Isolation, identification and bioactivities of abietane diterpenoids from Premna szemaoensis

Investigation of the leaves and stems of Premna szemaoensis resulted in the isolation of twelve new abietane diterpenoids, szemaoenoids A–L (1–12), together with four known abietane diterpenoids (13–16). The structures involved two rearranged-abietane skeletons: 17(15 → 16)-abeo-abietane (7, 10–12, 14 and 15) and 17(15 → 16),18(4 → 3)-diabeo-abietane (1–6, 13 and 16). The structures of the new compounds were established mainly by analyzing NMR and HRESIMS data. The absolute configurations of 1, 3 and 10 were confirmed by single crystal X-ray diffraction analysis. In bioactivity assays, compounds 11, 12, 14 and 15 were active against two human colon cancer cell lines (HCT-116 and HT-29) with IC50 values ranging from 8.8 to 34.3 μM, and compounds 10, 13 and 14 exhibited effective free radical scavenging activity with IC50 values ranging from 35.6 to 41.5 μM by DPPH experiment.


Introduction
The genus Premna (family: Verbenaceae) comprises approximately 200 species, which are mainly distributed in the tropical zone of Asia and Africa. 1 There are about 44 species and 5 varieties grown in the south of China, especially in Southwest China. The dried aerial parts of some Premna species have been used in traditional folk medicine for the treatment of pyogenic infections, trauma, fracture, dysentery, haemorrhoids, and rheumatic arthritis. 2 Previous phytochemical investigations of Premna have indicated the presence of diterpenoids, 3 avonoids, [3][4][5] iridoid glycosides, 4-7 xanthones, 8 phenylethanoid glycosides, 9 triterpenoids, 10,11 and lignins. 12 Their pharmacological effects, including neuroprotective, 13 analgesic, 14,15 antioxidative, cytotoxic, 16,17 anti-inammatory, 18 and a-glucosidase inhibition, 19 have been reported for crude extracts and pure compounds from Premna plants.
Premna szemaoensis Pei, locally called "simao dofu chai", is mainly distributed in the south of Yunnan province of China. 20 Table 1   This plant has drawn the attention of local farmers and has been cultivated to be an important commercial crop due to its various applications. Its fresh leaves can be rubbed and squeezed in water to yield a mucilaginous juice, which was used to prepare a food named "green tofu" by local people through addition of materials containing Ca 2+ . In addition, the local villagers also used the leaves of this plant to cure injuries and fracture. 20 However, the phytochemical investigation of this species was extremely rare, except a small number of avonoids.

Results and discussion
The aerial parts of P. szemaoensis were extracted three times with 70% acetone aqueous. Aer recycling acetone, the rest of portion was partitioned by liquid-liquid extraction between nbutanol and H 2 O. The n-butanol-soluble portion was repeatedly subjected to silica gel, Sephadex LH-20, and RP-C18 gel column chromatography (CC) and semi-preparative HPLC to afford 16 abietane diterpenoids, including 12 new compounds. The structures and stereochemistry of these isolates were elucidated mainly using spectroscopic analysis, X-ray diffraction analysis, and compared to data in the literature. Ultimately, the new compounds were named as szemaoenoids A-L (1-12), and known compounds were identied as teuvincenone F (13) Table 2) showed signals of 20 carbons of an aglycon, attributable to a ketone group at d C 200.7, three methyls (two tertiary), ve methylenes (one olenic), three methines (one olenic, one oxygenated), and eight quaternary carbons (six olenic, one oxygenated), along with signals for a hexose unit. These data In acetone-d 6 solution. suggested that 1 is a diterpene glycoside and in accordance with the characteristics of a 17(15 / 16),18(4 / 3)-diabeo-8,11,13abietatriene.
The proton and protonated carbon NMR signals of 1 were assigned unambiguously by the HSQC experiment. Partial structures and the whole connection were deduced from correlations observed in the 1 H-1 H COSY and HMBC spectra (Fig. 1). The HMBC correlations of CH 3 -18 (d H 1.41) to C-2 (d C 37.8), C-3 (d C 71.5, an oxygenated quaternary carbon), and C-4 (d C 153.7, an olenic quaternary carbon) and of H 2 -19 (d H 4.74, 5.20) to C-3/C-4/C-5 (d C 44.0) suggested the presence of a 18(4 / 3)-abeo-abietane structural unit, established the location of an OH group at C-3 and an exocyclic double bond at C-4 and C-19. The ketone group was placed at C-7 from correlations of H-5 ( 3) and C-15 (d C 40.9), were suggestive of 17(15 / 16)-abeo-abietane moiety in this structure, and an hydroxyl group at C-16. The location of the sugar moieties was determined by the HMBC correlation of the anomeric proton H-1 0 (d H 4.57) to C-12. In addition, 1 H-1 H COSY correlations: H 2 -1/H 2 -2, H-5/H 2 -6, and HMBC correlations of CH 3 -20 to C-1, C-9 and C-10, of H-5 to C-4 and C-10 were also key interactions to support this gross structure.
Compound 3 was obtained as white monoclinic crystals (MeOH). Its molecular formula assigned was determined to be C 26 H 36 O 11 based on the negative HRESIMS (m/z 523.2177 [M À H] À ). The NMR data for this compound were highly close to those of 2 (Tables 1 and 2   elds and ketone group (d C 207.1) obviously change in 13 C NMR spectrum vs. 2. Ultimately, the absolute conguration was conrmed by single crystal X-ray diffraction analysis, which assigned C-16 as R conguration (Fig. 3). Therefore, the structure of 3 was determined as (
Compound 11 and 12 were assigned to an identical molecular formula of C 20 H 24 O 5 by HRESIMS data, with one more degree of unsaturation then 10. As seen from 1 H NMR and 13 C NMR spectrum (Tables 1 and 2 In our cognition, many diterpenoides had been reported from plants of Premna genus, [33][34][35][36] but this was also the rst to report diterpenoids isolated from the Premna plants distributed in China, and the described rearranged-abietane skeletons were rstly isolated from Premna genus. Abietane diterpenoids represented a large group of secondary metabolites that have shown interesting biological activities. 31 But the rearranged abietane diterpenoids with 17(15 / 16)-abeo-abietane or 17(15 / 16),18(4 / 3)-diabeo-abietane were not common in nature. Structurally, the rearranged diterpenoids contain abundant hydroxyl groups and aromatic carbons, as well as trans-fused rings A and B according to biosynthetic pathway. However, the stereocenter of C-16 replaced with OH had never been established previously. Within this work, we rstly employed X-ray crystallography to assign absolute conguration of C-16 for a small series of 16-hydroxy-17(15 / 16)-abeo-abietane diterpenoids, which might assist future unambiguous identication of structurally related compounds.
All the above compounds, except 3, 4 and 10, were evaluated for cytotoxicity in vitro against two human colon carcinoma cell lines (HCT-116 and HT-29). As a result (Table 4), compounds 11-15 showed antiproliferative activity against HCT-116 cell line, and compounds 11, 12 and 15 also exhibited potent cytotoxicity on HT-29 colon carcinoma cell line. Impressively, the     Table 4). Almost all the diterpene aglycones showed effective cytotoxicity, but none of the diterpene glucosides exhibited remarkable activity. The reason might be that the glucosides with strong chemical polarity failed to penetrate the liposoluble cell membrane.
As most of natural products possessing phenolic hydroxy exhibited antioxidant activity, 37-39 some selective compounds of the diterpenosides were executed free radical scavenging activity assay in the DPPH experiment (Table 5). Among the tested compounds, diterpene aglycones 13, 14 showed strong free radical scavenging activity with IC 50 values of 41.5 AE 17.0 and 39.9 AE 12.9 mM respectively, and compound 10 was especially the strongest activity with IC 50 35.6 AE 9.8 mM (more potent than the positive control trolox and vitamin C). Compound 12 exhibited slightly weaken antioxidant activity with IC 50 values of 74.9 AE 6.9 mM. None of the tested diterpene glucosides (1, 2, 3, 5, 9 and 16) showed potent free radical scavenging activity. Concerning the structure-activity relationship, a reasonable conclusion was reasoned out that the more the structure possessed phenolic hydroxyl groups, the more its scavenging activity was strong.

Experimental section
General experimental procedures X-ray data were collected using a Bruker APEX DUO instrument. Optical rotations were measured with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained on a Tenor 27 spectrophotometer with KBr pellets. Onedimensional (1D) and two-dimensional (2D) NMR spectra were recorded on Bruker DRX-600 spectrometers with TMS as the internal standard. Chemical shis (d) were expressed in parts per million with reference to the solvent signals. HRESIMS was performed on an Agilent G6230 TOF MS. Semi-preparative HPLC was performed on an Agilent 1260 liquid chromatograph with a Zorbax SB-C18 (

Extraction and isolation
The air-dried and powdered aerial parts of P. szemaoensis (10 kg) were extracted with 70% aqueous acetone (40 L) four times (two days each time) at room temperature and then ltered. The ltrate was evaporated under reduced pressure at 40 C and then partitioned between n-butanol and H 2 O. The n-butyl alcohol soluble portion (600 g) was subjected to silica gel CC (2.5 kg, 100-200 mesh), eluted with a CHCl 3 -Me 2 CO gradient system (1:0-0:1) that afforded fractions A-E. The fractions were then decolorized using MCI gel and eluted with 95%

X-ray crystal structure analysis
Crystals of 1, 3 and 10 were obtained in MeOH, respectively. Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Ka radiation. Cell renement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97. 40 Renements were performed with SHELXL-97 and SHELXL-2014 using full-matrix least-squares, with anisotropic displacement parameters for all the nonhydrogen atoms. The H-atoms were placed in calculated positions and rened using a riding model. Molecular graphics were computed with PLATON. 41 (10) , g ¼ 90.00 , V ¼ 1271.53(4)Å 3 , T ¼ 100(2) K, space group P2 1 , Z ¼ 2, m(CuKa) ¼ 0.898 mm À1 , 10 232 reections measured, 3499 independent reections (R int ¼ 0.0328). The nal R 1 values were 0.0300 (I > 2s(I)). The nal wR(F 2 ) values were 0.0884 (I > 2s(I)). The nal R 1 values were 0.0300 (all data). The nal wR(F 2 ) values were 0.0885 (all data). The goodness of t on F 2 was 1.113. Flack parameter ¼ 0.17 (14). 29 The Hoo parameter is 0.10(6) for 1225 Bijvoet pairs. 30 Crystal data for szemaoenoid C (3). The goodness of t on F 2 was 1.058. Flack parameter ¼ 0.0(2). 29 The Hoo parameter is 0.01 (14) for 1718 Bijvoet pairs. 30 Acid hydrolysis of szemaoenoid A Compound 1 (4 mg) was hydrolyzed with 2 M HCl/dioxane (1 : 1, 4 mL) under reux for 8 h, respectively. The reaction mixture was partitioned between H 2 O and CHCl 3 (2 mL Â 3). The aqueous layer was neutralized with 2 M NaOH and then dried to give a monosaccharide. A solution of the sugar in pyridine (2 mL) was added to L-cysteine methyl ester hydrochloride (about 1.0 mg) and kept at 60 C for 1 h. Then trimethylsilylimidazole (about 1.0 mL) was added to the reaction mixture and kept at 60 C for 30 min. The mixture was subjected to GC analysis, run on a Shimadzu GC-14C gas chromatograph equipped with an H 2 ame ionization detector. The column was a 30 m Â 0.32 mm i.d. 30QC2/AC-5 quartz capillary column with the following conditions: column temperature, 180-280 C; programmed increase, 3 C min À1 ; carrier gas, N 2 (1 mL min À1 ); injector and detector temperature, 250 C; injection volume, 4 mL; and split ratio, 1/50. The conguration of the sugar moiety was determined by comparing the retention time with the derivatives of the authentic samples. The retention times of D-/L-glucose were 21.115/21.565 min. 43 The conguration of the sugar moiety from compound 1 was D-glucose (R t ¼ 21.117 min).

Cytotoxicity assay
Human colon adenocarcinoma cell lines, HCT-116 and HT-29 were obtained from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% FBS in a 5% CO 2 atmosphere. HCT-116 (3 Â 10 3 per well) and HT-29 (6 Â 10 3 per well) were seeded onto 96-well plates and allowed to grow for 24 h prior to treatment. Different concentrations of compounds were then added and further incubated for 3 days. Sorafenib (purity > 99%; Medchem Express) was used as positive control. The culture medium was replaced by fresh DMEM containing 0.5 mg mL À1 of MTT. Aer incubation for another 4 h, the medium was removed and the reduced formazan blue was solubilized by adding 100 mL DMSO to each well. The absorbance at 492 nm was measured using a microplate reader (Multiskan MK3, Thermo). The IC 50 values were calculated from concentration-response curves using Graphpad Prism soware.

Antioxidant activity assay
Trolox (purity > 98%; Sigma) and vitamin C (Ascorbic acid, purity > 98%; Sigma) were used as positive control. A 0.1 mM solution of DPPH radical in ethanol was prepared, and 100 mL of this solution was mixed with 100 mL of sample solution. The mixture was incubated for 5 min in a dark room at room temperature. Scavenging capacity was read spectrophotometrically by monitoring the decrease in absorbance at 517 nm. DPPH scavenging activity (%) ¼ [1 À (S À B)/(C À B)] Â 100%, where S, B and C are the absorbencies of the sample, the blank and the control, respectively. 44

Conclusions
In summary, we have rstly reported twelve new abietane diterpenoids (1-12) isolated from P. szemaoensis, together with four known compounds (13)(14)(15)(16). Structurally, these compounds involved two rearranged-abietane skeletons: 17(15 / 16)-abeoabietane and 17(15 / 16),18(4 / 3)-diabeo-abietane. Their structures with absolute congurations were characterized by a series of spectroscopic methods and X-ray diffraction. In bioactivity assays, compounds 11, 12, 14 and 15 were active against two human colon cancer cell lines (HCT-116 and HT-29) with IC 50 values ranging from 8.8 to 34.3 mM, and compounds 10, 13 and 14 exhibited effective free radical scavenging activity with IC 50 values ranging from 35.6 to 41.5 mM by DPPH experiment. In short, the current study adds to understanding of the chemical composition and biological effects of this plant prepared for green food and ethnodrugs.

Conflicts of interest
There are no conicts to declare.