ACYLATED ( + )-CATECHIN GLYCOSIDES FROM ULMUS PUMILA L . GROWING IN EGYPT

Extensive isolation work on the n-BuOH-soluble fraction obtained from the stem and root barks of Ulmus pumila L. afforded five compounds. Four were identified as (+)-catechin 1, (+)-catechin 7-O-gallate 2, (+)-catechin-5-O--D-apiofuranoside 3, and (+)-catechin-7-O--Dapiofuranoside 4. The structures of the remaining new compound was elucidated to be, (+)catechin-7-O-gallate-5-O-(5``-trans-caffeoyl)--D-apiofuranoside-3-O--D-apiofuranosyl(12)-glucopyranoside 5, by chemical and spectroscopic analysis. The ethanolic and n-butanol extracts and the isolated compounds (1-5) were tested for their antioxidant activity using DPPH radical scavenging assay, xanthine oxidase-induced generation of superoxide radical and lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat tissue homogenates.


INTRODUCTION
Ulmus pumila L. (Ulmaceae) is a deciduous tree, which is widely distributed in Korea.The barks of the stem and root of this plant have been used in oriental traditional medicine for the treatment of oedema, mastitis, gastric cancer, and inflammation (Lee and Korean 1996; College, 1977).Protecting neurons from glutamate-induced neurotoxicity (Dong, et al., 2006).The effect of the n-BuOH fraction of the methanol extract of this plant in inhibition of inflammatory diseases may be partially associated with the inhibition of NO synthesis and iNOS induction in murine macrophages (Chiranjit et al., 2012).Phytochemical studies of this species have resulted in the isolation of various sesquiterpenes (Chen, et al., 1972;Kim, et al., 1996), tritrpenes, (Dong, et al., 2006;Kim, et al., 1992;Moon, et al., 1995) and flavonoids (Son, et al., 1989), but there have been no previous phytochemical reports on the n-BuOH fraction of this plant.In this paper, I describe chemical components of the n-BuOH fraction of the methanol extract that led to the isolation and characterization of a series of (+)-catechin glycoside derivatives.
Compound 5 gave a molecular ion (FAB-MS, positive ion mode) at m/z 1031 [M + H] + , which together with the 1 H-and 13 C-NMR spectral data was consistent with the empirical formula C 47 H 50 O 26 .Bands for hydroxyl and chelated carbonyl functional groups were suggested by IR, and the UV absorptions at 282 nm were typical for a flavan-3-ol (Mabry, et al., 1970).Acid hydrolysis of 5 furnished 1, caffeic acid, gallic acid and monosaccharide components, identified as glucose and apiose by co-TLC with authentic samples.
13 C-NMR spectrum of 5 showed 47 carbon signals, of which 22 were similar to those of (+)-catechin-7-O-gallate 4 (11,12) and the remaining according to DEPT NMR analysis indicated 5 sp 3 methylenes, 5 sp 2 methines, 9 sp 3 methines, 4 sp 2 quaternary carbons and 2 sp 3 quaternary carbons atoms attributed to one glucosyl, two apiosyl and one caffeoyl moieties.Glycosidic substitutions for C-3 and C-5 were suggested by downfield shifts in the 13 C-NMR spectrum for C-3 ( 73.75) and C-5 ( 159.45) relative to the corresponding signals of 1.The disaccharide chain contained one -glucose and one -apiose units at C-3, and the interglycosidic linkage was established at C-2 of the glucose unit on the basis of the pronounced downfield shifts exhibited by this carbon resonance ( 79.13) and of H-2 of the glucose ( 4.53, d, J =9.7, 7.7 Hz) when compared to the respective shift in unglycosylated models (Hosny and Rosazza, 1998).Analysis of cross correlations by HMBC and ROESY established the interglycosidic connectivities, showing long-range correlations between H-1 ( 5.06) of the -D-glucose unit and C-3 of the aglycon ( 73.75), H-1 of -D-apiose unit ( 5.32) and C-5 ( 159.45) of aglycon; C-2 ( 79.13) of -D-glucose unit and H-1 ( 5.21) of the -D-apiose unit.The observed ROESY interaction between H-1 of -D-glucose and H-2, H-3 and H 2 -4 of the aglycon; and between H-1 and H-2 of the glucose unit and H-1 of -D-apiose unit and the absence of any glycoylation shifts for their carbon resonances indicated the attachment of the terminal sugar moiety to the aglycon.
All the tested samples had significant scavenging effects on the DPPH radical.The results recorded in (Table 2) indicated that compound 5 showed the highest activity among all the tested samples (84.8%), followed by the n-butanol fraction (70.3%) and the ethanolic extract (67.2%), and they were higher than α-tocopherol and BHT (66.5 and 55.3%, respectively).The results obtained for the n-butanol fractions and compound 5 indicated that the flavonoidal content, ester moieties and sugar units are responsible for the scavenging effects, and this could be attributed to their hydrogen donating activity.In general, the phenolic OH is considered a scavenger of free radicals, and it consequently exhibits antioxidative activity (Hosny, et al., 2002).b-Xanthine oxidase-induced generation of superoxide radical (Gongora, et al., 2003).
Phenolic compounds are known to inhibit generation of the superoxide anion radical (O 2•− ) in the hypoxanthine-xanthine oxidase system.The radical scavenging action of these phenolic compounds is through the formation of stable free radicals, which contribute to the inhibitory effects on lipid peroxidation and participate in the inhibition of (O 2•− ) generation.Since the n-butanol fractions is rich in (+)-catechin derivatives which were reported to have activities regarding inhibition of xanthine oxidase enzyme (Gongora, et al., 2003).Therefore, these fractions could be effective as natural antioxidants, through their double ability to inhibit xanthine oxidase activity and superoxide anion production, and could be considered an effective strategy in the treatment of inflammation.Uric acid production for controls was (61.0 ± 1.9 nmol/min).All tested samples and positive controls were tested at 100 μg/mL.Values are presented as mean ± SE of 3-test sample observation.P < 0.05 for all values.c-FeSO 4 /H 2 O 2 -stimulated lipid peroxidation in rat tissue homogenate (Hino, et al., 1998;Hosny and Rosazza, 2002).
For rat tissue homogenate (brain, heart and liver), the unstimulated control experiments i.e. the amount of thiobarbituric reactive substance (TBARS) formed in rat tissue homogenate (brain, heart and liver) were (0.44 ± 0.05, 0.25 ± 0.03 and 0.19 ± 0.02 nmol malondialdehyde, MDA/mg protein, respectively).After induction with FeSO 4 /H 2 O 2 , the amount of TBARS increased to 0.90 ± 2.55, 0.60 ± 2.16 and 0.52 ± 1.25 nmol malondialdehyde, MDA/mg protein, for brain, heart and liver, respectively.The tested samples significantly reduced malondialdehyde (MDA) formation in the presence of FeSO 4 -H 2 O 2 in tissue homogenates indicating anti-lipid peroxidation activities.The inhibition percentages were in the range of (55.10-80.78%),(48.75-71.50%)and (32.75-78.25%) in liver, heart and brain, rat tissue homogenates, respectively as recorded in (Table 3).It was interesting to note that the inhibition effects produced by the tested samples were more pronounced for liver tissue homogenates than heart and brain tissue homogenates, which could be especially beneficial in treatment of liver disease in cases with oxidative stress due to elevated levels of TBARS (Hino, et al., 1998;Hosny and Rosazza, 2002).The n-butanol extract and compound 5 showed the highest inhibition activity against FeSO 4 /H 2 O 2 -stimulated lipid peroxidation in liver rat tissue homogenate (75.60 and 80.78%), respectively, which was higher than both reference standards.Since DL-α-tocopherol is thought to be associated with lipid-rich membranes; its anti-oxidative ability is highly effective in protecting membranes against lipid peroxidation, as peroxyl and alkoxyl radicals.The data obtained from the present study indicates that the tested extracts have an anti-lipid peroxidative character with similar reaction mechanisms to those of DL-α-tocopherol.

CONCLUSIONS
This study provides strong evidence for the antioxidant activities of the n-butanol fraction obtained from the stem and root barks of Ulmus pumila L. Planch (Ulmaceae).The results obtained for the n-butanol fraction and the isolated catechin derivatives (1-5), indicated that the flavonoid compounds in studied plant are responsible for the scavenging effects through several mechanisms like hydrogen donating activity, inhibition of xanthine oxidase activity and superoxide anion production, as well as protection of membranes against lipid peroxidation.This could be potentially useful for the treatment of major free radicalinduced degenerative diseases including brain dysfunction, inflammation, and liver disorders.Further in-vivo studies could be done to support this point of view.

Experimental
General Procedures Optical rotations were measured with a JASCO P-1020 digital polarimeter.UV spectra were recorded using a Hitachi 340 spectrophotometer.IR absorbtion spectra (cm -1 ) were obtained with a Nicolet 205 FT-IR spectrometer connected to a Hewlett-Packard ColorPro plotter.HR-FAB-MS were recorded on a VG-ZAB-HF reversed geometry (BE configuration, where B is a magnetic sector and E is an electrostatic analyzer) mass spectrometer (MS) (VG Analytical, Inc.). 1 H-and 13 C-NMR spectra were obtained with a Bruker NMR 400 (Bruker Instruments, Billerica, MA), operating at 400 MHz for 1 H, and 100 MHz 13 C, respectively, in DMSO-d 6 using TMS as the internal standard.Chemical shifts are reported in parts per million on the  scale and coupling constants are in Hertz.Routine 1 H-and 13 C-NMR spectra DQF-COSY, ROESY, HMQC and HMBC-NMR experiments were carried out using a Bruker AMX-600 high-field spectrometer equipped with an IBM Aspect-2000 processor and with software VNMR version 4.1.Column chromatographies were performed with a high porosity polystyrene gel Diaion HP-20 (Mitsubishi Kasei Co., Ltd., Tokyo, Japan), flash silica gel (40 m) and Sepralyte C 18 (40 m) (J.T. Baker, Phillipsburg, NJ, USA), Sephadex Pharmacia Fine Chemical Co Ltd.).TLC was carried out on precoated silica gel 60 F 254 plates (0.2 mm thick, Merck) with solvent systems: A CHCl 3 -MeOH-H 2 O (80: 20: 2, v/v/v), B EtOAc-MeOH-H 2 O (100: 16.5: 13.5, v/v/v), C CHCl 3 -MeOH-H 2 O (61: 32 :7 v/v/v).Developed chromatograms were visualized by fluorescence quenching under 245-nm UV light and by spraying with 1% vanillin/H 2 SO 4 , followed by heating at 100 o C for 5 min.

Plant Material
The stem and root barks of U. pumila L. var.Japonica were collected from Al-Orman garden, Giza in May 2010, and identified by Dr. Mohammed El-Gibally, plant Taxonomist, Consultant of Egyptian Flora, Al-Orman garden, Giza, Egypt.

Animals
Male Wistar rats (250-300 g) were handled according to international regulations.They were allowed to take standard laboratory diet and water ad libitum, and the animals were maintained at 24 °C with 12 h light period.

DPPH radical scavenging activity
The ability of the extracts to scavenge free radicals was determined according to the method of Hosny, et al 2002 (19) .In a 96-well plate, 10 μL of each sample or standard dissolved in ethanol (100 μg/mL) was added to 190 μL of 316 μM/mL DPPH solution.A blank was prepared using ethanol.After incubation at 30 °C for 30 min, the absorbance of each solution was measured at 517 nm.DL-α-tocopherol and BHT were used as positive controls.The scavenging activity of the samples was calculated as a percentage of free radical inhibition according to the formula: % inhibition = A blank -A sample A blank × 100 where A blank is the absorbance of the blank at zero time and Asample is the absorbance of the sample after 30 min.All experiments were carried out in triplicate.

Table 2 .
Effects of the tested samples on the in vitro free radical generation

Table 3 .
Inhibition effects of the tested samples on FeSO 4 /H 2 O 2 -stimulated lipid peroxidation (MDA production) in rat tissue homogenates in vitro Values are presented as mean ± SE of 3-test sample observations, P < 0.05 for all values;