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EDITOR-IN-CHIEF DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA
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[email protected] PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China
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Natural Product Communications
Liquiritigenin Derivatives and Their Hepatotoprotective Activity
2010 Vol. 5 No. 8 1243 - 1246
Rashmi Gaur, Sunil Kumar, Priyanka Trivedi, Rajendra Singh Bhakuni*, Dnyaneshwar Umrao Bawankule, Anirban Pal and Karuna Shanker Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India
[email protected] Received: April 4th, 2010; Accepted: July 29th, 2010
Liquiritigenin (7,4’-dihydroxyflavanone), isolated from the roots of Glycyrrhiza glabra, was derivatized to liquiritigenin 7, 4’diacetate, liquiritigenin 4’-acetate, isoliquiritigenin, and liquiritigenin 7, 4’-dibenzoate. All these derivatives were evaluated for in vitro hepatoprotective activity against D-galactosamine–lipopolysaccharide(GalN/LPS) induced toxicity. In-vitro hepatotoxicity was manifested by a significant increase (P < 0.05) in liver toxicity biomarkers (SGPT, SGOT, ALKP, triglyceride, LPO, NO and LDH). The level of biomarkers in the treatment groups was significantly decreased ( P< 0.05) when compared with the GalN/LPS group. The results revealed that isoliquiritigenin exhibited better hepatoprotective activity than liquiritigenin and its derivatives. Key words: Glycyrrhiza glabra, liquiritigenin, isoliquiritigenin, in-vitro, hepatoprotective, rat.
Liquiritigenin (LTG), a flavonoid obtained from various kinds of licorice (Glycyrrhiza species; family Fabaceae), possesses hepatoprotective effects against cadmium-induced toxicity in rat-derived hepatocyte cell lines [1], and against acute injuries induced by acetaminophen [2]. Isoliquiritigenin (ISL), another constituent of the plant, is a chalcone that is a more potent hepatoprotective agent [3-5], which also has other biological activities [3,6]. In this study, we report the isolation of LTG and ISL from the roots of G. glabra. In addition, LTG was chemically transformed to ISL as well as to LTG 7,4’diacetate, LTG 4’-acetate, and LTG 7,4’-dibenzoate, all of which were evaluated for hepatoprotective effects against galactosamine-LPS induced toxicity in primary hepatocyte culture. Lipopolysaccharide causes endotoxemia, which occurs frequently in cases of liver failure [7,8] and is thought to play a role in the pathogenesis of liver diseases [9]. D-Galactosamine depletes UTP resulting in hepatocyte damage [10], and this model provides a practical tool for the evaluation of compounds that interfere with hepatic apoptosis, as well as chemical-induced acute hepatitis [11,12].
reaction mixture for 3 h, the diacetate was partially hydrolyzed to LTG 4’-acetate. In the third experiment, the monoacetate, upon further refluxing for 2 h, furnished a mixture of LTG and the active chalcone ISL. LTG was further benzoylated to LTG 7, 4’dibenzoate. This is the first report of the conversion of LTG to LTG 4’-acetate, ISL and dibenzoate. LTG, ISL and the diacetate were identified by comparison of their spectroscopic data with the reported literature [13,14]. The new monoacetate and dibenzoate derivatives were identified by comparison of their NMR and MS data with those of LTG [13] and reference literature [15]. Hepatoprotective evaluation of these compounds against GalN/LPS induced toxicity in primary murine hepatocytes culture revealed that ISL exhibited significant inhibition (P < 0.05) of the hepatotoxic markers (SGPT, SGOT, ALKP, triglyceride, LPO, NO, LDH) when compared with the GalN/LPS group. ISL was also found to be better than LTG and its derivatives, whereas LTG 7, 4’-dibenzoate exhibited significant hepatoprotective activity compared with other LTG derivatives (Table 1). Experimental
LTG and ISL were isolated from the roots of G. glabra. LTG upon acetylation gave LTG 7, 4’-diacetate at room temperature. In another experiment, by refluxing the
General: Column chromatography was performed on silica gel, 60-120 mesh. Buchi flash chromatography system was used for the purification of isolates.
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Gaur et al.
Table 1: Hepatoprotective activity of liquiritigenin and its derivatives using in-vitro rat hepatocytes bioassay model. Treatment Normal
SGPT (U/L)
SGOT (U/L)
ALKP (U/L)
Triglyceride (mg/dl)
LPO (µM/mL)
NO (µM/mL)
LDH (µM/mL)
39.6 ± 2.1
103.2 ± 7.8
49.2 ± 3.8
42.3 ± 4.1
9.0 ± 0.9
30.6 ± 3.3
61.8 ± 4.5
GalN/LPS
72.2 ± 4.7*
395.0 ± 33.4*
70.6 ± 4.6*
68.6 ± 7.9*
18.2 ± 1.3*
72.6 ± 5.3*
92.8 ± 4.1*
ISL
52.0 ± 4.2a
364.6 ± 26.4a
55.0 ± 2.2
51.0 ± 1.2a
12.5 ± 1.4a
50.2 ± 4.1a
71.8 ± 2.5a
55.5 ± 2.1
13.3 ± 2.0
a
51.6 ± 6.9
a
68.8 ± 2.6a
11.0 ± 0.8
a
55.8 ± 3.7
a
72.0 ± 2.1a
a
57.4 ± 4.2
a
68.2 ± 3.0a
LTG LTG 4’-acetate
60.4 ± 3.0 57.2 ± 5.1
365.0 ± 18.4 381.4 ± 22.4
56.0 ± 3.1
LTG 7,4’-diacetate
59.8 ± 3.7
354.4 ± 16.2
57.4 ± 4.0 59.4 ± 5.0
53.0 ± 1.6 54.8 ± 3.3
13.6 ± 2.0
LTG 7,4’-dibenzoate
56.6 ± 6.0
368.6 ± 10.9a
60.2 ± 2.4
51.2 ± 1.3a
13.4 ± 2.0
55.8 ± 3.0 a
67.2 ± 2.2a
Silymarin
51.4 ± 3.3a
339.6 ± 7.9a
53.4 ± 4.0
50.5 ± 2.2a
12.0 ± 0.8a
48.2 ± 3.5a
66.0 ± 1.0a
Results are presented as the means ± S.E.M. Statistical analysis was performed by analysis of variance (A NOVA) followed by tukey-kramer multiple comparison test using Instat-3 software. Differences with a P value < 0. 05 were considered significant. n= 05; *(Normal vs GalN); a (GalN vs Treatment )
Aluminum sheets precoated with silica gel 60 F254 were used as analytical TLC plates. The compounds were visualized by either exposure to I2 vapors or by spraying with vanillin-sulfuric acid- ethyl alcohol reagent, followed by heating at 110ºC for 15 mins. Solvents used were either of analytical grade or were pre-distilled. The purity of the compounds was determined by analytical HPLC using a Waters Symmetry C18 column (4.6 x 250 mm, 5 μm), a 10 μL sample injection volume, acetonitrile-water containing 1% acetic acid (70:30) as development solvent, a 1.0 mL/min flow rate, and UV detection at 254 nm. IR spectra were recorded on a Perkin Elmer 1719 FT-IR spectrophotometer. ESIMS spectra were recorded on a Perkin Elmer Turbo Mass/ Shimadzu LC-MS. NMR spectra were obtained in CDCl3 on a Bruker Avance, 300 MHz instrument using TMS as internal standard. Plant material: Glycyrrhiza glabra L. (Fabaceae) roots, collected from the research farm of the Regional Research Laboratory, Jammu, India were identified by Dr S.P. Jain and Dr S.C. Singh, Botany and Pharmacognosy Department, CIMAP, Lucknow where a voucher specimen (No 08801002) was deposited. Extraction and isolation: Dried roots powder (500 g) was extracted with ethanol (3 x 1000 mL, RT). The extract, after concentration, was fractionated with EtOAc (4 x 150 mL, 21.6 g). The EtOAc fraction (15 g) was column chromatographed (silica gel, 60-120 mesh 6.5 x 120 cm), eluting with CHCl3 (2 L), and CHCl3: MeOH 99:1 (2.5 L), 97:3 (3 L), and 94:6 (2 L). Similar CHCl3:MeOH (97:3) fractions, 250 mL of each, monitored by TLC provided LTG (320 mg from acetone-n-hexane). The mother liquor (1.2 g), through flash chromatography using CHCl3, with a flow rate of 2.5 mL/min and a 2 min per tube collection time, yielded ISL (55 mg), and LTG (122 mg). Derivatization of liquiritigenin: Acetylation of LTG (75 mg) with Ac2O-pyridine (0.5 mL each), at room temperature for 15 mins, followed by the usual workup,
and prep TLC (CHCl3-MeOH, 97:3), provided LTG 7,4’-diacetate in 81% yield as an amorphous powder. Refluxing the mixture for 3 h afforded an amorphous powder of LTG 4’-acetate in 53% yield. In a third experiment, refluxing the reaction mixture for a further 2 h, followed by workup/flash chromatography, provided ISL (11%) and LTG (23%). Benzoylation of LTG (75 mg) using PhCOCl/pyridine (0.5 mL each), at room temperature, afforded LTG 7, 4’-benzoate in 86% yield as light yellow crystals (CHCl3). The purity of the compounds was 95.9% (LTG 4’-acetate), 98.7% (ISL), 99.0% (LTG 7,4’-diacetate), 99.1% (LTG 7,4’-dibenzoate), and 99.9 (LTG), as determined by analytical HPLC with an UV detector (254 nm). Liquiritigenin 7, 4’-diacetate MP: 168-170ºC. 1 H NMR (CDCl3, 300 MHz) δ: 2.30, 2.31 (3H each, s, 2 x COCH3), 2.87 (1H, dd, J = 16.8, 3.0 Hz, H-3β), 3.04 (1H, dd, J = 17.1, 13.2 Hz, H-3α), 5.49 (1H, dd, J = 13.2, 3.0 Hz, H-2), 6.80 (2H, m, H-6 and H-8), 7.15 (2H, d, J = 8.4 Hz, H-3', H-5'), 7.47 (2H, d, J = 8.4 Hz, H-2', H-6'), 7.94 (1H, d, J = 8.1 Hz, H-5). 13 C NMR (CDCl3, 75 MHz) δ: 21.3 (2 x OCOCH3), 44.7 (C-3), 79.8 (C-2), 111.4 (C-8), 116.0 (C-6), 119.2 (C-4a), 122.3 (C-3', C-5'), 127.6 (C-2’, C-6'), 128.8 (C-5), 136.4 (C-1'), 151.4 (C-4’), 157.1 (C-7), 162.7 (C-8a), 168.6 (COCH3), 169.4 (COCH3), 190.7 (C-4). ESI-MS (positive): m/z 363 [M+Na]+, 341 [M+H]+ (negative) 339 [M-H]-(C19H16O6). Liquiritigenin 4’-acetate MP: 206-208ºC. 1 H NMR (CD3OD, 300 MHz) δ: 2.30 (3H, s, OCOCH3), 2.81 (1H, dd, J = 17.1, 3.0 Hz, H-3 β), 3.06 (1H, dd, J = 16.8, 12.9 Hz,H-3α), 5.54 (1H, dd, J = 12.9, 3.0 Hz, H-2), 6.41 (1H, d, J = 2.1 Hz, H-8), 6.54 (1H, dd, J = 8.7, 2.1 Hz, H-6), 7.17 (2H, dd, J = 6.9, 1.8 Hz, H-3', H-5'), 7.57 (2H, d, J = 8.7 Hz, H-2', H-6'), 7.76 (1H, d, J = 8.7 Hz, H-5). 13 C NMR (DMSO–d6, 75 MHz) δ: 21.6 (OCOCH3), 44.1 (C-3), 79.3 (C-2), 103.5 (C-8), 111.5(C-6), 114.5
Hepatoprotective activity of liquiritigenin derivatives
(C-4a), 122.7 (C-3',C-5'), 128.6 (C-2', C-6'), 129.2 (C-5), 137.4 (C-1'), 151.3 (C-4’), 163.8 (C-8a), 165.5 (C-7), 169.8 (COCH3), 190.3 (C-4). ESI-MS (positive): m/z 321 [M+Na]+, 299 [M+H]+, (negative) 297 [M-H]-(C17H14O5). Liquiritigenin 7, 4’-dibenzoate MP: 82-84ºC. 1 H NMR (CDCl3, 300 MHz) δ: 2.96 (1H, dd, J = 16.8, 2.7 Hz, H-3β), 3.14 (1H,dd, J = 16.8, 12.9 Hz,H-3α), 5.59 (1H, dd, J = 12.9, 2.7 Hz, H-2), 6.98 (1H, dd, J = 8.4, 1.8 Hz, H-6), 7.02 (1H, d, J = 1.8 Hz, H-8), 7.32 (2H, d, J = 8.4 Hz, H-3', H-5'), 7.56 (4H, m, m-protons of COBz), 7.63 (2H, d, J = 8.4 Hz, H-2', H-6'), 7.67 (2H, m, p-protons of COBz), 8.04 (1H, d, J = 8.4 Hz, H-5), 8.22 (4H, m, o-protons of COBz). ESI-MS(positive): m/z 487 [M+Na]+, 465 [M+H]+, (negative) 463 [M-H]- (C29H20O6). Hepatoprotective evaluation: The protocol using rats for the isolation of hepatocytes was approved by the Institutional Animal Ethics Committee (IAEC) followed by the Committee for the Purpose of Control, Supervision on Experiment on Animals (CPCSEA), Government of India. Hepatocytes were isolated from male Sprague Dawley rats using a collagenase perfusion technique that was a modification [16] of the method of Seglen [17], with minor variations. After the establishment of a monolayer, the culture was treated with GalN (10 µg/mL, Sigma, USA), LPS (1 µg/mL, Sigma, USA) and 100 µg/mL of all test compounds. The hepatocytes were incubated for 24 h after which the cell culture supernatant was collected for biochemical assays. Animals: Adult male Sprague Dawley rats, weighing 200-250 g, were used as hepatocyte donors. The rats were kept in polypropylene cages with sterile paddy husk as bedding material and wire mesh top. The animals were acclimatized to ambient temperature (22±2ºC) and humidity, and a natural light/ dark cycle. The animals had free access to standard pellet diet and drinking water and were raised in our facility. Primary cultures of rat hepatocytes: Briefly, rats were anesthetized with an i.p. injection of chloral hydrate (360 mg/kg). The peritoneal cavity was opened, cannulation of the portal vein was achieved and the
Natural Product Communications Vol. 5 (8) 2010 1245
inferior vena cava was severed. The liver was perfused first with an oxygenated solution (KCl 2.24 g/L, NaCl 7.5g/L, sodium phosphate monobasic 1.13 g/L, glucose 1.8 g/L, HEPES 2.3 g/L , EGTA 1.9 g/L ), pH 7.4 at 37ºC, followed by collagenase solution (KCl 2.24 g/L, NaCl 7.5 g/L, sodium phosphate monobasic 1.13 g/L, glucose 1.8 g/L, HEPES 2.3 g/L, collagenase-Himedia 0.3 g/L and Trypsin-Sigma, USA, 0.25%), pH 7.4 at 37ºC. Cells were suspended in Minimum Essential Medium(Sigma Aldrich, USA) supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL) and amphotericin B (0.25 μg/mL) containing 10% fetal calf serum (Hyclone), pH 7.4. Cell viability was greater than 90%, which was quantified by the Trypan blue dye exclusion method. Cells were seeded at a density of 1x106 cells/mL in collagen coated 12 well plates (NUNC) and incubated at 37ºC under a humidified environment of 5% CO2,/95% air (HERA cell 240). After incubation for 8-10 h, the cells were rinsed with MEM to remove unattached cells and debris. Then, the same media was added to the monolayer culture. After further incubation of 4 h at 37ºC, the monolayer culture was used for the different treatments. Lipid peroxidation: The cells harvested from the wells were washed with PBS. Malonaldehyde (MDA) concentration was quantified using a thiobarbituric acid method, which measures MDA reactive products. Briefly, 0.5 mL of the harvest was mixed with 0.5 mL of PBS and 0.5 mL of 25% trichloroacetic acid and centrifuged at 2000 rpm for 20 min. A 1 mL portion of protein free supernatant was mixed with 0.25 mL of 0.5% thiobarbituric acid and heated at 95ºC for 1 h. After cooling, the intensity of the pink color of the end fraction product was determined at 532 nm. The MDA concentration was calculated by regression analysis of the data obtained from the standard graph that was plotted using ODs from samples to which were added known concentrations of MDA (MP Bio, USA). Acknowledgements - The authors are grateful to the Director, CIMAP for providing necessary facilities for this work. Financial support from the Council of Scientific and Industrial Research (CSIR) Network Project “Biological and chemical transformation of plant compounds for production of value added products of therapeutic/aroma value (NWP-09)”, New Delhi, Govt. of India is duly acknowledged.
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Virucidal Activity and Chemical Composition of Essential Oils from Aromatic Plants of Central West Argentina Cybele C. García, Eliana G. Acosta, Ana C. Carro, María C. Fernández Belmonte, Renata Bomben, Claudia B. Duschatzky, Marina Perotti, Carola Schuff and Elsa B. Damonte
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Neolitsea sericea Essential Oil Attenuates LPS-induced Inflammation in RAW 264.7 Macrophages by Suppressing NF-κB and MAPK Activation Weon-Jong Yoon, Ji-Young Moon, Ji-Yong Kang, Gi-Ok Kim, Nam Ho Lee and Chang-Gu Hyun
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Qualitative Analysis of the Smoke-Stream of Different Kinds of Incense by SPME/GC-MS Antonietta Lombardozzi, Morela Strano, Manuela Cortese, Massimo Ricciutelli, Sauro Vittori and Filippo Maggi
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Essential Oil Composition and in vivo Volatiles Emission by Different Parts of Coleostephus myconis Capitula Guido Flamini, Pier Luigi Cioni, Simonetta Maccioni and Rosa Baldini
1321
Pesticide and Plasticizer Residues in Citrus Essential Oils from Different Countries Giuseppa Di Bella, Vincenzo Lo Turco, Rossana Rando, Gabriella Arena, Donatella Pollicino, Rosario Rocco Luppino and Giacomo Dugo Applying New Science for Old Medicines: Targeting Leukocyte-Endothelial Adhesions by Antiinflammatory Herbal Drugs Solomon Habtemariam
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Natural Product Communications 2010 Volume 5, Number 8 Contents Original Paper
Page
Phytochemical Investigation of Verbesina turbacensis Kunth: Trypanosome Cysteine Protease Inhibition by (–)-Bornyl Esters Ifedayo V. Ogungbe, Rebecca A. Crouch, William A. Haber and William N. Setzer
1161
Anti-herpetic Activities of Chemical Components from the Brazilian Red Alga Plocamium brasiliense Wilton José Ferreira, Rodrigo Amaro, Diana Negrão Cavalcanti, Claudia Moraes de Rezende, Viveca Antonia Giongo Galvão da Silva, Juliana Eymara Barbosa, Izabel Christina Nunes de Palmer Paixão and Valéria Laneuville Teixeira
1167
Chemical Constituents of the Soft Coral Sarcophyton infundibuliforme from the South China Sea Xue-Ping Sun, Chang-Yun Wang, Chang-Lun Shao, Liang Li, Xiu-Bao Li, Min Chen and Pei-Yuan Qian
1171
Metabolites from the Fungus Phoma sp. 7210, Associated with Aizoon canariense Jingqiu Dai, Hidayat Hussain, Siegfried Dräger, Barbara Schulz, Tibor Kurtán, Gennaro Pescitelli, Ulrich Flörke and Karsten Krohn
1175
Triterpenes from Protium hebetatum Resin Delcio Dias Marques, Ilmar Bernardo Graebner, Telma Leda Gomes de Lemos, Luciana Lucas Machado, Jõao Carlos Costa Assunção and Francisco José Queiroz Monte
1181
Cytotoxicity of 9,11-Dehydroergosterol Peroxide Isolated from Ganoderma lucidum and its Target-related Proteins Ya-Jun Cui, Shu-Hong Guan, Li-Xing Feng, Xiao-Yi Song, Chao Ma, Chun-Ru Cheng, Wen-Bo Wang, Wan-Ying Wu, Qing-Xi Yue, Xuan Liu and De-An Guo
1183
Polar Alkaloids from the Caribbean Marine Sponge Niphates digitalis Erik L. Regalado, Judith Mendiola, Abilio Laguna, Clara Nogueiras and Olivier P. Thomas
1187
A Short Stereoselective Synthesis of Racemic 2-Epicalvine Basem A. Moosa and Shaikh A. Ali
1191
Cytochrome P450 3A4 Inhibitory Activity Studies within the Lycorine series of Alkaloids James McNulty, Jerald J. Nair, Mohini Singh, Denis J. Crankshaw, Alison C. Holloway and Jaume Bastida
1195
Analysis of Amaryllidaceae Alkaloids from Zephyranthes robusta by GC-MS and Their Cholinesterase Activity Lucie Cahlíková, Andrea Kulhánková, Klára Urbanová, Irena Valterová, Kateřina Macáková and Jiří Kuneš
1201
Stereochemistry and NMR Data Assignment of Cyclopeptide Alkaloids from Zizyphus oxyphylla Muhammad Nisar, Waqar Ahmad Kaleem, Achyut Adhikari, Zulfiqar Ali, Nusrat Hussain, Inamullah Khan, Mughal Qayum and M. Iqbal Choudhary
1205
Geranylated Flavonols from Macaranga rhizinoides Mulyadi Tanjung, Didin Mujahidin, Euis H. Hakim, Ahmad Darmawan and Yana M. Syah
1209
A New Biflavonyloxymethane from Pongamia pinnata Anindita Ghosh, Suvra Mandal, Avijit Banerji and Julie Banerji
1213
Anti-inflammatory and Gastroprotective Properties of Hypericum richeri Oil Extracts Gordana Zdunić, Dejan Gođevac, Marina Milenković, Katarina Šavikin, Nebojša Menković and Silvana Petrović
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Production of Flavonoids in Organogenic Cultures of Alpinia zerumbet Cristiane P. Victório, Rosani do Carmo de O. Arruda, Celso Luiz S. Lage and Ricardo M. Kuster
1219
Phenolic Compounds in Leaves of Alchornea triplinervia: Anatomical Localization, Mutagenicity, and Antibacterial Activity Tamara R. Calvo, Diego Demarco, Fabio V. Santos, Helen P. Moraes, Taís M. Bauab, Eliana A. Varanda, Ilce M. S. Cólus and Wagner Vilegas
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Continued inside backcover
