Bioactive Compounds from the Leaves of Lumnitzera Racemosa

Home , Combretaceae, Lumnitzera, Lumnitzera racemosa

Ἃ₍◊✲沙漠研究 2626-3,-3, 183-186179 - 182 (2016) (2016) Journal of Arid Land Studies Journalhttp://dx.doi.org/10.14976/jals.26.3_183 of Arid Land Studies － DT12 Refereed Paper － http://dx.doi.org/10.14976/jals.26.3_179 㸫DT12 Refereed Paper㸫

Bioactive Compounds from the Leaves of Lumnitzera racemosa against Acetaminophen-induced Liver damage in vitro Ahmed Gomaa Gomaa DARWISH 1,2), Mamdouh Nabil SAMY 3) Sachiko SUGIMOTO 2), Hideaki OTSUKA 2,4), Hosni ABDEL-SALAM 1), Mohammady Ibrahim ISSA 1) Emad Sabry SHAKER 1) and Katsuyoshi MATSUNAMI*2)

Abstract: Phytochemical investigation of the mangrove plant Lumnitzera racemosa WILLD (Fam: Combretaceae) has resulted in the isolation of 8 compounds by various chromatographic techniques (silica gel, ODS column chromatography and HPLC). The structures of these compounds were determined by spectrometric analysis (UV, IR, HR-ESI-MS, 1D and 2D-NMR). All the isolated compounds were evaluated for their hepatoprotective activity. The Compound number 8 showed high hepatoprotective activity against acetoaminophen and compound 1 showed moderate activity compared to glycyrrhizin as the positive control using human Hep G2 cell line. The Compounds number 2, 3, 4, 5, 6 and 8 showed the highest DPPH radical scavenging activity (IC50: 14.74±5.66, 18.88±3.56, 5.93±2.1, 7.17±0.42, 7.38±1.03 and 15.72±1.18 µM, respectively), while compounds 1 and 7 showed lowest DPPH radical scavenging activity comparable with the standard trolox (IC50: 5.93±2.19 ȝM).

Key Words: Combretaceae; hepatoprotective, Lumnitzera racemosa, 1, 1-diphenyl-2-picrylhydrazyl (DPPH).

system to human liver toxicosis could be an effective way to 1. Introduction identify therapeutically applicable agents, using most widely drugs in the world APAP. Liver diseases are considered to be one of the most Lumnitzera racemosa Willd (Fam: Combretaceae) is a shrub or dangerous health problems around World. The incidence of a small tree found on the coast of Japan, India and on the liver cancer, including hepatocellular carcinoma, has been Andaman and Nicobar Islands. The reddish brown bark increased seriously in many countries (Ryder 2007). Major contains 15-19% tannins, while the leaves and wood contain etiological factors contributing to hepatocellular carcinoma are smaller quantities. A fluid obtained from incisions made in well established and include viral infections, excessive ethanol the stem was reported to be useful for the treatment of herbs consumption, environmental carcinogens, and hemochromatosis. and itches (The Wealth of India 1962). Antihypertensive Specifically, acetaminophen (APAP) is a well-known analgesic activity has been recently reported for the aqueous acetone agent in many European countries (Kaufman 2002, Blazer extract of the plant (Lin 1993). Marine halophytes, such as 2009, Wilcox 2005) and one of the most known drugs in the mangroves and related species, are known to have many and world. The data of the overdose of APAP showed its ability various metabolites possessing antibacterial, antifungal to increase the incidence of liver failure (Davidson 1966). (Abeysinghe 2006, Ravikumar 2009, Ravikumar 2010), Exposure of the liver to the free radicals derived from some antiviral (Zandi 2008), antidiarrheal (Rouf 2007), xenobiotics and drugs leads to oxidative stress, which is hepatoprotective (Ravikumar 2011), anti- feedant (Wu 2008), recognized to be an important factor responsible for liver injury insecticidal (Calderon 2001), cytotoxic (Han 2007), and or be involved in the pathogenesis of liver disorders antiplasmodial activities (Kim 1997). Chemical examination of (Bandaranayake 2002, Ashok 2001). Therefore, studies on this plant occurring in various parts of the world was reported scavenging free radicals or reactive oxygen species (ROS) as to give a large number of compounds, long chain rubber like well as reducing oxidative stress, and thereby avoiding polyisoprenoid alcohols in leaves (Skoczylas 1994), flavonoids hepatotoxicity, have received much attention. Biochemical and long chain fatty acids and low molecular weight studies suggested that toxic doses of APAP could cause carbohydrates (Popp 1984). Chemical examination of the changes in the morphology and function of liver mitochondrial Indian species was reported to give friedelin, ȕ-amyrin, (Placke 1987, Meyers 1988). APAP binds more frequently to taraxerol, betulin, ȕ-sitosterol and triacontanol (Majumdar mitochondrial proteins (Tirmenstein 1989), and causes 1980). The presence of trace elements was also reported mitochondrial oxidative stress (Jaeschke 1990). In the (Bhosale 1979). The present study deals with investigation of bioassay directed searching for hepatoprotective agents from the chemical constituents of L. racemosa plant collected from the natural sources, employing the closely relevant model marine regions in Japan, in addition to evaluation of * Corresponding Author: [email protected] 㸦Received, July 22nd, 2016; Accepted, November 29th, 2016㸧 1-2-3, Kasumi, Minami-Ku, Hiroshima 734-8553, Japan Tel:+ 81-82-257-5335 Fax: +81-82-257-5335 1) Department of Biochemistry, Faculty of Agriculture, Minia University 2) Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University 3) Department of Pharmacognosy, Faculty of Pharmacy, Minia University 4) Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women’s University 184 hepatoprotective and DPPH radical scavenging activities of the 16 fractions. All fractions were purified by RPCC and the isolated compounds. similar fractions have been combined. All fractions were purified by HPLC using different concentrations of MeOH affording one 2. Materials and Methods new compound (U-E-5-3-1) and seven known compounds. 3,4-dihydroxybenzoic acid, (U-E-10-1-1) (67.4 mg) and 2.1. General experiments procedures 3,4,5-trihydroxybenzoic acid methyl ester (U-E-10-1-2) (31.5 Optical rotation data were measured on JASCO P-1030 mg). Lyoniresinol (U-E-10-2-2) (5 mg), Loliolide (U-E-5-1-3), Polarimeter. IR and UV spectra were performed using a Sophoretin (U-E-13-5-4), Quercetin-3-O-(2''-O-galloyl)-Į- Horiba FT-710 Fourier transform infrared and a JASCO V-520 rhamnopyranoside (U-E-13-5-3) and Myricetin 3-O- UV/Vis spectrophotometers, respectively. 1H and 13C NMR (2''-O-galloyl)-Į-rhamnopyranoside (U-E-13-5-2). spectra were recorded on a JEOL JNM Į-400 spectrometer with tetramethyl silane as an internal standard. HR-ESI mass 3. Biological Assay spectrum was taken on a LTQ Orbitrap XL mass spectrometer. Silica gel column chromatography (CC) was performed on 3.1. Cell culture Silica gel 60 [(E. Merck, Darmstardt, Germany), 70-230 mesh]. Human liver hepatoma cells (HepG2) (RIKEN Cell Bank: Reversed-phase [octadecylsilanized silica gel (ODS)] open CC RCB1886), human cell line derived from hepatocyte (RPCC) was performed on Cosmosil 75C18-OPN (Nacalai carcinoma or hepatoblastoma of 15 years old male Caucasian, Tesque,Kyoto,Japan) (Ɏ=3 cm, , L=40 cm, 10 g fractions were cultured in RPMI1640 (R8758, SIGMA) containing being collected). High-performance liquid chromatography 10%FCS, and antibiotics cocktail. Amphotericin B (SIGMA (HPLC) was performed on an ODS column Inertsil ODS-3; A9528 100 mg) and Kanamycin sulfate 5 g (WAKO GL Science,Tokyo,Japan (Ɏ=6 mm, L=25 cm, flow rate:1.0 119-00703) are dissolved in 8.93 and 25 ml of sterilized MilliQ ml/min), using a refractive index and/or a UV detector. water, respectively. Equal volume of these solution, Precoated silica gel 60 F254 plates (E. Merck; 0.25 mm in amphotericin B solution (8.93 ml) and kanamycin sulfate thickness) were used for TLC analysis, visualized by spraying solution (8.93 ml) was combined, dispensed into microtube with a 10% H2SO4 solution in EtOH and heating to around with 500 ȝl and stored at -20 °C. 150°C on a hotplate. Human cell line (Hep G2) derived from MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium hepatocyte carcinoma or hepatoblastoma was obtained from 15 bromide) (23547-21 1 g, Nacalai tesque) was dissolved in years old male Caucasian (RIKEN Cell Bank: RCB1886) was 181.8 ml of sterilized MilliQ water (5.5 mg/ml), and then used. dispensed into microtube with 1ml of solution and store at -20°C. 2.2. Extraction and isolation The air dried and powdered leaves of L. racemosa were 3.2. Hepatoprotective activity assay extracted with 100% methanol till exhaustion and then After 3 days subculture, 100 ȝL of diluted cell suspension concentrated under reduced pressure to yield a viscous gummy were added to the wells of 96-well microtiter plates (3×104 material. This residue was dissolved in water and defatted cells/ well), then cultured in a 5% CO2 incubator at 37 °C for with n-hexane to yield hexane layer (58.8 g). The aqueous 24 h. The supernatant was aspirated off and then 100 ȝL of layer was evaporated to remove a trace amount of organic test compounds dissolved in DMSO in the presence of APAP solvent and then extracted with EtOAc and 1-BuOH. The (20 mM) were added to the each well of the 96-well microtiter EtOAc and 1-BuOH fractions were concentrated under plate at the final concentration of 1%, and then incubated at reduced pressure to give 139.07 g and 89.9 g of residues, 37°C for 36 h. After that a MTT solution was added to each respectively. The remaining aqueous layer was concentrated well and the plates were incubated for a further 1.5 h. Then to furnish a water- soluble fraction. the formazan precipitates were dissolved in 100 µL of DMSO. The EtOAc fraction (139.07 g) was fractionated by CC on a The absorbance was measured using a Molecular Device highly porous synthetic resin, Diaion HP-20 () = 60 mm, L = Versmax tunable microplate reader at 570 nm. Glycyrrhizin 70 cm). The column was eluted initially with 100% MeOH (6 was used as a positive control. L) then 100% Acetone (6 L). The MeOH fraction (94.30 g) %Viability was subjected to silica gel CC (2.8 kg), (Ɏ=80, L=96 cm). The 㸻 (Asample ̽ Abackground) / (ADMSO ̽ Abackground) 100 column was eluted initially with CHCl3, then with The concentration of vehicle (DMSO) in each well is adjusted

CHCl3-MeOH gradient mixture, 500 ml fractions being to contain 1%. collected. The similar fractions have been combined, affording 185

HO 4. Results and Discussion

HO COOH From the EtOAc fraction of a MeOH extract of the leaves of L. racemosa, eight compounds were isolated. One new U-E-10-1-1 compound (new cyclic compound) 1 and the structures of Chemical structure of Compound 3, 4-dihydroxybenzoic acid. known compounds were determined to be 3,4-dihydroxy benzoic acid, 3 (Ashok 2001), 3,4,5-trihydroxybenzoic acid methyl ester 2 (Kai-Jin 2007), Loliolide 7 (Xiudong 2011), Quercetin-3-O-(2''-O-galloyl)- Į-rhamnopyranoside 4, Myricetin 3-O-(2''-O-galloyl)-Į- rhamnopyranoside 5, Sophoretin 6, Lyoniresinol 8 (Guanggli 2012) (Fig. 1). The hepatoprotective activity and DPPH radical scavenging activity were evaluated in Figures 2-4.

Fig. 2. Hepatoprotective activity of L. rascemosa EtOAc compounds using HepG2 cell line induced by APAP. Viability percentages are expressed as mean values± S.D. of 3 experiments.

Fig. 3. DPPH radical scavenging activity of isolated compounds. Inhibition percentages are expressed as mean values± S.D. of 3 experiments.

Fig.1. Structures of the isolated compounds 1-8. Fig. 4. IC50 of different compounds against DPPH free radical. 186

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