Phytochemical Analysis and Antimicrobial activity of leaf and stem of Ipomoea staphylina Roem. & Schult

Ipomoea staphylina Roem. & Schult.A plant belonging to Convolvulaceae is commonly found on hedges and bushes in the forests and wastelands. It is a perennial, woody and glabrous shrub with pink lowers. Traditionally Ipomoea staphylina is used for respiratory disorders. Traditionally genus Ipomoea is used as purgative, dyspepsia, anthelmintic, bronchitis. Medicinal plants are the primary sources of medicines in Ayurvedha, Siddha, and Folk medicine systems. In India, about 95% of all modern drugs are derived from medicinal plants and very likely most of these medicines are used by people to cure many ailments. The Ayurvedic literature Sarangdhar Samhita” highlighted the concept of polyherbalism to achieve greater therapeutic ef icacy. The active phytochemical constituents of individual plants are insuf icient to meet the desirable therapeutic effects.When combining the multiple herbs in a particular ratio, it will give a better therapeutic effect and reduce the toxicity. Most of them are active even at a low dose and safe at a high dose. Thus they have superior risk to bene it ratio. Based on this the present study deals with physicochemical, phytochemical studies such as and biochemical estimation of medicinal plant of a combined mixture of polyherbal Ipomoea staphylina., also study evaluates the ethanol and methanol extracts of leaf and stem for their preliminary phytochemical analysis, antibacterial and antifungal activity. In the study of the phytochemical analysis reveals the presence of carbohydrate, protein, lavonoids, glycosides, alkaloids. The Ipomoea staphylina of ethanolic leaf and stem extract showed potent antimicrobial activity in all the tested concentrations against E.coli, Staphylococcus aureus, Bacillus subtilis and Aspergillus niger.Ipomoea staphylina could be exploited as a valuable source of antibacterial agent enrichingwith known antimicrobial compounds. Further studies needed for futuredrugdevelopment to treat various infectious diseases by microbes.


Ipomoea staphylina
Roem. & Schult, phytochemical screening, antifungal activity, leaf and stem extracts, antibacterial activity ABSTRACT Ipomoea staphylina Roem. & Schult.A plant belonging to Convolvulaceae is commonly found on hedges and bushes in the forests and wastelands. It is a perennial, woody and glabrous shrub with pink lowers. Traditionally Ipomoea staphylina is used for respiratory disorders. Traditionally genus Ipomoea is used as purgative, dyspepsia, anthelmintic, bronchitis. Medicinal plants are the primary sources of medicines in Ayurvedha, Siddha, and Folk medicine systems. In India, about 95% of all modern drugs are derived from medicinal plants and very likely most of these medicines are used by people to cure many ailments. The Ayurvedic literature Sarangdhar Samhita" highlighted the concept of polyherbalism to achieve greater therapeutic ef icacy. The active phytochemical constituents of individual plants are insuf icient to meet the desirable therapeutic effects.When combining the multiple herbs in a particular ratio, it will give a better therapeutic effect and reduce the toxicity. Most of them are active even at a low dose and safe at a high dose. Thus they have superior risk to bene it ratio. Based on this the present study deals with physicochemical, phytochemical studies such as and biochemical estimation of medicinal plant of a combined mixture of polyherbal Ipomoea staphylina., also study evaluates the ethanol and methanol extracts of leaf and stem for their preliminary phytochemical analysis, antibacterial and antifungal activity. In the study of the phytochemical analysis reveals the presence of carbohydrate, protein, lavonoids, glycosides, alkaloids. The Ipomoea staphylina of ethanolic leaf and stem extract showed potent antimicrobial activity in all the tested concentrations against E.coli, Staphylococcus aureus, Bacillus subtilis and Aspergillus niger.Ipomoea staphylina could be exploited as a valuable source of antibacterial agent enriching with known antimicrobial compounds. Further studies needed for future drug development to treat various infectious diseases by microbes.

INTRODUCTION
Plants are integral to nature. Nature re lects the creative power of living god. Plants are a source of medicine has been an ancient practice and is a crucial component of the health care system in India, Andaman, NicobarIsland, Eastern Himalayas and the Western Ghats were concentrated region in more than 45,000 medicinal plants available in India (Kumar and Masooda, 2013;Rao et al., 2012).
The plant produces a wide variety of phytochemical constituents, which are secondary metabolites and are used either directly or indirectly in the pharmaceutical industry.For centuries man has effectively used various components of plants or their extracts for the treatment of many diseases, including bacterial infections (Murugan et al., 2013).
Medicinal plants have played a pivotal role in primary healthcare and formed the basis of traditional systems of medicines.Plants have been bestowed us with food, spices, lavours, fragrances, medicines, etc.. plant is being used to treat many diseases or ailments viz. Infectious diseases, in lammatory disorders,skin diseases etc. since ancient time (F et al., 1993;Houghton, 1995).
According to the World health organization(WHO), more than 80% of the world's population relies on traditional medicine for their primary healthcare. Needs medical plants to contain large varieties of chemical substances, which possess essential therapeutic properties that could be utilized in the treatment of human diseases.
The potential of higher plants as a source of new drugs is still largely unexplored. Among the estimated 2, 50,000 -5,00,000 plant species only a small % has been investigated phytochemical and the fraction submitted to the biological or pharmacological investigation of a given plant will reveal only a very narrow spectrum of its constituents .historically pharmacological screening of compounds or synthetic origin have been the source of innumerable therapeutic agents. Random selection as a tool in discovering new biologically active molecule has been mostproductive in the area of antibiotics (Kviecinski et al., 2008).
Plants are considered not only as a dietary supplement to living organisms but also traditionally used for treating many health problems, and the medicinal value of many plants remains unexplored. Investigations of plants are carried out to ind novel drugs or templates for the development of new therapeutic agents.
Over 60% of the world human population, 80% in developing countries depends directly on plants for their medicinal purposes (Anushia et al., 2009).
Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties plant produces these chemicals to protect itself. Still, recent research demonstrates that many phytochemicals in fruits and herbs and each works differently (Dhillions et al., 2000). Many plant extracts have been shown to inhibit the growth of microorganisms.These extracts consist of chemicals and are usually considered to play a role in defence reactions of plants against infections by pathogenic micro-organisms (Argal and Pathak, 2006).
Plants are the vital storehouse of many chemical metabolites. The medicinal value of plants has assumed a more critical dimension in the past two decades mainly owing to the discovery that extracts from plants contain many minerals,primary metabolites and secondary metabolites with antioxidant potential chemical metabolites present in green plants are grouped into primary and secondary metabolites (phytochemicals). Phytochemicals are good source medicinal drugs, for instance, that with antimicrobial effect are used for the treatment of microbial infections (Alkinmoladun et al., 2007).
Since time immemorial plants have been widely used as sources of food, construction tools, clothing, spices, dyes and medicine. In various countries across the world, plants are used in primary healthcare either singly, or speci ic combinations by traditional medicinal practitioners countries, e.g. India, China, Bangladesh, Pakistan, Bhutan, Thailand and several European, American and African countries widely employ several plant species as medicine to cure several ailments or disorders. Plant-based medicines are prevalent in developing and underdeveloping countries as the high cost of modern drugs and no access to advanced medications to remote places. Plants have also been an indispensable component of traditional medicinal systems, namely Ayurveda,Siddha, (TCM)Traditional Chinese medicine and Unani drugs viz.. Aspirin, Quinine, Digoxin, Artemisinin, codeine and several others are from plant origin.
Plant-based drugs have been used worldwide in traditional medicines for the treatment of various diseases.India is the largest producer of medicinal herbs and appropriately called the botanical garden of the world (Jaiswal et al., 2016). The pharmacology provides an alternative approach for the discovery of antimicrobial (activity) agents, namely the study of medicinal plants with a history of traditional use as a potential source of substance with signi icant pharmacological and biological activities pharmacological and biological activities such as antioxidant anti-cancerous and hepatoprotective.
The systemic screening of antimicrobial plant extracts represents a continuous effort to ind new compound with the potential to act against multi-resistant pathogenic bacteria and fungi .phytochemical studies have attracted the attention of plant scientists due to the development of new and sophisticated techniques. These techniques played a signi icant role in the search for additional resources of raw material for the pharmaceutical industry.
Phytochemical such as lavonoids,terpenoids,alkaloids are plant-derived natural products, which have received considerable attention in recent years due to their diverse pharmacological properties, including cytotoxic and cancer chemoprotective effects (Kviecinski et al., 2008). over 50%of, the drugs isolated from the natural source were used in clinical trials for antitumour activity (Seifert et al., 2008).In India, about 65% of the population relies on ethnomedicine for their primary health care needs, which is the only source of medicines.
Knowledge of herbs has been handed down from generation to generation for thousands of years. The revival of interest in natural drugs started in the last decade, mainly because of the widespread belief that green medicine is healthier than synthetic products. In the recent past, there has been a tremendous increase in the use of plant-based health products in developing as well as developed countries resulting in an exponential growth of herbal products globally. According to WHO, about 80% of the population in the world rely on traditional medicine for the treatment of various diseases (Padmaa et al., 2018).
Medicinal plants have been tested for various kinds of activities such as antimicrobial, hypoglycemic, anthelminthic, hepatoprotective, antioxidant, analgesic, antipyretic activities. Medicinal plants such as Ocimumgratissimum and Eugenia uni lora are rich in volatile oils. It is also reported that they contain 75% thymol which has an antimicrobial effect against Staphylococcus sp.., E. coli and Shigella sp. They are also used in treatments like diarrhoea human ear infection.
Herbal medicines also offer therapeutics for agerelated disorders like memory loss, osteoporosis, immune disorders etc. for which no modern medicine is available. The chemical constituents present in them are a part of the physiological functions of living lora, and hence they are believed to have better compatibility with the human body.
Therapeutic plants are generously available throughout the world. Therapeutic plants are now much more motivated than ever before because they are capable of producing numerous bene its to the community indeed to humanity, particularly inside the type of medication. These naturally occurring compounds are believed to form the building blocks of modern-day prescription medication as we recognize today (Sahu and Sharmisthag, 2014;Owolabi et al., 2007)

Collection of plant
Collection of plant material fresh and healthy, disease-free leaves of Ipomoea staphylina were collected from Mandya Figure 1.

Preparation of leaf and stem extract
The aerial parts of plant fresh leaves and stem of Ipomoea staphylina leaves were two times washed thoroughly under running tap water and at last with sterile distilled water to remove the dirt and dried under shade dried at room temperature.
The shaded dried leaves were powdered in a blender or mixer grinder until ine, coarse powder obtained and stored in an airtight container at stored in room temperature and they used for curd extraction Figure 2.

Preparation of solvent extract
Ipomoea staphylina leaves, and stem extract was obtained by using three types of solvents (Petroleum ether,Ethanol,Aqueous). Take 40 gms of leaf and stem of ine powder in a250 ml conical lask and add 100 ml of Ethanol, Petroleum ether,Aqueous in a conical lask and kept in a rotary shaker for 24 hrs. The extract was iltered through Whatman ilter paper no.1, and the extract was kept for evaporate. Further, the dried residue was preserved in an airtight container or glassware for further analysis.

Screening of Antibacterial activity
The Petroleum ether, ethanol and aqueous extracts of the plant were subjected to antimicrobial activity assay, by disk method. The bacteria inoculums were spread uniformly on the sterile nutrient agar plates with a sterile swab moistened with the bacterial suspension. Each organic solvent extract of Ipomoea staphylina(10, 20, 30, 40, 50µl from 100mg/ml stock solution) was taken in a micropipette separately, and they are loaded to the sterile discs.
Discs impregnated with organic solvent extracts at different concentrations, positive controlwas aseptically placed over bacteria seeded medium with the help sterile forceps. The plates were incubated in an upright position at 37 o C for 24 hrs, and the zone of inhibition was measured and expressed in mm.
Antimicrobial activity was recorded for each concentration of different extracts, including positive control by regulating the zone of inhibition in diameter surrounding the discs and each experiment performed in triplicates.

Antifungal activity study
Antifungal activity was studied by is using the paper disc diffusion method. The PDA medium was poured into a sterile Petriplates and allowed to solidify. The test fungal cultures were spread evenly over the media by sterile Cotton swads. The discs were soaked in extracts of different concentration, and they are air-dried and stored in the refrigerator.
The pre-soaked disc was placed on the agar plate. Standard antibiotics disc and negative control were placed on the agar Petri plate in each set for each fungus which served as standard Petri plate were incubated at roomtemperature for 48 hrs. After the incubation period, the petriplates were observed for the occurrence of inhibitions, and the diameters were measured.

PHYTOCHEMICAL TEST
Different extracts of Ipomoea staphylina were subjected to phytochemical analysis to detect the pres-ence of the chemical Constituents. The chemical test was carried out to identify the presence of Phyto constituents in petroleum ether, ethanol and aqueous extract of I.staphylina using the standard procedure.

TEST FOR CARBOHYDRATES
Benedict's test 1ml of the extract was treated with benedict's reagents and heated gently orange-red precipitate indicates the presence of reducing sugar.

Fehling's test
The plant extract was dissolved in 5ml of distilled water and ilter. The iltrate was treated with 1ml of Fehling's solution [A&B] and heat in a boiling water bath. A reddish-orange precipitate is obtained.

Foam test
Extract was treated with distilled water to 20ml, and this was shaken in a graduated cylinder for 15 min resulting in the formation of 1cm layer of foam indicates the presence of saponins.

Iodine test
Leaves and bark extract were treated with diluted Iodine solution and appearance of transient red colour indicates the presence of tannins.

Nitric acid test
Leaf and bark extract were treated with diluted Nitric acid and formation of reddish to yellowish colour indicates the presence of tannins.

Biuret test
Leaf and bark extract were treated with 1ml of NaOH [10%] solution, and 1-2 drops of CuSo4 solution is added. A violet colour indicates the presence of proteins.

Alkaline test
Extract was treated with a few drops of lead acetate solution. After a few minutes appearance of yellow colour, precipitate indicates the presence of lavonoids.

Ferric chloride test
To a small quantity of the extract dissolved in methanol and a few drop of neutral ferric chloride solution was added.If phenols and enols are present, it gives a green, blue, violet colour.

TEST FOR GLYCOSIDE
0.2g of the extract is dissolved in 1ml of glacial acetic acid containing one drop of ferric chloride solution then 1ml of concentrated sulphuric acid is added slowly. A brown ring obtained at the interface indicates the presence of glycosides.

Liebermann-Burchardt reagent
Few mg of extract was dissolved in chloroform. To this 1-2ml of chilled acetic anhydride was added and mixed well. Then 2-3 drops of chilled concentration sulphuric acid were added along the sides of the test tube. The colour developed at the junction of the two liquids was observed. Steroids/triterpenoids gives a characteristic colour [red/pink/violet].

TEST FOR ALKALOIDS
The extracts were stirred with a few ml of dilute Hydrochloric acid and iltered. The iltrate was tested with various alkaloid reagents as follows:

Mayer's test
To a few ml of iltrate, 2-3 drops of Mayer's reagent was added along the sides of the test tube. A white (or) creamy precipitate indicates the presence of alkaloids.

Hager's test
To a few ml of iltrate, 2-3 drops of Hager's reagent was added along the sides of the test tubes. A yellow precipitate indicates the presence of alkaloids.

RESULTS
In the present study, the results were recorded for the phytochemical and antimicrobial activity of Ipomoea staphylina. The phytochemical analysis of Ethanol, Petroleum ether, Aqueous extracts of Ipomea staphylina of leaf and stem showed in

Antibacterial activity of Ipomoea staphylina by paper disk diffusion method
In the present investigation, the antibacterial activity of Ipomoea staphylina was carried out by disk diffusion method, against three bacterial strains viz., B. subtilis, S. aureus were Gram-positive, and E. coli were Gram-negative bacteria. Antimicrobial activity against all the test pathogens was conducted using three different solvents, namely Ethanol, Petroleum ether and aqueous extracts. The inhibition zone of different extract of I. staphylina was observed at a various concentration (10,20,30,40,50 µg/ml) after 24 hrs of incubation. An antibiotic Streptomycin is used as a positive control.

Antibacterial activity of Ipomoea staphylina of different solvents against B.subtilis
In the petroleum ether extract of leaf and stem, no zones are formed.In the ethanol extract of leaf 20µl concentration shows the 19 mm of the zone. In the 30µlconcentration shows the 20 mm. In the 40 µl concentration shows the 18 mm. In the stem extract, 10 µl concentration shows the 21 mm. In the 20 µl concentration shows the 19 mm. In the 40 µl concentration shows the 18 mm. In the 50µl concentration show the 15 mm.In the aqueous extract of leaf and stem, no zones are formed, as shown in Figure 3 & Table 2.

Antibacterial activity of Ipomoea staphylina different solvents against S. aureus
In the petroleum ether extract of leaf 10µl, con .shows the 18 mm zone. In the 30µl con. Shows 27 mm. in the 40µl con. Shows 19 mm. In the 50 µl con. Shows 18 mm. In the stem extract, no zones are formed. In the Ethanol extract of leaf 10µl con. shows 30 mm. in the 20 µl con. Shows 39 mm. In the 30µl con. shows 50 mm. In the 40µl con. shows 49 mm. In the 50µl con. shows 49 mm. In the stem extract of the stem, no zones are formed. In the aqueous extract of leaf 40µl con. shows 53 mm. In the 50µl con. shows the 52 mm. In the stem extract, no zones are formed, as shown in Figure 4 & Table 3.

Antibacterial activity of Ipomoea staphylina different solvents against E.coli
In the petroleum ether extract of leaf and stem shows no zone, in the ethanol extract 30µl con. shows the 27 mm. In the 40µl con. shows 25 mm. In the stem extract 10µl con. shows 20 mm. In the 20µl con shows the 24 mm. In the 30µl con. shows the 27 mm. In the 50µl con. shows 18 mm.In the aqueous extract of leaf and stem, no zones are formed, as shown in Figure 5 & Table 4.

Antifungal activity of Ipomoea staphylina against Aspergillus niger
The positive control of 100µl con. shows no zones. The leaf extract of ethanol of 50 µl con. shows 9mm, the stem extract of negative control in 50µl con. show 12 mm, as shown in Figure 6 & Table 5.

DISCUSSION
Herbal preparations are other supplement systems of medicine for the treatment of disease caused by bacteria (Amani et al., 2014). Considering the vast potentiality of plants as sources for antibacterial drugs with references to antimicrobial activity from the plant I. staphylina. Members of Convolvulaceae are medicinally essential plants. Compounds present in I. staphylina are known to be biologically active and therefore aid the antimicrobial activities.The cold extraction method was carried out dried powdered leaf and stem I. staphylina by soaking the powder in 3 different solvents viz., ethanol, petroleumether, aqueous. Alkinmoladun et al. (2007) showed that the antimicrobial activity of leaf extract I .asarifolia against gram +ve and -ve bacteria. The gram -vebacteria E.coli of aqueous leaf extract shows the high inhibition 21 mm. The gram +veS. aureus of aqueous leaf extract shows the low inhibition 20 mm. According to our study in Ipomoeastaphylina the gram +ve bacteria S. aureus of aqueous leaf extract shows the 17.6 mm of inhibition in E. coli absent of inhibition. Kiruthiga et al. (2014) showed that the antimicrobial activity from the latex I. staphylina against gram +ve and -ve bacteria. The gram +ve bacterial S. aureus shows the very high inhibition at 1 ml con. And the gram -ve bacterial the 700 ml and 1 ml both the con. shows very high inhibition.
According to our study, the different solvent extract of different bacteria among them the E.coli and B. subtilis of leaf and stem extract of ethanol shows the high inhibition at 30 µl con. The S .aureus of the aqueous leaf has shown the inhibition at 40 µl con.
Antifungal activity against Aspergillusniger the stem extract of -ve ethanol shows 12 mm at 50µl con.The leaf extract of ethanol shows 9 mm at 50µl con. Phytochemical analysis conducted on the plant extracts revealed the presence of constituents which are known to exhibit therapeutic as well asphysiological activities. Analysis of the plant extract revealed the presence of phytochemicals such as alkaloids,carbohydrate, glycosides,tannins, lavonoids.
The result obtained in this study thus suggests the identi ied phytochemical compound compounds may be the bioactive constituents and these plants are proving to be an increasingly valuable reservoir of bioactive compounds of substantial medicinal merit.

CONCLUSION
Nature has gifted the humankind with plant wealth which has been used for traditional medicines as they contain components of therapeutic values. Plants are natural sources of antimicrobial agents. They contain a wide range of metabolites that can be extracted from them and used to treat infectious and chronic diseases. Most of the aromatic and medicinal plants are botanical raw material also knows as herbal drugs, that are primarily used for therapeutic, aromatic and culinary purposes as compounds of cosmetics, medicinal products, health foods and other natural health products.
The present study was formulated to study the phytochemical analysis and antimicrobial activity. In the study of the phytochemical analysis revealed the presence of carbohydrate, protein, lavonoids, glycosides, alkaloids.
The Ipomoea staphylina of leaf and stem extract showed potent antimicrobial activity against E.coli, Staphylococcus aureus, Bacillus subtilis and Aspergillus niger.The plant extract could be used as drugs for various ailments.