Proceedings of The 9th Joint Conference on Chemistry ISBN 978-602-285-049-6

Phenolic Compounds from the of blossfeldiana()

Yenny Febriani Yuna,b,*, Lilis Siti Aisyaha,b, Tri Reksa Saputraa, Arif Rahman Hakima, Tati Herlina, Euis Julaehaa, Achmad Zainuddina, Unang Supratmana

Abstract

Kalanchoe blossfeldiana(Crassulaceae) is a belonging to the Kalanchoe. Various species of Kalanchoe have been widely used as raw material for traditional medicine and also as an ornamental plant. This study is a continuation searching for secondary metabolites from Kalanchoe Indonesia plants. Fresh K.blossfeldiana leaves 5.7 kg was extracted with methanolat room temperature to obtain a concentrated extract as much as 155.74 g. Concentrated methanol extract was dissolved in water and then partitioned successively within- hexane and ethylacetate. Ethylacetate extract (20 g) was separated by a combination of chromatography on silica and ODS and produced compound 1 (7.1 mg) in the form of a yellow solid, compound 2 (4 mg), and compound 3 (15.4mg) in the form of a white solid. The chemical structures of compounds 1, 2, and 3, were determined by UV spectroscopy, IR, MS, 1H-NMR, 13C-NMR spectroscopy and comparison of data obtained from the literature and identified as phenolic compounds, namely 3,3',4',5,7 pentahydroksi flavonorquercetin (1), epigallocatechingallate (2), and gallic acid (3).

Keywords:Kalanchoe blossfeldiana, phenolic, 3,3',4',5,7 pentahydroksi flavon, epigallocatechingallate, gallic acid a Natural Product Chemistry Group, Department of Chemistry, The Faculty of Mathematics and Natural Sciences, Padjadjaran University, Bandung-Sumedang Km21, Jatinangor,Sumedang 45363 bDepartment of Chemistry, The Faculty of Mathematics and Natural Sciences, Jenderal Achmad Yani University, Cimahi 40528

Corresponding author email addresses: * [email protected],**[email protected]

Introduction This study is a continuation searching for secondary metabolites from Kalanchoe Indonesia plants, specially Indonesia has high plant biodiversity and its Kalanchoe blossfeldiana(Crassulaceae). ecosystems (Walujo, 2011).Some of the plants mostly used by Indonesian society, especially as herbal Methodology medicines, are from the genus Kalanchoe.Usually they are used as ornamental plants, with the characteristics Materials of thick and watery leaves. These plants are very popular because of easy propagation,low water Plant needer, and the flower colours. from the point of The leaves of K. blossfeldiana were collected from ethnopharmacology Kalanchoe plants are used as Lembang, Western Bandung area, West Java, traditional medicines to cure headache, cough, chest Indonesia, and identified at Herbarium Bogoriense, pain, ulcer, and other skin deseases. They overcome Biology Research Centre of LIPI Lembaga Ilmu fever, fix the irregular menstruation, heal wound and Pengetahuan Indonesia or LIPI (The Indonesian boil, not only in Indonesia but also almost everywhere Institute of Sciences), Cibinong, Bogor, West Java, in the world (Quazi et al., 2011). Some researches Indonesia. reported that Kalanchoe plants contain bufadienolide (Supratman et al., 2000; Supratman et al., 2001), Chemical triterpenoid (Gaind and Gupta, 1972), and flavonoid (Muzitano et al., 2006; Okwu and Nnamdi, 2011), and The chemicals needed were both the various technical biological activities like antileismanial, antiinflamatory, solvents (redistilled), i.e.n-hexane, ethyl acetate, cytotoksic, and inhibiting tumor cell growth methanol, acetone, andpro-analysis (Supratman et al., 2001; Costa, 2006; Biswas et al., i.e.dichloromethane and chloroform. Silica GF254 was 2011). used in Thin Layer Chromatography (TLC), Silica-

Green Chemistry Section 4: Organic Chemistry,Yenny Febriani Yun, et al. P a g e | 359 This Proceedings©Chemistry Department, FSM, Diponegoro University 2015 Proceedings of ISBN 978-602-285-049-6 The 9th Joint Conference on Chemistry

G60(10-40m)with the surface area (500 m2/g) was 7.79 (1H, d, J = 1.95 Hz, H-2’), 7.79 (1H, d, J = 1.95 and used in vacuum liquid chromatography, andsilica G60 8,45Hz, H-6’); 13C-NMR (Acetone-D6, 125.76 MHz)  (70-230 and 230-400 mesh) were used in the open ppm: 164.9 (C-2), 136.7 (C-3), 176.5 (C-4), 164.9 (C-5), column chromatography, and the solution ofAlCl3 (10% 99.1 (C-6), 164.9 (C-7), 94.5 (C-8), 157.8 (C-9), 104.1 (C- in ethanol) as stain-display reagent. 10), 123.7 (C-1’), 115.7 (C-2’), 145.8 (C-3’), 148.3 (C-4’), 116.2 (C-5’), 121.5 (C-6’); ESIMS m/z [M-1]+301.43, Equipments [M]+302.43.

The equipments used were glass wares common in The data of UV showed that thecompound (1) has the organic chemistry laboratory, macerator,rotary absorbance at the region 휆maks 370.8 nm (band 1) and evaporator R-200 Buchi with vacuum pump Vac V-500 255.4 nm (band 2) which are the characteristics of Buchi and water heater B-490 Buchi, open flavonoid compound. Band 1 indicated the absorbance chromatography column with various sizes, UV lamp which was related to the resonance of sinamoil group VilbertLuomart (λ 254 nm dan λ 365 nm), that involved ring B.Band 2 indicated the absorbance spectrophotometer FTIR Spectrum One Perkin Elmer, which was related tothe resonance of benzoil group Spectrometer Nuclear Magnetic Resonance (NMR) that involved ring Aof flavonoid.The addition of AlCl3 JEOL JNM ECA-500 with TMS as internal standard, caused the shifting is from 370.8 to 340.0 nm,and from Preparative HPLC. 255.4 to 268.4.The batochromic shift took place, with the decrease of the intensity of band 1 which was Methods caused by the formation of chelatbetweenAlCl3 and the ring B. Extraction and Isolation The data of IR showed the absorbance of wide band at The 18 kg of fresh K. blossfeldianaleavewas grinded, 3400 cm-1, which showed the existence of hydroxyl extracted, and then concentrated.The 385.83 gof group (the spread of O-H), 2000-1750 cm-1(overtone methanol extract obtained was dissolved in water and aromatic), 1650 cm-1(the spread of C=O partitioned respectively using n-hexane and ethyl carbonil),1600-1400 cm-1 (the spread of C=C aromatic), acetate, yielding in n-hexane extract (32 g) and ethyl 1092 cm-1 (the spread of C-O ether) and 800-750 acetate extract (25 g). The ethyl acetate extract was cm-1(distributed benzene). fractioned using liquid vacuum chromatography, resulted in 7 combined fractions.The fraction The data of13C-NMR showed thatthe compound (1) has combination was performed through the thin layer 15 carbon signals, which consist of 1 carbonil at δc chromatography guiding under the UV lamp 254 nm 176.55 ppm, 12 aromatic carbon atom, which with stain-displaying reagent10% AlCl3in ethanol. Out appeared in the signal range δc 100 – 160 ppm, and 2 of the 7 combined fractions, the fraction 5 was further shielded aromatic carbon atom, which appeared at the fractioned.Fraction 5 was purified using column signals δc 94.48 ppm and 99.09 ppm, because they chromatography using silica G60, with the eluent n- were in close range with carbon group bonded by hexane:ethyl acetate (3:7), isocratially, which resulted hydroxyl group. in yellow solid of 95.5 mg.This solid was further fractioned using column chromatography using the The data of1H-NMR on the compound(1)showed it ODS silica, with the eluent methanol:water:acetone consisted of 5 protons which were bonded at Csp2 , (1:2:1), isocratically, which resulted in yellow solid which could be calculatedJ or coupling constant. This J isolate of 7.1 mg (1) andthewhiteamorphous solid value(1.95) showed that both H6 and H8 had meta isolate of 15.4mg (3). Fraction 3 was purified using position. The locations of H2’ dan H6’ were inmeta column chromatography using silica G60, with the positions, because they had the same J values,1.95, eluentn-hexane:ethyl acetate; CHCl3:MeOH while the H5’ had the position of ortho against H6’ , stepwise,which resulted fraction 3’. Fraction 3’ was because theJ value was 8.45 , which indicated the purified using preparative HPLC, acetonitrile:water ortho in aromatic system. to know the molecular (4:6) which resulted in white solid isolate of 4 mg (2). weight of compound (1), the measurement of MS was performed. The data of MS using the negative ion Results and Discussion method resulted the [m/z [M+H]-] of 301.04, which meant the real molecular weight was 302.04. The ethyl acetate was fractionedusing various chromatography methods, and resulted the From the data of UV, IR, NMR and MS, it could be compound (1), (2),and (3). The compound (1) in predicted that the compound (1) had the molecular theyellow solid (7.1 mg),UV (MeOH) maks: 255.4; formula C15H10O7.Knowing the predicted formula, the 370.8, 268.4; 340.0 nm; IR (KBr) maks: 3400 (OH), 2000- DBE value was determined 11, with 8 double bondings 1750 (overtone aromatic), 1650 (C=O ketone), 1600, and 3 cyclics. It was predicted that the structure of the 1500, 1400 (derivative of benzene), 1092 (C-O ether), compound (1)was the 3,3’,4’,5’7 pentahydroxyflavon 800-750 cm-1(distributed benzen) ; 1H-NMR (Acetone, or known as Quercetin. 500.0 MHz)  ppm:6.23 (1H, d, J = 1.95 Hz, H-6), 6.49 (1H, d, J = 1.95 Hz, H-8), 6.97 (1H, d, J = 8.45 Hz, H-5’),

360|P a g e Green Chemistry Section 4: Organic Chemistry, Yenny Febriani Yun, et al. This Proceedings©Chemistry Department, FSM, Diponegoro University 2015 Proceedings of The 9th Joint Conference on Chemistry ISBN 978-602-285-049-6

The compound (2) were identified by 1H-NMR, 13C- pentahydroxyflavonorquercetin (1), epigallocate NMR, and comparisson with literature values as chingallate(2), and gallic acid(3). epigallocatechin gallate (EGCG) (4 mg)(Peres et al., 2010; Eldahshan, 2011; Fu et al., 2011): the white solid, Acknowledgments 1 H-NMR (500 MHz, MeOH-d3) δ: 2.88 (eq. H, dd, J = 16.4, 3.5 Hz, H-4), 3.02 (ax. H, dd, J = 16.4, 4.4 Hz, H-4), We thank SimlitabmasDikti for funding this research in 5.05 (1H, d, J = 7.5 Hz, H-2), 5.56 (1H, m, H-3), 5.95 (1H, the program Desentralization Grant, scheme d, J = 1.8 Hz, H-6), 5.97 (1H, d, J = 1.8 Hz, H-8), 6.75 (2H, Competitive Grant 2013-1014.We also thank s, H-2’ & H-6’), 7.00 (2H, s, H-2’’ & H6’’), 13C-NMR (500 Herbarium Bogoriense, Biology Research Centre of MHz, MeOH-d3) δ: 27.11 (C-4), 69.97 (C-3), 79.23 (C-2), Lembaga Ilmu Pengetahuan Indonesia or LIPI (The 96.00 (C-6), 96.71 (C-8), 99.44 (C-10), 105.38 (C-2’ & C- Indonesian Institute of Sciences), Cibinong, Bogor, 6’), 110.26 (C-2” & C-6”), 121.46 (C-1”), 130.64 (C-1’), West Java, Indonesia, and Chemistry Research Centre 136.24 (C-4”), 139.98 (C-3’ & C-5’), 146.60 (C-3’’ & of LIPI Serpong, Tangerang for their support in C5”), 149.00 (C-5), 157.38 (C-9), 158.00 (C-7), 167.32 measuring the spectra NMR.The staff of Medical (C-7” & C=O). Pharmacokinetic Laboratory of Padjadjaran University, thank you for measuring the MS. The compound (3) was identified by UV,1H-NMR, 13C- NMR, and compared with literature values as gallic References acid (5 mg) (Binutu and Cordell,2000; Saxena et al., 2008; Liu et al., 2008; Eldahshan, 2011): thewhite Binutu, A., Cordell, G. A. (2000). Gallic Acid Derivatives 1 amorphous solid, λmax(MeOH) 271 nm, H-NMR (500 from Mezoneuron benthamianum Leaves. 13 MHz, MeOH-d3) δ: 7.05 (1 H, s,H-2, 6), C-NMR (500 PHARMACEUTICAL BIOLOGY, 38(4):284-286; MHz, MeOH-d3) δ: 122.12 (C-1), 110.40 (C-2 & C-6), 146.48 (C-3 & C-5), 139.66 (C-4), and 170.55 (C-7). Biswas, S. K. et al. (2011), Literature Review on Pharmacological Potentials of Kalanchoepinnata(Crassulaceae), African J. Pharmacy (1) and Pharmacology, 5(10): 1258-1262;

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