IN-VITRO ANTIOXIDANT PROPERTIES OF DELONIX ELATA WHOLE PLANT EXTRACTS

IN-VITRO ANTIOXIDANT PROPERTIES OF DELONIX ELATA WHOLE PLANT EXTRACTS

B. S. Sharvana Bhava ¹, K. Umasankar ¹ and A. Kottai Muthu ^{* 2}

Department of Pharmacology ¹, Vaagdevi College of Pharmacy, Warangal - 506001, Telangana, India.

Department of Pharmacy ², Faculty of Engineering & Technology, Annamalai University, Annamalai Nagar, Chidambaram - 608002, Tamil Nadu, India.

ABSTRACT: Delonix elata (D. elata) whole plant was chosen to evaluate in-vitro anti-oxidant activities. The total polyphenols and flavonoids were determined after the extraction. DPPH (2, 2'-diphenyl-1-picrylhydrazy), H₂O₂ (Hydrogen peroxide), NO (Nitric oxide) and TBARS (Thiobarbituric acid) methods were used to evaluate antioxidant activities of D. elata whole plant. The results showed that the highest levels of polyphenols were recorded in DEM (15.32 ± 0.32 mg of GA/g of extract). Total flavonoid contents of extracts/fractions showed that DEM (15.57 ± 0.69 mg of Qu/g of extract) contains the highest level. The DEM extract shows more inhibition of DPPH (92.56), NO (143.88), H₂O₂ (182.28) and TBARS (121.94 µg/ml), respectively when compared with other extracts. These findings suggest that the extracts obtained from D. elata stem have active substances contributing to the increase in natural antioxidant potential. It can be concluded from the findings that D. elata extracts have potent antioxidant potential.

Delonix elata, Anti-oxidant, DPPH, Hydrogen peroxide, Nitric Oxide, TBARS, Total flavonoids

INTRODUCTION: The substance even in lower concentrations which oppose or delay oxidation of a substrate is called an anti-oxidant. Normal cellular metabolisms form reactive oxygen species (ROS) and reactive nitrogen species (RNS), which cause many diseases due to oxidative stress. Thus research on various anti-oxidants with great anti-oxidant potential is worth of importance ^{1, 2}. Two mechanisms viz., A) Obstructing the free radical production  B) protecting the cell membrane by free radical scavengers such as catalase (CAT), glutathione peroxidase (GPx) and super oxide dismutase (SOD) are postulated for antioxidant activity ³.

The major source of antioxidants till today is the medicinal plants ⁴. Delonix elata belongs to the family Caesalpiniaceae, and it is widely found in south India. Presence of glycosides, flavonoids, phenols, saponins, tannins, and steroids were found in the preliminary phytochemical screening of the extract of the aerial parts. The motto of this work was to substantiate the in-vitro antioxidant activities of aerial parts of Delonix elata petroleum ether, ethyl acetate, and methanolic extracts respectively.

MATERIALS AND METHODS:

Reagents and Standards: Petroleum ether, ethyl acetate, methanol, gallic acid, quercetin, aluminum-chloride, 1, 1-diphenyl-2-picrylhydrazyl (a a-diphenyl-b-picrylhydrazyl; DPPH), H₂O₂, griess reagent, trichloroacetic acid, thiobarbituric acid.

Plant Materials: The aerial parts of D. elata were collected in parts of Kerala. The collected whole plant was authenticated by Dr. V. Chelladurai, Research Officer- Botany (Scientist -C) and Authentication no. is BSI/SRC/5/23/2017/Tech/727 Central Council for Research in Ayurveda & Siddha, AYUSH, Government of India.

Preparation of Extracts: The collected material was washed with distilled water and dried at room temperature to constant weights and ground to powder. The powder (UNIT Quantity) was sealed in a muslin cloth and Soxhlated with a pet. ether, ethyl acetate, and methanol as solvents respectively according to the increasing order of polarity. Each extract was concentrated to dryness under reduced pressure at 40 °C using a rotary evaporator.

Determination of Total Phenolic Content (TPC): The three extracts were evaluated for total phenolic content with Folin-Ciocalteu method ⁵. Gallic acid calibration curve was used for estimation of the phenolic content of three extracts. Sample determinations were performed in triplicate. Total phenolic content was expressed as milligrams of gallic acid equivalent (GAE) per g of extract.

Determination of Total Flavonoid Content (TFC): Total flavonoid content in the extracts was determined by aluminum-chloride colorimetric assay ⁶. The determination of total flavonoid content in the three extracts and standard were performed thrice. The result was determined from the calibration curve and expressed as milligram of quercetin equivalent (QE) per gram of extracts ⁷.

In-vitro Antioxidant Activity:

DPPH Radical Scavenging Activity: The anti-oxidant potential of the three extracts was measured based on the scavenging of DPPH (1, 1-diphenyl-2-picrylhydrazyl (an a-diphenyl-b-picrylhydrazyl)).

According to Manzocco et al., the sample extract (0.2 mL) is diluted with methanol, and 2 mL of DPPH solution (0.5 mM) is added.  After 30 min, the absorbance is measured at 517 nm using UV-Visible spectrophotometer. The percentage inhibition of DPPH radical can be extracted from the equation as follows:

% inhibition of DPPH radical = {(Abr -Aar)/ Abr} × 100

Where Abr is the absorbance before reaction and Aar is the absorbance after the reaction has taken place ⁸.

Hydrogen Peroxide Scavenging (H₂O₂) Assay: The ability of the extract to scavenge hydrogen peroxide is estimated by following the method of Ruch et al. ⁹

Sample extracts are added prepared hydrogen peroxide solution, and absorbance is determined after 10 min at 230 nm against a blank solution (Phosphate buffer without hydrogen peroxide). The percentage scavenged by the sample can be determined by:

% scavenged (H₂O₂) = {(Ai -At)/ Ai} × 100

Where Ai is the absorbance of the control and At is the absorbance of the test.

Nitric Oxide Scavenging Activity: 2 mL of 10 mM sodium nitroprusside dissolved in 0.5 mL phosphate buffer saline (pH 7.4) is mixed with 0.5 mL of sample. After 150 min of incubation at 25 °C, 0.5 ml of the incubated solution is withdrawn and mixed with 0.5 mL of Griess reagent [(1.0 mL sulfanilic acid reagent (0.33% in 20% glacial acetic acid at room temperature for 5 min with 1 mL of naphthyl ethylenediamine dichloride (0.1% w/v)]. This is then incubated at room temperature for 30 min, and its absorbance was measured at 546 nm. The percentage inhibition of NO radical can be obtained by:

% inhibition of NO radical = {(A0-A1)/ A0} × 100

Where, A0 is the absorbance before reaction, and A1 is the absorbance after the reaction has taken place with Griess reagent ¹⁰.

TBARS Assay: The final sample concentration of 0.02% w/v, as described by Ottolenghi et al., was used in this method. To 1 ml of the sample solution, 2 mL of 20% trichloroacetic acid and 2 mL of 0.67% of thiobarbituric acid were added and kept in a boiling water bath for 10 min. The mixture was cooled and then centrifuged for 20 min at 3000 rpm. The supernatant absorbance was measured at 552 nm and recorded after it has reached its maximum ¹⁰.

Statistical Analysis: The results were expressed as Mean ± SD. The data were analyzed using one-way analysis of variance (ANOVA), and the differences between samples were determined by Dunnett’s test, P<0.05 were regarded as significant.

RESULTS AND DISCUSSION:

Total Phenolic and Flavonoid Content: The total phenolic and flavonoid content of the petroleum ether, ethyl acetate and methanolic extracts of Delonix elata (DE) were done by Folin-Ciocalteu colorimetric and AlCl₃ methods, respectively. The total phenolic contents were expressed as mg gallic acid equivalent per gram of dry extract. In Table 1, phenolic content in the examined whole plant extracts of DE ranges from 0.32 to 15.32. The DEM has very high values of total phenolic contents (15.32) when compared with remaining extracts like DEP (0.32) and DEE (5.72). In the analysis of results for the concentrations of total phenolic compounds extracts, it is noticed that the highest concentration of phenolic compounds is in the extracts obtained using solvents of high and moderate polarity. The high dissolubility of phenols in polar solvents provides high concentration of these compounds in the extracts obtained using polar solvents for the extraction ^{11, 12}.

TABLE 1: TOTAL PHENOLIC CONTENTS IN THE PLANT EXTRACTS EXPRESSED IN TERMS OF GALLIC ACID EQUIVALENT (mg of ga/g of extract)

The concentration of flavonoids in DE whole plant extracts were determined using the spectrophotometric method with AlCl₃. The content of flavonoids is expressed in terms of Quercetin equivalent (QuE), mg of Qu/g of extract. The summary of quantities of flavonoids identified in the tested extracts is shown in Table 2.

TABLE 2: TOTAL FLAVONOID CONTENTS IN THE PLANT EXTRACTS EXPRESSED IN TERMS OF GALLIC ACID EQUIVALENT (mg of Qu/g of extract)

The flavonoids content in the examined different extracts ranges from 0.78 to 15.57 mg Qu/g. The DEM extract (15.57) have very high values of total flavonoids contents when compared with DEP (0.78) and DEE (5.14). Based on the obtained values for the concentration of flavonoids in the observed leaf, bark and fruit extracts, it has been found that the highest concentration of these compounds is in the extracts obtained using solvents of moderate polarity and non-polar solvents. The concentration of flavonoids in plant extracts depends on the polarity of solvents used in the extract preparation ¹³.

DPPH Radical Scavenging Activity: The comparison of the antioxidant activities of the extracts and ascorbic acid is shown in Table 3. The IC₅₀ values for scavenging of free radicals were 35.28, 92.56, 334.82 and 457.51 µg/ml for ascorbic acid, methanol, ethyl acetate, and petroleum ether extracts respectively, which indicate the efficient DPPH scavenging activity. The order of DPPH scavenging activity was in the order of methanol>ethyl acetate > pet. ether extracts.

TABLE 3: DPPH RADICAL SCAVENGING ACTIVITY OF DELONIX ELATA EXTRACTS

Hydrogen Peroxide (H₂O₂) Radical Scavenging Assay: The comparison of the antioxidant activities of the extracts and ascorbic acid is shown in Table 4. The IC₅₀ values for scavenging of free radicals were 56.35, 182.28, 304.33 and 397.26 µg/ml for ascorbic acid, methanol, ethyl acetate, and petroleum ether extracts respectively, which indicate the efficient H₂O₂ scavenging activity.

TABLE 4: HYDROGEN PEROXIDE (H₂O₂) SCAVENGING ASSAY OF DELONIX ELATA EXTRACTS

Nitric Oxide (NO) Radical Scavenging: The comparison of the antioxidant activities of the extracts and ascorbic acid is shown in Table 5.

TABLE 5: NITRIC OXIDE (NO) SCAVENGING ACTIVITY OF DELONIX ELATA EXTRACTS

The IC₅₀ values for scavenging of free radicals were 67.97, 143.88, 176.83 and 386.49 µg/ml for ascorbic acid, methanol, ethyl acetate, and petroleum ether extracts respectively, which indicate the efficient NO scavenging activity.

Thiobarbituric Acid Reactive Substances (TBARS) Assay: The comparison of the antioxidant activities of the extracts and ascorbic acid is shown in Table 6. The IC₅₀ values for scavenging of free radicals were 46.99, 121.94, 187.24 and 392.57 µg/ml for ascorbic acid, methanol, ethyl acetate, and petroleum ether extracts respectively, which indicate the efficient TBARS assay.

TABLE 6: THIOBARBITURIC ACID (TBARS) ASSAY OF DELONIX ELATA EXTRACTS

CONCLUSION: The Delonix elata whole plant methanolic extract showed a strong antioxidant activity by inhibiting DPPH, NO, H₂O_2, and TBARS activities when compared with the standard L-ascorbic acid. In addition, the Delonix elata was found to contain a noticeable amount of total phenols and flavonoids, which play a major role in controlling oxidation. The results of this study show that the Delonix elata whole plant can be used as an easily accessible source of natural antioxidant. Therefore, a further study is needed to determine the mechanism behind the antioxidant activity of this plant. Further studies are needed to elucidate whether Delonix elata whole plant could be useful in the management of human diseases resulting from oxidative stress.

ACKNOWLEDGEMENT: The authors are grateful to Secretary, Vishwambhara Educational Society, and Warangal for providing the necessary facilities to carry out the study.

CONFLICTS OF INTEREST: No conflicts of interests.

REFERENCES:

1. Gupta RK, Patel AK, Shah N, Chaudhary AK, Jha UK, Yadav UC, Gupta PK and Pakuwal U: Oxidative stress and antioxidants in disease and cancer. Asian Pacific Journal of Cancer Prevention 2014; 15: 4405-9.
2. Kapadia GJ, Azuine MA, Takayasu J, Konoshima T, Takasaki M, Nishino H and Tokuda H: Inhibition of Epstein Barr virus early antigen activation promoted by 12-O-tetradecanoylphorbol-13-acetate by the non-steroidal anti-inflammatory drugs. Cancer Letters 2000; 161: 221-9.
3. Violi F and Cangemi R: Antioxidants and cardiovascular disease. Current Opinion in Investigational Drugs 2005; 6: 895-00.
4. Peng KT, Hsu WH, Shih HN, Hsieh CW, Huang TW and Hsu RW: The role of reactive oxygen species scavenging enzymes in the development of septic loosening after total hip replacement. The Journal of Bone and Joint Surgery 2011; 93: 1201-1209.
5. Edeoga HO, Okwu DE and Mbaebie BO: Phytochemical constituents of some Nigerian medicinal plants. African Journal of Biotechnology 2005; 4: 685-8.
6. John BI, Sulaiman CT, George S and Reddy VR: Total phenolics and flavonoids in selected medicinal plants from Kerala. International Journal of Pharmacy and Pharmaceutical Sciences 2014; 6: 406-8.
7. Kostiæ DA, Dimitrijeviæ DS, Mitiæ SS, Mitiæ MN, Stojanoviæ GS and Živanoviæ AV: Phenolic content and antioxidant activities of fruit extracts of Morus nigra (Moraceae) from Southeast Serbia. Tropical Journal of Pharmaceutical Research 2013; 12: 105-10.
8. Da Porto C, Calligaris S, Celotti E and Nicoli MC: Antiradical properties of commercial cognacs assessed by the DPPH test. Journal of Agricultural and Food Chemistry 2000; 48: 4241-5.
9. Ruch RJ, Cheng SJ and Klaunig JE: Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 1989; 10: 1003-8.
10. Alam MN, Bristi NJ and Rafiquzzaman M: Review on in- vivo and in-vitro methods evaluation of the antioxidant activity. Saudi Pharmaceutical Journal 2013; 2: 143-52.
11. Zhou K and Yu L: Effects of extraction solvent on wheat bran antioxidant activity estimation. Food Science and Technology 2004; 37: 717-721.
12. Mohsen MS and Ammar SMA: Total phenolic contents and antioxidant activity of corn tassel extracts. Food Chemistry 2008; 112: 595-98.
13. Min G and Hun-Zhao L: Comparison of techniques for the extraction of flavonoids from cultured cells of Saussurea medusa Maxim. World Journal of Microbiology and Biotechnology 2005; 2: 1461-63.
    

    ---

    How to cite this article:

    Bhava BSS, Umasankar K and Muthu AK: In-vitro antioxidant properties of Delonix elata whole plant extracts. Int J Pharm Sci & Res 2019; 10(12): 5386-90. doi: 10.13040/IJPSR.0975-8232.10(12).5386-90.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---