Inhibitory Effect on β-Hexosaminidase Release from RBL-2H3 Cells of Extracts and Some Pure Constituents of Benchalokawichian, a Thai Herbal Remedy, Used for Allergic Disorders

Introduction. Benchalokawichian (BCW), a Thai traditional herbal formulation, has long been used as antipyretic and to treat skin disorders. It comprises roots from five herbs: Ficus racemosa, Capparis micracantha, Clerodendrum petasites, Harrisonia perforata, and Tiliacora triandra. This polyherbal remedy has recently been included in the Thailand National List of Essential Medicines (Herbal Products list). Methodology. A Bioassay-guided fractionation technique was used to evaluate antiallergy activities of crude extracts, and those obtained by the multistep column chromatography isolation of pure compounds. Inhibitory effect on the release of β-hexosaminidase from RBL-2H3 cells was used to determine antiallergic activity. Results. Two pure compounds from BCW formulation showed higher antiallergic activity than crude or semipure extracts. Pectolinarigenin showed the highest antiallergic activity, followed by O-methylalloptaeroxylin, with IC50 values of 6.3 μg/mL and 14.16 μg/mL, respectively. Moreover, the highest activities of pure compounds were significantly higher than chlorpheniramine (16.2 μg/mL). Conclusions. This study provides some support for the use of BCW in reducing itching and treatment of other skin allergic disorders. The two isolated constituents exhibited high antiallergic activity and it is necessary to determine their mechanism of action. Further phytochemical and safety studies of pure compounds are required before development of these as antiallergy commercial remedies.


Introduction
Allergic diseases are manifested as hyperresponsiveness to allergenic environmental substances in the various target organs of the body (skin, nose, lung, gastrointestinal tract, etc.) and involve both IgE-mediated and non-IgE-mediated components [1,2]. Exposure to allergenic materials results in production, by B cells, of a multitude of antibodies, collectively called immunoglobulins (Ig) that are antigen-specific. The allergic reaction starts when immunoglobulin E binds to specific receptors (FcRI) on the surface of mast cells and basophils [3,4], which in turn induces degranulation of the cells and release of mediators such as histamine, leukotrienes, serotonin, and platelet activating factors [5][6][7]. Histamine is the main cause of many of the symptoms of allergies, such as runny nose, sneezing, and itching. Histamine also contributes to the progression of allergic-inflammatory responses by enhancement of the secretion of proinflammatory cytokines [8]. Although antihistamines are the first drugs of choice for treatment of many types of allergic disorders, they do have certain side effects. A large proportion (70-95%) of the world's population still relies on herbal medicines for primary health care [9]. Therefore, there is a continuous search for newer and better drugs for allergy treatment, including evaluation of traditional herbal remedies [10]. Since IgEs play an important role in the allergic reaction, it has been 2 Evidence-Based Complementary and Alternative Medicine suggested that the way to treat and prevent allergic diseases is to block the activity of IgE response [11][12][13].
Benchalokawichian (BCW) is a Thai traditional medicine formulation containing parts from roots of five plants in equal amounts: Ficus racemosa Linn. (Moraceae), Capparis micracantha DC. (Capparidaceae), Clerodendrum petasites S. Moore. (Lamiaceae), Harrisonia perforata Merr. (Simaroubaceae), and Tiliacora triandra Diels. (Menispermaceae). It has long been used for relief of fever and to treat skin rash. This formulation is included in the Thailand National List of Essential Medicines [14]. It has also been used for prevention of influenza H1N1 infections and in recent years this remedy has been used to treat acne, skin rashes, and other similar skin disorders.
Currently the antipyretic and anti-inflammatory activities of BCW have only been studied in vivo in rats [26,27]. The antimicrobial activity of ethanolic and water extracts of BCW has recently been reported [28,29]. There are no other in vitro studies on antioxidant, antiallergy, or anti-inflammatory activities on BCW. However, a recent study on fruits of H. perforata has demonstrated that organic extracts exhibited high antioxidant activity by the DPPH method but failed to show any cytotoxicity against human myelogenous leukemia (K562) and human cancer (SGC-7901), cell lines in vitro by the MTT method [30]. Another report has also described the antioxidant activity in extracts of fruits of H. perforata by the DPPH method [31]. The results of in vivo rats suggest that H. perforata bark aqueous extracts does not cause acute and subchronic toxicities [32].
Benchalokawichian remedy has yet not been thoroughly studied, either in vitro or in vivo. Its recent inclusion in the Thailand National List of Essential Medicines has encouraged us to carry out systematic phytochemical and bioactivity assessment of crude extracts. Singharachai et al. studied morphological characters including macroscopic, microscopic examination, and pharmacognostic parameters and investigated 3D-HPLC fingerprint profile [33]. We also report the use of bioactivity-guided isolation of semipure and some pure constituents from this polyherbal remedy. In this preliminary study we have concentrated on antiallergic activity and on limited number of pure compounds. It is hoped that furthermore detailed studies of this type, along with safety studies in animals, will provide data that may allow the investigation of its clinical efficacy in controlled clinical trials for some of the conditions it is currently used for in Thailand by Thai Traditional medicine (TTM) practitioners. . Calcium chloride dehydrate, magnesium chloride 6H 2 O, potassium chloride, and sodium carbonate were purchased from Merck (Darmstadt, Germany). Piperazine-N,N -bis(2-ethanesulfonic acid) (PIPES) was purchased from Amresco (OH, USA). Sodium chloride and sodium hydroxide (analytical grade) were purchased from Univar (Ajax Finechem, Australia). Sodium bicarbonate was purchased from BDH (Poole, U.K.). The solvents for analysis, hexane, chloroform, ethyl acetate, and methanol (analytical grades) were purchased from RCI Labscan (Bangkok, Thailand). Sterile water was obtained by purification using a Milli Q system from Millipore (Bedford, MA, USA). Silica gel 60 grade numbers 1.07734 and 1.09385 (70-230 mesh and 230-400 mesh) and TLC silica gel 60 F 254 were purchased from Merck (Darmstadt, Germany). Chromatographic column (4.5 × 54 cm, glass) was purchased from Becthai (Bangkok, Thailand). CO 2 humidified incubator was purchased from Shellab (OR, USA). Laminar air flow cabinet was purchased from Boss tech (Bangkok, Thailand). Microplate reader was purchased from BioTek (VT, USA).

Preparation of the Extracts.
The roots from each of these five plants were cleaned, cut in small pieces, and dried at 50 ∘ C 24 h. Each dried plant material was powdered using an electric grinder (40 mesh particle size). Five plant powders in the same ratio were mixed to provide the BCW remedy. BCW and each of the five plants were macerated with 95% ethanol, filtered with whatman number 1 and solvent removed using a rotary evaporator under reduced pressure (40 ∘ C) to obtain the dry ethanolic extracts. These extracts were further dried Evidence-Based Complementary and Alternative Medicine 3 to constant weight in a vacuum desiccator. All extracts were kept at −20 ∘ C until required for further use.

Preparation of Semipure Extracts and Isolation of
Pure Compounds 2.4.1. Vacuum Liquid Chromatography (VLC). Bioassayguided fractionation was used to isolate the pure compounds by the following modified method of Tewtrakul and Itharat [34]. The BCW ethanolic extract (50.46 g) was subjected to silica vacuum liquid chromatography (VLC), using five solvent systems of increasing polarity; hexane (1500 mL), hexane : chloroform (1 : 1, 2000 mL), chloroform (2500 mL), chloroform : methanol (1 : 1, 2500 mL), and methanol (2000 mL). The VLC column was packed with silica gel 60 (mesh 230-400), the crude extract was applied on top of the column, then they were eluded with these five different solvents. The solvent in each fraction (fractions 1-5) was removed by rotary evaporator (40 ∘ C), and the dry semipure extract further dried to constant weight.

Identification of Compounds 1 and 2. TLC (silica gel 60
GF 254 aluminium sheets, Merck) were used to demonstrate the purity of compounds, with three different solvent systems of varying polarity and detection with anisaldehyde reagent. The structures of the isolated compounds were determined by their NMR data [ 1 H and 13 C on a Varian Unity Inova 500 spectrometer (500 MHz for 1 H; 125 MHz for 13 C)], UV spectra [SPECORD S 100 (Analytikjena) spectrometer], and ESI mass spectra, both HRMS and LRMS, were obtained from a Agilent Technologies 1200 Binary LC System coupled to a Bruker microtof mass spectrometer.

Determination of Antiallergic
Activity. Inhibitory effects on the release of -hexosaminidase from Rat Basophilic Leukemia cell line (RBL-2H3) were evaluated by the following modified method [35]. RBL-2H3 cells were cultured in MEM medium supplemented with 15% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 g/mL). The cells were seeded in 24-wells plate (5 × 10 5 cells/mL) and incubated to adhere at 37 ∘ C in 5% CO 2 for 1.5 hour. RBL-2H3 cells were sensitized with anti-DNP IgE (antidinitrophenyl-immunoglobulin E) (0.45 g/mL), and incubated at 37 ∘ C in 5% CO  (F1-F5). Yields (% w/w) of 95% ethanolic crude extracts of BCW remedy and its constituent were low, in range of 2-4% (see in Table 1). The ethanolic extract of BCW (50.46 g) was subjected to vacuum liquid chromatography (VLC) to obtained five semipure extracts (F1-F5). Fraction 4 (chloroform : methanol elution) showed the highest yield, followed by fraction 3 and fraction 5, with yields being 65.40%, 13.20%, and 10.52%, respectively. The yields are shown in Table 2. All extracts and fractions were test inhibitory effects on release ofhexosaminidase. The fractions which showed highest antiallergy activity were further subjected to purification to isolate pure compounds.

Isolation and Identification of Isolated Pure Compounds 1 and 2.
The semipure extract from fraction F3 of VLC was purified using silica gel chromatogram (70-230 mesh) using solvents of increasing polarity for elution, with gravity feed of solvents. This step of purification resulted in the isolation of two pure compounds. All collected fractions were examined by TLC using anisaldehyde (in H 2 SO 4 ) as detection spray. Compound 1 appeared as a yellow spot on TLC plates on heating and compound 2 appeared as yellow spot with no heating required. Compounds 1 and 2 were further identified by 1 H and 13 C NMR, and the structure identity confirmed by mass spectroscopy; compound 1 was pectolinarigenin and compound 2 was O-methylalloptaeroxylin.

Inhibitory Effects on
Pectolinarigenin and O-methylalloptaeroxylin exhibited the highest antiallergic activity, with IC 50 values of 6.3 and 14.2 g/mL (20.1 and 51.8 M), respectively. These two compounds show higher inhibitory effect than chlorpheniramine, positive control (IC 50 = 16.2 g/mL, 58.8 M).

Discussion and Conclusions
This study has isolated for the first time two pure compounds from the ethanolic extract of BCW formulation and identified them as pectolinarigenin (compound 1) and Omethylalloptaeroxylin (compound 2), which have not presented on 3D-HPLC chromatogram in previous study [33]. Compound 2 has previously been isolated from branches of H. perforata [36,37]. In a recent study Choodej et al. [20] have investigated the effects of compound 2 on NO production in LPS-stimulated macrophages and showed it had potent activity, with IC 50 value of 66.41 ± 5.21 M. Although this structure was described more than ten years ago, research on inhibitory effect against release of -hexosaminidase in RBL-2H3 cell lines has not been described to date. This is, therefore, the first report on antiallergic effect of compound 2.
The study on TLC chromatogram of two compounds (Figure 3), we found that compound 1 (pectolinarigenin) is presented on H. perforata, whereas compound 2 (O-methylalloptaeroxylin) is presented on C. petasites in the same as previous reviews. Both of pure compounds show exhibited higher antiallergic activity than all fractions, BLW extract and positive control, represented in Figure 4. This preliminary study provides some supports for the use of BCW for treatment of allergic skin rash in Thai traditional medicine. This is the first report on the two isolated compounds from BCW ethanolic extract and their antiallergic activities. The ethanolic extract of BCW could be further developed into 6 Evidence-Based Complementary and Alternative Medicine  commercial formulations for the treatment of allergic dermal diseases, whereas the two pure compounds can serve as bioactive markers for the analysis and standardization of any new formulated products.