Phytochemical Evaluation of Polyherbal Formulation of Clinacanthus nutans and Elephantopus scaber to Identify Flavonoids

Background: Modern healthcare system recognizes herbal medicine as a form of alternative medicine and also identify as holistic approach. Everyone in life experiences different kind of wound.Clinacanthus nutans and Elephantopus scaber are well known traditional wound healing herbs. Objective: To develop a new polyherbal formulation in the treatment of wound and identify flavonoid by means of chromatography, chemical method and spectroscopic method. Preliminary phytochemical and fluorescent evaluation of Clinacanthus nutans,Elephantopus scaber and herb-herb combination. Methods and Material: Preliminary phytochemical and fluorescent evaluation of Clinacanthus nutans, Elephantopus and polyherbal formulation of both herb by chemical method. Identification of flavonoids by thin layer chromatography and fourier transform infrared spectroscopy. Results: Flavonoids have found inside polyherbal formulation by comparing the colour change after chemical analysis, Fluorescence analysis, retention time by thin layer chromatography and functional groups by fourier transform infrared spectroscopy. Conclusions: Flavonoids may responsible for its activity as wound healing. It may work with other bioactive compounds as synergistic effect.


INTRODUCTION
The trend of using herbal medicines has increased in a tremendous way in the last decade.As a result, World health Organization (WHO) has taken a broader step of including Phytotherapy.2][3] Many commercial and non-commercial polyhedral formulations exist in different part of World.Scientific evidence such as pharmacognostic evaluation, phytochemical study, and pharmacological evaluation Lacks in most of the polyherbalformulation. 1 Clinacanthus nutans Lindau is known as snake grass belonging to the Acanthaceae family.This plant has diverse and potential medicinal uses in traditional herbal medicine for treating skin rashes, insects and snake bites, lesions caused by herpes simplex virus, diabetes, and gout in Malaysia, Indonesia, Thailand, and China. 4Elephantopus scaber Linn. is known as Prickly-leaves elephant's foot, tutup bumi in Malay or di dan tou in Chinese.It has been used in traditional medicine to stimulate diuresis, reduce fever and eliminate bladder stones, as well as to treat nephritis, edema, dampness, chest pain, pneumonia, scabies, arthralgia and leukemia. 5,6It was used in number of poly herbal formulations.The selection of both herb was based upon its activity as wound healing.
The objective of our study was development of a new formulation and phytochemical evaluation of new herbal formulation.

Plant Material
The leaves of Clinacanthus nutans and Elephantopus scaber were collected from Institute Of Sustainable Agro technology, Sg.Chuchuh, University Malaysia Perlis (UniMAP) and washed using clean water.After that, the leaves were dried inside a dryer at the temperature of 35-40ºC for two days.Once dried, the leaves were grinded into fine powder by using mechanical grinder.

Preparation of Plant Extract
Soxhlet extraction was used in this experiment to extract the herbs.For Soxhlet extraction, a powder sample is weighted approximately 10 g for each herb and put into the extract chamber of the Soxhlet extractor.In each running experiment, 100 ml of aqueous ethanol 50% was used as solvent for 12 hour extraction.The extract solution was then evaporated by using rotary evaporator to remove the solvent in the extract solution and dried in oven at 35-40ºC for 12 hour.In order to study the synergistic effects of the herb pairs, a powdered mixture containing equal proportions of two herbs (5 g each) was extracted according to the above method.The final samples were stored at 4°C until it analysed and the percentage of yield was calculated by using Equation Y extract = m extract /m herb ×100 where m extract is the crude extract mass (g) and m herb is the extracted herb mass.The extract was fractionated using different solvents viz.hexane, chloroform, ethyl acetate, n-butanol and water.The supernatant was filtered using Whatman No. 1 sheet, pooled and concentrated using vacuum rotary evaporator.The concentrated solutions were then lyophilized to get the dry form of respective fractions.

Phytochemical test
Phytochemical analysis for each sample's crude extract ware carried out according to the standard procedure methods. 7,8st for alkaloids 1 mL of crude extract was mixed with 5 mL of diluted hydrochloride acid (HCL) and was placed in a water bath at 60°C for 15 minutes. 1 mL of Wagner's reagent was added into 1mL of filtered suspension.
Appearance of reddish-brown precipitate showed the presence of alkaloids.

Test for cardiac glycoside
Crude extract was treated with 1mL of glacial acetic acid diluted (3%) ferric chloride (FeCl 2 ) follow by adding concentrated sulphuric acid (H 2 SO 4 ) at the side of the test tube.The formation of brown ring indicated the presence of cardiac glycoside.

Test for flavonoid
Intense yellow color formed when 1 mL of sodium hydroxide (NaOH) solution was added into 1 mL of crude extract.The yellow color turned colorless upon addition of diluted H 2 SO 4 , which indicated the presence of flavoniod.

Test for phenolic compounds and tannin
2 mL of crude extract in test tube and add 3% FeCl 2 drop by drop.Appearance of bluish black precipitate indicates the presence of phenolic compounds and tannin.
Test for saponin 0.5 mL ofcrude extract was diluted with 5 mL of distill water.The suspension was shaken vigorously for a few minutes.Development of foam which is able to persist for 10 minutes shown the presence of saponin.

Salkowski Test for phytosterols
Crude extract was treated with 0.5 mL of chloroform, add 1 mL of concentrated H 2 SO 4 from the sides of test tube.Appearance of of reddish brown color in chloroform layerindicates the prescene of phytosterol.
Liebermann-Burchard's Test for triterpenoid 0.5 mL of crude extract was treated with few drops of acetic anhydride, boil and cool.1mL of concentrated H 2 SO 4 was added from the sides of test tube.Formations of reddish brown at the junction of two layer and formation of deep red color confirm the presence of terpenoid.

Fluorescence analysis
Fluorescence is an important phenomenon exhibited by various chemical constituents present in plant material.If the substances themselves are not fluorescent they may often be converted into fluorescent derivatives by reagents, hence some crude drugs are often assessed qualitatively in this way and it is an important parameter of pharmacognostical evaluation 9 TLC Analysis for flavonoids About 2 µg of extracts was loaded on TLC plates (Merck).The plates were developed in toluene: chloroform: methanol (4:4:1, v/v/v) to separate flavonoid compounds of the extracts.The developed plate was air dried.Then anisaldehyde sulfuric acid was sprayed on the surface of the plate and incubated for 20 min at 100°C.The present flavonoid compound of this extracts was detected as blue spot on developed TLC plate.The R f value of the bands were also determined. 8urier Transform Infrared Spectrophotometer Fourier Transform Infrared Spectrophotometer (FTIR) is perhaps the most powerful tool for identifying the types of chemical bonds (functional groups) present in compounds.The wavelength of light absorbed is characteristic of the chemical bond as can be seen in the annotated spectrum.By interpreting the infrared absorption spectrum, the chemical bonds in a molecule can be determined.10 The sample of each plant specimen was loaded in FTIR spectroscope (PerkimElmer Spectrum 65, USA), with a Scan range from 650 to 4000 cm -1 using Attenuated Total Reflection (ATR) Method.Result were obtain using Perkim Elmer Spectrum version 10.03.06.

RESULTS
Preliminary phytochemical analysis of newly polyherbal formulation is of significant importance as scientist need to understand the change upon extraction of equal portion of two different medicinal herb (Clinacanthus nutans and Elephantopus scaber).The combined herbal extract possess the bioactive compound of both herb.Results have showed shows the strong presence of Flavonoid, phenol, tannin, saponin, alkaloid.Whereas it possess moderate presence of Triterpenoid, phytosterol, cardiac glycoside.The phytochemical analysis of Clinacanthus nutans shows the strong presence of flavonoids, phenolic compounds, alkaloids and tannin.Whereas it possess moderate presence of triterpenoid and minimum level of phytosterol and cardiac glycoside.The phytochemical analysis of Elephantopus scaber shows the strong presence of phytosterol and saponin.Whereas it possess moderate presence of cardiac glycoside.Flavonoids, phenol, triterpenoid.All the result was based upon the qualitative analysis such as color change after reaction (Table 2).Fluorescent Analysis of Clinacanthus nutans, Elephantopus scaber and Clinacanthus nutans + Elephantopus scaber were performed by different solvents.Different colours gives an idea about nature of compound inside sample.An interesting observation have found during the evaluation of polyherbal powder that the colour after reaction is similar with the colour after Reaction of reagent with Elephantous scaber.This may be due to dark colour of powder of Elephantopus scaber as compare to Clinacanthus nutans.Clinacanthus nutans is light greenish powder whereas Elephantous scaber powderis dark green in nature.Fluorescent Analysis identify possible bioactive compounds by observing the colour present inside the sample as mentioned in Table 3. TLC Analysis of flavonoids of Clinacanthus nutans, Elephantopus scaber, Clinacanthus nutans + Elephantopus scaber and their fractions have confirm the presence of flavonoids.Blue colour spots indicates that flavonoid present inside the sample (Figure 1 and 2).Retention factor shows possible bioactive compound inside the sample (Table 7).