Diterpenoids from the needles and twigs of the cultivated endangered pine Pinus kwangtungensis and their PTP1B inhibitory effects

Phytochemistry Letters 20 (2017) 239–245

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Diterpenoids from the needles and twigs of the cultivated endangered pine Pinus kwangtungensis and their PTP1B inhibitory effects

MARK

⁎

Chang-Ling Hua, Juan Xionga, , Pei-Pei Wangb, Guang-Lei Maa, Yu Tanga, Guo-Xun Yanga, Jia Lib, ⁎ Jin-Feng Hua, a b

Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China

A R T I C L E I N F O

A B S T R A C T

Chemical compounds studied in this article: Lambertianic acid (PubChem CID: 11869599) Cassipourol (PubChem CID: 11500301)

Pinus kwangtungensis is an endangered pine species native to China. In the present study, 15 diterpenoids including three new labdane-type analogs were isolated and characterized during a pioneer phytochemical investigation on a mass-limited sample of the needles and twigs of this plant, which is growing in a Cantonese garden. The new structures, (4S,5R,9S,10R)-6-oxo-labd-7,13-dien-16,15- olid-19-oic acid (1), 15(S)-n-butoxypinusolidic acid (2), and β-D-glucopyranosyl- (4S,5R,9S,10R)-labda-8(17),13-dien-15,16-olid-19-oate (3), were established by extensive spectroscopic methods and some chemical transformations. Among the isolates, lambertianic acid (10) and cassipourol (15) showed inhibitory activities against human protein tyrosine phosphatase 1 B (PTP1B), a target for the treatment of type-II diabetes and obesity, with IC50 values of 25.5 and 11.2 μM, respectively.

Keywords: Pinus kwangtungensis Pinaceae Endangered plant Diterpenoids PTP1B inhibition

1. Introduction Naturally occurring compounds from the rare and endangered plants have been documented to show greater potential for drug discovery (Ibrahim et al., 2013; Zhu et al., 2011). It is an urgent need to prioritize protection and utilization of these fragile plants at risk and facing extinction. Since 2013, we have launched a new program to systematically identify novel bioactive natural products from the rare and endangered plants endemic to China (Hu et al., 2016a,b; Ma et al., 2016; Wang et al., 2015, 2016; Wu et al., 2016; Xiong et al., 2015, 2016). In particular, great attentions have been paid to the rare and endangered species in the Pinaceae family, which ranks among the top20 privileged drug-prolific families that produced high numbers of approved drugs (Fig. 1, Zhu et al., 2011). In our previous studies, a number of novel terpenoids with interesting bioactivities were obtained, e.g., dabeshanensins A–K from the endangered plant Pinus dabeshanensis (Hu et al., 2016a), and beshanzuenones A–D from the critically endangered plant Abies beshanzuensis (Hu et al., 2016b). Among the 39 Pinaceae plants recorded in the China Plant Red Data Book (CPRDB) (Fu and Jin, 1992), Pinus kwangtungensis Chun ex Tsiang is characterized as a five-needled pine that generally inhabits the summits, cliffs or slopes of some remote mountains in southern China (Tian et al., 2008). Global warming and long-term deforestation threaten its existence and genetic integrity, and this species is listed

⁎

Corresponding authors. E-mail addresses: [email protected] (J. Xiong), [email protected] (J.-F. Hu).

http://dx.doi.org/10.1016/j.phytol.2017.05.006 Received 28 October 2016; Received in revised form 28 April 2017; Accepted 8 May 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

as “threatened” in the CPRDB (Fu and Jin, 1992). To our knowledge, only a composition of pimaric-type resin acids from this plant has been previously analyzed (Chen et al., 2008). As part of our ongoing research towards the discovery of novel bioactive agents from the wild and/or cultivated endangered Pinaceae plants (Hu et al., 2016a,b), three new (1–3) and 12 known (4–15) diterpenoids were isolated from the masslimited (290 g, dried) needles and twigs of the title plant, which is growing in the Foshan Botanical Garden at Canton (kwangtung) of China. Herein, we report their isolation and structure elucidation, as well as their inhibitory activities against the enzyme of human protein tyrosine phosphatase 1 B (PTP1B). 2. Results and discussion From the 90% MeOH extract of the needles and twigs of P. kwangtungensis, ten labdane-type (1–10), two abietane-type (11, 12), two podocarpane-type (13, 14), and one retinane-type (15) diterpenoids were obtained and characterized (Fig. 2). By comparison of their spectroscopic data and physicochemical properties with those reported in the literature, the known ones were identified as 15(R)-n-butoxypinusolidic acid (4, Kim et al., 2012), adenanthoside C (5, Wu et al., 2015), pinosolide acid (= pinusolidic acid, 6, Calderón et al., 1987; Fang et al., 1989; Yang and Han, 1998), pinusolide (7, Hu et al., 2016a; Raldugin et al., 1970), 15ξ-hydroxypinusolidic acid [= 15-oxo-

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Fig. 1. Pinaceae ranks among the top-20 privileged drug-prolific families that produced high numbers of approved drugs (Zhu et al., 2011).

for 1, which was concluded to be Δ7,8-6-one from the key HMBC correlations from H-5 to C-6, from H-7 to C-5/C-17, and from Me-17 to C-7/C-8 (Fig. 3). The relative configuration of 1 was assigned as shown in Fig. 3 by the observed NOE correlations of Me-18/H-5, H-5/H-9, and Me-20/Hb11. In consistency with this deduction, the 13C chemical shift of Me-18 (δC 28.3) is typical of an equatorial disposition. In general, the axialoriented methyl is resonating at a higher field (Δ = ca. −10 ppm) than such a group at the equatorial position due to the δ-syn axial effect with Me-20 (Barrero and Altarejos, 1993; San Feliciano et al., 1993). Its absolute configuration was then deduced by analysis of the electronic circular dichroism (ECD) spectrum, from which a diagnostic negative Cotton effect at 243 nm (Δε −13.4) due to the π−π* transition of the conjugated enone group was observed. As summarized by Burgstahler et al., the Cotton effect in the region of the ultraviolet maximum (230–260 nm) in a cyclic conjugated enone is usually dominated by allylic axial perturbation(s) of the double bond (Burgstahler and Barkhurst, 1970). Thus, the absolute configuration at C-9 in 1 could be assigned as S due to the left-handed allylic axial chirality contribution of H-9 adjacent to the unsaturated ketone chromophore (Burgstahler and Barkhurst, 1970). Consequently, the structure of compound 1 was characterized as (4S,5R,9S,10R)-6-oxo-labd-7,13dien-16,15-olid-19-oic acid. Compound 2 was assigned the molecular formula C24H36O5 by the positive mode HRESIMS (m/z 405.2636 [M + H]+) and its 13C NMR data. The IR absorption bands at 1766 and 1722 cm−1 gave hints of the presence of α,β-unsaturated γ-lactone and carboxyl functionalities. Similar to pinosolide acid (6), the presence of two singlet methyls [δH 1.25 (s, Me-18), δC 29.0; 0.61 (s, Me-20), δC 12.8], an exocyclic double bond [δH 4.58 and 4.90 (each br s, H2-17), δC 106.9], an α,β-

8(17),13-labdadiene-16,19-dioic acid, lactol form, 8, Asili et al., 2004], 16-hydroxy-8(17),13-labdadien-15,16-olid-19-oic acid [= 16-oxo8(17),13- labdadiene-15,19-dioic acid, lactol form, 9, Asili et al., 2004], lambertianic acid (10, Asili et al., 2004; Dauben and German, 1966; Fang et al., 1991), abieta-7,13-diene-18-oic acid (11, Wang et al., 2008; Yang et al., 2010), 7ɑ,15-dihydroxy-8,11,13-abietatrien-18-oic acid (= 7ɑ,15-dihydroxy-dehydroabietic acid, 12, Prinz et al., 2002; Yang et al., 2010), 8(14)-podocarpen-13-on-18-oic acid (13, Yang et al., 2008), abiesanordine E (14, Yang et al., 2008), and cassipourol (15, Chaturvedula et al., 2006), respectively. Compound 1 was obtained as a colorless oil and its molecular formula was determined to be C20H26O5 from a pseudo-molecular ion at m/z 347.1853 ([M+H]+, calcd 347.1853) in its positive HRESIMS and from its 13C NMR data, requiring eight degrees of unsaturation. The IR spectrum of 1 showed the existence of α,β-unsaturated γ-lactone (1756 cm−1), carboxyl (1721 cm−1) and conjugated enone (1678 cm−1) functionalities. Its 1H NMR data (Table 1) contained signals typical for two tertiary methyls (δ 1.45, s, Me-18; 0.81, s, Me20), one vinylic methyl (δ 2.10, s, Me-17), one oxymethylene (δ 4.83, 2H, br s, H2-15), and two olefinic protons (δ 6.00, s, H-7; 7.21, br s, H14). The 13C NMR spectrum of 1 (Table 1) exhibited twenty carbon resonances, which were classified by DEPT and HSQC NMR experiments as three methyls, six methylenes (one oxygenated at δ 70.3), four methines (two olefinic at δ 127.0 and 145.0), four quaternary carbons (two olefinic at δ 133.5 and 164.0), and three carbonyl groups (δ 174.0, 176.4, and 205.3). The above spectroscopic data resembled those of pinosolide acid (6, Calderón et al., 1987; Fang et al., 1989), suggested that compound 1 is also a bicyclic labdane-type diterpenoid featuring an α,β-unsaturated γ-lactone group. Differing from the exocyclic double bond Δ8,17 presented in 6, an α,β-unsaturated ketone group was found

Fig. 2. Chemical structures of diterpenoids 1–3.

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CHCl3)} and 4 {[α]D20 +42.0 (c 0.05, CHCl3)} have similar positive specific rotations. The above data suggested that these two compounds should be a pair of C-15 epimers. This inference was confirmed by the change in sign of the Cotton effects around 250 nm [(15S)-2: Δε246 (+3.42); (15R)-4: Δε250 (−1.06), Fig. 4] for the π−π* transition of the unsaturated lactone due to the different configuration at C-15 (Kim et al., 2012). Thus, the structure of 2 was determined as 15(S)-nbutoxypinusolidic acid. Indeed, the alcoholysis (e.g., methanolysis, ethanolysis, or butanolysis) of natural hemiacetals to form the corresponding acetals during isolation and purification is commonly occurred when alcohol solvents (e. g., MeOH, EtOH, and n-BuOH) are used (Chien et al., 2004; Kim et al., 2012; Wu et al., 2013). Compound 3 showed an [M + Na]+ ion peak at m/z 517.2413 in the positive mode HRESIMS, which in conjunction with its 13C NMR data indicated a molecular formula of C26H38O9. Among the twenty-six carbon resonances observed in the 13C NMR spectrum of 3, twenty ones accounted for the labdane backbone. The remaining six oxygen-bearing carbons (δ 95.6, 78.8, 78.5, 74.1, 71.2, 62.5) were typical of a monosugar unit (Table 1). This revealed a closely structural similarity between compound 3 and adenanthoside C (5) (Wu et al., 2015), a known labdane-type diterpene glycoside previously isolated from the roots of Isodon adenantha. The only difference between the two glycosides is that the 16,15-olide moiety presented in 5 was replaced by a 15,16-olide group in compound 3. This was confirmed by the key HMBC correlations from H-12 to C-16 (δ 75.0), C-14 (δ 115.2), and C13 (δ 174.9), along with the absence of a correlation from H-12 to the carbonyl group at δ 177.1 (C-15) (Fig. 5). The relative configuration of the bicyclic diterpene core in 3 was assumed to be the same as that of 5 by analyses of the proton–proton coupling constants JH-5/Hβ-6 (13.6 Hz) and JH-9/Ha-11 (12.6 Hz) (Table 1), and the NOE correlations of Me-20/ Hb-11 (δ 1.70), Me-18/H-5 and H-5/H-9 (Fig. 5). Similar to adenanthoside C (5), the glycosidic linkage position at C19 in 3 was determined by the distinctive HMBC correlation from H-1′ (δ 5.42) to C-19 (δ 177.5) (Fig. 5). The β-orientation of the anomeric proton (H-1′, δ 5.42) was evident from its J value (7.8 Hz). The identification of the glucose moiety in the mass-limited 3 and its absolute configuration could be determined by analogy with cooccurring 5 based on their similar NMR data (Table 1). Finally, acid hydrolysis of 5 gave a monosaccharide (Scheme 1), which was identified to be D-glucose by the direct comparison of its HPLC profile and optical rotation datum with an authentic sample (see Experimental section). Interestingly, the ESI–MS and 1H NMR spectroscopic data of the obtained aglycone (5a) revealed the presence of an unexpected Δ8,9 double bond instead of the exocyclic methylene group (i. e., Δ8,17) in 5. This might be resulted from a double-bond isomerization reaction under acidic conditions (Chan et al., 1971; Hu and Zhou, 1982). A supplementary enzymatic hydrolysis of 5 with β-glucosidase gave the desired aglycone 5b (Scheme 1), which was found to be identical to pinosolide acid (6) by comparing their spectroscopic (NMR, MS) and [α] data. In particular, 5b was recognized as a normal labdane-type diterpenoid (the Me-20 group is β-oriented) based on its positive specific rotation, which was similar to that of 6 {[α]D20 +54.5° (c 0.1, CHCl3)} (Hu et al., 2016a; Yang and Han, 1998) but much different from the negative value reported for the ent-6 {[α]D20 −33.0° (c 0.74, MeOH)} (Waridel et al., 2003). Furthermore, considering that the specific rotation of 3 {[α]D20 +10.0° (c 0.1, CHCl3)} was much closer to that of 5 {[α]D20 +14.0° (c 0.1, CHCl3)}, the absolute configuration of the aglycone in both compounds should be the same. Based on the above evidence, compound 3 was identified as β-D-glucopyranosyl(4S,5R,9S,10R)-labda-8(17),13-dien-15,16-olid-19-oate. All the isolated compounds were evaluated for their inhibitory activity against PTP1B, a target for the treatment of type-II diabetes and obesity (Alonso et al., 2004; Johnson et al., 2002; Hu et al., 2016a). Among the 15 compounds tested, cassipourol (15) exhibited the most potent PTP1 B inhibitory activity, with an IC50 value of 11.2 ± 0.9 μM. Lambertianic acid (10), a labdane-type diterpenoid featuring a furan

Table 1 1 H and 13C NMR Data (δ in ppm, J in Hz) of Compounds 1-3a. No.

1β 1α 2β 2α 3β 3α 4 5

1

b

2

7α 8 9 10 11a 11b 12a 12b 13 14 15 16

δH

δC

δH

δC

1.98, br d (13.0) 1.29, dd (13.0, 13.0) 1.91, m 1.54, m 2.33, br d (13.0) 0.95, dd (13.0, 13.0)

39.0

1.83, m

39.2

1.87, m

40.5

1.04, m 18.5 39.1

43.6 64.4 205.3

6.00, s

2.27, br d (13.0) 1.75, 1.73, 2.59, 2.41,

m m m m

7.21, br s 4.83, br s, 2H

127.0

164.0 55.3 44.6 25.7 27.4 133.5 145.0 70.3 174.0 22.5

18 19 20 1′

1.45, s

28.3 176.4 13.4

b c

0.81, s

1.86, m 1.52, m 2.16, br d (13.0)

1.14, m 19.8 38.0

1.04, m

2.10, s

a

c

δC

17

2′ 3′ 4′ 5′ 6′

3

δH

2.63, s

6β 6α 7β

b

1.33, 3.6) 1.85, 1.99, 1.99, 4.0) 2.42,

dd (13.4, m m dd (13.0,

m m m m

6.78, br s 5.80, br s

4.90, br s 4.58, br s 1.25, s 0.61, 3.86, 6.8) 3.66, 6.8) 1.62, 1.40, 0.94,

26.1 38.6

m

1.62, dd, overlapped 1.80, 1.52, 2.48, 2.10,

44.1 56.2

s dt (16.0,

1.95, m 1.51, m 2.20, br d (13.0) 1.10, m

1.42, dd (13.6, 3.6) 1.82, m 2.02, m 1.93, m

21.1 39.1

45.7 57.7 27.3 39.8

2.41, m 147.2 55.7 40.5 21.6 24.5 139.0 141.8 101.8 171.6 106.9 29.0 182.3 12.8 70.2

1.68, dd (12.6, 3.6) 1.84, 1.70, 2.58, 2.29,

m m m m

5.87, br s 4.83, 2H 4.90, 4.54, 1.24,

d (1.7), br s br s s

0.64, s 5.42, d (7.8)

149.0 57.0 41.7 22.7 28.6 174.9 115.2 177.1 75.0 107.1 29.3 177.5 13.8 95.6

dt (16.0, m, 2H m, 2H t (6.8)

31.5 19.1 13.8

3.36, m 3.33, m 3.33, m 3.33, m 3.80, dd (12.8, 2.4) 3.66, dd (12.8, 4.8)

74.1 78.8 71.2 78.5 62.5

Assignments were made by a combination of 1D and 2D NMR experiments. Recorded in CDCl3. Recorded in methanol-d4.

unsaturated γ-lactone moiety [δH 6.78 (br s, H-14); δC 139.0 (C-13), 141.8 (C-14), 171.6 (C-16)], and a carboxyl group (δC 182.3) could be readily recognized from the 1H and 13C NMR data of 2 (Table 1). Besides, signals characteristic for an acetal group [δH 5.80 (br s, H-15), δC 101.8], along with resonances attributable to an additional n-butoxy substitute were also observed for 2 (Table 1). The above spectroscopic data were found to be almost identical to those of 15(R)-n-butoxypinusolidic acid (4), a known labdane-type diterpenoid previously isolated from the pine cone of Thuja orientalis (Kim et al., 2012). Further analysis of the NOESY spectrum (see Supporting information) of 2 confirmed that the relative configurations at C-4, C-5, C-9, and C10 were consistent with those of compound 4. Nevertheless, compound 2 (tR = 23.0 min) has a different retention time from that of 4 (tR = 25.7 min) when comparing their reverse-phase HPLC profiles by employing a normal ODS column (for details, see Experimental section and Supporting information). Meanwhile, 2 {[α]D20 +52.5 (c 0.04, 241

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Fig. 3. 1H–1H COSY, key HMBC and NOE correlations of compound 1.

limited sample (290 g, dried) of the cultivated endangered pine P. kwangtungensis. Unlike P. dabeshanensis, for which abietane-type diterpenoids were found to be the major secondary metabolites (Hu et al., 2016a), P. kwangtungensis is rich in labdane-type diterpenoids. In our previous reports, naturally occurring pimarane- and abietane-type diterpenoids were found to show remarkable PTP1 B inhibitory effects (Hu et al., 2016a; Xiong et al., 2015). A literature survey revealed that, kaurane- and labdane-type diterpenoids also tend to be potent PTP1 B inhibitors (Jung et al., 2012, 2013; Li et al., 2012). In this study, only one labdane- (10) and one retinane-type (15) diterpenoids were found to show PTP1 B inhibitory effects. To our knowledge, cassipourol (15) is reported herein as the first retinane-type diterpene from the whole Pinaceae family, and it represents the first retinane derivative with PTP1 B inhibitory activity. Our findings may contribute to the therapeutic potential of structurally diverse diterpenoids in the treatment of type-II diabetes and obesity. Fig. 4. Experimental ECD spectra of compounds 2 and 4.

3. Experimental ring, also showed an inhibitory effect (IC50 = 25.5 ± 1.5 μM) against PTP1B. Such an inhibitory potency of 10 had been also described in a previous report (Li et al., 2012). The rest were judged inactive (IC50 > 100 μM). In summary, 15 diterpenoids (including three new labdane-type derivatives 1–3) with diverse structures were obtained from a mass-

3.1. General experimental procedures Optical rotations were acquired with an Autopol IV-T polarimeter. UV and IR spectra were recorded on a Shimadzu UV-2550 and an Avatar 360 ESP FTIR spectrometer, respectively. NMR spectra were recorded on a Bruker Avance DRX-500 or a Bruker Avance 600 MHz

Fig. 5. Key HMBC and NOE correlations of compound 3.

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Scheme 1. Acid and enzymatic hydrolyses of compound 5.

spectrometer. Chemical shifts are expressed in δ (ppm), and referenced to the residual solvent signals. ESI–MS were measured on an Agilent 1100 series mass spectrometer; HRESIMS were measured on an AB Sciex TripleTOF 5600 mass spectrometer. Semi-preparative HPLC was performed on a Waters e2695 apparatus equipped with a 2998 photodiode array detector, a 2424 evaporative light-scattering detector (ELSD), a SunFire ODS column (5 μm, 250 × 10 mm) and a TOSOH TSK-GEL column (300 × 7.5 mm). Column chromatography (CC) was performed using silica gel (200–300 mesh, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Silica gel-precoated plates (GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, China) were used for TLC detection. Spots were visualized using UV light (254 and/or 365 nm) and by spraying with 15% (v/v) H2SO4-EtOH followed by heating to 120 °C.

tR = 25.2 min) and 13 (8.0 mg, tR = 21.4 min). The n-BuOH fraction (ca. 13.0 g) was transferred to a silica gel column, eluted with an EtOAc–MeOH gradient (15:1, 10:1, 5:1, v/v) to give five fractions (Fr. D–H). Fr. D was subjected to semipreparative HPLC [MeOH-H2O (containing 0.05% TFA, v/v) 80:20, v/v] to afford 2 (4.0 mg, tR = 23.0 min) and 4 (3.0 mg, tR = 25.7 min). Fr. E was separated by semipreparative HPLC [MeOH-H2O (containing 0.05% TFA, v/v) 70:30, v/v] to furnish 6 (8.0 mg, tR = 26.0 min) and 7 (4.0 mg, tR = 30.0 min). Compound 1 (9.0 mg, tR = 16.2 min) was purified from fraction F by semipreparative HPLC [MeOH-H2O (containing 0.05% TFA, v/v) 45:55, v/v]. Fr. G was applied to CC over Sephadex LH-20 (in MeOH) and then semipreparative HPLC [MeOHH2O (containing 0.05% TFA, v/v) 70:30, v/v] to yield 12 (10.0 mg, tR = 20.2 min) and 14 (15.0 mg, tR = 10.5 min). Compounds 3 (1.5 mg, tR = 25.0 min) and 5 (6.0 mg, tR = 27.0 min) were isolated from Fr. H by CC over Sephadex LH-20 (in MeOH) and further purified by semipreparative HPLC [MeOH-H2O (containing 0.05% TFA, v/v) 55:45, v/v].

3.2. Plant material The needles and twigs of P. kwangtungensis were collected in November 2014 from the Foshan Botanical Garden, Guangdong Province of China. The plant was identified by Mr. Huan Ke (Foshan Municipal Forestry Research Institute, China). A voucher specimen (No. 20141118) was deposited at the Herbarium of the Department of Natural product chemistry, School of Pharmacy at Fudan University.

3.3.1. (4S,5R,9S,10R)-6-Oxo-labd-7,13-dien-16,15-olid-19-oic acid (1) Colorless oil; [α]D20 +9.0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 242 (1.56) nm; ECD (c 1.70 × 10−3 M, MeOH) λmax (Δε) 315 (+3.5), 243 (−13.4); IR (KBr) νmax 2960, 2856, 1756, 1721, 1678, 1542, 1441, 1216, 1046, 908, 806 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 1; (+) ESIMS m/z 347 [M + H]+, 369 [M + Na]+, (−) ESIMS m/z 345 [M-H]−; HRESIMS m/z 347.1853 [M + H]+ (calcd for C20H27O5, 347.1853, Δ = 0.1 ppm).

3.3. Extraction and isolation The air-dried and pulverized needles and twigs (ca. 290.0 g) were extracted three times with 90% MeOH (5 × 1.0 L) at room temperature. The solvent was removed under reduced pressure to give a brown residue (ca. 30.0 g, semi-dry). The entire crude extract was suspended in water and partitioned with petroleum ether (PE), EtOAc and n-BuOH, successively. The EtOAc fraction (ca. 7.0 g) was subjected to silica gel CC, eluted with a PE–EtOAc gradient (20:1, 10:1, 5:1, 3:1, 1:1, v/v) to give three fractions (Fr. A–C). Fr. A (600.0 mg) was chromatographed on silica gel with a PE–EtOAc gradient (10: 1 to 3: 1, v/v) to provide two subfractions, A1 and A2. Fr. A1 was separated by semipreparative HPLC [MeOH-H2O (containing 0.05% TFA, v/v) 90:10, v/v] to furnish 15 (8.0 mg, tR = 15.0 min). Fr. A2 was further purified by Sephadex LH-20 (in MeOH) to obtain 10 (100.0 mg). Fr. B (1.9 g) was fractionated by a silica gel column with a gradient elution of PE–EtOAc (10:1, 5:1, 2:1, v/v), to provide three subfractions, B1–B3. Compound 11 (9.0 mg) was purified from Fr. B1 by Sephadex LH-20 eluted with MeOH. Fr. C was subjected to Sephadex LH-20 (in MeOH) and then semipreparative HPLC [MeOH-H2O (containing 0.05% TFA, v/v) 65:35, v/v] to yield compounds 8 (5.0 mg, tR = 28.4 min), 9 (6.0 mg,

3.3.2. 15(S)-n-Butoxypinusolidic acid (2) Colorless oil; [α]D20 +52.5 (c 0.04, CHCl3); UV (CHCl3) λmax (log ε) 217 (1.28) nm; ECD (c 1.24 × 10−3 M, MeOH) λmax (Δε) 336 (+0.17), 307 (−0.18), 246 (+3.42); IR (KBr) νmax 3076, 2955, 2928, 2872, 1766, 1722, 1646, 1448, 1326, 1256, 1156, 1064, 1021, 926, 809, 722 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 1; (+) ESIMS m/z 405 [M + H]+, 427 [M + Na]+, (−) ESIMS m/z 403 [M−H]−; HRESIMS m/z 405.2636 [M + H]+ (calcd for C24H37O5, 405.2636, Δ = 0.2 ppm). 3.3.3. β-D-Glucopyranosyl-(4S,5R,9S,10R)-labda-8(17),13-dien-15,16olid-19-oate (3) Pale yellow oil; [α]D20 +10.0 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 217 (1.42) nm; IR (KBr) vmax 3424, 3220, 2980, 2872, 1762, 1722, 1606, 1526, 1440, 1208, 1156, 1014, 990, 906, 806, 782 cm−1; 1H (500 MHz) and 13C (125 MHz) NMR data, see Table 1; (+) ESIMS m/z 495 [M + H]+, 517 [M + Na]+; HRESIMS m/z 517.2413 [M + Na]+ (calcd for C26H38O9Na, 517.2408, Δ = 0.9 ppm). 243

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3.3.4. β-D-Glucopyranosyl-labda-8(17),13-dien-16,15-olid-19-oate (adenanthoside C, 5) Pale yellow oil; [α]D20 +14.0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 217 (1.32) nm; IR (KBr) vmax 3424, 3228, 2982, 2876, 1758, 1724, 1621, 1524, 1442, 1321, 1206, 1146, 1056, 906, 807 cm−1; 1H and 13C NMR data in methanol-d4, see ref. (Wu et al., 2015); 1H NMR (600 MHz, in CDCl3): δ 0.55 (3H, s, Me-20), 1.06 (2H, m, H-1ɑ, H-3ɑ), 1.23 (3H, s, Me-18), 1.32 (1H, dd, J = 13.6, 3.6 Hz, H-5), 1.50 (1H, m, H-2ɑ), 1.60 (1H, m, H-11a), 1.62 (1H, dd, J = 12.4, 3.5 Hz, H-9), 1.75 (1H, m, H11b), 1.81 (1H, m, H-2β), 1.84 (1H, m, H-1β), 1.86 (1H, m, H-6ɑ), 1.98 (1H, br d, J = 12.8 Hz, H-7ɑ), 2.03 (1H, m, H-6β), 2.13 (1H, m, H-12a), 2.17 (1H, m, H-3β), 2.43 (1H, dd, J = 12.8, 12.8 Hz, H-7β), 2.46 (1H, m, H-12b), 3.45 (3H, m, H-2′, H-3′, H-5′), 3.55 (1H, br d, J = 8.6 Hz, H4′), 3.71 (1H, br d, J = 12.8 Hz, H-6′), 3.80 (1H, br d, J = 12.8 Hz, H6′), 4.61 (1H, br s, H-17), 4.79 (2H, br s, H-15), 4.89 (1H, br s, H-17), 5.46 (1H, d, J = 7.8 Hz, H-1′), 7.18 (1H, br s, H-14); 13C NMR (150 MHz, in CDCl3): δ 39.2 (C-1), 19.9 (C-2), 38.0 (C-3), 44.6 (C-4), 56.4 (C-5), 26.0 (C-6), 38.6 (C-7), 147.4 (C-8), 55.6 (C-9), 40.4 (C-10), 21.9 (C-11), 24.7 (C-12), 134.8 (C-13), 144.5 (C-14), 70.4 (C-15), 174.6 (C-16), 107.0 (C-17), 28.8 (C-18), 176.4 (C-19), 12.9 (C-20), 93.8 (C1′), 72.4 (C-2′), 76.5 (C-3′), 69.6 (C-4′), 76.4 (C-5′), 61.9 (C-6′); (+) ESIMS m/z 495 [M+H]+, 517 [M+Na]+; HRESIMS m/z 517.2413 [M +Na]+ (calcd for C26H38O9Na, 517.2408, Δ = 0.9 ppm).

containing 50 mM 3-[N-morpholino]-propanesulfonic acid (MOPS), pH 6.5, 2 mM p-NPP, and 30 nM recombinant PTP1B, activities were continuously monitored and the initial rate of the hydrolysis was determined using the early linear region of the enzymatic reaction kinetics curve. The non-enzymatic hydrolysis of the substrate was corrected by measuring the control without addition of the enzyme (negative control). The IC50 was calculated using Prism 4 software (GraphPad Software, San Diego, CA) from the non-linear curve fitting of the percentage of inhibition (% inhibition) versus the inhibitor concentration [I] by using the following equation: % Inhibition = 100/[1 + (IC50/[I])k], where k is the Hill coefficient. Oleanolic acid (purity ≥ 98%), a known PTP1 B inhibitor, was used as the positive control (IC50 = 3.3 μM) (Hu et al., 2016a).

3.4. Acid and enzymatic hydrolysis of 5 and sugar analysis

Appendix A. Supplementary data

Acid hydrolysis of 5: A solution of compound 5 (1.5 mg) in MeOH (0.6 mL) and 1N HCl (1.0 mL) was refluxed at 80 °C for 1 h. After removal of MeOH, the hydrolysate was diluted with H2O and extracted with EtOAc three times. The aqueous layer was neutralized with NaHCO3 and then worked up as usual to afford a mono-sugar (0.8 mg). The EtOAc layer was combined and concentrated to give a residue, which was further purified by HPLC to give 0.5 mg of 5a (yield: 33.3%). Labda-8,13-dien-16,15-olid-19-oic acid (5a): 1H NMR (400 MHz, CDCl3): δ 0.89 (3H, s, Me-20), 1.25 (3H, s, Me-19), 1.64 (3H, s, Me17), 4.78 (2H, br s, H-15), 7.14 (1H, br s, H-14); (+) ESIMS m/z 333 [M + H]+, 355 [M + Na]+, 665 [2M + H]+. Enzymatic hydrolysis of 5: A solution of 5 (1.2 mg) in acetic acid and sodium acetate buffer (pH 5.5, 1.0 mL) was treated with lyophilized almond β-glucosidase (2.0 mg, Sigma), and was then stirred at 37 °C for 8 h. After cooling, the reaction mixture was partitioned into CHCl3. The aqueous layer was filtered and concentrated under reduced pressure to give a mono-sugar. The CHCl3 layer was filtered and concentrated under reduced pressure to furnish the aglycone 5b (0.5 mg), which was purified by semipreparative HPLC [MeOH-H2O (containing 0.05% TFA, v/v) 70:30, v/v]. The spectroscopic data of 5b were found to be identical to those of pinosolide acid (6, Calderón et al., 1987; Fang et al., 1989; Yang and Han, 1998). Sugar analysis: The residue was analyzed by HPLC [column: TOSOH TSK-GEL; column temperature: 70 °C; detector: Waters 2424 (ELSD); mobile phase: H2O; flow rate: 1.0 mL/min]. Comparison of the retention time (tR) in the HPLC-ELSD chromatogram and the optical datum {[α]D20＋80.0°(c 0.1, H2O)} of the purified sugar with those of an authentic sample confirmed that the sugar unit in 5 was D-glucose (Li et al., 2011; Ma et al., 2016 Ma et al., 2016).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.05.006.

Acknowledgements This work was supported by NSFC grants (Nos. 21472021, 81273401, 81202420) and the National Basic Research Program of China (973 Program, Grant No. 2013CB530700). The authors are grateful to Mr Xian-Cong Hu (Head of Foshan Municipal Forestry Research Institute, Guangdong, PR China) and his colleague Mr Huan Ke for the permission and the assistance in the plant collection and identification.

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