INTRODUCTION:

Free radicals alters the form and nature of several biological molecules which leads to the origin of lifestyle related, ageing and chronic diseases in the form of oxidative stress.¹

Oxidative stress is prominent in pathogenesis of several cardiac diseases and neurodegenerative diseases like Alzheimer’s disease (AD), Parkinson’s disease etc.² Reactive oxygen species (ROS) responsible for oxidative stress are produced enormously due to endogenous and exogenous stimuli leads to mitochondrial dysfunction and cell death.³ Adding to these inflammation is also one of the evident process of oxidative stress, the formation of inflammation mediators, such as interleukins and adhesion molecules are having significant role in diseases like diabetes, neurodegenerative diseases, arthritis and cardiovascular diseases. Inhibition of inflammation is important in treating such diseases.⁴ Dietary phytochemical antioxidants like phenolic and polyphenolic compounds, such as flavonoids and catechins exhibit potent antioxidant activities and are capable of removing free radicals like superoxide and hydroxyl radicals and inhibit oxidative modification of low density lipoprotein. Dietary supplementation of polyphenols decrease serum concentrations of total cholesterol and malondialdehyde and increase serum concentrations of high density lipoprotein in humans. Bioactive compounds with multichannel ability to reduce the free radicals formation, with antioxidant capacity and membrane stabilizing potential is of great use in treating most of diseases.⁵ Seaweeds are rich in bioactive compounds like sulfated polysaccharides, phlorotannins and diterpenes which are benefit for both human and animal health applications and are considered to be a rich source of antioxidants.⁶ The potential antioxidant compounds like fucoxanthin, astaxanthin, carotenoid and polyphenols like phenolic acid, flavonoid, tannins are widely distributed in seaweeds and are known to exhibit antioxidative activities via reactive oxygen species scavenging activity and the inhibition of lipid peroxidatio.^7-10Turbinaria ornata, the spiny leaf seaweed has been studied for its anti-oxidant, antiulcer, wound healing and hepatoprotective activities.¹¹ They are also the key raw materials in the production of Algin (Sodium Alginate) used widely in manufacturing industries like textiles, food, medicine, etc. in the present study the preliminary photochemical screening, free radicals scavenging activity, anti-inflammatory activity and anti-hemolytic activity of Turbinaria ornata have been investigated in search of new drug from natural bioactive compounds.

MATERIALS AND METHODS:

Chemicals:

Hydrogen peroxide, 2, 2-diphenyl-1-pricrylhydrazyl (DPPH), gallic acid, and ascorbic acid were obtained from Himedia laboratory Ltd., Mumbai, India. Dichlofenac sodium was purchased from Sigma Aldrich, Bangalore and all other chemicals were of reagent grade and organic solvents were of spectral grade.

Collection of samples:

The marine brown alga Turbinaria ornata was collected by hand picking from the intertidal waters of the Mandapam coast (Longitude 78⁰ 8’E, Latitude 9⁰ 17’ N) in the Gulf of Mannar during the early hours in the month of May 2015. The algal material was identified and authenticated in BSI coimbatore, Tamilnadu and a voucher specimen was maintained in our research Laboratory (BSI/SRC/5/23/2015/Tech./1304), collected algal material was washed with sea water and then with fresh water and freed from sand, salts and epiphytes.

Preparation of Seaweed Extracts:

The dried seaweed samples (25 g) were milled and extracted using 250 ml of various solvents such as ethanol, methanol and aqueous for 24 hours by using Soxhlet apparatus. Each filtrate was concentrated to dryness under reduced pressure using rotary evaporator. The samples were lyophilized by using freeze dryer (Lark, Penguin Classic Plus, India) and stored in a refrigerator at 2-8^∘ C for use in subsequent experiments.

Preliminary phytochemical Screening:

Preliminary phytochemical screenings of three different extracts were carried out as per the standard protocols of Harborne¹².

FTIR analysis of TOME:

Fourier transform infrared spectroscopy (FTIR) analysis was done with the solid samples of TOME¹³. TOME (10mg) were mixed with 100mg of dried potassium bromide (Kbr) and compressed to prepare as a salt disc. The disc was then read spectrophotometrically (Bio-Rad FTIR-40, USA). The frequencies of different components present in each sample were analyzed. The FTIR spectrum of TOME was compared with the standard gallic acid FTIR spectrum which was determined previously.

Total antioxidant activity:

The antioxidant activity of the seaweed extract was evaluated by the phosphomolybdenum method¹⁴. The assay is based on the reduction of molybdenum by the extract and subsequent formation of a green complex at acidic pH. 2 ml of extract was combined with 1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4mM ammonium molybdate), methanol was used in place of sample as blank. The tubes containing the reaction solution were capped and incubated in water bath at 95⁰ for 90 minutes. After the samples were incubated at room temperature, the absorbance of the solution was measured at 635 nm using a spectrophotometer against blank. The antioxidant activity was expressed as an equivalent of gallic acid (mg GA/g dried extract). All the measurements were measured in triplicates.

In vitro Free Radical Scavenging Assay:

TOME was screened for its free radical scavenging and antioxidant potential by the following assays 2, 2-diphenyl-1-pricrylhydrazyl (DPPH) scavenging assay ¹⁵, reducing power assay ¹⁶, Nitric oxide (NO) scavenging assay¹⁷, hydrogen peroxide scavenging assay ¹⁸ and superoxide scavenging assay¹⁹. All these assays were carried out in triplicates and compared with ascorbic acid as a standard.

Antihemolytic study on human RBC:

To determine the effect of TOME on H₂O₂ induced hemolysis in the light of the previous study²⁰ with slight modifications, the following sets of spectrophotometric tubes (each four tubes) prepared:

Group I:

Control tubes:

Theses tubes were contained 1.0 ml RBC suspension. Final volume was made to 4.0ml with normal saline

Group II:

TOME tubes :

1ml (100µg/ml) TOME was mixed with 1.0ml RBC suspension .Final volume was made to 4.0ml with normal saline

Group III :

H₂O₂ treated tubes :

8mM H₂O₂ solution (0.5ml) was mixed with 1.0ml RBC suspension and the final volume was made to 4.0 ml with normal saline.

Group IV:

H₂O₂ and TOME treated tubes:

1 ml of RBC suspension treated with 0.5ml of 8mM H₂O₂ solution and TOME (100µg/ml concentration) of 0.5ml, 0.75ml and 1ml, final volume was made to 4.0ml with normal saline. All the tubes were incubated at 37^oC for 4 hr with intermittent shaking. Absorbance of the supernatants were obtained after centrifuging the incubated tubes at 1000rpm for 10 minutes and read spectrophotometrically at 540 nm.

Percentage of hemolysis was calculated by the formula given below

Percentage of hemolysis=

Absorbance of individual tubes

---------------------------------------- x 100

Absorbance with 100% hemolysis

In vitro anti-inflammatory activity on human RBC membrane stabilization method:

The human RBC membrane stabilization has been used as a method to study the anti inflammatory activity ^[21]. Blood was collected from healthy volunteers. The principle involved here is stabilization of human red blood cell membrane by hypotonicity induced membrane lysis. The collected blood was mixed with equal volume of sterilized Alsever solution (2 % dextrose, 0.8 % sodium citrate, 0.05% citric acid and 0.42 % sodium chloride in water). The blood was centrifuged at 3000 rpm and packed cells were washed with isosaline (0.85%, pH 7.2) and a 10 % v/v suspension was made with isosaline. The assay mixture contains 1 ml phosphate buffer [pH 7.4, 0.15 M], 1 ml hypo saline [0.36%], 1 ml HRBC suspension [ 10 % v/v] with 1 ml of plant extracts of various concentrations (62.5, 125, 250 µg/ ml), standard drug diclofenac sodium (62.5, 125, 250, 500µg/ml) and control [distilled water instead of hypo saline to produce 100% hemolysis] were incubated at 37^o c for 30 min and centrifuged respectively. The hemoglobin content in the suspension was estimated using spectrophotometer at 560nm. The percentage hemolysis produced in the presence of distilled water taken as 100%. percentage of HRBC membrane stabilization or protection was calculated using the formula

Anti-inflammatory activity =

100- (Test OD/Control ODx100)

RESULTS AND DISCUSSION:

Phytochemical analysis of methanolic extract of Turbinaria ornata:

Table 1 shows the phytochemicals present in the aqueous, methanolic and ethanolic extracts of Turbinaria ornata. Methanolic extract of Turbinaria ornata (TOME) constitutes carbohydrates, alkaloids, saponins, phenolic compounds, flavonoids, tannins, coumarines, steroids and terpenoids. Our findings are supported by the earlier study,²² therefore TOME was chosen for further invitro free radical scavenging effects.

Table 1. Phytochemical analysis of Turbinaria ornata extracts in different solvents

S. No	Phytochemicals	Aqueous	Ethanol	Methanol
1	Carbohydrates	-	-	+
2	Alkaloids	+	-	+
3	Saponins	-	-	+
4.	Phenolic compounds	-	+	+
5.	Flavonoids	-	+	+
6.	Tannins	-	+	+
7.	Coumarines	-	+	+
8.	Proteins	-	-	-
9.	Steroids	-	+	+
10.	Anthroquinones	-	-	-
11.	Terpenoids	-	+	+

FTIR analysis of TOME:

Fig.1 depicts the FTIR analysis of TOME. The FTIR analysis of the samples was done and the associated functional groups were determined, the FTIR spectrum of the TOME sample showed the peaks at range of 3886.27, 3854.88, 3844.25, 3752.03, 3333.78, 2918.03, 1605.92, 1415.39 and 1230.54 which are near similar to the FTIR spectrum of standard gallic acid showed thirteen major peaks at the range of 3 407.05, 2 960.40, 2 931.51, 2 865.75, 1 639.70, 1 509.58, 1 420.99, 1 153.78, 1 097.08, 777.58, 663.54, 601.28, 462.89cm-1 in a different study13. The large absorption peak at 3752.03 - 3886.27 cm-1 was C-H stretching vibration of alkenes and asymmetrical O-H stretching absorption band at range of 3752.03 cm-1 was found to be OH stretching. Absorption peak around 3333.78 was N-H stretching of hetero aromatic compounds. C-H stretching peak of methyl group (Alkanes) was around 2918.03 cm-1. FTIR spectrum strongly elucidates the presence promising bioactive compounds in TOME.

Fig.1 FTIR Spectrum of TOME

Table 2. In vitro free radical scavenging assay of TOME

Concentration of TOME/Ascorbic acid µg/ml	NO scavenging capacity (%)	H₂O₂ scavenging capacity (%)	SOD scavenging capacity (%)	NO scavenging capacity of Ascorbic acid (%)	H₂O₂ scavenging capacity of Ascorbic acid (%)	SOD scavenging capacity of Ascorbic acid (%)
10	22.46±2.24	28.03±3.76	14.03±3.28	26.16±1.11	32.09±2.67	16.11±1.21
20	28.46±1.21	34.46±4.20	28.16±3.88	33.16±2.18	38.60±2.94	32.10±2.71
30	35.22±2.18	39.22±4.50	32.12±4.18	39.11±3.22	43.11±3.21	39.13±2.15
40	41.65±2.71	41.65±4.93	49.25±4.77	44.35±1.21	51.62±3.47	52.35±2.27
50	47.27±5.09	53.57±5.25	53.33±5.09	52.10±2.19	59.13±2.12	61.31±2.14
60	53.14±5.43	58.80±5.74	62.45±5.43	57.24±1.23	64.64±2.01	67.18±2.32
70	58.11±3.24	65.18±6.17	69.11±2.69	61.22±1.20	70.92±2.12	71.18±3.19
80	62.32±4.11	71.60±6.61	78.46±6.21	67.12±1.21	77.43±2.17	82.21±5.21
90	68.25±2.37	83.65±6.93	81.58±4.77	74.13±1.25	89.93±3.11	84.65±3.16
100	77.37±5.27	89.57±7.33	86.23±3.21	79.37±3.11	97.20±2.18	89.57±7.17

Total antioxidant activity and free radical scavenging activity of TOME:

Fig. 2 shows the total antioxidant activity of TOME by phosphomolybdenum method, TOME at the concentration of 100µg shows 89.11% of total antioxidant activity as compared with the standard gallic acid which is supported by the previous report.²³ Fig. 3 and Fig. 4 shows the DPPH radical scavenging and reducing power of TOME respectively. DPPH is a stable radical and is widely used to evaluate antioxidant activities of bioactive substances. The antioxidant activity of a samples was carried out by measuring the absorbance of DPPH radical in the sample at 517 nm as against to that of control. The scavenging ability of TOME was concentration dependent. The TOME shows the greatest DPPH antioxidant activity (~80%) at 100 µg/ml, the reducing power was evaluated in the samples on the basis of their abilities to reduce Fe (III) complex to Fe (II). Greater the absorbance value, higher is the reducing power of the TOME.²⁴TOME having appreciable DPPH radical scavenging activity and reducing power as the standard ascorbic acid. Table 2 depicts the free radical scavenging capacity of TOME and the standard ascorbic acid. Concentrations of TOME were chosen as per the earlier report,²⁵with slight modifications. The antioxidant properties of phenolics are as a result of their ability to act as reducing agents, hydrogen donors, and free radical quenchers. Phenolics can also act as metal chelators which prevent the catalytic function of metal in the process of initiating radicals. Also, it is reported that Turbinaria ornata have high phenolic content which is responsible for their respective antioxidant activity. The free radicals NO, H₂O₂ and SOD scavenging activity are increased with increase in the concentration of TOME and the results are well supported by the previous study²³.

Fig.2 The total antioxidant activity of TOME

Fig.3 The DPPH free radical scavenging activity of TOME

Fig.4 The reducing power of TOME

Anti-hemolytic activity of TOME on human RBC:

The anti-hemolytic activity of TOME on human RBC hemolysis is depicted in Table 3. The rupturing of RBC cells is known as hemolysis. Hemolysis is an indicator of cytotoxicity. Hydrogen peroxide affects the integrity of the erythrocyte membrane. Thus Group III H₂O₂ treated shows high level hemolysis compared to control Group I. Group II TOME treated RBC models shows no significant hemolysis compared to Group III H₂O₂ treated which is similar to the earlier different study²⁶. Whereas Group IV treated with different concentrations of TOME (0.5 mg/ml, 0.75 mg/ml, 1mg/ml) and H₂O₂shows significant reduction in hemolysis compared to Group III H₂O₂ treated. TOME maintains the stability of human red blood cell membrane and also inhibits the hydrogen peroxide assault on RBC membrane.

Anti-inflammatory activity of TOME:

Inflammation is the primary defense mechanism but uncontrolled inflammation in chronic diseases leads to adverse effects, anti-inflammatory action is one of the important diseases treating strategy. Table 4 shows the anti-inflammatory activity of TOME in different concentrations of 62.5, 125, 250 and 500 µg/ml. Increase in TOME concentrations increased the human RBC membrane stabilization, the higher concentration level (500µg/ml) shows about 81 % of anti-inflammatory activity compared to 100% lysis induced in control (data not shown), Which is significant as the standard Diclofenac, as similar to the earlier report.²⁷ Since, the human red blood corpuscle (RBC) membrane is similar to lysosomal membrane components, the prevention of hypo tonicity induced human RBC membrane lysis is taken as a measure of anti-inflammatory activity of drug. Human RBC membrane stabilization by TOME reveals the anti-inflammatory activity of TOME.

Table 3. Anti hemolytic activity of TOME on human RBC

S. No	RBC Suspension (5X10⁸RBC/ml)	H₂O₂Concentration	TOME 1mg/1ml	Hemolysis %
Group 1	1 ml	0.0	0.0	0.11±0.01
Group 2	1 ml	0.0	1 ml	0.12±0.02
Group 3	1 ml	8mM	0.0	93.75±7.50
Group 4	1 ml 1 ml 1 ml	8mM 8mM 8mM	0.5 ml 0.75 ml 1.00 ml	62.49±5.0 32.47±2.60 20.83±1.67

Table 4. Anti inflammatory effect of TOME by human RBC membrane Stabilization

S. NO	RBC suspension 5X10⁸RBC/ml	Concentration of TOME (µg/ml)	Percentage of human RBC membrane stabilization by TOME	Diclofenac sodium (µg/ml)	Percentage of human RBC membrane stabilization by Diclofenac sodium
1	1	62.5	51.00±4.08	62.5	64.80±5.18
2	1	125	68.00±5.44	125	77.0±6.16
3	1	250	72.23±5.78	250	86.00±6.88
4	1	500	81.13±2.18	500	93.17±2.23

CONCLUSION:

In the present study preliminary phytochemicals, antioxidant analysis of methanolic extract of Turbinaria ornata leaves have been done and investigated further for its anti-hemolysis and anti-inflammatory properties in RBC model of blood collected from healthy volunteers of 22-25 years age. The phytochemical analysis elucidates the presence of significant bioactive compounds in the methanolic extract of Turbinaria ornata, similarly the FTIR analysis also denotes the similarity of TOME with gallic acid which is a potent antioxidant. Thus, Turbinaria ornata with potent bioactive compounds shows appreciable antioxidant activity prevents H₂O₂induced hemolysis in human RBC model. The anti-inflammatory activity of Turbinaria ornata is revealed by its ability of HRBC membrane stabilization as much as appereciable to standarad dichlofenac. Further invivo studies, NMR assay, drug leads and molecular level investigations will be done to bring out the medicinal value of the Turbinaria ornata in treating different diseases due to the assault of free radicals and inflammation.

ACKNOWLEDGEMENT:

The authors are Thankful to Dr. T. Manivasagam, Department of Biochemistry and Biotechnology, Annamalai University and Rev. Jacob Memorial Christian College for necessary assistance.

CONFLICT OF INTEREST:

Author declared no Conflict of interest.

REFERENCES:

1. Usha V, Suriyavathana M. Free radical scavenging activity of ethanolic extract of Desmodium gangeticum. Journal of Acute Medicine. 2012; 2:36-42

2. Klein JA, Ackerman SL. Oxidative stress, cell cycle, and neurodegeneration. J Clin Invest. 2003; 111:785-793.

3. Nusrat Jahan, Shahnaj Parvin Mst., Nandita Das et al., Studies on the antioxidant activity of ethanol extract and its fractions from Pterygota alata leaves. Journal of Acute Medicine. 2014; 4:103-108.

4. Parameswari G, Suriyavathana M. Evaluation of total phenolic content and free radical scavenging activity of Boerhavia erecta. Journal of Acute Medicine. 2013; 3:103-109

5. Yun-Zhong Fang, Sheng Yang, and Guoyao Wu. Free Radicals, Antioxidants, and Nutrition. Nutrition. 2002; 18:872- 879.

6. Cahyana AH, Shuto Y, Kinoshita Y. Pyropheophytin a as an antioxidative substance from the marine alga, Arame (Eisennia bicyclis). Biosci Biotechnol Biochem. 1992; 56:1533-1535.

7. Yoshie Y, Wang W, Petillo D, Suzuki T. Distribution of catechins in Japanese seaweeds. Fish Sci. 2000; 66:998-1000.

8. Athukorala Y, Lee KW, Song CB, Ahn CB, Shin TS, Cha YJ, Shahidi F, Jeon YJ. Potential antioxidant activity of marine red alga Grateloupia filicinaextracts. J Food Lipids. 2003; 10: 251-265.

9. Heo SJ, Park EJ, Lee KW, Jeon YJ. Antioxidant activities of enzymatic extracts from brown seaweeds. Biores Technol. 2005; 96: 1613-1623.

10. Siriwardhana N, Lee KW, Kim SH, Ha JW, Jeon YJ. Antioxidant activity of Hizikia fusiformison reactive oxygen species scavenging and lipid peroxidation inhibition. Food Sci Technol Int. 2003; 9:339-346.

11. Kulandhaisamy, Arul Senthil, Annappan Murugan. Antiulcer, wound healing and hepatoprotective activities of the seaweeds Gracilaria crassa, Turbinaria ornata and Laurencia papillosafrom the southeast coast of India. Brazilian Journal of Pharmaceutical Sciences. 2013; 49:669-678.

12. Harborne. Methods of extraction and isolation, in Phytochemical Methods. 3^rd ed. PA: Chapman&Hall; 1998.

13. Selvaraju Meenakshi., Shanmugam Umayaparvathi., Muthuvel Arumugam., and Thangavel Balasubramanian., In vitroantioxidant properties and FTIR analysis of two seaweeds of Gulf of Mannar. Asian Pacific Journal of Tropical Biomedicine., (2012) S66-S70

14. Prieto CD, Calligaris S, Celloti E, Nicoli MC. Antiradical properties of commercial cognacs assessed by the DPPH test. J of Agri Food Chem. 1999; 48: 4241- 4245.

15. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958; 181:1199-1200.

16. Yen GC, Duh PD, Chuang DY. Antioxidant activity of anthroquinones and anthrone. Food Chem. 2000; 70:437-441.

17. Gulcin I. Antioxidant and antiradical activities of L- Carnitine. Life Sciences. 2006; 78:803-811.

18. Gulcin T, Irfan KO, Kufrevioglu M, Oktay M, Buyukokuroglu ME. Antioxidant, antimicrobial, antiuclcer and analgesic activities of nettle (Urtica dioica L.). J. Ethnopharmacol. 2004; 90:205-215.

19. Nishimiki M, Rao NA, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun. 1972; 46:849-853.

20. Neeta Mathuria, Ramtej Jayram Verma. Aflatoxin induced hemolysis and its amelioration by turmeric extracts and curcumin in vitro. Acta Poloniae Pharmaceutica - Drug Research. 2007; 64:165-168.

21. Gandhidasan R, Thamaraichelvan A, Baburaj S. Anti inflammatory action of Lannea coromandelica by HRBC membrane stabilization. Fitoterapia. 1991; 62:81-83.

22. John Peter Paul J, Shridevi S.D.K. Preliminary phytochemical analysis of Sargassum myriocystum J.Ag. and Turbinaria ornata (Turner) J.Ag. from the South-east coast of Tamil Nadu. India Journal of Biochemical and Pharmaceutical Research. 2013; 2:37-43.

23. Vijayabaskar P, Shiyamala V. Antioxidant properties of seaweed polyphenol from Turbinaria ornata (Turner) J. Agardh, 1848. Asian Pacific Journal of Tropical Biomedicine. 2012; S90-S98.

24. Dharmesh R Chejara, Stalin Kondaveeti, Ramavatar Meena, Siddhanta AK. Antioxidant activity and phytochemical analysis of a few Indian seaweed species. Indian Journal of Geo-Marine Sciences. 2014; 43:507-518.

25. Kayalvizhi, Vasuki Subramanian, Sithranga Boopathy, Kathiresan. Antioxidant properties of brown seaweeds (Turbinaria ornata (Turner) J.Agardh, 1848 and Padina tetrastromatica (Hauck). Journal of Biotechnological Sciences. 2014; 2:29- 37.

26. Lakshmi G, Smitha N, Ammu S V, Priya C L, Bhaskara rao KV. Phytochemical profile, In Vitro antioxidant and hemolytic activities of various leaf extract of Nymphaea nouchali linn: an in vitro study. Int j pharm pharm sci. 2014; 6:548-552.

27. Ananthi, Gayathri, Chandronitha, Lakshmisundaram, Hannah. Free radical scavenging and anti-inflammatory potential of a marine brown alga Turbinaria ornata (Turner) J.Agardh. Indian Journal of Geo-Marine Sciences. 2011; 40:664-670.

Received on 22.05.2017 Modified on 30.05.2017

Research J. Pharm. and Tech. 2017; 10(7): 2243-2248.

DOI: 10.5958/0974-360X.2017.00397.3