Evaluation of Total Phenolic Content, Flavonoid Content and Antioxidant Activity of Stem Bark of Careya arborea Roxb

 

 

KA Wadkar* and CS Magdum

Appasaheb Birnale College of Pharmacy, Sangli.416416

 

ABSTRACT:

In this study we have determined the total phenolic, flavonoid content and antioxidant activity of pet. ether,   aqueous and alcoholic extract of the stem bark of Careya arborea Roxb. (Lecythidaceae). Folin-ciocalteu method was used to determine the total phenolic content and flavonoid contents of these extract were assessed by aluminium chloride method. The antioxidant activities were determined by DPPH method. Aqueous extract and alcoholic extract showed presence of phenolic and flavonoid content and hence possessed greater antioxidant activity. Pet. ether extract containing very less amount of phenol and flavonoids,  did not showed any antioxidant activity. The results suggest that the plant is good source of antioxidants and support their use in various diseases.

 

KEYWORDS: Careya arborea, total phenol, flavonoids, antioxidant.

 

INTRODUCTION:

Careya arborea (Lecythidaceae) is a medium sized deciduous tree; bark is dark grey exfoliating in thin strips, alternate leaves 15-30 by 7.5- 15 cm., broadly obovate or obovate-oblong, rounded or shortly acuminate, crenate-denticular, rather membranous, glabrous, lateral nerves 10-12 pairs; petiole 0-1.8 cm. long , stout, margined, flowers 6.3-9 cm. across, white, ill-smelling, sessile1. Methanolic extract of bark is potential source of natural antimicrobial and antioxidant agents2. Bark of Careya arborea showed analgesic 3 and antidiarrhoeal4 activities. When it was tested against carbon tetrachloride induced liver damage in rats, it showed hepatoprotective and in vivo antioxidant effects5.

 

It is well known that free radicals play a fundamental role in several diseases. Free radicals contribute to more than hundred disorders in humans including atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, cancer, diabetes mellitus and AIDS6,7. Recently there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing such free radical induced tissue injury. Besides well known and traditionally used natural antioxidants from tea, wine, fruits, vegetables and spices, some natural antioxidant (e.g. rosemary and sage) are already exploited commercially either as antioxidant additives or a nutritional supplements. Many other plant species have also been investigated in the search for novel antioxidants, but generally there is a continuous demand to find more information concerning the antioxidant potential of plant species occurring naturally. Phenolic compounds including flavonoids, tannins and phenolic acids are currently of growing interest due to their biological effects in human health. It has been recognized that flavonoids show antioxidant activity and their effects on human nutrition and health are considerable. Phenolic compounds are a class of antioxidant agents which act as free radical terminators8.

 

Our study was aimed in determining the total phenolic and flavonoid content and evaluating the antioxidant activity of pet. ether, alcoholic and aqueous extracts of stem bark of Careya arborea Roxb. An easy, rapid and sensitive method for the antioxidant screening of plant extracts is free radical scavenging assay using 1,1-diphenyl-2-picryl hydrazyl (DPPH) stable radical by  spectrophotometrically.

MATERIAL AND METHODS:

The stem bark of the Careya arborea Roxb.. was collected from Dajipur jungle (Radhanagri wild life sanctuary), Kolhapur, Maharashtra. The said plant was authenticated in the botany department of Willigdon College, Sangli and also by Dr. S. S. Sathe. The voucher specimen has been preserved in our laboratory for future reference.

 

DPPH and quercetin were purchased from Hi-media laboratories Pvt. Ltd. Mumbai, Folin-ciocalteu was purchased from loba chemie Pvt. Ltd Mumbai, then gallic acid, ascorbic acid and aluminium chloride were purchased from Research Labs Mumbai. All the chemicals used were of AR grade. Double beam UV-Vis Spectrophotometer (Jasco V-550) was used in the study.

 

Preparation of extract:

The dried barks were subjected to size reduction to obtain a coarse powder. Pet. ether and alcoholic extract of the bark powder was obtained by soxhlet extraction while for aqueous extract the powder was macerated for seven days using distilled water. The extracts were concentrated with the help of rotary vacuum evaporator (make- medica, model – roteva)

 

Table 1 Phenol and Flavonoids content

Extracts

Phenols*

(GAE g/100gms)

Flavonoids*

(QE g/100gms)

Pet.ether  extract

1.12 ± 0.28

0.19  ± 0.004

Aqueous

6.38 ± 0.72

2.13 ± 0.10

Alcoholic

5.59 ± 0.51

3.34 ± 0.33

* Each value is average ± SD (n = 3)

 

Table 2: % inhibition of pet ether, aqueous and alcoholic extracts

conc. (µg/ml)

% Inhibition

Pet. ether

Aq.

Alcoholic

0

0

0

0

1

0

13.63±2.36

7.55±2.11

10

0

48.51±2.22

54.13±2.63

50

0.303±0.12

58.37±2.16

71.43±2.19

100

10.891±.4

62.96±1.44

74.08±3.27

500

42.49±2.31

74.27±1.90

77.50±1.68

 

Determination of total phenol content:

Total phenolic content was determined by Folin ciocalteu reagent9. The dilute extracts (0.5ml of 1mg/ml) were mixed with Folin ciocalteu reagent (5ml, 1:10 diluted with distilled water) and aqueous sodium carbonate (4 ml, 1 M). The mixtures were allowed to stand for 60 min and total phenols were determined using double beam UV-Vis spectrophotometer at 765nm. Total phenolic values were expressed as gallic acid equivalent in g/100g of extract which is a common reference compound. The concentration of polyphenols in samples were derived from a standard curve of gallic acid ranging from 10 to 50 µg/ml.

 

Total flavonoids determination:

Aluminum chloride colorimetric method was used for flavonoids determination10. Alcoholic and aqueous extracts of the bark (2ml) were mixed with 0.1ml of 10% w/v aluminium chloride, 0.1ml of 1M potassium acetate and 2.8ml of distilled water. It was kept at room temperature for 30 minutes and the absorbance of the reaction mixture was measured at 415nm using double beam UV-Vis spectrophotometer. The calibration curve was plotted using quercetin solutions at concentrations 12.5 to 100 µg/ml in methanol.

 

Figure 1: Anti oxidant effect of Stem bark of Careya arbora

 

 

Free radical scavenging activity determinations:

The free radical scavenging capacity of the extracts was determined using DPPH method11. Plant extracts and ascorbic acid were weighed and dissolved in methanol to obtain six different concentration (1, 5, 10, 50, 100 and 500 µg/ml). Aliquots  were prepared suitably by diluting with methanol.

 

DPPH was weighed and dissolved in methanol to make 0.004% w/v solution. 3ml of 0.004% DPPH solution was added to each test tube with the help of calibrated pipette to obtain the desired concentrations. The prepared mixtures were incubated at 37°C for 30 min. The absorbance value of each test tube was determined using UV-Visible spectrophotometer at 517nm. The percentage inhibition values were calculated using equation.

 

C 50 was determined from % inhibition vs concentration graph.  IC 50 expressed the antioxidant activity defined as the concentration in ml that inhibits the formation of DPPH radicals by 50 %.

 

RESULTS AND DISCUSSIONS:

Total phenol and flavonoid contents of the extracts:

The results of total phenolic contents was obtained from regression equation of calibration curve (y= 0.0041x + 0.0062, r2= 0.9888). The Values are expressed in gallic acid equivalents (GAE) for phenols (table.1).

 

The flavonoids contents expressed in quercetin equivalents (QE)/100g of extract, were determined from regression equation of calibration curve  (y = 0.0165x -0.0235, r2=0.9973). Values were expressed in quercetin equivalents (QE) (table.1)

 

Antioxidant activity:

DPPH is the best, easiest and widely used method for testing preliminary free radical scavenging activity of a compound or a plant extract12. In the present study IC50. values of all three extracts were calculated and compared with IC50 value ascorbic acid as a standard ( IC50= 12.42 µg/ml).

 

Table 3: Free radical scavenging activity

Extract

Pet.ether extract

Aqueous extract

Alcoholic extract

IC50 (µg/ml) for DPPH scavenging activity

--

17 ug/ml

9 ug/ml

 

This investigation was based on the measurement of the relative inhibitory effect of extract tested at different concentrations. Table 2 represents the % inhibition of all three extracts. Figure 1 is graphical representation of % inhibitory activity Vs Concentration.

 

Table 3 shows capacity of alcoholic and aqueous extract to scavenge the DPPH radical. Both the extracts showed antioxidant activity.

 

CONCLUSION:

The result of the present study showed that the alcoholic and aqueous extracts of B. retusa, exhibited the greater antioxidant activity as compared to Pet. ether extract. Polyphenols, tannins and flavonoids are very valuable plant constituents in the scavenging action due to their several phenolic hydroxyl groups13. Hence it is clear that there is direct relationship between phenolic, flavonoids content of B. retusa and its antioxidant activity.

 

ACKNOWLEDGMENT:

We are grateful to the Principal and Management of Appasaheb Birnale College of Pharmacy, Sangli for providing the necessary facilities to carry out this work.

 

REFERENCES:

1.     Kirtikar KR, Basu BD, Indian medicinal plants, Vol. II, Periodical   exports book agency, Delhi. 1991, Edn 2, pp.1061-2.

2.     Kumar RS et al, Antimicrobial and Antioxidant activities of Careya arborea Roxb. stem bark , Indian journal of pharmacology and therapeutics. 2006; 5 : 35-41.

3.     Ahmed M, Rahman MW, Rahman MT, Hossain CF., Analgesic principles from the bark,   Pharmazie., 2002 ;57: 698-701.

4.     Rahman MT, Khan OF, Saha S, Alimuzzaman M., Antidiarrhoeal activity of the bark extract of Careya arborea Roxb. Fitoterapia., 2003;74: 116-118.

5.     Kumar, RS, et al., Hepatoprotective and in vivo Antioxidant effects of Careya arborea againt Carbon tetrachloride Induced Liver Damage in Rats, International Journal of Molecular Medicine and Advance Sciences. 2005 ;1: 418-424.

6.     Kumpulainen J.T., Salonen J.T.,  Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease,  The Royal Society of Chemistry, UK. 1999: 178- 187.

7.     Cook N.C, Samman S, Flavonoids- chemistry, metabolism, ardioprotective effects, and dietary sources. Nutritional Biochemistry, 1996;7: 66- 76.

8.     Shahidi F, Wanasundara, PKJPD Phenolic antioxidants. Critical Reviews in food Science and Nutrition, 1992; 32:67-103.

9.     Mcdonal S, et al; Phenolic content and antioxidant activity of olive extracts. Food chemistry, 2001; 73:73-84.

10.   Chang C, Yang M, Wen H; Chern J Estimation of total flavonoids content in propolis by two complementary colorimetric methods. J. Food Drug Analysis, 2002;10: 178-182.

11.   Braca A, Nunziatina, De Tommasi, Lorenzo, Di Bari, Cosimo, Pizza, Mateo, Politi, and Ivano, Morelli Antioxidant principles from Bauhinia terapotensis. Journal of Natural Products, 2001; 64: 892–895.

12.   Uddin S.N, Ali and Yesmin M.N.; Antioxidant and Antibacterial Activities of Sennatora Roxb. American J.Plant Physiol., 2008; 3:096-100.

13.   Tolmasoff J.M.,  Ono T, Cutller R.G.; Superoxide dismutase: corre-lation with life-span and specific metabolic rate in primate spe-cies. Proc Natl Acad Sci U.S.A. 1980: 77: 2777-81.

 

 

Received on 08.11.2009

Accepted on 11.12.2009

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(1): Jan.-Feb. 2010, 49-51