COMPOSITIONS AND METHODS OF ANTIALLERGIC PHORBOL ESTER AND PHORBOL DERIVATIVES AS THE MAIN ACTIVE INGREDIENTS FROM THE SEEDS OF AQUILARIA MALACCENSIS

Abstract

The present invention relates to phorbol esters from the seeds of Aquilaria malaccensis by a series of chromatographic processes, and compositions containing these congeners for the treatment or prevention of allergic responses. Furthermore, compositions are provided which comprise these phorbol esters in combination with at least one to assess the effects for preventing or treating allergies.

Claims

1. A method for preparing the effective compositions from Aquilaria malaccensis seeds to prevent or treat allergies and related hypersensitive immuno-responses, including following steps: (a) after air-dried and powdered, the seeds of A. malaccensis were extracted with 90% ethanol (EtOH) at room temperature in triplicate and then concentrated under reduced pressure to afford an ethanolic (EtOH) extract. (b) the EtOH extract was suspended in water and partitioned with equivalent ethyl acetate in triplicate, and an organic (EtOAc) layer was separated. (c) the EtOAc layer was further partitioned with n-hexane (Hexane) and 90% aqueous methanol (MeOH) to obtain Hexane layer and MeOH layer. (d) the MeOH layer was subjected to a column chromatography over silica gel (23 cm×4 cm, silica gel 60, 0.063˜0.200 mm, Merck) under a gradient elution of n-hexane/CH.sub.2Cl.sub.2/MeOH to yield six fractions (AM1, 6:3:1; AM2, 6:4:1; AM3, 6:6:1; AM4, 6:8:1; AM5, 6:10:1 and AM6, 6:10:2). (e) fraction AM4 was further fractionated over a Sephadex LH-20 column (CH.sub.2Cl.sub.2/MeOH, 1:1) to obtain eight sub-fractions (AM4-1 to AM4-8). (f) fraction AM4-3 was subjected to column chromatography (17 cm×4 cm, Geduran Si 60, 0.040˜0.063 mm, Merck) under gradient elution of EtOAc/n-hexane (from 1:10 to 4:1) yielding 15 fractions AM4-3-1˜AM4-3-15. (g) fraction AM4-4 was further separated by column chromatography on silica gel (30 cm×1.5 cm, Geduran Si 60, 0.040˜0.063 mm, Merck) under gradient elution of EtOAc/n-hexane (from 1:15 to 4:1) to obtain 12 subfractions AM4-4-1˜AM4-4-12.

2. The method as claimed in claim 1, wherein a new phorbol ester of Formula I, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, was isolated from AM4-4-9. ##STR00010##

3. The method as claimed in claim 1, wherein a new phorbol ester of Formula II, 12-deoxy-13-O-acetoylphorbol-20-octadec-9-enoate, reported by now was isolated from AM4-3-6 and AM4-4-3. ##STR00011##

4. The method as claimed in claim 1, wherein a new phorbol ester of Formula III, 12-O-(2E,4E)-6-oxohexa-2,4-dienoylphorbol-13-acetate, reported by now was isolated from AM4-3-13. ##STR00012##

5. The method as claimed in claim 1, wherein a new phorbol ester of Formula IV, 12-O-(2E,4E)-6,7-dihydroxytetradeca-2,4-dienoylphorbol-13-acetate, reported by now was also isolated from AM4-3-13. ##STR00013##

6. A new phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, being pharmaceutically prepared for preventing and treating antiallergies and related hypersensitive immuno-responses, being represented by Formula I: ##STR00014##

7. An ethanolic (EtOH) extract of Aquilaria malaccensis seeds being pharmaceutically prepared for preventing and treating allergies and related hypersensitive immuno-responses, obtained by air-dried and powdered seeds of A. malaccensis extracted with 90% ethanol at room temperature in triplicate and then concentrated under reduced pressure.

8. An Aquilaria malaccensis seeds extract prepared for preventing and treating allergies, an ethyl acetate (EtOAc) layer, which is obtained by air-dried and powdered seeds of A. malaccensis extracted with 90% ethanol 5 L in triplicate and partitioned with water and ethyl acetate after concentration under reduced pressure.

9. A n-hexane (Hexane) layer and a methanolic (MeOH) layer, which are obtained via liquid-liquid partition of EtOAc layer of Aquilaria malaccensis seeds prepared for preventing and treating allergies in claim 8 by n-hexane and aqueous methanol.

10. The ethylacetate (EtOAc) layer of Aquilaria malaccensis as claimed in claim 9, wherein the phorbol ester-rich fraction AM4 of Aquilaria malaccensis seeds is pharmaceutically prepared for preventing and treating allergies and related hypersensitive immuno-responses, yielded by a silica gel column chromatography (23 cm×4 cm, silica gel 60, 0.063˜0.200 mm, Merck) of the MeOH layer with a gradient elution of n-hexane/CH.sub.2Cl.sub.2/MeOH (AM1, 6:3:1; AM2, 6:4:1; AM3, 6:6:1; AM4, 6:8:1; AM5, 6:10:1 and AM6, 6:10:2).

11. The ethylacetate (EtOAc) layer of Aquilaria malaccensis as claimed in claim 10, wherein the phorbol ester-rich fractions AM4-3 and AM4-4 of Aquilaria malaccensis seeds are pharmaceutically prepared for preventing and treating allergies and related hypersensitive immuno-responses, separated by a Sephadex LH-20 column (CH.sub.2Cl.sub.2/MeOH, 1:1) of AM4.

12. The ethylacetate (EtOAc) layer of Aquilaria malaccensis as claimed in claim 11, wherein the phorbol ester-rich fractions AM4-3-6 and AM4-3-13 of Aquilaria malaccensis seeds are pharmaceutically prepared for preventing and treating allergies and related hypersensitive immuno-responses, separated by a silica gel column chromatography (17 cm×4 cm, Geduran Si 60, 0.040˜0.063 mm, Merck) of AM4-3 with a gradient elution of EtOAc/n-hexane (from 1:10 to 4:1).

13. The ethylacetate (EtOAc) layer of Aquilaria malaccensis as claimed in claim 11, wherein the phorbol ester-rich fractions AM4-4-7, AM4-4-8, and AM4-4-9 of Aquilaria malaccensis seeds are pharmaceutically prepared for preventing and treating allergies and related hypersensitive immuno-responses, separated by a column chromatography on silica gel (30 cm×1.5 cm, Geduran Si 60, 0.040˜0.063 mm, Merck) of AM4-4 with a gradient elution of EtOAc/n-hexane (from 1:15 to 4:1).

14. A phorbol ester, 12-deoxyphorbol 13-decanoate, being pharmaceutically prepared for preventing and treating antiallergies and represented by the Formula V: ##STR00015##

15. A phorbol ester, 12-deoxyphorbol 13-octanoate, being pharmaceutically prepared for preventing and treating antiallergies and represented by the Formula VI: ##STR00016##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1. shows preparation scheme of the present invention.

[0017] FIG. 2. shows isolation scheme of AM4-3 to afford 15 subfractions.

[0018] FIG. 3. shows isolation scheme of AM4-4 to afford 12 subfractions.

[0019] FIG. 4. shows .sup.1H NMR spectra comparison of selected compositions from Aquilaria malaccensis seeds, the ethanolic extract (EtOH), 90% aqueous methanol layer (MeOH), AM-4, and AM4-4.

[0020] FIG. 5. shows .sup.1H NMR spectrum of compound represented by Formula I.

[0021] FIG. 6. shows .sup.13C NMR spectrum of compound represented by Formula I.

[0022] FIG. 7. shows .sup.1H NMR spectrum of compound represented by Formula II.

[0023] FIG. 8. shows .sup.13C NMR spectrum of compound represented by Formula II.

[0024] FIG. 9. shows .sup.1H NMR spectrum of compound represented by Formula III.

[0025] FIG. 10. shows .sup.13C NMR spectrum of compound represented by Formula III.

[0026] FIG. 11. shows .sup.1H NMR spectrum of compound represented by Formula IV.

[0027] FIG. 12. shows .sup.13C NMR spectrum of compound represented by Formula IV.

[0028] FIG. 13. shows .sup.1H NMR spectrum of compound represented by Formula V.

[0029] FIG. 14. shows .sup.13C NMR spectrum of compound represented by Formula V.

[0030] FIG. 15. shows .sup.1H NMR spectrum of compound represented by Formula VI.

[0031] FIG. 16. shows antiallergic activities of the compositions from Aquilaria malaccensis seeds by inhibition of β-hexosaminidase release.

[0032] FIG. 17. shows Antiinflammatory effects of the compositions from Aquilaria malaccensis seeds on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.

[0033] FIG. 18. shows Cytotoxic activities of the compositions from Aquilaria malaccensis seeds against HepG2, MDA-MB231, and A549 carcinoma cell lines.

[0034] FIG. 19. shows Activity of phorbol ester-rich fraction (AM4) and phorbol ester I on stimulant-free degranulation in RBL-2H3 cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention is further illustrated by the preparation schemes (FIG. 1˜3), the NMR spectra of phorbol esters represented by Formulas I to VI (FIG. 4˜15), and the bioactive results (Tab. 1˜4), and the skills and methods used herein are described in detail:

(a) After air-dried and powdered, the seeds of A. malaccensis (462 g) were extracted with 90% EtOH (5 L) at room temperature in triplicate and then concentrated under reduced pressure to afford an ethanolic extract (27.7 g).
(b) The ethanolic extract was suspended in water (1 L) and partitioned with equivalent ethyl acetate in triplicate, an organic (EtOAc) layer (25.6 g) and an aqueous (H.sub.2O) layer were separated.
(c) The EtOAc layer was further partitioned with n-hexane and 90% aqueous MeOH to obtain n-hexane layer (7.1 g) and MeOH layer (16.2 g) whereas the H.sub.2O layer was further partitioned with n-butanol to divide into an n-butanol (BuOH) layer and an aqueous (Water) layer.
(d) The MeOH layer was subjected to a column chromatography over silica gel (23 cm×4 cm, silica gel 60, 0.063˜0.200 mm, Merck) under a gradient elution of n-hexane/CH.sub.2Cl.sub.2/MeOH (6:3:1, 6:4:1, 6:6:1, 6:8:1, 6:10:1, and 6:10:2) to yield six fractions (AM1, 2917.0 mg; AM2, 1320.0 mg; AM3, 6834.0 mg; AM4, 3212.2 mg; AM5, 1703.0 mg; and AM6, 97.9 mg).
(e) Fractions AM4 was further fractionated over a Sephadex LH-20 column (CH.sub.2Cl.sub.2/MeOH, 1:1) to obtain eight sub-fractions (AM4-1, 688.0 mg; AM4-2, 688.0 mg; AM4-3, 762.0 mg; AM4-4, 173.7 mg; AM4-5, 609.0 mg; AM4-6, 253.5 mg; AM4-7, 80.0 mg; and AM4-8, 80.0 mg). Of which subfractions, AM4-3 and AM4-4 are phorbol ester-rich fractions. The above scheme are shown in FIG. 1.
(f) Fraction AM4-3 was subjected to column chromatography (17 cm×4 cm, Geduran Si 60, 0.040˜0.063 mm, Merck) under gradient elution of EtOAc/n-hexane (from 1:10 to 4:1) yielding 15 fractions AM4-3-1˜AM4-3-15 (FIG. 2). (AM4-3-1, 3.4 mg; AM4-3-2, 25.3 mg; AM4-3-3, 345.6 mg; AM4-3-4, 49.2 mg; AM4-3-5, 16.4 mg; AM4-3-6, 39.5 mg; AM4-3-7, 8.4 mg; AM4-3-8, 3.8 mg; AM4-3-9, 42.7 mg; AM4-3-10, 23.0 mg; AM4-3-11, 42.5 mg; AM4-3-12, 43.5 mg; AM4-3-13, 23.5 mg; AM4-3-14, 7.9 mg; and AM4-3-15, 38.3 mg) Of which subfractions, AM4-3-6 and AM4-3-13 are two phorbol ester-rich fractions.
(g) Fraction AM4-4 was further separated by column chromatography on silica gel (30 cm×1.5 cm, Geduran Si 60, 0.040˜0.063 mm, Merck) under gradient elution of EtOAc/n-hexane (from 1:15 to 4:1), and 12 subfractions AM4-4-1˜AM4-4-12 were obtained (FIG. 3). (AA/14-4-1, 2.2 mg; AM4-4-2, 16.3 mg; AM4-4-3, 3.5 mg; AM4-4-4, 5.7 mg; AM4-4-5, 7.7 mg; AM4-4-6, 11.5 mg; AM4-4-7, 37.6 mg; AM4-4-8, 6.8 mg; AM4-4-9, 43.9 mg; AM4-4-10, 3.3 mg; AM4-4-11, 3.5 mg; and AM4-4-12, 23.5 mg) Of which subfractions, AM4-4-3, AM4-4-7, AM4-4-8 and AM4-4-9 are four phorbol ester-rich fractions.
(h) From AM4-4-9, a new phorbol ester named 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate (43.9 mg) was isolated. This compound possesses a molecule of C.sub.36H.sub.50O.sub.8 and is represented by Formula I:

##STR00006##

(i) From AM4-3-6 and AM4-4-3, a new phorbol ester named 12-deoxy-13-O-acetoylphorbol-20-octadec-9-enoate (8.8 mg) was afforded. This compound possesses a molecule of C.sub.40H.sub.62O.sub.7 and is represented by Formula II:

##STR00007##

(j) From AM4-3-13, two new phorbol esters were given. One new phorbol ester named 12-O-(2E,4E)-6-oxohexa-2,4-dienoylphorbol-13-acetate (0.7 mg) with a molecule of C.sub.28H.sub.34O.sub.9 is represented by Formula III. Another new phorbol ester named 12-O-(2E,4E)-6,7-dihydroxytetradeca-2,4-dienoylphorbol-13-acetate (0.9 mg) possesses a molecule of C.sub.36H.sub.52O.sub.10 and is represented by Formula IV.

##STR00008##

(k) From AA/14-4-7 and AA/14-4-8, two known phorbol esters, 12-deoxyphorbol 13-decanoate (8.5 mg) and 12-deoxyphorbol 13-octanoate (1.4 mg), were isolated. These two compounds possess molecules H of C.sub.30H.sub.46O.sub.6 and C.sub.28H.sub.42O.sub.6 and are represented by Formulas V and VI, respectively.

##STR00009##

[0036] Analysis of the .sup.1H NMR spectra comparison of the ethanolic extract (EtOH), 90% aqueous methanol layer (MeOH), AM-4, and AM4-4 (FIG. 4), the EtOH extract, MeOH layer, AM-4, and the subfraction AM4-4 all comprise phorbol esters, and the efficacy of anti-allergy is in proportion to the signal strength of phorbol esters.

[0037] In accordance with the preparation methods, phorbol ester I is 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate with a molecular formula of C.sub.36H.sub.50O.sub.8 deduced from HRESIMS data m/z 633.33980 [M+Na].sup.+ (calcd for C.sub.36H.sub.50O.sub.8Na, 633.33979); [α].sup.D.sub.25: 3.75±1.97 (c 0.0667, CHCl.sub.3); UV (MeOH)λ.sub.max (log ε): 303 (2.78), 233 (2.75) nm; and IR (neat) ν.sub.max: 3413, 2965, 2922, 1710, 1615, 1377, 1258, 1092, 802 cm.sup.−1. The .sup.1H and .sup.13C NMR spectra of phorbol ester I are shown in FIG. 5 and FIG. 6, respectively.

[0038] In accordance with the preparation methods, phorbol ester II is 12-deoxy-13-O-acetoylphorbol-20-octadec-9-enoate with a molecular formula of C.sub.40H.sub.62O.sub.7 deduced from HRESIMS data m/z 677.43884 [M+Na].sup.+ (calcd for C.sub.40H.sub.62O.sub.7Na, 677.43878); [α].sup.D.sub.25: +2.91±0.49 (c 0.3333, CHCl.sub.3); UV (MeOH) λ.sub.max (log ε): 285 (2.78), 250 (2.83) nm; and IR (neat) ν.sub.max: 3409, 2922, 2855, 1717, 1375, 1332, 1152, 1021 cm.sup.−1. The .sup.1H and .sup.13C NMR spectra of phorbol ester II are shown in FIG. 7 and FIG. 8, respectively.

[0039] In accordance with the preparation methods, phorbol ester III is 12-O-(2E,4E)-6-oxohexa-2,4-dienoylphorbol-13-acetate with a molecular formula of C.sub.28H.sub.34O.sub.9 deduced from HRESIMS m/z 537.20959 [M+Na].sup.+ (calcd for C.sub.28H.sub.34O.sub.9Na, 537.20950); [α].sup.D.sub.25: +4.20±0.82 (c 0.1667, CHCl.sub.3); UV (MeOH) λ.sub.max (log ε): 295 (2.80), 249 (2.84) nm; and IR (neat) ν.sub.max: 3413, 2925, 2855, 2360, 2339, 1625, 1597, 1261, 1184, 755 cm.sup.−1. The .sup.1H and .sup.13C NMR spectra of phorbol ester III are shown in FIG. 9 and FIG. 10, respectively.

[0040] In accordance with the preparation methods, phorbol ester IV is 12-O-(2E,4E)-6,7-dihydroxytetradeca-2,4-dienoylphorbol-13-acetate with a molecular formula of C.sub.36H.sub.52O.sub.10 deduced from HRESIMS m/z 667.34515 [M+Na].sup.+ (calcd for C.sub.36H.sub.52O.sub.10Na, 667.34527); [α].sup.D.sub.25: +10.44±1.45 (c 0.1667, CHCl.sub.3); UV (MeOH) λ.sub.max (log ε): 289 (2.79), 249 (2.83) nm; and IR (neat) ν.sub.max: 3392, 2925, 2851, 1710, 1632, 1455, 1375, 1261, 1024, 802, 755 cm.sup.−1. The .sup.1H and .sup.13C NMR spectra of phorbol ester IV are shown in FIG. 11 and FIG. 12, respectively.

[0041] In accordance with the preparation methods, phorbol ester V is 12-deoxyphorbol 13-decanoate with a molecular formula of C.sub.30H.sub.46O.sub.6 deduced from HRESIMS m/z 525.31921 [M+Na].sup.+ (calcd for C.sub.30H.sub.46O.sub.6Na, 525.31921); [α].sup.D.sub.25: +8.55±0.63 (c 0.200, CHCl.sub.3); UV (MeOH) λ.sub.max (log ε): 325 (0.14), 249 (1.31) nm; and IR (neat) ν.sub.max: 3392, 2925, 2356, 1710, 1629, 1335, 1155 cm.sup.−1. The .sup.1H and .sup.13C NMR spectra of phorbol ester V are shown in FIG. 13 and FIG. 14, respectively.

[0042] In accordance with the preparation methods, phorbol ester VI is 12-deoxyphorbol 13-octanoate with a molecular formula of C.sub.28H.sub.42O.sub.6 deduced from ESIMS m/z 475 [M+H].sup.+ (calcd for C.sub.28H.sub.43O.sub.6); [α].sup.D.sub.25: +4.75±1.27 (c 0.200, CHCl.sub.3); UV (MeOH) λ.sub.max (log ε): 311 (0.01), 250 (2.85) nm; and IR (neat) ν.sub.max: 3377, 2922, 2858, 1710, 1625, 1332, 1018 cm.sup.−1. The .sup.1H NMR spectrum of phorbol ester VI is shown in FIG. 15.

[0043] In the present specification and the scope of the patent application, the provided chemicals with formulas I˜VI should comprise all of their optical isomers and stereoisomers, therefore, all of these optical and stereo isomers are included within the scope of the present invention.

[0044] For the proposes of this invention, the compositions of antiallergic phorbol ester and phorbol derivatives as the main active ingredients from the seeds of Aquilaria malaccensis were evaluated by the following assays.

[0045] 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.sub.2 in air.

[0046] Sample preparations: In accordance with the preparation methods of this invention, the compositions from the seeds of Aquilaria malaccensis, the ethanolic extract (EtOH), n-butanol layer (BuOH), aqueous layer (Water), ethyl acetate layer (EtOAc), n-hexane layer (Hexane), 90% aqueous methanol layer (MeOH), the divided AM-4, AM4-4-7, AM4-4-8, AM4-4-9 (phorbol ester I), were prepared for bioactive assays.

[0047] Cell viability assay: A methylthiazol tetrazolium (MTT) assay was used to measure the potential toxic effects of the samples on RBL-2H3 cells (Chen et al., J. Nat. Prod., 72, 950-953, 2009). Briefly, RBL-2H3 cells (2×104 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). 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.

[0048] 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 (Chen et al., J. Nat. Prod., 72, 950-953, 2009; Matsuda et al., Bioorg. Med. Chem., 12, 5891-5898, 2004) with following modifications. RBL-2H3 cells were seeded in a 96-well plate (2×10.sup.4 cells/well) for A23187-induced and in 48-well plate (3×10.sup.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.sub.2, 1.0 mM MgCl.sub.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 Na2/NaHCO3, 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.

[0049] 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 (2×10.sup.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 (the section of Cell viability assay). All experiments were repeated three times.

[0050] 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 (4×10.sup.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 (the section of Cell viability assay). A23187 (1 μM) was used as a positive control. All experiments were repeated three times.

[0051] 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 (Boyum et al., Scand. J. Clin. Lab. Invest., 97, 77-89, 1968). Purified neutrophils containing >98% viable cells, as determined by the trypan-blue exclusion method (Jauregui et al., In Vitro, 17, 1100-1110, 1981), were resuspended in a Ca.sup.2+-free Hank's buffered salt solution (HBSS) at pH 7.4 and were maintained at 4° C. prior to use.

[0052] 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 (Babior et al., J. Clin. Invest., 52, 741-744, 1973; Hwang et al., Free Radical Bio. Med., 41, 1433-1441, 2006). Degranulation of azurophilic granules was determined by measuring the elastase release as described previously (Hwang et al., Free Radical Bio. Med., 41, 1433-1441, 2006). All experiments were repeated at least three times.

[0053] Cytotoxic assay: MTT assay was used to according to the used in a previous manuscript. HepG2 (1×10.sup.4 cells), A549 (5×10.sup.3 cells), and MDA-MB-231 (1×10.sup.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.

[0054] In accordance with the assays, antiallergic activity of the compositions from Aquilaria malaccensis seeds and A23187 (a positive control) was concluded in Table 1 whereas antiinflammatory effects of the compositions from A. malaccensis seeds on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils were summarized in Table 2. Furthermore, activity of phorbol ester-rich fraction (AM4) and phorbol ester I on stimulant-free degranulation in RBL-2H3 cells was shown in Table 4. The RBL-2H3 cells were treated with AM4 (10 μg/ml) and phorbol ester I (10 μg/ml) for 10 h. In the stimulant-free degranulation assay, Tyrode's buffer supplemented with glucose, bovine serum albumin (BSA) and glutamine was used as a medium. A23187 (1 μM) was used as a positive control.

[0055] In accordance with antiallergic activity of the compositions from Aquilaria malaccensis seeds (Table 1), the ethanolic extract (A-EtOH) showed potent antiallergic activity (IC.sub.50 0.92 and 3.9 μg/mL in A23187 and antigen-induced β-hexosaminidase assay, respectively. To clarify that antiallergic activity of the samples was due to inhibition of β-hexosaminidase release, and not false positive as a result of direct inhibition of β-hexosaminidase enzymatic activity (Wang et al., Biol. Pharm. Bull., 30, 388-392, 2007), the enzyme was extracted and tested with the active samples. None of the samples inhibited the enzymatic activity of β-hexosaminidase. As the methanol layer (A-MeOH) proved the best antiallergic activity (IC.sub.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. Among them, fraction AM4 showed the most remarkable antiallergic activity inhibiting β-hexosaminidase release from mast cells induced by either A23187 (IC.sub.50 0.0034 μg/mL) or antigen (IC.sub.50 0.0065 μg/mL).

[0056] In accordance with antiinflammatory effects of the compositions from Aquilaria malaccensis seeds on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils (Table 2), the ethanolic extract (A-EtOH) showed antiinflammatory activity (90.1% and 85.3% inhibition of superoxide generation and elastase release at 10 μg/mL, respectively). All partitioned fractions except the water layer (A-Water) displayed significant antiallergic and antiinflammatory activities.

[0057] In accordance with cytotoxic activities of the compositions from Aquilaria malaccensis seeds against HepG2, MDA-MB231, and A549 carcinoma cell lines (Table 3), only some of the compositions showed cytotoxic activities at 20 μg/mL level, (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 the compositions towards RBL-2H3 cells, the antiallergic active fraction AM4 exerted therapeutic index up to 28000. To further rule out the possibility that AA/14 causes direct mast cell activation, we examined the capacity of AA/14 to elicit degranulation by itself. Results showed that the AA/14 treatments did not cause significant degranulation as compared with untreated control (Table 1).

[0058] Particularly, in accordance with antiallergic activity of the compositions from Aquilaria malaccensis seeds (Table 1), AA/14-4 (IC.sub.50 4.8×10.sup.+5 μg/mL, therapeutic index 1477328, A23187-induced; and IC.sub.50 6.8×10.sup.+4 μg/mL, therapeutic index 103776, antigen-induced (3-hexosaminidase assay) afforded the most active fraction AA/14-4-8 (IC.sub.50 7.6×10.sup.−6 μg/mL, therapeutic index 9645374, A23187-induced; and IC.sub.50 8.0×10.sup.+5 μg/mL, therapeutic index 9645374, antigen-induced degranulation assay), and a new compound, phorbol ester I (IC.sub.50 values of 0.0017 μM, therapeutic index 71538, A23187-induced; and IC.sub.50 0.011 μM, therapeutic index 10550, antigen-induced degranulation assay).

[0059] In accordance with the above bioactive results in this invention, the phorbol ester I really possesses the antiallergic activity and can be pharmaceutically applied for preventing and treating allergies. Similarly, AA/14-4-9 subfraction comprising phorbol ester I also really possesses the antiallergic activity and can be pharmaceutically applied for preventing and treating allergies. Therefore, AA/14-3-13, AA/14-4-3, and AA/14-4-7 subfractions have the antiallergic activity correspondingly. In accordance with the results of this invention, the extractions and the fractionated fractions (phorbol ester-contained fractions) from Aquilaria malaccensis seeds can be pharmaceutically applied for preventing and treating allergies and show the potent antiallergic activity.

REFERENCES

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[0082] Outstandingly, this invention is characteristic for the advantages of easy collection, simple procedure, and plant growth no-effect due to the phorbol esters preparations from the seeds of Aquilaria malaccensis rather than the stems and barks. Besides, in accordance with the preparation methods of this invention, four new phorbol esters I˜IV were isolated.

[0083] This invention is unprecedented and filled with novelty and progressiveness, thus it is prudential to meet the requirement invention patents, and the patent application is made according to the law.

[0084] All above described is used only for illustrating possible embodiments of the present invention, and therefore it can not limit the protection scope of this invention. All skilled equal changes or modifications in accordance with the present invention are still under the terms of the specification and the scope of the present invention.