Title Science and Technology Indonesia e-ISSN:2580-4391 p-ISSN:2580-4405 Vol. 4, No. 3, July 2019 Research Paper Cytotoxic Activity of Tahongai (Kleinhovia hospita Linn.) Leaves Extracts Using Brine Shrimp Lethality Test Indah Solihah1*, Herlina1, Khairunnisa1, Riana Sari Puspita Rasyid2, Tri Suciati2 1Faculty of Mathematics and Natural Sciences, Sriwijaya University, Ogan Ilir 30662, South Sumatera, Indonesia 2Faculty of Medicine, Sriwijaya University, Ogan Ilir 30662, South Sumatera, Indonesia. *Corresponding author: indahsolihah26614@gmail.com Abstract Tahongai (Kleinhovia hospita Linn.) leaves had been known contain alkaloids, flavonoids, saponins, steroids, and tannins. At Komering, South Sumatera province, tahongai leaves had been known to treat tumor, cancer, polyps, acne, and dysmenorrhea. The study of cytotoxic activity of tahongai bark and stem were done. This study aims to determine the cytotoxic activity of tahongai leaves extracts using BSLT method. Tahongai leaves were extracted using gradual maceration with n-hexane, ethyl acetate, and ethanol 96%. Each extract was tested cytotoxic activity towards Artemia salina L. larvae. The yield of n-hexane, ethyl acetate, and ethanol extracts are 2,686%, 7,033%, and 7,933% respectively. Ethanol extract of tahongai leaves had the best cytotoxic activity with lethality value 76,667% at 500ppm. Statistical analysis with two way ANOVA showed extract and concentration had a significant (p<0,05) e�ect on larvae lethality percentage. Keywords Kleinhovia hospita Linn., Artemia salina L., cytotoxic, BSLT. Received: 4 June 209, Accepted: 17 July 2019 https://doi.org/10.26554/sti.2019.4.3.60-63 1. INTRODUCTION Tahongai (Kleinhovia hospita Linn.) is a tropical plant. Tahongai has a therapeutic e�ect as antidiabetic, antioxidant, hepatopro- tective, anticancer, and antihyperlipidemic (Paramita, 2016). Re- search on cytotoxic e�ects of tahongai plant has been carried out by Morilla et al. (2015) using bark and stem ethanol extract with an LC50 value 452,03 �g/mL. Methanol extract of tahongai leaves, stem, and bark had been studied to have cytotoxic activity in hepatoma cells and can inhibit murine leukemia cells (P388) with IIC50 56 �g/mL (Nurhidayah et al., 2013). Decoct of tahongai bark and stem also have cytotoxic activity with an LC50 value of 698.54 �g/mL Morilla et al. (2015). The leaves and wood of Kleinhovia hospita Linn. found to contain �avonols, kaempferol, and quercetin (Arung et al., 2012).Pieme et al. (2010) found that quercetin and kaempferol had anticancer activity. Quercetin and kaempferol are one of the compounds of �avonoid that have anticancer e�ects both in vitro and in vivo test (Baghel et al., 2012). Based on the study above, in this study, gradual maceration was carried out to obtain active compounds from various polarity levels using n-hexane (non- polar), ethyl acetate (semi-polar) and ethanol (polar) solvents. Each extract was done to compare the cytotoxic activity using Brine Shrimp Lethality Test (BSLT). 2. EXPERIMENTAL SECTION 2.1 Materials Plant materials used for this study comes from the same source used by Solihah et al., (2018). The chemicals used of this study were n-hexane (Brataco®), ethyl acetate (Brataco®), ethanol (Brataco®), aquadest (Brataco®), Mayer reagent (Merck®), Wag- ner reagent (Merck®), Dragendor� reagent (Merck®), sulfuric acid (Merck®), ammonia (Merck®), chloroform (Merck®), hy- drochloric acid (Merck®), magnesium powder (Merck®), sodium hydroxide (Merck®), iron (III) chloride (Merck®), anhydrous acetic acid (Merck®), DMSO (Merck®), quercetin (FitoPure®), kaempferol (FitoPure®), and shrimp larvae Artemia salina Leach (Supreme®). 2.2 Instruments The instruments used for this study are blender (Philips®), ro- tary evaporator (IKA-C Mag®), maceration chamber, water bath (Memmert™), glass tools (Pyrex®), silica TLC plate G60 F254 (Merck®), vaporizer plate (RRC®), micropipette (Dragon Lab®), vortex (Corning LSETM), 40-60 W (Itsuyama®) lighting, analyti- cal scale of readability 0,01 and 0,0001 g (Electronic Scale® and Ohaus®), magnetic stirrer (IKA C-Mag®). https://doi.org/10.26554/sti.2019.4.3.60-63 Solihah et. al. Science and Technology Indonesia, 4 (2019) 60-63 2.3 Extraction A total of 1 kg dried leaves of tahongai were soaked with 6 L n-hexane where every two days the solvent was replaced until clear about ten days. Powdered pulp was macerated with 6 L ethyl acetate solvent and 6 L 96% ethanol using the same method. Filtrate evaporated using a rotary evaporator to obtain a thick extract. 2.4 Phytochemical Screening 2.4.1 Flavonoid identi�cation The extracts of 0,5 g were added with 5 mL of hot ethanol for 5 minutes. The extract are �ltered so that the �ltrate was obtained and then a few drops of concentrated HCl are added, and 0,2 mg of magnesium powder was added. The red color indicates the presence of �avonoid group compounds. 2.4.2 Saponin identi�cation The extracts of 1 g were added with distilled water and boiled for 2 minutes and shaken vigorously. The existence of saponins was characterized by the formation of a stable foam in a test tube. 2.4.3 Alkaloids identi�cation The extracts of 2 g were taken then a little chloroform and sand were added and ammonia in chloroform. The mixture was shaken and then �ltered. The �ltrate was added with H2SO4 2N and then shaken. Two layers were formed. The top layer in the form of a water phase was separated and then tested by Mayer, Wagner, and Dragendor� reagents. The presence of alka- loids was characterized by red sediment with Wagner reagents, white sediment with Mayer reagents, and red or orange color with Dragendor� reagent (Al-Daihan and Bhat, 2012). 2.4.4 Steroids and Triterpenoids identi�cation The bottom layer from alkaloid identi�cation was separated and dropped onto the drop plate, allowed to dry. Anhydrous acetic acid was two drops, and one drop of concentrated sulfuric acid (Lieberman-Buchard reagent) was added to the residual. The green or red color formed indicates the presence of steroid or triterpenoid compounds (Al-Daihan and Bhat, 2012). 2.4.5 Identi�cation of Tannins An amount of 2 g tahongai leaves extracts were added with 100 mL of water then boiled for 15 minutes then cooled and �ltered so that �ltrate was obtained. An amount of 1% iron (III) chloride solution was added to the �ltrate. The presence of tannin compounds was characterized by the formation of dark blue or blackish green (Al-Daihan and Bhat, 2012). 2.5 Identi�cation of quercetin and kaempferol using TLC Identi�cation of quercetin compounds on tahongai (Kleinhovia hospita Linn.) leaves extracts using TLC method. TLC plates were measured at 6 x 10 cm2 with the upper and lower limits of 1 cm. Tahongai leaves extracts, quercetin, and kaempferol were made at a concentration of 5% and then dripped along with the silica TLC plate G60 F254. The TLC plate was eluted using chloroform : ethyl acetate (5:2) eluent. Comparison of eluents was obtained from the results of the experiment. The TLC results were aerated and detected under UV light at a wavelength of 254 and 366 nm. Then sprayed with 5% aluminum (III) chloride spray reagent in ethanol (Colegate and Russel, 1993). Results of TLC were stains or spots that glow greenish-yellow (Nuria et al., 2011). 2.6 Cytotoxic Activity test 2.6.1 Preparation of stock solution Each tahongai leaves extracts were dissolved in 4 drops of DMSO 10%, and 1000 ppm (mother liquor) concentration was made then a test solution with a concentration of 500; 250; 125; 62,5; and 31,25 ppm. DMSO 10% control solution was used without the addition of extracts (Morilla et al., 2015). 2.6.2 Preparation of Artemia salina Leach Larvae An amount of 1 L of arti�cial seawater (20 g of salt in 1 L of water) was needed for 1 g of Artemia salina Leach. eggs. Artemia salina Leach. egg put into a hatchery that has contained seawater or synthetic seawater then illuminated with a 40 – 60 W lamp for 24 – 48 hours. Eggs that have hatched into larvae were moved to another place, and 24 hours later, the larvae were used as test subjects (Morilla et al., 2015). 2.6.3 Cytotoxic test Six pieces of vials were used in testing each variation of the extract, which was divided into �ve vials for each test concen- tration and one vial for control. Each concentration was taken according to the calculation while for the negative control, 5 mL of seawater was added. A total of 10 larvae of Artemia salina Leach. The larvae put into seawater, which has been mixed with the test solution. Observations were made for 24 hours (Lisdawati et al., 2016). 2.7 Statistical analysis The data of percentage of larvae lethality are expressed as the mean ± SD. Percentage of larvae lethality used as a dependent factor, whereas the type of extract and concentration used as an independent factor. The di�erence among the means has been analyzed by two way ANOVA. 3. RESULTS AND DISCUSSION 3.1 Extraction and phytochemical screening results Extraction of tahongai leaves by gradual maceration method using n-hexane, ethyl acetate, and ethanol 96%. The yield of extracts can be seen in Table 1. Table 1. The yield of extracts Extract Total (g) % Yield N-Hexane 26,86 g 2686% Ethyl Acetate 70,33 g 7033% Ethanol 79,330 g 7933% © 2019 The Authors. Page 61 of 63 Solihah et. al. Science and Technology Indonesia, 4 (2019) 60-63 Yield states the percentage of raw material obtained from the total raw material in the extraction process. The higher the yield value, more the opportunity for the raw material to be utilized. Percent of yield can be in�uenced by the duration of extraction time and the degree of �neness of the simplicia particle size. Each extract was macerated for ten days. The smaller the size of the simplicia, the higher the surface area of the particles so that the solvent is more attract compounds in the sample but if the simplicia powder is too �ne, it can broken cell walls and unwanted substances (ballasts) will enter the maceration results. This is not desirable in the extraction process. The percentage of yield obtained showed that ethanol extract had the highest percentage of yield compared to other extracts of 7,933%. Ethanol as a polar solvent, which can attract polar components. This is indicates that the most polar component was found in tahongai leaves so the percentage of yield highest than another. Phytochemical screening is an examination of the chemical content qualitatively to ensure the presence of the desired sec- ondary metabolites contains in plant or extract. The data reveal the presence of various constituents in each extract. Phytochem- ical test results can be seen in Table 2. Table 2. Phytochemicals screening test of tahongai leaves ex- tracts Chemical Screening Result of extracts Substance n-Hexane Ethyl acetate Ethanol Alkaloid + + + Flavonoid - + + Saponin - + + Tanin - + + Triterpenoid - - - Steroid + + + All tahongai leaves extracts do not contain triterpenoids. N- hexane extract only contains alkaloid and steroid compounds. While ethyl acetate and ethanol extracts contain alkaloids, �avonoids, tannins, saponins, and steroids. 3.2 Identi�cation of quercetin and kaempferol with TLC Identi�cation of quercetin and kaempferol in tahongai leaves extracts was used thin-layer chromatography. Stationary phase used was silica while the mobile phase used is chloroform: ethyl acetate (5:2). Spots produced by each component were observed in UV light 254 nm and UV 366 nm. UV light 254 shows that the compound has at least two conjugated double bonds while the �uorescence under UV light 365 nm shows that the component has a chromophore and has an auxochrome group in its structure (Alen et al., 2017). KLT plate was sprayed with aluminum (III) chloride (AlCl3) reagent. The resulting color shows a yellow color. The results showed in �gure 1. Quercetin and kaempferol compounds based on the results of TLC testing contained in ethyl acetate and ethanol extract. Quercetin and kaempferol are both polyhydroxy �avonol com- Figure 1. The chromatogram of Tahongai leaves extracts (K = kaempferol; Q = Quercetin; 1 = n-hexane extract; 2 = ethyl acetate extract; 3 = ethanol extract) (a) under UV light 366 nm (b) under UV light 254 nm (c) sprayed with AlCl3 pounds. Leaves of Kleinhovia hospita L. had been reported con- tains scopoletin, kaempferol, quercetin, eleutherol and kaempferol 3-O-B-D glucoside compounds (Arung et al., 2012) 3.3 Cytotoxic activity The test of cytotoxic activity of tahongai leaves extracts were used BSLT (Brine Shrimp Lethality Test) method. The cytotoxic e�ect of tahongai leaves extracts was determined within 24 hours after administering each extract with various concentration us- ing Artemia salina as a test subject. Each test was carried out on 10 larvae then replicated 3 times. Results obtained are calculated as the percentage value. Based on the data in table 3 it can be seen that the higher concentration of extract can cause the mortality of the larvae was greater. While the extract which has the most potential as a cytotoxic agent was the ethanol extract of tahongai leaves. In the ethanol extract of tahongai leaves contained polar compounds, such as polyhydroxy �avonoids. The mechanism of larvae lethality of Artemia salina L is estimated to be related to the compounds contained in tahongai leaves extract such as �avonoids as cytotoxic agents. From the results of phytochemical screening showed that ethyl acetate and ethanol extract contained �avonoids while �avonoids were not found in n-hexane extract. Flavonoids at certain levels have the potential for acute toxicity (Carballo et al., 2002). The presence of �avonoids in the cell environment causes hydroxy groups in �avonoids to bind to cell membrane integral proteins. This causes the blocking of the active transports Na+ and K+, the active transport stops, causing uncontrolled inclusion of Na+ ions into the cell. This causes the rupture of cell membranes (Scheuer, 2004). Rupture of cell membranes causes cell death. Data analysis was used two way ANOVA method. Two way ANOVA was conducted to test the di�erences in several groups based on two independent variables. Data on percent lethality of larvae as a dependent factor, while the type and concentration of extracts as an independent factor. Data is declared homoge- neous if the signi�cance value is ≥ 0,05. The homogeneity value obtained is 0,238 (p> 0,05) so the data has the same variation. © 2019 The Authors. Page 62 of 63 Solihah et. al. Science and Technology Indonesia, 4 (2019) 60-63 Table 3. The e�ect of tahongai leaves extract in larvae lethality Concentration Larvae lethality (mean ± SD) (%) (ppm) n-Hexane Ethyl acetate Ethanol 31,25 6,667±0,577 13,333±1,155 16,667±0,577 62,5 13,333±0,577 30,000±1,000 40,000±1,000 125 16,667±0,577 43,333±0,577 56,667±0,577 250 23,333±0,577 53,333±0,577 70,000±0,000 500 26,667±0,577 66,667±0,577 76,667±0,577 At ANOVA values, there was type of extract variable, concen- trations, and type of extract-concentration interactions. If the signi�cance value is <0.05, the data is signi�cant. The value obtained is a correction model of 0,000 the data is signi�cantly di�erent. The signi�cance of type of extract, concentration, and type of extract-concentration interaction variables was 0,000 (p <0,05). This states that type of extracts, concentrations, and interactions between type of extracts and concentrations have a signi�cant e�ect on percent lethality of Artemia salina L. larvae. 4. CONCLUSIONS Based on the results of this study, it can be concluded that the ethanol extract of tahongai leaves has the greatest yield and contain the greatest phytochemical groups compound. This correlates with its activity as a cytotoxic agent. Tahongai leaves ethanol extract has the largest cytotoxic activity compared to n-hexane and ethyl acetate extract. 5. ACKNOWLEDGEMENT Authors would like to express their gratitude toward Sriwijaya University PNBP Sateks Research Grant that made this research possible. REFERENCES Al-Daihan, S. and R. Bhat (2012). Antibacterial activities of extracts of leaf, fruit, seed, and bark of Phoenix dactylifera. Afr J Biotechnology, 11(42); 10021–10025 Alen, Y., F. Agresa, and Y. Yuliandra (2017). Thin-layer chro- matography (TLC) analysis and antihiperuricemia activity of Schizostachyum brachycladum Kurz (Kurz) bamboo shoot ex- tract in male white mice. J of Sains & Clinical Pharm, 3(2); 146 – 152 Arung, E., I. Kusuma, S. Purwatiningsih, S. Roh, C. Yang, and S. Jeon (2012). Antioxidant activity and cytotoxicity of the traditional indonesian medicine tahongai (Kleinhovia hospita L.) extract. J Acupunct Meridian Stud, 2(4); 306–308 Baghel, S., N. Shrivastava, R. Baghel, P. Agrawal, and S. Rajput (2012). A review of quercetin: Antioxidant and anticancer properties. World J Pharm Pharmaceut Sci, 1(2); 146–160 Carballo, J., Z. Inda, P. Perez, and M. Gravalos (2002). A com- parison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BMC Biotechnology, 2(17); 1–5 Lisdawati, V., S. Wiryowidago, and L. Kardono (2016). Brine shrimp lethality test (BSLT) of various fractions of fruit �esh and seed skin mahkota dewa (Phaleria macrocarpa). Medical Res Bulletin, 34(3); 111-118 Morilla, L., O. Nuñeza, and M. Uy (2015). Brine shrimp lethality test of Kleinhovia hospita stem and bark from agusan del sur. ELBA Bio�ux, 7(1); 61-66 Nurhidayah, N., M. Minarti, A. Pratama, and I. Imran (2013). Test the activity of terpenoid steroid and phenolic derivative com- pounds from extracts of stem tissue from ndokulo (Kleinhovia hospita L.) plants to cancer cell growth (Leukemia P-388). Pro- ceeding PIMNAS PKM-P, Kendari, Indonesia Nuria, M., Wahyono, and R. Susidarti (2011). Identi�cation of kaempferol from jangkang leaves (Homalocladium platy- cladum (F.Muell) Bailey) and their antibacterial activity. In- donesian Journal of Pharmacy, 22(1); 1–8 Paramita, S. (2016). Tahongai (Kleinhovia hospita L.): A review of herbal medicine from East Kalimantan. Science, 9(1); 29–35 Pieme, C., V. Penlap, J. Ngogang, and M. Costache (2010). In vitro and antioxidant activities of �ve medicinal plants of malvaceae family from Cameroon. Environmental Toxicology and Pharmacology, 29; 223-228 Scheuer, P. (2004). Ciguatera and its o�shoots: Encounters en route to a molecular structure. Tetrahedron, 50; 3-18 © 2019 The Authors. Page 63 of 63 INTRODUCTION EXPERIMENTAL SECTION Materials Instruments Extraction Phytochemical Screening Flavonoid identification Saponin identification Alkaloids identification Steroids and Triterpenoids identification Identification of Tannins Identification of quercetin and kaempferol using TLC Cytotoxic Activity test Preparation of stock solution Preparation of Artemia salina Leach Larvae Cytotoxic test Statistical analysis RESULTS AND DISCUSSION Extraction and phytochemical screening results Identification of quercetin and kaempferol with TLC Cytotoxic activity CONCLUSIONS ACKNOWLEDGEMENT