The search of novel anti-inflammatory agent is not considered as an ending process, since most of the clinically used anti-inflammatory drugs such as NSAIDs, Coxibs, GCs etc. are allied with considerable toxicity. However, numerous approaches were used to overcome the toxicity level such as co-administration with suitable agent/substance which provides protection against toxicity as well to synthesise new potent and safe anti-inflammatory drug. Although the drug treatment has been improved steadily but yet, it is still a challenge for the medicinal chemists to identify more potent therapeutic agents to treat or reduce the symptoms of inflammatory diseases [1]. In addition, it is well documented that bacterial infections often produce pain and inflammation and therefore chemotherapeutic agent along with anti-inflammatory compounds are prescribed simultaneously to treat bacterial infections having inflammatory disorders. Unfortunately, none of the drugs possesses these two activities in a single component. Therefore, our aim is to find a compound having dual antimicrobial and anti-inflammatory activities.

      Moreover, medicinal plants are the richest bio-resources for their wide variety of chemical compounds which have important biological functions and enables as an herbal medicines having beneficiary role in the pharmacology [2]. The bioactive compounds (alkaloids, tannins, flavonoids etc.) present in traditionally used medicinal plants possess definite physiological action on the human body [3]. In addition, most of the all bioactive constituents are known to have therapeutic value as anti-constipative, spasmolytic, antibacterial, antifungal, anti-inflammatory, insecticidal and antioxidant [4].

      The plant Martynia annua Linn. (family: Martyniaceae), is a herbaceous erect, branched, grandular hairy annual herb. Fruits are hard, woody with two sharp re-curved hooks and seeds are oblong [5]. The common names of plant include Devil’s Claw (English), Bichhu (Hindi), Kakanasika (Sanskrit) and Vichchida (Gujarati). The leaves and fruits together contributes the most active biological part of the plant [6-7]. In Ayurveda the plant is known as kaakanassikaa and the ayurvedic pharmacopoeia of India recommended the seed of plant for arresting of graying of hair [8]. According to literature survey, the plant was found to possesses anticonvulsant [8], anthelmintic [9], analgesic and antipyretic [10], antibacterial [11], antifertility [12], antinociceptive and CNS depressant [13], antioxidant [14] and wound healing activity [15]. Therefore, the present study was carried out to investigate in vitro antibacterial and antiinflammatory activity of different organic extracts of leaves of Martynia annua Linn.

      Plant material: The leaves of Martynia annua plant were collected between the months of August-September 2017, from the local area of Yamuna Nagar district of Haryana state, India. After collection, the leaves were washed thrice with water to remove dust or debris and air dried. After total dryness, the leaves were ground to coarse powder using a blender, passed through the 40-mesh sieve and stored in well-closed container.

      Chemicals and drugs: The drug amoxicilline and diclofenac sodium were obtains as gift sample from Oscar Remedies Pvt. Ltd., Badi Majra,, Yamuna Nagar, Haryana 135001. All other chemicals were of analytical grade obtained commercially.

      Extraction procedure: A weighed quantity (100gm) of the coarsely powdered plant material was sequentially extracted with petroleum ether (60-80°C), chloroform, ethylacetate and methanol using ultra sonicator technique. All the extracts obtained were concentrated and completely dried. The collected extracts were used for phyto-chemical screening and in vitro anti-bacterial and anti-inflammatory activity.

      Analysis of phytochemicals: Quantitative analysis of all prepared extracts were carried out to investigate various phytoconstituents such as alkaloids, tannins, phenols, steroids, glycosides, saponins and flavonoids as per standard procedures and protocols [16-17].

In vitro antibacterial activity

      The in vitro antimicrobial potential of all the prepared extracts was carried out at Microbiology Laboratory, guru Gobind Singh College of Pharmacy, Yamuna Nagar-135001 (India) using the agar plate diffusion method using microorganisms cell suspension whose concentration was equilibrated to 0.5 McFarland. The bacterial strains used in this present study are Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia and Escherichia coli . All the bacterial strains were pre-cultured in nutrient broth overnight and then grown in Muller Hinton agar medium. The inoculums of microorganisms were prepared from bacterial culture. About 15-20 ml of Muller-Hinton agar medium was poured in the sterilized glass petridishes and allowed to solidify. One drop of each strain was spread over the medium by a sterile rod. Wells of 5 mm in diameter and about 2 cm apart were punctured in the cultured media using sterile cork borer. About 1 ml of plant extract was added to the wells. Inoculated plates were then incubated at 37°C for 24 hrs. Antibacterial activities were evaluated by measuring the diameters of inhibition zones. The Minimum Inhibitory Concentration (MIC) of petroleum ether, chloroform, ethylacetate and ethanol extract was determined as the lowest concentration of the plant extract inhibiting the visible growth of organism.

In vitro anti-inflammatory activity:

      The reaction mixture (5 mL) consisted of 0.2 mL of egg albumin (from fresh hen’s egg), 2.8 mL of phosphate buffered saline (PBS, pH 6.4) and 2 mL of varying concentrations of different organic extracts of Martynia annua so that final concentrations become 31.25, 62.5, 125, 250, 500, 1000 µg/mL [16]. Similar volume of double-distilled water served as control. Then the mixtures were incubated at (37±2) °C in a BOD incubator (Labline Technologies) for 15 min and then heated at 70 °C for 5 min. After cooling, their absorbance was measured at 660 nm (SHIMADZU, UV 1800) by using vehicle as blank. Diclofenac sodium at the final concentration of (78.125, 156.25, 312.5, 625, 1250, 2500 µg/mL) was used as reference drug and treated similarly for determination of absorbance. The percentage protection from denaturation is calculated by using the formulae and it is tabulated.

      % inhibition = (1- Vt / Vc) x 100

      Where, Vt = absorbance of test sample, Vc = absorbance of control.

Phytochemical screening

      The phytochemical screening of different extracts of Martynia annua revealed the presence of medicinally active constituents as depicted in Table 1. The prelimininary phytochemical analysis of Martynia annua leaves indicated the presence of alkaloids, glycosides, saponins, phenolic compounds, tannins, proteins and terpenoids in the solvents ethylacetate and ethanol. However, chloroform extract shows the presence of alkaloids, glycosides, saponins, phytosterol, quinones and terpenoids. In addition, Petrolium ether extract shows only presence of phytosterol, quinones and terpenoids.

Table 1

Table 1: Phytochemical screening of the leaf extracts of Martynia annua Linn.

In vitro antimicrobial activity

      The antibacterial activity of the tested extracts of Martynia annua showed significant dose dependent reduction in bacterial growth in terms of zone of inhibition (Table 2). In the present study, maximum growth of inhibition (28 mm) was observed in Martynia annua ethanolic leaf extracts at 100 μg/mL against Staphylococcus aureus which was followed by Bacillus subtilis (27 mm), Klebsiella pneumonia and Escherichia coli (25 mm).

Table 2

Table 2: Antibacterial activity of leaf extracts of Martynia annua L. against human pathogenic microorganisms.

      Similarly, ethyl acetate extracts showed maximum growth inhibition (22 mm) at the concentration of 100 μg/mL against Staphylococcus aureus, followed by Bacillus subtilis (20 mm), Klebsiella pneumonia (16 mm) and Escherichia coli (14 mm).

      In addition, chloroform extracts at the concentration of 50 and 100 μg/mL showed significant antibacterial activity, against all the tested organisms the petroleum ether extract of Martynia annua leaf were found to be inactive at 25 and 50 μg/mL and showed minimum zone of inhibition against all the tested microorganisms.

In vitro anti-inflammatory activity

      The in vitro anti-inflammatory potential of different organic extracts of Martynia annua was evaluated against denaturation of egg albumin as depicted in Table 3. The present findings exhibited a concentration dependent inhibition of protein (albumin) denaturation by different extracts throughout the concentration range from 62.5µg/mL to 1000µg/mL. Diclofenac sodium at same concentration range was used as reference drug. However, out of all prepared extracts, the anti-inflammatory potential of ethanol, ethyl acetate and chloroform extracts was found to be more when compared with the standard drug diclofenac sodium. In addition, petroleum ether extracts also exhibited concentration dependent inhibition of protein denaturation; however, their effect was found to be less as compare to diclofenac sodium.

Table 3

Table 3: Anti-inflammatory activity of different organic extracts of leaves of Martynia annua Linn.