Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 1, April 2023 | Pages: 273-280 | DOI: 10.14421/biomedich.2023.121.273-280 ISSN 2540-9328 (online) Phytochemical and Antioxidant Activity of Blumea balsamifera and Cordyline fruticosa Based on Ethnopharmacology Knowledge of Muara Tae Tribe, East Kalimantan Nur Maulida Sari1, Farida Aryani2,*, Wartomo1, Muhammad Fikri Hernandi1, Erna Rositah3, Joko Prayitno1 1Department of Forest Product Processing; 2Department of Plantation Products Technology; 3Department of Forest Management, Samarinda State Agriculture Polytechnic, Kampus Gunung Panjang Jalan Samratulangi, Samarinda 75131, East Kalimantan, Indonesia. Corresponding author* faridaaryani@politanisamarinda.ac.id Abstract Plant use as traditional medicine is still widely practiced in Indonesia. Muara Tae tribe people, West Kutai regency are one of the regions that still rely on Blumea balsamifera and Cordyline fruticosa plants as traditional medicine. This study aims to determine the potential of Blumea balsamifera and Cordyline fruticosa leaves as medicinal plants with phytochemicals and antioxidants. Phytochemical analysis was tested using Harborne and Kokate methods. Antioxidant activity was evaluated b y DPPH radical scavenging assay with slight modification. The results of the phytochemical analysis showed that the extracts of n-hexane, ethyl acetate, and ethanol from the leaves of Blumea balsamifera and Cordyline fruticosa contained alkaloids, tannins, and triterpenoids. Antioxidant activity of Blumea balsamifera leaves extract showed that the n-hexane extract display an ability to inhibit DPPH free radical by 50% at 100 ppm concentration, while ethyl acetate and ethanol extracts display an ability by 77% and 81% at 50 ppm concentration. IC 50 value of ethyl acetate and ethanol extracts of Blumea balsamifera leaves sequentially were 23.68 µg/mL and 17.59 µg/mL. Antioxidant activity of Cordyline fruticosa leaves extract showed that the n-hexane and ethyl acetate extract display an ability to inhibit DPPH free radical by 45% and 56% at 100 ppm concentration, while ethanol extracts display an ability by 76% at 50 ppm concentration. IC50 value of ethyl acetate and ethanol extracts of Cordyline fruticosa leaves sequentially were 73.72 µg/mL and 20.17 µg/mL. Based on the results, Blumea balsamifera and Cordyline fruticosa leaves extracts had the potential to develop as natural antioxidants. Keywords: Blumea balsamifera; Cordyline fruticosa; DPPH; Ethnopharmacology; Phytochemical. INTRODUCTION Indonesia has unique flora and fauna that complement its, cultural and ethnic diversity. These ethnic groups occupy certain areas of the country (Sreekeesoon & Mahomoodally, 2014). Each ethnic group has its own lifestyle and traditions, including foods, herbs, and spices. It is also very capable of using and preserving organic and ecological diversity. Medicinal plants are used by the local community to treat illnesses, mainly due to health restrictions. The original knowledge of the use of medicinal herbs was probably passed down from generation to generation. This approach has so far succeeded in keeping knowledge alive (Yaseen et al., 2015). Therefore, local knowledge of medicinal plants has always been a source of research in testing the effects of plants and developing new therapeutic means (Bolson et al., 2015). Indonesia has the second highest biodiversity in the world after the Amazon forests and a population of, people from more than 300 nationalities (Elfahmi et al., 2014). Most of the studies focused on the prevention of diseases or specific medicinal herbs in Indonesia (SILALAHI et al., 2014). Although Indonesian researchers have studied traditional knowledge about the use of plants as medicine, most studies have not been published in international journals. About 30,000 medicinal plants grow and develop in Indonesia, which covers 90% of medicinal plants in the Asian region (Syamsiah et al., 2016). The use of traditional medicine is increasing with population growth, healthy lifestyles, and degenerative diseases (Triratnawati, 2016). Also up to 21,4% of the Indonesian population used traditional medicine to self-treat health problems. Herbal medicine and massage are the most common treatments offered by traditional healers in Indonesia (Peltzer & Pengpid, 2019). The consumption of traditional medicines in Indonesia increased by 5,4% per year. Efficiency, lower cost, easy availability, and dissatisfaction with traditional treatment methods are some of the reasons for choosing traditional treatment (Riptanti et al., 2018). Several studies in recent years Manuscript received: 15 January, 2023. Revision accepted: 14 March, 2023. Published: 23 March, 2023. https://doi.org/10.14421/biomedich.2023.121.273-280 274 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 273-280 about medicinal plants use, informed that plants were the best sources of natural antioxidants such as phenolics, alkaloids, and flavonoids compound (Zhao et al., 2018). An antioxidant agent from the plants was a huge resource to scavenging free radicals naturally. Medicinal plants also prevent oxidative stress, maintain health, and disease and delay aging processes (NGUYEN et al., 2018). Oxidative stress of human lipids is known to have various human health problems such as cardiovascular disease, cancer, diabetes, and others (Truong et al., 2019). Generally, antioxidants are divided into 2 types natural and synthetic antioxidants. Natural antioxidants are widely used as free radical inhibitors, while synthetic antioxidants had a negative effect with long-term used (Stoia & Oancea, 2022). Muara Tae was local tribe exist in West Kutai, East Kalimantan, Indonesia. The Muara Tae community known as a sub-tribe of Dayak used medicinal plants as alternative drugs for healthcare treatment and diseases such as fever, diabetes, external wound, stomachache, and others. Several studies about potential plant use by Dayak people informed the plant used as natural antifungal, antioxidant, and antibacterial agents by Bentian Tribe (Kusuma et al., 2016). Blumea balsamifera and Cordyline fruticose were the plant’s belief as medicinal plants by Muara Tae’s people. These plants had only limited attempts to explore the biological properties of plants regards their uses as medicine by the local people. The present study aims to explore the potential of Blumea balsamifera and Cordyline fruticosa leaves extract for its antioxidant activity from the Muara Tae tribe in Indonesia (Figure 1). MATERIALS AND METHODS Plant collection The leaves of Blumea balsamifera and Cordyline fruticosa were collected from Muara Tae Village, West Kutai, East Kalimantan, Indonesia. The samples were washed thoroughly with water to remove the extemporaneous and dried for about 3 days in the laboratory with air conditioning (A.C.) set for 20-25ºC. The samples were kept in A.C. room to keep the moisture content stable and milled with a blender. The powdered samples were prepared for further analysis. Figure 1. Morphology of Blumea balsamifera and Cordyline fruticosa Plants (Photo source: personal doc.). Procedures Maceration About 30 gr powdered samples of Blumea balsamifera and Cordyline fruticosa were extracts with n-hexane, ethyl acetate, and ethanol solvent at room temperature with continuous shaking on a shaker for 48 hours. Following filtration of the suspension through Whatman paper No.2 (Maidstone, UK), the crude extracts of Blumea balsamifera and Cordyline fruticosa were evaporated in a rotary evaporator at 38-40 ºC and put in a vacuum over near dryness to yield the plant extract. Preliminary Phytochemical Analysis The n-hexane, ethyl acetate, and ethanol extracts of Blumea balsamifera and Cordyline fruticosa leaves were subjected to preliminary screening of phytochemical such as alkaloids, flavonoids, tannin, steroids, triterpenoids, carbohydrate, and saponin using some following standard procedures (Harborne, 1998; Kokate, 2001). Alkaloids determination: 5 mL of the n-hexane, ethyl acetate, and ethanol extracts of Blumea balsamifera Sari et al. – Phytochemical and Antioxidant Activity of … 275 and Cordyline fruticosa leaves were added to 2 mL Hydrochloride Acid, then 1 mL of Dragendorff solution was added. The color changes in the solution indicated the presence of alkaloids (Kokate, 2001). Flavonoids Determination: About 1 mL of the n- hexane, ethyl acetate, and ethanol extracts of Blumea balsamifera and Cordyline fruticosa leaves were drops of 1% Sodium Hydroxide. The presence of yellow color at extracts solution and were colorless after the addition of 1 % Hydrochloride Acid indicated the presence of flavonoids (Kokate, 2001). Tannin Determination: 10 mL of the n-hexane, ethyl acetate, and ethanol extracts of Blumea balsamifera and Cordyline fruticosa leaves were added 1 % Lead II Acetate. The yellow precipitate reaction in the solution indicated the presence of tannin (Kokate, 2001). Steroids Determination: 1 mL of the n-hexane, ethyl acetate, and ethanol extracts of Blumea balsamifera and Cordyline fruticosa leaves dropped about 10 Acetic Acid Anhydride and 2 drops of Sulfuric Acid, sequentially. The green or blue color changes in the solution indicated the presence of steroids (Harborne, 1998). Triterpenoids Determination: 1 mL of the n- hexane, ethyl acetate, and ethanol extracts of Blumea balsamifera and Cordyline fruticosa leaves dropped about 10 Acetic Acid Anhydride and 2 drops of Sulfuric Acid, sequentially. The red or purple color changes in the solution indicated the presence of steroids (Harborne, 1998). Carbohydrate Determination: About 1 drop of Molisch solution were added to 1 mL of the n-hexane, ethyl acetate and ethanol extracts of Blumea balsamifera and Cordyline fruticosa leaves. Then 1 mL of Sulfuric Acid was added thourgh the tube glass wall, slowly. The formation of purple ring between 2 layers indicated the presence of carbohydrates (Harborne, 1998). Saponins Determination: 10 mL of hot distilled water was added to 1 mL of the n-hexane, ethyl acetate, and ethanol extracts of Blumea balsamifera and Cordyline fruticosa leaves. The solution was then cooled and shaken vigorously (10 seconds). A stable froth upon standing for 10 minutes after adding 1 drops of Hydrochloride Acid 2N indicated the presence of saponins (Harborne, 1998). Antioxidant Assay Test of antioxidants using 5 concentration samples was grouped into 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, and 6.25 ppm times of dilution, respectively. Further, 3 mg of Ascorbic acid was weighed, then dissolved in 1000 µL of ethanol solvent and regarded as a positive control. While the ethanol solvent was used as a negative control. About 33 µL sample was mixed in a glass tube with 467 µL of ethanol added, and 500 µL of 2,2-diphenyl-1- picryhydrazyl (DPPH) radical scavenging activity (Shimizu et al., 2001). The mixing of the sample was stopped while the volume reached 1000 µL (1 mL). Samples were incubated for 20 minutes with minimum light and A.C. set for 27-30 ºC. The antioxidant activity was determined by decolorization of DPPH with a wavelength of 517 nm using a Spectrophotometer. Measurement was performed in the triplicate examination. The percentage of DPPH free radical was calculated using the following equation: % DPPH radical scavenging activity = ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙− ∆𝑠𝑎𝑚𝑝𝑙𝑒 ∆𝑐𝑜𝑛𝑡𝑟𝑜𝑙 x 100 (1) The actual decrease in absorbance caused by the test was compared with positive controls. The values of IC50 (concentration giving 50% of inhibition) were calculated by a dose-inhibition curve over a linear range of by plotting the extract concentration and the corresponding washout effect (Tuldjanah et al., 2021). RESULTS AND DISCUSSION Plant extracts Blumea balsamifera and Cordyline fruticosa leave were extracted using 3 different solvents n-hexane, ethyl acetate, and ethanol. Maceration is done with yielded 2.18-4.60% extracts on the basis of sample dry weight from Blumea balsamifera leaves, while Cordyline fruticosa leaves yielded 1.09-5.20% extracts (Table 1). Table 1. Yield of Blumea balsamifera and Cordyline fruticosa Leaves Extract. Plants Solvent Extract yield (%) Blumea balsamifera n-hexane 2.18 ethyl acetate 3.18 ethanol 4.60 Cordyline fruticosa n-hexane 1.09 ethyl acetate 3.03 ethanol 5.20 The results showed the ethanol extracts of Blumea balsamifera and Cordyline fruticosa produce an extractive content more concentrated than the n-hexane and ethyl acetate extracts. Phytochemical screening The n-hexane, ethyl acetate, and ethanol leaf extracts of Blumea balsamifera and Cordyline fruticosa were analyzed as the secondary metabolites. Plants known to contain multiple phytochemical molecules such as terpenoids, lignins, phenolics, vitamins, tannins, and others metabolites as antioxidant agents. Several studies showed many phytocompounds possess antidiabetic, antimicrobial, and anti-inflammatory activities (Campbell-Tofte et al., 2012). The pharmacological activities of secondary plant metabolites are known as large compounds as a source of medicinal agents (Muthukrishnan & Manogaran, 2018). Phytochemical analysis of the n-hexane, ethyl acetate, and ethanol leaves extracts of Blumea balsamifera and Cordyline fruticosa is presented in Table 2. 276 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 273-280 Table 2. Phytochemical Analysis of Blumea balsamifera and Cordyline fruticosa Leaves Extract. Compounds Blumea balsamifera Cordyline fruticosa N-hexane Ethyl Acetate Ethanol N-hexane Ethyl Acetate Ethanol Alkaloids + - + + - + Flavonoids - - - - - - Tannins - + + - - + Steroids - - - - - - Triterpenoids + - - + - - Carbohydrate - - - - - - Saponins - - - - - - The phytochemical screening of Blumea balsamifera revealed the presence of alkaloids and triterpenoids in the n-hexane extract, and tannins in the ethyl acetate extract, while the ethanol extract contained alkaloids and tannins. The n-hexane extract of Cordyline fruticosa revealed the presence of alkaloids and triterpenoids, while the ethanol extract contained alkaloids and tannins. The bioactivity potency of plants was indicated based on the secondary metabolites compounds. The presence compounds of tannins, flavonoids, and alkaloids are known as antitumor, antibacterial, antivirus also had antioxidant activity, antimicrobial and anticancer (Taşkın & Taşkın, 2017). Phenolic known as the largest compounds were found in plants and it’s an important compound for free radical scavenging, also contains the hydroxyl group compound. Flavonoid, phenol, and tannin compound are known as the largest group of phenolic compounds and also had the abilities as antioxidant agents (Sen et al., 2013). Antioxidant activity Free radical DPPH scavenging of the n-hexane, ethyl acetate, and ethanol leaves extracts of Blumea balsamifera and Cordyline fruticosa was determined by its hydrogen donating abilities. The inhibition of Blumea balsamifera n-hexane leaves extracts displayed the ability to inhibit free radical DPPH formation by 50% at 100 ppm concentration, while the ethyl acetate and ethanol extracts by 77% and 81% at 50 ppm concentration, respectively. The antioxidant activity of the n-hexane, ethyl acetate, and ethanol leaf extracts of Blumea balsamifera is presented in Figure 2. Figure 2. Antioxidant Activity of Blumea balsamifera leaves extracts against DPPH. Several studies about Blumea balsamifera plants informed the plant extracts contained steroids, alkaloids, phenolic and saponins compounds used as traditional medicine for eczema, rheumatic, menstrual pain relief, flu, fever, asthma, diabetes cough, and diarrhea (Pang et al., 2014). Its also has biological activity as an anti- inflammatory, anticancer, and antioxidant and also functions as an antimicrobial (Nessa et al., 2010). The inhibition of Cordyline fruticosa n-hexane and ethyl acetate leaf extract displayed the ability to inhibit Sari et al. – Phytochemical and Antioxidant Activity of … 277 free radical DPPH formation by 45% and 56% at 100 ppm concentration, while the ethanol extracts by 76% at 50 ppm concentration. Antioxidant activity of the n- hexane, ethyl acetate, and ethanol leaves extracts of Cordyline fruticosa is presented in Figure 3. Figure 3. Antioxidant Activity of Cordyline fruticosa leaves extracts against DPPH. Several studies about Cordyline fruticosa leaves plants, informed the plant contained saponins, tannins, flavonoids, polyphenol, and alkaloids compound which has biological activity as antioxidant, anti-inflammatory, antibacterial, anti-allergic, anticancer, and antivirus (Dyary et al., 2014). This plant is also known to accelerate the wound healing process, the function of tannins compound as astringents uses shrink skin pores and bleeding minor stopped. The antioxidant activity of Blumea balsamifera and Cordyline fruticosa leaves extracts indicated plants had a source to inhibit free radical DPPH. Refers to the results, the IC50 of the ethyl acetate and ethanol leaves extracts of Blumea balsamifera and Cordyline fruticosa extracts were determined as shown in Table 3. Table 3. The IC50 of Blumea balsamifera and Cordyline fruticosa Leaves Extract. Blumea balsamifera Cordyline fruticosa Ethyl Acetate Ethanol Ethyl Acetate Ethanol 23.68±19.69 17.59±18.71 73.72±14.42 20.17±17.59 The antioxidant activity of IC50 had a classification of the antioxidant activities compound based on the acquisition of the IC50 value as shown as Table 4 (Analianasari et al., 2022). Table 4. The Antioxidant Activity of IC50 Classification. Inhibition Value (ppm) Category <50 Extreme 50-100 Strong 100-150 Moderate 150-200 Weak >200 Fragile 278 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 273-280 Figure 4. Antioxidant Activity of Cordyline fruticosa leaves extracts against DPPH. Based on Figure 4, the linear equation value for Blumea balsamifera ethyl acetate extract y = 1.0237x+25.762, the calculation of the IC50 value for Blumea balsamifera ethyl acetate extract obtained the following values: y = 1.0237x+25.762 (for y = 50), then the x value is 23.68 ppm. Furthermore, based on the calculation of the regression curve in the ethanol extract of Blumea balsamifera, the regression equation y = 0.964x + 33.039 (assuming the value of y = 50) is obtained, and the x value is 17.59 ppm. The IC50 value of the Blumea balsamifera ethyl acetate and ethanol extracts had extreme IC50 antioxidant activity (<50 ppm). Several studies about Blumea balsamifera, especially the leaves part informed fresh and dry leaves had antioxidant activity with boiling water method, antidiabetic activity with hydro-ethanol extract, antitumor activity with essential oil extract and also anticancer activity of ethyl acetate extract fraction (Widhiantara & Jawi, 2021). The linear equation value for Cordyline fruticosa ethyl acetate extract y = 0.3628x + 23.253 (assuming the value of y = 50), then the x value is 73.72 ppm. The IC50 value of Cordyline fruticosa ethyl acetate extract is strong (50-100 ppm). While the regression of Cordyline fruticosa ethanol extract equation y = 0.902x + 31.804 (with the value of y = 50) is obtained, the x value is 20.17 ppm. The IC50 value of Cordyline fruticosa ethanol extract had extreme IC50 antioxidant activity (<50 ppm). The bioactive compounds of Cordyline fruticosa methanol leaf extract have been isolated. The active compound had antitumor activity, antibacterial activity, and cytotoxic activity (Assyifa et al., 2022). CONCLUSIONS An ethnobotanically-selected medicinal plant, Blumea balsamifera, and Cordyline fruticosa has been investigated for their antioxidant activities. The results informed that the leaves extract of ethyl acetate and ethanol of the plant showed good antioxidant properties by the extreme-good category of IC50. Further investigation is needed to find the responsible compound in the Blumea balsamifera and Cordyline fruticosa plants and explored the possibility of bioproduction for the active compounds, also developed for healthcare in the future. Acknowledgments: The research was funded by the Ministry of Education, Culture, Research, and Technology through Penelitian Dosen Pemula Scheme (Junior Lecturer Research Grants) 2022 under contract number 012/PL21.G/PG/2022. 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