potential of methanolic extract of M. Indica. The scavenging effect of M. indica bark extract was evaluated against DPPH (1,1-diphenyl,2-picryl hydrazyl), nitric oxide, super oxide, hydroxy radical, and inhibition of lipid peroxidation, and total phenolic content was also determined.

  Materials and Methods Top

Plant material

Madhuca indica (Sapotaceae) was purchased from a local market and identified by Dr. K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. A voucher specimen (BIT/ Anu/2009/MI-520) was deposited in the herbarium of School of Pharmacy, Bharat Institute of Technology, Meerut, UP, India.

Preparation of plant extract

Madhuca indica bark sample was dried under shade and then powdered with a mechanical grinder to obtain a coarse powder. Equal quantity of powder was passed through 40 mesh sieve and extracted with methanol in Soxhlet apparatus at 60 o C. The solvent was completely removed by rotary vacuum evaporator. The extract obtained with methanol was 0.39% w/w. The extract was freeze-dried and stored in vacuum desiccators.


All chemicals used were of analytical grade. Rutin was obtained from Acros organics, New Jersey, USA. DPPH (1,1-diphenyl,2-picryl hydrazyl), NBT (nitro blue tetrazolium), NADH (nicotinamide adenine dinucleotide phosphate reduced), PMS (phenazine methosulphate), TCA (trichloro acetic acid), BHT (butyl hydroxy toluene), and quercetin were obtained from Sigma Chemical Co., USA. Ascorbic acid and vitamin E were obtained from SD Fine Chemicals Ltd, Biosar, India. TBA (thiobarbituric acid) and pyridine were obtained from Loba Chemie, Mumbai, India. Acetic acid, EDTA (ethylene diamine tetra acetic acid disodium salt), and hydrogen peroxide (H 2 O 2 ) were obtained from Qualigens Fine chemicals, Mumbai, India. Naphthyl ethylene diamine dihydrochloride was obtained from Roch-light ltd, Suffolk, England. Sodium nitro prusside was obtained from Ranbaxy Lab, Mohali, India. Potassium ferric cyanide was obtained from May and Bakers, Dagenham, UK. 2-Deoxy-2-ribose was obtained from Fluka (Buchs, Switzerland) and Folin-ciocalteu's phenol reagent was obtained from SISCO Research Laboratories Pvt. Ltd, Mumbai, India.

In vitro antioxidant activity

Methanolic extract of M. indica bark was tested for its free radical scavenging activity using different in vitro models. All experiments were performed in triplicate and the results averaged.

DPPH radical scavenging activity

The free radical scavenging capacity of the extracts was determined using DPPH. Briefly, 0.15% solution of DPPH in ethanol was prepared and 1 ml of this solution was added to 3 ml of MMI in methanol at different concentrations (1-40μg/ml). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min in dark. Then the absorbance was measured at 515 nm using a spectrophotometer (Perkin-Elmer Lambda 20 UV-visible spectrophotometer). The inhibition curve was plotted and IC 50 values were obtained. [20]

Nitric oxide radical scavenging activity

Nitric oxide radical inhibition was estimated by the use of Griess Illosvoy reaction. [21] In this investigation, Griess Illosvoy reagent was modified by using naphthyl ethylene diamine dihydrochloride (0.1% w/v) instead of 1-napthylamine (5%). The reaction mixture (3 ml) containing sodium nitroprusside (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and MMI (10-320 μg) or standard solution (rutin, 0.5 ml) was incubated at 25°C for 150 min. A control without test compound but equivalent amount of methanol was taken. After incubation, 0.5 ml of the reaction mixture was mixed with 1 ml of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthyl ethylene diamine dihydrochloride was added, mixed, and allowed to stand for 30 min at 25°C. The concentration of nitrite was assayed at 540 nm and calculated with reference to the absorbance of the standard nitrite solutions.

Superoxide anion radical scavenging activity

The superoxide scavenging activity was determined by the PMS-NADH superoxide generating system [22] and slightly modified. About 1 ml of nitroblue tetrazolium (NBT) solution (156 μM NBT in 100 mM phosphate buffer, pH 7.4) 1 ml NADH solution (468 μM in 100 mM phosphate buffer, pH 7.4) and 0.1 ml of sample solution of MMI (5-80 μg) in water were mixed. The reaction was initiated by adding 100 μl of phenazine methosulphate (PMS) solution (60 μM PMS in 100 mM phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against blank samples. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity.

Inhibition of lipid peroxidation

The effect of crude extract on Fe 2 SO 4 -H 2 O 2- induced lipid peroxidation in rat liver homogenate was determined by malondialdehyde (MDA)-TBA adducts formation. [23] The reaction mixture containing 0.5 ml of liver homogenate, 0.05 ml of potassium phosphate buffer (pH 7.4), 0.025 ml of 5nm FeSO 4 , 0.025 ml of 0.3% H 2 O 2 , and different concentration of MMI (50-200 μg) were incubated for 1 h at 37° C. After incubation, TBA (0.4% in 0.2 M HCl) and BHT (0.2% in 95% ethanol) at a ratio of 1:2:0.3 were added, and the mixture was heated at 90°C for 30 min. After cooling, 5 ml of n-butanol was added, and the mixture was separated by centrifugation at 1000 Χ g for 10 min, and MDA production was measured at 532nm. Using the external standard tetramethoxy propane, the ability to inhibit MDA formation by test extracts was calculated using the following formula:

%Inhibition = MDA in liver homogenate without test - MDA in liver homogenate with test/MDA in liver homogenate without test

Inhibition of hydroxyl radical

The assay was performed as described by Halliwell [24] with minor changes. All solutions were prepared freshly. The reaction mixture (1 ml) contained 100 μl of 28 mM 2-deoxy-2-ribose (dissolved in KH 2 PO 4 -K 2 HPO 4 buffer pH 7.4), 500 μl solution of various concentrations of MMI (10-80 μg), 200 μl of 200 μM FeCl 3 and 1.04 mM EDTA (1:1 v/v), 100 μl H 2 O 2 (1.0 mM), and 100 μl ascorbic acid (1.0 mM). After an incubation period of 1 h at 37°C, the extent of deoxyribose degradation was measured by the TBA reaction. [25],[26] The absorbance was measured at 532 nm against the blank solution. Vitamin E was used as a positive control.

Determination of total phenolic content

The contents of total phenolic compounds present in the MMI and MRI were determined using the FolinCiocalteu's phenol reagent and the absorbance was measured at 760 nm according to the method of Slinkard and Singleton. [27] All the tests were performed in triplicate and the results averaged. The concentration of total phenolic compounds in MMI was expressed as gallic acid (μg) equivalent by using the following equation, which was obtained from the standard gallic acid graph. [28]

Absorbance = 0.001 Χ gallic acid (μg) + 0.0033

  Results and Discussion Top

The extract showed significant antioxidant activity in vitro by free radical scavenging models. In all models MMI showed dose-dependent results. The percentage of inhibition in various models, viz., DPPH, nitric oxide, super oxide radical, hydroxy radical, and lipid peroxidation are shown in [Table 1],[Table 2],[Table 3],[Table 4],[Table 5], respectively. Determination of total phenolic compounds showed that 1 mg of MMI contains 98.48 μg equivalent of gallic acid.
Table 1: Scavenging effect of MMI on the DPPH radical

Click here to view
Table 2: Nitric oxide scavenging activity of different concentrations of MMI

Click here to view
Table 3: The effects of different concentrations of MMI on the scavenging activities of superoxide anion

Click here to view
Table 4: The effect of different concentrations of MMI on FeSO4-H2O2-induced lipid peroxidation in rat liver homogenate in vitro

Click here to view
Table 5: The effects of different concentrations of MRM and MMI on the scavenging activities of hydroxyl radical

Click here to view

MMI as well as ascorbic acid produced significant quenching of DPPH radical due to its scavenging ability [Table 1]. Free radical scavenging activity increased with an increase in concentration. MMI had strong hydrogen donating ability with an IC 50 value of 21.7 μg/ml and the value was found to be less than the standard vitamin C (IC 50 value of 3.1 μg/ml). The percentage inhibition of 40 mg/ml concentration of MMI in DPPH radical scavenging model was found as 74.1%.

The scavenging of nitric oxide by the plant extract was concentration dependent as illustrated in [Table 2]. The IC 50 value of MMI was found to be 192.8 μg/ml. The IC 50 value of rutin was 161.7 μg/ml.

MMI elicited significant and concentration-dependent superoxide radical scavenging effect in PMS-NADH-NBT system [Table 3]. MMI and standard curcumin exhibited IC 50 values 38.1 and 5.84 μg/ml, respectively.

MMI inhibited the OH radical-mediated lipid peroxidation by the FeSO 4 -H 2 O 2 system in a concentration-dependent manner that was determined by the amount of MDA formed in liver homogenate as given in [Table 4]. The percentage inhibition of MDA formation by 200 mg/ml of MMI was found to be 58.8%. IC 50 of MMI was 178.9 μg/ml and that of vitamin E was 119.2 μg/ml.

MMI demonstrated significant scavenging activity of OH - radical generated from Fe 2+ -ascorbate-EDTA-H 2 O 2 system [Table 5]. The IC 50 values of MMI and vitamin E were 71.4 and 32.5 μg/ml, respectively.

The high content of phenolic components in the extract may be a contributing factor toward antioxidant activity because the phenolic compounds are known to have direct antioxidant property due to the presence of hydroxyl groups, which can function as hydrogen donor. [29],[30],[31]

  Conclusion Top

This study shows that the methanolic extract of M. indica possesses significant antioxidant effects. It may be assumed that the antioxidant property of the methanolic extract of M. indica could be due to the presence of various phenolic compounds. These preliminary results lend to support the use of this plant in folk medicine as an antioxidant.

Thus, the radical scavenging activity, reductive capability, and anti-lipoperoxidant activity strongly suggests that MMI has antioxidant potential. Further studies are needed to evaluate the in vivo antioxidant potential of this extract in various animal models and to isolate the active component.

  Acknowledgement Top

The authors are grateful to the Director (P and D) Prof. R.L. Khosa, Bharat Institute of Technology, Meerut, for the necessary facilities provided to carry out the research work.

  References Top

1.Halliwell B. The antioxidant paradox. Lancet 2000;355:1179-80.  Back to cited text no. 1
2.Metodiewa D, Koska C. Reactive oxygen species and reactive nitrogen species: Relevance to cyto(neuro)toxic events and neurologic disorders: An overview. Neurotox Res 2000;1:197-233.  Back to cited text no. 2
3.Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol 2001;54:176-86.  Back to cited text no. 3
4.Maxwell SR. Prospects for the use of antioxidant therapies. Drugs 1995;49:345-61.  Back to cited text no. 4
5.Larson RA. The antioxidants of higher plants. Phytochemistry 1988;27:969-78.  Back to cited text no. 5
6.Gazzani G, Papetti A, Massolini G, Daglia M. Anti and prooxidant activity of water soluble components of some common diet vegetables and the effect of thermal treatment. J Agric Food Chem 1998;46:4118-22.  Back to cited text no. 6
7.Velioglu YS, Mazza G, Gao L, Oomah BD. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J Agric Food Chem 1998;46:4113-7.  Back to cited text no. 7
8.Awasthi YC, Mitra CR. Madhuca latifolia: Constituents of fruit pulp and nut shell. Phytochemistry 1967;6:121-5.  Back to cited text no. 8
9.Kirtikar KR, Basu BD. Indian medicinal plants, Allahabad India: Sudhindra Nath Basu; 1918.747.  Back to cited text no. 9
10.Khaleque A, Wahed Miah MA, Huq MS, Khan NA. Madhuca latifolia I. Constituents of the seeds. Sci. Res 1969;6:227-8.  Back to cited text no. 10
11.Dhingra DR, Seth GL, Speers PC. Indian seed fat-mowha (Bassia latifola) and tamal (Garcinia morella) fats. J. Soc. Chem. Ind. 1933;52:116-8.  Back to cited text no. 11
12.Hilditch TP, Ichaporia MB. The fatty acids and glycerides of solid seed fats. III. The seed fat of madhuca (Bassia latifolia) (mowrah fat). J. Soc. Chem. Ind. 1938;57:44.  Back to cited text no. 12
13.Heywood BJ, Kon GA, Ware LL. Sapogenin. Part V. Bassic acid. J. Chem. Soc. 1939;33:1124-9.  Back to cited text no. 13
14.Awasthi YC, Mitra CR. Madhuca latifolia: Triterpenoid constituents of the trunk bark. Phytochemistry 1968;7:1433-4.  Back to cited text no. 14
15.Bhargava PN, Singh RP. Studies of mahua sterol from Bassia latifola. J. Indian Chem. Soc. 1958;35:763-7.  Back to cited text no. 15
16.Bhatnagar SC, Awasthi YC, Mitra CR. Steroidal and other constituents of Madhuca latifolia leaves. Phytochemistry 1972;11:1533.  Back to cited text no. 16
17.Banerji R, Mishra G, Nigam SK. Madhuca indica leaf saponin and its biological activity. Fitoterapia 1985;56:186-8.  Back to cited text no. 17
18.Hariharan V, Rangaswami S, Sarangan S. Saponins of the seeds of Bassia latifolia. Phytochemistry 1972;11:1791-5.  Back to cited text no. 18
19.Subramanian SS, Nair AG. Myricetin and myricetin-3-O-Lrhamnoside from the leaves of Madhuca indica and Achras sapota. Phytochemistry 1972;11:3090-1.  Back to cited text no. 19
20.Viturro C, Molina A, Schmeda-Hischmann G. Free radical scavengers from Mutisia friesiana (Asteraceae) and Sanicula graveolens (Apiaceae). Phytother Res 1999;13:422-4.   Back to cited text no. 20
21. Garrat DC. The Quantitative analysis of Drugs. Japan: Chapman and Hall Ltd.; 1964. p. 456-8.  Back to cited text no. 21
22.Nishimiki M, Rao NA,Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochem Biophys Res Comm 1972;46:849-53.  Back to cited text no. 22
23.Yang CS, Tsai PJ, Wu JP, Lin NN, Chou ST, Kuo JS. Evaluation of extracellular lipid peroxidation in brain cortex of anaesthetized rats by microdialysis perfusion and high-performance liquid chromatography with fluorimetric detection. J Chromatogr B 1997;693:257-63.  Back to cited text no. 23
24.Halliwell B, Gutteridge JM, Aruoma OI. The deoxyribose method: A simple'test tube' assay for determination of rate constants for reaction of hydroxyl radicals. Anal Biochem 1987;165:215- 9.   Back to cited text no. 24
25. Houghton PJ, Zarka R, Heras BD, Hoult JR. Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med 1995;61:33.  Back to cited text no. 25
26.Aruoma OI, Laughton M, Halliwell B. Carnosin, homocarnosin and anserine: Could they act as antioxidants in vivo? Biochem. J. 1989;264:863-9.   Back to cited text no. 26
27. Slinkard K, Singleton VL. Total phenol analyses; automation and comparision with manual methods. Am J Enol Vitic 1977;28:49-55.  Back to cited text no. 27
28.Madhujith T, Naczk M, Shahidi F. Antioxidant activity of common beans (Phaseolus vulgaris L.). J. Food Lipids 2004;11:220-33.  Back to cited text no. 28
29.Duh PD, Tu YY,Yen GC. Antioxidant activity of water extract of Chyrsanthemum morifolium (Ramat.). Lebens on Wiss Technol. 1999;32:269-77.  Back to cited text no. 29
30.Dreosti IE. Antioxidant polyphenols in tea, cocoa, and wine. Nutrition 2000;16:692-4.  Back to cited text no. 30
31.Arnason T, Hebda RJ, Johns T. Use of plants for food and medicine by native people of eastern Canada. Can J Bot 1981;59:2189-325.  Back to cited text no. 31


  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

This article has been cited by
1 Antiplasmodial activity of medicinal plants from Chhotanagpur plateau, Jharkhand, India
Niharika Singh,Naveen Kumar Kaushik,Dinesh Mohanakrishnan,Santosh Kumar Tiwari,Dinkar Sahal
Journal of Ethnopharmacology. 2015;
[Pubmed] | [DOI]
2 Anti-Inflammatory and Antioxidant Effects of Aloe Saponaria Haw in a Model of UVB-Induced Paw Sunburn in Rats
Mariane Arnoldi Silva,Gabriela Trevisan,Carin Hoffmeister,Mateus Fortes Rossato,Aline Augusti Boligon,Cristiani Isabel Bandeṛ Walker,Jonatas Zeni Klafke,Sara Marchesan Oliveira,Cássia Regina Silva,Margareth Linde Athayde,Juliano Ferreira
Journal of Photochemistry and Photobiology B: Biology. 2014;
[Pubmed] | [DOI]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Materials and Me...
Results and Disc...
Article Tables

 Article Access Statistics
    PDF Downloaded309    
    Comments [Add]    
    Cited by others 2    

Recommend this journal