EVALUATION OF TOTAL PHENOLICS, FLAVONOID CONTENTS AND IN-VITRO ANTIOXIDANT PROPERTIES OF CATHARANTHUS PUSILLUS (APOCYNACEAE)

EVALUATION OF TOTAL PHENOLICS, FLAVONOID CONTENTS AND IN-VITRO ANTIOXIDANT PROPERTIES OF CATHARANTHUS PUSILLUS (APOCYNACEAE)

P. Y. Nithya ¹, S. M. Jelastin Kala ² and V. R. Mohan ^*3

Department of  Chemistry ¹, A.P.C. College for women, Thoothukudi, Tamilnadu, India.

Department of  Chemistry ², St. Xavier’s College, Palaymkottai Tamilnadu, India.

Ethnopharmacology Unit ³, Research Department of Botany, V.O. Chidambaram College, Tuticorin-628008, Tamilnadu, India.

ABSTRACT: Objective: Present study was aimed to evaluate the total phenolic, flavonoid content and in vitro antioxidant activity of petroleum ether, benzene, ethyl acetate, methanol and ethanol extracts of whole plant of Catharanthus pusillus. Methods: Antioxidant activity have been tested using various antioxidant model systems viz., DPPH, hydroxyl, superoxide, ABTS and reducing power. Results: Methanol extract of Catharanthus pusillus is found to possess higher DPPH, hydroxyl, superoxide radical scavenging activity, while ethyl acetate extract is found to possess higher ABTS radical cation scavenging activity. Methanol extract of whole plant of Catharanthus pusillus shows the highest reducing ability. Conclusion: The study indicates the significant free radical scavenging potential of Catharanthus pusillus whole plant which can be experimented for the treatment of various free radicals mediated aliments

Ethanol, Catharanthus pusillus, DPPH, antioxidant, phenol, flavonoid

INTRODUCTION: The World Health Organization (WHO) has reported that approximately 80% of the World’s population currently used as herbal medicine. The herbal drugs with easily accessible liquid such as water, milk to safe and reliable for human beings ^{1, 2}. Medicinal plants are of great importance to the health of individuals and communities in general. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic compounds ^{3, 4}.

Antioxidant is a chemical compound or substance that inhibits oxidation. They are found in many foods. They work to keep our cells healthy by protecting them from damage by free radicals (molecules responsible for aging, tissue damage, and some disease). Oxidation results from everyday body functions such as breathing or walking, but certain processed and fatty foods, toxic substances, and sunlight can increase its effects. Antioxidants help repair damaged cells, which can prevent diseases, including cancer. A diet rich in a variety of plant-based foods provides all of the antioxidants the body needs. Research shows that vitamins, minerals, and phytochemicals from whole foods interact to boost their disease-fighting effects⁵.

Catharanthus pusillus belonging to family Apocynaceae is known with various names in India and all over the world. It is widely used as various treatments of diseases and traditionally used as herbal medicine ⁶. The roots, leaves and latex of these plants are used to treat skin and liver diseases, leprosy, dysentery, worms, ulcers, tumor and ear aches. The leaf powder of C.pusillus were mixed with coconut oil and used for treat the antidandruff activity and also used to kill the lice ⁷.

The main purpose of this study was to evaluate total phenolic, flavonoids contents and in vitro antioxidant activity in whole plant of C.pusillus. However there is no information available on antioxidant activity of this plant.

 MATERIALS AND METHOD:

Collection of plant material:

The whole plant of C.pusillus were collected from Pechiparai, Kanayakumari District, Tamil Nadu. With the help of local flora, voucher specimens were identified and preserved in the Ethnopharmacology unit, Research department of Botany, V.O. Chidambaram College, Thoothukudi, Tamil Nadu for further references.

Prepation of plant Extract:

The dried whole plant of Catharanthus pusillus were powdered in a Wiley mill.100 gm of whole plant powder was packed in a soxhlet apparatus and extracted with petroleum ether, benzene, ethyl acetate, methanol and ethanol. All these extacts were concentrated in a rotary evaporator. The concentrated extracts were used for antioxidant activity, methanol extract was used for the estimation of total phenolics and flavonoids.

 Estimation of total phenolic content:

Total phenolic content was estimated using Folin-Ciocalteau reagent based assay as previously described ⁸ with little modification. To 1mL of each extract (100µg/mL) in methanol, 5mL of Folin-Ciocalteau reagent (diluted ten-fold) and 4mL (75g/L) of Na₂CO₃ were added. The mixture was allowed to stand at 20ºC for 30min and the absorbance of the developed colour was recorded at 765nm using UV-VIS spectrophotometer. 1mL aliquots of 20, 40, 60, 80, 100 µg/mL methanolic gallic acid solutions were used as standard for calibration curve. The absorbance of solution was compared with gallic acid calibration curve. The total phenolic content was expressed as gallic acid equivalents (GAE g/100g dry weight of extract).

Estimation of flavonoids:

The flavonoid content was determined according to Eom et al ⁹. An aliquot of 0.5ml of sample (1mg/mL) was mixed with 0.1mL of 10% aluminium chloride and 0.1mL of potassium acetate (1M). In this mixture, 4.3ml of 80% methanol was added to make 5mL volume. This mixture was vortexed and the absorbance was measured spectrophotometrically at 415nm. The value of optical density was used to calculate the total flavonoid content present in the sample.

 DPPH radical scavenging activity:

The DPPH is a stable free radical and is widely used to assess the radical scavenging activity of antioxidant component. This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen donating antioxidant due to the formation of the non radical form DPPH-H.

The free radical scavenging activity of all the extracts was evaluated by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) according to the previously reported method ¹⁰. Briefly, an 0.1mM solution of DPPH in methanol was prepared, and 1mL of this solution was added to 3 mL of the solution of all extracts at different concentration (50,100,200,400 &800μg/mL).

The mixtures were shaken vigorously and allowed to stand at room temperature for 30 minutes. Then the absorbance was measured at 517 nm using a UV-VIS spectrophotometer (Genesys 10S UV: Thermo electron corporation). Ascorbic acid was used as the reference. Lower absorbance values of reaction mixture indicate higher free radical scavenging activity. The capability to scavenging the DPPH radical was calculated by using the following formula.

DPPH scavenging effect (% inhibition) = {(A₀ –A₁)/A₀)*100}

Where, A₀ is the absorbance of the control reaction, and A₁ is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged

Hydroxyl radical scavenging activity:

The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell ¹¹. Stock solutions of EDTA (1mM), FeCl₃ (10mM), Ascorbic Acid (1mM), H₂O₂ (10mM) and Deoxyribose (10 mM) were prepared in distilled deionized water. The assay was performed by adding 0.1mL EDTA , 0.01mL of FeCl₃,0.1mL H₂O₂, 0.36mL of deoxyribose, 1.0mL of the extract of different concentration (50,100,200,400 &800μg/mL) dissolved in distilled water,0.33mL of phosphate buffer (50mM, pH 7.9), 0.1mL of ascorbic acid in sequence. The mixture was then incubated at 37⁰C for 1 hour. 1.0mL portion of the incubated mixture was mixed with 1.0mL of 10%TCA  and 1.0mL of 0.5% TBA (in 0.025M NaOH containing 0.025% BHA) to develop the pink chromogen measured at 532nm. The percentage inhibition was calculated by comparing the results of the test with those of the control using the above formula.

Superoxide radical scavenging activity:

The superoxide anion scavenging activity was measured as described by Srinivasan et al ¹². The superoxide anion radicals were generated  in 3.0 ml of Tris – HCL buffer (16 mM, P^H 8.0), containing  0.5 mL of NBT (0.3mM), 0.5 mL NADH (0.936mM) solution, 1.0 mL extract of different concentration (50,100,200,400 & 800μg/mL), and 0.5 mL Tris – HCl buffer (16mM, P^H 8.0). The reaction was started by adding 0.5 mL PMS solution (0.12mM) to the mixture, incubated at 25^oC for 5 min and the absorbance was measured at 560 nm against a blank sample, ascorbic acid.  The percentage inhibition was calculated by comparing the results of the test with those of the control using the above formula

Antioxidant activity by radical cation (abts. +):

ABTS assay was based on the slightly modified method of Huang et al ¹³. ABTS radical cation (ABTS+) was produced by reacting 7mM ABTS solution with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. The ABTS + Solution were diluted with ethanol to an absorbance of 0.70+0.02 at 734 nm. After addition of sample or trolox standard to 3.9 mL of diluted ABTS+ solution, absorbance was measured at 734 nm by Genesys 10S UV-VIS (Thermo scientific) exactly after 6 minutes. Results were expressed as trolox equivalent antioxidant capacity (TEAC). The percentage inhibition was calculated by comparing the results of the test with those of the control using the above formula.

Reducing power:

The reducing power of the extract was determined by the method of Kumar and Hemalatha ¹⁴. 1.0 mL of solution containing 50,100,200,400 &800μg/mL of extract was mixed with sodium phosphate buffer (5.0 mL, 0.2 M, pH6.6) and potassium ferricyanide (5.0 mL, 1.0%): The mixture was incubated at 50^oC for 20 minutes. Then 5mL of 10% trichloroacetic acid was added and centrifuged at 980 g (10 minutes at 5^oC) in a refrigerator centrifuge. The upper layer of the solution (5.0 mL) was diluted with 5.0 mL of distilled water and ferric chloride and absorbance read at 700 nm. The experiment was performed thrice and results were averaged.

Statistical analysis:

Antioxidant activities like DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical activity, ABTS radical cation scavenging activity and reducing powers were estimated in triplicate determinations. Data were analyzed using the statistical analysis system SPSS (SPSS software for windows release 17.5; SPSS Inc., Chicago IL, USA) Estimates of mean, standard error for aforesaid parameters were calculated.

RESULT:

Total phenolic content and total flavonoid content: The total phenolic content and total flavonoid content of the methanol extract of whole plant were found to be 0.621 A and 0.996 A.

DPPH radical scavenging activity:

DPPH radical scavenging activity of petroleum ether, benzene, ethyl acetate, methanol and ethanol extracts of whole plant C. pusillus was shown in Fig.1. The scavenging effect increases with the concentration of standard and plant extracts. Among the solvent tested methanol extract exhibited highest DPPH radical scavenging activity.

At 800µg/mL concentration methanol extract of C. pusillus whole plant possessed 113.96% scavenging activity a DPPH. IC₅₀ values of methanol extract whole plant C. pusillus and standard ascorbic acid were 38.72µg/mL and 31.75µg/mL respectively. (Table 1)

FIG. 1: DPPH RADICAL SCAVENGING ACTIVITY OF DIFFERENT EXTRACTS OF WHOLE PLANT OF C .PUSILLUS

Hydroxyl radical scavenging activity:

Hydroxyl radical scavenging activity of petroleum ether, benzene, ethyl acetate, methanol and ethanol extract of C. pusillus whole plant was shown in Fig. 2. Methanol extract showed very potent activity. At 800µg/ml concentration, methanol extract of C.pusillus whole plant possessed 131.16 % scavenging activity on hydroxyl radical. IC₅₀ values of methanol extract of whole plant of C. pusillus and standard ascorbic acid were 31.22 µg/mL and 43.93µg/mL respectively.

FIG. 2: HYDROXYL RADICAL SCAVENGING ACTIVITY OF DIFFERENT EXTRACTS OF WHOLE PLANT OF C .PUSILLUS

Superoxide radicial scavenging activity:

The different solvent extracts of C. pusillus whole plant extract were subjected to the superoxide radical scavenging activity and the results were shown in Fig. 3. It indicated that methanol extract of whole plant (800µg/ml) exhibited the maximum superoxide radical scavenging activity of 136.81%. The IC₅₀ values of methanol extract and standard ascorbic acid were 38.55µg/mL and 31.75µg/mL respectively.

FIG. 3: SUPEROXIDE RADICAL SCAVENGING ACTIVITY OF DIFFERENT EXTRACTS OF WHOLE PLANT OF C.PUSILLUS

ABTS radical cation Scavenging activity:

The Catharanthus Pusillus whole plant extract were subjected to the ABTS radical cation scavenging activity and the results were presented the Fig.4. The ethyl acetate extract exhibited potent ABTS radical cation scavenging activity in concentration dependent manner. At 800µg/mL concentration, C.pusillus whole plant possessed 98.33% scavenging activity in ABTS. The IC₅₀ values of methanol extract and standard trolox were 39.63µg/mL and 37.64µg/mL respectively.

FIG. 4: ABTS RADICAL SCAVENGING ACTIVITY OF DIFFERENT EXTRACTS OF WHOLE PLANT OF C. PUSILLUS

Reducing Power:

Fig.5 shows the reducing ability of different solvent extracts of C. pusillus whole plant compared to ascorbic acid. Absorbance of the solution was increased when the concentration increased. A highest absorbance indicated a higher reducing power. Among the solvent tested, methanol extract exhibited higher reducing activity. (0.593 OD).

FIG. 5: REDUCING POWER OF DIFFERENT EXTRACTS OF WHOLE PLANT OF C. PUSILLUS

TABLE1: IC₅₀ VALUES OF DIFFERENT SOLVENT EXTRACTS OF WHOLE PLANT OF C. PUSILLUS

 DISCUSSION: The antioxidant properties of the different solvent extracts of C. pusillus whole plant were significantly corroboralated by the phytochemical  constituents of the extracts. In general the phenolic compound are also found in many plants, These compounds composes of an aromatic ring bearing one or more hydroxyl groups and their structures may range from that of a simple phenolic molecule to a complex high-molecular weight polymer ¹⁵. The antioxidant activity of phenolic compounds depends on their structure, in particular the number and positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. Antioxidant activity of phenolic compounds is based on their ability to donate hydrogen atoms to free radicals.

Flavonoids are important secondary metabolites of plant modulating lipid peroxidation involved in atherogenesis, thrombosis and carcinogenesis. It has been confirmed that pharmacological effects of flavonoids is correlating with their antioxidant activities ¹⁶. Free radicals and other reactive species are thought to play an important role in many human diseases. Radical scavenging activities are very important due to the deleterious role of free radicals in biological systems. Many secondary metabolites which include flavonoids, phenolic compounds etc serve as sources of antioxidants and do scavenging activity ¹⁷. The presence of high phenolic and flavonoid content in the methanol extract of C. pusillus whole plant has contributed directly to the antioxidant activity by neutralizing the free radicals.

In the present study, the different solvent extracts of whole plant C.pusillus  possess free radical  scavenging activity with different in vitro models  viz DPPH, hydroxyl , superoxide and ABTS radical cation scavenging activity.

The DPPH test provided information on the reactivity of test compounds with a stable free radical. Because of its odd electron, DPPH gives a strong absorption band at 517nm in visible spectroscopy. The efficacies of antioxidants are often associated with their ability to scavenge stable free radicals ¹⁸. In the DPPH assay, the methanol extract of C.pusillus exhibited high scavenging activity (113.96%). A higher DPPH radical scavenging activity is associated with the lower IC ₅₀ value (38.72µg/ml). Hydroxyl radical is one of the potent reactive oxygen species in the biological system. It reacts with polyunsaturated fatty acid moieties of cell membrane phospholipids and causes damage to cell ¹⁹. IC₅₀ values of methanol extract of whole plant of C. pusillus and standard ascorbic acid were 31.22 µg/mL and 43.93µg/mL respectively. The hydroxyl radical scavenging activity may be due to the presence of various phytochemicals including polyphenols and flavonoids in C. pusillus whole plant extract.

Although superoxide anion is a weak oxidant, it gives rise to generation of powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative stress ²⁰. Numerous biological reactions generate superoxide anions which are highly toxic species. Therefore, whole plant C. pusillus was undertaken to test whether it has scavenging activity of superoxide anions. The IC₅₀ values of methanol extract and standard ascorbic acid were 38.55 µg/mL and 31.75µg/mL respectively. The results clearly indicate that the C. pusillus whole plant extracts have a noticeable effect as scavenging superoxide radical.

The decolorization of ABTs radical reflects the capacity of an antioxidant species to donate electrons or hydrogen atoms to inactivate this radical species. The ABTs radical cation is generated from the reaction of ABTs with potassium persulfate overnight in water ²¹. The ABTS chemistry involves direct generation of ABTS radical mono cation with no involvement of any intermediary radical. The IC₅₀ values of ethyl acetate extract and standard ascorbic acid were 39.63µg/mL and 37.64µg/ml respectively.

The reducing capacity of a compound may serve as an important indicator of its potential antioxidants activity ^{22 .}The reducing activity of a compound generally depends on the presence of reductases which have been exhibited antioxidant potential by breaking the free radical chain, donating a hydrogen atom ²³. The reducing capacity of C. pusillus whole plant is a significant indicator of this potential antioxidant activity.

CONCLUSION: The methanol extracts of C. pusillus whole plant showed strong antioxidant activity of inhibiting DPPH, hydroxyl and superoxide scavenging activity when compared with standard ascorbic acid. In addition, the C. pusillus whole plant found to be contain a noticeable amount of total phenolics and flavonoids which plays a major role in controlling antioxidants. Although the antioxidant activities found in vitro experiments were only indicative of the potential health benefit, these results remain important as the first step in screening antioxidant activity of C. pusillus whole plant.

 REFERENCES:

1. Okwu, Flavoring properties of spices of Cassava Fu Fu. AFr. J. Root Tuber Crops, 1999; 3(2): 19-21.
2. Okwu, Evaluation of Chemical composition of indigenousspices and Flavoring agents. Global J.Pure and applied sciences, 2001; 7(3): 445-449.
3. Julsing KM, Quax JW, Kayser O. The Engineering of Medicinal Plants: Prospects and Limitations of Medicinal Plant Biotechnology. In Oliver Kayser and Wim J Quax, ed. Medicinal Plant Biotechnology. WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim, 2007; 978-3-527- 31443-0.
4. Kalidass C, Daniel A, Mohan VR. Rapid propagation ofPlumbago zeylanica An important medicinal plant. J. Am. Sci, 2010; 6 (10): 1027-1031
5. Gale CR, Hall NF, Phillips DI, Martyn CN. Plasma antioxidant vitamins and carotenoids and age-related cataract. Ophthalmology, 108, 2001, 1992-1998
6. Don G. Catharanthus Roseus In: Ross I.A (Ed), “Medical Plants of The World”, Human Press, Totowa, NJ. 1999; 109-118
7. Balaji rao, N.S. Rajasekhar, D. and Chengal raju, D (1996).Folklore Remedies for Dandruff from Tirumala hills of Andhra Pradesh. Ancient Science of life. 1996; XV (4), P: 296 -300.
8. Wireman H and Halliwell B 1993 Carcinogenic antioxidants: Diethylstiboestrol, hexoestrol and 17 alpha-ethynyl-oestradiol FEBS Left 322:159-163
9. Eom SH, Cheng WJ, Hyoung JP, Kim EH, Chung MI, Kim MJ, Yu C, Cho DH. Far infra red ray irradiation stimulates antioxidant activity in Vitis flexuosa Thunb. Berries. Kor. J. Med. Crop Sci 2007; 15: 319-323.
10. Huang MH, Huang SS, Wang BS, Sheu MJ, Hou WC. Antioxidant and anti-inflammatory properties of Cardiospermum halicacabum and its reference compounds ex vivo and in vivo. J. Ethnopharmacology. 2011; 133: 743-750.
11. Halliwell B, Gutteridge JMC, Aruoma OI. The deoxyribose method: a simple test to be assay for determination of rate constants for reaction of hydroxyl radicals. Ana. Biochem 1987; 65: 215-219
12. Srinivasan R, Chandrasekar MJN, Nanjan MJ, Suresh B. Antioxidant activity of Caesalpinia digyna root. J Ethnopharmacology 2007; 113: 284-291
13. Wireman H and Halliwell B 1993 Carcinogenic antioxidants: Diethylstiboestrol, hexoestrol and 17 alpha-ethynyl-oestradiol FEBS Left 322:159-16316
14. Kumar RS, Hemalatha S. In vitro antioxidant activity of alcoholic leaf extract and subfractions of Alangium lamarckii Thwaites. J Chem and Pharm Res. 2011; 3: 259-267.
15. Lattanzio, M.Veronica, T. Lattanzio and A. Cardinali. Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects .Phytochemistry: Advances in Research, 2006: 23-67
16. Mbaebie, BO, Edeoga HO, Afolayan AJ. Phytochemical analysis and antioxidants activities of aqueous leaf bark extract of Schotia latifolia Asian Pac J Trop Biomed 2012; 2: 118-124
17. Ghasemi, K., Ghasemi, Y., Ebrahimzadeh, M.A., Antioxidant activity, phenol and flavonoid contents of 13 Citrus species peels and tissues. Pak. J. Pharm. Sci., 2009, 22, 277-281.
18. Basama, A., Zakania, Z., Latha, L., Saridhasan, S., Antioxidant activity and phytochemical screening of the methanol extracts of Euphorbia hirta L. Asian Pac J Trop Med 2011, 4, 386-390.
19. Battu, C.R., Ethadi, S.R., Vede, P.G., Swathi, P.K., Chandrika, K., Rao, V.A., Evaluation of antioxidant and anti-inflammatory activity of Euphorbia heyneana Spreng. Asian Pac J Trop BioMed 2011,1, 191- 194.
20. Naskar, S., Islam, A., Mozumdes, U.K., Saha, P., Haldar, P.K., Gupta, M., In vitro and in vivo antioxidant potential of hydromethanolic extract of Phoenix dactylijera fruits. J. Sci. Res 2010, 2, 144-157.
21. Mbaebie, B.O., Edeoga, H.O., Ajelayan, A.J., Phytochemical analysis and antioxidant activities of aqueous bark extract of Schoba lahtolic JACQ. Asian Pac J Trop Biomed 2012, 2, 118-124.
22. Dub, P.D., Tu, Y.Y., Yen, G.C., Antioxidant activity of water extract of Harng lyur (Chrysabthemum moifolium Ramat). Lebnsm Wiss Technol 1999, 32, 269-277.
23. Gordon, M.H., The mechanism of the antioxidant action in vitro. In: Hudson BJF, editor, Food antioxidants: London: Elsevier; 190; p: 1-18
    

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    How to cite this article:

    Nithya PY, Jelastin Kala SM and Mohan VR: Evaluation of Total Phenolic, Flavonoid Contents and in-vitro antioxidant Properties of Catharanthus Pusillus (Apocynaceae). Int J Pharm Sci Res 2016; 7(7): 3021-27.doi: 10.13040/IJPSR.0975-8232.7(7).3021-27.

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