PRELIMINARY STUDIES ON PHYTOCHEMICALS AND ANTIMICROBIAL ACTIVITY OF SOLVENT EXTRACTS OF MEDICINAL PLANT LAWSONIA INERMIS.

Medicinal plants comprise bioactive compounds called phytochemicals which are used for curing of various human diseases. Phytochemicals are non-nutritive, chemical compounds that occur naturally on plants and have diverse protective properties. These compounds are produced by plants for their protection. Recent research demonstrates the protective role of such compounds against human diseases in several medicinal plants. In this present study the compositional analysis of the medicinal plant Lawsonia inermis was documented. Solvent extraction of leaf samples with Chloroform, Acetone, Ethanol, Methanol, and water extracts of leaf samples revealed the phytochemical constituents in the plants. In this study, antimicrobial activity was performed by disc diffusion and well diffusion method. The maximum activity was showed in methanol extraction against pathogens. The The results of the present study revealed the presence of phytochemicals in the extract of Lawsonia inermis. It plays a major role in pharmacological activity spectrum. In moderndays people looking for nontoxic medication so it can used as traditional medicine and without any toxicity and side effects. Important medicinal phytochemicals such as terpenoids, reducing sugar, flavanoids and alkaloids were present in the samples. By considering these results we can carried out for further experiment to get more medicinal and industrial products. secondary metabolites active these secondary metabolites result of phytochemical analysis shows that the experimental plant is rich in phytochemicals. The phytochemical screening of medicinal plant Lawsonia inermis Due to the presence of several chemical components it can be used as analgesic, hypoglycemic, hepatoprotective, immunostimulant, anti-inflammatory, antibacterial, antimicrobial, antifungal, antiviral, antiparasitic, antitrypanosaomal, antidermatophytic, antioxidant, antifertility, tuberculostic and anticancer properties. It clearly shows that the plant Lawsonia inermis is considered as valuable source for traditional and industrial products. The results obtained from the phytochemical test shows correlation with further studies in some ways and contradiction in some other ways. In the present investigation, the phytochemical screening of ethanolic extract of Lawsonia inermis shows the presence of phytochemicals such as Aminoacids, Phenolic compounds, Reducing sugar, Terpenoids, Saponins and Steroids out of eleven for which screening has performed. Similarly, the methanolic extract shows the presence of Alkaloids, Aminoacids, Phenolic compounds, Reducing sugar, Terpenoids, Glycosides, Flavonoids,

Medicinal plants comprise bioactive compounds called phytochemicals which are used for curing of various human diseases. Phytochemicals are non-nutritive, chemical compounds that occur naturally on plants and have diverse protective properties. These compounds are produced by plants for their protection. Recent research demonstrates the protective role of such compounds against human diseases in several medicinal plants. In this present study the compositional analysis of the medicinal plant Lawsonia inermis was documented. Solvent extraction of leaf samples with Chloroform, Acetone, Ethanol, Methanol, and water extracts of leaf samples revealed the phytochemical constituents in the plants. In this study, antimicrobial activity was performed by disc diffusion and well diffusion method. The maximum activity was showed in methanol extraction against pathogens. The main objective of the research work was to check the presence or absence of the phytochemical constituents in the selected medicinal plant.

ISSN: 2320-5407
Int. J. Adv. Res. 7 (10), 887-896 888 pathological processes such as in ageing, neurodegenerative disorder, atherosclerosis and inflammation (Kawoand Kwa, 2011). In addition to protective effects, they contribute to the plant's color, aroma and flavor. Phytochemicals are found to be accumulated in different parts of the plants, such as in the roots, stems and leaves. Phytochemicals are categorized as primary and secondary constituents in which proteins, chlorophyll and common sugars are included in primary constituents and terpenoid, alkaloids and phenolic compounds are grouped into secondary constituents (Krishnaiah et al., 2007), Terpenoids exhibit various important pharmacological activities (Mahato and Sen, 1997), Alkaloids are used as anaesthetic agents and are found in medicinal plants (Hérouart et al., 1988). These compounds are known as secondary plant metabolites and have biological properties such as antioxidant activity, antimicrobial effect, modulation of detoxification enzymes, stimulation of the immune system, decrease of platelet aggregation and modulation of hormone metabolism and anti cancer property (Saidulu et al., 2014).
Once such medicinal plant commonly known as henna, mehendi available in tropical and subtropical areas has a long history of traditional medicine in India due to its diverse uses and appreciable role in ayurvedic and natural herbal medicines (Lavhate and Mishra, 2007).
Henna is a flowering plant, having a height of 5 meters, natal to subtropical and tropical regions of world including South Asia, Africa, and oases of Sahara Dessert and even in northern regions of Australia. Leaves of henna plant are entire, opposite, sub-sessile, oval-shaped and smooth (Ashnagar et al., 2011). Leaves have length of 2-3 cm with 1-2 cm width (Basirian Mina et al., 2012). Henna shrub is highly branched and has greyish-brown barks (Chang et al., 2006). Main chemical constituents of henna are Lawsone (2-hydroxynaphthoquinone), mucilage, mannite, gallic acid and tannic acid (Nisa et al., 2013). Henna is known to be used as a cosmetic agent for dyeing hair, nails and skin (Kawoand Kwa, 2011).
In traditional medicine, henna plant is used to treat many diseases like oedema, bronchitis, menstrual disorder, rheumatism, hemorrhoids and even in jaundice, leprosy, pain, spleen enlargement, dysentery and skin problems (Rahmoun et al., 2010;Mahato and Sen, 1997;Bhuvaneshwari et al., 2002;Warrier et al., 1995). Henna can also be used as an astringent and antihemorragic agent and is also known for its hypotensive, cardio inhibitory and sedative effects (Rahmoun et al., 2010). In addition, henna is reported to show some other properties including hypoglycemic (Syamsudin and Winarno, 2008), immunostimulant (Mikhaeil et al., 2004), hepatoprotective (Chaudary et al., 2012), anti-inflammatory (Singh et al., 1982), tuberculostatic (Sharma, 1990), anti-cancer and antioxidant properties (Kamal and Jawaid, 2010). Lawsonia inermis is a small tree or large shrub growing to six meters high. It has lateral branches with leaves that grow in pairs, two or four centimeters. Throughout India, plant parts commercially cultivated in Punjab, Gujarat, Madhya Pradesh, Rajasthan and Tamilnadu (Olopade et al., 1992). Leaf is deciduous shrub with lateral branches often ending in spines; leaves are simple, opposite, entire, lanceolate, petioles very short or absent. Flower is white or rose coloured fragrant in large terminal pyramidal panicled cymes, stamens 8 in 4 pairs inserted on the calyx tube. Fruits is globose capsules, tipped with the style and supported by the persistent calyx, seeds numerous, smooth, pyramidal. Genus Lawsonia consists of one species, Lawsonia inermis (Henna, mehendi, shudi, Madurang, Mendi, Manghati, Madayantika and goranti), (Gupta, 2003). It is biennial dicotyledons herbaceous herb. The origin of henna plant is North Africa and south west Asia. It is grown for ornamental as well as dye plant. It is much branched glabrous shrub or small tree (2 to 6 m in height. Leaves are small, opposite in arrangement along the branches, sub-sessile, about 1.5 to 5 cm long, 0.5 to 2 cm wide, greenish brown to dull green, elliptic to broadly lanceolate leaves with entire margin, petiole short and glabrous and acute or obtuse apex with tapering base. Young branches are green in colour and turn red with age. Bark is greyish brown, unarmed when young but branches of older trees are spine tipped. Inflorescence is a large pyramid shaped cyme. Flowers of henna are small about 1cm across, fragrant, white or rose coloured with four crumbled petals. Calyx is 0.2 cm tube and 0.3 cm spread lobes. Fruit is a small brown coloured round capsule. Fruit opens irregularly and splits into four sections at maturity and is many seeded. Seeds are about 3mm across, numerous, smooth, pyramidal, hard, thick seed coat with brownish coloration (Chauhan and Pillai, 2007).
Henna plant has several ethno botanical uses. It is widely used as medicinally and cosmetically. Henna leaves consist of 0.5-1.5% lawsone which is responsible for orange red dye color. It has an orange red dye and leaf paste or powder is used for decorating hands, nails and feet. It can also be used as a hair dye. Henna plant leaves are used to cure jaundice, skin diseases, veneral diseases, smallpox, and spermatorrhoea. Henna plant leaves, flowers, seeds, stem bark, roots are used as agent to treat ailments as leprosy, fever, eucorrhea, diabetes, cardiac disease, rheumatoid arthritis, headache, ulcers, diahorrea, hepatoprotective and coloring (Vasudevan and Laddha, 2003). Based on this 889 view, the present study was aimed to analyze the chemical constituents present in the solvent extracted leaves of Henna using biochemical studies.

Methodology:-Collection and processing of plant material:
The leaves of Lawsonia inermis (Plate 1) were collected from the local area for its freshness, healthy and free from any deformation. The collected leaves were transferred immediately to the laboratory for analysis. The samples were shadow dried for a week and blended into powder by mixture blender. Then it was passed through the sieve to get the equal size particles. The powder was kept in air tight container at moisture free environment for further analyses. 890

Plant Extract Preparation using Hot Method
The powdered leaves were extracted with different solvents by hot method using soxhlet apparatus. Briefly, 10 gm of powdered plant leaves were taken in clean sterile soxhlet apparatus and extracted with 150 ml of different solvents such as Chloroform, Acetone, Ethanol, Methanol, and water (low polar to high polar). After extraction, the extracts were dried at room temperature until reaches the solid (powder) form. Subsequently, the extracts were dissolved in Dimethyl sulfoxide (DMSO) to the concentration of 10 µg ⁄ml for further analysis.

Phytochemical Analysis
The preliminary phytochemical analyses of the different extracts were determined using the standard methods ( Anthraquinones 1ml of plant extract is boiled with 10% HCL for few minutes in a water bath. It is filtered and allowed to cool. Equal volume of CHCL 3 is added to the filtrate few drops of 10% Ammonia are added to the mixture and heat. Formation of rose pink colour indicates the presence of anthraquinones.

Screening of Antibacterial Activity
The pathogenic cultures of bacterial strains were used. The bacterial strains were obtained from clinical laboratory. Five pathogens were chosen for the present investigation. The cultures were prepared separately in nutrient broth. The culture was stored at 4 0 C and it was taking just before performing the experiment. Name list of the strains used in the present study, 1. Staphylococcus aureus 2. Escherichia coli 3. Pseudomonas aeruginosa 4. Proteus vulgaris 5. Streptococcus mutans

Disc Diffusion method (Perz et al., 1990)
Antibacterial activity of the extracts was tested by Disc-diffusion method. 0.2 ml bacterial cultures (4hrs) were uniformly spread on solidified nutrient agar medium. The filter paper discs prepared with plant extracts were carefully placed over the spreaded cultures by using sterile needle and incubated at 370°C for 24hrs. After the incubation period, the plates were examined for inhibitory zones (including the diameter of the disc).

Well diffusion assay
It was used to test the antagonistic activities of Lawsonia inermis extract against various test organisms.The selected strains of bacteria were inoculated in to 5ml of sterile nutrient broth, and incubated at 37°C for 16-18 hrs. The cotton swab and agar plates were prepared. Using the sterile cotton swabs, the nutrient broth cultures were swabbed on the surface of sterile nutrient agar plates. The well was created on the agar plates by using sterilized cork borer with 10mm diameter. 100μl of different solvents extract of Lawsonia inermis were transferred into each well. The plates were incubated for 24 hrs at 37°C and examined for clear inhibitions zone around the well. The diameter of inhibition zones was measured in mm and the results were recorded (Baues et al., 1996).

Results and Discussion:-
The herbal medicines serve the health needs of about 80% of the world's population, especially for millions of people in the vast rural areas of developing countries; more than 65% of the global population uses medicinal plants as a primary health care modality (WHO, 2001). In recent years, many possible sources of natural antibiotics have been in use for several infectious diseases, mostly bacterial and fungal. In view of this, the searches for new antimicrobial agents from medicinal plants are even more urgent in the countries like India where infectious diseases of the commonly used antibiotics (Abebe et al., 2003). Considering the high costs of the synthetic drugs and their various side effects, the search for alternative products from plants used in folklore medicine is further justified. It is believed that plant chemical classes such as sterols, alkaloids, glycosides, saponins, flavonoids, tannins, and carbohydrates are generally superior in their antimicrobial activities (Cowan, 1999).

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The results of the present study revealed the presence of phytochemicals in the extract ofLawsonia inermis. It plays a major role in pharmacological activity spectrum. In moderndays people looking for nontoxic medication so it can used as traditional medicine and without any toxicity and side effects. Important medicinal phytochemicals such as terpenoids, reducing sugar, flavanoids and alkaloids were present in the samples. By considering these results we can carried out for further experiment to get more medicinal and industrial products.
The secondary metabolites mainly attribute the antimicrobial activity to plants (Gonzalez-Lamothe et al., 2009). The active constituents of these secondary metabolites include phenolic compounds and tannins (Edwin, 1996). The result of the phytochemical analysis shows that the experimental plant is rich in phytochemicals. The phytochemical screening of medicinal plant Lawsonia inermis is shown in (Table 1). Due to the presence of several chemical components it can be used as analgesic, hypoglycemic, hepatoprotective, immunostimulant, anti-inflammatory, antibacterial, antimicrobial, antifungal, antiviral, antiparasitic, antitrypanosaomal, antidermatophytic, antioxidant, antifertility, tuberculostic and anticancer properties. It clearly shows that the plant Lawsonia inermis is considered as valuable source for traditional and industrial products. The present study has been under taken to find out the effectiveness of the different extracts of Lawsonia inermis leaves against some bacterial pathogens. Out of five strains selected three strains showed good antimicrobial potential against our plant extract.
The antimicrobial potency of Lawsonia inermis leaf extracts were tested against some bacterial pathogens was quantitatively assessed for the presence or absence of zone of inhibition. The results relative to antibacterial activity was observed by measuring the diameter of the zone of inhibition (Figure 1). By disc diffusion method the activity of methanol extracts of Lawsonia inermis against Pseudomonas aeruginosa the zone of inhibition obtained at 75% (3.3±3.16mm) followed by 100% (2.6±2.16mm), 50% (2.2±2.4mm) and 25% (1.7±2.1mm) showed maximum activity when compared to all the three strains ( The important phytochemical properties identified by our study in the local plant of Lawsonia inermis will be helpful in the copping different diseases. Lawsonia inermis samples demonstrated antibacterial activity against all isolates but the highest susceptibility was against Pseudomonas aeruginosa of this particular region. Thus we concluded that the medicinal plants could be used as drug to treat the infections caused by bacteria without any side effects.

Acknowledgements:-
If words are considered as symbols of approval and token of acknowledgement, then words play the role of thanks to exhibit the deeply embedded feelings of gratitude. The authors express their gratitude to Karpagam academy of higher education, Coimbatore for providing laboratory facilities to carry out the research.

Conflicts of Interest
The authors declare that they have no conflict of interest.