Centella asiatica is a popular medicinal plant that has a long history of usage as a traditional medicine that is used for neurological disease, skin disease, immune disease, and others. Nowadays, there is a wide array of Centella asiatica -based products. Although Centella asiatica was claimed to be having an established profile on the free radical scavenging activity, total phenolic content and antimicrobial activity of Centella asiatica but there is still a lack of detailed study that focuses on those properties. This study aims to fill the gap in the previous research, especially on the antimicrobial activity of C. asiatica. This study is worth examining because it helps to assure the effectiveness of C. asiatica medicinal products on the market. The antioxidant activity and total phenolic content of the plant  extract were examined by using DPPH assay and Folin-Ciocalteu method respectively. Based on the findings, C. asiatica was found to be having concentration-dependent antioxidant activity with the highest radical scavenging activity recorded at 52.7% at 100 μg/mL. For total phenolic concentration wise, 1mg/ml maceration and soxhlet extract of C. asiatica were found to be having 20.74 and 27.48 μg/mL Gallic acid equivalent (GAE) respectively. The antimicrobial activity was determined by using the modified broth dilution method. Based on the findings, C. asiatica was found to be effective against E. coli, K. pneumonia, and S. epidermidis with the MIC recorded at 4mg/ml. On the other hand, B.pumilus did not portray

sensitivity against C. asiatica at the tested concentration. This study is significant as it verifies the antioxidant activity, total phenolic content, and antimicrobial activity of the plant extract successfully.

Centella asiatica is an herbaceous, perennial plant that belongs to family Apiaceae. The other scientific name of Centella asiatica include Centella erecta, Hydrocotyle asiatica L., Hydrocotyle erecta, and Centella repanda. It also has a wide variety of common names include Asiatic pennywort, Indian Pennywort, and Goyu Kola. The Chinese common name of Centella asiatica is Ji Xue Cao. In India, it is known as Brahmi or Thankuni. It is widely found in tropical region such as China, India, Sri Lanka, Africa, Malaysia, Myanmar and Vietnam. Centella asiatica has a long history of usage in these countries. Apart from medicinal use, Centella asiatica has been used as an ingredient of local cuisine in these countries. For example, in Malaysia, Centella asiatica is one of the ingredients used to prepare “Ulam”, which is a type of salad taken by Malaysian. The taxonomical classification of Centella asiatica is depicted in Table 1 [1].

Table 1 : Botanical features of Centella asiatica

The plant prefers relatively shady and damp habitat such as wetland, riversides, ponds, wet

Meadows, forest and can adapt to an altitude from 300m to 1800m [2]. Figures 1-2 shows the leaves and flowers of Centella asiatica.

Figure 1: Centella asiatica asiatica asiatica leaves

Figure 2: Top View of Centella asiatica flower

It was also traditionally being used to treat gastrointestinal disease, sleep disorder, tuberculosis and diabetes [3].

Centella asiatica is known to have a rich phytochemical content. Majority of compound found in this plant belong to a chemical class known as triterpene saponosides. For instance, asiatic acid, madecassic acid, asiaticoside, madecassoside, madasiatic acid, betulinic acid, thankunic acid and isothankunic acid. These triterpene molecules are responsible for the biological properties such as increasing cellular hyperplasia, collagen production and also the anti-inflammatory properties of Centella asiatica. Flavonoid derivative such as quercetin, kaempferol, patuletin, rutin, apigenin, castilliferol and others are also being found in Centella Asiatica. Other compound such as polysaccharides, polyacetylenes, sterol, tannis, fatty acid has also been successfully isolated from the plant [4–6]. Centella asiatica also displayed a wide array of pharmacological properties. It has prominent anti-inflammatory, anti-oxidant and antimicrobial properties, which will be discussed further in this paper. Apart from that, it is also claimed to have anti-rheumatic, antipyretic, diuretic and antiviral properties which is evidenced from its wide array of traditional use [3].

Collection of Centella asiatica

Dried Centella asiatica were purchased from traditional herbal shop. The dried leaves were identified and authenticated. Then the dried leaves were cut into small pieces and grinded  into fine pieces by using an electronic grinder. After that, the grinded leaves of C. asiatica were weighed by using a digital weightbalance. The weightof the dried, grinded Centella asiatica leaves was found to be 207.3 gram and the weight was recorded. The grinded dried leaves were placed into a basket under room temperature for 1 day to ensure all the extracts were completely dried and free from moisture.

Preparation of Centella asiatica extract Maceration

75 grams of Centella asiatica leaves powder were added into a 1-liter conical flask and 600 ml ethanol.. The procedure above was repeated in order to have 2 set of maceration setup. Thus, a total of 150 gram of C. asiatica leaves and 1200 ml of ethanol were used for the maceration process of C. asiatica. After that, the mixture was allowed to stand for 7 days at room temperature in a dark cabinet. The mixture was shaken daily in order to create a homogenous mixture and ensure that all the contents of C. asiatica were extracted into the ethanol solution. After 7 days, the mixture was subjected to filtration by using muslin clothes. The ethanolic extract was then be subjected to evaporation process in order to get a concentrated extract.

Hot percolation (Soxhlet extraction)

25 grams of Centella asiatica powder was packed into thimble made from a strong filter paper or cellulose. Solvent ethanol was poured into round-bottom flask and connected to the lower opening of Soxhlet extractor. The temperature was controlled at around 70- 75°C as the boiling point of ethanol was around 78.3. This can ensure that only ethanol evaporated and minimum deterioration occurs to the plant phytochemical. Extraction occurred through direct contact between extraction solvent and the plant power. The entire process was continued for around 12 hours [7].

Evaporation of ethanolic extract

The temperature was set at 60°C and the rotation per minutes (rpm) of the evaporator was controlled at 100. The process was continued until a thick, viscous extract was obtained. The products were then put into evaporating dish and the evaporating dish was covered with aluminium foil. The temperature of the hot air oven was set at 45 °C to allow further evaporation of the ethanol solvent to occur gently. This process was continued until a viscous, thick paste of the extract was obtained [8].

Test for free-radical scavenging activity DPPH assay

DPPH assay is a method that deploy stable free radical which is 2, 2-diphenyl-1- picrylhydrazyl (C18H12N5O6) to determine the antioxidant activity of the tested compound. In this test, the scavenging capacity of antioxidant towards DPPH will be measured. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidant to the corresponding hydrazine. Therefore, DPPH assay is actually related to the hydrogen donating ability of the tested extract. DPPH will produce violet color in methanol solution but fades to shades of yellow color upon scavenged. The procedure for conducting DPPH assay was mentioned below [9].

Preparation of different concentration of extract and ascorbic acid solution

1 mg/ml C. asiatica stock solution was prepared by dissolving 100 mg of C. asiatica extract obtained from maceration process, which was in viscous paste form, in 100 ml of ethanol solution. After that, serial dilution process was carried out to prepare six different concentration of C. asiatica solution which were 10, 20, 40, 60, 80 and 100 μg/ml respectively. The different concentration of C. asiatica solution was prepared by mixing different volume of the stock solution with different volume of ethanol as shown in the Table2.

Table 2: Formulae for different dilution of C.asiatica extract

The total volume of different dilution should be 10 ml.  The C. asiatica solution was stored in a 10 ml volumetric flask for further use. The process was repeated by using the Soxhlet extract of C. asiatica and also ascorbic acid (9).

Preparation of 1,1-Diphenyl-2-picryl hydrazyl (DPPH) solution

0.3 mM of DPPH reagent was prepared by dissolving 11.83 mg in 100 ml of ethanol.

DPPH assay procedure

2.5 ml of different concentrations of maceration extract of C. asiatica were added into 6 separate test tubes. All the test tubes were then covered with aluminium foil as DPPH is a photosensitive reagent. After that, 1 ml of 0.3 mM of alcoholic reagent of DPPH was added into the test tubes. The test was performed in triplicate for each concentration of maceration extract of C. asiatica. The procedure mentioned above was repeated with the Soxhlet extract of C. asiatica and ascorbic acid solution. Similarly, control solution was prepared by  replacing the extract with ethanol and addition of 1ml of 0.3 mM of alcoholic solution of DPPH. A blank solution was prepared only with 3ml of 95% ethanol. All the test tubes containing the preparation mentioned above were allowed to stand for 30 minutes in dark cupboard. The absorbance at 518nm of different preparations was studied by using UV- Visible spectrophotometer. The test needed to be performed in a dark condition. The absorbance values of each different concentration of standard ascorbic acid, C. asiatica extract, control and blank were recorded. The free radical scavenging activity of different concentration of C. asiatica extract was calculated by using the formulae given below (10,11).

DPPH Radical Scavenging Activity (%) = (Acontrol - Asample)/Acontrol ×100

Result of DPPH assay

Table 4: Result of DPPH assay of C. asiatica maceration extract

Graph 1: Free radical scavenging against concentration of maceration extract of C. asiatica

Table 5: Result of DPPH assay of C. asiatica soxhlet extract

Graph 2: DPPH free radical scavenging against concentration of soxhlet extract of C. asiatica

Table 6: Result of DPPH assay of ascorbic acid

Graph 3 : DPPH free radical scavenging activity of maceration extract of ascorbic acid

Result of Total Phenolic Content

Table 7: Absorbance value of extract. Gallic acid, control and blank

Graph 4 : Standard curve of Gallic acid

Calculation of total phenolic content in C.asiatica extract

Given,

y = 0.0144x - 0.0297, where x=concentration of phenolic compound, y = absorbance value Maceration extract, absorbance =0.269,

Concentration of phenolic compound = (0.269+0.0297) /0.0144

=20.74 μg/ml Gallic acid equivalent

Soxhlet extract, absorbance = 0.366

Concentration of phenolic compound = (0.366+0.0297)/0.0144

= 27.48 μg/mL Gallic acid equivalent

Result for antimicrobial activtiy

Antimicrobial acitivity against Escherichia coli

Antimicrobial acitivity against K. Pneumoniae

Figure 4 Growth of K. pneumoniae at 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 mg/ml C. asiatica extract, blank, positive control and negative control

This study was conducted to evaluate the antimicrobial activity of C. Asiatica on 4 bacteria strains which are Escherichia coli, Bacillus pumilus, Klebsiella pneumoniae and Staphylococcus epidermidis. These bacterias were chosen because they are the most common that can be easily encountered in our daily life and can cause disease to individuals. Besides, this study also studies the free radical scavenging activity of the extract. Total phenolic content in C.asiatica was also studied as phenolic compounds are often the components that are responsible for the antioxidant properties of this medicinal plants.

C. asiatica was proved to have free radical scavenging radical effect in the research. This study discovered that the free radical scavenging activity of maceration ethanol extract of C asiatica at 100, 80, 60, 40, 20, 10 μg/mL are 52.7%, 48.8%, 46.4%, 41.6%, 40.2%, and 36.3% respectively. From the findings, it was found that C.asiatica did possess concentration- dependent free radical scavenging activity. As the concentration of C.asiatica extract increases, the absorbance value decreases, and the free radical scavenging activity of the extract increases. Another study conducted by Kulkarni et al. supports the  finding  that ethanol extract of C.asiatica did possess free radical scavenging activity. Although the study conducted by Kulkarni et al. was based on higher concentration, which are 200, 400, 600, 800, 1000 and 1200 μg/mL, however, from the graph plotted, it was found that there is the finding by this study is in coherent with the study performed by Kulkarni et al. . It is because the graph obtained from this study is consistent with that obtained by Kulkarni et al.. This study also portrayed that C.asiatica possess antioxidant activity even at a low concentration of 10 μg/ml, which imply that C.asiatica has a strong antioxidant activity. The study by Kulkarni et al. also shows that C.asiatica has higher antioxidant activity compared to Bacopa monnieri, which is another widely used traditional Ayurvedic medicine that is used for Alzheimer’s disease and improves brain function (14).

The free radical scavenging activity between the maceration extract and soxhlet extract of C.asiatica were found to be slightly different. The soxhlet extract of the C.asiatica was found to be having comparatively lower free radical scavenging activity at all concentrations compared to maceration extract. This finding is in agreement with the study by Mohapatra et al. which found out that maceration extraction method is superior to the heat reflux method in extraction of bioactive compounds from the C.asiatica. This may be due to exposure of the phytochemical compounds to continuous, high heat during the extraction method which will subsequently lead to deterioration of the antioxidant substances in C.asiatica. In the study for total phenolic content, Gallic acid was used as the standard. A standard curve was constructed based on the absorption value of Gallic acid at 760 nm for various concentrations which are 10, 20, 40, 60, 80, and 100 μg/ml, respectively. An equation was derived based on the standard curve constructed which was found to be Y=0.0144X-0.0297. The correlation coefficient R2 of the standard graph was found to be 0.9942. The value indicates that the absorbance value of Gallic acid at 760nm was highly related to the concentration. The absorbance value of maceration extract of C.asiatica was found to be equal to 0.269 at 760nm. By using the formulae derived from the standardcurve, the total phenolic contentof C. asiatica maceration extract was found to be 20.74 μg/ml Gallic acid equivalent. On the other hand, the absorbance value of Soxhlet extract was found to be 0.366 at 760nm. By using the formulae derived from the standard curve, the total phenolic content in C. asiatica Soxhlet extract was found to be 27.48 μg/mL Gallic acid equivalent(14,15).

According to the result of the study, C.asiatica was found to be having antimicrobial activitiy against E.coli. This is consistent with the finding of Dash et al. whereby the C.asiatica was suggested to be effective against the E.coli. Although there was a slight difference between the methodology used by Dash et al. and this study, there is still validity in the correlation between this study and study performed by Dash et al. The minimum inhibitory concentration (MIC) of C.asiatica against E.coli was found to be 4 mg/ml. It is because at 4mg/ml, the growth of E.coli was obviously inhibited to only around 100 colonies while in previous concentration which is 2mg/ml, there is turbidity shown all over the petri plate, which indicates visible growth of E.coli all over the plate. Based on the MIC, The minimum bactericidal concentration (MBC) was predicted to be 8mg/ml. In control wise, there is lack  of inhibitory effect of chloramphenicol against E.coli most probably due to the development of antibiotic resistance in E.coli [16]. K.Pneumoniae was found to be sensitive to the antimicrobial activity of C. asiatica. This is validated by the study performed by Suresh et al. wherein the ethanol extract of C.asiatica showed positive inhibitory effect on the growth of K. Penumoniae. The minimum inhibitory concentration (MIC) of C.asiatica against K.pneumoniae was found to be 4mg/ml in this study. According to the observation,  the growth of K.pneumoniae was suppressed to a few colonies at 4mg/ml while at 2mg/ml, there is still turbidity that indicates the growth of bacteria cells all over the plate. Based on MIC,  the minimum bactericidal concentration (MBC) of C.asiatica against K.penumoniae was speculated to be 8mg/ml. The number of coloniesof K.pneumoniae found on the surface of petri plate was almost the same compared with E.coli. Thus, it is safe to conclude that C.asiatica is having comparable activity against E.coli and K. Pneumoniae, which are both gram-negative bacteria [17]. C.asiatica was found to be effective against S. Epidermidis. This finding can be supported by the study conducted by Sieberi et al.. It is because the bacterial growth was suppressed into colonies at 4mg/ml. However, if compared to the inhibitory effect found in E. coli and K pneumoniae, the inhibitory effect of C.asiatica and S.epidermidis was less significant as there are more bacteria colonies found on the surface of plate. Thus, it is coherent to S.epidermidis is less vulnerable to C.asiatica although it shares the same MIC with E.coli and K.pneumoniae [18,19]. C.asiatica did not portrayed inhibitory effect on B.pumilus with the concentration given which are 4mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml , 0.25mg/ml ,0.125 mg/ml and 0.0625 mg/ml. The positive control in which chloramphenicol was added to the nutrient broth containing B.pumilus confirmed the viability of the B.pumilus bacteria used in this study. There is still lack of study and evidence about the antimicrobial activity of C.asiatica on B.pumilus. Thus, there is lack of evidence to conclude that C.asiatica does not possess antibacterial activity against B.pumilus. According to comparative studies performed by Idris and Mohd Nadzir, other studies that investigated the activity of Casiatica on the Bacillus species did yield positive results. However, those studies utilize Bacillus cereus and Bacillus subtilis in their studies. Moreover, most of those studies were based on disc diffusion method, which only studies on the sensitivity of bacteria against the plant extract and did not give a value on the inhibitory concentration of C.asiatica on Bacillus species. There is also a possibility that C.asiatica only inhibit B.pumilus at a higher concentration [8].

It was found that C.asiatica was more potent on Gram-negative bacteria than on Gram- positive strains, thus acting as a broad spectrum. This can be validated by less number of bacterial colonies for E.coli and K.pneumoniae compared to B.pumilus and S. Pneumoniae at their respective minimum inhibitory concentration. These findings are consistent with other reported research works such as the study by Sieberi et al. and Ashok et al., and this could be attributed to the presence of a lipophilic outer membrane consisting of lipopolysaccharide molecules with an affinity for lipophilic molecules (18,20).

This study found that the % free radical scavenging of maceration ethanol  extract  of  C asiatica at 100, 80, 60, 40, 20, 10 μg/mL were 52.7%, 48.8%, 46.4 Percentage, 41.6 Percentage, 40.2 Percentage, and 36.3 Percentage

respectively. The soxhlet extract of C asiatica also showed a similar result as the maceration

extract. This study shows that C asiatica possesses high potential of antioxidant activity. This study also showed that the total phenolic content in C.asiatica maceration and soxhlet extract at 1 mg/ml is 20.74 and 27.48 μg/mL Gallic acid equivalent (GAE) respectively. The study on the antimicrobial activity showed that C. asiatica has antimicrobial activity against E. coli,

K. pneumonia, and S. Epidermidis. On the other hand, B. pumilus was not inhibited by C. asiatica at the concentration tested. The MIC of C. asiatica for E. coli, K. pneumoniae and S. epidermidis was 4mg/ml.

The authors are highly thankful to the Faculty of Pharmacy, AIMST University, Bedong, Kedah D.A., Malaysia, for funding and providing facilities to carry out this research project. 

Authors declare that there is no conflict of interests.