In-Vitro Antimicrobial and Antioxidant Activity of Securinega leucopyrus (Willd) Muell

To evaluate antimicrobial and antioxidant activity of leaf and bark of Securinega leucopyrus for anti-microbial activity against clinically important bacterial and fungal strains using agar disc diffusion method and for antioxidant activity. The methanolic extracts of leaf and stem bark were tested for antimicrobial activity against four gram positive bacteria, four gram negative bacteria, antifungal activity and for their antioxidant activity using total antioxidant capacity, total reducing power, ferric reducing antioxidant power and DPPH free radical scavenging activity. Securinega leucopyrus (Willd) Muell. is traditionally used for the management of boils and wounds in Sri Lanka and India. This shrub is clinically evaluated for wound healing as case reports and some clinical trials also showed encouraging results in diabetic wounds. So it is need of an hour to evaluate its antimicrobial as well as antioxidant activity for scientific validation. The highest antimicrobial activity was exhibited by 250μg/ml methanolic extracts of both leaf and bark. The extracts at the 250μg/ml concentration shows maximum antifungal activity against Aspergillus flavus used in the study. Methanolic extracts of leaves exhibited highest radicals scavenging activity, that is, 88.14 percent. Similar results were observed in total antioxidant capacity, total reducing power and ferric reducing assay determination. Methanolic extract of leaf and stem bark at different concentrations showed in-vitro antimicrobial and antioxidant activity.

Plant derived drugs are generally nontoxic, effective at low concentrations, easily affordable and environment friendly. It has been reported that some fruits, vegetables and herbs are effective for certain chronic diseases because they may show their bactericidal, antiviral, analgesic, antiinflammatory, anti-carcinogenic, and/or antioxidant actions. 1,2 Traditional medicines have a long history of serving the people all over the world. The use of medicinal plants to maintain public health and treat diseases in many countries with cultures of different nations is highly prevalent. 3,4 Nowadays, bioactive photochemical found in traditional plants have gained significant consideration because these compounds retard the lipids degradation, prevent microbial deterioration and improve the food quality. 5,6 Medicinal plants are rich sources of antimicrobial agents. Many infectious diseases have been known to be treated with herbal extracts. The clinical efficacy of many existing antibiotics is being threatened by the emergence of multidrugresistant pathogens. Although many plant species have been tested for antimicrobial properties, the majority of them have not been sufficiently evaluated 7 . As an alternate source to the existing antibiotics, there is an urgent need to discover new antimicrobial compounds from various medicinal plants which can be used to treat many infectious diseases.
Reactive oxygen species (ROS) are class of highly reactive molecules derived from the metabolism of oxygen. Rapid production of free radicals may lead to oxidative damage to bio-molecules and results in disorders such as degenerative disorders, cancer, diabetes, neural disorders and ageing.8 These free radicals occur in the body during an imbalance between ROS and antioxidants. Many medicinal plants have large amount of antioxidants such as Vitamin C, Vitamin E, polyphenols etc. Natural antioxidants increase the antioxidant capacity of the plasma and reduce the risk of certain diseases like heart disease, cancer. 9 There are many synthetic antioxidants but they have side effects, hence there is a need for more potent and less toxic antioxidants. 10 Securinega leucopyrus (Willd) Muell. belonging to family Euphorbiaceae known as Katupila (in Sri Lanka), Thumri (Sanskrit name), Humari (in Hindi), Shinavi (in Gujarat) and also called as "Spinous fluggea" in English. 11 Tribal people in Sri Lanka used various parts of this plant against cancer, wounds and ulcers in diabetes mellitus. 12 S. leucopyrus leaves act as an antiseptic and its paste is used in folklore to extract any extraneous materials from body tissues. Some clinical case studies reported the wound healing potential of this herb, [13][14][15] However no reports are available on its antimicrobial activity and antioxidant activity. Hence the study was planned to evaluate the in-vitro antimicrobial activity of S. leucopyrus leaf and bark.

Collection of Plant Materials
The plant was identified from its natural habitat of Kaduwela, Colombo, Sri Lanka and its morphological characters and comparing them with the characters mentioned in various floras. 11,16 The stem bark was collected, washed properly under running water, to make them free from foreign matter like sand, soil etc. and dried under shade. Herbarium voucher No. Phm 6126/2013/14 was also prepared and submitted to Pharmacognosy museum of IPGT & RA, Jamnagar, for future reference. sample Preparation For analysis S. leucopyrus leaf and stem bark was coarsely powdered to a sieve of 60 mesh size and then tests were performed.

antimicrobial activity extract Preparation
Methanol extract was used for the study, for this 1g of material was extracted in 50 ml of methanol by sonicating it for 10 min and then keeping it overnight. Next day after filtration, methanol evaporated, then by taking weight of residue 5 different concentrations 5 µg/ml, 25 µg/ ml, 50 µg/ml, 100µg/ml, 250µg/ml of each sample prepared. These are used for determination of antimicrobial activity.

antimicrobial assay
Sterile soybean casein digest agar (25 ml/plate) used for antibacterial activity and sterile sabouraud agar (25ml per plate) used for antifungal activity 17 . Medium obtained from Himedia laboratories. Sterile 20 ml medium poured in sterile plates aseptically and let them solidified. Then inoculate 0.5 ml of culture in 5 ml sterile melted and cooled medium and poured them on solidified agar plates aseptically. After solidification made well with the help of cup borer and inoculate 0.3 ml of each sample in the well and for antibiotic discs there is no need to make wells and directly place disc on agar surface aseptically. For diffusion purpose plates were placed in refrigerator for 20-25 minutes. Then Incubate plates at 370 C for 24 hrs except sabouraud agar plates and plates containing K. pneumoniae organism, they incubated at 300 C for 24-48 hrs. After incubation zone of inhibition was measured with Himedia antibiotic zone scalec. antioxidant activity total antioxidant Capacity 10% w/v methanolic extracts of both leaves and stem bark samples prepared by macerating overnight, diluted further with methanol were used for estimation of total antioxidant activity 18 . An aliquot of 0.1 ml of sample solution containing a reducing species in methanol was combined in an Eppendorf tube with one ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a thermal block at 950C for 90 min. After those samples were cooled to room temperature, the absorbance of the solution was measured at 695 nm against a blank. A typical blank solution contained 1 ml of reagent solution and the appropriate volume of same sample used for the sample, and it was incubated at the same conditions as the rest of the samples. Ascorbic acid solution (concentration range: 20 to 80 µg) was taken as the known standard. Total antioxidant capacity of the samples calculated in terms of ascorbic acid µg equivalents corresponding to 1 mg of original sample using the standard curve. total reducing Power 10% w/v methanolic extracts of the samples prepared by macerating overnight, diluted further with 1 ml methanol, were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of potassium hexacyanoferrate solution (1% w/v)19. The mixture was incubated at 500C for 20 min, 1.5 mL of trichloroacetic acid (10% w/v) was added to the mixture and filtered. 2.5 mL of filtrate was mixed with equal volume of distilled water and 0.5 mL of ferric chloride solution (0.1% w/v) and the absorbance was measured at 700 nm after 40 min. Ascorbic acid solution (concentration range: 10 to 40 µg) was taken as the known standard.
Increased absorbance of the reaction mixture indicates stronger reducing power. A calibration curve was plotted for absorbance of ascorbic acid standard against its concentration in micrograms and the equivalent values for samples were extrapolated from the slope equation of line against their respective absorbance. The results were expressed in microgram equivalents of ascorbic acid with respect to the original sample. Total reducing power of the samples calculated in terms of ascorbic acid µg equivalents corresponding to 1 mg of original sample using the standard curve.

Ferric reducing antioxidant Power (FraP assay)
FRAP assay was performed according to the methods of Benzie and Strain (1999) with slightly modification20. It is based on the principle of reduction of Fe3+-TPTZ to Fe2+-TPTZ complex at low pH which gives blue colour and can be measured at 593 nm. 0.1 ml of 10% w/v methanolic extracts of the samples prepared by macerating overnight, diluted further with methanol was added to 3.0 ml of FRAP working reagent, mixed well and absorbance was measured after 10 minutes. Freshly prepared aqueous ascorbic acid solution (0.1 mg/ml) was used as standard. Different volumes of ascorbic acid solution (equivalent to 2-8 µg) were used in same manner for calibration of standard curve and quantification was done in terms of mg equivalents of ascorbic acid. The blank was prepared by using distilled water in place of sample/standard. dPPh Free radical scavenging activity (dPPh assay) This assay measures the free radical scavenging capacity of the extract under investigation. DPPH is a molecule containing a stable free radical [21][22][23] . In the presence of an antioxidant, which can donate an electron to DPPH, the purple colour which is typical for free radical decays and change in absorbency at 517 nm was measured using PerkinElmer Lambda 25 UV-Visible spectrometer. 0.3 mM DPPH solution was prepared in methanol (freshly prepared), for standard Ascorbic acid solution -1 mg/ml in methanol was prepared. Different volumes (100, 200, 250 and 300 µg) of test samples/standard were taken in a set of test tubes and methanol was added to make the volume to 3 ml. To this, 1 ml of DPPH reagent was added mixed thoroughly  24 Antibiotic treatment is a preferred choice to treat bacterial infections; however, emergence of antimicrobial resistance and toxicity issues subside the use of antibacterial agents. 25,26 Safety-and efficacy-related limitations to antibiotics augment biological research on the antimicrobial role of plants due to comparable toxicity and efficacy. 24 In the present study, we have investigated the antibacterial activity of different concentrations   (Table-2).The zone of inhibition around the disc impregnated with plant extract over the lawn of bacterial and fungal culture plates determined the antimicrobial activity as quantitatively. The result showed that the antimicrobial activities of plant extracts were increased with increasing concentration. Both, sample-A which was methanolic extract of leaves and sample-B which was methanolic extract of bark, at 250µg/ml showed highest inhibition of growth of all four Gram-positive bacteria with inhibition zone ranging from 11 to 17 mm and all four Gram-negative bacteria with inhibition zone ranging from 13 to 18 mm. Gentamycin (10 µg/ ml), Cifpodoxime (10 µg/ml) and Streptomycin (30 µg/ml) were used as a positive control which showed highest inhibition against Bacillus subtilis with inhibition zone of 28 mm, Staphylococcus aureus with inhibition zone of 23 mm and Bacillus subtilis with inhibition zone of 27 mm respectively. The reason accounting for antimicrobial activity of methanolic extracts might be firstly due to Both extracts at 250µg/ml concentration shows maximum antifungal activity against Aspergillus flavus used in the study (Table-3). Antifungal activity exhibited may attributes to the presence of these secondary metabolites. These compounds can combat with pathogens by different mode of action. 29

antioxidant activity
In recent years much attention has been devoted to natural antioxidants and their health benefits. Antioxidant-based drug formulations are used for the prevention and treatment of many complex diseases. Plants are a major source of natural antioxidants; they produce a wide range of secondary metabolites with antioxidative activities that have therapeutic potential. Polyphenols are the most abundant antioxidant compounds of plant raw material. Their antioxidant activity is based on to their redox properties, which facilitate their activity as reducing agents, hydrogen donors, singlet oxygen quenchers, metal chelators and reductants of ferryl hemoglobin. The reducing ability is generally associated with the presence of reductants which exert antioxidant action through breaking the free radical chain by donating a hydrogen atom or preventing peroxide formation. 30 Medicinal plant tissues are commonly rich in phenolic compounds such as flavonoids, phenolic acids, stilbenes, tannins, coumarins, lignans and lignins. These compounds have multiple biological effects including antioxidant activity. 31,32 The methanol extracts of plant showed a high effective total antioxidant capacity, high effective free radical scavenging in DPPH assay, total reducing power and Ferric reducing antioxidant power.

total antioxidant Capacity
Total antioxidant capacity is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of green phosphate/Mo (V) complex at acid pH. TAC of the phosphomolybdenum model evaluates both watersoluble and fat-soluble antioxidant capacity (total antioxidant capacity). The results indicate a total antioxidant capacity in both samples (Table-4). It means that the methanol extracts of leaf and stem bark will have to contain as much quantity of antioxidants compounds as equivalents of ascorbic acid to effectively reduce the oxidant in the reaction matrix33. Antioxidant capacity of ascorbic acid has been used as a reference standard from which plant extracts with potential antioxidant activity are compared (Figure-1).

total reducing Power
For the measurements of the reductive ability it has been investigated from the Fe3+ to Fe2+ transformation in the presence of extract samples using the method described by 0yaizu (1986) . 19 The data shows that leaves of S. leucopyrus have a moderate antioxidant activity as far as this in vitro method is concerned (Table-4). The values are expressed in ascorbic acid microgram equivalents with respect to original sample (Figure-2). The reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain or by donating a hydrogen atom. 34

FraP assay
FRAP assay method depends upon the reduction of ferric tripyridyltriazine [Fe (III)-TPTZ] complex to ferrous tripyridyltriazine [Fe (II)-TPTZ] by a reductant at low pH. Fe (II)-TPTZ has an intensive blue colour and can be monitored at 593 nm. FRAP values are obtained by comparing the absorbance change at 593 nm in test  35 Ferric reducing antioxidant power of the samples calculated in terms of ascorbic acid µg equivalents corresponding to 1 mg of original sample using the standard curve. 21 The ascorbic acid standard curve for FRAP at a concentration range of 2 to 8 µg (Figure-3). Methanolic extracts of leaves showed mean 51.93 ascorbic acid µg equivalent/mg of sample and stem bark showed mean 29.45 Ascorbic acid µg equivalent/mg of sample (Table-4).

dPPh assay
The DPPH assay has been largely used as a quick, reliable and reproducible parameter to search for the in-vitro antioxidant activity of pure compounds as well as plant extracts. It was observed that leaves of S. leucopyrus had higher free radical scavenging activity than that of stem bark. At a concentration of 300 µg, the scavenging activity of methanol extract of the leaves reached 88.14 %, while at the same concentration, that of the stem was 47.25 %. The usual trend to show scavenging activity is by indicating IC50 (Half-maximal Inhibitory concentration). IC50 for leaf was 153.66 µg and for stem bark 308.89 µg (Table-5). The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability. 36,37 The study showed that the extracts have the proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants. 38

ConClusion
The result obtain in the present study are in agreement to a certain degree with the traditional use of the plant. In this screening work, the test drug extract at different concentrations was found to be comparatively highly effective against all organisms such as Gram positive, Gram negative and single fungal strain. From the above results the activity of all extracts showed significant antibacterial and antifungal activity, however the results shown by leaf extract were better than stem bark. The present study justified the claimed ethnic uses of Katupila (Securinega leucopyrus) leaf and stem bark externally in ringworms, scurvy, snakebite, sprains, bruises, rheumatic swelling, wounds and to treat various infectious diseases caused by the microbes.

aCknowledgeMent
The authors wish to thank Director Prof. AB Thakar, Institute of Postgraduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar for providing facilities and consulting staff.