INTRODUCTION:

Cyclooxygenase is the predominant Inflammatory enzyme is induced in mast cell after exposure to cytokines, particularly IL-1β which catalyzed the synthesis of prostaglandin E_{2 ,}which plays a key role in inflammation and its associated diseases such as cancer, arthritis and diabetes¹. Thus, at this site inhibition of COX-2, an inflammatory key enzyme is an appropriate target².

There has been a noticeable spurt in recent times in the number of studies aimed at characterizing the health-promoting properties of many phenolic compounds which exhibit antioxidant properties. They are useful in the treatment and management of cancer, cardiovascular and neurodegenerative diseases or for use in anti-aging or cosmetic products. Phenolic compounds are ubiquitous in plants which collectively synthesize several thousand different chemical structures and are characterized by hydroxylated aromatic ring(s)³. In the past decade, there has been a resurgence of interest in bioactive components of natural origin. Most of the anti inflammatory activities of plants from Indian System of Medicine (ISM) were attributed to their bio active marker. Herbs of ISM have been used in Asia for thousands of years to treat inflammatory conditions for variety of diseases and symptoms including RA and related diseases⁴. It has been reviewed that natural polyphenols have been found exert its anti-inflammatory effects either by scavenging free radicals and decreasing formation of reactive oxygen species⁵ or by modulation of pro-inflammatory gene expression of cyclooxygenase, Lipooxygenase and cytokines mainly by acting through Nuclear factor – kappa B and Mitogen Activated Protein Kinase signaling⁶. This accounts for markedly reduced occurrence of gastric acidity with the use of selective inhibitors of COX-2. Which are needed in the present scenario for treating inflammatory problems? Some evidence showing a greater effectiveness of phytoextracts and its fractions compared with isolated compounds has lately moved the attention towards bioguided fraction from natural source⁷. .With this information we have selected the plants Terminalia chebula [fruits], Sida rhombifolia [root] and Leucas cephalote [entire plant].from Indian System of Medicine for the present work. The above said plants are used in Ayurveda for treatment of inflammatory associated diseases such as gout, rheumatism, arthritis, wounds and ulcer⁸ and are included in the official monograph.

MATERIALS AND METHODS:

The selected plant materials were collected from in and around places of Chennai, Tamil nadu and authenticated by renowned botanist and its record, voucher specimen, herbarium was maintained and deposited in the Department of Pharmacognosy, School of Pharmaceutical Sciences, and VELS University.

Isolation of Tannin fraction:⁹

The sample Terminalia chebula fruits were extracted with acetone: water mixture (70:30) by cold maceration method. The solvent was filtered. The filtrate was treated with a saturated sodium chloride solution and a few ml of 0.1% ascorbic acid. After that filtrate was dissolved in petroleum ether, petroleum ether layer was removed and aqueous layer was concentrated under vacuum tested for tannins.

Test for Tannins:¹⁰

A pinch of dried extract was dissolved in water, was treated with gelatin solution. A white precipitate was formed.

Isolation of flavonoid fraction:

The dried roots sida rhombifolia was separately extracted with 80% methanol in soxhlet for 24 hrs. The extract was concentrated in petroleum ether (40°- 60°C) (fraction-I), diethyl ether (fraction-II) and ethyl acetate (fraction-III) in succession. Each of the steps was repeated three times to ensure complete extraction in each case. Fraction I and II were rejected since it was rich in fatty substances whereas fraction III was analysed for the free flavonoids.

Fraction III was hydrolyzed by refluxing with 7% H₂SO₄ (10 ml/gm residue) for 5 hours. The mixture was filtered and the filtrate was extracted with ethyl acetate in a separating funnel. The ethyl acetate layer was separated and washed with distilled water till neutrality and dried in vacuum. The yellow amorphous residue obtained was tested for flavonoid.

Test for flavonoids:¹⁰

Shinoda test:

A pinch of dried extract was dissolved in ethanol, mixed thoroughly and filtered. The filtrate was treated with magnesium metal pieces and concentrated hydrochloric acid and heated gently.

Appearance of magenta colour indicates the presence of flavonoids.

· Test with Sodium Hydroxide solution:

A pinch of dried extract was treated with sodium hydroxide solution.

Formation of yellow colour indicates the presence of flavones and yellow to orange colour indicates the presence of flavonones.

Isolation of steroid fraction:

Dried sample of Leucas cephalotes (Entire plant) was macerated with N-hexane for one hour and then was extracted by cold maceration method. It was filtered and concentrated under vacuum the extract thus obtained was tested for steroid.

Test for Steroid:

Liberman Burchard Test:

Two ml extract was mixed with chloroform and 1-2 ml anhydrous acetic acid then 1-2 drops concentrated sulphuric acid was added. A green colour change to blue was observed.

In Vitro Inhibition of COX-2 Enzymatic Activity:

Sample tested:

Sample1: Tanninfractionof Terminala Chebula + steroid fraction of Leucas cephaetus at 1:1 ratio.

Sample2; Tannin fraction of Terminalia chebula+ flavonoid fraction of Rubia cardifolia

at 1:1 ratio.

Method:

COX-1, COX-2, initial activity tubes were prepared taking 120µl of reaction buffer, 10µl of heme and 10µl of COX-1 and COX-2 enzymes in respective tubes. Similarly, COX-1, COX-2 inhibitor tubes were prepared by adding 20µl of inhibitor (sample) in each tube in addition to the above ingredients. The inhibitory assays were performed in the presence of sample at different concentrations (10 to 1000 µg/mL). The background tubes correspond to inactivated COX-1 and COX-2 enzymes obtained after keeping the tubes containing enzymes in boiling water for 3 min. along with vehicle control. Reactions were initiated by adding 20µl of arachidonic acid in each tube and quenched with 50µl of 1M HCl. PGH2 thus formed was reduced to PGF2α by adding 20µl TMPD. The prostaglandin produced in each well was quantified using broadly specific prostaglandin antiserum that binds with major prostaglandins and reading the 96-well plate at 590 nm. The wells of the 96-well plate showing low absorption at 590 nm indicated the low level of prostaglandins in these wells and hence the less activity of the enzyme. Therefore, the COX inhibitory activities of the combined fractions could be quantified from the absorption values of different wells of the 96-well plate¹¹. The indomethacin (selective COX-1 inhibitor) and celecoxib (selective COX-2 inhibitor) were used as positive controls in the study. The test extract concentration causing 50% inhibition of PGE₂ release (IC₅₀) was calculated from the concentration-inhibition response curve by regression analysis. The results were tabulated in Table 1.The Bioactive fractions were standardized by Spectrophotometry. Estimation, TLC and High Performance Thin Layer Chromatography Technique (HPTLC).

Table 1: In vitro percentage inhibition of Bioguided fraction for COX-1 and COX-2 enzymes by

S. No	Sample	Concen-tration (µg/ml)	% Inhibition
S. No	Sample	Concen-tration (µg/ml)	COX-1	COX-2
1	Tannin fraction: steroid fraction [1:1]	10	26.8±1.54**	40.50±0.60*
		50	20.4±0.79**	41.3±1.06**
		100	9.37±0.90**	42.37±2.04**
		500	4.43±0.20**	45.9±0.80**
		1000	1.54±0.70**	47.8±0.70**
2.	Tannin fraction: Flavonoid fraction [1:1]	10	--	50.30±.04**
		50	4.3±0.30**	53.61±1.06**
		100	10.32±0.90**	54.82±1.84**
		500	13.46±1.90**	65.31±1.04**
			15.65±2.90**	72.16±2.04**
3	Indo-methacin	30	96.8±1.08**	56.66±1.81**
4	Celecoxib	30	13.90±0.38**	93.36±0.90**

Values are mean ± SEM of 6 parallel measurements. Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s‘t’ test (n=6). **P< 0.01, when compared against control.

Estimation of tannin by UV spectrophotometry method:¹²

500 mg of tannin fraction was placed in 50 ml of distilled and shaken for 1 hr in a mechanical shaker. Into a 50 ml volumetric flask and made up to the mark. Then, 5 ml of the filtrate was pipetted out into a tube and mixed with 3 ml of 0.1m Fecl₃ in 0.1N HCl and 0.008M potassium Ferro cyanide. The absorbance was measured in a spectrophotometer at 630 nm wavelength. A blank sample was prepared and the color developed was red at the same wavelength. Standard curve was plotted using different concentrations of tannic acid as standard.

Determination of Total Flavonoids:¹³.

Total flavonoid content in the fraction was estimated by spectrometric method (Perkin-Elmer UV-Vis spectrometer Lambda 16 (Germany). The flavonoid fraction was dried over anhydrous sodium sulphate, concentrated under vacuum up to a concentration of 1 gm/ml extract. It was further diluted with ethyl acetate to obtain 0.01 gm/ml solutions. About 10 ml of the solution was transferred into a 25 ml volumetric flask, 1 ml of 2% AlCl₃ was added and the solution was made up to volume with methanol-acetic acid and was kept aside for 30 min. The absorbance was measured at 390 nm against the same solution without AlCl₃ being blank. Quercetin [Sigma Aldrich. India] was used as standard to construct the calibration curve in the concentration range 1.0-10.0 μg/ml.

Thin layer Chromatography of Tannin Fraction:

Preparation of test sample:

10 mg of test sample was dissolved in 5ml of 95% ethanol. 1μl was applied as a spot.

Preparation of standard sample:

10 mg of Gallic acid (pure sample) was dissolved in 5 ml of ethanol. 1μl was applied as a spot.

Chromatographic chambers, conditions of saturation and the development of TLC plates:

Developing solvent system:

A number of developing solvent systems were tried, but the satisfactory resolution was obtained in the solvent systems mentioned in the Table. After development of plates, they were air-dried and number of spots were noted and R_fVlaues were calculated. Spots were visualized by day light after spraying with ferric chloride solution as the detecting agen¹⁴ .The results of TLC presented in Table.2.

Thin layer Chromatography of Flavonoid Fraction:

Preparation of test sample:

10 mg of test sample was dissolved in 5ml of methanol. 1μl was applied as a spot.

Preparation of standard sample:

10 mg of Quercetin (pure sample) [Sigma Aldrich. India] was dissolved in 5 ml methanol. 1μl was applied as a spot.

Chromatographic chambers, conditions of saturation and the development of TLC plates;

Developing solvent system:

A number of developing solvent systems were tried, but the satisfactory resolution was obtained in the solvent systems mentioned in the Table. After development of plates, they were air-dried and number of spots were noted and R_fVlaues were calculated. Spots were visualized by UV light after spraying the detecting agent. The results of TLC presented in Table 2.

HPTLC:

The combined fraction was subjected to HPTLC for the conformation of the active constituents. HPTLC was performed on 10 cm × 10 cm aluminum backed plates coated with silica gel 0F254 (Merck, Mumbai, India). Standard solution of rutin, Gallic acid and sample solution were applied to the plates as bands 8.0 mm wide, 30.0 mm apart, and 10.0 mm from the bottom edge of the same chromatographic plate by use of a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a 100-μL Hamilton (USA) syringe. Ascending development to a distance of 80 mm was performed at room temperature (28 ± 2°C), with Toluene: Ethyl acetate: Formic acid (5 : 4: 1) (v/v/v), as mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapor for 20 min. After development, the plates were dried with a hair dryer and then scanned at 254 nm with a Camag TLC Scanner with WINCAT software, using the deuterium lamp (Rich and Schibli). A stock solution of standard sample (100μg/mL) were prepared in methanol Different volume of stock solution 1, 2 and 3μL, were spotted on to TLC plate to obtained concentration 100 ng, 200 ng and 300 ng/spot of rutin. Calibration curve range (100 - 300 ng/spot) for rutin was found to be linear. The data of peak areas, height and Rf were shown in Table 3. and in Figs 3 and 4

Table 3: HPTLC Peak table

Track	Sample ID	Rf	Height	Area	Assigned substance
1	Reference	0.53	63.9	488.2	Quercetin
2	Reference	0.53	61.7	478.0	Quercetin
3	Reference	0.53	58.4	430.2	Quercetin
4	Reference	0.53	57.5	420.4	Quercetin
5	Reference	0.53	55.6	390.6	Quercetin
7	Test [Flavonoid fraction]	0.52	54.1	363.6	Quercetin

RESULTS:

The chemical analysis of total tannin and total flavonoid content were 14.80% w/w and 1.2 % w/w respectively. To support of phytochemical test, the flavonoid and tannin fractions were subjected to thin layer chromatography. The tannin fraction showed very clear single blue coloured spot in Toluene: acetone: Glacial acetic acid ( 3:1:2) and 5% ferric chloride as detecting agent . The Rf value of one spot matches with the R_fvalue of standard [Gallic acid].

Whereas the flavonoid fraction showed 11 well separated spots with R_f values 0.07, 0.13, 0.15, 0.23, 0.36, 0.43, 0.53, 0.57, 0.72, 0.85, 0.94 in Toluene: Ethyl acetate: Ethanol (70%) (7: 1: 0.5) solvent system and 10 % solution of sodium hydroxide solution as detecting agent shown in Table 2. The R_f value of one spot matches with the R_fvalue of standard sample [Quercetin]. In the present investigation, flavonoids were quantified at 254 nm using peak area by comparison to a calibration curve derived from the Quercetin and Tannins were quantified at 254 nm using peak area by comparison to a calibration curve derived from the Gallic acid. The Rf value of standard Quercetin was found to be 0.53 shown in Fig.1 and its peak area was 5535.6 The tannin fraction showed seven peaks in the mobile phase Toluene: ethyl acetate: formic acid. The sixth peaks showed Rf values of 0.19 which are almost coinciding with standard Rf values. The Rf value of standard Gallic acid was found to be 0.2 shown in Fig 2 and peak area 39005.8.Results showed that tannin and steroid combined fraction showed only 47.33% of inhibition of COX -2 at 1000µg/ml concentration. IC50 value was found to be >1000 µg/ml whereas tannins and flavanoid fraction exhibitioned 72.16% of inhibition COX- 2 activity with IC50 value is <50µ.

Fig 1 Rf value of test extract compared with that of standard quercetin

Fig 3 Chromatogram of Standard Quercetin

DISCUSSION:

Many people today are using herbal medicine as complementary and alternative medicine along with conventional medicine¹⁵. As synthetic compounds may exaggerate the levels of inflammatory mediators via COX pathway, So many researchers are focusing in search of natural compound and its bioguided fraction for COX- 2 inhibitor. From the literature review it is evident that natural polyphenols such as flavonoid and tannins have been found exert its anti-inflammatory effects either by decreasing formation of reactive oxygen species⁵ or by modulation of pro-inflammatory gene expression of cyclooxygenase, Lipooxygenase and cytokines mainly by acting through Nuclear factor – kappa B (NF-kappa) and mitogen activated protein kinase signaling⁶. Earlier literature revealed that Terminalia chebula fruits, sida rhombifolia (root) Leucas cephalotes entire plant were found to contain polyphenolic compounds and steroid and are used in traditional system of medicine for inflammatory associated diseases ant its problems. The crude extract of the above plants were screened for anti-inflammatory activity in vivo method Hence the present study is focused to isolate the bioactive fraction from the above mentioned Indian Medicinal plants and screen it in combined fractions to achieve synergistic effect for inhibition of inflammatory key enzymes .Further the bioactive fraction was standardized by HPTLC and spectroscopy as per standard protocol. COX -1 is a constitutive enzyme is normally present in cells, tissues. Whereas COX -2 is an inducible enzyme, induced by mitogenic agents such as LPS, Interleukins and phorbolesters.COX -2 is responsible for the biosynthesis of prostaglandins (these are the main mediators for inflammation). So the inhibition of COX- 2 is a target for the anti inflammatory activity. In inflammatory cells IL-1 induced the synthesis of the enzyme cyclooxygenase-2 (COX-2) through the activation and translocation of the transcription factor, nuclear factor kappa (NF-κB)¹⁶. LOX products play a role in a variety of disorders such as allergies, asthma, psoriasis, and atherosclerosis; also have profound influence on the development of several human cancers¹⁷. Hence inhibitors against this group of enzymes have great potential for rational drug design and discovery in health sectors in the management of inflammatory problems. But poly phenolic compounds from plants has the ability to scavenge the free radicals to prevent the formation of COX products, blocking of inflammatory mediators are needed in the present scenario for treating inflammatory associated diseases. COX (cyclooxygenase) is a bifunctional enzyme exhibiting both COX and peroxidase activity. This assay utilizes the peroxidase component of COX. This peroxidase activity is assayed calorimetrically by observing the appearance of oxidised N, N, N’, N’-tetramethyl-p-phenyline diamine (TMPD). It is measured at 610 or 590 nm. It is mainly based on the oxidation of TMPD during the reduction of PGG2 to PGH2.More recently, human clinical trials with COX-2 inhibitor drugs have shown similar anti-inflammatory and analgesic efficacy to traditional NSAIDs, yet with significantly less gastrotoxicity¹⁸. The present investigation showed bioactive combined fraction [Tannin and Flavonoid combination in 1:1 ratio] selectively inhibited COX-2 with minimum IC₅₀ value.

CONCLUSION:

From the above study it was concluded that, the anti-inflammatory activity of combined bioactive fraction could be at least in part due to COX-2 enzyme inhibition which may be attributed to the presence of flavonoids and tannins that are known to have direct antioxidant property due to the presence of hydroxyl groups, which can function as hydrogen donor. The outcome of the present work would be exploration of traditional plants and exploitation of its phyopharmacophore in Indian system of medicine for management of inflammatory problems.

ACKNOWLEDGEMENTS:

Authors are extremely thankful to Vels University (VISTAS) and its management for providing research facilities and encouragement.

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Received on 21.08.2016 Modified on 10.09.2016

Research J. Pharm. and Tech. 2017; 10(1): 233-238.

DOI: 10.5958/0974-360X.2017.00049.X

S.No	Sample	Solvent system	Detecting agent	Number of spots	R_fValue
1.	Standard (Gallic acid )	Toluene: acetone :Glacial acetic acid ( 3:1:2)	5% Fecl₃	1	0.9
2.	Tannin Fraction	Toluene: acetone :Glacial acetic acid ( 3:1:2)	5% Fecl₃	1	0.91
3.	Flavonoid fraction	Toluene: Ethyl acetate: Ethanol (70%) (7: 1: 0.5).	Vanillin in sulphuric acid	11	0.07 - 0.94