EVALUATION OF ANTI-OXIDANT, ANTI-INFLAMMATORY AND CYTOTOXICITY POTENTIAL OF HEMIGRAPHIS COLORATA

EVALUATION OF ANTI-OXIDANT, ANTI-INFLAMMATORY AND CYTOTOXICITY POTENTIAL OF HEMIGRAPHIS COLORATA

T.T. Akhil* and Punieethaa Prabhu

Department of Biotechnology, K.S. Rangasamy College of Technology, Tiruchengode- 637 215, Nammakkal, Tamil Nadu, India

ABSTRACT: Plants have been used as a source of natural products for various therapeutic processes from a long time. The proposed work illustrates the study of the importance of natural products in terms in-vitro approaches. On account of this study the phytochemical constituents of various extracts of Hemigraphis colorata (Blume) are evaluated. The plant was traditionally known for its wound healing activities. Crude plant extract was prepared by percolation method using a Soxhlet apparatus. The solvents used were Hexane, Chloroform, Acetone and Ethanol .The in-vitro antioxidant capacity was analysed through DPPH and reducing power assays. Anti-inflammatory properties of various extracts from the whole plant are analyzed through human RBC membrane stabilisation method. The anti-oxidant and anti-inflammatory activities were more for the ethanolic extracts of Hemigraphis colorata when compared to acetone, chloroform and hexane extracts.The in-vitro cytotoxicity analysis of ethanolic extract was also carried out in the present study using trypan blue method

DPPH, cytotoxicity, Anti-inflammatory, RBC

INTRODUCTION:  Natural products especially which are derived from plants have been used as a source for various therapeutic processes for long times from the human civilisation. The most common strategy of drug development from plants is careful observation of use of natural resources in flock medicine in different cultures by ethanopharmacology ¹.Free radicals have been implicated in numerous diseases, cancer, inflammation and neurodegenerative disorders ⁶. The current drugs which are used as anti-inflammatory drugs can cause severe side effects.

Ethanopharmacological approach of analysing the phytochemical properties of various extracts of Hemigraphis colorata (Family: Acanthaceae) is used in the present study. The anti-oxidant, anti-inflammatory potential of this plant is exploited in the present study. Hemigraphis colorata plant is claimed in flock medicine for its very good wound healing activity ².

Hemigraphis colorata (Blume) H.G. Hallier leaf paste on the wound promotes wound healing in mice but the oral administration was in effective ². The phytochemical constituents on Hemigraphis colorata were identified by examining the crude extracts of its leaves and stem using various solvents, screened the antibacterial activity against selected pathogens ⁷. The crude aqueous, acetone, benzene, chloroform, ethanol, and petroleum ether extracts of the plant had phenols, carbohydrates, steroids, saponins, coumarins, tannins, proteins, flavonoids, alkaloids.The hypoglycaemic and anti-diabetic properties of H. colorata was identified for the first time using wistar rats and Swiss albino mice by  ³.

Glucose lowering effect and antidiabetes activity studied using glucose tolerance test with different extracts .n-Hexane and ,to some extent ethanolic extracts were found to lover the levels of blood glucose in glucose feed rats. The pharmacological actions of ethanolic extracts of leaves of Hemigraphis colorata and Clerodendron phlomoides were studied by ⁴ which reviewed the anti-inflammatory effects, antioxidant activities; wound healing properties, effects on arthritis by analysing Phytochemical profiles.

The preliminary photochemical studies of methanolic extract of H. colorata to evaluate wound healing activity of the methanolic extract ointment of dried leaves of H. colorata in albino rats and found that increase in tensile strength of treated wounds may be due to the increase in collagen concentration per unit area and stabilisation of fibers⁵.An attempt to screen various compounds responsible for the anti-oxidant and anti-inflammatory properties by in vitro assays is carried out in the present study.

MATERIALS AND METHODS:

Collection of Plant: Hemigraphis colorata plant species were collected from Mannuthy, Thrissur district of Kerala during the month of July to September 2012 was dried at room temperature and powdered in a mixer grinder for further analysis.

Preparation of Extracts: Crude plant extract was prepared by percolation method using a Soxhlet apparatus ⁸. About 10 grams of plant material was packed uniformly into a thimble and extracted with 150 ml of different solvents separately based on the increasing order of polarity n-Hexane, chloroform, acetone, and ethanol were the solvents used. The extraction process was continued till the solvent in siphon tube of the apparatus became colourless. The crude extract was filtered and concentrated under vacuum and controlled temperature. The extract was stored in refrigerator at 4 °c until further use.

Qualitative Phytochemical Analysis: The preliminary phytochemical analyses were carried out using the whole plant extracts of Hemigraphis colorata followed by ⁹Phenols and tannins were identified by ferric chloride test, test for flavonoids were performed using lead acetate solution, saponins were identified by froth test, glycosides were identified by adding concentrated H₂SO₄, steroids by Salkowski’s test, alkaloids by adding Dragendroff’s reagent, and reducing sugars with Fehling’s test.

Determination of Anti-Oxidant Activity

1. DPPH Free Radical Scavenging Assay: The free radical scavenging capacity of the Hemigraphis colorta whole plant extract was determined using DPPH according to the method of Blioswith slight modifications¹². DPPH solution (0.004% w/v) was prepared freshly in 80% methanol and was added to 1ml sample solutions containing various extracts ranging from concentration 100μg/ml -500μg/ml .The mixture was allowed to stand at room temperature in dark for 30 minutes and vortexed. The absorbance was read at 517nm using spectrophotometer. Reference standard used was Ascorbic acid while DPPH solution without any extract served as control.80% methanol solution was used as blank solution.All tests were performed in triplicates. Percentage scavenging of the DPPH free radical was measured using the following equation,

Radical scavenging activity (%) =

(A_control ‐ A_Test)/A_control X 100

Where A_control is the absorbance of the control reaction and A_Test is the absorbance in the presence of the extracts.

2. Reducing power (FRAP) Assay: The Fe³⁺ reducing power of the whole plant extract of Hemigraphis colorata was determined by the method of Oyaizhu with slight modifications ¹². Different concentrations (100 - 500μg/ml) of the  various extracts of were mixed with  phosphate buffer (0.2 M, pH6.6) and made up to 1ml , 0.5 ml potassium ferrocyanide (1%), followed by incubation at 50°C in a water bath for 20 min. After incubation, 0.5 ml of Trichloroacetic acid (10%) was added to terminate the reaction. The upper portion of the solution (1ml) was mixed with 1 ml distilled water, and 0.1 ml FeCl₃ solution (0.1%) was added. The reaction mixture was left for 10 min at room temperature and the absorbance was measured at 700 nm against an appropriate blank solution in UV spectrophotometer. All tests were performed in duplicates. A higher absorbance of the reaction mixture indicated greater reducing power. Standard: Ascorbic acid
3. Anti-inflammatory properties: The anti-inflammatory properties of H. colorata whole plant extracts were analysed by Human red blood cell membrane stabilization method¹⁰.Fresh 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 sterilized Alsever solution (containing 2% dextrose, 0.8% sodium citrate, 0.05% citric acid and 0.42% sodium chloride) and were centrifuged at 3000 rpm and the packed cells obtained were washed with isosaline (0.85% NaCl, pH 7.2) thrice and 10% (v/v) suspension was made. Solutions of different concentrations ranging from 50 -300 μg/ml of the solvent extracts of H. colorata were prepared.

Assay mixture contained the active drug, 1 ml of phosphate buffer (0.15 m pH 7.4) 2 ml of hyposaline (0.25% Nacl) and 0.5 ml of 10% RBC suspension. Control tube contained 2 ml of distilled water instead of hyposaline. All the tubes were incubated at 37°C for 30 min. They were centrifuged at 3000 rpm for 20 minutes and the haemoglobin content in the supernatant was estimated using UV spectrophotometer at 560 nm. The percentage inhibition of haemolysis or membrane stabilization was calculated.

% inhibition of haemolysis =

[OD₁-OD₂/OD₁] X 100

Where, OD₁ = Optical density of hypotonic-buffered saline solution, OD₂ = Optical density of test sample in hypotonic solution.

4. Cytotoxicity screening: The in vitro short term cytotoxicity of ethanolic extract of H. coloratawas assessed using DLA (Daltons Lymphoma Ascites) tumor cell lines by Trypan blue exclusion method. The cell lines were obtained from Amala Cancer Research Institute, Thrissur, Kerala, India

Cell viability was determined by trypan blue exclusion method ¹³.Viable cell suspension (1×10⁶ cells in 0.1 ml) was added to tubes containing various concentrations of the ethanolic extracts of H. colorata was dissolved in DMSO and the volume was made up to 1ml using phosphate buffered saline (PBS). Control tube contained only cell suspension .These assay mixture were incubated for 3 hours at 37°C. Further cell suspension was mixed with 0.1 ml of % trypan blue and kept for 2-3 minutes and loaded on a haemocytometer. Dead cells take up the blue colour of trypan blue while live cells do not take up the dye. The number of stained and unstained cells was counted separately.

RESULTS AND DISSCUSSION:

Plant collection and extraction: The H. colorata plant collected and the dried whole plant parts was powdered and extracted successively using with hexane, acetone, chloroform and ethanol in the increasing order of polarity and their colour and consistency were studied. The ethanolic extract showed maximum average extractive yield of 2.4% (w/w) and least value for acetone extract of yield 0.5% (w/w). The results were tabulated in table 1.

TABLE 1: COMPARISON OF VARIOUS SOLVENT EXTRACTS OF H. COLORATA BY CONTINUOUS HOT PERCOLATION METHOD BY SOXHLET APPARATUS

Qualitative Phytochemical Analysis: The phytochemical characteristics of H. colorata revealed the presence of active compounds. The results were summarized in table 2. Analysis of the plant extracts revealed the presence of phyto-constituents which are known to exhibit medicinal as well as physiological activities ⁹.

Phytochemicals such as phenols, tannins, alkaloids, glycosides, steroids, saponins etc. were found in the extracted compounds. Ethanolic extract showed the presence of flavonoids, steroids, phenols and tannins, saponins, glycosides and reducing sugars. The observations were in accordance with ⁷.

TABLE 2: PHYTOCHEMICAL ANALYSIS OF H. COLORATA

+ indicates presence, - indicates absence

DPPH Radical Scavenging Assay: Antioxidants react with DPPH, a stable free radical which is reduced to DPPH‐H and as a consequence the absorbance is decreased from the DPPH radical to the DPPH‐H. The degree of discolouration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability ¹². Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. All tests were performed in triplicates. Percentage scavenging of the DPPH free radical was measured and expressed in graph along with error value at 5% significance as in fig. 1.

FIGURE 1: DPPH RADICAL SCAVENGING ASSAY OF VARIOUS EXTRACTS WITH STANDARD ERROR AT 5% OF SIGNIFICANCE

The percentage of radical scavenging property was more for ethanolic extract per 100μg of extract was 91.10 ± 1.71% and that of the control was 97.76 ± 0.70 %. The radical scavenging activities of extracts of leaf was expressed as the IC₅₀ value (μg/ml), i.e., the concentration necessary to decrease the DPPH concentration by 50%. The results revealed that ethanolic extract of H. colorata has maximum activity when compared with the standard ascorbic acid. The concentration necessary to decrease the DPPH concentration by 50% of ethanolic extract was found to be 54.88mg/ml. The present study revealed that ethanolic extracts of H. colorata exhibits proton donating ability and could serve as good antioxidants by scavenging free radicals ¹³.

Reducing Power (FRAP) Assay: Reducing power is the measure to test the reductive ability of anti-oxidant and is evaluated by the transformation of Fe³⁺ to Fe²⁺ in the presence of various plant extracts ¹⁵.

The formation of Prussian blue colour was measured at 700 nm against an appropriate blank solution in UV spectrophotometer. All tests were performed in triplicates and the result is expressed in fig. 2 along with error value at 5% significance. A higher absorbance of the reaction mixture indicated greater reducing power. The results obtained shows ethanolic extract of H. colorata have more reducing power when comparing with other extracts of the plant. Hexane extract showed lower absorbance while ethanolic extract had good increasing value for increasing concentration ¹⁴.

FIGURE 2: RESULTS OF FRAP ASSAY WITH STANDARD ERROR AT 5% OF SIGNIFICANCE

Anti-inflammatory analysis: The in vitro anti-inflammatory assay was carried out by HRBC membrane stabilization method. The ethanolic extract showed good percentage of membrane stabilisation when compare to hexane, acetone, and chloroform extracts. Diclofenac sodium was used as the reference standard. The absorbance of human blood treated with the assay mixture at 560 nm is tabulated in Table 3. The percentage inhibition of haemolysis or membrane stabilization was calculated and plotted in fig. 3 by mentioning 5% significance of error value. Ethanolic extract of H. colorata showed a good percentage of inhibition of haemolysis when compared to the other extracts. The results were comparable with ¹².

TABLE 3: RESULTS OF ABSORBANCE VALVES HRBC MEMBRANE STABILIZATION TEST AT 560 NM

All readings are represented as mean± standard error value at 5% level of significance

FIGURE 3: RESULTS OF HAEMOLYSIS DISPLAYED WITH ERROR VALUE OF 5% SIGNIFICANCE

The reference drug Diclofenac sodium showed 76.79 % of haemolysis while it was 39.34%, 34%, 20.4%, 34% for ethanolic, acetone, chloroform, and hexane extracts respectively, in the concentration of 150μg/ml. The anti-inflammatory effects of the H. colorata extracts may be due to the presence of flavonoids, tannins, phenols etc. The enzyme phospholipase A2 is responsible for the formation of mediators of inflammation such as prostaglandins. By attracting polymorphonuclear leucocytes to the site of inflammation, they can lead to tissue damage probably by the release of free radicals. Phospholipase A2 converts phospholipids in the cell membrane into arachidonic acid, which is highly reactive and is rapidly metabolized by cyclooxygenase (prostaglandin synthesis) to prostaglandins. Prostaglandins are main compounds responsible for inducing pain during inflammation.

Cytotoxicity assay:The trypan blue exclusion method was used to study the  cytotoxicity when the DLA cells obtained  from the carrier mice were treated with different concentrations of the ethanolic extract  of H. colorata for 3 hours the relative cell survival progressively decreased in a dose dependent manner .The cytotoxic effect of ethanolic extract of H. colorata against the activity against DLA cell lines is relatively poor which is in accordance with the report on H. colorata which exhibit low cytotoxicity against DLA cell lines up to a concentration of 200 µg/mL in a short-term bioassay. The results were tabulated in table 4.

TABLE 4:    IN VITRO CYTOTOXICITY STUDY

CONCLUSION: The results obtained suggests that the ethanolic extracts of Hemigraphis colorata possess good antioxidant capacity to scavenge various free radicals   which may be due to the presence of phytochemicals like flavonoids, tannins, saponins, and phenolic compounds .The anti-inflammatory properties of the various extracts of Hemigraphis colorata comparatively less in the human RBC membrane stabilization test.

The present study has been found useful in the identification of several phytoconstituents like phenols, tannins, steroids, flavanoids, saponins, reducing sugars; glycosides were present in the ethanolic extract of whole plant of Hemigraphis colorata. , which may be responsible for the anti-oxidant and anti-inflammatory actions. The active fraction obtained from the ethanolic extract of whole plant can be used for further studies leading to possible drug development process involving the in silico process.

The cytotoxicity studies on the ethanolic extracts had poor cytotoxic effects which revealed that the extracts were not too toxic to the cells, which adds advantage to the drug development process. Development of phytomedicines is relatively inexpensive and less time consuming. Various environmental factors may lead to variations in the phytochemical content of the plant materials.

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    How to cite this article:

    Akhil TT and Prabhu P: Evaluation of anti-oxidant, anti-inflammatory and cytotoxicity potential of Hemigraphis colorata. Int J Pharm Sci Res 2013: 4(9); 3477-3483. doi: 10.13040/IJPSR. 0975-8232.4(9).3477-83

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