Phytochemical Analysis and Antibacterial Activities of Kyllinga nemoralis extracts against the Growth of some Pathogenic Bacteria

this study aimed to screen the phytochemical contents and investigate antibacterial activities of the aqueous and methanolic extracts of Kyllinga nemoralis . extraction was done using the whole plant of K. nemoralis except the root. the phytochemical screening was carried out on both aqueous and methanolic extracts of K. nemoralis . the aqueous extract showed the presence of saponin and high amount of steroid, while the methanolic extract showed high amount of terpenoid and steroid. the antibacterial activities of K. nemoralis extracts were tested against five Gram positive bacteria ( Staphylococcus aureus , methicillin-resistant Staphylococcus aureus (MRSA), S taphylococcus epidermidis, Streptococcus pyogenes, and Bacillus thuringiensis ) and four Gram negative bacteria ( Escherichia coli, Shigella sonnei, Salmonella typhi and Klebsiella pneumoniae ). K. nemoralis extracts were subjected to testing of their antibacterial activities by the disk diffusion method. Furthermore, the minimum inhibitory concentrations of the extracts were determined. the results indicate that the aqueous extract of K. nemoralis exhibits more antibacterial activities than the methanolic extract. the aqueous extract of K. nemoralis showed efficacy against S. aureus and MRSA while the methanolic extract of K. nemoralis was found to exert antibacterial activity against MRSA. the results proved the potency of K. nemoralis extracts as natural antibacterial and supported the potential of use in the medication of the diseases caused by the tested bacteria.


iNtROduCtiON
In the past two decades, the discovery of new molecules (with antimicrobial properties) from microorganisms has declined drastically.Scientists began to seek potential novel antibacterial compounds within medicinal plants.There are a lot of methods that are used to screen the antibacterial properties of medicinal plants, with different types of principles.However, with the different types of principles that are used, the results become varied.The results can be influenced by the method selection, the microorganisms that are used, and the degree of solubility of the test compound. 1edicinal plants have long been used in traditional and complementary medicine worldwide.The information that the ancient people used plant materials as medicinal sources has been discovered in archaeological finds and old literature.In the Malay Peninsula, the record shows that not less than 1,300 plants have been used in the traditional medicine practice.This is due to Malaysia's tropical rainforest which is rich with biological and chemical resources.The plants in the tropical rainforest are very diverse biologically, and they synthesize a lot of chemicals to protect themselves from pests, predators, and diseases. 2However, Malaysia is yet to develop the rich plant resources to their full potential as pharmaceutic agents.This is due to the high cost of research and development, coupled with the lack of expertise and technology in the natural product research.Ostensibly, Malaysia needs to have a collaboration with international researchers or pharmaceutical companies to explore and develop the product from the rich medicinal plant resources that can be found in Malaysia. 3he medicinal plant that is used in this study is Kyllinga nemoralis.The common name for K. nemoralis is white water sedge, whitehead spike sedge, white kyllinga, white-flowered kyllinga, and poverty grass.The family of the K. nemoralis is Cyperaceae and the genus is Kyllinga Rottb.The description of this plant is grass-like in the habitat, and it propagates by seeds and creeping rhizomes.The plant is considered invasive because when the plant is dispersed in a new place, it will reproduce and then out-compete the native plants.Moreover, this plant also has the characteristics of a successful invasive species because it has an asexual spreading and has positive respond to human-caused disturbance. 4. nemoralis has been known to be widely used in the traditional medicine practice throughout the world.The parts that are usually used are the leaves and the rhizomes, which contain a lot of biologically active chemicals.The leaves are traditionally used to relief malarial chills, pruritis of the skin, diabetes, and thirst due to fever. 5In India, the leaves are used as anti-venom for snake poisoning. 6However, the mechanism of action of the plant is not direct inhibition of the action of the venom, but rather symptomatic relief by the anti-inflammatory, tranquilizing, or analgesic properties of the plant.The rhizomes of K. nemoralis has a sweet fragrant.The rhizomes are traditionally used as antidiarrhea, expectorant, refrigerant, stomachic, and diuretic.The rhizomes can also be used to treat worm infection by mixing the rhizomes paste with milk. 7The current study was carried out to screen the phytochemical contents and investigate antibacterial activities of K. nemoralis extracts towards selected Gram positive and Gram negative bacteria.

Plant Material
In this study, the plant samples were collected in Kuala Nerus, Terengganu, Malaysia.Whole plants of K. nemoralis except root (500 g) were washed with water and then drained at ambient temperature until it dried completely.The plants were further oven-dried for three days at 60°C.After the drying process, the plants were cut into small pieces and ground using the electric blender (Kenwood Ltd, Havant, United Kingdom).The coarse powder was kept in air-tight container.The coarse powder of the plants was split into two portions which were used for the aqueous and methanolic extracts. 8

extraction Methods
For preparing aqueous extract of the K. nemoralis, 100 g of the coarse powder of the plants was mixed with 1.5 L of distilled water and stirred using glass rod to dissolve the powder properly.The mixture was boiled on the hot plate for 20 minutes in a glass beaker.Then, the mixture was cooled off to room temperature before being filtered with the filter paper.The filtering process was repeated twice to finely filter the powder.After that, the filtrate was frozen at -20°C for seven hours.Then, the frozen filtrate was transferred into the freeze dry machine (SP VirTis Genesis Pilot Lyophilizer, USA).The freeze dry process was to stabilize and preserve the chemical structure of the plants extract; the process took 31 hours to complete.The resulting extract was weighed using analytical balance and kept in the chiller at 4°C. 9 The methanolic extract of the K. nemoralis was prepared by immersing 100 g of the coarse powder of the plants with 1 L of methanol for 96 hours at ambient temperature.Then the mixture was filtered using filter paper.After that the powder was re-immersed in 1 L of methanol for another 96 hours and this process was repeated three times.All of the three repeated filtrates were separately collected.The filtrates were then evaporated using rotary evaporator (Eyela N-1110, Japan) until almost dry.The crude extract was oven dried at 50°C for three days to completely dry the methanol solvent.After that, the extract was weighed and the extract was preserved at 4°C. 10

Phytochemical Screening test for Alkaloid
One mL of 25% ammonia was added into 10 mg of extract in a test tube and the mixture was set aside for a minute.After that, 5 mL of chloroform was added into the mixture and the test tube was shaken for three minutes.Then, three drops of Mayer's reagent were added to the mixture.The development of creamy whitecolored precipitate indicated the presence of alkaloid in the mixture. 11detection of Saponin 10 mg of extract was dissolved in 1 mL of distilled water.Then the mixture was shaken for 5-15 minutes.The formation of soap-like foam layer shows that there was presence of saponin in the extract solution. 12est for terpenoid.10 mg of extract was added to 2 mL of chloroform followed with 3 mL of concentrated sulphuric acid.The formation of a reddish-brown indicated the existence of terpenoid in the extract. 13termination of Steroid 10 mg of extract was added to 2 mL of chloroform.Then the mixture was added with 2 mL of concentrated sulphuric acid and shaken well.If there was presence of steroid, the chloroform layer would appear red or reddish-brown after a few minutes. 14

Antibacterial Activity test Bacteria
The

Culture Media and inoculums Preparation
The Gram positive and Gram negative bacteria were cultured in both nutrient agar and broth.The bacteria inoculums were prepared from the 24 hours, 37°C culture of bacteria isolates in nutrient broth.

Antibacterial test
Antibacterial test is to measure how much the aqueous and methanolic extracts of K. nemoralis can inhibit the bacteria.The antibacterial test was the agar disk diffusion method.The Mueller-Hinton agar plates were inoculated with a bacteria strain with 0.5 MacFarland standard.Then filter paper disc (6 mm in diameter) containing the extracts with concentrations of 500, 250 and 125 mg/mL were placed on the agar surface.Chloramphenicol was used as a positive control and 10% methanol was used as a negative control.The Mueller-Hinton agar plates were incubated aerobically at 37°C for 24 hours.Subsequently, the diameter of the zone of inhibition surrounding the extract disk, positive control, and negative control were measured and the data were recorded. 15

Minimum inhibitory Concentration (MiC)
The MIC test was performed in 96-well microtiter plate.The aqueous and methanolic extracts of K. nemoralis were subjected to twofold serial dilutions to a final volume of 100 μL at final concentrations of 125, 250 and 500 mg/ mL.Next, 100 μL of stock solutions of S. aureus, S. epidermidis, B. thuringiensis, MRSA, S. pyogenes, S. sonnei, E. coli, K. pneumoniae and Salmonella Typhi (0.5 MacFarland) was added to each well to obtain a final volume of 200 μL.Wells containing MHB with aqueous and methanolic extracts of K. nemoralis were used as negative control while wells containing MHB and bacteria inoculum were used as viability control.Wells containing MHB alone acted as sterility control.Well with two-fold serial dilutions of 1 mg/mL to 512 mg/mL chloramphenicol were used as positive control.All tests were performed in triplicates and incubated at 37°C for 24 hours.The determination of MIC was based on the clear appearance of the MHB indicated that there is no visible growth after being incubated with the tested microorganisms. 16

ReSultS
The phytochemical screening of aqueous extract of K. nemoralis showed the existence of saponin and steroid, while methanolic extract of K. nemoralis showed terpenoid and steroid.The results for the alkaloid test are negative for both extracts, as given in Table 1.
A varying antimicrobial activities was shown by both extracts when tested against the nine pathogenic bacteria (Tables 2 and 3).Aqueous extract of K. nemoralis was active against Gram positive bacteria, while totally inactive against Gram negative bacterial strains.The methanolic extract of K. nemoralis exhibited the highest zone of inhibition on MRSA.
MIC values for aqueous extract of K. nemoralis ranged from 15.63-500 mg/mL against tested bacterial strains and MIC values for methanolic extract of K. nemoralis ranged from 0.98-3.91mg/mL against tested bacterial strains (Table 4).

diSCuSSiON
The presence of phytochemical constituents seen in this study like saponin, terpenoid, and steroid was in good agreement with a previous report, 17 while the presence of carbohydrates, phenols, flavonoids, flavones, tannins, saponins, alkaloids, steroids, terpenoids, coumarins, quinones, lignins, and fats and oils in all the plant parts have been reported. 18This difference could be because of the geographical difference of the plant material.Phytochemical constituents such as saponins might potentially be antimicrobial, antiviral, antioxidant, and antiinflammatory. 19aponins are one of the most diverse and numerous groups of plant natural products.The function of saponins in plants is as the defense against disease, herbivores, and can be also allelopathic agents in interactions with other plants.The saponin compounds are present in the aqueous extract of K. nemoralis, thus this extract has antibacterial activity towards Gram positive bacteria.The result from our study is supported by the results of another study of saponin substances that have showed suppressive effect on Gram positive organism nevertheless not on Gram negative. 20erpenoids are the substantial and also constitutionally most varied group of the secondary metabolites extracted from plants.Previous studies on plant methanolic extracts showed terpenoids might potentially be responsible for the therapeutic value of the plant material extract. 21It is known that terpenoids have various therapeutic effects which includes antifungal, antihyperglycemic, anti-inflammatory, antimicrobial, antiviral, antiparasitic, antioxidants, and immunomodulatory. 22The presence of terpenoid in the methanolic extract of K. nemoralis may influence the antibacterial activity of the extract.From a previous study, the terpenoids have been shown to have a strong antibacterial effect. 23In another study, terpenoids were capable to suppress the essential activities needed for microbial viability which is oxidative phosphorylation and oxygen intake. 24n plants, steroids play a lot of roles that cannot be replaced by other substances such as controlling fluidity, sustaining membrane semi-permeability, and also serve as biosynthetic precursors for steroid hormones. 25In one study, stigmasterol, the plant-based steroid, showed an antibacterial activity against S. aureus even at low concentrations.The antibacterial effect of the steroid and cholesterol derivatives may come from the disruption of cell integrity and permeability. 26n our study, steroids are present in both extracts.The steroids are soluble in both polar and nonpolar solvents.However, polar solvents such as methanol and water are more preferred for steroid extraction. 27aponin, terpenoid, and steroid contents in aqueous and methanolic extracts of K. nemoralis may lead their antibacterial property.The significant antibacterial activity against Gram positive bacteria is perhaps because of a single or joined effect of these secondary metabolites.
The natural products from the plants have been reported to target and act on a few bacterial targets such as the bacterial cell wall, bacterial cell division, pyruvate kinase, DNA topoisomerase, and efflux pump. 28In one study, the antibacterial activities of the natural products were in a positive correlation with the amount of the phenolic compounds that can be found in the plants. 29hus, K. nemoralis might have an active phenolic compound that can inhibit the growth of the S. aureus and also MRSA.However, antibacterial activities of medicinal plants were observed by a researcher to be least effective against the Gram negative bacteria compared to Gram positive bacteria. 30This is unsurprising because the Gram negative bacteria have been shown to be more resistant to antibiotics. 31Probably due to the characteristic of their cell walls, Gram positive bacteria were more susceptible to the plant substances than Gram negative bacteria.Gram negative bacteria have phospholipid membranes containing lipopolysaccharide elements that make their cell wall impassable to certain natural or synthetic antimicrobial drugs.
The results show that the methanolic extract exhibited antibacterial activities on more bacteria than the aqueous extract.Even though the freeze dry process can preserve phytochemical substances better than any other extraction method, the extraction yield and the biological activity of the extracts are also affected by the extraction solvent.The phytochemical processing of the raw plant materials is important to optimize the concentration of the constituents and to maintain the antibacterial activities. 32here are a few solvents that are usually used in the plant extraction such as distilled water, aqueous mixture of ethanol, ethanol, methanol, and acetone.The selection of the suitable extraction method and the solvent are important, and the selection is based on the sample chemical properties of the analytes, efficiency, desired properties, matrix properties and matrix-analyte interaction. 33Besides that, the chemical characteristics and polarities of the substances in the natural products may be or may not be dissolved in a certain solvent.
Methanol, acetone, and ethyl acetate are examples of polar solvent frequently used in the process of recovering polyphenols from the natural products.Production of lower molecular weight of polyphenols is known to be more effective by using methanol as solvent; while aqueous acetone is known to extract higher molecular weight of flavanols. 34However, distilled water is still useful in extracting other molecules.In a study, distilled water was found to be a better solvent in extracting tea catechins than methanol or ethanol. 35IC is the minimal concentration of antimicrobial drug that inhibit the growth of microorganisms after 24 hours incubation.The MIC is used to estimate the antimicrobial efficacy of potential antimicrobial drug by estimating the impact of descending antimicrobial doses.Antimicrobial drug with lower MIC is thought to be more potent.The MIC values derived from the

CONCluSiON
The aqueous and methanolic extracts of K. nemoralis exhibited a significant antibacterial activity.K. nemoralis extracts exhibited more effective antibacterial properties against Gram positive bacteria than Gram negative at low concentrations, which indicates that K. nemoralis contain special bioactive substances that may provide some benefits in the treatment of infectious diseases caused by Gram positive bacteria.
Shigella sonnei (ATCC 25931), Escherichia coli (ATCC 10536), Klebsiella pneumoniae (ATCC 10031) and clinical isolate Salmonella Typhi.The first five organisms are Gram positive, while the rest are Gram negative.All the stock cultures were obtained from the Microbiology Laboratory, Faculty of Medicine, Universiti Sultan Zainal Abidin.

table 2 .
Zone of inhibition (mm) for disc diffusion method of aqueous extract of K. nemoralis against selected bacteria

table 3 .
Zone of inhibition (mm) for disc diffusion method of methanolic extract of K. nemoralis against selected bacteria

table 4 .
36C value of aqueous and methanolic extracts of K. nemoralisTests were performed in triplicate current study proved that K. nemoralis aqueous extract was more potent against S. aureus and MRSA, which comply with the agar disk diffusion method result.Potent antibacterial activity was also recognized against S. aureus and MRSA at low concentrations of K. nemoralis extracts.The dissimilarities in bacterial susceptiveness are perhaps due to differences in essential strength of bacteria, or the physicochemical effects of phytochemicals contained in the plant materials.36