Division of City SchoolsCity of Mandaluyong Science High SchoolE. Pantaleon St., Hulo Mandaluyong City

In Vitro Antimicrobial Activity of Extracts fromTakip kohol (Centella asiatica) leaves asAntibacterial Gauze Dressing

A Research presented tothe faculty ofCity of Mandaluyong Science High School

A Research projectin Partial Fulfillment ofResearch II

Submitted by:Almodiente, Allayza R.Bernardo, Claire L.Jacobe, Reaiah Anne G.Parganel, Daniel A.Pelagio, Maria Mikaela V.Retizos, Alexandra Denise M.Group I

IPSF OFFICIAL ABSTRACT FORMTITLE: In Vitro Antimicrobial Activity of Extracts from Takip kohol (Centellaasiatica) leaves as Antibacterial Gauze Dressing

Name:Almodiente, Allayza R.; Pelagio, Maria Mikaela V.; Bernardo, Claire L.; Jacobe, Reaiah Anne G.; Retizos, Alexandra Denise M.; Parganel, Daniel A.Address: E, Pantaleon St., Brgy. Hulo, Mandaluyong CitySchool: City of Mandaluyong Science High School

Asiaticoside is a triterpene glycoside from the plantCentellaasiatica or Takip kohol. Asiaticoside has been reported to elevate collagen synthesis in fibroblasts. It also facilitates wound healing through an increase in peptidichydroxyproline content, tensile strength, collagen synthesis and epithelialization. The bacterium used in this study is S. aureus. These bacteria can enter the wound if the skin is punctured or broken for any reason. It most commonly causes skin infections like folliculitis, boils, impetigo, and cellulitis that are limited to a small area of a person's skin.Ground Takip kohol leaves were mixed with methanol solution and undergone a series of processes of centrifugation, molisch test and HPLC in order to extract the Asiaticoside. In an in vitro process, the bacteria sample, S. aureus, was used to test the effectiveness of Asiaticoside. After testing, it was made to be an antibacterial gauze pad.The result of this study proves that Asiaticoside is an effective to set as an antimicrobial antiseptic. The product would provide an alternative wound healer as gauze pad which is all natural and organic.

Category:Pick one only mark an X in box at right: Biochemistry Botany Chemistry Computer Science Earth & Space Engineering Environmental Science Mathematics Medicine & Health Microbiology Physics Zoology Gerontology

We hereby certify that the above is the result of work done in our research

___________________________________________Signature of Team LeaderDate

ACKNOWLEDGEMENTThe researchers would like to give their utmost gratitude to Mr. Chris Patron, as their Investigatory Project Adviser and Mr. Benjamin Ola, their Research II teacher whose encouragement, counsel and support from the initial to the final part of the study enabled them to develop an understanding of the subject.To Mrs. Roxanne Villanueva, Mrs. Odette Fabon, Mrs. Digna Bal of the school faculty, and Ms. Zarah Almodiente for giving their recommendations for this study.This study would not also be possible without the help of their principal, Mr. Henry A. Sabidong for giving them permissions and assistance to conduct their research.To their parents, who provided them overflowing moral and financial support. Their classmates who gave them joy and became inspiration to continue this study.Lastly, the researchers would like to thank the Almighty Lord for giving them courage wisdom and knowledge all throughout the study.

TABLE OF CONTENTSTITLE1ABSTRACT2ACKNOWLEDGEMENT3TABLE OF CONTENTS4INTRODUCTION5BACKGROUND OF THE STUDY7SIGNIFICANCE OF THE STUDY9STATEMENT OF THE PROBLEM10SCOPE AND LIMITATIONS10DEFINITION OF TERMS10REVIEW OF RELATED LITERATURE12METHODOLOGY17Materials/Equipment17Methods undertaken to answer the research questions18Flowchart21PRESENTATION, INTERPRETATION AND ANALYSIS OF THE RESULTS22SUMMARY OF FINDINGD, CONCLUSIONS AND RECOMMENDATIONS26BIBLIOGRAPHY27

INTRODUCTIONCentella asiatica or also called Takip kohol in Philippine term is a small herbaceous annual plant of the family Mackinlayaceae or subfamily Mackinlayoideae of family Apiaceae. Takip kohol has been used for hundreds of years in many Asian countries as a traditional medicine to improve wound healing.It is also a pantropical plant used over two thousand years in India. The primary constituents reported in the literature for Takip kohol are known as the triterpenoid compounds. There are three main chemical constituents in Takip kohol. The first is Asiaticoside, which is a triterpene glycoside and classified as an antibiotic. It aids in wound healing and has been used in the treatment of leprosy and tuberculosis. The second constituent is a pair of saponins, Brahmoside and Brahminoside, which are saponin glycosides. These are diuretic in nature and have a slightly sedative action in large doses. Finally, there is Madecassoside, a glycoside that is a strong anti-inflammatory agent. The plant is also a source of Vitamin K, magnesium, calcium and sodium. C. asiatica also contains a number of alkaloids. Its ability to heal wounds, improves mental clarity, and treat skin conditions such as leprosy and psoriasis were important reasons for its extensive use.Asiaticoside (C48H78O19) is a triterpene glycoside, an active chemical compound, from the plant Centella asiatica is an essential ingredient in treating open wounds and burns.Asiaticoside has been reported to elevate collagen synthesis in fibroblasts. In vitro histological findings also revealed enhanced proliferation of fibroblasts, thereby supporting the biochemical results.

Enhanced healing activity has been attributed to increased collagen formation and angiogenesis.Asiaticoside is used in many skin treatments aside from healing wounds. It stimulates the collagen synthesis for dermis reconstruction. It reinforces the bio-mechanic properties of mature skins. In addition, it helps the normalization and regulation of the dermis. It also facilitates wound healing through an increase in peptidic hydroxyproline content, tensile strength, collagen synthesis and epithelialization. Staphylococci or staph are a common type of bacteria that live on the skin and mucous membranes of humans. Staph bacteria are usually associated with open wounds because these bacteria are not only present in the environment but also on the skin. These are opportunistic bacteria and can cause infection when skin has cut or open wound. The researchers used Staphylococcus aureus (S. aureus) bacterium. It is the most important of these bacteria in human skin diseases especially to open wounds. Other staphylococci, including S. epidermidis, are considered commensals, or normal inhabitants of the skin surface.Therefore, Asiaticoside can be one of the best microbial treatments for open bacterial wounds for it is a trisaccharide triterpene, identified as the most active compound in the plant associated with the healing of wounds. It helps in the increase of collagen in the skin and can perform different functions and help the skin to be healthier and more resistant to different ailments present in the skin.

BACKGROUND OF THE STUDYCentella asiatica or commonly known as Takip kohol(Philippine term) has many medicinal benefits but this paper will mainly focus on its wound healing capability. Takip kohol consists of active chemical components which are good for curing wounds. One of these is Asiaticoside (C48H78O19) a trisaccharide triterpene, which has been identified as the most active compound in the plant associated with the healing of wounds and duodenal ulcers, while the triterpene saponins are also reported to possess immunomodulatory properties. Asiaticoside is one of the most active compounds which can serve as a marker and is used for its standardization. Asiaticoside has shown promising wound-healing activity.Asiaticoside was found to induce cell-cycle progression proliferation and collagen synthesis in human dermal fibroblast. Itis also has been reported to decrease fibrosis in wounds, thus preventing new scar formation. Angiogenesis plays an important role in wound healing, and newly formed blood vessels comprise 60% of the repair tissue. Neovascularization helps hypoxic wounds attain normoxic conditions. Asiaticoside promotes angiogenesis in both in vitro and in vivo models as indicated by histological studies. Asiaticoside also accelerates the healing of gastric kissing ulcers. Gastriculcer healing is a complex orchestrated process involving resolution of inflammation and repair of gastric tissues through granulation tissue formation, re-epithelialization, and extracellular matrix remodeling. Asiaticoside suppresses myeloperoxidase, which is a marker of neutrophil infiltration during inflammation.

Staph is the shortened name for Staphylococcus, a type of bacteria. These bacteria can live harmlessly on many skin surfaces, especially around the nose, mouth, genitals, and anus. These bacteria can enter the wound if the skin is punctured or broken for any reason. The bacterium used in this study in this is S. aureus. It most commonly causes skin infections like folliculitis, boils, impetigo, and cellulitis that are limited to a small area of a person's skin. S. aureus can also release toxins (poisons) that may lead to illnesses like food poisoning or toxic shock syndrome.Gauze is a thin, transparent fabric with a loose open weave, commonly used as dressing for open lacerations. It allows air to circulate around the site, promoting rapid healing. It was originally made from silk and was used for clothing. When used a medical dressing, gauze is generally made of cotton instead of fabrics where it might stick to the burn or laceration. This study would be assessing in Asiaticoside as an effective active ingredient for antibacterial gauze dressing.

SIGNIFICANCE OF THE STUDYThis study aims to develop and provide inexpensive antibacterial gauze dressing with the chemical compound Asiaticoside from Takip kohol as its active ingredient for healing open wounds. Although there are many existing antiseptic products available, the result and product of this study would provide an alternative wound healer which is all natural and organic. Most people who would benefit from this study are those who cannot afford to buy expensive antiseptic products. This would also be beneficial to those are health conscious individual who prefer herbal medicine and treatment rather than the chemical based products. Because today we are facing economic problems that affect our way of living, this study was conducted to produce a new antiseptic product that can be afford by the lower economic class people.

STATEMENT OF THE PROBLEMThe present study deals with investigating the in vitro antimicrobial activity of extracts from Takip kohol, specifically, it sought to answer the following questions:

1. In what percentage is Asiaticosde present in the leaves of Takip kohol (Centella asiatica)?2. What effect does Asiaticoside have in the growth and reproduction of staphylococcus bacteria?3. What amount of Asiaticoside applied as an antisepetic as an antiseptic ingredient in gauze dressing is most effective in inhibiting bacterial growth?

SCOPE AND LIMITATIONSIn this study, the researchers primarily focused in creating an antiseptic gauze pad from Asiaticoside found in Takip kohol plant. The plant material was limited to 500 grams due to its availability in the country. Its maturity was not a factor in choosing the leaves for extraction. The use of Asiaticoside is limited only as a compound to cure Staphylococcus bacteria found in open wounds. The amount of bacteria used was measured before it was tested with Asiaticoside. It was limited according to the ratio given. The gauze pad which will be used as a medium for the Asiaticoside solution was limited to the commercialized gauze pad sold in the market.DEFINITION OF TERMS Chromatography-A technique for the separation of a mixture by passing it in solution or suspension through a medium in which the components move at different rates. Centrifuge-A machine with a rapidly rotating container designed to apply centrifugal force to its contents, usually to separate liquids from solids, or fluids of different densities. Hydroxyproline-Is a nonessential amino acid, which means that it is manufactured from other amino acids in the liver; it does not have to be obtained directly through the diet. Pantropical-Occurring or distributed throughout the tropical regions of the earth. Proliferation-Increase rapidly in numbers; produce rapidly. Triterpene-Areterpenesconsisting of sixisoprene unitsand have the molecular formula C30H48. Trisaccharide-A carbohydrate that yields three monosaccharides upon hydrolysis. Staphylococcus aureus-Also known asgolden staph, is the most common cause ofstaph infections. It is a sphericalbacterium, frequently living on the skin or in the nose of a person. Mackinlayaceae- is a subfamily ofplantscontaining about 67 species in six genera. In theAPG II systemit was treated at family rank asMackinlayaceae,but since then it has been demoted to a subfamily ofApiaceae. Triterpene glycoside- is used for fever,menstrual cramps, arthritis, and insomnia. Epithelialization- The process of covering a denuded surface with epithelium. Commensals-living on or within another organism, and deriving benefit without harming or benefiting the host;a parasite that causes no harm to the host.

REVIEW OF RELATED LITERATURETakip kohol (Centella asiatica) , also known as Centella and Indian pennywort, is a creeping vine found growing in tropical and subtropical climates in Africa, Asia, Australia, India and South America, and even the southeastern United States. It has been used for thousands of years in Ayurvedic medicine to improve memory and maintain a healthy central nervous system, and recent clinical studies support this tradition. Takip kohol has a long history of use to promote wound healing and treat skin diseases. In Europe, extracts of Centella asiatica are used as drugs for the treatment of wound healing defects. (Michael Castleman, 1991)Historically, Takip kohol has also been used to treat syphilis, hepatitis, stomach ulcers, mental fatigue, epilepsy, diarrhea, fever, and asthma. Today, American and European herbalists use Takip kohol most often to treat chronic venous insufficiency (a condition where blood pools in the legs). It's also used in ointments to treat psoriasis and help heal minor wounds. Takip kohol should not be confused with kola nut (Cola nitida). Unlike kola nut, Takip kohol has no caffeine, and is not a stimulant. The plant is reported to contain glycosides like brahmosides, indocentelloside, asiaticoside, theankuniside and isotheankuniside. Asiaticoside, a trisaccharide triterpene, has been identified as the most active compound in the plant associated with the healing of wounds and duodenal ulcers, whilst the triterpene saponins are also reported to possess immune-modulatory properties. Asiaticoside is one of the most active compounds which can serve as a marker and is used for its standardization. A few methods such as gravimetric and column chromatography have been suggested for the quantitative estimation of Asiaticoside which are not very precise, sensitive and require multiple step extraction and purification. Literatures reveal that HPLC methods are also available.Extracts of Centella asiatica, as well as various preparations of its prime constituent, Asiaticoside, have been shown in numerous studies to effectively promote the healing of skin wounds, particularly burns. Extracts of Centella asiatica have successfully treated surgical wounds, skin grafts, gangrene (life-threatening condition), and traumatic injuries, including perineal lesions from childbirth.Asiaticoside, a Trisaccharide triterpene (terpenesconsisting of sixisoprene unitsand have the molecular formula C30H48), has been identified as the most active compound in plant associated with the healing of wounds, specifically, 1st to 2nd degree burns and duodenal ulcers, while the Triterpene saponins are also reported to possess immune-modulatory properties. Triterpenoid saponinsaretriterpeneswhich belong to the group ofsaponincompounds. Triterpenes belong to a large group of compounds arranged in a four or five ring configuration of 30 carbons with several oxygens attached. Triterpenes are assembled from a C5 isoprene unit through the cytosolic mevalonate pathway to make a C30 compound and are steroidal in nature. Asiaticoside is one of the most active compounds which can serve as a marker and is used for standardization. A few methods such as gravimetric and column chromatography have been suggested for the quantitative estimation of Asiaticoside which are not very precise, sensitive and require multiple step extraction and purification.Shukla and colleagues studied the activity of Asiaticoside from Takip kohol in normal and delayed-type wound healing in guinea pigs. Results suggest that both topical (0.2% and 0.4% solutions of Asiaticoside) and oral (1mg/kg) administration of Asiaticoside exhibit significant wound healing activity in normal and delayed healing models.A tincture of Centella asiatica containing Asiaticoside as the main ingredient healed 4% of soiled wounds and chronic or recurrent atony that was resistant to standard treatment regimens. Asiaticoside was found to induce cell-cycle progression, proliferation and collagen synthesis in human dermal fibroblasts. Scientists thought, this extracellular matrix synthesis might be related to certain gene expression.Interestingly, Asiaticoside was shown to inhibit healing of experimental corneal wounds in rabbits (Callizoet al.1996). Studies of the effect of Asiaticoside in corneal wounds and ulcers have been documented by AbouShousa& Khalil (1967) and by Mekkawi (1968). Asiaticoside appears also to be able to inhibit collagen production in such a way as to inhibit formation of hypertrophic scars, keloids, and stretch marks (striaegravidarum / striaedistensae) (Bosseet al.1979, Qiet al.2000, Widgerowet al.2000, Young & Jewell 2003). Extracts from the plant have been formulated into several commercial products including Collaven, Emdecassol, Madecassol, Centelase, Marticassol, Blastoestimulina, and Trofolastin. Reports of allergic contact dermatitis, confirmed by patch testing have appeared sporadically following topical use of Madecassol, (Eun& Lee 1985), Centelase (Santucciet al.1985, Vena &Angelini 1986, Daneseet al.1994), and Blastoestimulina (Izuet al.1992, Aguirreet al.1993, Bilbaoet al.1995, Gonzalo Garijoet al.1996). Asiaticoside, asiatic acid, and madecassic acid appear to be the sensitisers (Huriez& Martin 1969, Hausen 1993). The possibility of sensitivity to other components of the formulations (for example, propylene glycol) should not be overlooked (Eun& Lee 1985, Hausen 1993).Asiaticoside induced elevation of antioxidant levels in healing wounds. Since antioxidants have been reported to play a significant role in the wound healing process we studied the effect of asiaticoside on the levels of certain antioxidants in the wound so as to explore the possible involvement of such a mechanism in the asiaticoside induced wound healing. Asiaticoside application (0.2%, topical) twice daily for 7 days to excision-type cutaneous wounds in rats led to increased enzymatic and non-enzymatic antioxidants, namely superoxide dismutase (35%), catalase (67%), glutathione peroxidase (49%), vitamin E (77%) and ascorbic acid (36%) in newly formed tissues. It also resulted in a several fold decrease in lipid peroxide levels (69%) as measured in terms of thiobarbituric acid reactive substance. However, continued application for 14 days showed no significant difference in these antioxidants compared with their values in vehicle treated wound tissue. It appears from the present study that Asiaticosides. Enhanced induction of antioxidant levels at an initial stage of healing which may be an important contributory factor in the healing properties of this substance.(Chuen Lung Cheng, 2003)Antibiotic-resistant bacteria continue to be a major health concern worldwide. In particular, Staphylococcus aureus, both methicillin-resistant and -sensitive, are of concern in their ability to cause difficult skin and underlying tissue infections.Staphylococcus aureus, also calledS. aureusor staph, is abacteriumthat frequentlycolonizesthe human skin and is present in the nose of about 25-30% of U.S. adults.S. aureuscan exist in this form without harming its host or causing symptoms. However, if there is a break in the patients skin from a wound or surgery, or if there is a depression in the persons immune system, then colonizingS. aureuscan cause an infection. Staph frequently causes localized skin infections, such asfolliculitis, furuncles, andimpetigo. It can also causeabscessesand spread into the bones (osteomyelitis), lungs (staphylococcal pneumonia), blood (bacteremiaorsepsis), heart (endocarditis), and other organs. Staph may also infect others as it can be passed from both infected and colonized people to other people through skin contact or through sharing contaminated objects, such as towels or razors.Methicillin-ResistantStaphylococcus aureus(MRSA) has become a headline concern due to its emergence as a common infection in a new age of antibiotic resistance. Infection control measures are especially important in crowded school and childcare settings because MRSA is spread by skin-to-skin contact which occurs more easily in such conditions. The following questions introduce information regarding transmission, treatment, and prevention of these now common bacteria.The majority of MRSA infections occur among patients in hospitals or other healthcare settings; however, it is becoming more common in the community setting. Data from a prospective study in 2003 suggests that 12% of clinical MRSA infections are community-associated, but this varies by geographic region and population.Community-Associated Methicillin-ResistantStaphylococcus Aureus(CA-MRSA) are staph bacteria that are resistant to some antibiotic treatments, specifically to the antibiotic called methicillin. Until recently it was only found in hospital settings. However, now it can be found commonly outside of hospitals; thus it is called community-associated. These unique clones of CA-MRSA are increasingly responsible for community-associated infections in healthy children and adults. The most frequent manifestation of these community-associated MRSA infections is skin and soft tissue infection, but invasive disease and pneumonia can also occur. In the present investigation, effects of Asiaticoside from Centella asiatica or Takip kohol on the bacteria S. aureus to reveal its antimicrobial potential.

METHODOLOGYThe part presents the materials used and the methods utilized in investigating the effects of Asiaticoside on S. aureus.I. MATERIALS/EQUIPMENTThe procedure carried out in this investigation was divided into two parts: preparation, extraction, and identification of the Asiaticoside, and the antibacterial activity test. The first part involved the collection and treatment of the Takip kohol (Centella asiatica) samples, homogenizing of the sample, preparation for centrifugation, and identification sing the High Performance Liquid Chromatography. The second part chiefly focused on preparation of the Asiaticoside extract and the S. aureus for the antibacterial test.The table summarizes the materials used in this investigation, with focused on which part they were used, and the amount of research time devoted to using them.Table no 1. Materials used in the different phases on the investigationResearch PhaseMaterial UsedTime Spent

A. Preparation, Extraction, and Identification of the Asiaticoside from Takip kohol samples.

a. Collection and treatment of Takip kohol (Centella asiatica) samples500g of Takip kohol leavesdigital weighing scalesmall bowlaspiratorscissorswatch glassplastic used for air drying15 days (including time for cultivation of Takip kohol)

b. Homogenizing of the sampleMortar and pestleMethyl alcohol (methanol solution)Evaporating dishLaboratory gownTest tube50 ml graduated cylinder

1 hour

c. Preparation for Centrifugationcentrifugecentrifuge tubepipetteaspiratortest tube racktest tube1 hour

d. Identification of the Extract through HPLCvolumetric flaskcentrifuged sample of Takip kohol leavesHPLC2 weeks

B. Preparation for and Conduct of the Antibacterial Activity test100gm of Staphylococcus aureuspetri dishmannitol salt agarcoagulase solution 4 hours

II. METHODS UNDERTAKEN TO ANSWER THE RESEARCH QUESTIONSThe present investigation deals with the effects of Asiaticoside from Takip kohol on the activity of the bacteria S. aureus. Methods used to answer the research problems posed were divided into two: preparation, extraction, and identification of the Asiaticoside and determination of its effects on the activity of S. aureus. In detail, the following steps were undertaken:A. Preparation, Extraction, and Identification of the Asiatiocoside from Takip kohol1. Preparation of the leaves of the Takip kohol for the extraction of the Asiaticoside

Figure 1. Determining the mass of the Takip koholTakip kohol was home-grown for two weeks. Only the leaves were selected and used. Then the leaves were cut with the use of a pair of scissors. The leaves in good condition (i.e., fresh, green, no evidence of discoloration or withering) were selected and separated from other leaves. The leaves were weighed and 500g of the leaves were selected for air drying. The leaves were then placed in a clean medium-sized bowl and were tightly covered with a plastic with holes. To dry then, the leave swere exposed and dried under the sun for thirty minutes. 2. Centrifugation of the Dried Takip kohol leaves

Figure 2.Takip kohol leaves were ground using a mortar and pestleThe Takip kohol leaves were then ground using a mortar and pestle until they became very fine. It was then mixed in a 25ml methanol solution. 4mil of the solution was poured each in a centrifuge tube. Five samples were prepared and placed inside the centrifuge. The centrifuge was set to five (5) rotations per minute for twenty minutes. The process was repeated three times to remove impurities.After centrifugation, the sample was subjected to molisch test, a test to determine the presence of carbohydrates. Appositive result was indicated by a purple ring on the solution.

Figure 3. Dried Takip kohol leaves dissolved in methanol were placed inside the centrifuge3. Identification of the Asiaticoside from the Takip koholTen milligram of Asiaticoside reference standard was weighed accurately in to a 10mL volumetric flask. This was then dissolved in 5mL of methanol. Conversely, 10 mL of sample in 5 mL of methanol. The HPLC equipment was then adjusted as per conditions prescribed. HPLC analysis of

Figure 4. HPLC equipment for deteminig the Asiaticoside presence in the Takip kohol leavesthe standard or reference and the samples were carried out using mentioned protocol and the chromatograms were recorded. The retention time and the peak area of the standard and the sample were noted and compared. Amount of the Asiaticoside present in the sample was then determined by comparing the sample peak with that of the standard.B. Preparation of the S. aureus for the Test of Antibacterial Activity1. Bacteria CultureS. aureus was cultured as used as a reference strain. The reference strain was touched with a needle loop and growth was transferred to blood agar and incubated at 37C. The strain was confirmed using coagulase solution and mannitol salt sugar.

Figure 5.Sample of S. Aureus used in the study2. Antibacterial Activity TestThe bacteria were placed in a Petri dish. A drop (approximately 2mL) of Asiaticoside and observations were made. This method was carried out to determine the effects of Asiaticoside in cultured bacteria.

500 grams of Takip kohol leaves were collectedMETHODOLOGY FLOW CHART

Conduct High Performance Liquid Chromatography.

Leaves were air-dried for thirty minutes

4mL of the methanol solution with air dried ground leaves were placed in the centrifuge tube

With the use of mortar and pestle, leaves were ground until fine

The powdered leaved were diluted in 25mL methanol solution

Centrifuge was set to 5 rotations per minute for twenty minutes. This step was repeated three times.

Molisch test was conducted to test presence of carbohydrates

Antimicrobial property of the Asiaticoside was determined with the used of S.aureus bacteria

Figure 6. Flowchart of the Steps undertaken in this InvestigtionPRESENTATION, INTERPRATATION AND ANALYSIS OF THE RESULTS

This study aimed at determining the presence of Asiaticoside in Takip kohol and at investigating its effects in S. aureus to underline its potency as antimicrobial agent in a gauze pad. The steps undertaken involved reparation and identification of the Asiaticoside from the Takip kohol leaves using HPLC and determination of its antimicrobial potential when incorporated in a gauze pad. 1. Determining the Presence of the Asiaticoside in the Takip kohol leavesHigh Performance Liquid Chromatography was used to determine the presence of Asiaticoside in Tahip kohol leaves. In the said process, the sample was compared to standard to reveal its presence in terms of retention time and peak time. The figure below shows the result of the process:

Figure 7. HPLC Chromatogram of the Standard AsiaticosideThe chromatogram shows that the peak at retention rate of the standard Asiaticoside was 11.65min. The figure below shows the chromatogram of the sample Asiaticoside

Figure 8. Chromatogram for the methanolic sample

Table 2. Estimation of the Asiaticoside through the HPLCFigure 8 showed the chromatogram of methanolic extract of Centella asiatica. The quantification yielded 1.7mg / 100mg of the test extract. It shows the peak retention time of the sample to be 11.95min, which is comparable to the value obtained for the sample, which was 11.65min. The estimation of the Asiaticosie through HPLC is shown in the table below:

The estimation of Asiaticoside in Centella asiatica showed in Table 2. The signal to noise ratios 3:1 and 10:1 were considered as LOD and LLOQ respectively. The LOD and LLOQ were found to be 50 and 200 mg/spot. Calibration graph, a plot of peak area obtained versus Asiaticoside concentration, showed good linearity in the concentration range of 32 - 1027 g/mL, (Y = 2055.5X +27975), with a correlation coefficient of 0.999.

1 2 3 4 5 6 7 8 9 10 11 12

Asiaticoside

Figure 9. Chromatogram of the Asiaticoside extract from C. asiatica leave powder extracted with 95% methanol

The figure above shows another chromatogram of the sample Asiaticoside extract from the C. asiatica. It reveals how the standard Asiaticoside (numbered 2 and 4 in the figure) compared with the sample (number 4 in the figure). The table below shows the significant differences between extract and standard for the different amounts of the C. asiatica leaves.2. Antibacterial Activity of the Asiaticoside ExtractThe antimicrobial activity of the Asiaticoside was determined by placing approximately 2mL of Asiaticoside in concentration 20%, 40%, 60%, and 80% in the Petri dish containing cultured S. aureus. Three trials were done per concentration. The mean was determined, as well as the standard deviation. Table 3 shows the results obtained.

Table 3. Antimicrobial activity of the Asiaticoside in different concentrationsConcentration of Asiaticoside extract in percentage

20%40%60%80%

Zone of Inhibition (mm)Trial 17.216.45.814.85

Trial 27.226.495.765.01

Trial 37.47.016.134.96

Mean7.286.75.94.94

SD0.0870.270.1640.067

The table shows that the Asiaticoside extract from the Takip kohol possesses antimicrobial activity as determined by the decrease in the zone of inhibition. At higher concentrations, the zone of inhibition became smaller. It can be seen from the table that at a concentration of 80%, the zone of inhibition is least, making it the concentration that is most effective.

SUMMARY OF FINDINGS, CONCLUSION, AND RECOMMENDATIONSA. The following are the salient findings of this study:1. The presence of the Asiaticoside in Takip kohol was determined using the HPLC. Results obtained showed that the amount was at 32-1027mg/mL.2. The growth of the S. aureus bacteria was inhibited upon the application of the Asiaticoside at different concentrations. In these investigations, concentrations of 20%, 40%, 60%, and 80% yielded zones of inhibition represented by means of 7.28mm, 6.67mm, 5.9mm and 4.94mm respectively.3. The results showed that the concentration of 80% was most potent in the antimicrobial activity.CONCLUSIONThe study considered investigating the effect of Asiaticoside on the S. aureus bacteria. The presence of Asiaticoside in Takip kohol was determined using HPLC. Its effects on bacteria were demonstrated by the decrease or the bacterias zone on inhibition as the concentration of Asiaticoside was raised. The concentration that was most effective in inhibiting the growth of the bacteria was at 80%.RECOMMENDATIONSIt is recommended that better statistical techniques be used to interpret the date obtained to a richer extent. Furthermore, it is recommended that other ways be explored in determining the presence and the amount of Asiaticoside in Takip kohol and it varieties. Lastly, it is recommended that other kinds of bacteria be used in the test of antimicrobial activity.

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