BIBECHANA 18 (2) (2021) 18-25 18 Phytochemical analysis, antioxidant and antibacterial efficacy of methanol and hexane extract of Centella asiatica Shanta Pokhrel*, Prabha Neupane Department of Chemistry, Tri-Chandra Multiple Campus, Tribhuvan University, Kathmandu, Nepal *E-mail: shantabhattarai2014@gmail.com Article Information: Received: August 22, 2020 Accepted: November 08, 2020 Keywords: Antibacterial Antioxidant DPP IC50 Phytochemical screening ABSTRACT Centella asiatica is well known for its anti-inflammatory, anticancer, antioxidant, antimicrobial, analgesic, diuretic properties. Its hexane and methanol extracts were screened for the presence of phytoconstituents as well as their antibacterial and antioxidant activity. The phytochemical screening showed the presence of carbohydrate, glycosides, flavonoids, phenolic compound, alkaloids, and saponins. The methanol extract showed more effective antibacterial activity against Salmonella typhi. Antioxidant activity of methanol extract was evaluated by 2, 2- diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity showed potent antioxidant activities with IC50 value 2.57 μg/mL slightly higher than standard ascorbic acid (IC50 = 3.74 μg/mL). The present study indicates that the tested plant can be an important source of antibacterial agents and recommends that the active phytoconstituents be isolated, identified, and screened individually for activities and also subjected further for in vivo and toxicological studies. DOI: https://doi.org/10.3126/bibechana.v18i2.30760 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons.org/licenses/by-nc/4.0/ 1. Introduction Centella asiatica (family Umbelliferae) commonly referred to as water pennywort or Indian pennywort or Asiatic pennywort or Ghodtapre in Nepal. It is a common perennial herbaceous creeper flourishing abundantly in moist areas and distributing widely in tropical and subtropical countries including Bangladesh, Nepal, South Africa, India etc [1]. The plant of Centella asiatica is a quite aromatic, prostrate, perennial, stoloniferous, creeper herb, which height up to 15 cm. Stem is striated, glabrous, rooting at the nodes. Several pharmaceutical companies are utilizing such plant based formulations in treatment of various diseases and disorders world around [2]. A large number of people in developing countries depend on medicinal plants as their primary source of medication [3]. Centella asiatica has been used by Ayurvedic medical practitioners for almost 3000 years [4] to treat wounds, mental and neurological disorders, atherosclerosis, microbial infections, and cancer [5]. Traditionally it is used to treat a broad range of diseases such as diarrhea, hepatitis, measles, toothache, syphilis, leucorrhoea etc [6]. It also possesses ulcer-preventive [7, 8a, b], BIBECHANA ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher: Department of Physics, Mahendra Morang A.M. Campus, TU, Biratnagar, Nepal https://doi.org/10.3126/bibechana.v18i2.30760 https://creativecommons.org/licenses/by-nc/4.0/ http://nepjol.info/index.php/BIBECHANA Shanta Pokhrel and Prabha Neupane / BIBECHANA 18 (2) (2021) 18-25 19 antioxidant, and antidepressive effects and improves venous insufficiency [9, 10]. Pittella reported that Centella asiatica prevents the oxidative damage that takes place in neuropathological disorders including stroke, Parkinson’s disease and Alzheimer’s disease by increasing the antioxidant neurological state related to aging [11]. Therefore, this study was conducted to investigate the bioactive compounds of methanol and hexane extracts, their antioxidant and antibacterial activities against different strains of bacteria. 2. Materials and Methods 2.1 Plant materials The plants (Centella asiatica) were collected from Hamsapur, Gorkha District, Western Development Region of Nepal in June, 2018 and authenticated by Central Department of Botany, Tribhuvan University, Kathmandu, Nepal. The collected plants were washed, air dried in shade for about a month then ground to powder and stored in clean plastic bag. 2.2 Chemicals and equipment DPPH, Ascorbic acid and Ofloxacin were purchased from Merck, Darmstadt, Germany. DMSO, methanol, n-hexane etc. are of analytical grade reagent were brought from Thermo Fisher Scientific India Pvt. Ltd. (India). Spectrophotometer [SL177] was used for the absorption for DPPH assay. 2.3 Extraction The dried and powdered form of whole plant (65 g) were extracted successively with hexane (300 mL, 8 h), methane (300 mL, 8 h) by Soxhlet extraction. The extracts were concentrated by rotary evaporator and solid and semi-solid mass obtained was kept in freezer for further analysis. 2.4 Phytochemical screening The freshly prepared crude extracts were subjected to analyze the presence of main classes of phytoconstituents in hexane and methanol extract using standard protocol [12]. 2.5 Antibacterial screening The antibacterial assay of the crude extract of Centella asiatica was carried out by agar well diffusion method. Effectiveness of antibacterial substance was evaluated by determination of zone of inhibition (ZOI) as given in Ref [13, 14]. The microbial strains staphylococcus aureus cocci ATCC 25923 (gram-positive) and Escherichia coli ATCC 25922 and Salmonella typhi (gram-negative) bacteria were obtained from MED-MICRO Nepal Lab, Kathmandu. The 50 μL of the working solution of the plant extract, DMSO as negative control (NC) and 25 μL of Ofloxacin (antibiotic- ear and eye drop) as positive control (PC) at the same time in separated well (6 mm) were loaded into the respective wells with the help of micropipette. The plates were incubated overnight at 37 ℃. After 24 hours of incubation, the plates were observed for the presence of inhibition of bacterial growth that indicated by a clear zone around the wells. The size of the zone of inhibition was measured and the antibacterial activity expressed in term of the average diameter of zone of inhibition in millimeters. 2.6 Antioxidant activity Antioxidant activity of the methanol extract was assessed using DPPH free radical [15, 16]. Initially, 10 mg of the sample to be tested was dissolved in 10 mL methanol to get the stock solution of concentration of 1 mg/mL (1000 µg/mL). Different concentrations of test samples of 20, 40, 60, 80 and 100 µ/mL were made from stock solutions. Then 2 mL of all the concentration of test solution were mixed with 2 mL of DPPH solution. The test tubes were shaken vigorously for the uniform mixing then the solutions was kept for 30 minutes in dark place at room temperature. The control was prepared as above but without the plant extracts (methanol + DPPH). After 30 minutes, absorbance of the entire sample was measured at 517 nm using a UV-visible spectrophotometer. Ascorbic acid of same concentration was prepared as a standard and its absorbance was also taken at 517 nm and calibration curve was constructed. Percent radical scavenging Shanta Pokhrel and Prabha Neupane / BIBECHANA 18 (2) (2021) 18-25 20 activity by sample treatment was determine by comparison with methanol treated control group, ascorbic acid was used as positive control. The radical scavenging activity was expressed as the radical scavenging percentage using the equation (1) [16]: 𝑅𝑎𝑑𝑖𝑐𝑎𝑙 𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 (%) = 𝐴0−𝐴𝑠 𝐴0 × 100 (1) where, A0 = Absorbance of the control As = Absorbance of test sample The IC50 value is the concentration of sample required to scavenge 50% of DPPH free radical and was calculated from the plotted graph of radical scavenging activity against the concentration of extracts. 3. Results and Discussion 3.1 Phytochemical screening Results of the phytochemical screening are summarized in below Table 1. The phytochemical screening of methanol extract of Centella asiatica leaves revealed that carbohydrate, glycosides, phenols, flavonoids, and saponin were present but alkaloids, protein and xanthoprotein were absent. Except alkaloid, carbohydrate, glycosides, other tested phytochemicals were found to be absent in hexane extract. The most important bioactive compounds (phytochemicals) such as alkaloids, saponins, flavonoids, tannins, sterols and phenolic compounds [17, 18] are plant derived metabolites naturally occurring in medicinal plant leaves, stem and roots where they are used as defense mechanism to protect the plant from various diseases. The presence of these phytochemicals in Centella asiatica is an indication of its medicinal potential. The results presented in the above table are slightly different than the data present in the literature of plants. Flory Shobana et al. reported that the plant part contains most of the important secondary metabolites like carboxylic acid, flavanoids, saponin, steroids, resins, xanthoprotein, coumarins etc [17]. Saranya et al. reported the presence of carbohydrates, tannins, steroids, terpenoids, alkaloids, flavanoids, cardiac glycosides, saponins etc [18]. The outputs of this study are consistent with Saranya et al. and Flory Shobana et al. with slight different results. These slight differences are due to variation in altitude of plants, different environmental conditions. 3.2 Antibacterial susceptibility assay Results obtained from the antibacterial assay of methanol and hexane extracts of Centella asiatica are tabulated as Table 2. Hexane extracts against Escherichia coli, Staphylococcus aureus and Salmonella typhi gives ZOI value 13 mm, 0 mm, and 10 mm respectively in 10% concentration. Hence, the hexane extracts showed more effective towards E. coli. Figure 1 (a, b, c) show the results of antibacterial activity of methanol and hexane extract. Hexane extracts against Escherichia coli, Staphylococcus aureus and Salmonella typhi showed ZOI value 13, 0, and 10 mm respectively in 10% concentration. However that of 1% hexane extracts against Escherichia coli, Staphylococcus aureus and Salmonella typhi was found 11 mm, 0 mm, and 12 mm respectively. Thus hexane extracts was found more effective against Escherichia coli and Salmonella typhi. On the other hand hexane extract did not show any antibacterial activity against Staphylococcus aureus. Methanol (10% concentration) extracts showed moderately effective against Escherichia coli, Staphylococcus aureus and Salmonella typhi with ZOI 11, 10, and 14 mm respectively whereas 1% methanol extracts proved more effective against Salmonella typhi with 16 mm zone of inhibition (ZOI). Flory Shobana et al. reported that the methanol leaf extract showed maximum inhibition against Staphylococcus aureus 16 mm and the 50% and 75% methanol extract also showed inhibition zone 10 and 11 mm respectively [17]. Zaidan et al. explored in vitro antibacterial activity of the plant extract against Staphylococcus aureus showed a zone of inhibition of 5 mm [19]. Therefore, the result of antibacterial assay was also supported by other study [17, 19]. Staphylococcus aureus causes infections including Shanta Pokhrel and Prabha Neupane / BIBECHANA 18 (2) (2021) 18-25 21 superficial skin lesion, localized abscesses, and food poisoning [19]. Centella asiatica has traditional claims for antibacterial activity and this finding is in line with Table 1: Phytochemical screening of different extracts of Centella asiatica S.N Phytochemical Hexane Methanol 1 Alkaloids + - 2 Carbohydrate + + 3 Glycosides + + 4 Phenol - + 5 Flavanoids - + 6 Proteins - - 7 Xanthoproteins - - 8 Saponins - + (+) indicates present and (–) indicates absent Table 2: Antibacterial analysis results of methanol and hexane extracts of Centella asiatica S.N. Plant extract Bacteria ZOI (mm) of crude extract ZOI (mm) of positive control 1. Hexane 10% 1% E. coli 13 11 28 S. aureus 0 0 30 S. typhi 10 12 28 2. Methanol E. coli 11 0 28 S. aureus 10 0 30 S. typhi 14 16 28 Table 3: Percentage of radical scavenging with different concentration % of radical scavenging Concentration (µg/mL ) Sample 20 40 60 80 100 Methanol extract 26.68 34.25 61.98 74.44 88.94 Ascorbic acid 13.21 19.38 27.50 58.66 72.59 Shanta Pokhrel and Prabha Neupane / BIBECHANA 18 (2) (2021) 18-25 22 Fig. 1: Antibacterial screening of methanol and hexane extract of (a) Centella asiatica against (b) Escherichia coli, Staphylococcus aureus and (c) Salmonella typhi. their indication as curative properties for antibacterial as claims. The result of this study can form the basis for further studies to evaluate against a wider range of bacteria strains. Figure 1 (a, b, c) show the results of antibacterial activity of methanol and hexane extract. 3.3 Antioxidant activity The antioxidant activity of extract was calculated as their capacity to scavenge free radicals of DPPH, which has been widely used to evaluate the antioxidant activity of natural products from plant and microbial sources [20]. The DPPH radical assay was carried out for the methanol extract of Centella asiatica by using ascorbic acid as standard. The absorbance values were measured at 517 nm for different concentrations of extracts and the control. These values were used to calculate the percentage inhibitions of DPPH radicals against the samples. IC50 value was calculated from the calibration curve constructed by measuring the absorbance of ascorbic acid (Table 3). The absorbance with the different concentration of ascorbic acid was taken as the reference from our previous paper [14]. The comparison of percentage radical scavenging at different concentration between plant extract and ascorbic acid as standard was shown in Fig 2. a b c Shanta Pokhrel and Prabha Neupane / BIBECHANA 18 (2) (2021) 18-25 23 Fig. 2: A plot of percentage radical scavenging activity vs. concentration of methanol extract and ascorbic acid. Table 4: IC50 values of methanol extract and ascorbic acid S.N. Sample IC50 (µg/ mL) 1. Standard Ascorbic acid 3.74 2. Methanol extract 2.576 The IC50 value of methanol extract and ascorbic acid was observed 2.576 µg/mL and 3.74 µg/mL respectively supported by Ref [14]. The antioxidant potential is in an inverse relation with IC50 value, lower value of IC50 indicates high antioxidant potential. Anand et al. reported 0.07 mg/mL and 500 μg/mL IC50 values of DPPH and hydroxyl radical scavenging activity of methanol extract respectively [21]. Since the phytochemical screening of methanol extract showed the presence of phenol and flavonoids which are responsible for the antioxidant activity. Singh et al. mentioned antioxidant activity of methanol extracts ranged from 72.0 to 85.7% and proved that there is a significantly positive correlation with anthocyanin, total phenol, flavonoids, and tannin [22]. Phenolic contents are very important constituents because they act as reducing agent, hydrogen donors and metal chelator. They also act as radical scavenger due to their hydroxyl groups. Funde reported that flavonoids can show their antioxidant action through scavenging or chelating process [23]. Alkaloids have been associated with medicinal uses for centuries and one of their common biological properties is their cytotoxicity [23]. y = 15.803x - 9.145 y = 16.471x + 7.845 0 20 40 60 80 100 20 40 60 80 100 % R a d ic a l sc a n v en in g Concentration (µg/mL) Shanta Pokhrel and Prabha Neupane / BIBECHANA 18 (2) (2021) 18-25 24 4. Conclusions The results of this study revealed that the methanol extract of Centella asiatica possesses pharmacologically active substances like carbohydrate, glycosides, phenols, flavonoids, saponins. The antibacterial assay of hexane extract showed moderate activity against Salmonella typhi and Escherichia coli but methanol extract has proved most effective against Salmonella typhi. Similarly, antioxidant activity of methanol extract showed potent antioxidant activities with IC50 value 2.57 μg/mL slightly higher than standard ascorbic acid (IC50 = 3.74 μg/mL). It is concluded that Centella asiatica extract possess pronounced antioxidant and antibacterial biological properties and recommends that active phytoconstituents can be isolated, identified, and screened individually for activities and also subjected further for in vivo and toxicological studies. Acknowledgements The authors are grateful to Department of Chemistry, Tri-Chandra Multiple Campus, Tribhuvan University, Kathmandu, Nepal for providing laboratory facilities to conduct experimental work. We are thankful to Med-Micro Nepal Lab, Thapathali, Kathmandu, Nepal for antibacterial tests. References [1] G.E. Trease, W.C. Evans. Pharmacognosy, 13th edition; ELBS Oxford University. Press, London, UK, (1989) 245-263. [2] A.V. Khan, M.M. Alam, and V.K. Singh, Ethanomedical Uses of Citrullus colocynthis (L.) Schard, In Rural Areas of Aligarh District of UP, India, 7 (2002) 383-388. [3] K. Kitonde, D.S. Fidahusein, C.W. Lukhoba, and M.M. Jumba, Antimicrobial Activity and Phytochemical Study of Vernonia glabra (steetz) Oliv. & Hiern in Kenya, Afr. J. Tradit. Complement Altern. Med. 10 (2012) 149–157. [4] R. Arora, R. Kumar, A. Agarwal, K.H. Reeta, and Y.K. Gupta, Comparison of Three Different Extracts of Centella asiatica for Anti-Amnesic, Antioxidant and Anticholinergic Activities: In Vitro and In Vivo Study, Biomed Pharmacother 105 (2018) 1344–1352. [5] https://doi.org/10.1016/j.biopha.2018.05.156. [6] E.O. Omwenga, P.O. Okemo, P.K. Mbugua, and C. Ogol, Ethnobotanical Survey and Antimicrobial Evaluation of Medicinal Plants used by the Samburu Community (Kenya) for treatment of Diarrhorea, Phcog Mag 5 (2009) 165-175. [7] K. Zahara, Y. Bibi, and S. Tabassum, Clinical and Therapeutic Benefits of Centella asiatica. Pure Appl Biology 3 (2014) 152-159. [8] C.L. Cheng, J.S. Guo, J. Luk, and M.W.L. Koo, The Healing Effects of Centella extract and Asiaticoside on Acetic Acid Induced Gastric Ulcers in Rats. Life Sci. 74(18) (2004) 2237-2249. [9] a) S.A. Polash, T. Saha, Hossain M.S., and S.R. Sarker, Phytochemical Contents, Antioxidant And Antibacterial Activity of the Ethanolic Extracts of Centella asiatica (L.) Urb. Leaf and Stem, Jahangirnagar University J. Biol. Sci. 6 (2017) 51- 57. b) S.A. Polash, T. Saha, M.S. Hossain, and S.R. Sarker, Investigation of the Phytochemicals, Antioxidant, and Antimicrobial Activity of the Andrographis paniculata Leaf and Stem Extracts, Advances in Biosci. Biotechnol. 8 (2017) 149-162. [10] B.M. Sieberi , G.I Omwenga, R.K. Wambua, J.C. Samoei, and M.P. Ngugi, Screening of the Dichloromethane, Methanolic Extract of Centella asiatica for Antibacterial Activities Against Salmonella typhi, Escherichia coli, Shigella sonnei, Bacillus subtilis, and Staphylococcus aureus, Sci. World J. (2020) https://doi.org/10.1155/2020/6378712 [11] F. Pittella,, R.C. Dutra, D. D. Junior, M.T.P. Lopes and N.R. Barbosa, Antioxidant and Cytotoxic Activities of Centella asiatica (L). Urb, Int. J. Mol. Sci. 10(9) (2009) 3713-3721. https://doi.org/10.3390/ijms10093713 [12] J.B. Harborne. Phytochemical Methods, Chapman and Hall, Ltd., London, (1973) 49-188. [13] S.J. Cavalieri, R.J. Harbeck,, Y.S. Mc Carter, J.H. Ortez,, I.D. Rankin, R.L. Sautter, S.E. Sharp, and https://doi.org/10.1155/2020/6378712 Shanta Pokhrel and Prabha Neupane / BIBECHANA 18 (2) (2021) 18-25 25 C.A. Spiegel, Manual of Antimicrobial Susceptibility Testing. Washington DC, American Society for Microbiology (2005) 53-62. [14] S. Pokhrel, and K. Chaulagain, Phytoconstituents and Biological Analysis of Acorus calamus Rhizome of Sindhupalchowk District, Nepal, BIBECHANA 17 (2020) 104-109. [15] G. Funde, Phytochemicals Evaluation, Anticancer, Antioxidant and Antimicrobial Activity of Acorus Calamus Different Solvent Extracts, J Chem Pharm Res 7 (2015) 495-504. [16] S. Jamuna, S. Paulsamy, and K. Karthika, Screening of In Vitro Antioxidant Activity of Methanolic Leaf and Root Extracts of Hypochaeris radicata L. (Asteraceae), J. Appl. Pharm. 2 (2012) 149-154. [17] M. Flory Shobana, R. Ravikumar, P. Sivasankari, S.N. Suresh, and V. Subha Priya, Phytochemicial Screening and Antibacterial Property of Centella Asiatica (Linn), World J. Pharm. Biotech. 1 (2014) 60-70. [18] S. Saranya, A.V. Nair., and S. Kumar, Phytochemical Analysis of Centella asiatica L. Leaf Extracts, Int. J. Adv. Res. 5 (2017) 1828- 1832. [19] M.R. Zaidan, A. Noor Rain, A.R. Badrul, A. Adlin, A. Norazah, and I. Zakiah, In vitro Screening of Five Local Medicinal Plants for Antibacterial Activity Using Disc Diffusion Method, Trop Biomed 22 (2005) 165-170. [20] S.T. Chang, J.H. Wu, S.Y. Wang, P.L. Kang, N.S. Yang, and L.F. Shyur, Antioxidant Activity of Extracts from Acacia confusa Bark and Heartwood, J. Agric. Food Chem. 49 (2001) 3420- 3424. [21] T. Anand, M. Naika, P. Kumar, and G.F. Khanum, Antioxidant and DNA Damage Preventive Properties of Centella asiatica (L) Urb, Pharmacogn. J. 2 (2010) 53-58. [22] S. Singh, D.R. Singh, V. Shajeeda Banu, and N. Avinash, Functional Constituents (Micronutrients and Phytochemicals) and Antioxidant Activity of Centella asiatica (L.) Urban Leaves, Ind Crops Prod 61 (2014) 115–119. [23] D. Chaudhuri, N.B. Ghate, R. Sarkar, and N. Mandal, Phytochemical Analysis and Evaluation of Antioxidant and Free Radical Scavenging Activity of Root. Asian J. Pharmaceut. Clin. Res. 5 (2012) 193-199.