Aphanamixins A–F, Acyclic Diterpenoids from the Stem Bark of Aphanamixis polystachya

Abstract

Six new acyclic diterpenoids named Aphanamixins A–F (1–6), together with two known compounds of nemoralisin and nemoralisin C, were isolated from the stem bark of Aphanamixis polystachya (WALL) J. N. BARKER. Their structures were established through a comprehensive analysis of NMR spectroscopic data and high resolution mass spectrometric data. The absolute configurations of carbon stereocenters were determined by means of auxiliary chiral α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA) derivatives and circular dichroism (CD), respectively. All the new isolates were tested for their antiproliferative activity against HepG2, AGS, MCF-7, and A-549 cancer cell lines and they exhibited weak cytotoxicities (IC₅₀>10 µM). Moreover, we highlighted that the six new diterpenoids characterized by acyclic skeleton was rarely seen in nature.

Secondary metabolites obtained from genus Aphanamixis (Meliaceae) were well documented mainly as triterpenes, typically limonoids. Some of them with diverse carbon skeletons displaying cytotoxic, antimalarial, insecticidal and insect growth regulatory activities have attracted considerable attention from medicinal chemists and pharmacologists in recent years.¹⁾ Aphanamixis polystachya (WALL) J. N. BARKER is a timber tree mainly growing in the tropical areas of Asia, such as China, India, Malaysia, and Indonesia.²⁾ Previous chemical investigations on this plant species have led to the isolation of a series of new limonoids.^3,4) In addition, phytochemical studies also revealed the occurrence of unusual diterpenoids from this plant.^5,6) As part of an ongoing investigation on discovery of naturally occurring bioactive agents from genus Aphanamixis, six new acyclic diterpenoids, named Aphanamixins A–F (1–6), together with two known compounds of nemoralisin and nemoralisin C,⁷⁾ were obtained from the stem bark of A. polystachya. This paper mainly deals with the isolation, structural characterization, and in vitro cytotoxic evaluation of the new isolates against HepG2, MCF-7, A-549, and AGS cancer cell lines. Additionally, the six new diterpenoids characterized by acyclic skeleton with rare occurance in nature should be highlighted.

Fig. 1. Chemical Structures of Compounds 1–6

Results and Discussion

Repeated chromatography of the EtOAc-soluble extract over silica gel, Sephadex LH-20, followed by semi-preparative HPLC purification afforded the six new acyclic diterpenoids (1–6) and two known compounds, nemoralisin and nemoralisin C.

Aphanamixin A (1), obtained as colorless oil, was determined to have a molecular formula of C₂₀H₂₈O₄ based on the pseudo-molecular ion peak at m/z 355.1887 [M+Na]⁺ (Calcd 355.1885) in the positive high resolution-electrospray ionization (HR-ESI)-MS. Its IR spectrum displayed characteristic absorption bands corresponding to hydroxyl (3432 cm⁻¹) and carbonyl (1702, 1693 cm⁻¹) groups. The ¹H-NMR spectrum of 1 contained signals were attributed to five methyl groups (two tertiary at δ_H 1.37, and three vinylic at δ_H 1.66, 1.89, 1.92), four olefinic protons which are grouped into two singlet at δ_H 5.59 (s), 5.67 (s) and two triplet at δ_H 6.66 (t, J=7.2 Hz), 5.23 (t, J=7.2 Hz). The ¹³C-APT spectrum of 1 displayed carbon signals inlcuding five methyl groups, four methylene groups (δ_C 27.9, 28.3, 34.4, 39.5), one ketone group (δ_C 209.4), four trisubstituted olefinic bonds at δ_C 99.4, 118.4, 126.1, 127.2, 135.9, 140.9, 160.8, 187.6, one oxygenated quaternary carbon at δ_C 89.8.

The establishment of the two partial structural units a (C-4 to C-6) and b (C-8 to C-10) as shown in boldface in Fig. 2 was elucidated based on the ¹H–¹H correlation spectroscopy (COSY) spetrum. The planar structure of 1 was elucidated based on the heteronuclear multiple bond connectivity (HMBC) spectrum. The linkages of units a and b were determined mainly based on HMBC correlations between δ_H 5.23 (H-6) and δ_C 135.9 (C-7), δ_C 39.5 (C-8); δ_H 1.66 (H₃-19) and δ_C 126.1 (C-6), 135.9 (C-7), 39.5 (C-8). The HMBC spectrum also defined the attachment from C-1 to C-4, since the methyl group (δ_H 1.89, H₃-20) showed correlations to δ_C 118.4 (C-2), 160.8 (C-3), and 34.4 (C-4). Additionally, HMBC correlations indicated the connectivities of the five methyl groups to the diterpene skeleton and the linkage from C-10 to C-15 as shown in Fig. 2. The geometry of the double bonds were determined as 2Z, 6E, 10E, 12Z by observed nuclear Overhauser effect spectroscopy (NOESY) cross peaks (Fig. 2) and comparison of NMR chemical shifts with similar compounds.^7,8) Therefore, the structure of 1 was unequivocally established as shown in Fig. 1 with a given name of Aphanamixin A.

Fig. 2. Key ¹H–¹H COSY (━), HMBC (→) and NOESY () Correlations of 1

Aphanamixin B (2) was purified as colorless oil. Its molecular formula C₂₀H₃₀O₄ was determined by the HR-ESI-MS data, evidencing two hydrogen atoms more than 1. ¹H- and ¹³C-NMR data were superposable upon those of 1, apart from the absence of Δ²⁽³⁾ double bond in 2, which was supported by the HMBC correlations between H-11 (δ_H 2.70) and C-10 (δ_C 34.9), C-12 (δ_C 199.5), C-18 (δ_C 18.3). Further COSY and HMBC correlations confirmed the structure 2 as depicted. NOESY correlations of 2 allowed the assignment of the olefinic geometries. However, the configuration of C-11 could not be determined from the available data.

Aphanamixin C (3), obtained as a colorless oil, has the molecular formula C₂₀H₂₈O₅ as determined by HR-ESI-MS and NMR data. According to its molecular formula and NMR spectroscopic data, 3 was determined to have the same carbon framework as 1, differing only in that the methylene of C-8 in 1 was assigned as an oxygen bearing methine carbon in 3, as confirmed by the HMBC cross peaks from H-8 (δ_H 4.10) to C-6 (δ_C 128.2), C-7 (δ_C 137.7), C-9 (δ_C 35.7), C-10 (δ_C 138.7), C-19 (δ_C 13.4). Similarly, the olefinic geometries were established in the same manner as for 1.

The configuration at C-8 could not be assigned from the NOESY spectrum because of the flexibility of aliphatic chain. Therefore, the Mosher ester method was applied for determination of the absolute configuration of 3.⁹⁾ Treatment of 3 with R-(−)- and (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) gave the (S)- and (R)-MTPA esters 3a and 3b, respectively. The ¹H-NMR signals of the two MTPA esters were assigned unambiguously based on ¹H–¹H COSY spectra, and the Δδ_H (S–R) values were then calculated. The results indicated the 8S absolute configuration as shown in Fig. 3.

Aphanamixin D (4), with the molecular formula C₂₀H₃₀O₅, was assigned as a derivative of 3 from its spectroscopic data. Its ¹H- and ¹³C-attached proton test (APT) data were compatible with those of 3 except the absence of Δ²⁽³⁾ double bond in 4. This was unambiguously confirmed by the observed HMBC cross peaks from H-20 (δ_H 0.96) to C-2 (δ_C 42.8), C-3 (δ_C 31.4), and C-4 (δ_C 37.6). The absolute configuration of C-8S was resolved by applying the Mosher method as shown in Fig. 3. However, the configuration of C-3 could not be assigned from the available data.

Fig. 3. Key Values of Δδ_H (S–R) for the MTPA Esters of 3 and 4 (in CD₃OD)

Aphanamixin E (5) was obtained as coloress oil. Its molecular formula was determined to be C₂₀H₃₀O₅ as based on the results of HR-ESI-MS with a quasi molecular ion peak of [M+Na]⁺ at m/z 373.1979 (Calcd 373.1991). Its ¹H- and ¹³C-NMR data suggested that 5 was an analogue of 1. The differences were the presence of a hydroxyl group at C-3 and the absence of Δ²⁽³⁾ double bond in 5, which could be demonstrated by the HMBC cross peaks from H-20 (δ_H 1.27) to C-2 (δ_C 46.7), C-3 (δ_C 72.2), and C-4 (δ_C 42.9).

The chiral center of C-3 was successfully resolved by analyzing the CD data of the in situ formed [Rh₂(OCOCF₃)₄] complex, with the inherent contribution subtracted. The Rh-complex of 5 displayed a positive Cotton effect at λ_max 342.5 nm (the E band), correlating with a 3S absolute configuration on basis of the bulkiness rule for tertiary alcohols.^10,11)

Aphanamixin F (6) gave the molecular formula C₂₀H₃₂O₅, with two more hydrogen atoms than 5, as shown by HR-ESI-MS. Comparison of the NMR data between compounds 5 and 6 indicated the absence of Δ¹⁰⁽¹¹⁾ double bond in 6. This was unambiguously confirmed by the observed HMBC cross peaks from H-18 (δ_H 1.21) to C-10 (δ_C 37.0), C-11 (δ_C 34.9) and C-12 (δ_C 199.4). Furthermore, the stereochemistry of C-3 was tentativley determined to be 3S, the same as compounds 5, on the biogenic consideration.

Experimental

General Experimental Procedures

Optical rotations were obtained on a Perkin–Elmer 341 digital polarimeter. UV spectra were recorded on a Shimadzu UV2550 spectrometer. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectropolarimeter. IR spectra were measured on a FTIR-8400S spectrometer. One-dimensional (¹H, ¹³C-APT) and two-dimensional (¹H–¹H COSY, HSQC, HMBC) NMR experiments were performed on Bruker AV III 600 spectrometers operating at 600 MHz for ¹H and 150 MHz for ¹³C, respectively (tetramethylsilane (TMS) an internal standard). Chemical shifts are expressed in δ (ppm) referenced to solvent peaks at δ_H 3.31 and δ_C 49.2 for CD₃OD, and coupling constants are in Hz. HR-ESI-MS spectra were obtained from a Thermo Scientific LTQ-Obitrap XL instrument (Thermo Scientific, Bremen, Germany). Silica gel (200–300 mesh, Qingdao Marine Chemistry Co., Ltd.) and Sephadex LH-20 (Amersham Pharmacia Biotech AB) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F₂₅₄, 0.25 mm) were used for TLC analyses. HPLC chromatography was performed on a lumtech K1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and a column of ZORAX SB-phenyl (250×10 mm, 5 µm, Agilent Technologies Co., Ltd.). Mixtures of MeOH–H₂O were used as the eluents. All solvents used were of analytical grade (Beijing Chemical Works, Beijing, P. R. China).

Sample Collection

The stem bark (4.0 kg) of Aphanamixis polystachya (WALL) J. N. BARKER (Meliaceae) was collected in July 2012 from Tunchang County, Hainan Province, and was identified by Prof. Jianpin Tian, School of Pharmaceutical Science, Hainan Medical University, Haikou City, Hainan Province, P. R. China. A voucher specimen (No. AP21020720-B) was deposited in the Herbarium of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College.

Extraction and Isolation

The air-dried stem bark of A. polystachya (4.0 kg) was extracted with 95% EtOH (3×10.0 L) under reflux for 1.5×3 h. The ethanol extract was filtered and concentrated under reduced pressure to yeild a crude extract (500.0 g), which was suspended in distiled H₂O (5.0 L) and then successively partitioned with hexane (3×2.0 L) and ethyl acetate (3×2.0 L) to afford EtOAc-souble fraction (150.0 g). The EtOAc-souble fraction was subjected to silica gel chromatography (1000 g, 200–300 mesh, 10×110 cm) eluting with a gradient of EtOAc in hexane (0 : 1, 20 : 1, 10 : 1, 5 : 1, 2 : 1, v/v) to give five fractions (Frs. A–E) monitored by TLC. Compound 6 (5.0 mg) was obtained from Fr. B (3.5 g) by a silica gel chromatography (100 g, 200–300 mesh, 5×20 cm) eluted with a mixture of hexane–acetone (85 : 15, v/v), followed by preparative HPLC eluting with MeOH–H₂O (65 : 35). Fraction C (2.8 g) was further chromatographed over a Sephadex LH-20 column (2.5×120 cm) using CHCl₃–CH₃OH (50 : 50, v/v) as a mobile phase, yielding three fractions (Frs. C1–3). Fraction C2 (850.0 mg) was purified by preparative HPLC eluting with MeOH–H₂O (65 : 35, v/v, flow rate 2.0 mL/min) to afford compouds 1 (25.0 mg) and 4 (5.0 mg). Fraction D (1.5 g) was subjected to Sephadex LH-20 column chromatography (2.5×120 cm) eluting with CHCl₃–CH₃OH (50 : 50, v/v) to give four fractions (Frs. 4A–D). Fraction 4D (85.0 mg) was passed over preparative column eluting with MeOH–H₂O (60 : 40, v/v, flow rate 2.0 mL/min) to afford compoud 3 (7.0 mg). Fraction D (1.7 g) was subjected to a reversed-phase C₁₈ silica gel medium-pressure column (MeOH–H₂O, v/v, 1 : 1→1 : 0, flow rate 5.0 mL/min) to give four fractions (Frs. 4A–D). Fraction E (2.8 g) was purified over a column chromatography of Sephadex LH-20 (2.5×120 cm) eluting with CH₃OH followed by preparative HPLC chromatography eluting with MeOH–H₂O (55 : 45, v/v, flow rate 2.0 mL/min) to yield compounds 2 (8.0 mg) and 5 (10.0 mg).

Aphanamixin A (1): Colorless oil (MeOH). ¹H- and ¹³C-NMR (CD₃OD) data: see Tables 1 and 2; IR (KBr) ν_max 3432, 2975, 2932, 1702, 1693, 1581, and 1381 cm⁻¹; UV (MeOH) λ_max (log ε) 202 (2.52) nm, 261 (2.30) nm; (+)-HR-ESI-MS m/z 355.1887 [M+Na]⁺ (Calcd for C₂₀H₂₈O₄Na, 355.1885).

Table 1. ¹H-NMR Data for Aphanamixins A–F (1–6) (600 MHz, CD₃OD)

Position	1	2	3	4	5	6
2	5.67 br s	5.66 br s	5.70 br s	a 2.28 dd (14.4, 6.6)	2.45 s	2.43 s
				b 2.08 ma)
3	—	—	—	1.94, m	—	—
4	2.64 t (7.2)	2.65 t (7.2)	2.66 t (7.2)	a 1.26 m	1.57 t (7.2)	1.56 t (7.2)
				b 1.42 m
5	2.20 q (7.2)	2.20 q (7.2)	2.24 q (7.2)	2.08 ma)	2.10 q (7.2)	2.08 q (7.2)
6	5.23 t (7.2)	5.18 t (7.2)	5.46 t (7.2)	5.42 t (7.2)	5.19 t (7.2)	5.17 t (7.2)
7	—	—	—	—	—	—
8	2.15 t (7.2)	2.00 dt (7.2, 2.4)	4.10 t (7.2)	4.11 t (7.2)	2.16 t (7.2)	2.00 t (7.2)
9	2.39 q (7.2)	1.44 m	2.50 q (7.2)	2.50 q (7.2)	2.39 q (7.2)	1.44 m
10	6.66 t (7.2)	a 1.63 m	6.68 t (7.2)	6.67 t (7.2)	6.67 t (7.2)	a 1.63 m
		b 1.47 m				b 1.47 m
11	—	2.70 m	—	—	—	2.70 m
12	—	—	—	—	—	—
13	5.59 s	5.42 s	5.59 s	5.60 s	5.59 s	5.42 s
14	—	—	—	—	—	—
15	—	—	—	—	—	—
16	1.37 s	1.35 s	1.37 s	1.37 s	1.37 s	1.34 s
17	1.37 s	1.35 s	1.37 s	1.37 s	1.37 s	1.34 s
18	1.92 s	1.22 d (7.2)	1.93 s	1.93 s	1.92 s	1.22 d (6.6)
19	1.66 s	1.60 s	1.66 s	1.65 s	1.66 s	1.60 s
20	1.89 s	1.89 s	1.90 s	0.96 d (6.6)	1.27 s	1.26 s

a) Signals were overlapped.

Table 2. ¹³C-NMR Data for Aphanamixins A–F (1–6) (150 MHz, CD₃OD)

Position	1	2	3	4	5	6
1	—a)	—	—	—	—	—
2	118.4	118.0	118.2	42.8	46.7	46.7
3	160.8	161.3	161.0	31.4	72.2	72.2
4	34.4	34.4	34.2	37.6	42.9	43.0
5	27.9	27.9	35.7	26.3	23.8	23.8
6	126.1	125.5	128.2	127.6	126.8	126.2
7	135.9	136.5	137.7	138.0	135.2	135.8
8	39.5	40.6	77.5	77.5	39.5	40.5
9	28.3	26.4	35.7	35.7	28.2	26.4
10	140.9	34.9	138.7	137.7	141.0	34.9
11	127.2	37.1	126.9	128.2	127.2	37.0
12	187.6	199.5	187.5	187.5	187.6	199.4
13	99.4	100.8	99.5	99.5	99.4	100.8
14	209.4	210.7	210.2	210.2	210.2	210.5
15	89.8	90.0	89.9	89.9	89.8	90.0
16	23.5	23.2	23.5	23.4	23.5	23.2
17	23.5	23.2	23.5	23.4	23.5	23.2
18	13.2	18.3	11.7	13.4	13.3	18.3
19	16.2	16.1	13.4	11.8	16.1	15.9
20	25.5	25.5	25.8	20.3	27.4	27.3

a) Signals were undetected.

Aphanamixin B (2): Colorless oil (MeOH). ¹H- and ¹³C-NMR (CD₃OD) data: see Tables 1 and 2; IR (KBr) ν_max 3428, 2931, 1702, 1556, and 1382 cm⁻¹; UV (MeOH) λ_max (log ε) 203 (2.24) nm, 261 (3.16) nm; (+)-HR-ESI-MS m/z 357.2032 [M+Na]⁺ (Calcd for C₂₀H₃₀O₄Na, 357.2042); CD (0.008 M, MeOH) λ_max (Δε) 212 (−10.6), 232 (−0.48) nm; [α]_D²⁵ −6.0 (c=0.1, MeOH).

Aphanamixin C (3): Coloress oil (MeOH). ¹H- and ¹³C-NMR (CD₃OD) data: see Tables 1 and 2; IR (KBr) ν_max 3420, 2974, 2933, 1705, 1695, 1581, 1383, and 1250 cm⁻¹; UV (MeOH) λ_max (log ε) 202 (2.47) nm, 262 (2.18) nm; (+)-HR-ESI-MS m/z 371.1822 [M+Na]⁺ (Calcd for C₂₀H₂₈O₅Na, 371.1834); [α]_D²⁵ −14.0 (c=0.1, MeOH).

Aphanamixin D (4): Colorless oil (MeOH). ¹H- and ¹³C-NMR (CD₃OD) data: see Tables 1 and 2; IR (KBr) ν_max 3425, 2974, 2933, 1710, 1690, 1583, and 1382 cm⁻¹; UV (MeOH) λ_max (log ε) 201 (2.47) nm, 260 (2.27) nm; (+)-HR-ESI-MS m/z 373.1978 [M+Na]⁺ (Calcd for C₂₀H₃₀O₅Na, 373.1991); [α]_D²⁵ −24.0 (c=0.1, MeOH).

Aphanamixin E (5): Colorless oil (MeOH). ¹H- and ¹³C-NMR (CD₃OD) data: see Tables 1 and 2; IR (KBr) ν_max 3445, 2934, 1702, 1690, and 1580 cm⁻¹; UV (MeOH) λ_max (log ε) 201 (1.45) nm, 294 (3.10) nm; (+)-HR-ESI-MS m/z 373.1979 [M+Na]⁺ (Calcd for C₂₀H₃₀O₅Na, 373.1991); [α]_D²⁵ −22.0 (c=0.1, MeOH); CD (0.007 M, MeOH) λ_max (Δε) 211 (−9.6), 230 (−0.34) nm; [Rh₂(OCOCF₃)₄]-induced CD (CDCl₃, Δε) λ_max 342.5 (+0.0984) nm.

Aphanamixin F (6): Colorless oil (MeOH). ¹H- and ¹³C-NMR (CD₃OD) data: see Tables 1 and 2; IR (KBr) ν_max 3434, 2931, 1706, 1692, 1580, and 1384 cm⁻¹; UV (MeOH) λ_max (log ε) 201 (1.47) nm, 260 (3.27) nm; (+)-HR-ESI-MS m/z 375.2137 [M+Na]⁺ (Calcd for C₂₀H₃₂O₅Na, 375.2147); CD (0.007 M, MeOH) λ_max (Δε) 211 (−8.6), 233 (−0.38) nm; [α]_D²⁵ −21.0 (c=0.1, MeOH).

Preparation of Mosher Esters

Aphanamixins C, D (1.0 mg in 1.0 mL of CH₂Cl₂, respectively) were sequentially added pyridine (0.1 mL), 4-(dimethyl-amino)pyridine (0.1 mg), and 15 mg of (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride. The mixture was stirred at rt from 4 h to overnight and passed through a disposable pipette (0.4×4 cm) containing silica gel (100–200 mesh) and eluted with 3.0 mL of CH₂Cl₂. The CH₂Cl₂ residue, dried in vacuo, was redissolved in CH₂Cl₂ and washed in 1% NaHCO₃ (5.0 mL) and distilled H₂O (2×5.0 mL), the CH₂Cl₂ layer was dried in vacuo to give the (S)-Mosher esters. Using (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride gave the (R)-Mosher esters.

Absolute Configuration of the Tertiary Alcohol Moiety in 5

Compound 5 (0.5 mg) was dissolved in a dry solution of [Rh₂(OCOCF₃)₄] complex (1.0 mg) in CDCl₃ (300 µL), respectively. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary. The inherent CD was subtracted. The observed sign of the E band near 350 nm in the induced CD spectrum was correlated to the absolute configuration of tertiary alcohol moiety.^10,11)

Cytotoxicity Bioassays

The following human tumor cell lines were used: HepG2, A-549, AGS, and MCF-7. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% fetal bovine serum and cultured at a density of 6×10⁴ cells/mL per well in a 96-well microtiter plate. The cytotoxicity assay was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method in 96-well microplates. In short, adherent cells (100 µL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, and suspended cells were seeded just before drug addition with an initial density of 1×10⁵ cells/mL. Each tumor cell line was exposed to the tested compounds at different concentrations in triplicate for 48 h. Doxorubicin (Sigma, St. Louis, MO, U.S.A.) was used as positive control. After treatment, cell viability was measured and the cell growth curve was plotted. Half maximal inhibitory (IC₅₀) values were calculated by the Reed and Muench method.

Table 3. In-Vitro Antiproliferative Activity of Compounds 1–6

Compounds	IC50(µM)
	HepG2	A-549	MCF-7	AGS
1	23.2±3.2 a)	38.6±6.7	34.5±5.9	28.4±3.8
2	32.6±6.2	25.8±7.2	38.5±8.3	25.8±2.8
3	35.8±6.3	38.8±7.4	40.5±9.2	15.8±2.7
4	45.5±8.4	34.8±5.7	36.2± 7.4	14.6± 3.4
5	25.4±4.2	26.6±6.1	32.8±7.4	26.8±6.5
6	26.8±6.4	24.8±5.1	26.2± 6.4	34.3± 7.4
Doxorubicinb)	1.2±0.02	0.74±0.01	0.59±0.13	0.94±0.08

a) Values present mean±S.D. of triplicate experiments. b) Positive control substance.

Acknowledgment

The authors acknowledge the financial assistance by the National Natural Science Foudation of China (No. 81202994, 31000137). This work was also financially supported from the technological large platform for comprehensive research and development of new drugs in the Twelfth Five-Year “Significant New Drugs Created” Science and Technology Major Projects (No. 2012ZX09301-002-001-026), and the chemical composition of the digital library of traditional Chinese medicine of drug discovery in the Twelfth Five-Year “Significant New Drugs Created” (No. 2011ZX09307-002-01).

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