Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis

The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia against diseases such as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC50 value less than 1 µg/mL. Therefore, the present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structure of 1 was assigned by means of 1D and 2D NMR data and high-resolution mass spectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulation assay with IC50 values of 1.7 and 11 nM, respectively, with a therapeutic index up to 71,000. The antiallergic activities of A. malaccensis seeds and aquimavitalin (1) have never been revealed before. The results indicated that A. malaccensis seeds and the pure compound have the potential for use in the treatment of allergy.

As the methanol layer proved the best antiallergic activity (IC 50 0.0089 and 0.069 µg/mL in A23187 and antigen-induced degranulation assay, respectively), it was further separated using silica gel column chromatography to yield six fractions, AM1-AM6 (subfractions of methanol layer from Aquilaria malaccensis seeds). Among them, fraction AM4 showed the most remarkable antiallergic activity inhibiting β-hexosaminidase release from mast cells induced by either A23187 (IC 50 0.0034 µg/mL) or antigen (IC 50 value 0.0065 µg/mL).
In cytotoxicity assay against a panel of three cancer cell lines (human hepatocellular carcinoma cells HepG2, human breast adenocarcinoma cells A549, and human lung adenocarcinoma cells MDA-MB231), only some of the AMS fractions showed cytotoxic activities at a 20-µg/mL level (Table 3) (A-BuOH 57.1% against A549, AM4 56.5% against MDA-MB231 and 79.3% against A549, AM6 56.0% against MDA-MB231 cell line). Moreover, considering weak cytotoxicity of AMS towards RBL-2H3 cells, the antiallergic active fraction AM4 exerted therapeutic index up to 28,000. To further rule out the possibility that AM4 causes direct mast cell activation, we examined the capacity of AM4 to elicit degranulation by itself. Results showed that the AM4 treatments did not cause significant degranulation as compared with untreated control (Figure 1). These data implied that AM4 is the best target for further phytochemical analysis.
In general, phorbol esters, particularly phorbol-12-myristate-13-acetate (PMA), are well-known as irritant, proinflammatory and cocarcinogenic. Nevertheless, phorbol esters were previously reported to exert antiinflammatory, anti-HIV, antiparasitic and anticancer activities [26]. Both free C-20 hydroxy, and C-12 and/or C-13 ester moieties were important for the activities of phorbol esters [26]. Importantly, it was suggested that unsaturation of ester functionality may play a crucial role in bioactivity of phorbols [26,27]. Previously, 12-O-(2Z,4E,6E)-deca-2,4,6-trienoylphorbol-13-acetate, a phorbol ester possessing similar conjugated fatty acid moiety as 1, was isolated from A. malaccensis bark and exerted cytotoxic activity in P-388 lymphocytic leukemia cells in vitro [18]. In structure-activity relationship study on phorbol esters containing fatty acids with different level of unsaturation and carbon chain length, phorbol esters carrying conjugated unsaturated fatty acid as acyl group showed irritant but very weak tumor-promoting activities. [27]. This is the first study to report on the antiallergic potential of pure phorbol ester with the therapeutic index up to 71,000. The antiallergic activity of AMS together with identification of its active component provides scientific support for the folk use of A. malaccensis against asthma.

Plant Material
The seeds of A. malaccensis were obtained from Hsue-Yin Hsu, Tzu Chi University, Hualien, Taiwan, in November 2014. The plant material was identified by Hsue-Yin Hsu, Department of Life Sciences, Tzu Chi University, Hualien, Taiwan. A voucher specimen (code no. KMU-AMS 1) was deposited in the Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.

Extraction and Isolation
Air-dried and powdered seeds of A. malaccensis (462 g) were extracted with 90% EtOH at room temperature (3ˆ5 L) and then concentrated under reduced pressure. The combined extracts were concentrated and obtained crude ethanolic extract (A-EtOH, 27.7 g) was suspended in water and partitioned with ethyl acetate (3ˆ1 L). The water layer was partitioned with n-butanol (3ˆ1 L) to yield water layer (A-Water, 1654.0 g) and n-butanol layer (A-BuOH, 398.2 g). The EtOAc layer (A-EtOAc, 25.6 g) was further partitioned with n-hexane and 90% aqueous MeOH to obtain n-hexane layer (A-Hexane, 7.

Cell Culture
The mucosal mast cell-derived rat basophilic leukemia (RBL-2H3) cell line was purchased from the Bioresource Collection and Research Center (Hsin-Chu, Taiwan). Cells were grown in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin plus 100 µg/mL streptomycin. Cells were cultured in 10 cm cell culture dishes at 37˝C in a humidified chamber with 5% CO 2 in air.

Cell Viability Assay
A methylthiazol tetrazolium (MTT) assay was used to measure the potential toxic effects of the samples on RBL-2H3 cells [28]. Briefly, RBL-2H3 cells (2ˆ10 4 cells/well) were seeded in a 96-well plate overnight and treated with various concentrations of samples (10-100 µg/mL) for 24 h. MTT solution (0.5 mg/mL) was added to the wells (80 µL per well) and incubated for 1 h. The formed formazan crystals were dissolved in DMSO (80 µL). The absorbance at 595 nm was measured using microplate reader (Multiskan Ascent, Thermo Scientific, Waltham, MA, USA). The degree of cell viability of each sample was calculated as the percentage of control value (untreated cells). The maximal tolerated dose of DMSO was 0.5%. All experiments were repeated at least two times.

Degranulation β-Hexosaminidase Assay Induced by A23187 or Antigen
The degree of A23187-and antigen-induced degranulation in RBL-2H3 cells was determined by a β-hexosaminidase release assay as described previously [28,29] with following modifications. RBL-2H3 cells were seeded in a 96-well plate (2ˆ10 4 cells/well) for A23187-induced and in 48-well plate (3ˆ10 4 cells/well) for antigen-induced experiment. Cells were treated with various concentrations of the samples for 20 h. Dexamethasone (10 nM) was used as a positive control. The cells for the antigen-induced experiment were first sensitized with anti-DNP IgE (5 µg/mL) for at least 2 h. After thorough washing by pre-warmed Tyrode's buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 5.6 mM glucose, 20 mM HEPES at pH 7.4), the cells were stimulated by either calcium ionophore A23187 (1 µM) or antigen DNP-BSA (100 ng/mL) in Tyrode's buffer for 1 h. Unstimulated cells were either lysed with 0.5% Triton X-100 solution for the total amount of β-hexosaminidase release or left untreated for spontaneous release of β-hexosaminidase. Then aliquots of supernatants (50 µL) were incubated with equal volume of 1 µM of p-NAG (50 µL) prepared in 0.1 M citrate buffer (pH 4.5) serving as a substrate for the released β-hexosaminidase. After 1 h of incubation at 37˝C, the reaction was quenched by the addition of 100 µL of stop buffer (0.1 M Na 2 /NaHCO 3 , pH 10.0). Absorbance was measured at 405 nm on a microplate reader (Multiskan Ascent, Thermo Scientific). The inhibition percentage of β-hexosaminidase release was calculated as the percentage of control value (untreated stimulated cells). The maximal tolerated dose of DMSO was 0.5%. All experiments were repeated three times.

Effect on Enzymatic Activity of β-Hexosaminidase
To test the possible effect of the sample on enzymatic activity, following assay was performed. The cell suspension (2ˆ10 6 cells) in 2 mL of Tyrode's buffer was sonicated for 5 min. The solution was then centrifuged, and the supernatant was diluted with 8 mL of Tyrode's buffer. The enzyme solution (45 µL) and test sample solution (5 µL) were transferred into a 96-well microplate and enzyme activity was examined as described above (Section 3.7). All experiments were repeated three times.

Direct Degranulation β-Hexosaminidase Assay Induced by the Sample
The degree of β-hexosaminidase release triggered by the sample in RBL-2H3 cells was determined by a modified β-hexosaminidase release assay. Briefly, RBL-2H3 cells (4ˆ10 4 cells/well) were seeded in a 48-well plate and treated with the samples for 10 h. Tyrode's buffer supplemented with 5.6 mM glucose, 2 mg/mL BSA and 2 mM glutamine was used to prepare the samples and treat the cells. Then, 50 µL of supernatants were transferred into a 96-well microplate and examined as described above (Section 3.7). A23187 (1 µM) was used as a positive control. All experiments were repeated three times.

Preparation of Human Neutrophils
Human neutrophils from venous blood of healthy, adult volunteers (20-30 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll-Hypaque gradient and hypotonic lysis of erythrocytes [30]. Purified neutrophils containing >98% viable cells, as determined by the trypan-blue exclusion method [31], were resuspended in a Ca 2+ -free Hank's buffered salt solution (HBSS) at pH 7.4 and were maintained at 4˝C prior to use.

Superoxide Anion Generation Assay and Elastase Release Inhibition Assay
Neutrophil superoxide anion generation was determined using superoxide dismutase (SOD)-inhibitory cytochrome reduction according to described procedures [32,33]. Degranulation of azurophilic granules was determined by measuring the elastase release as described previously [33]. All experiments were repeated at least three times.

Cytotoxic Assay
MTT assay was used according to the method described before [34]. Briefly, HepG2 (1ˆ10 4 cells), A549 (5ˆ10 3 cells), and MDA-MB-231 (1ˆ10 4 cells) were seeded into 96-well plates, followed by treatment with the AMS samples at concentration of 20 µg/mL. After 72 h, the medium was removed and 100 µL of MTT solution (0.5 mg/mL) was added to each well. The plates were then incubated at 37˝C for 1 h and then, the MTT dye was detected by the addition of DMSO (100 µL). The absorbance was recorded at 550 nm. Doxorubicin was used as a positive control.

Statistics
The results were expressed as mean˘SD unless otherwise specified. The IC 50 values were calculated using the Microsoft Office (linear function). Statistical significance was calculated by one-way analysis of variance (ANOVA), followed by Dunnett's test (SigmaPlot, Jandel Scientific, San Rafael, CA, USA). Values with * p < 0.05, ** p < 0.001 were considered statistically significant.

Conclusions
The present investigation revealed bioactive fractions and pure principle from the extract of AMS. It resulted in the isolation of the active pure compound, aquimavitalin (1). The remarkable inhibitory activity of 1 on mast cell degranulation with nanomolar IC 50 values provides evidence that phorbol ester could possess antiallergic activity.
Moreover, high potency of phorbol esters may shed light on the use of A. malaccensis seeds in the treatment diseases related to allergy. However, further studies are needed to examine the safety of these materials in therapy.