Int J Pharm Pharm Sci, Vol 7, Issue 7, 340-343Original Article


EVALUATION OF IN VITRO ANTI-OXIDANT AND ANTI-ARTHRITIC ACTIVITY OF METHANOLIC EXTRACT OF MARINE GREEN ALGAE CAULERPA RACEMOSA

SUMANYA H, LAVANYA R*, UMAMAHESWARA REDDY C

Department of Pharmacology, Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai, Tamil Nadu 600116
Email: [email protected]

Received: 06 Dec 2014 Revised and Accepted: 30 May 2015


ABSTRACT

Objective: The present study was aimed to evaluate in vitro anti-oxidant activity and in vitro anti arthritic activity of methanolic extract of Caulerpa racemosa.

Methods: The in vitro anti oxidant activity was evaluated by using following method viz; DPPH scavenging activity, Nitric oxide scavenging activity, Total anti-oxidant activity, Determination of reducing power. The in vitro anti arthritic activity was evaluated by using protein denaturation method.

Results: Methanolic extract of Caulerpa racemosa showed a very good anti-radical activity in scavenging DPPH radical and nitric oxide radical with maximum % inhibition of 88.87±1.05% at 2000μg/0.1 ml concentration and 80.49±1.43% at 2000μg/ml respectively. Total anti-oxidant activity and reducing power of Caulerpa racemosa was found to be 32μg equivalents of ascorbic acid at 500 μg/ml and 88.80±0.98% at 2000μg/0.05 ml respectively. The methanolic extract showed 49.33±0.597% of percentage inhibition at 1000μg/0.05 ml by protein denaturation method.

Conclusion: From the results obtained, it can be concluded that the methanolic extract of Caulerpa racemosa possesses significant anti-oxidant and anti-arthritic activity. Further studies are required to signify the mechanism of action of these pharmacological activities and to mark them out for their significant pharmacological actions.

Keywords: Caulerpa racemosa, Anti-oxidant, Diclofenac sodium, Anti-arthritic activity, Protein denaturation.


INTRODUCTION

Anti-oxidant is a molecule that inhibits oxidation of other molecules which produce free radicals. These radicals in turn produce chain reactions thereby cause damage to the cells, resulting in development of various ailments [1]. Anti-oxidants terminate these chain reactions by removing free radicals and inhibiting oxidative reactions. Therefore, antioxidants with free radical scavenging effect will be of greater importance in the prevention and therapeutics of the disease. Rheumatoid arthritis is a chronic inflammatory auto immune disease that exerts its greatest impact on the joints of the body that is lined with synovium, a specialized tissue responsible for maintaining the nutrition and lubrication of the joint [2, 3]. It is characterized by multiple conditions that cause pain, swelling, stiffness and loss of function in joints [4].

Caulerpa racemosa, marine green algae is a pan-tropical to temperate warm water species widely distributed throughout the world, found in many areas of shallow sea [5]. It consists of a number of branches linked to stolons of more than, with up to 200 fronds, which are anchored to sandy substrate by rhizoids and branches grown up to a height of 30 cm. Many spherical or ovate side shoots branch off and gives the seaweed its name ‘bunch of grapes’. Caulerpa racemosa is used to lower blood pressure and to treat rheumatism [5]. It also contains compounds which function as a mild anesthetic, which gives the seaweed clinical value. On photochemical screening of methanolic extract, revealed the presence of various pharmacologically active compounds like steroids, triterpenoids, alkaloids, tannins, glycosides, carbohydrates, flavonoids and proteins. Therefore, the study was carried out to investigate the anti-oxidant and anti-arthritic activity of methanolic extract of Caulerpa racemosa.

MATERIALS AND METHODS

Chemical and reagents

Chemicals used in the studies were sodium nitroprusside, quercetin, Griess reagent, 1% sulphanilamide, 2% phosphoric acid, 0.1% napthyl ethylene diamineHcl, 1,1,-dipHenyl-2-picryl hydrazine (DPPH), dimethylsulphoxide (DMSO), potassium ferrricyanide, trichloro acetic acid (TCA), ferric chloride, ascorbic acid, sulphuric acid, sodium phosphate, ammonium molybdate, bovine serum albumin, diclofenac sodium, 1N hydrochloric acid, methanol, phosphate buffer saline (PBS).

Plant material

Collection and authentication of plant materials

Caulerpa racemosa is a green marine algae of the family Caulerpaceae, which spread from tropical to warm water regions, was collected from Mandapam of Rameshwaram, Tamil Nadu in the month of October 2011. The plant was identified and authenticated by Dr. V. Krishnamurthy, Director, Krishnamurthy Institute of Algalogy, Chennai.

Preparation of extract

The Caulerpa racemosa was washed with sea water and then with tap water to remove the epiphytes and were shade dried, macerated with methanol exhaustively. The crude extract obtained after distillation of solvent at a lower temperature was used for the study.

Preliminary phytochemical screening

A portion of methanolic extract of Caulerpa racemosa was analyzed for the presence of various phytoconstituents [6]. Photochemical analysis exhibited the presence of steroids, triterpenoids, alkaloids, tannins, glycosides, carbohydrates, flavonoids and proteins.

In-vitro anti-oxidant activity

DPPH scavenging activity

DPPH scavenging activity was measured by the spectrophotometric method. A stock solution of DPPH (150μm) was prepared in ethanol. 0.1 ml of various concentrations (31.25, 62.5, 125, 250, 500, 1000, 2000 μg/0.1 ml) of methanolic extracts and DPPH (1.9 ml) was added. Control is prepared in the same manner without the test compound. In blank, ethanol replaced DPPH. The mixtures were kept in dark for 20 mins, for the reaction to complete and absorbance was read at 517 nm. Lower the absorbance of the reaction mixture higher the scavenging activity. Quercetin (31.25, 62.5, 125, 250, 500, 1000, 2000 μg/0.1 ml) was used as standard. The percentage inhibition of DPPH was calculated as follows [7].

Nitric oxide scavenging activity

Nitric oxide scavenging activity of the extract was determined according to the method (green. et. al., 1982). Aqueous solution of sodium nitroprusside generates nitric oxide (NO) spontaneously at physiological pH. NO interacts with oxygen to produce nitrate ions which were measured colorimetrically. 3 ml of reaction mixture containing 2 ml of sodium nitroprusside (10 mM) in PBS and 1 ml of various concentration (62.5, 125, 250, 500, 1000, 2000 μg/ml) of methanolic extracts were incubated at 37˚C for 4 hours. Control without test compound was kept in the same manner. After incubation 0.5 ml of Griess reagent was added. The absorbance of the chromophore formed was read at 546 nm [8, 19]. Quercetin (50, 100, 200μg/ml) was used as standard.

The percentage inhibition of nitric oxide scavenging was calculated using the formula,

Total anti-oxidant activity

Total anti-oxidant activity is a spectroscopic method used for quantitative determination of anti-oxidant capacity. Sample solution/ascorbic acid equivalent to 500μg/0.1 ml was mixed with 1 ml of reagent solution (0.6M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). In blank, 0.1 ml of methanol was used instead of sample. The reaction mixtures were incubated in a boiling water bath at 95˚C for 90 minutes. Samples were allowed to cool to room temperature. The absorbance of the reaction mixtures were measured at 695 nm against blank. The anti-oxidant activity was expressed as equivalents of ascorbic acid (μg/g) [20].

Determination of reducing power

For the measurement of reductive ability, transformation of Fe3+-Fe2+was investigated according to the method (Oyaizu. et. al., 1986) in the presence of 2.5 ml of phosphate buffer (pH 6.6), 2.5 ml of 1% potassium ferrricyanide and extracts at different concentrations (31.25, 62.5, 125, 250, 500, 1000, 2000 μg/ml) were incubated at 50˚C for 20 minutes. To the mixture, 2.5 ml of 10% TCA were added and centrifuged at 3000 rpm for 10 mins. After centrifugation, 2.5 ml of the supernatant was diluted with 2.5 ml of water and added 0.5 ml freshly prepared 0.1% ferric chloride. The absorbance was measured at 700 nm [9]. The control solution consists of water in the place of sample. The increase in absorbance indicates higher reductive ability [10].

In-vitro anti-arthritic activity

0.5 ml of the reaction mixture consisted of 0.45 ml of 5%W/V bovine serum albumin in aqueous solution and 0.05 ml of various concentrations of crude extracts (31.25, 62.5, 125, 250, 500, 1000mcg/0.05 ml). 1 N HCl was used to adjust the pH to 6.3. The samples were incubated for 20 min at 37˚C and heated for 3 mins (57˚C). Cool the sample, add 2.5 ml of phosphate buffer. Turbidity produced was measured at 416 nm [11]. For test control, 0.05 ml distilled water was added. Bovine serum albumin was not present in the Product control [4]. The percentage inhibition of protein denaturation was given by

Control indicates 100% protein denaturation. Diclofenac sodium was taken as standard.

Statistical analysis

Experiments were carried out in triplicate and results are given as the mean±S.D. deviation. All the data in all tests were analyzed using Microsoft Excel 2007.

RESULTS AND DISCUSSION

Anti-oxidant activity

DPPH scavenging activity

DPPH assay is rapid, easy and cost effective method to measure an antioxidant effect, which involves the use of the free radical [12], 2, 2-Diphenyl-1-picrylhydrazyl (DPPH). DPPH is commonly used to test the ability of compounds to act as free radical scavengers or hydrogen donors and it is based on the ability of DPPH to decolorize in the presence of antioxidant [5, 13]. Methanolic extract of Caulerpa racemosa showed a very good anti-radical activity in scavenging DPPH radical and showed a maximum % inhibition of 88.87±1.05% at 2000μg/0.1 ml concentration. DPPH scavenging activity of Caulerpa racemosa is shown in table 1.

Table 1: DPPH scavenging activity of Caulerpa racemosa

Concentration

(μg/0.1 ml)

Percentage inhibition (%)
Caulerpa racemosa
2000 88.87±1.05
1000 77.96±1.40
500 68.84±0.80
250 67.23±1.40
125 62.59±0.64
62.5 54.37±0.45
31.25 10.40±0.57

μg/ml: Microgram/milliliter

Nitric oxide scavenging activity

Nitric oxide scavenging activity is based on the inhibition of nitric oxide radical generated from sodium nitroprusside, which interacts with oxygen to produce nitrate ions and measured colorimetrically by the use of griess reagent [8, 14]. The methanolic extract of green algae confirmed the scavenging of nitric oxide radical, formed as a result of addition of sodium nitroprusside. NO scavenging activity was found to be increased with increase in concentration. It showed the maximum inhibition of 80.49±1.43% at 2000μg/ml. NO scavenging activity may be due to the anti-oxidative effects of flavonoids and tannins. Nitric oxide scavenging activity Caulerpa racemosa is shown in table 2.

Total anti-oxidant activity

Total anti-oxidant activity was assayed by Phosphomolydenum method through formation of Phosphomolydenum complex [15]. The assay is based on the reduction of Mo (VI) to Mo (V) by the sample analyte and the subsequent formation of green phosphate/Mo (V) complex at acidic pH [16, 17]. It is a quantitative method since the total antioxidant activity is expressed as the number of equivalents of ascorbic acid [18]. Total anti-oxidant activity of Caulerpa racemosa was found to be 32μg equivalents of ascorbic acid. From the results obtained, it is clear that the methanolic extract of Caulerpa racemosa possess free radical scavenging activity through total anti-oxidant activity. The total anti oxidant activity of Caulerpa racemosa is given in table 3.

Table 2: Nitric oxide scavenging activity Caulerpa racemosa

Sample Concentration (μg/ml) Percentage inhibition (%)
Caulerpa racemosa 2000 80.49±1.43
1000 75.69±1.18
500 71.06±0.78
250 61.44±1.01
125 60.94±0.81
62.5 20.7±0.41
Quercetin 200 36.48±1.45
100 21.99±0.82
50 11.48±0.59

Table 3: Total anti oxidant activity of Caulerpa racemosa

Sample Concentration (μg/ml) Absorbance
Ascorbic acid 2000 0.0475±0.0001
1000 0.0462±0.0011
500 0.0426±0.0032
250 0.0406±0.0005
125 0.0275±0.0007
62.5 0.0267±0.0006
31.25 0.0233±0.0005
Caulerpa racemosa 500 0.0151±0.0008

Table 4: Effect of Caulerpa racemosa on reductive ability

Concentration (μg/0.05 ml) Percentage inhibition (%)
Caulerpa racemosa
2000 88.80±0.98
1000 79.70±0.99
500 74.582±1.0
250 65.44±0.65
125 56.41±0.89
62.5 43.37±0.34
31.25 6.64±0.26

Table 5: In vitro anti arthritic activity

Concentration (μg/0.05 ml) Percentage inhibition (%)
Caulerpa racemosa
1000 49.33±0.597
500 44.91±0.805
250 43.36±0.405
125 18.87±0.341
62.5 18.01±0.208
31.25 14.33±0.575

Determination of reducing power

The reducing capacity of a compound may serve as a vital recorder of its potential anti-oxidant activity. For the estimation of reductive ability, transformation of Fe3+-Fe2+was investigated. The change in the optical density of the mixture is measured at 700 nm [9]. An increase in optical density indicates higher reductive ability [10]. Thus, the results obtained revealed reductive ability of Caulerpa racemosa and it was found to be increased with rise in concentration. Table 4 shows the reductive ability of Caulerpa racemosa

Anti-arthritic activity

The methanolic extract of Caulerpa racemosa produced a significant inhibition of protein denaturation and its effect was compared with standard Diclofenac sodium. It showed maximum percentage inhibition of 49.33±0.597% at 1000mcg/0.05 ml. Denaturation of proteins and production of autoantigens is the major cause of rheumatoid arthritis [4]. From the results obtained, it can be stated that the methanolic extract of Caulerpa racemosa is capable of controlling the production of autoantigens and thereby inhibit the denaturation of proteins. The in vitro anti arthritic activity of Caulerpa racemosa is given in table 5.

CONCLUSION

The in vitro activity of methanolic extract of Caulerpa racemosa revealed that it has potential dose dependent anti-oxidant and anti-arthritic properties. Steroids, triterpenoids, alkaloids, tannins, glycosides, carbohydrates, flavonoids and proteins are present in Caulerpa racemosa and these might be responsible for bringing out its potentiality in having these properties. The results obtained are encouraging to carry out the structural exploration of the anti-oxidant and anti-arthritic components present in them. Further studies are required to signify the mechanism of action of these pharmacological activities and to mark them out for their significant pharmacological actions.

ACKNOWLEDGEMENT

I express my heartfelt gratitude to the Department of pharmacology, Faculty of Pharmacy and University management for providing all necessary facilities to carry out the work.

CONFLICTS OF INTERESTS

Declared None

REFERENCES

  1. Heo SJ, EJ Park, KW Lee, YJ Jeon. Antioxidant activities of enzymatic extract from brown sea weeds. Bioresour Technol 2005;96:1613-23.
  2. Heinz Lullmann, Albrecht Ziegler, Klaus Mohr. Color atlas of pharmacology. 2nd ed; 2000. p. 320.
  3. Goodman, Gilman. Manual of Pharmacology and Therapeutics. McGraw-Hill companies; 2008. p. 54.
  4. L Shravan Kumar N, Kishore G, Siva Kumar G, SindhuPriya ES. In-vitro anti-inflammatory and anti-arthritic activity of leaves of Physalis angulata. Int J Pharm Ind Res 2011;1(03):211-3.
  5. https://seaweedindustry.com/seaweed/type/caulerpa-racemosa
  6. Blois Ms. Antioxidant determination by the use of stable free radical. Nature 1958;181:1199-200.
  7. Sanchez Moreno C, Larraurij A, Saura-calixto FA. Free radical scavenging capacity of selected of selected red rose and white wines. J Sci Food Agric 1990;79:1301-4.
  8. R Lavanya, S Umamaheshwari, G Harish, J Bharath Raj, S Kamali, D Hemamalini, et al. Uma Maheshwara Reddy. In-vitro anti-oxidant studies of Anisomeles malabarica and Coldenia procumbene L. Res J Pharm Biol Chem Sci 2010;1(4):737-44.
  9. Polterait O. Antioxidants and free radical scavengers of natural origin. Curr Org Chem 1997;1:415-40.
  10. Repetto MG, Liesuy SF. Antioxidant properties of natural compounds used in popular medicine for gastric ulcer. Braz J Med Biol Res 2002;35:523-34.
  11. Satoskar RS, Bhandarkar SD, Anipure SS. Pharmacology and pharmacotherapeutics, In, Pharmacotherapy of gout. 18th edi. Rheumatoid arthritis and osteoarthritis; 2003. p. 1022-7.
  12. Richaupadhyay, Jitendra kumar chaurasia, Kavindra Nath Tiwari, Karuna Singh. Antioxidant property of aerial parts and root of phyllanthus fraternus webster, an important medicinal plant. Scientific World J 2014;2014:2-5.
  13. MR Saha, SMR Hasan, R Akter, MM Hossain, MS Alam, MA Alam, MEH Mazumder. In vitro free radical scavenging activity of methanol extract of the Leaves of mimuso pselengi linn. Bangl J Vet Med 2008;6(2):197–202.
  14. Palash Mandal, Tarun Kumar Mishra, Mitali Ghosal. Free radical scavenging activity and phytochemical analysis in the leaf and stem of Drymaria diandra Blume. Int J Integr Biol 2009;7(2):80-4.
  15. Mediacano A, Alcaraz O, Arnao MB. Free radical scavenging activity of indolic compounds. Anal Bioanal Chem 2003;376:33-7.
  16. Oyaizu M. Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine. Japan J Nutr 1986;44(6):307–15.
  17. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of Vitamin E. Anal Biochem 1999;269:337-41.
  18. Raghu KL, Ramesh CK, Srinivasa TR, Jamuna KS. Total antioxidant capacity in aqueous extracts of some common vegetables. Asian J Exp Biol Sci 2011;2(1):58-62.
  19. S Banerjee, A Chanda, A Ghoshal, R Debnath, S Chakraborty, R Saha, et al. Nitric oxide scavenging activity study of ethanolic extracts of from two different areas of kolkata ixora coccinea. Asian J Exp Biol Sci 2011;2(4):595-9.
  20. Abubakar B Aliyu, Mohammed A Ibrahim, Aliyu M Musa, Aisha O Musa, Joyce J Kiplimo, Adebayo O Oyewale. Free radical scavenging and total antioxidant capacity of root extracts of Anchomanes difformis Engl. (araceae). Acta Pol Pharm 2013;70(1):115-21.