Inhibition effects of the classical pathway complement of isolated compounds from Quercus glauca

Abstract

Species of the Quercus species is an evergreen broadleaf tree found not only in Korea but also in China, Taiwan, and Japan. Quercus species is the most commonly occurring plant among the 50 native species of the family Fagaceae in Korea, China, and Taiwan. Quercus species have been used for diarrhea, dysentery, dermatitis, and hemorrhagia in Korean folk medicine. The present study evaluated the anticomplement effect of constituents from Quercus species (Fagaceae) in classical pathway complement system. We have evaluated leaves of five species of the Quercus genus with regard to its anticomplement activity and have identified its active principles following activity-guided isolation. Bioactivity-guided fractionation of the 80% methanol extracts of the stem barks of Quercus glauca Thunberg has led to the isolation of galloyl derivatives, displaying high anticomplement activity. Four galloyl derivatives isolated from the leaves of Q. glauca, namely 6′-O-galloyl salidroside (1), methyl gallate (2), 1,2,3,6-tetragalloylglucose (3), and 1,2,6-trigalloylglucose (4). 1, 2, 3 and 4 showed inhibitory activity against complement system with 50% inhibitory concentrations (IC₅₀) values of 224 μM, 362.4 μM, 32.3 μM, and 138.3 μM. Among the compounds tested, 3 showed the most potent anticomplement activity (IC₅₀, 32.3 μM). This is the first report of the isolation and anticomplement activity from Q. glauca.

Introduction

The serum complement (C) system consists of at least 19 proteins, mostly in preactivated enzymatic forms, activated in a multi-step cascade reaction via either the classical or alternative pathway. The invertebrate complement system can be activated by a cascade mechanism of the classical pathway (CP), an alternative pathway (AP), or the mannan binding lectin/MBL-associated serine protease (MBL/MASP) pathway. The complement system is important against infectious agents and in the pathogenesis of autoimmune diseases, such as rheumatoid arthritis and lupus erythematosis. ¹ Plant products are generally considered to be less toxic with fewer side effects than synthetic products. Consequently, plant-derived materials have received increased attention as biochemical active agents in inflammatory diseases therapies. The study of such medicines might offer a natural key to unlock the medical pharmacy for the future. So, some herbal drugs are a good source of natural inflammatory agents. ² Quercus species have been used for diarrhea, dysentery, dermatitis, and hemorrhagia in Korean folk medicine. Specially Quercus salicina Blume, which is distributed through the southern parts of the Korean Peninsula and Japan, has been used for diuretic, anti-inflammatory, antiedemic and litholytic agent. ^3,4 Compounds such as stenophyllanin A,B,C, salidroside gallate, scyllo quercitol gallate, quinic acid gallate, stenophynin A,B, grandinin, acuttissimin A,B, guaiacyl glycerol are reported from Quercus salicina Blume. ^5-7 However, this species biological activity has not been investigated in detail. From this point of view, we have screened the local plants in Korea for their abilities to anticomplement activity in vitro. This work compared almost quantitatively the magnitude of the anticomplement activity actions of five species of the Quercus genus in South Korea and also located some source plants where potential anticomplement phytochemicals could be characterized. In this paper, isolation of the 80% methanol extracts from the leaves of Quercus salicina yielded four galloyl derivatives. The active galloyl derivative exhibited potent inhibitory activity against anticomplement activity. In the context of our natural product chemistry program dealing with the development of new potent anti-inflammation agents, in this paper we describe the anticomplement activity of compounds from this plant.

Material and methods

General

Thin-layer chromatography (TLC) was performed on precoated silica gel G and GP uniplates from Analtech and visualized with 254–nm UV light. Vacuum liquid chromatography was carried out on silica gel 60 (Scientific Adsorbents Incorporated [SAI], particle size 32–63 µm, pore size 60 Å). HPLC system, a Shimadzu SPD-M10A HPLC system with a photodiode array detector (Tokyo, Japan) equipped with a Midas autoinjector. ¹ H NMR and ¹³C NMR spectra were recorded on a Bruker DPX 500 at 500 MHz and 125 MHz; respectively. The chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane, and J–values are in Hz. Sheep red blood cell was obtained from the Agricultural Technology Center, Jangheung-gun, Jeolranamdo, South Korea. Normal human serum was collected from a healthy 23-year old man in city hospital (Dong Tan, Kyung-gi Do, South Korea). Rosmarinic acid, Hemolysin, gelatin, MgCl₂, CaCl₂, sodium barbital and barbituric acid were purchased from Sigma-Aldrich Co (St. Louis, Missouri, USA). Tiliroside was purchased from Santa Cruz Biotechnology (California, USA).

Plant materials and isolation of compounds

The plant materials were collected or provided by the Dr Jong-Jin Kim on Kon Kuk University (Seoul, South Korea). Sources of plant materials are given in Table 1 . Voucher specimens are deposited at the herbarium of Department of Applied Life science, Kon Kuk University (Seoul, South Korea). The botanical identification was made by one of the authors, Dr Jong-Jin Kim on Kon Kuk University (Seoul, South Korea). The indeciduous Quercus species studied were Quercus salicina Blume, Quercus acuta Thunberg, Quercus phillyraeoides A. Gray, Quercus glauca Thunberg, and Quercus myrsinaefolia Blume, which were collected from southern coast and southern island of Korea. The collection of shoots and leaves from this tree species was conducted from 10 May 2009 to 15 May 2009. In particular, those from uercus salicina Blume were collected from an experimental forest in the Warm-Temperate Research Center of the Korea Forest Research Institute on Jeju island. Those from other species were collected from an experimental forest in the Southern Forest Center throughout the southern coast of Korea. This experimental forest consisted of 30−40 year-old forest; for this study, we collected leaves and shoots from 2−3 year-old shoots. For anticomplement bioassay from 80% methanol extract, two sets (5 g each) of roughly ground, air-dried plant materials of every selected species were separately extracted twice with 80% methanol (50 mL for each extraction) by refluxing for 4 h on a sonication bath at 35°C. In vacuo evaporation of solvents from the filtered 80% methanol extracts gave residues, which were subjected to an immediate hydrophilization into dry powders with the yields given in Table 1. For the bioassay, the fractions were dissolved in dimethyl sulfoxide and further diluted with incubation buffer. These were again pooled by 80% methanol extracts, of which Q. glauca was the most active (Table 1). Therefore, active compound was isolated from Q. glauca. The air-dried leaves of Q. glauca (200 g) were extracted with 80% methanol by refluxing for 4 h (three times × 1 L) on a sonication bath at 35°C. The 80% methanol extract was filtered through a Buchner funnel using Whatman No. 1 filter paper. The combined methanol extract (13 g) was concentrated on Amberlite XAD-16 (Sigma) and eluted with methanol, converting the aqueous extract to a methanol fraction. The methanol fraction (8 g) was chromatographed on a vacuum liquid chromatography of silica gel (200 g). The column was eluted using a gradient of CHCl₃, MeOH, and H₂O to give four fractions (QG-1−4: 0.2 g, 2.2 g, 0.8 g, and 3.3g, respectively). Repeated column chromatography of QG-2 on silica gel (CHCl₃–MeOH, 8:1), Sephadex LH-20 (MeOH9), and ODS column (50% aqueous MeOH), followed by HPLC on RP-18 (50% aq. MeOH and 60% aq. CH₃CN) afforded 1 (19 mg) and 2 (13 mg). Repeated column chromatography of QG-4 on Sephadex LH-20 (MeOH and CHCl₃, 9: 1), silica gel (CHCl₃-MeOH, 5:1), and ODS column (40% aq. MeOH), followed by HPLC on RP-18 (40% aq. MeOH and 80% aq CH₃CN) furnished 3 (23 mg) and 4 (9 mg).

Table 1.

In vitro anticomplement activity of 80% methanol extracts of five Quercus species from South Korea

Species	Local name	Parts	Inhibition (%) a	Yield (%)	Voucher number
Quercus salicina Blume	Cham-Ga-Si-Na-Moo	Leaves	18.3 ± 1.3	11.2	KKU-2010-1
Quercus acuta Thunberg	Bul-Ga-Si-Na-Moo	Leaves	25.2 ± 1.2	13.1	KKU-2010-2
Quercus phillyraeoides A. Gray	Gol-Ga-Si-Na-Moo	Leaves	2.1 ± 0.02	12.3	KKU-2010-3
Quercus glauca Thunberg	Jong-Ga-Si-Na-Moo	Leaves	36.9 ± 1.0	11.3	KKU-2010-4
Quercus myrsinaefolia Blume	Ga-Si-Na-Moo	Leaves	14.2 ± 1.9	12.1	KKU-2010-5

^a A full response experiment was performed on 80% methanol ether extract that showed the percentage of anticomplement inhibition at 50 μg/mL.

Anticomplement assay

A hemolytic assay was used to determine the inhibition of the alternative (AP) and classical pathways (CP) of complement activation as described. ⁸ Assays were done using barbital buffer containing 145 mM NaC1, 4mM diethylbarbituric acid, 0.25 mM Ca²⁺, 0.83 mM Mg²⁺, 0.1% gelatin, and 0.02% NaN₃, pH 7.3 for the CP or 145 mM NaCI, 4 mM diethylbarbituric acid, 7 mM MgCI₂, 10 mM EGTA, 0.1% gelatin, and 0.02% NAN₃, pH 7.3 for the AP. Test samples contained twofold dilutions of isolated compounds containing final concentrations that ranged from 1 to 500 μM. Sensitized sheep erythrocytes (CP) or rabbit erythrocytes (AP) were incubated with the test sample for 15 min at 37°C. A complement dilution (SRBC complement), CP (Sigma) or human plasma collected in the presence of EDTA as an anticoagulant (AP), was then added and incubated for 30 (AP) or 60 (CP) min at 37°C. The reaction was stopped by adding barbital buffer containing 10 mM EDTA. Erythrocytes were removed by centrifugation at 1600 × g for 10 min and the released hemoglobin from complement-lysed erythrocytes was measured at 412 nm using a Shimazhu UV/VIS spectrophotometer. The influence on complement activity was evaluated by the effect of the test compound on CH₅₀ and AH₅₀ values that denote the amount of complement necessary to lyse 50% of the erythrocytes for CP and AP, respectively. Anticomplement activity was determined as a mean of triplicate measurements and expressed as the 50% inhibitory concentrations (IC₅₀) values from complement-dependent hemolysis of the control. Tiliroside and rosmarinic acid were used as positive Controls. ⁹

Results and discussion

The human complement system plays an important role in the host defense system against foreign invasive organisms, i.e. viruses, bacteria, and fungi, as well as an external wound. Its effects are normally beneficial to the host, but it can also cause adverse effects depending on the site, extent, and duration of complement activation. Activation of the system may lead to pathologic reactions in a variety of inflammatory and degenerative diseases such as multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, dermatological disease, rheumatoid arthritis, and gout. ¹⁰ Through these analyses, it was verified that for the Quercus species the 80% methanol extract was the most active. The results of in vitro anticomplement activity at each level of purification are presented in Tables 1 and 2. In an aim to identify the active substances, the extracts were submitted to column chromatography in silica gel using elution mixtures of several solvents of increasing polarity. Various chromatography of the aqueous methanol fraction of Q. glauca on Amberlite XAD-16, ODS, and Sephadex LH-20 led to the isolation of four galloyl derivatives (1-4). 6′-O-galloyl salidroside (1), ¹¹ methyl gallate (2), ¹¹ 1,2,3,6-tetragalloylglucose (3), ¹² and 1,2,6-Trigalloylglucose (4) ¹² complete spectral data set corresponded fully with literature values with the exception of optical rotation: Literature values for four compounds were levorotatory at various wavelengths in these same solvents (Figure 1). Inhibition of the classical complement pathway and activation of the alternative pathway of complement fixation was shown by compounds obtained from the leaves of Q. glauca. 1, 2, 3 and 4 showed inhibitory activity against complement system with 50% inhibitory concentrations (IC₅₀) values of 224 μM, 362.4 μM, 32.3 μM and 138.3 μM. 3 showed the strongest inhibition of the classical pathway at a concentration of 32.3 μM (Table 2). Further work will isolate higher concentrations of the 3 from Q. glauca and the molecular mechanism of the active substances will be determined. Since inhibition of the classical pathway and activation of the alternative pathway by different organic solvent extracts were exclusive events, it is possible that different compounds were responsible for this observed activity. Although the cellular and molecular mechanisms that underlie the anticomplement action of galloyl derivatives revealed in the present study should be studied further, these findings about galloyl derivatives from Q. glauca containing anticomplement activities may provide some ideas for the development of agents to ameliorate the cellular dysfunction due to inflammatory diseases.

Figure 1.

Chemical structures of compounds from the leaves of Quercus glauca.

Table 2.

Inhibition effects of the classical pathway complement from compounds

Compounds	IC50 (μM) a
6′-O-galloyl salidroside	224.2 ± 11.3
Methyl gallate	362.4 ± 42.8
1,2,3,6-tetragalloyglucose	32.3 ± 5.8
1,2,6-trigalloylglucose	138.3 ± 32.4
Rosmarinic acid b	174.4 ± 28.4
Tiliroside b	98.7 ± 16.5

^a Results are the mean (n = 5).

^b This compound was used as a positive control.