INTRODUCTION:

India has a rich culture of medicinal herbs and spices, which includes about more than 2000 species and has a vast geographical area with high potential abilities for Ayurvedic, Unani, Siddha, traditional medicines but only few have been studied chemically and pharmacologically for their potential medicinal value ( Gupta et al., 2005).

Human beings have used plants for the treatment of diverse ailments for thousands of years. According to the World Health Organization, most populations still rely on traditional medicines for their psychological and physical health requirements, since they cannot afford the products of western pharmaceutical industries, together with their side effects and lack of healthcare facilities.

Rural areas of many developing countries still rely on traditional medicine for their primary healthcare needs and found a place in day –to-day life. These medicines are relatively safer and cheaper then synthetic or modern medicine (Ammara et al., 2009 ).

Inflammation is a reaction of living tissues towards injury and it comprises systemic and local responses. Modern medicine and the tremendous advances in synthetic drugs, a large number of the world populations (80% of people) cannot afford the products of the pharmaceutical industry and have to rely upon the use of traditional medicines, which are mainly derived from plant material. The fact is well recognized by the WHO which has recently compiled an inventory of medicinal plants listing over 20,000 species (Ejebe et al., 2010).

Inflammation is considered as a primary physiologic defense mechanism that helps body to protect itself against infection, burn, toxic chemicals, allergens or other noxious stimuli. An uncontrolled and persistent inflammation may act as an etiologic factor for many of these chronic illnesses. Although it is a defense mechanism, the complex events and mediators involved the inflammatory reaction can induce, maintain or aggravate many diseases. Currently used synthetic anti-inflammatory drugs are associated with some severe side effects. Therefore, the development of potent anti-inflammatory drugs with fewer side effects is necessary from medicinal plants origin (Mohammad et al., 2012).

Tephrosia purpurea L. belongs to family Fabaceae, commonly known as Kattu Kolingi in Tamil and Sharapunka in Sanskrit. It is one of the most important plants used in the traditional system of medicine. Roots and seeds are used an insecticidal and pesticidal. Decoction of roots given in dyspepsia, diarrhea, rheumatism, asthma and urinary disorders, and also used in elephantiasis. The roots smoked for relief from asthma and cough, decoction of pods used as a vermifuge and to stop vomiting (Kritikar and Basu, 1980) and respiratory disease and applied on leprosy and wounds (Maheshwari, 2000). Though many pharmacological works has been undertaken in T. purpurea work was subjected to anti-inflammatory study using Carrageen an induced model. In this study was aimed to evaluate in-vitro anti-inflammatory activity of T. purpurea seed extracts.

MATERIALS AND METHODS:

Collection of plant material:

The seed of Tephrosia purpurea (L) were collected during November 2015, from Therizhandur, Nagapattinum (District), Tamilnadu, India.

Extract Preparation:

The seed of Tephrosia purpurea (L) were air dried at room temperature for 3 weeks. The dried parts were later ground well to powder. 15g of plant powder materials was soaked with 150 ml of solvent in a sealed container for 3 days. Then the mixture was filtered through a Whatman no. 1 filter paper. Crude extract were obtained by evaporating the solvent in a water bath at low temperature (40-50ºC) and stored in a refrigerator at 4 ºC - 8 ºC.

Aqueous extract:

15g of seed powder was extracted with water. To one part of the plant material three parts of water was added, boiled and then reduced to one third and filtrate was evaporated to dryness. Paste from of the extract obtained was subjected to screening test.

QUALITATIVE PHYTOCHEMICAL ANALYSIS:

The preliminary chemical tests were carried out for the extracts of Tephrosia purpurea (seed) to identify the presence of various phytoconstituents.

QUALITATIVE METHOD OF PHYTO CHEMICAL SCREENING (Sofowara (1993):

The Tephrosia purpurea seed extracts were analyzed for alkaloids flavanoids, pholabatannins, glycosides, phenols, saponins, lipids and fat, tannins, anthraquinones, quinines, cardiac glycosides, coumarines, acids, steroids, phytosterols, proteins, carbohydrates etc.

Detection of Alkaloids:

About 50 mg of Solvent free extract was stirred with 3 ml of dilute hydrochloric acid and then filtered thoroughly. The filtrate was tested carefully with various alkaloid reagents as follows:

Mayer’s test:

To a 1 ml of filtrate, few drops of Mayer’s reagent are added by the side of the test tube. The white or creamy precipitate indicated test as positive.

Wagner’s test:

To a 1 ml of filtrate, few drops of Wagner’s reagent are added by the side of the test tube. The color change was observed. A reddish-brown precipitates confirms the test as positive.

Dragendorff’s test:

To a 1 ml of filtrate, 2 ml of Dragendorff’s reagent are added and the result was observed carefully. A prominent yellow precipitate confirms the test as positive

Detection of Carbohydrate:

Fehling’s test:

One ml of extract was boiled on water bath with 1 ml each of Fehling solutions A and B. The color change was observed. A red precipitates indicated presence of sugar.

Barfoed’s test:

To 1 ml of extract, 1 ml of Barfoed’s reagent was added and heated on a boiling water bath for 2 minutes. The color change was noted and recorded. A red precipitates indicated presence of sugar.

Benedict’s test:

To 0.5 ml of extract, 0.5 ml of Benedict’s reagent was added. The mixture is heated on a boiling water bath for 2minutes and the result was observed. A red precipitates indicated presence of sugar.

Detection of Glycosides:

Legal’s test:

Chloroform (3ml) and ammonia solution (10%) was added to 2ml plant extract. Formation of pink color indicated the presence of glycosides.

Detection of Proteins:

The extract was dissolved in 10 ml of distilled water and filtered through Whatman No.1 filter paper and the filtrate is subjected to tests for proteins and amino acids.

Million’s test:

To 2 ml of filtrate, few drops of Millon’s reagent are added. The result was observed. A white precipitates indicated presences of proteins.

Biuret test:

An aliquot of 2 ml of filtrate was treated with drop of 2% copper sulphate solution. To this, 1 ml of ethanol (95%) was added, followed by excess of potassium hydroxide pellets. The pink color in ethanol layer indicated presences of proteins.

Detection of amino acid:

Ninhydrin test:

Two drops of ninhydrin solution (5 mg of ninhydrin in 200 ml of acetone) are added to two ml of aqueous filtrate. The color change was observed. A characteristic purple color indicated the presence of amino acids.

Detection of Phytosterols:

Liberdmann-Burchard’s test:

The extract (5 mg) was dissolved in 2 ml acetic anhydride and one or two drops of concentrated sulphuric acid was added slowly along the sides of the test tube. The formation of blue green color indicated the presence of triterpenoids and Phytosteroids.

Detection of Tannins:

Ferric chloride test:

The extract (5 mg) was dissolved in 5 ml of distilled water and few drops of neutral 5% ferric chloride solution were added. The formation of blue green color indicated the presence of tannins.

Detection of Phenols:

Lead acetate test:

The extract (5 mg) was dissolved in distilled water and 3 ml of 10% lead acetate solution was added. A bulky white precipitates indicated the presence of phenols.

Detection of flavonoids:

An aqueous solution of the extract was treated with ammonium hydroxide solution. The yellow fluorescence indicated the presence of flavonoids.

Detection of coumarines:

10% NaOH (1ml) was added to 1 ml of the plant extracts formation of yellow color indicated presence of coumarines.

Detection of Saponins:

Distilled water 2ml was added of each plant extracts and shaken in a graduated cylinder for 15 mins lengthwise. Formation of 1cm foam indicates the presence of saponins.

Detection of Quinone:

Concentrated sulphuric acid (1ml) was added to 1ml of each of the plant extract. Formation of red color indicated the presence of Quinones.

Detection of Cardiac glycosides:

Glacial acetic acid (2ml) and few drops of 5% ferric chloride were added to 0.5% of the extract. This was under layered with 1ml of concentrated sulphuric acid. Formation of brown ring at the interface indicated presence of cardiac glycosides.

Detection of Terpenoid:

Chloroform (2ml) and concentrated sulphuric acid was added carefully to 0.5 ml of extract. Formation of red brown color at the interface indicated the presence of terpenoid.

Detection of Phlobatannins:

Few drops of 10% ammonia solution were added to 0.5 ml of root extract. Appearance of pink color precipitates indicated the presence of phlobatannins.

Detection of Anthraquinones:

Few drops of 2% HCL were added to 0.5 ml of seed extract. Appearance of red color precipitate indicated presence of anthraquinones.

Detection of steroids and Phytosteroids:

To 0.5 ml of the plant extract equal volume of chloroform was added and subjected with few drops of concentrated sulphuric acid. Appearance of brown ring indicates the presence of steroids and appearance of bluish brown ring indicated the presence of Phytosteroids.

In-vitro anti-inflammatory activity:

The blood was collected from healthy human volunteer who had not taken any NSAIDS for 2 weeks prior to the experiment and mixed with equal volume of Alsever solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.42% NaCl) and centrifuged at 3,000 rpm. The 10 % packed cells were washed with isosaline. Various concentrations of extracts were prepared (100, 200, 300, 400 and 500 µg/ml) using distilled water and 1 ml of plant extracts, 1 ml of phosphate buffer, 2 ml hyposaline and 0.5 ml of HRBC suspension were added. It was incubated at 37⁰C for 30 min and centrifuged at 3,000 rpm for 20 min. and the hemoglobin content of the supernatant solution was estimated spectro photometrically at 560 nm. Diclofenac (1 mg/ml) was used as reference standard and a control was prepared by omitting the extracts (Gandhisan et al., 1991). The percentage of HRBC membrane stabilization or protection was calculated by using the following Formula,

Optical density of drug treated sample

% Protection = 100 - --------------------------------------------------- x100

Optical density of control

Protein denaturation method:

The anti-inflammatory activity of Tephrosia purpurea (seed) was studied by using inhibition of protein denaturation method (Sakat et al.,2010). The reaction mixture (5ml) consist of 0.2 ml of egg albumin (from fresh hen’s egg), 2.8ml phosphate buffered saline (pH: 6.4) and 2ml of varying concentration of plant extracts. Similar volume of double distilled water served as control. Then the mixtures were incubated at 37±2°C in an incubator for 15 minutes and then heated at 70ºC for 5 minutes. After cooling, their absorbance was measured at 660nm by using vehicle as blank. Diclofenac at the final concentration of (1mg/ml) was used as reference drug and treated similarly for determination of absorbance.

The Percentage inhibition of protein denaturation was calculated as follows:

(Abs Control - Abs Sample)

% Inhibition = -------------------------------------------------- X 100

Abs Control

RESULTS AND DISCUSSION:

The different solvent extracts of seed of Tephrosia purpurea showed the presence of alkaloids, carbohydrate, glycosides, protein, aminoacid, phytosteroids, tannins, phenols, flavanoids, coumarins, quinine, cardiac glycosides, terpenoid, steroids and phytosteroids, where as absence of saponin, anthraquinones, Phlobatannins in the three extracts.

Plants generally produce many secondary metabolites which are biosynthetically derived from primary metabolites. Secondary metabolites that are used commercially as biologically active compounds were steroids, quinines, alkaloids, terpenoids and flavonoids, which are used in drug manufacture by the pharmaceutical industries. From a long period of time medicinal plants or their secondary metabolites have been directly or indirectly playing an important role in the human society to combat diseases (Wink et al., 2005).

The study revealed the presence of steroids, triterpenes, alkaloids, tannins, glycoside and flavanoids were present in all solvent extracts where as polyphenols were present in methanol, ethanol and aqueous extracts, of Tephrosia purpurea seed (Anuradha et al., 2013).

Qualitative phytochemical analysis of seed of Tephrosia purpurea species showed the presence of all the biological active compounds like carbohydrate, alkaloid, phytosterol, tannins and phenols, saponins and flavanoids. Plant sample show absence of fixed oil, fats, gums and mucilages (Nivedithadevi et al., 2012). Tephrosia purpurea showed the presence of almost all phytochemicals except any one either in tannins, saponins, steroids (Gnanaraja et al., 2014).

Table 1: Phytochemical screening of Tephrosia purpurea seed extracts

Phytochemical analysis	Plant extracts
Phytochemical analysis	Aqueous	Ethyl acetate	Hexane
1. Alkaloids	+++	+++	+++
2. Carbohydrate	++	+++	++
3. Glycosides	_	_	_
4. Proteins	_	+	+
5. Amino acid	+	++	_
6. Phytosterols	++	++	++
7. Tannins	+	++	+
8. Phenols	++	+++	++
9. Flavanoids	+	++	+
10. Coumarins	++	+++	+++
11. Saponin	+++	_	_
12. Quinone	++	+++	+++
13. Cardiac glycosides	++	+++	+++
14. Terpenoid	++	+++	++
15. Phlobatannins	_	_	_
16. Anthraquinones	+	_	_
17. Steriods and phytoSteriods	+++	+++	+++

Highly present (+++), Moderate (++), Mild (+), Absence ( -)

In -vitro anti-inflammatory activity:

Inflammation is a common phenomenon and it is a reaction of living tissues towards injury. Steroidal anti-inflammatory agents will lyse and possibly induce the redistribution of lymphocytes, which cause rapid and transient decrease in peripheral blood lymphocyte counts to affect longer term response (Matpal et al., 2013).

Aqueous, ethyl acetate and hexane extracts exhibited anti inflammatory activity in all the concentration, but the highest RBC membrane protection was observed in the aqueous extract of Tephrosia purpurea (seed) at maximum protection showed in 500µg of plant extract. Whereas ethyl acetate extract possess higher protection when compared to hexane extract of Tephrosia purpurea (seed). Diclofenac as a drug showed the maximum protection 90.3±5.21 at the concentration of 1 mg/ml.

Figure 1:

Epidemiological evidence suggests that a high intake of plant foods is associated with lower risk of chronic diseases. However, the mechanism of action and the components involved in this effect have not been identified clearly. In recent years, the scientific community has agreed to focus its attention on a class of secondary metabolites extensively present in a wide range of plant product: the flavonoids, suggested as having different biological roles. The anti-inflammatory actions of flavonoids in vitro or in cellular models involve the inhibition of the synthesis and activities of different pro-inflammatory mediators such as eicosanoids, cytokines, adhesion molecules and C-reactive protein (Serafini et al., 2010).

Inflammation is involved in increasing number of diseases necessitating the development of new, effective and safe treatments. Non steroidal anti-inflammatory drugs (NSAIDs) have been helpful in many instances, but they only inhibit cyclooxygenase (COX), but not the generation or actions of cytokines. Instead, some natural flavonoids have multiple anti-inflammatory effects, including COX inhibition, and a much safer profile. Increasing evidence indicates that inflammation plays a critical role in the pathogenesis of many diseases that also involve mast cells. Consequently, the need for new, effective and safe anti-inflammatory drugs is all the more urgent (Conti et al., 2013).

The HRBC membrane stabilization has been used as a method to study the in-vitro anti-inflammatory activity because the erythrocyte membrane is analogous to the lysosomal membrane (Gandhidasan et al., 1991) and its stabilization implies that the extract may well stabilize lysosomal membranes. Stabilization of lysosomal membrane is important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated neutrophil, such as bacterial enzymes and proteases, which causes further tissue inflammation and damage upon extra cellular release. The lysosomal released during inflammation produce a various disorders. The extra cellular activity of these enzymes are said to be related to acute or chronic inflammation. The non steroidal drugs act either by inhibiting these lysosomal enzymes or by stabilizing the lysosomal membrane (Shenoy et al., 2010).

The exact mechanism of the membrane stabilization by the extract is not known yet; hypo tonicity-induced hemolysis may arise from shrinkage of the cells due to osmotic loss of intracellular electrolyte and fluid components. The extract may inhibit the process, which may stimulate or enhance the efflux of these intracellular components. Flavonoids are referred to as nature’s biological response modifiers because of their inherent ability to modify the body’s reaction to allergens, viruses and carcinogens. They show anti-allergic, anti-inflammatory, antimicrobial and anticancer activity (Vadivu et al., 2008).

Protein denaturation method:

There are certain problems in using animals in experimental pharmacological research, such as ethical issues and the lack of rationale for their use when other suitable methods are available or could be investigated. Hence, in the present study the protein denaturation bioassay was selected for in-vitro assessment of anti-inflammatory property of different extracts of T.purpurea (seed).

As part of the investigation on the mechanism of the anti-inflammatory activity, ability of plant extract to inhibit protein denaturation was studied by albumin denaturation. The highest inhibition was observed in aqueous extract of seed of Tephrosia purpurea at all the concentration (50 ± 6. 04 / 100 µg, 54 ± 6.19/ 200 µg, 63.8 ± 6.80 / 300 µg, 65.2 ± 7.88/ 400 µg, 73.1 ± 8.78 / 400 µg ) when compared to ethyl acetate and hexane extracts. Diclofenac a standard anti-inflammatory drug showed the maximum inhibition 89.3±5.21 at the concentration of 1 mg/ml.

Figure 2:

Protein denaturation is a process in which proteins lose their tertiary structure and secondary structure by application of external stress or compound, such as strong acid or base, a concentrated inorganic salt, an organic solvent or heat. Most biological proteins lose their biological function when denatured. It is a well documented cause of inflammation (Jolly et al., 2014).

This anti-denaturation effect was further supported by the change in viscosities. It has been reported that the viscosities of protein solutions increase on denaturation. This decrease in viscosities may be due to decrease in concentration of test extract/drug in reaction mixture, which resulted in decreased viscosity; and/or other uncertain physico-chemical factors. Nevertheless, the viscosity data indicated inhibition of protein (albumin) denaturation. The effect of concentration of test agent on viscosity behavior of denatured protein dispersion requires further studies (Sangita et al., 2012). Denaturation of protein is one of the causes of lipodystrophy, hyperlipidaemia, diabetes mellitus type 2, kidney stones and rheumatoid arthritis that are documented (Fantry, 1999). Agents that can prevent denaturation of protein inhibition therefore, would be worthwhile for anti-inflammatory drug development.

Protein structure is very specific and their three dimensional structure is disrupted; the protein loses its functionality and is said to have undergone denaturation. The interactions, such as hydrogen bonding, that dictate the tertiary structure of proteins are not as strong as covalent chemical bonds. Because these interactions are rather weak, they can be disrupted with relatively modest stresses. The melting temperature varies for different protein, but temperature above 41ºC will break the interactions in many proteins and denature them (Branden 1998).

The anti-inflammatory activity of Canthium parviflorum extract found may be due to the presence of therapeutically active phytocompound flavonoids. The therapeutic applications of flavonoids on inflammation have previously been reported (Middleton, 2000). In-vitro anti-inflammatory studies of Anthracephalus cadamba demonstrated the suppression of both inflammations. The cause of rheumatoid arthritis is denaturation of proteins (Brown et al., 1968) and inhibition denaturation is one of the in-vitro tests to screen anti-inflammatory drugs (Grant et al., 1970).

CONCLUSION:

The result of present study, phytochemical analysis of different extracts of Tephrosia purpurea (seed) revealed the presence of various bioactive phytochemical compounds were found in all extracts. In-vitro anti- inflammatory activity of T. purpurea (seed) extracts were screened against HRBC membrane stabilization and protein denaturation. In-vitro anti-inflammatory activity of T. purpurea may be due to responsible for the presence of biological active compound such as flavonoids and tritrepenoids and related polyphenols. Further and detailed studies are in process for the isolation of active constituent responsible for this property and to identification of the possible mechanism of its anti- inflammatory property.

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Received on 23.03.2015 Accepted on 11.04.2015

Asian J. Pharm. Res. 5(2): April-June 2015; Page 83-89

DOI: 10.5958/2231-5691.2015.00012.X

S.No	Concentration of plant extract (µg / ml )	Different extracts / % Protection
S.No	Concentration of plant extract (µg / ml )	Aqueous	Ethyl acetate	Hexane	Standard Diclofenac
1.	Control	_	_	_	90.3 ± 5.21
2.	100	45.3 ± 6.96	36.3 ±5.38	9 ± 5.52	_
3.	200	63.3 ± 7.90	44.3 ±6.67	17 ± 6.08	_
4.	300	70.6 ± 8.63	54.3 ±6.96	26 ± 7.64	_
5.	400	80.6 ± 8.86	63.3 ±7.90	35 ± 8.24	_
6.	500	85.6 ± 9.48	73.3 ±8.94	53 ± 9.21	_

S.No	Concentration of plant extract (µg / ml )	Different extracts / % Inhibition of protein denaturation
S.No	Concentration of plant extract (µg / ml )	Aqueous	Ethyl acetate	Hexane	Standard diclofenac
1.	Control	_	_	_	89.3 ± 5.21
2.	100	50 ± 6. 04	46.4 ± 5.00	25.8± 2.80	_
3.	200	54 ± 6.19	51.7 ± 5.52	32.4± 3.66	_
4.	300	63.8 ± 6.80	59 ± 6.08	39 ± 3.75	_
5.	400	65.2 ± 7.88	64.7 ± 6.19	45.4± 4.19	_
6.	500	73.1 ± 8.78	69.3 ± 6.67	52.1 ± 4.65	_

*Corresponding Author E-mail: flowervidu@gmail.com