In vitro biological screening and chemical constituents of Euphorbia perbracteata Gage

To assess the in vitro biological activity and chemical constituents of Euphorbia perbracteata using different polar solvents, the phytochemical constituents were screened and total phenols, antioxidants, flavonoids content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and IC50 values were calculated. Antimicrobial assay was carried out by using disc diffusion method and minimum inhibitory concentration (MIC) was determined against Gram-positive (4), Gram-negative (4) and fungal (1) human pathogenic strains. The results showed that the extracts possessed a broad spectrum of antimicrobial activity with significant MIC values which ranged from 156 to 312 μg/ml. Total phenols (5.163 ± 0.573), antioxidants (2.589 ± 0.115 mg/g Dw) and IC50 values for DPPH free radical scavenging activity (12.953 μg/ml) was calculated. The observations substantiate the folk claims and the significant biological properties prompted to carry out further studies to isolate the bioactive principles involved in the property which is under progress.


INTRODUCTION
Euphorbiaceae is the largest family among the anthophyta, represented with 300 genera and 5,000 species all over the world.The genus Euphorbia, with characteristic flower structure, has strong representation in the humid tropics of both hemispheres and consists of 2,000 species ranging from annuals to trees (Binojkumar and Balakrishnan, 2010;Pullaiah, 2006).Euphorbia species are used for the treatment of various human ailments such as skin diseases, gonorrhoea, migraine, intestinal parasites, antiviral (Ramezani et al., 2008), antimicrobial (Al Younis et al., 2009;Papp, 2004) and also involved as one of the common constituents in popular pharmaceutical preparations.The latex was used as fish poison and insecticide.In the present study, the crude drug samples of Euphorbia perbracteata were screened for qualitative and quantitative analysis for secondary metabolites like total phenol content, total flavonoids, total antioxidants and free radical scavenging activity.The extract was subjected for biological activites including antitumor activity, antimicrobial and brine shrimp assay.
ethnomedicinal properties were recorded based on intensive interviews conducted with adivasi herbal practitioners, bare foot doctors from tribal communites, inhabited in the forests and other adjoining rural areas.The voucher specimen was deposited in the Sri Krishnadevaraya University Herbarium (SKU), Anantapur.

Preparation of extracts
The plant material was air dried and ground to moderately fine powder and 25 g of each plant part was extracted with 200 ml of hexane, ethyl acetate by using soxhlet unit.The excess of solvents was recovered from the extract under reduced pressure with the Rotoflash evaporator.

Qualitative analysis of phytoconstituents
The semi-arid solvent extracts were subjected to preliminary phytochemical tests (Amarasingham et al., 1964;Gibbs, 1974;Harborne, 1996) and the positive reactions were observed for 30 different groups of secondary metabolic compounds.

Total phenol content
The extraction of total phenolics was performed using the Folinciocalteu assay (Saxena et al., 2007;Harborne, 1984) with some modifications.In total, 100 µl of each extract (1 mg/ml) was added to a test tube containing 50 µl of the phenol reagent (1 M).Further 1.85 ml of distilled deionized water was added to the solution and allowed to stand for 3 min after vortexed and then 300 µl Na2Co3 (20% in water, v/v) was added and made into a final volume of 4 ml with 1.7 ml of deionized water.The final mixture was vortexed and incubated for 1 h in dark at room temperature.Thereafter, absorbance was measured at 725 nm on shimadzu UV-spectrometer (V-2.43 model).A standard curve was prepared using 0.65, 125 and 250 mg/l gallic acid in methanol (50:50 v/v).Total phenolic values were expressed in terms of gallic acid equivalents (GAE) in milligrams per gram plant extract.All determinations were performed in triplicate.

Total flavonoids
The total flavonoid content in extracts was determined as described by Moreno et al. (2000).Briefly, 0.5 ml sample (1 mg/ml) was mixed with 0.1 ml each of 10% aluminium nitrate and potassium acetate (1 M) and 4.3 ml of 80% ethanol was added to make a total volume of 5 ml.The mixture was vortexed, incubated for 40 min at room temperature and absorbance was measured by spectrophotometry at 415 nm.All determinations were performed in triplicate.Total flavonoid values were expressed in terms of quercetin equivalents (QE) per gram of plant extract.A standard curve was prepared using 0.5, 10 and 100 mg/l solutions of quercetin.

Total antioxidants
For total antioxidant activity assay, 0.1 ml extract (10 mg/ml) was dissolved in dimethyl sulphoxide (DMSO) combined with 1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in an eppendrof tube and incubated in a thermal block at 95°C for 90 min.After cooling to room temperature, the absorbance was measured at 695 nm.Ascorbic acid was used as the standard and the total antioxidant activity was expressed as equivalents of ascorbic acid (Kim et al., 2007;Kumar et al., 2010).

DPPH free radical scavenging activity
The hydrogen atom and electron donation abilities of the corresponding extracts were measured from the bleaching of the purple colored methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH).This spectrophotometric assay uses the stable radical DPPH as a reagent (Burits and Bucar, 2000).A volume of 1 ml of various concentrations (2, 4, 6, 8, 10, and 12 µg/ml) of the extracts in methanol was added to 4 ml of 0.004% methanol solution of DPPH.After a 30 min incubation period at room temperature, the absorbance was read against blank at 517 nm.The DPPH free radical activity was calculated by percent inhibition using the following formula.
Where A blank is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compound.The extract concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against extract concentration.Tests were conducted in triplicate to confirm the activity.The lower absorbance of the reaction mixture indicates higher free radical scavenging activity.Ascorbic acid was taken as standards.

Brine shrimph assay
The procedure described by Meyer et al. (1982) was adopted.In brief, a rectangular dish (22 × 32 cm) was compartmentalized into two unequal halves with plastic divider of 2 mm with several holes and filled with artificial sea water 28 g sea salt/L (Sanders, grate salt lake, South ogdon, Utah).Approximately 25 mg eggs (Artemia salina) were sprinkled in the larger compartment, which was darkened, while the smaller compartment was illuminated.After 24 h, phototropic nauplii (brine shrimp larvae) were collected by pipette from the lightened side.0.5 ml of 10, 50, 100, 500, 1000, 10,000 µg/ml concentrations of the methanol extract was poured in vials and kept at room temperature until the evaporation of solvent.About 2 ml of sea water was added and 10 shrimps were transferred to each vial.The vials were placed under the illumination at room temperature (25 to 28°C) and after 24 h the number of survivors was counted to calculate LC50 using probit analysis (Kumar et al., 2010).

Potato disc tumor bio assay
For antitumor activity, the procedure described by Galsky et al. (1980) and Kahl and Schell (1982) was followed.In brief, Agrobacterium tumefaciens virulent strains were grown for 48 h in Lauria broth medium containing rifampicin (10 µg/ml).Red skinned potatoes were surface sterilized in 0.1% mercuric chloride solution for 10 min and thoroughly washed with autoclaved distilled water.Potato discs (5 mm × 8 mm) were made with cork borer and placed on agar (2%) plates (10 discs per plate).Agrobacterium culture (2 ml) mixed with 50 µl each of 10, 100, 1000 µg/ml of plant extract (prepared in DMSO) and applied on the surface of each disc of respective concentration.Petri plates were then placed at 28°C for % of tumor inhibition = 100 -ns/nc × 100 Where ns = number of tumors in sample; nc = number of tumors in control.

Antimicrobial evaluation
Preparation of extracts and filter paper discs: Dry powder of 100 g of test material was loaded onto soxhlet apparatus and fractionated in 250 ml of different polar solvents successively.The extracts obtained were filtered and concentrated under reduced pressure, below 40°C to dryness and the residue was subjected for antimicrobial activity.The crude extracts of each sample dissolved in dimethyl sulfoxide (DMSO) and the concentrations of 25 to 75 mg/ml were prepared.Whatman's no.1 filter paper discs of 6 mm diameter were prepared and autoclaved by keeping in a clean and dry petri plate.The filter paper discs were saturated with 20 to 25 μl of different concentrations of stock solutions prepared in DMSO.
Later, the filter paper discs were carefully dried on the laminar airflow bench and used for antimicrobial studies.

Minimum inhibitory concentration (MIC)
The extracts demonstrating the significant zones of inhibition which were used for determining the minimum inhibitory concentration (MIC) of crude extracts determined by the broth-micro-dilutions (Koneman, 1995) using 96 well microtitre plates.Initially the extracts were dissolved in DMSO to create a concentration of 10 mg/ml of stock solution.100 μl of this solution was serially diluted (two fold dilution) to obtain different concentrations.10 μl of the previously prepared different microbial suspensions (10 5 CFU/ml) were added to each well.Plates were incubated for 18 h at 37°C and then were examined with Elisa reader (TECAN, Sunrise -China) at 620 nm and the lowest concentration of each extract showing growth was taken as its minimum inhibitory concentration (MIC).The bacterial suspension was used as positive control, extracts and DMSO solution in broth were used as negative control.All samples were tested in triplicates to confirm the activity.

RESULTS
Phytochemical analysis of n-hexane, ethyl acetate, methanol and water extracts of whole plants revealed that the alkaloids were observed in all the extracts, while flavonoids and phenols were present only in methanol and aqueous extracts, followed by glycosides, catacholic compounds and coumarins (Table 1).

Quantitative analysis
Plant phenols are the most widely spread secondary metabolites in plant kingdom and have received much attention as potential natural antioxidants in terms of their ability to act as both efficient radical scavengers and metal chelators.The yield of the extract was analysed for quantity of total phenols and falvonoids, and antioxidant content mg/g of dry material were provoded (Table 2).

Free radical scavenging activity (DPPH)
Scavenging activity for free radical of 2,2-diphenyl-1picrylhydrazyl (DPPH) has been widely used to evaluate the antioxidant activity of natural products from plant sources.Test extracts were quantitatively determined

Bio assay studies
Bio assays offer special advantages for identification of medico-botanical extracts.Most often a desired biological response is not due to one component but rather to a mixture of bioactive plant components.Therefore, crude extracts were screened for biological activity followed by fractionation to analyse active extract and directed with bio-assays to exploit the bioactive compounds.

Brine shrimp toxicity
The brine shrimp lethality assay is one of the con-venient probes for the pharmacological evaluation of plant extract.Different concentrations were were subjected to examine the activity and the results were tabulated (Table 3).None of the plant extracts were effective at probability level 0.05.LC 50 values describe that the methanolic extract was more effective with LC 50 value 944.20 followed by n-hexane while ethyl acetate extract failed to show the activity.

Potato disc assay
1995).In the present study, the ethyl acetate extracts were assessed for antitumor activity using the potato disc assay which is a well accepted assay for the preliminary screening of crude extracts for potential anticancer and antitumor activity.The extracts have exhibited the activity as dose-dependent as inhibition of tumors.The percent of inhibition at various concentrations (10, 100, 1,000 µg/ml) with their level of antitumor activity was analysed (Table 4 and Figure 1).

Antimicrobial activity
The test solvent extracts of E. perbracteata exhibited significant inhibition which ranged from 9 to 17 mm against all test microorganisms except Candida albicans, Salmonella typhimurium and Micrococcus luteus.The observations revealed that Klebsiella pneumoniae, Pseudomonas aeruginosa and Bacillus cereus were more susceptible than other test pathogens.The MIC values (156 to 312 µg/ml) were identical for all polar solvents (Table 5).
Crown gall is a plant neoplastic disease induced by the Gram-negative bacterium Agrobacterium tumefaciens (Kahl and Schell, 1982;Koneman,

DISCUSSION
Medicinal plants contain different antimicrobial compounds which are responsible in killing the pathogen or inhibit their growth.Several studies revealed similar results in genus Euphorbia

Graph-1
Euphorbia perbracteata (Wp) against Gram positive and Gram negative bacteria.The qualitative analysis of the extracts from whole plant of Euphorbia perbracteata showed the presence of phytochemical constitutents such as alkaloids, flavonoids, phenolic compounds, glycosids, catacholic compounds and coumarins which are responsible for antimicrobial activities.
The phenol compounds are known to be toxic to microorganisms.The site and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to the microorganisms.The probable mechanism responsible for phenolic toxicity to microorganisms include enzyme inhibition by oxidized compounds possibly through reaction with sulfhydryl groups or through more nonspecific interaction with proteins.Flavonoids are also hydroxylated phenolic substences but occur as a C 6 -C 3 unit linked to an aromatic ring, since they are known to be synthesized by plants in response to microbial infection and found in vitro to be effective antimicrobial substences against a wide array of microorganisms.
Free radicals are produced in our body as a result of oxidative biochemical metabolisms which causes damage to cells and are responsible for ageing and other ailments such as cancer.Flavonoids are flavone-like substances that are usually antioxidants.Flavonoids scavenge free radicals by forming a stable radical that can react with another flavonoid radical to produce two non-radicals.
It is concluded that the test material plant contains potential antibacterial, antifungal and antitumor components that may be useful for evaluation of pharmaceutical property against the reported ailments.Further studies have been conducted in order to isolate, identify, characterize and elucidate the structure of bioactive principles to develop new antimicrobial and antitumor medications.

Table 2 .
Total amount of Phenolic compounds, flavonoids and antioxidants of E.perbracteata (wp).

Table 4 .
Effect of the ethyl acetate extract of E. perbracteata on Agrobacterium tumefaciens.