Antimicrobial Activity of Cymhopogon Citratus ( Lemon Grass ) and It ' s Phytochemical Properties

The cold maceration and agar diffusion technique were emp loyed to assess phytochemical propert ies and the antimicrobia I potency of Cy mbopogon citratus (lemongrass) against selected microbial pathogens using hexane, ch loroform and methanol as extracting solvents. The mean zones of inhibition of the chloroform leaf and corresponding root extracts for the test organisms were Staphylococus aureus (l1.33±1.l5,11.66±2.52), Salmonella typhi (I 1.33±1.53,I 3.66±0.58), Escherichia coli (16.33±0.58,15.66±2.31) and Candida albicans (7.66±0.58,8.66±\.53) respectively. He xane and methanol extracts showed no activity against the test organisms. The min imu m inhib itory concentration (MIC) and the corresponding minimum bactericidal concentration (MBC) for chloroform leaf and root extracts were: Staphylococus aureus (24~g/ml, 28~g/ml), Salmonella typhi (20~g/ml, 28~g/ml), Escherichia coli (14~g/ml, l Sug/rnl), Candida albicans (32~g/ml, 38J.!g/ml) and Staphylococus aureus (20~g/ml, 26J.!g/ml), Salmonella typhi( l Sug/rnl, 24J.!g/ml), Escherichia coli (14~g/ml, l6J.!g/ml), Candida albicans (28J.!g/ml, 32J.!g/ml) respectively. Phytochemical screening on Cymbopogon citratus showed that five active ingredients: Tannins, Flavonoids, Phenols, Carbohydrates and volatile oil were present in both the root and leaf parts. The mean zones of inhibit ion showed that Cymbopogon citratus exh ibited an intermediate antimicrobial activity against the bacteria species while C. albicans was resistant. Higher dose of C. citratus may be recommended to exert a remarkable antimicrobial activity against the test organisms.


Introduction
Nature has been a source of medicinal agents for thousands of years and since the beginning of man. In Nigeria, almost all plants are med icinal and the application of medicinal plants especially in traditional medicine is currently well acknowledged and established as a viable profession [l]. In addition to providing the animal kingdom it's food, fuel and shelter, plants accumulate other phytochemical constituents -the secondary metabolites which are produced as by-product and are not directly useful to them. These secondary metabolites gives plants their medicinal value some of which include Alkaloids, Tannins, Saponins, Flavonoids, Antraquinones, Glycosides, Volatile oils, Terpenes, Essential oils, Resins [2].
Medicinal plants have therefore been described as one in which one or more of its organs contain substances that can be used for therapeutic purpose [19]. It may be in the form of vegetable drugs which may either be organized (material wh ich posses a cellular structure e.g. Leaf, bark petal, flower, stem, root, etc) or unorganized drugs (a cellular structural medicinal agents such as gums, balsams and Latex), such plants materials may be utilized in the form of decoctions in cold water or warm water, concoctions, preparations of soups, drinks etc made fully from many ingredients. They can also be used as infusions often made by pouring water on a specified plant material and allowing the mixture to stand for about 15 minutes [18].
For the past two decades, there has been an increasing interest in the investigation of different extracts obtained from traditional medicinal plants as potential sources of new antimicrobial agent [l]. Although it has been estimated that about one in four of all prescribed drugs' and almost 7,000 different medicaments contain compounds of plant origin or their derivatives with their commercial value being put at about $40 billion annually [5]. Indicated that about 33%0 f drugs produced in the developed countries are derived from plants [6]. Cy mbopogon citratus of the Poaceae family is a tall, monocotyledonous aromatic perennial plant with slender sharp-edged green leaves, pointed apex that is native to tropical Asia. C. citratus is known as Guatemala in West Indian, or Madagascar lemongrass [9]. C. c itratus is cu Itivated in Africa, the West Indies, Central and South America, and tropical regions. The linear leaves can grow up to 90 cm in height and 5 mm in width [9]. Research and Development (NIPRD) Abuja. The plant samples were crushed and blended into smaller pieces to enhance the penetration of the extracting solvents into the plant cells, thus facilitating the release of the active principles.The cold maceration method as described by [25] was used. Two hundred grams (200g) of powdered Cymbopogon citratus samples (leaf and root) were weighed using a weighing balance into two 1000ml capacity conical flask. One litre each of the solvents (Hexane, Chloroform and Methanol) was added to each of the samples respectively. The conical flasks containing the mixtures were placed on a shaker for 24 hours. After 24 hours of shacking and mixing, it was next filtered using muslin cloth. The filtrates were then filtered again using suction pressure with the aid of a vacuum pump. The filtered extracts were concentrated using the rotary evaporator equipment after which they were dried on an evaporating dish at a temperature of 50'C to 60'C to a semi-solid form. A sticky semi-solid greenish substance was obtained for both samples. The extracts were stored in a well corked universal bottle.

Phytochemical
analysis was performed to screen the extracts for the presence of the following active principles: Tannins, Flavonoids, Volatile oils, Phenol, Carbohydrates. All procedures were as described by Sofowora (1984

Test for Volatile Oils
Volatile oils are characterized by their odour, oil-like appearance and ability to volatilize at room temperature. The plant materials were distilled with water by steam distillation and the distilates were collected in a graduated tube. The aqueous portion which separates automatically was returned to the distillation flask. The formation of emulsion which floats on top of the aqueous phase owing to its low density is indicative of the presence of volatile oils[2I].

Test for Carbohydrates
Three grams of powdered samp les each of Cymbopogon citratus leaf and root were boiled separately in 50ml of distilled water on a water bath for 3 minutes. The mixtures were filtered wh ile hot and the resulting filtrate allowed to cool. A few drops of Molisch 's reagent was added to 2ml of the leaf extract, a small quantity of concentrated sulphuric acid was added and allowed to form a lower layer. The procedure was repeated with the root extract. A purple ring at the interface of the liquids indicated the presence of carbohydrates.
The mixtures were then shaken allowed to stand for 2 minutes and then diluted with 5ml of water. A purple precip itate also indicated the presence of carbohydrate[2l] .

Preparation of Test Organisms
The microorganis ms used namely Staphylocuccus aureus, Escherichia coli, Salmonella typhi, Candida albicans were obtained from stock cultures in the Microbiology Laboratory of the Nat ional Institute for Pharmaceutical Research and Development (NIPRD), Idu Abuja,Nigeria. They were subcultured and identified based on their colonial morphology, microscopic appearance and specific biochemical reactions. The test organisms were sub cultured in I Oml broth each and incubated at 37'C for 18 to 24 hours. After24 hours, the organisms were sub cultured into a fresh Mueller Hinton Broth and incubated for 3hours which was used for all analyses.

Preparation of Extracts
Zero point one grams (O.lg)o fthe hexane, chloroform and methanol extracts were weighed and dissolved in 5ml each of sterile distilled water. This gives 20mg/ml concentration each.

2.S.Antibacterial Assay of Crude Extracts
Mueller Hinton Agar med ia was streaked uniformly according to the number of test organisms and was labelled appropriately; sterile cup borer (6mm) was used to bore holes in the culture media. The base of each wells were sealed with a drop of molten agar to prevent unwanted spread of the extracts. In a drop-wise manner, I ml of the prepared extracts was added into each well and the cultures were allowed to stand for 30 minutes before they were transferred in to the incubator. The cultures were incubated for 24 -48 hours at' 37'C before final read ings were taken. Control plates were also prepared for each test organisms without the addition of extracts. Zones of inhibition were measured to the nearest millimetre [7] .

Antifungal Assay of Crude Extracts
The agar diffusion method [4] was employed. The test organism Candida albicans was inoculated into test tubes containing sabouraud dextrose broth and incubated at room temperature for 72 hours. The organisms were subcultured into sabouraud dextrose agar by the pour plate method. A sterile cork borer (6mm) was used to bore holes in the cu lture med ia and the base of the wells was sealed with a drop of mo lten agarto prevent unwanted spreading ofthe extracts. In a drop-wise manner, I ml 0 fthe extract was added into each of the well and the cultures were allowed to stand for 30 minutes before incubation at room temperature for 48 hours. After48 hours of active growth, the zones of inhibition were measured with the aid of a meter ru le considering the diameter of the cork borer. A control plate was also prepared for each test organism without the addition of extracts.

Column chromatography
The micro scale flash column chromatographic method according to [lO] was used to separate the fractions of the chloroform extracts. The column was prepared by plugging a Pasteur pipette with a small amount of cotton using a simp le dry-pack method and with a wood applicator stick it was tamped down lightly. One hundred milligram silica gel was added as the stationary phase. The column was next pre-eluted by the addition of solvent (Chloroform) and it was allowed to flow slowly down the column by gravity and the flow was also slightly aided by the application of air at the top of the column with the aid ofa pipette bulb. The column was loaded with the sample by the wet method which involves dissolving lOOmg of the extract in the solvent before addition. It was then eluted as necessary by forcing more solvent through the column with the aid of a Pasteur pipette bulb which also prevents the silica gel from going dry. The fractions were collected in test tubes according to their colour development.

Antimicrobial Analysis ofC.citratus Fractions
The same method for the determination of the antimicrobial activ ity of the crude extracts against the test organisms (both for the fungi and bacteria species) was used for the fractions obtained fro m the colu mn chro matography. The only difference is that in this case the fractions were used in place of the crude extract and each culture was incubated appropriately based on the species under test. to l> 108c fu/ml. The inoculated broths were incubated at 3TC and 2S-30'Cfor bacteria and fungi respectively for 24 hours. After 24 hours, the tubes were observed for growth and recorded as the minimum inhibitory concentration (MIC). The tubes with no growth after 24 hours were sub-cultured on freshly prepared Mueller -Hinton agar and sabouraud dextrose agar by the streaking method for bacteria and fungi respectively. The culture media were incubated appropriately for 24 hours and then observed for growth. After 24 hours, the lowest concentration from which the microorganisms did not recover and grow when transferred to the fresh media was recorded as the minimum bactericidal concentration (MBC) [7].

Standard Antibiotic Susceptibility Test
Disc diffusion method was used in this test as described by [24]. Six millimetre (6mm) commercially prepared antibiotic paper discs were used. The antibiotic discs used and their concentrations are Ciprofloxacin (30Ilg), Erythromycin (LSug), Tetracycline (30Ilg) and Ketoconazole (l Sug) for Staphylococcus aureus, Salmonella typhi, Escherichia coli and Candida albicans respectively.
The discs were applied in accordance to the National committee for clinical laboratory standard. The antibiotics used in the test were chosen after a preliminary survey and to reflect the range of drugs commonly prescribed for the treatment caused by the test organisms 2.11. Susceptibility Test Sterile swab sticks were used to transferthe test organisms into tubes containing physiological normal saline to form a suspension. Prepared Mueller Hinton Agar and sabouraud dextrose agar were inoculcated appropriately with the test organisms tStaphylococus aureus, Salmonella typhi, Escherichia coli and candida albicans) by dipping the sterile swab sticks into the suspension and removing excess inoculum by pressing and rotating the swab firmly against the side of the tube. The inoculu ms were streaked all overthe surface of the mediu m rotating the plates through an angle of 60°after each application [24]. The inoculated plates were allowed to dry for a few minutes at room temperature with the lid closed. The antibiotic discs were then placed ascetically on the inoculated plates using a pair of sterile forceps. Each disc was gently pressed down to ensure even contact with the medium. The plates were incubated at 37'C for 24 hours in the case of bacteria while that of fungi were incubated at room temperature for 48 hours. At the end of the incubation period, the results were recorded as sensitive or resistance based on the occurrence of zone of inhib ition respectively.

3.1.Antimicrobial Assay of Crude Extracts of C.citratus
The mean zones of inhib ition of the leaf and root extracts of Cymbopogon Citratus against Staphylococus aureus, Salmonella typhi, Eschenchia coli and Caudida albicans are shown in were from the root extract wh ile 3 (colourless, orange and pale yellow) were from the leaf extract (Table 3.3). The fractions showed higher antimicrobial activity than the crude extract against the organisms which indicates a pure form of the crude extract after passing through the column chromatography process (Table 3.3).

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of C.citratus Extracts
The result of minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of chloroform extract of Cymbopogon citratus root is shown in Table 3.4. The minimum concentration of Cymbopogon citratus causing inhibition and total death of microorganism was 14.0J.1gfml and 38 J.1g fml respectively.  LG=light; OR=orange.

Phytochemical Analysis
The result of phytochemical screening on Cymbopogon Citratus showed that five active ingredients were present in both the root and leaf parts. These include Tannins

Discussion
Extraction and Phytochemical screening of b ioactive agents from medic inal plants permits the demonstration of their physiological activities. The phytochemical analyses showed that Flavonoids and Vo latile 0 il are present in hexane extract; Tannins, Flavonoids, Phenol, Carbohydrates and Volatile oil were present in chloroform extract; Tanins, Flavonoid'S and Carbohydrates were present in methanol leaf extract while only Tannins and Carbohydrates were present in methanol root extract (Table 5). Phytochemical screening of Cymbopogon citratus also revealed the presence of volatile oil, also call essential oil and according to [ll], the presence of volatile oil gives plant their specific aromas which is confirmed by the aroma produced by this plant and are extracted by solvent Extraction. The presence ofvolatiIe oil also confirms the report ofl20] of the application of Cymbopogon citrarus in perfumery, cosmetics and soap industry .
According to [12], tannins and phenolic compounds have been found to inhibit bacterial and fungal growth and also capable of protecting certain plants against infection. According to the report of[l S], that phytochemical component has antifungal properties which were confirmed in this study. The presence of tannins in the plant extract agrees with the report of[ll] that tannins are important in herbal medic ine and they are applied in arresting bleeding and wound healing. Tannins and tannic acid own their stringent action to the fact that they precipitate protein and render them resistant to attack by proteolytic enzymes, internally; they form a pellic le of coagulated protein over the lining of the alimentary tract. The antifungal activity against Caudida albicans is low with mean zone of inhibition of 7.66±0.S8 and 8.66 ± I.S3 with an MIC of 28Jlg/ml and 32Jlg/ml for leaf and root extract respectively which suggest that the use of this plant for therapeutic purpose against infections of Caudida albicans may not be fully successful as the fungi may show resistance to the plant. This in no doubt confirm part Iy the report that Cymbopogon citratus has been used against gastrointestinal disturbances [16] but might require high dosage due to the level 0 f antimicrobial activity it showed in this research res ult.
The root of Cymbopogon citratus showed more antimicrobial activity than the leaf extracts (Table I) despite the presence of bioactive agents in both, which showed that there are more different types of active ingredient in different plant species and different plant parts even though they have the same generic name i.e. the type oftannins present in one plant orplant part may be different from the tannins present in another plant species or plant part [II]. Tannins have also been reported to have antidiarrheal, homeostatic and antihemorrhagal activity, [3]. According to [14] there are three different types of tannins; Hydrolysable tannins, Non-Hydrolysable tannins or condensed tannins and Pseudo tannins. Various volatile oils in plant have been reported to have med icinal values ranging fro m skin treat ment to remedy for cancer [13].
The isolation of volatile oils in Cymbopogon citratus confirms the activity showed against the test organisms by this plant and also in part confirms the report of [4] of the oils isolated from same plant by distillat ion to exhib it great antibacterial activ ity and also confirms the potency of this particular plant against skin cancer prevention as reported by [16).
In a 13-oil studies, lemongrass oil was found to be among the most active against human dermatophyte strains inhibiting 80% of strains as reported by [IS] and [3], this is confirmed by the antifungal activity of Cymbopogon Citratus against strains offungi species used as test organism and also confirm reports by traditional users of lemongrass against ring worm infections. According to the phytochemical result, phenol was not found in both hexane and methanol extract but only in chloroform extract. This may contribute to the high antimicrobial activ ity showed by the chloroform extract wh ich is absent in the other two so Iven ts extracts.
Methanol extracts shows no antimicrobial activity which is in agreement with the report 0 f [3] Table 3). The fractions had antimicrobial activity higherthan the crude extracts of same plant. Th is may be as a result of the fact that the chromatography procedure purified the extract. On the other hand the crude extract may still contain some impurities which may likely prevent their activity. Therefore purification is very important because the presence of some ingredients wh ich reduce the activity of the bioactive components is eliminated, concentrating the active component hence, increasing the activity of the active components ..

Conclusions
The results ofthis study on Cymbopogon citratus have led to the following conclusions: The extracts of Cymbopogon citratus leaf  The MIC of Cymbopogon citratus reveals that a higher dose of the plant extract is required to bring about a significant activity in the body. Five active ingredients were identified in the plant (root and leaf) which include flavonoids, tannins, phenol, volatile oil and carbohydrates.