Enhancement of an In Vivo Anti-Inflammatory Activity of Oleanolic Acid through Glycosylation Occurring Naturally in Stauntonia hexaphylla

Stauntonia hexaphylla (Lardizabalaceae) has been used as a traditional herbal medicine in Korea and China for its anti-inflammatory and analgesic properties. As part of a bioprospecting program aimed at the discovery of new bioactive compounds from Korean medicinal plants, a phytochemical study of S. hexaphylla leaves was carried out leading to isolation of two oleanane-type triterpene saponins, 3-O-[β-d-glucopyranosyl (1→2)-α-l-arabinopyranosyl] oleanolic acid-28-O-[β-d-glucopyranosyl (1→6)-β-d-glucopyranosyl] ester (1) and 3-O-α-l-arabinopyranosyl oleanolic acid-28-O-[β-d-glucopyranosyl (1→6)-β-d-glucopyranosyl] ester (2). Their structures were established unambiguously by spectroscopic methods such as one- and two-dimensional nuclear magnetic resonance and infrared spectroscopies, high-resolution electrospray ionization mass spectrometry and chemical reactions. Their anti-inflammatory activities were examined for the first time with an animal model for the macrophage-mediated inflammatory response as well as a cell-based assay using an established macrophage cell line (RAW 264.7) in vitro. Together, it was concluded that the saponin constituents, when they were orally administered, exerted much more potent activities in vivo than their sapogenin core even though both the saponins and the sapogenin molecule inhibited the RAW 264.7 cell activation comparably well in vitro. These results imply that saponins from S. hexaphylla leaves have a definite advantage in the development of oral medications for the control of inflammatory responses.


Introduction
Immunity is the body's defense system against infectious agents or tumor formation. Proper magnitudes and durations of the responses are, however, critical for the health of the body as unnecessarily overactive or long-lasting immune responses can cause various immunological disorders compromising the quality of life. For example, while macrophages play a critical role in defense against bacterial infections, they can also cause inflammatory disorders when their responses persist for too long [1]. Thus, it is often necessary to suppress macrophage activation to prevent chronic inflammatory disorders. Medicinal herbs have been identified as rich sources of natural products with anti-inflammatory activities [2].
Diverse proinflammatory cytokines and mediators such as nitric oxide (NO), and tumor necrosis factor α (TNF-α) are mainly released by activated macrophages, which emerge in response to exogenous stress or endogenous signals such as external pathogen and damage-associated molecular patterns (DAMPs) [3]. These are key mediators to induce inflammatory disorders and may have potential as the therapeutic targets [4].
Stauntonia hexaphylla (Lardizabalaceae) has been used as a traditional herbal medicine in Korea, China and Japan for its analgesic and diuretic properties [5]. The genus Stauntonia produces various phytochemicals, such as oleanane triterpenoids [6], phenolic glycosides [7] and triterpene saponins [8], which exhibit diverse pharmacological effects including anti-HIV-1 protease [9], anti-gout [10,11], anti-diabetic [12] and anti-osteoporotic [13] effects. In the previous study, we had identified several triterpene glycosides that inhibited the activation of an established macrophage cell line called RAW 264.7 in vitro [14,15]. Previously, oleanane triterpene saponins have shown anti-inflammatory inhibitory effects, such as aster saponin C2 [16], and kalopanaxsaponin B [17]. In this study, we identified two major derivatives of oleanolic acid saponins and examined their pharmacological activities for inhibition of the activation of the RAW 264.7 cell line in vitro, relative to those of oleanolic acid aglycon ( Figure 1). In this study, the preliminary comparison of anti-inflammatory activity of these saponins with their aglycon was also discussed. Additionally, to investigate the in vivo efficacies of the saponins and the aglycon, we performed an animal experiment widely used for determining macrophage-dependent immune response, i.e., the lipopolysaccharide (LPS)-induced mouse sepsis model, administered orally with either the saponin or the aglycon beforehand [14,15,18].  with C-2 (δ C 81.5). The HMBC correlation between the anomeric Glc H-1 (δ H 6.22) and C-28 of the aglycon (δ C 178.4) confirmed that β-d-glucopyranosyl was at C-28. Additionally, the strong HMBC signals of Glc H-1 (δ H 5.15; d, J = 7.5 Hz) with C-6 (δ C 70.6) confirmed that the sugar linkages were 28-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside. Based on this evidence, the structure of compound 1 was identified as: [19].  [19]. To the best of our knowledge, this is the first time that compounds 1 and 2 have been reported for the Lardizabalaceae family.

Quantitative Analysis of Compounds 1, and 2 in the EtOH Extract of S. hexaphylla Leaves
Several studies have reported using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to measure the content of saponins in herbal medicines for better accuracy [20]. To quantify the contents of compounds 1 and 2 in S. hexaphylla, an accurate method was developed using a mass spectrometer equipped with an electrospray ionization (ESI) source.

Cytotoxic Properties of Compounds 1 and 2
Cytotoxicity is an undesirable property of immune modulators. To determine the cytotoxicity of compounds 1-2, and oleanolic acid aglycon, we treated RAW 264.7 cells with serially diluted concentrations of individual molecules for 3 days and examined the numbers of cells in the culture by an MTT assay. The results showed that none of the molecules exerted noticeable cytotoxicity even at 20 µM ( Figure 4).

Inhibition of the Activation of the RAW 264.7 Cell Line by the Oleanolic Acid Saponins In Vitro
Nitric oxide (NO) is a key mediator for the inflammatory response and pathogenesis. We examined the inhibitory activities of compounds 1 and 2 for the activation of macrophage RAW 264.7, leading to the production of NO. When RAW 264.7 cells were treated for 24 h with either compound 1 or 2 before incubation with LPS, the levels of NO produced by those cells decreased in a concentration-dependent manner. The extent of inhibition caused by 2 was noticeably higher than that caused by 1 and oleanolic acid aglycon at the same concentrations ( Figure 5). The difference in the extent of inhibition caused by 1 and oleanolic acid aglycon was marginal, if at all.

Inhibition of the Activation of Macrophage by Orally Administered Oleanolic Acid Saponins In Vivo
TNF-α is a well-characterized, proinflammatory cytokine that plays a crucial role in host defense and the inflammatory response. TNF-α has been implicated in the pathogenesis of both acute and chronic inflammatory diseases. Thus, we also examined the effects of compounds 1 and 2 on the activation of macrophage in vivo. Oleanolic acid aglycon and compounds 1 and 2 were administered orally using an oral gavage technique for 4 days before macrophage activation in vivo with LPS ( Figure 6). When mice were administered either compound 1 or 2 in saline (1% Tween 20) before LPS injection (i.p.) to cause sepsis, the levels of TNF-α in the blood were diminished markedly relative to those of TNF-α in the blood of sham-treated mice. In contrast, the treatment with oleanolic acid aglycon caused no significant decrease. , respectively, LPS was injected i.p., and the blood was drawn for the analysis of the concentration of TNF-α. The concentration of TNF-α was determined with ELISA (* p <0.05, ANOVA). Dexamethasone (Dexa # ) was used as the positive control. Sham means a negative control group that is injected with LPS to induce inflammation but not treated.
Previous studies demonstrated that the extract of S. hexaphylla leaves has an anti-inflammatory effect through suppression of nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression [5,15].  (2). Their structures were determined by the analysis of their spectroscopic data, IR, NMR, ESI-MS and chemical reaction. Furthermore, the quantitative analysis of their compounds were first time reportedly. The potential anti-inflammatory effects of isolated compounds were also reported by the in vitro and in vivo study. The structure-activity relationship (SAR) in triterpenoid saponins may be deduced from anti-inflammatory effects. Consideration of the structure-activity relationship (SAR) of these triterpenoid saponins suggested that the presence of the sugar units at C-3 and/or C-28 of the aglycon might play an important role in the anti-inflammatory inhibitory activity of these compounds. Thus, in this study, the preliminary comparison anti-inflammatory activity of these saponins with their aglycon was also discussed.
Efforts have been made to identify the specific chemical components responsible for the immunomodulatory activities of S. hexaphylla [5]. As a result, various oleanolic acid-based saponins, as well as its aglycon, were isolated preferentially as pure forms, suggesting that oleanolic acid-based glycosides are, at least in part, responsible for the pharmacological activities of S. hexaphylla.
It is well accepted that saponins have superior pharmacological activities relating to their aglycon [29]. In line with this notion, compound 2 showed a somewhat more potent inhibition of the activation of the RAW 264.7 cell line in vitro than oleanolic acid aglycon. The difference in the extent of the inhibition by 2 and oleanolic acid aglycon seemed more conspicuous at the higher concentrations (i.e., 3.3 and 10 µM) than at lower concentrations ( Figure 5). Notably, however, the difference was, at most, about 15%. These results indicate that oleanolic acid aglycon can fully exert immunomodulatory activities and that the sugar constituents may have only some additive effect.
Given the results from the in vitro assay, it was rather surprising that the in vivo assay results indicated that compounds 1 and 2 exerted much stronger macrophage activation inhibition than oleanolic acid aglycon. The difference between the in vitro and in vivo assay results is not readily reconciled. However, it is postulated that the poor solubility of oleanolic acid aglycon in aqueous solution may be responsible for the poor pharmacological activities shown in the in vivo assay. It is known that the addition of sugar moieties to a triterpene compound increases its solubility. As the molecules were given orally during the in vivo assays, it is conceivable that the poor aqueous solubility of oleanolic acid aglycon resulted in poor absorption in the gastrointestinal tract.
The results that the olanolic acid saponins exerted better effects than their aglycon, when they were formulated in an aqueous solution and administered orally, have important implications.
Considering that traditional oriental medicines are generally prepared by simmering medicinal herbs in water (H 2 O) and administered orally, it is likely that saponins play more important roles than their aglycons in the actions of traditional oriental medicines. It is possible that the pharmacological properties of the sapogenin core may change depending on the composition and arrangement of the sugar moieties to be attached. Thus, further research is warranted to understand the structure-activity relationship between the sugar moieties in saponins and their pharmacological properties, particularly in vivo. Our study suggests that oleanane triterpene saponins from S. hexaphylla leaves could be developed as a therapeutic agent for treating inflammatory diseases.

Chemical and Reagents
The optical rotation values were recorded with a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). FT-IR spectra were performed on a JASCO Report 100 infrared spectrophotometer (JASCO, Tokyo, Japan). All NMR spectra were carried out on Bruker Avance III 600 spectrometers (Bruker, Billerica, MA, USA) in pyridine-d 5 with TMS as the internal standard. NMR data analysis was conducted with MestReNova version 12.0.4 (Metrelab Research SL, Santiago de Compostela, Spain). LC-MS/MS data were performed by using a Shimadzu LCMS-8040 system (Shimadzu, Kyoto, Japan) in positive and negative mode [14]. All solvents used for isolation were provided by the SK chemical Company, Korea. Silica gel (70-230, 230-400 mesh, Merck, Whitehouse Station, NJ, USA) and YMC RP-18 resins (75 µm, Fuji Silysia Chemical Ltd., Kasugai, Japan) were used as absorbents in the column chromatography. Thin layer chromatography (TLC) plates were purchased from Merck KGaA (silica gel 60 F 254 and RP-18 F 254 , 0.25 µm, Merck, Darmstadt, Germany). Spots were detected under UV radiation (254 and 365 nm, Ultra-Violet Products Ltd., Cambridge, UK) and by spraying the plates with 10% H 2 SO 4 followed by heating with a heat gun. Chemical reagents and standard compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Plant Material
The plant material S. hexaphylla (Lardizabalaceae) leaves were collected in the area of Jangheung, Jeollanam-do, Korea and authenticated by the National Institute of Biological Resources (Supporting data, Figures S1 and S2). A voucher specimen (CNU 17002) was deposited at the Herbarium of College of Pharmacy, Chungnam National University (Daejeon, Korea).

Biodirected Isolation of the Active Compounds
The dried leaves of S. hexaphylla (2.0 kg) were extracted with 95% aqueous EtOH (10 L × 3 times) under reflux condition. Evaporation of the solvent under reduced pressure gave the EtOH extract (700 g). The EtOH extract was suspended in H 2 O and successively separated with dichloromethane (CH 2 Cl 2 ) and ethyl acetate (EtOAc) to yield the CH 2 Cl 2 extract (300 g), EtOAc extract (210 g) and H 2 O layer, respectively. Low-range polarity fraction (DCM and EtOAc extracts, notably contained chlorophyll, essential oil and fatty substances). Furthermore, the H 2 O fraction showed potent NO inhibitory activity, which was greater than that in the presence of the EtOH extract. Thus, this H 2 O fraction of S. hexaphylla leaves was chosen for subsequent study.
The H 2 O layer was separated using a Diaion HP-20 column and was eluted with a gradient solvent mixture of MeOH-H 2 O (from 25:75 to 50:50, 75:25 and pure MeOH, stepwise) to yield four fractions (W-1 to W-4, respectively), based on the thin-layer chromatography (TLC) analysis. The fraction W-4 showed stronger NO inhibitory activity than the fractions W-1-W-3. Thus, fraction W-4 was chosen as the focus for active compound isolation. Fraction W-4 was subjected to silica gel column chromatography (CC), eluting with the gradient solvent system of CH 2

LC-MS/MS Analysis of the Isolated Compounds
LC-MS/MS analysis were performed by using a Shimadzu LCMS-8040 system (Shimadzu, Kyoto, Japan) in positive and negative mode. Electrospray (Shimadzu, Kyoto, Japan) was operated under the following condition, the interface voltage was −3.5 kv for the negative mode and the positive mode was 4.5 kv, the nebulizing gas flow rate was 3 L/min, drying gas flow rate was 15 L/min, a desolvation line temperature was 250 • C, and the heat block temperature was 400 • C. The stationary phase was Hector M 18 (250 mm × 4.6 mm, 5 µm, RSTech, Daejeon, Korea). The mobile phase consisted of (A) 0.1% formic acid and (B) acetonitrile-formic acid (99.9:0.1) using a gradient elution of 28-50% B at 0-60 min under the flow rate of 0.5 mL/min. The optimized collision energy in the MRM mode for compounds 1-2 were 18 V and 22 V, respectively. 4.6. Biological Studies 4.6.1. Cytotoxicity Assay Using MTT RAW 264.7 cells were maintained in DMEM medium with 10% fetal bovine serum (FBS) in a humidified CO 2 incubator. Cell cytotoxicity was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Briefly, RAW264.7 cells suspended in DMEM medium (10% FBS, Sigma-Aldrich, St. Louis, MO, USA) were seeded onto a 96-well culture plate at a density of 5 × 10 3 cells/100 µL. The next day, 100 µL of the fresh medium was supplemented to the culture, and then 2 µL of compounds 1 and 2, oleanolic acid and dexamethasone in DMSO were added to the culture at 20 µM, 5 µM, 1.25 µM, 0.32 µM and 0.08 µM, respectively. Three days (72 h) after culture with the compound at 37 • C, MTT assays were performed as described previously. The assays were run in triplicate. For animal experiments, all of the compounds were first pulverized with Tween 20 and then diluted with saline (1:99) to maximize the solubility of compounds in water [30]. The compounds in the saline/Tween 20 solution (2.5, 0.83 and 0.28 mg/mL, respectively) was administered via oral gavage (200 µL per injection). Mice (male BALB/C, 8 weeks; Samtako, Osan, Korea) were pretreated orally with individual compounds for 4 days. Two hours after the last treatment, the mice were injected i.p. with LPS (0.1 mg/kg) [31]. Ninety min after the LPS injection, the blood was collected, and the concentration of TNF-α in the blood was measured with TNF-α ELISA kit (Abcam, Cambridge, MA, USA). The experiment was performed with 3 mice per group.

Statistical Analysis
The significance of the difference among separate experimental groups was determined with a nonparametric one-way ANOVA (Kruskal-Wallis test), and Bonferroni correction was used as post hoc analysis. p < 0.05 was considered statistically significant.
Supplementary Materials: The following are available online. Figure S1: Certification of S. hexaphylla leaves from National Institute of Biological Resources, Figure S2

Conflicts of Interest:
The authors declare no conflict of interest.