Pharmacognostic, Phytochemical and In vitro Biological Evaluation of Blepharis repens (Vahl) Roth

Blepharis repens (Vahl) Roth of Acanthaceae family is edible herb and commonly known as Hadsan in Marathi and Haridachchu in Kannada.1,2 It iscommonly found in rocky regions of Tamil Nadu, Delhi, Chota-nagpur and in Srilanka. The whole plant is of good medicinal value and has been used as folk remedy for treating ailments like wounds, bone fracture, skin disease, urinary infections, cancer, diarrhea and leaves are useful in syphilis and dysentery.3-6 Traditionally whole plant powder with milk and or wheat or black gram floor is commonly used in healing bone fracture in various parts of India. Tribal people in southern India uses leaf juice heated in gingelly oil topically for wound healing.7,8 Very little information for both phytochemistry and pharmacological efficacy is available on this plant.9 Pharmacologically, all parts of plant have been reported to possess antidiabetic, antihyperlipidaemic, hepatoprotective and bone and wound healing activity.10


INTRODUCTION
Blepharis repens (Vahl) Roth of Acanthaceae family is edible herb and commonly known as Hadsan in Marathi and Haridachchu in Kannada. 1,2 It iscommonly found in rocky regions of Tamil Nadu, Delhi, Chota-nagpur and in Srilanka. The whole plant is of good medicinal value and has been used as folk remedy for treating ailments like wounds, bone fracture, skin disease, urinary infections, cancer, diarrhea and leaves are useful in syphilis and dysentery. [3][4][5][6] Traditionally whole plant powder with milk and or wheat or black gram floor is commonly used in healing bone fracture in various parts of India. Tribal people in southern India uses leaf juice heated in gingelly oil topically for wound healing. 7,8 Very little information for both phytochemistry and pharmacological efficacy is available on this plant. 9 Pharmacologically, all parts of plant have been reported to possess antidiabetic, antihyperlipidaemic, hepatoprotective and bone and wound healing activity. 10 The objective of the present study is to establish pharmacognostic and in-vitro biological like antioxidant, anticancer, antimicrobial efficacy parameters. The results will be helpful in identification, preventing possible adulteration and primary evaluation as part of primary stage of quality control standardization of herbal drugs.

Plant material collection, identification and authentication
Plant material was collected from surrounding open field in hill, rocky region in district Hingoli taluka Sengaon. Identification and authentication was done by Dr. Arvind S. Dhabe, Professor, Department of Botany, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad. (Accession number is 0564).

Plant material extraction
The plant was shade dried for 7 days and cleaned, introduced in cutter mill and powder is obtained. This powder is stored in air tight container. From that 100 gm of powder wrapped in thimble which isexhaustively extractedusing petroleum ether, chloroform, methanol, water and hydro-alcohol (70%) as solvent in successive solvent extractor. Extraction continues till the solvent in sample tube became clear at required temperature to particular solvent. The extract was obtained which subjected to concentrate to one fourth volume under reduced pressure for further use wherever required. 11

Morphological, microscopical and powder microscopical evaluation
Morphological evaluation of various parts of plant like root, leaf, stem and flower was performed. Colour, odour, taste, shape is evaluated by visual observation and size is measured for width and length. For microscopical evalaution, stem, root and leaf were kept in water for some time and sections were taken with the help of blade. The sections were hydrated with glycerin and covered with cover slip and focused under digital microscope. Photographs under normal bright field at different magnifications were taken. Similarly, powder was evaluated for different microscopic parameters. 12

Physicochemical evaluation
The determination of various physicochemical constants such as ash values, extractive values, element detection and loss on drying determined as per World Health Organization guidelines. All experiments were carried out in triplicates and expressed as mean value. [12][13][14] Fluorescence analysis of powder drug Powdered plant material was treated with various chemical reagents and exposed to visible, ultraviolet light (Short UV) to explore their fluorescence behavior. A small amount of powder drug was placed on a micro slide and treated with 1 N NaOH in methanol, 1 N HCl, 1 N NaOH in water, nitric acid (1:1), sulphuric acid (1:1), glacial acetic acid, 1 N potassium hydroxide and observed under UV 366 nm, UV 254 nm and in daylight under moist condition. 15,16 Phytochemical evaluation

Preliminary phytochemical screening
Preliminary phytochemical screening of pure extract for various class of phyto-constituent such as alkaloid, glycoside, tannin, carbohydrate, saponin, protein, flavonoids, steroids, gum, mucilage and oil is completed by chemical evaluation using various reagents. 13 The osazone test for carbohydrate lead to formation of various shapes of crystals. Cube shape crystals may be of glucose or fructose, needle shape may be of monosaccharides and flower shape crystal may be of maltose sugar.

Quantitative estimation of phytochemicals
Various phytochemicals present in the hydro-alcoholic (70%) extract were quantified for total carbohydrate content by anthrone method, total tannin content by Folin-Denis method, total flavonoid content by aluminium chloride method, total alkaloid content by Bromocresol-Green method using glucose, tannic acid, quercetin and atropine as standard drug respectively. 13 Thin layer chromatography analysis TLC separation and evaluation for different phytoconstituent of four extracts is carried out using silica gel GF254 as stationary phase and different mobile phases as well as spraying reagents as summarised in Tables 1-5. 13,17 Extracts are dissolved in their respective solvents and then spots are applied by using microcapillary on TLC plates.  Loss on drying 0.43 ± 0.02%  Alkaloids

In-vitro biological activity
Cynogenetic Glycoside --7 Coumarin Glycoside --8 Flavonoids Tannins Steroid or Triterpenoid --11 Saponin Essential Oil --  test tubes. To this 5 ml of methanolic solution of DPPH was added, shaken well and covered with aluminium foil, mixture was incubated at 37 0 C for 30 min. The absorbance was measured against methanol as blank at 517 nm. Absorbance of DPPH solution was taken as control. Percent scavenging activity was calculated by formula: Scavenging activity (%) = absorbance control-absorbance sample / absorbance control × 100. IC50 values were determined from the graphs of percentage scavenging activity against concentration of extracts. These values are defined as inhibitory concentration of the extract necessary to decrease the initial DPPH radical concentration by 50% and are expressed in µg/ml. [18][19][20][21] In-vitro anticancer activity by SRB assay method

where + Present and -Absent
The monolayer cell culture was trypsinized and the cell count was adjusted to 0.5-1.0 x 10 5 cells/ml using medium containing 10% new born sheep serum. To each well of the 96 well microtitre plate, 0.1ml of the diluted cell suspension (approximately 10,000 cells) was added. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off, washed once and 100 µl of different test compound concentrations were added to the cells in micro titre plates. The plates were then incubated at 37 0 C for 72 hours in 5% CO 2 incubator, microscopic examination was carried out, and observations recorded every 24 hours. After 72 hours, 25 µl of 50% trichloro acetic acid was added to the wells gently such that it forms a thin layer over the test compounds to form overall concentration 10%. The plates were incubated at 4 0 C for one hour. The plates were flicked and washed five times with tap water to remove traces of medium, sample and serum, and were then air-dried. The air-dried plates were stained with 100μl SRB and kept for 30 minutes at room temperature. The unbound dye was removed by rapidly washing four times with 1% acetic acid. The plates were then air dried. 100 µl of 10mM Tris base was then added to the wells to solubilize the dye. The plates were shaken vigorously for 5 minutes. The absorbance was measured using microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using following formula, % cell inhibition= 100-{(At-Ab)/ (Ac-Ab)} x100. 22,23 In-vitro antimicrobial activity by well diffusion method In this method the extract diffused from well on solidified agar plate which extend such that growth of added microorganism is preventedin Anisaldehyde sulphuric acid Treatment after spraying: heated on hot plate at 110 0 C till color developed and intensified. circular area known as zone of inhibition.Using pipette 0.2 ml of each of the seeded broth containing 10 6 -10 7 cfu/ml was inoculated on the plate of solidified agar and speeded uniformly by glass spreader. Then five well cut out in plate by using borer of diameter 5mm. to the five well the 0.2 ml solution of four extract and standard was added to each well. all work carried out under aseptic condition. This plate is incubated at 37 0 C±1 0 C for 24 hours. After incubation period diameter of the zone of inhibition in mm obtained around the well was measured. [24][25][26] In vitro antimicrobial activity by bioautographic method

RESULTS AND DISCUSSIONS
Pharmacognostic standardization helps for correct identification, authentication and detection of adulteration and routine quality control of crude drugs. Physicochemical numerical standards reported in this work will be useful for correct identification, doubtful specimen evaluation and compilation of a suitable monograph.
Thin layer chromatography ( Figure 2)  In vitro antioxidant by DPPH (Table 6) reveals that both methanolic and aqueous extracts of B.repens possess significant free radical scavenging properties in concentration-dependent manner. Hence, it can be concluded that the B.repens could be further pharmaceutically exploited for antioxidant properties by in vivo models. Methanolic extract showed more potent antioxidant activity than aqueous extract which might be due to presence of polyphenolic compound and flavonoids.
In vitro anticancer activity of four extracts and alkaloid fraction of B.repens is performed on HL 60 cancer cell lines at ACTREC Mumbai, India. The cell viability was measured using SRB assay. As IC50 values of all extracts found above 80 μg/ml, hence it is concluded that evaluated extracts of B.repens do not have anticancer potential against selected leukemia cell line. Finally, it can be concluded that extracts of B. repens have strong antioxidant activity but no anticancer activity against human leukemia cell line HL-60.
Antimicrobial activity study of methanolic, chloroform, petroleum ether extract and alkaloid fraction has shown maximum activity against S. aureus, medium potency for E. coli and very weak activity against A. niger. Aqueous extract showed weak activity against E.coli and S.aureu ( Figure 3).     compounds due to their reaction with the spray reagent used (vanillin/ sulphuric acid). In bioautographic method, the appearance of white areas against a purple-red background on the chromatograms denotes inhibition of growth of the bacteria or fungi (Figure 4)

CONCLUSION
Folk claim of use of B. repens in wound healing along with secondary skin infections, skin and urinary infections, diabetes and cancer can be explored scientifically with present paper results. Antimicrobial and anti-oxidant potential is responsible for wound healing along with associated secondary infections, skin and urinary infections. Antioxidant potential is also responsible for treatment of cancer and diabetes. In present paper, B. repens extracts are not found anticancer against leukemia cell lines but same extracts might be useful against other cancer cell lines. Finally it can be concluded that morphological, microscopical, physicochemical, chemical and few biological parameters like antioxidant, anticancer, antimicrobial efficacy of plant B.repens established for the first time.