In-vitro Antioxidant activity of Premna integrifolia Linn. Roots

Sanjay Jain*, Mamta Singh, Rakesh Barik, Neelesh Malviya

Department of Pharmacognosy, Smriti College of Pharmaceutical Education, 4/1, Pipliya Kumar Kakkad, Mayakhedi Road, Nipania, Indore 452010, Madhya Pradesh.

ABSTRACT:

Free radicals are fairly unstable and highly reactive substances, causing oxidation and sometimes irreversible damage to cells. This has led to an increase in intake of antioxidants. Medicinal plants are being studied extensively for their antioxidant properties. The present study was aimed to study the in-vitro antioxidant activity of aqueous and methanolic extracts of Premna integrifolia Linn roots (Verbenaceae). Both the extracts were subjected to various in-vitro antioxidant activity screening models such as DPPH, DMSO, ABTS, Nitric oxide and Iron chelation assay. Ascorbic acid was used as the standard. In all the models studied, the aqueous extract showed IC₅₀ values of 111.009, 101.369, 99.976, 109.827, 105.239 µg/ml and the methanolic extract showed IC₅₀ values of 98.252, 96.559, 88.163, 100.631, 95.005µg/ml for DPPH, DMSO, ABTS, Nitric oxide and Iron chelation assay, respectively.

KEYWORDS: Free radicals, Antioxidants, Premna integrifolia.

INTRODUCTION:

Free radicals are continuously produced by the body’s normal use of oxygen such as respiration and some cell mediated immune functions. During the process of oxygen utilization in normal physiological and metabolic processes, approximately 5% of oxygen gets univalently reduced to oxygen derived free radicals like superoxide, hydrogen peroxide, hydroxyl and nitric oxide radicals^1-3. Naturally, there is a dynamic balance between the amount of free radicals generated in the body and the antioxidants to quench and/or scavenge them and protect the body against their deleterious effects⁴. Oxidative stress is believed to be one of the major factors behind several acute and chronic diseases and may also be associated with ageing. Excess formation of free radicals in miscellaneous body environment may originate from endogenous response to cell injury, and exposure to a number of exogenous toxins. When the antioxidant system is overwhelmed, cell damage occurs⁵. There is an inverse relationship between dietary intake of antioxidant-rich foods and incidence of a number of human diseases^6,7. Many plants often contain substantial amount of antioxidant including vitamin C and E, carotenoids, flavonoids and tannins etc. and thus can be utilized to scavenge the excess free radicals from human body⁸.

Premna integrifolia Linn. (Verbenaceae) popularly known as “Arni” is a large shrub or small tree, commonly found in plains of Assam and Khassi hills in India along the Andaman coasts. In P. integrifolia (PI) alkaloids premnine, ganikarine and premnazole are reported from roots, while flavonoid luteolin, sterols and triterpene are reported from the leaves. The roots are used in the treatment of diabetes, inflammation, liver disorders, piles, constipation and fever^9-11.

MATERIALS AND METHODS:

All chemicals used were of analytical grade. 1,1-diphenyl-2-picryl hydrazyl (DPPH) and {2,2-azinobis (3 ethyl benzothiazolin 6 sulphonic acid)} (ABTS) were obtained from Sigma Chemicals, USA. O-phosphoric acid, sulphanilamide, nitro blue tetrazolium chloride, sodium nitro prusside, naphathyl ethylene diamine di-hydrochloride, ferrous sulphate, tris hydrochloride, sodium chloride, di-sodium hydrogen phosphate, potassium di-hydrogen phosphate, ascorbic acid and potassium per sulphate were obtained from Loba chemie, India. Di-methyl sulphoxide was obtained from Ranbaxy Laboratory, India.

Plant material:

Premna integrifolia roots were collected from the Pharmacognosy Garden of Timba Ayurvedic Pharmacy College, Timba, Gujarat. They was identified and authenticated at Department of Botany, Govt. Agriculture College, Indore. A voucher specimen (No. SCOPE/Phcog/07-09/06) has been deposited in the museum of our department for further references.

Plant extract:

The roots were dried under shade, coarsely powdered and passed through Sieve No. 18. The powder was extracted using methanol and water with the help of soxhlet and reflux respectively. The extracts were lyophilized and stored in vacuum desiccator for experimental use.

In-vitro antioxidant studies:

The aqueous and methanolic extracts of P. integrifolia roots were tested for free radicals scavenging property using different in-vitro models. All experiments were performed in triplicate and the results averaged.

DPPH radical scavenging activity:

To 2 ml of various concentration of extract, 2 ml solution of DPPH (0.1mM) was added and incubated for 20 min. in dark. An equal amount of methanol and DPPH were served as control. The absorbance was measured at 517 nm using spectrophotometer^12,13.

Superoxide radical scavenging activity:

To the reaction mixture containing 0.2 ml of nitroblue tetrazolium (NBT), and 0.6 ml of the various concentration of extract, 2 ml of alkaline DMSO was added to give a final volume of 2.8 ml. For control methanol was used instead of the test compound. The absorbance was measured at 560 nm using spectrophotometer¹⁴.

ABTS radical cation scavenging activity:

To the various concentration of extract (1ml), 0.6 ml of ABTS radical cation and 3.4 ml of phosphate buffer saline (pH 7.4) were added. For control methanol was used, instead of the test compound. The absorbance was measured at 734 nm using spectrophotometer¹⁵.

Nitric oxide radical scavenging activity –

Nitric oxide (NO) radicals were generated from sodium nitroprusside solution at physiological pH. To 2.5 ml of various concentration of test compound, sodium nitroprusside (5 mM) 0.75 ml was added and incubated at 25⁰C for 5 hours. After 5 hours, 0.5 ml of Griess reagent was added. For control methanol was used, instead of the test compound. The absorbance was measured at 546 nm using spectrophotometer¹⁶.

Iron chelation assay:

150 µl of freshly prepared 2 mM FeSO₄ was added to a reaction mixture containing 168 µl of 0.1 M Tris-HCl (pH 7.4), 218µl saline and the test extract. The reaction mixture was incubated for 5 min and after incubation 13µl of 0.25% 1,10 phenantroline (w/v) was added. For control instead of extract methanol was taken. The absorbance was measured at 510 nm using the spectrophotometer^17,18.

In all the models studied, the percentage scavenging was calculated by using the following formula.

Absorbance of Control - Absorbance of Test

% Scavenging = ---------------------------------------------- × 100

Absorbance of Control

Linear regression analysis was used to calculate IC₅₀ values.

RESULTS:

Preliminary phytochemical screening of the roots revealed the presence of alkaloids, carbohydrates, tannins, phenolic compounds, flavonoids and saponins. Several concentration ranging from 5-160µg/ml of aqueous and methanolic extracts of P. integrifolia were tested for the antioxidant activity in different in vitro models such as DPPH, DMSO, ABTS, Nitric oxide and Iron chelation assay. It was observed that free radicals were scavenged by the test compounds in a concentration dependent manner. In all the models studied, the aqueous extract showed maximum activity with IC₅₀ values 111.01, 101.37, 99.98, 109.83, 105.24 µg/ml and the methanolic extract showed maximum activity with IC₅₀ values 98.25, 96.56, 88.16, 100.63, 95.01µg/ml for DPPH, DMSO, ABTS, Nitric oxide and Iron chelation assay, respectively. Ascorbic acid was used as standard and IC₅₀ of ascorbic acid in DPPH, DMSO, ABTS, nitric oxide and iron chelation assay were found to be 10.06, 15.55, 7.72, 7.93 and 14.86µg/ml respectively. The IC₅₀ values are presented in Table 1.

DISCUSSION:

Oxidative stress is one of the causes of certain diseases such as diabetes, cardiovascular diseases, inflammatory conditions, cancer and ageing. Antioxidant may offer resistance against the oxidative stress by scavenging free radicals and thus prevent diseases¹⁹.

DPPH forms relatively stable free radicals and its assay determines the ability of methanolic and aqueous extracts of P. integrifolia to reduce the radicals to corresponding hydrazine by converting the unpaired electrons to paired ones^20-22. Both extracts showed inhibition of lipid peroxidation in a concentration dependent manner.

Alkaline DMSO is used as superoxide generating system. The generated superoxide will react with NBT to give coloured diformazan²³. Diformazan being insoluble in water slowly precipitate out. Therefore the spectral measurement was done immediately after the reaction was carried out in the presence of scavenger.

ABTS assay involves the scavenging of ABTS [2,2’ azino bis (3- ethylbenzthiazoline- 6- sulfonic acid) diammonium salt] radical cation. The principle behind the technique involves the reaction between ABTS and potassium persulphate to produce the ABTS radical cation, a blue green chromogen. In the presence of antioxidant reductant, the intensity of the coloured radical is decreased²⁴.

Nitric oxide (NO) is an important chemical mediator generated by endothelial cells, macrophages, neurons etc. and involved in the regulation of various physiological processes²⁵. Excess concentration of NO is associated with several diseases²⁶. Oxygen reacts with the excess nitric oxide to generate nitrite and peroxy nitrite anions which act as free radicals²⁷. In the present study, the extracts compete with oxygen to react with nitric oxide and thus inhibit the generation of the anions.

1,10 phenantroline may exert prooxidant effects by interacting with iron. 1,10 phenantroline quantitatively forms complex with Fe²⁺, which gets disrupted in the presence of chelating agents. The antioxidants interfere with the formation of ferrous-1,10 phenantroline complex, thereby suggesting that the extracts may have metal chelating activity²⁸.

In all the models tested, the antioxidant activity of aqueous and methanolic extract of P. integrifolia was studied in relation to that of ascorbic acid, a known potent antioxidant. Even though found to be less potent than ascorbic acid, the data of present study showed increasing free radical scavenging activity in various in vitro models in a concentration dependent manner. The phytoconstituent like flavonoids and polyphenolic compounds have been found to be responsible for antioxidant activity in many earlier reported studied^29,30. Hence the presence of these phtoconstituent in the roots studied may be responsible for these activity.

REFERENCES:

1. Shirwaikar A, Prabhu K S and Punitha I S R, In vitro antioxidant studies of Sphaeranthus indicus (Linn), Ind J Exp Biol, 44 (2006) 993.

2. Mondal S K, Chakraborty G, Gupta M and Mazumder U K, In vitro antioxidant activity of Diospyros malabarica Kostel bark, Ind J Exp Biol, 44 (2006) 39.

3. Yu B P, Cellular defenses against damage from reactive oxygen species, Physiol Rev, 74 (1994) 139.

4. Halliwell B and Gutteridge J M C, Free Radicals in Biology and Medicine, 2^nd ed (Clarendon Press, Oxford) 1988, 1.

5. Basu S, The enigma of in vivo oxidative stress assessment: isoprostanes as an emerging target, Scandinavian J Food Nut, 51 (2007) 48.

6. Malpure P P, Shah A S and Juvekar A R, Antioxidant and anti-inflammatory activity of extract obtained from Aspergillus candidus MTCC2202 broth filtrate, Ind. J. Exp. Biol, 44 (2006) 468.

7. Rice-Evans C A, Sampson J, Bramley P M and Hoiloway D E, Why do we expect carotenoids to antioxidants in vivo, Free Radic Res, 26 (1997) 381.

8. Lu Y R and Foo L Y, Antioxidant and radical scavenging activities of polyphenols from apple pomace, Food Chem, 68 (2000) 81.

9. Gokhani R H, Lahiri S K, Santani D D and Shah M B, Evaluation of immunomodulatory activity of Clerodendrum phlomidis and Premna integrifolia roots, Int J Pharmacol, 3 (2007) 352.

10. Dash G K, Patro Ch P and Maiti A K, A study on anti-hyperglycaemic effect of Premna Corymbosa Rottl. roots, J Nat Rem, 5 (2005) 31.

11. Rajendran R, L Suseela, R Sundaram M and N Basha S, Cardiac stimulant activity of bark and wood of Premna serratifolia, Bangladesh J Pharmacol, 3 (2008)107.

12. Shirwaikar A, Shirwaikar A, Rajendran K and Punitha I S R, In vitro antioxidant studies on the benzyl tetra isoquinoline alkaloid berberine, Biol Pharm Bull, 29 (2006) 1906.

13. Singh R, Singh S, Kumar S and Arora S, Studies on antioxidant potential of methanol extract/fraction of Acacia auriculiformis A. Cunn, Food Chem, 103 (2007) 505.

14. Srinivasan R, Chandrasekar M J N, Nanjan M J and Suresh B, Free radical scavenging activity of Ipomoea obscura (L.) Ker-Gawl, J Nat Rem, 7 (2007) 184.

15. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic Biol Med, 26 (1999) 1231.

16. Govindaranjan R, Vijayakumar M, Rawat A K S and Mehrotra S, Free radical scavenging potential of Picrorhiza kurrooa Royle ex Benth, Ind. J. Exp. Biol, 41 (2003) 875.

17. Sabir S M and Rocha J B T, Water-extractable phytochemical from Phyllanthus niruri exhibit distinct in vitro antioxidant and in vivo hepatoprotective activity against paracetamol induced liver damage in mice. Food Chem, 111(2008) 845.

18. Puntel R L, Nogueira C W and Rocha J B T, Kreb cycle intermediates modulate thiobarbituric acid reactive species (TBARS) production in rat brain in vitro, Neurochemical Research, 30 (2005) 225.

19. Marx J L, Oxygen free radicals linked to many diseases, Science, 235 (1987) 529.

20. Vani T, Rajani M, Sarkar S and Shishoo C J, Antioxidant properties of ayurvedic formulation Triphala and its constituents, Int J Pharmacognosy, 35 (1997) 313.

21. Sanchez Moreno C, Methods used to evaluate the free radical scavenging activity in foods and biological systems, Food Sci Tech Int, 8 (2002) 122.

22. Sanchez Moreno C, Larrauri J and Saura-Calixto F, Free radical scavenging capacity of selected red and white wine, J Sci Food Agric, 79 (1999) 1301.

23. Khanam S, Shivprasad H N and Kshama D, In-vitro antioxidant screening models: a review, Ind J Pharm Edu, 38 (2004) 180 – 183.

24. Youdim K A and Joseph J A, A possible emerging role of phytochemicals in improving age-related neurological dysfunctions: a multiplicity of effects, Free Rad Biol Med 30 (2001) 583.

25. Lata H and Ahuja G K, Role of free radicals in health and disease, Ind J Physio and Allied Sci, 57 (2003) 124.

26. Ialenti A, Moncada S and Di Rosa M, Modulation of adjuvant arthritis by endogenous nitric oxide, Br J Pharmacol, 110 (1993) 701.

27. Sainani G S, Manika J S and Sainani R G, Oxidative stress: a key factor in pathogenesis of chronic diseases, Med Update, 1 (1997) 1.

28. Marcocci L, Packer L, Droy-Lefaiz M T, Sekaki A and Gardes-Albert M, Antioxidant action of Ginko biloba extract, Meth Enzymol, 234 (1994) 462.

29. Pietta P G, Flavonoids as antioxidants, J Nat Prod, 63 (2000) 1035.

30. Kahkonem M P, Antioxidant activity of plant extracts containing phenolic compounds, J Agri Food Chem, 47 (1999) 3954.

Received on 29.08.2013

Modified on 30.09.2013

Accepted on 15.10.2013

Research J. Pharmacology and Pharmacodynamics. 5(5): September–October 2013, 293-296

Extract/ standard	IC₅₀ value (µg/ml)
Extract/ standard	DPPH	DMSO	ABTS	Nitric oxide	Iron chelation
PI Aqueous extract	111.01	101.37	99.98	109.83	105.24
PI Methanolic extract	98.25	96.56	88.16	100.63	95.01
Ascorbic acid	10.06	15.55	7.72	7.93	14.86