Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 1, April 2023 | Pages: 329-334 | DOI: 10.14421/biomedich.2023.121.329-334 ISSN 2540-9328 (online) Phytochemical, Antibacterial and Antioxidant Activities of Schefflera elliptica Leaves I Gede Yoga Ayuning Kirtanayasa1, Anak Agung Gede Indraningrat2,*, I Putu Candra1 1Food Science and Technology Study Program, Faculty of Agriculture, Warmadewa University 2Departement of Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Warmadewa University Jl. Terompong No 24 Denpasar 80235, Tel. +62 361 240727, Indonesia. Corresponding author* indraningrat@warmadewa.ac.id Abstract Schefflera elliptica or locally called kayu tulak, is one of the Balinese herbal plants that have traditionally be used to reject (tulak) negative influences that exist in the human body. Althoug, S. elliptica has been routinely used as a part of a ritual in Bali, only a limited study has been reported on its bioactivity. This study was designed to analyze the phytochemical content, antibacterial activity, and antioxidant activity of S. elliptica. In this study, simplisia of S. elliptica was extracted using n-hexane and ethyl acetate solvents, then the viscous extracts of the two solvents were carried out for phytochemical tests, antibacterial activity tests with the Kirby-Bauer method and antioxidant activity tests with based on DPPH method. Phytochemical screening showed that the n-hexane extract contains active compounds in the form of phenols and steroids while ethyl acetate contains active compounds in the form of phenols, tannins, and steroids. Antibacterial screening showed ethyl acetate extract of S. elliptica displayed a diameter zone of inhibition of 10.72±0.71 mm against Staphylococcus aureus, 12.17±2.80 mm against Streptococcus mutans, 12.40±1.65 mm against Escherichia coli and 15.20±2.44 mm against Klebsiella pneumoniae. The DPPH analysis showed percentages of 61.17% and 67.42% from n-hexane and ethyl acetate extracts respectively, which indicated the antioxidant properties of S. elliptica. Overall, this research provides a preliminary report on the bioactivity potential of S. elliptica mainly in term of antibacterial and antioxidant properties which open up possibilities for future drug development. Keywords: antibacterial; antioxidant; phytochemical; schefflera elliptica. INTRODUCTION Indonesia is one of the countries with high plant diversity. The biological diversity of flora in Indonesia, especially seeded plant species reaches 30,000–40,000 types, or equivalent to 15.5% of the total number of plants in the world (Widjaja et al., 2014). Furthermore, more than 2,039 plant species are categorized as medicinal plants (Zuhud, 2009). Among many islands in Indonesia, Bali is one of the islands with rich biological diversity and Balinese people have applied herbal plants for traditional medicine (Sutomo and Iryadi, 2019). In general, information about traditional Balinese medicine has been recorded in a manuscript called Lontar Usada Bali, which explains the function of each herbal function and the procedure to use it (Oktavia et al., 2017). Schefflera elliptica or locally called kayu tulak is one of the types of local Balinese plants listed in Usada Tiwang (Arsana et al., 2020). This plant is commonly found in the traditional Balinese offerings called banten byakala has a function to reject (tulak) any impurities or negative influences that exist in the human body (Puspa et al., 2019). More specifically, the leaf part of kayu tulak is used as a repellent for disasters or bad luck in human body (Hanum, 2011). Schefflera elliptica leaves can also traditionally be used as a remedy for skin diseases and fractures (Sivaperuman et al., 2018). Methanol and ethyl acetate extracts of S. elliptica leaves have also been reported to have antibacterial activity against Staphylococcus aureus (Purwantoro et al., 2009). However, apart from antibacterial against S. aureus, no other studies have been published on the antibacterial activities of S. elliptica extracts against other Gram-positive and Gram- negative bacterial species. In addition, rather limited information is available on other aspects of the bioactivities of S. elliptica e.g. phytochemicals and antioxidant activities. This present study aimed to assess the bioactivities S. elliptica leaves focusing on three main aspects namely phytochemical, antibacterial, and antioxidant bioactivities. The obtained information is expected to give further knowledge on the bioactivities of S. elliptica which could be the basis to develop the ethnomedicine and drug development purposes of the plant. Manuscript received: 04 March, 2023. Revision accepted: 24 March, 2023. Published: 01 April, 2023. https://doi.org/10.14421/biomedich.2023.121.329-334 330 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 329-334 METHODS Sample Collection and Determination Leaves samples of S. elliptica plant were obtained from Gerih Village, Bali, Indonesia on February 2022 (Figure 1). Samples were selected by taking 3-5 leaves of S. elliptica which were calculated from the leaflets, provided that it was in full bloom, fresh, without hollow, and free of insect infections. Plant determination was performed by sending fresh and dried vouchers to the Characterization Laboratory of the Botanical Garden "Eka Karya" Bali – National Research and Innovation Agency (BRIN), Candikuning, Tabanan, Bali. The purpose of plant determination is to obtain a clear identity of the plant under study and avoid errors in collecting the main research material. Sample Preparation and Extraction Two kilograms of S. elliptica leaves were washed in running tap water to remove debris. Subsequently, leaves samples were drained, and dried using an oven at 40oC. The dried simplisia was dried and were sorted by separating foreign objects that occurred during drying. The dried simplisia was turned into powder using a blender and was sieved with a 60-mesh sieve. Finally, the powder was stored in a clean glass jar to prevent contamination and other impurities before extraction. Two types of crude extracts were prepared using two different chemical solvents namely ethyl acetate (Smart- Lab) dan n-hexane (Merck). For each of solvent, 100 gram of dry powder leaves of S. elliptica was macerated with a ratio of 1:5 (w/v) (Wijaya and Indraningrat, 2021). Maceration was carried out for 24 hours and each mixture was stirred every five minutes with a time span of six hours. For each of the solvent, remaceration was carried out after 24 hours. Subsequently, after maceration and remaceration were completed, each macerate was separated from the residue using a vacuum filter, followed by evaporation in a rotary evaporator (Ika RV 10, Germany) at a speed of 100 rpm at a temperature of 40ºC. Figure 1. S. elliptica plant (left), and S. elliptica leaves (right). Phytochemical Screening Each the ethyl acetate and n-hexane S. elliptica crude extracts was tested to detect the presence of a group of compounds based on the following methods. Phenol One mL of each S. elliptica extract was transferred into a test tube followed by the addition of 2 – 3 drops of iron (III) chloride (FeCl3) 5%. The presence of phenols was indicated by a blue-black color (Friany et al., 2017). Flavonoids One mL of each S. elliptica extract was transferred into a test tube. Subsequently, 2 mg of magnesium powder and 3 drops of concentrated HCl were added to the tube. The mixture was shaken and the formation of a red, yellow, or orange color on the solution indicated the presence of flavonoids (Purwati et al., 2017). Tannins One mL of each S. elliptica extract was mixed with a few drops of 10% iron (III) chloride (FeCl3) solution. A dark blue or greenish-black color indicates the presence of tannins in a solution (Baud et al., 2014). Alkaloids Two mL of each S. elliptica extract was mixed with 3 drops of concentrated HCl and 5 drops of Mayer reagent. A white precipitate indicates that a sample contains alkaloids (Ergina and Pursitasari, 2014). Steroid/Terpenoid Two mL of each S. elliptica extract were mixed with Liebermann Burchard reagent, a mixture of concentrated HCl and concentrated H2SO4. Positive results were indicated by the presence of a red-orange color for triterpenoids and blue for steroid (Sangi et al., 2008; Ergina and Pursitasari, 2014). Saponin Two mL of each S. elliptica extract were mixed with 10 mL of aqua dest. The mixture was shaken for 1 minute and subsequently, two drops of HCl 1 N was added. The presence of a stable foam for approximately 7 minutes indicated that the mixture contains saponins (Mondong et al., 2015). Antibacterial Activity Screening For each ethyl acetate and n-hexane extract, paper discs with a diameter of 6 mm were prepared. Each paper disc was soaked into a viscous extract and allowed to dry for 15 minutes until the extract was evenly absorbed. Paper discs containing extracts were transferred into Luria Bertani agar which already contained one of the following test bacteria namely Staphylococcus aureus ATCC 25923, Streptococcus mutans FNCC 0405, Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603. Triplicate paper discs of Kirtanayasa et al. – Phytochemical, Antibacterial and Antioxidant Activities of … 331 each extract were used for each of the tested bacteria. Nalidixic Acid (Oxoid) paper discs were used as a positive control, while ethyl acetate and n-hexane were used as negative controls. Antibacterial activities were calculated based on the triplicate average zone of inhibition (ZOI) that were formed on each of the lawn bacterial species. Antioxidant Assay The radical inhibition activity of the sample was carried out based on its inhibition against free radicals 1, 1- diphenyl-2-picrylhydrazyl (DPPH) (Pangestuty, 2016). Sample absorbance was measured with a UV-Vis spectrophotometer. The magnitude of antioxidant activity was measured by the following formula: Antioxidant Activity (AA%) = 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100 RESULT AND DISCUSSION Phytochemical Screening Results Phytochemical screening is a preliminary test and qualitative analysis that can be used as initial information about a group of compounds present in a plant. In this research, phytochemical screening of n-hexane and ethyl acetate extracts of S. elliptica were summarized in Table 1. In general, there were discrepancies in the presence of specific groups of compounds by comparing the phytochemical constituents of each extract. Alkaloids tests showed clean deposits were formed in both extracts. This result indicated the absence of alkaloids in ethyl acetate and n-hexane. A positive result was indicated by the presence of white deposit (Ergina and Pursitasari, 2014) Table 1. Phytochemical composition of S. elliptica extracts. Test Extract N-hexane Ethyl Acetate Alkaloid - - Flavonoid - - Tannin - + Phenol + + Saponin - - Steroid + + Terpenoid - - The presence of flavonoids in n-hexane and ethyl acetate extracts was tested using two methods, namely by adding concentrated Magnesium (Mg) + Hydrochloric Acid (HCl) and using H2SO4. Positive signs were indicated by a change of color in the mixture to red or orange. In this study, n-hexane and ethyl acetate samples did not change color to red or orange, so it was concluded that flavonoids were absent in both extracts (Friany et al., 2017; Purwati et al., 2017). A positive test of tannin was indicated by the formation of blackish-green color (Baud et al., 2014). The occurrence of this green color change is due to the reaction between Fe metals and tannins to form complex compounds due to the presence of coordination covalent bonds between metal ions or atoms and non-metallic atoms (Effendy, 2007). Our result indicated that tannin was present in ethyl acetate extract based on the color change that was observed. Meanwhile, no color change was observed from the n-hexane extract. This could happen mainly because n-hexane is a solvent that has non-polar properties while tannins are polar compounds and tend to dissolve in polar or semi polar solvents (Muthmainnah, 2017). The presence of phenol was screened by adding 10% of FeCl3 reagent and positive signs were indicated by a color change to blackish-green (Friany et al., 2017). Color changes to green-black were observed from both S. elliptica extracts to indicate both extracts contained phenol compounds. The discoloration of n-hexane and ethyl acetate extracts of S. elliptica was because phenol compounds can dissolve in polar solvents as well as non- polar solvents (Wongso, 2014). In the saponin test, n-hexane and ethyl acetate extracts of S. elliptica were heated followed by addition of 10 ml of aquadest. The mixture was shaken strongly and a positive sign was indicated by the presence of a stable foam of 1-10 cm high which was stable after the addition of 1 drop of HCl 1N (Mondong et al., 2015). The appearance of foam was present because saponin compounds have physical properties that are easily hydrolyzed in water so that saponin compounds will cause foam when shaken. In both n-hexane and ethyl acetate extracts of S. elliptica the saponin test did not show positive results because the foam formed after shaking only lasted for a few seconds. The presence of steroids and terpenoids were screened by the addition of Liebermann-Burchard reagents. The positive result for steroid compounds was indicated by the color change to blue or purple while for triterpenoid compounds was indicated by a brownish-red color (Sangi et al., 2008; Ergina and Pursitasari, 2014). The test results showed that n-hexane and ethyl acetate of S. elliptica extracts underwent a blue color change so that they were positive for containing steroid compounds. Antibacterial Activities Screening The antibacterial screening showed that the crude extractof n-hexane displayed lower antibacterial activity against test bacteria compared to the crude extract of ethyl acetate (Figure 2, Table 2). Ethyl acetate extract has an average ZOI of 10.72±0.71 mm against S. aureus. Ethyl acetate extract had an average ZOI of 12.17±2.80 mm against S. mutans. Meanwhile, in the ethyl acetate extract has an average ZOI of 12.40±1.65 mm against E. coli and an average ZOI of 15.20±2.44 mm against K. pneumoniae. 332 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 329-334 Figure 2. Antibacterial activities of n-hexane and ethyl acetate extract againts bacteria. Annotations: H = n-hexane, E= Ethyl acetate; SA = Staphylococcus aureus ATCC 25923; SM = Streptococcus mutans FNCC 0405; EC= Escherichia coli ATCC 25922; KP=Klebsiella pneumoniae ATCC 700603 Table 2. Antibacterial activities of S. elliptica extract against testing bacteria. Diameter zone of inhibition of each treatment (ethyl acetate, n- hexane, positive and negative controls) were calculated from triplicate samples. Bacterial Strains Samples Solvents ZOI (mm) S. aureus S. elliptica n-Hexane - Ethyl acetate 10,72±0,71 + control Nalidixic acid 15,34±0,03 - control n-Hexane - Ethyl acetate - S. mutans S. elliptica n-Hexane - Ethyl acetate 12,17±2,80 + control Nalidixic acid 16,29±0,12 - control n-Hexane - Ethyl acetate - E. coli S. elliptica n-Hexane - Ethyl acetate 12,40±1,65 + control Nalidixic acid 16,58±0,04 - control n-Hexane - Ethyl acetate - K. pneumoniae S. elliptica n-Hexane - Ethyl acetate 15,20±2,44 + control Nalidixic acid 15,50±0,13 - control n-Hexane - Ethyl acetate - When compared to the results of the study of Purwantoro et al, (2009) which stated that the ethyl acetate extract of S. elliptica at a concentration of 50 μg/ml, 100 μg/ml, and 200 μg/ml had a ZOI with an average of 7.5 mm against S. aureus and E. coli bacteria, the crude extract of ethyl acetate with a concentration of 100% had a bigger ZOI against S. aureus and E. coli bacteria. The difference in inhibitory power can occur due to the difference in the concentration of the extract where the higher the concentration of the extract, the higher the inhibitory power will be. The same thing is also stated by (Zuhud et al., 2001) who mentioned that the higher the concentration of the extract, the more the amount of antimicrobial compounds released will be, thus facilitating the penetration of compounds into cells. Davis and Stout, (1971) classified the diameter zone of inhibition into four categories, namely weak, medium, strong, and very strong. Zones of inhibition with a diameter of ≤5 mm are categorized as weak, 6-10 mm are categorized as medium, 11-20 mm are categorized as strong, and above 20 mm are categorized as very strong. Based on these categories, the ZOI formed by ethyl acetate extracts of S. elliptica could be considered as a strong activity. However, the observed antibacterial activity was still lower compared to positive control nalidixic acids. Nevertheless, the observed antibacterial activity from ethyl acetate extracts provides a valuable insight on bioactive compounds that present in S. elliptica leaves. The presence of antibacterial activities in the ethyl acetate extract of S. elliptica may occur due to the content of its secondary metabolites. The mode of action of phenol compounds is in general by denaturing cell proteins. Hydrogen bonds formed between phenol compounds could damage protein layers in cell structures (Bontjura et al., 2015) Meanwhile, tannin was also reported to display have antibacterial activity against Gram-positive and Gram-negative bacteria by entering the cell wall (Kaczmarek, 2020). Tannin forms hydrogen bonds with bacterial cell’s proteins and subsequently hydrogen bonds formed between tannins and proteins will disrupt bacterial cell walls (Mailoa et al., 2014). Antibacterial screening showed that the zone of inhibition of ethyl acetate extract of S. elliptica against Gram-positive bacteria (S. aureus and S. mutans) was smaller compared to Gram-negative bacteria (E. coli and K. pneumoniae). Such discrepancies could probably happen because the cell wall of Gram-negative bacteria is thinner compared to Gram-positive bacteria so the protein structure in the cell wall of Gram-negative bacteria was damaged by the presence of tannin compounds (Mailoa et al., 2014). Furthermore, Gram- positive bacteria have a thick and rigid peptidoglycan layer while Gram-negative bacteria have a thinner peptidoglycan layer (Sudarmi et al., 2017). The absence of ZOI observed in n-hexane extracts even though qualitatively the extract contained phenol compounds could be influenced by differences of the polarity of solvents in extraction which affecting the total content of bioactive compounds in the extract (Santoso et al., 2012). The total content of phenol compounds according to Hidayah et al., (2017) has an influence on antibacterial activity as the higher the levels of oxidized phenol compounds, the stronger the antibacterial activity Kirtanayasa et al. – Phytochemical, Antibacterial and Antioxidant Activities of … 333 will be. The extract of n-hexane S. elliptica did not display a zone of inhibition power even though it qualitatively contained steroid compounds. Such a condition could happen because lipids have large sizes of molecules which interfere with the diffusion process and consequently, n-hexane extracts are unable to inhibit bacterial growth (Naufalin et al., 2005). Antioxidant Activities Test Results Antioxidant activities were analyzed based on the 2,2- diphenyl-1- picrilhydrazyl (DPPH) method. In theory, the higher the concentration of antioxidant activity added to the DPPH solution, the more the absorbance value will decrease (Sapri et al., 2013). The absorbance value of n- hexane and ethyl acetate S.elliptica samples (Table 3) showed that the antioxidant activities (AA) of n-hexane extract was 61.17% while for ethyl acetate extract was 67.42%. According to Rahmawati (2004) in (Parwata et al., 2009) the inhibition value of 0% means that an extract has no antioxidant activity and the inhibition value of 100% means total dampening. An extract could be classified as an active antioxidant when its inhibition percentage is more than or equal to 50%. Therefore, based on this criteria, crude extracts of n-hexane and ethyl acetate of S. elliptica are classified as active antioxidants. Table 3. Antioxidant percentages of S. elliptica crude extracts. Sample Crude extracts Concentrations AA% S. elliptica N-Heksan 1000 ppm 61,17 Etil Asetat 1000 ppm 67,42 Factors that affect the absorbance are the type of solvent, the pH of the solution, the temperature, the high concentration of the solution, and the presence of a disruptive substance. The amount of antioxidant activity of n-hexane and ethyl acetate extracts from S. elliptica is due to the content of secondary metabolites such as phenols, steroids, and tannins in the extract. Tannins are compounds composed of polyphenols that have free radical capture activity. The more tannin content contained in the extract, the more antioxidant activity (Malangngi et al., 2012). According to (Amarowicz, 2007) tannins not only function as primary antioxidants but also function as secondary antioxidants. Phenolic compounds have the ability to contribute hydrogen atoms or electrons to free radicals. This process converts phenols into phenoxyl radicals that can stabilize themselves so that there is no radical formation reaction (Pangestuty, 2016; Diniyah and Lee, 2020). CONCLUSIONS In conclusion, this study confirmed antibacterial and antioxidant activities from S. elliptica crude extracts. In addition, S. elliptica leaves contain phytochemicals in the form of phenol compounds, tannins, and steroids. The selection of solvents seems to play an important role in extracting bioactive compounds from S. elliptica leaves. In terms of antibacterial activity, ethyl acetate which is a semipolar solvent seems more suitable to extract the active antibacterial substances compared to n-hexane which is a non-polar solvent. Further studies should be focused to explore the ideal solvent for chemical extraction. Comparison of polar, semipolar, and non- polar solvents for extraction of S. elliptica should also be done to provide an ideal comparison of bioactivities. In addition, antibacterial screening should also be expanded against multi-drug resistance bacteria and fungi. Further studies should also explore other aspects of bioactivity screenings such as cytotoxicity, anti-larvicide, and anticancer tests of S. elliptica to elucidate possible untapped bioactivities of the plant. 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