Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 DOI: 10.22144/ctu.jen.2021.043 Primary investigating chemical constituents of bioactive extract from Centrostachys aquatica (R.Br.) Wall. ex Moq.-Tand. Ngo Quoc Luan1*, Ta Thao Cuong1, Tran Thi Manh Huynh1, Le Minh Dan1, Ngo Khac Khong Minh2, Ngo Trong Nghia3, Nguyen Diep Xuan Ky4,5, Phan Nhat Minh4,5 and Nguyen Tan Phat4,5 1 School of Education, Can Tho University, Viet Nam Medical Faculty, Nam Can Tho University, Viet Nam 3 Faculty of Environment and Natural Resource, Kien Giang University, Viet Nam 4 Institute of Chemical Technology, Vietnam Academy of Science and Technology, Viet Nam 5 University of Science and Technology, Vietnam Academy of Science and Technology, Viet Nam *Correspondence: Ngo Quoc Luan (email: ngoquocluan@ctu.edu.vn) 2 Article info. ABSTRACT Received 21 Jul 2021 Revised 20 Aug 2021 Accepted 21 Oct 2021 This study is aimed to screen the biological activities and chemical composition to find evidences for potential medicinal applications of Centrostachys aquatica in the Mekong Delta. Crude methanol extract and subextracts in n-hexane, ethyl acetate, and acetone from Centrostachys aquatica were tested bioactivities. The methanol extract, n-hexane and ethyl acetate subextracts exhibited antimicrobial activity with corresponding MIC values of 200, 100 and 200 µg/mL, respectively. The ethyl acetate subextract was inhibited cytotoxicity against cancer cell line LU-1 with IC50 of 27.66 µg/mL. None of the extracts showed antioxidant ability. Three known secondary metabolites including oleanolic acid (1), 20-hydroxyecdysone (2), and b-spinasterol (3) were isolated for the first time from the bioactive (ethyl acetate) subextract of Centrostachys aquatica. Their structures were elucidated by modern spectra as MS, NMR and comparison with published data. Keywords Antimicrobial activity, antioxidant activity, Centrostachys aquatica, 20hydroxyecdysone, oleanolic acid, b-spinasterol a local name of “Cỏ Xước nước” in Vietnamese, it means “water scratch grass”. It is only one aquatic species belonging to the genus Centrostachys in the family Amaranthaceae (Figure 1). 1. INTRODUCTION Centrostachys aquatica (R.Br.) Wall. ex Moq.Tand. or Achyranthes aquatica R.Br. (Ho, 1999) has 71 Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 Figure 1. Centrostachys aquatica (R.Br.) Wall. ex Moq.-Tand. In Viet Nam, Centrostachys aquatica distributed in wetlands such as swamps, alluvial flats along rivers and canals in the Mekong Delta. People use this plant as a kind of vegetable and there is no information about its use in traditional medicine. evaporating was accomplished by using RE-52A rotary evaporator system (China). Thin layer chromatography (TLC) was carried out on pre-coasted silica gel 60F254 (0.25 mm) aluminium sheet (Merck). Traces of compounds were detected by illuminating under UV light (254/365 nm) or spraying 10% H2SO4 solution in ethanol and then heating at 105°C for 1-2 min on electric stove. In recently researched documents on Centrostachys aquatica were found, there was only one report about isolating a compound called loliolide; the aqueous methanol extract and loliolide from this extract both have the ability to inhibit the growth of roots and hypocotyls of cress (Lepidium sativum) (Bich & Kato-Noguchi, 2014). This could be the basis for a convincing explanation for phytotoxicity and allelopathic activity of Centrostachys aquatica. For common phase column chromatography (CPCC), silica gel 60 (0.040-0.063 mm, Merck), increasing polarity solvent systems including nhexane (H), chloroform (C), ethyl acetate (E) and methanol (M) was used. Compounds were purified by re-crystallization in pure solvents. To find out if Centrostachys aquatica had other beneficial activities, extracts from this plant were tested some of the bioactivities. The results showed that the ethyl acetate extract has good antimicrobial and cytotoxic activity against lung cancer cells (section 3.1). This is also the reason for studying the chemical composition of this bioactive extract with three natural compounds were initially isolated. 2.2.2. Structural elucidation and identification Melting point (mp.) was recorded by a melting point meter (Electrothermal 9100, UK), using capillary at Can Tho University. 1H-NMR, 13C-NMR, DEPT, HSQC, COSY, HMBC spectra were recorded on a Bruker AM500, 600 FT-NMR spectrometer; Mass spectrum (MS) was recorded on mass spectrometer (HP 1100 series, LC/MSD Trap, Agilent) at Vietnam Academy of Science and Technology. 2. EXPERIMENT 2.1. Plant material 2.2.3. Antimicrobial, cytotoxic and antioxidant activity testing The whole plants of Centrostachys aquatica were collected in Can Tho city in May, 2021. Voucher specimens have been identified at Deparment of Biology, School of Education, Can Tho University. After cleaning, poor quality parts were removed. Good material was dried at 50°C in order to decrease the humidity to less than 2%, followed by crushing into fine powder. Biological activity assays were conducted at the Experimental Biology Department, Institute of Chemistry of Natural Compounds, Vietnam Academy of Science and Technology. The antimicrobial activity assay was followed the disc diffusion agar method of Vanden-Berghe and Vlietinck (1991), Mckane and Kandel (1996), improved by using 96-well microplate and ELISA reader. 2.2. General experimental procedures 2.2.1. Extraction and purification Solid-liquid extractions were used with methanol, nhexane, ethyl acetate and acetone. Solvent 72 Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 5:5 to 25:75, ending with methanol) to afford 6 subfractions (CAE5.1-5.6). The subfraction CAE5.2 (H:E 5:5, 4.21 g) was cleaned up by E:M 9:1 and recrystallized two times in methanol to yield compound 2 (7.2 mg). The cytotoxic activity assay was performed according to the method of Skehan et al. (1990) and Likhiwitayawuid and Angerhofer (1993) has been applied at the USA National Cancer Institute (NCI) and the College of Pharmacy, University of Illinois, Chicago, USA. The fraction CAE1 (H:E 9:1; 8.34 g) was taken CPCC with eluent of H:E (gradient, 0 to 100% E) to give 6 subfractions (CAE1.1-1.6). The subfraction CAE1.4 (H:E 9:1, 1.42 g) was continued to perform CP-CC with H:E 95:5 to get 4 subfractions (CAE1.4.1-1.4.4). The subtraction CAE1.4.2 (H:E 95:5, 0.12 g) was re-crystallized in n-hexane to obtain compound 3 (9.2 mg). The antioxidant activity assay was based on its ability to trap free radicals generated by 1,1diphenyl-2-picrylhydrazyl (DPPH) of BrandWilliams et al. (1995), Shela et al. (2003) and Kumar et al. (2013). 2.3. Extraction and isolation The dried plant powder (7.0 kg) was exhaustedly extracted with methanol 70%vol (> 20 L) and then evaporated under reduce pressure to remove the solvent to give dry dark-green residue of crude methanol extract (CAMe, 305 g). 2.4. Physical characteristic and spectral data Oleanolic acid (1): A white amorphous powder, mp. 306-308°C, lotus purple chromatographic stain, no luminescence under UV lamp. ESI-MS m/z 455 [M-H]-;. 1H-NMR (CDCl3, 600 MHz, δH ppm, J Hz) and 13C-NMR (CDCl3, 150 MHz, δC ppm) (Table 4). The CAMe extract (300 g) was distributed with nhexane (10 L), ethyl acetate (15 L), and acetone (10 L), respectively, and the solvents were evaporated under poor pressure to obtain subextracts CAHe (50.6 g), CAEt (93.7 g), CAAc (40.3 g) and the remainder was insoluble in distributed solvents (CAW, 101 g). 20-hydroxyecdysone (2): White needle-shaped crystals, mp. 242-244°C, purple pink TLC stain, luminescence under UV lamp. ESI-MS m/z 481 [M+H]+; . 1H-NMR (DMSO-d6, 500 MHz, δH ppm, J Hz) and 13C-NMR (DMSO-d6, 125 MHz, δC ppm) (Table 4). The CAEt subextract (90 g) was subjected to CP-CC with H:E (gradient, 0 to 100% E) and final with E:M (9:1) solvent mixtures as eluent to give 7 fractions (CAE1-7). b-Spinasterol (3): White needle-shaped crystals, mp. 164-166°C, red purple TCL stain, no luminescence under UV lamp. ESI-HRMS m/z 395.3689 [M-H2O+H]+;. 1H-NMR (CDCl3, 500 MHz, δH ppm, J Hz) and 13C-NMR (CDCl3, 125 MHz, δC ppm) (Table 4). The fraction CAE2 (H:E 8:2; 13.12 g) was treated with CP-CC (H:E, gradient, 0 to 100% E) to afford 6 subfractions (CAE2.1-2.6). The subfraction CAE2.4 (H:E 85:15, 3.45 g) was continued to take CP-CC (C:M, gradient, 0 to 100% M) to get 5 subfractions (CAE2.4.1-2.4.5). The subfraction CAE2.4.3 (C:M 95:5, 0.55 g) was washed with H:E 9:1 and then re-crystallized in methanol to produce compound 1 (10.1 mg). 3. RESULTS AND DISCUSSIONS 3.1. Bioactivities of extracts Results of antimicrobial activity, anticancer and antioxidant activities of 4 extracts from Centrostachys aquatica were presented in Table 1, 2 and 3, respectively. The fraction CAE5 (H:E 25:75, 23.52 g) was performed CP-CC with H:E solvent systems (from 73 Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 Table 1. Results of antimicrobial activity of extracts from Centrostachys aquatica Minimum inhibitory concentration (MIC, µg/mL) Initial Gram (+) Bacteria concentration Gram (-) Bacteria No. Code Comments Escherichia Pseudomonas Bacillus Staphylococcus (µg/mL) coli aeruginosa subtillis aureus (-)-Control (-) (-) (-) (-) 1 CAAc 400 (-) (-) (-) (-) Negative Positive 2 CAHe 400 (-) (-) 100 (-) (1 strain) Positive 3 CAMe 400 (-) (-) 200 (-) (1 strain) Positive 4 CAEt 400 (-) (-) 200 (-) (1 strain) It can be seen from the Table 1 that CAHe, CAMe 200 µg/mL, respectively. The CAAc extract was and CAEt samples exhibited resistance to the test completely negative for 4 tested microorganism strain of B. subtilis with MIC values of 100, 200 and strains. Table 2. Results of anticancer activity of extracts from Centrostachys aquatica Cell survival rate (CS value, %) IC50 value (µg/mL) Cell trains Cell trains Hep-G2 LU-1 Hep-G2 LU-1 DMSO 100 100 0,32 0,27 (+) Control 3,14±0,71 1,89±0,60 1 CAAc 98,88±1,09 88,15±1,64 2 CAHe 63,59±2,04 66,39±0,51 3 CAMe 93,48±2,25 93,38±1,62 27,66 4 CAEt ± 53,36±1,52 40,49 0,48 remaining samples did not show cytotoxic activity As the results in Table 2, only the sample CAEt of 2 cancer cell lines including Hep-G2 (liver showed inhibitory activity on LU-1 cell line (lung cancer) and LU-1 at the tested concentration. cancer) with IC50 value of 27.66 µg/mL. The No. Code Initial concentration (µg/mL) 5 40 40 40 40 Table 3. Results of antioxidant activity of extracts from Centrostachys aquatica No. 1 2 3 4 Code (+) Control (-) Control CAAc CAHe CAMe CAEt Initial concentration (µg/mL) Scavenging capacity (SC, %) 50 200 200 200 200 79,24±1,02 0 12,21±2,01 10,98±0,41 11,40±0,66 24,63±1,50 (-) Control: DPPH/EtOH + DMSO. A half scavenging concentration (SC50, µg/mL) 12,02 - Comments Positive Negative Negative Negative Negative Negative (+) Control: DPPH/EtOH + ascorbic acid. Table 3 showed the test samples did not exhibit antioxidant activity on the DPPH system at the test concentrations. 3.2. Chemical structure elucidation of isolated compounds Three isolated compounds had some similar characteristics as being white solids, no producing positive reaction to FeCl3 reagent; it can be inferred that they do not belong to the group of phenolic substances. Typical signals of protons and carbons In summary, the bioactivity assay showed that the CAEt extract was the most active of the tested extracts. So this extract was chosen to investigate its chemical composition. 74 Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 carbons. In which, 2 carbons at δC 122.7 and 143.6 exhibited the presence of a double bond; carbon at δC 182.8 allowed to predict having a carboxylic group; and carbon at δC 79.1 was oxygenated methine group (Table 4). in 1D-NMR showed that they had the patterns of triterpene and sterol backbones. 3.2.1. Compound 1 Compound 1 was obtained as a white amorphous powder, its mp. was about 306-308°C. It had lotus purple chromatographic spot and no luminescence under UV lamp. From mentioned 1D-NMR data, compound 1 gave the characteristic spectra pattern of a pentacyclic triterpene. Based on the spectral data analysis, comparison with those given in the literature (Zuhal et al., 2009) and checking with its own 2D-NMR spectra, compound 1 was identified as oleanolic acid (Figure 2). The molecular formula of compound 1 was speculated to be C30H48O3 (456 amu, seven degrees of unsaturation) on the basis of ESI-MS (m/z 455 [M-H]-). The 1H-NMR spectrum of compound 1 revealed 7 singlet signals of methyl protons at δH [0.76 (3H, s), 0.77 (3H, s), 0.90 (3H, s), 0.91 (3H, s), 0.93 (3H, s), 0.99 (3H, s) and 1.13 (3H, s)]; one oxygenated methine proton at δH 3.22 (1H, dd, 11.4, 4.2); one double-bonded methine proton at δH 5.28 (1H, t, 3.6) and about 25 other protons of methine, methylene, hydroxyl groups (Table 4). Oleanolic acid could be used to prevent the majority of the most common diseases of civilization i.e. cancer, cardiovascular diseases, atherosclerosis or diabetes (Paszel-Jaworska et al., 2014). 3.2.2. Compound 2 Compound 2 was isolated as white needle-shaped crystals, its mp. was about 242-244°C. It had purple pink TLC stain and luminescence under UV lamp, that proved there was a conjugate system in its chemical structure. The 13C-NMR and DEPT spectra of compound 1 appeared signals of total 30 carbons including 7 methyl, 10 methylene, 5 methine and 8 quaternary bFigure 2. Chemical structures of isolated compounds 1 The molecular formula of compound 2 was H-NMR spectrum of compound 2 appeared typical established as C27H44O7 (480 amu, six degrees of proton signals of 5 methyl groups at δH [0.76 (3H, unsaturation) by ESI-MS (m/z 481.1 [M+H]+). s), 0.84 (3H, s), 1.06 (6H, s), and 1.08 (3H, s)]; 3 oxygenated methine proton at δH [3.12 (1H, d, 5.0), 75 Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 hydroxyecdysone (Figure 2) given in the literature (Vokac et al., 1998). Moreover, all correlation signals between protons and carbons in HSQC and HMBC spectral data of compound 2 conformed with the mentioned chemical structure, so compound 2 was determined to be 20hydroxyecdysone. 3.56 (1H, s), and 3.77 (1H, s)]; one double-bonded methine proton at δH 5.63 (1H, s) and about 25 other protons of methine, methylene, hydroxyl groups (Table 4). 13 C-NMR and DEPT spectra exhibited signals of total 27 carbons containing 5 methyl, 8 methylene, 7 methine and 7 quaternary carbons. There were figurative signals as one ketone carbon at δC 202.6; 6 oxygenated methine and quaternary carbons at δC [66.6, 66.7, 68.7, 75.7, 76.2 and 82.9]; 5 methyl carbons at δC [17.1, 20.9, 23.8, 29.0 and 29.9]. In addition, methine carbon at δC 120.4 and quaternary carbon at δC 165.2 confirmed the presence of a C=C double bond (Table 4). A special available activity of 20-hydroxyecdysone was reducing moult cycle duration of the edible freshwater crab Travancoriana schirnerae (Raghavan and Ayanath, 2019). 3.2.3. Compound 3 Compound 3 was also received as white needleshaped crystals, its mp. was 164-166°C. It had red With the above analyzed spectral characteristics, it purple TCL spot and no luminescence under UV is possible to predict compound 2 belonging to the light. steroid group. The 1D-NMR spectral data of compound 2 were similar to those of 20Table 4. 1D-NMR spectral data of isolated compounds C-position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Compound 1 H 1.60, 0.95 1.75, 1.56 3.22 (1H, dd, 11.4, 4.2) 1 0.73 (1H, s) 1.52, 1.33 1.43, 1.29 1.54 1.89, 1.87 5.28 (1H, t, 3.6) 1.13, 1.06 1.95, 1.99 2.82 (1H, dd, 9.6, 4.2) 1.64, 1.18 1.38, 1.24 1.78, 1.55 0.99 (3H, s) 0.77 (3H, s) 0.91 (3H, s) 0.76 (3H, s) 1.13 (3H, s) 0.90 (3H, s) 0.93 (3H, s) 13 C 38.4 27.2 79.1 38.8 55.2 18.3 32.6 39.3 47.6 37.1 23.4 122.7 143.6 41.6 27.7 23.0 46.5 41.0 45.9 30.7 33.8 32.4 28.1 15.6 15.3 17.1 25.9 182.8 33.1 23.6 Compound 2 H 1.59, 1.29 3.56 (1H, s) 3.77 (1H, s) 1.57, 1.48 2.20 (1H, dd, 13.0, 30.0) 1 5.63 (1H, s) 3.01 (1H, s) 1.86,1.64 1.79, 1.52 2.01 (1H, d, 8.5), 1.72 1.62 (2H) 2.26 (1H, t, 8.5) 0.84 (3H, s) 0.76 (3H, s) 1.06 (3H, s) 3.12 (1H, d, 5.0) 1.50, 1.12 1.66, 1.24 1.08 (3H, s) 1.06 (3H, s) 13 C 36.6 66.7 66.6 31.5 50.1 202.6 120.4 165.2 33.1 37.6 20.0 30.3 46.8 82.9 30.8 20.2 48.7 23.8 17.1 75.7 20.9 76.2 26.1 41.4 68.7 29.9 29.0 Compound 3 13 H C 1.09, 1.82 37.2 1.39, 1.77 31.5 3.56-3.62 (1H, m) 71.1 1.27, 1.70 38.0 1.40 40.3 1.22, 1.74 29.7 5.15 (1H, brs) 117.5 139.6 1.65 49.5 34.2 1.48 21.6 1.99, 2.02 39.5 43.3 1.81 55.1 1.40, 1.52 23.0 1.25 28.5 1.25 55.9 0.81 (3H, s) 13.0 0.55 (3H, s) 12.1 2.04 40.8 1.03 (3H, d, 6.5) 21.4 5.17 (1H, dd, 15.0, 8.5) 138.2 5.03 (1H, dd, 15.0, 8.5) 129.5 1.55 51.3 1.55 31.9 0.85 (3H, d, 6.5) 21.1 0.82 (3H, s) 19.0 1.18, 1.42 25.4 0.80 (3H, t, 2.0) 12.2 1 Note: Compounds 1 and 3 were recorded in CDCl3; compound 2 was recorded in DMSO-d6. 76 Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 bioactivity towards human applications and studied chemical components for the first time. The molecular formula of compound 3 was speculated to be C29H48O (412 amu, six degrees of unsaturation) on the basic of ESI-HRMS m/z 395.3689 [M-H2O+H]+. The CAEt extract exhibited resistance to the test strain of B. subtilis, inhibited for LU-1 cell line. CAHe and CAMe extracts showed resistance to the test strain of B. subtilis. Other above-declared experiments were negative. Most of 1D-NMR spectral signals of compound 3 were similar to those of compound 2 (Table 4). However, compound 3 had two carbons (one methyl group) more than compound 2. There were only one oxygenated methine group at δC 71.1 and with it the disappearance of the carbonyl group (Table 4). Oleanolic acid, 20-hydroxyecdysone and bspinasterol were isolated for the first time from CAEt extract of Centrostachys aquatica. Spectral data of compound 3 were compared with those given in the literature (Ragasa and Lim, 2005), 2D-NMR spectra of 3 were also used to check the fit of the predicted structure. As a result, compound 3 was identified as b-spinasterol (Figure 2). The interesting result was ability to inhibit human lung cancer cell of CAEt extract. Therefore, it is necessary to further isolate and test the anticancer activity of the purified compounds from this extract in order to be used as a medicinal plant. This research is still going on; the next results will be published as soon as possible. b-Spinasterol was well evaluated its antiproliferative activity against human cancer cell lines HeLa and murine cancer cell line RAW 264.7 (Meneses-Sagrero et al., 2017). ACKNOWLEDGMENT This study is funded by the Can Tho University, project code T2021-110. The authors also thank Dang Minh Quan for plant identification. 4. CONCLUSIONS Extracts from the whole plant of Centrostachys aquatica collected in Can Tho city were tested Meneses-Sagrero, S. E., Navarro-Navarro, M., RuizBustos, E., Del-Toro-Sánchez, C. L., JiménezEstrada, M., & Robles-Zepeda, R. E. (2017). Antiproliferative activity of spinasterol isolated of Stegnosperma halimifolium (Benth, 1844). Saudi Pharmaceutical Journal, 25(8), 1137– 1143. https://doi.org/10.1016/j.jsps.2017.07.001 Paszel-Jaworska, A., Romaniuk, A., & Rybczynska, M. (2014). Molecular Mechanisms of Biological Activity of Oleanolic Acid - A Source of Inspiration for A New Drugs Design. Mini-Reviews in Organic Chemistry, 11(3), 330–342. https://doi.org/10.2174/1570193X1103140915111839 Ragasa, C. Y., & Lim, K. (2005). Sterols from Cucurbita maxima. Philippine Journal of Science, 134(2), 83–87. Raghavan, S. D. A., & Ayanath, A. (2019). Effect of 20OH ecdysone and methyl farnesoate on moulting in the freshwater crab Travancoriana schirnerae. Invertebrate Reproduction & Development, 1–10. https://doi.org/10.1080/07924259.2019.1653387 Shela, G., Ratiporn, H., Yong-Seo, P., Soon-Teck, J., Zofia, Z., Zenon, J., Elena, K., Simon, T., & Olga, M. B. (2004). Bioactive compounds and antioxidant potential in fresh and dried Jaffa sweeties, a new kind of citrus fruit. Journal of the Science of Food and Agriculture, 84, 1459–1463. https://doi.org/10.1002/jsfa.1800 Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., … & Boyd, M. R. REFERENCES Bich, T. T. N., & Kato-Noguchi, H. (2014). Isolation and identification of a phytotoxic substance from the emergent macrophyte Centrostachys aquatica. Botanical Studies, 55(59), 1–5. https://doi.org/10.1186/s40529-014-0059-1 Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. L.W.T. Food Science and Technology, 28(1), 25–30. https://doi.org/10.1016/S0023-6438(95)80008-5 Ho, P. H. (1999). “Vietnamese plants”- An illustrated flora of Vietnam. Tre Publisher (in Vietnamese). Kumar, G. P., Navyaa, K., Ramya, E. M., Venkataramana, M., Anand, T., & Anilakumar, K. R. (2013). DNA damage protecting and free radical scavenging properties of Terminalia arjuna bark in PC-12 cells and plasmid DNA. Free Radicals and Antioxidants, 3, 35–39. https://doi.org/10.1016/j.fra.2013.04.001 Likhitayawuid, K., & Angerhofer, C. K. (1993). Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Sephania evecta. Jounal of Natural Products, 56(1), 30–38. https://doi.org/10.1021/np50091a005 Mckane, L., & Kandel J. (1996). Microbiology – Essentials and Applications (2nd ed.). McGraw-Hill Companies. 77 Can Tho University Journal of Science Vol. 13, No. 3 (2021): 71-78 (1990). New colorimetric cytotoxicity assay for anticancer-drug screening. JNCI Journal of the National Cancer Institute, 82(13), 1107– 1112. https://doi.org/10.1093/jnci/82.13.1107 Vanden Berghe, D. A., & Vlietinck, A. J. (1991). Screening methods for antibacterial and antiviral agents from higher plants. In K. Hostettmann (ed.), Methods in Plant Biochemistry, Vol. 6, Assays for Bioactivity, Academic Press, London (pp. 47–69). Vokac, K., Budesnsky, M., Harmatha, J., & Kohoutova, J. (1998). Ecdysteroid constituents of the mushroom Tapinella panuoides. Phytochemistry, 49(7), 2109– 2114. https://doi.org/10.1016/S0031-9422(98)00448-8 Zuhal, G., Hilal, O., Ay，se K., Cavit K., & Omur, L. D. (2009). Secondary metabolites from Nepeta heliotropifolia. Turk Journal of Chemistry, 33, 667–675. 78