New triterpenes from Cimicifuga yunnanensis down-regulating the mRNA expression of CD147, MMP-2, and MMP-9

Eleven new 9,19-cycloartane triterpenes (1–9, 11–12) and one undescribed lanostane-type aglycone (10) were identified from the aerial parts of Cimicifuga yunnanensis. The new structures were elucidated by analysis of spectroscopic data. Compounds 3–5, 7–9, and 11, without obvious cytotoxicity at 50 μM, were evaluated for inhibiting the mRNA expressions of atherosclerosis-related factors of CD147 (extracellular matrix metalloproteinase inducer, EMMPRIN), matrix metalloproteinase 2 (MMP-2) and MMP-9 in phorbol-12-myristate-13-acetate (PMA) induced Human monocytic THP-1 cells by using a quantitative real-time PCR method (q-PCR). Among them, aglycones 7 and 8 showed potent activities, whereas all tested glycosides were inactive. Compounds 7 and 8 suppressed the mRNA expression of CD147 in a dose-dependent manner, with an IC50 value of 3.38 ± 0.27 μM and 8.25 ± 0.33 μM, respectively. Besides, 7 dose-related down-regulated the mRNA expression of MMP-2, and MMP-9, having an IC50 value of 6.32 ± 0.31 μM and 11.57 ± 0.23 μM, respectively. Meanwhile, 8 at 10 μM reduced the mRNA expression of MMP-2 and MMP-9 by 35% and 25%, respectively. Significantly, the migration ability of the induced THP-1 cells was potently and dose-dependently inhibited by 7, with an IC50 value of 5.87 ± 0.27 μM.


Introduction
Cardiovascular diseases (CVDs) are leading causes of death globally, by which more than 17.0 million people die every year. 1,2 Pathologically, atherosclerosis, a chronic inammatory disease, is a critical causing factor of CVDs. 1,3 Patients who can maintain stability of atherosclerotic plaques will adapt to stable angina pectoris. Otherwise, a life-threatening acute coronary syndrome, including acute myocardial infarction (AMI) and unstable angina pectoris (UA) would happen to them. 4-6 CD147, extracellular matrix metalloproteinase inducer, and metalloproteinases (MMPs), such as MMP-2 and MMP-9 (a novel marker of AMI), are overexpressed in advanced atherosclerotic plaques by monocytes/macrophages and have been shown to contribute to AMI and UA through degradation of the extracellular matrix. [7][8][9][10] Of note, CD147 stimulates macrophages to produce MMP-2 and MMP-9 in a paracrine or autocrine way. 4,11 Therefore, to suppress the expression of CD147 and MMPs represents a promising strategy for antiatherosclerosis.
Plants of Cimicifuga genus (C. racemosa, C. foetida, and C. simplex) are famous herb medicines in Europe, the United States, and East Asia. 12,13 These herbs mainly contain 9,19cycloartane triterpenes (CTs) with diverse bioactivities, such as cytotoxicity, 14,15 anti-angiogenic, 16 anti-inammatory, 17,18 and neuro-protective. 19,20 Recently, we identied two CTs, yunnanterpene G (YG) and 12b-hydroxycimiacerol (HC), with antiatherosclerosis potentials by potently suppressing the mRNA expressions of CD147 and MMPs. 21,22 As a part of our successive program to explore bioactive CTs from Cimicifuga spp, nine unreported CTs glycosides (1- 6, 9, and 11-12) and two new aglycones (7 and 8), together with one undescribed lanostanetype triterpene (10) were identied from the aerial parts of C. yunnanensis (Fig. 1), an indigenous species distributed in the southwest region of China. 16 Signicantly, the q-PCR experiments showed that aglycones 7 and 8 dose-dependently attenuated the mRNA expression of CD147, with an IC 50 value of 3.38 AE 0.27 mM and 8.25 AE 0.33 mM, respectively, in PMA-induced THP-1 cells. Of note, the CD147 mRNA inhibitory effect of 7 at 10 mM is more potent than that of YG (positive control). While, 8 has comparable activity as YG at this concentration. Moreover, 7 dose-dependently down-regulated the mRNA expression of MMP-2, and MMP-9 and suppressed the migration ability of the induced THP-1 cells, having an IC 50 value of 6.32 AE 0.31 mM, 11.57 AE 0.23 mM, and 5.87 AE 0.27 mM, respectively. Conversely, all tested glycosides (3-5, 9 and 11) were inactive at 10 mM. Described herein are the isolation, structure elucidation, and biological activities of compounds 1-12.
group (d C 171.1), and the 1 H-1 H COSY correlation of the olenic proton resonance (d H 5.15) and H-6 (d H 1.54 and 1.82).
In the ROESY spectrum ( Fig. 2), cross-peaks of H-3 with H-5 (biogenetically a-oriented), H-16 and H-17 with CH 3 -28 (biogenetically a-oriented), and H-20 with CH 3 -18 (biogenetically boriented) were observed, which helped to establish the relative conguration of the core structure of 1. Moreover, the characteristic ROESY correlations of H-21/H-24 and H-24/CH 3 -27 further decided the conguration of ring F and G, as well as the ternary epoxy ring and ring G as shown (Fig. 2). Intensive analysis of 1 D and 2D NMR spectra demonstrated that compound 2 had the same core structure as that of 1, with the major differences being at C-24, C-25 and C26. Diagnostically, the ROESY correlation of H-21/H-24 was absence in 2, instead, the association of H-22a/H-24 was observed. Thus, the conguration of ring F and G of 2 was determined as same to . 23 Finally, the structure of 1 and 2 were determined as 7(8)-en-acteol-3-O-a-L-arabinopyranoside and 7(8)-en-23-epi-acteol-3-O-a-L-arabinopyranoside, respectively.  -17, s). Aforementioned data indicated that 3 was a CTs glycoside. The sugar unite was determined as L-arabinose by the same way as that of 1. The NMR data of aglycone part of 3 resembled that of 20 except that the acetoxy group was changed to C-12 and the OH-16 was replaced by a carbonyl group. HMBC correlations of H- 12 (d H 5.74) with the ester carbonyl group (d C 170.8), and CH 3 -28 (d H 1.31) with the carbonyl carbon (d C 207.3) further conrmed these elucidations. ROESY cross-peaks of H-3/H5, H-12/CH 3 -28, and H-20/CH 3 -18 in 3 suggested the a-   J in Hz) in Pyridine-d 5   orientation of H-3, H-12, and CH 3 -21. The b-orientation of H-24 was deduced by the ROESY correlation of H-24/H-20. In addition, identical to isodahurinyl-type molecules, H-24 of 3 was a singlet in 1 H NMR spectrum, suggesting S conguration of C-24 (the coupling constant of H-24 and H-23 of dahurinyl-type compounds, with R conguration of C-24, is around 6-9 Hz). 15 Therefore, the structure of 3 was determined as 12b-acetoxy-16 (17) (23)-ene, the characteristic ROESY association of CH 3 -18/CH 3 -26 was observed in 4, indicating it shares the same conguration at C-24 as R in AC (As shown in Fig S100, † when conguration of C-24 is S, it is impossible to see the cross-peak of CH 3 -18/CH 3 -26). Thus, the structure of 4 was determined as 12b-acetoxy-22 (23)-en-15deoxy-isodahurinyl-3-O-a-L-arabinopyranoside.
Compound 5 was puried as white powder, with the molecular formula C 37 H 58 O 11 , given by the HREIMS ([M] + m/z 678.3990, calcd 678.3979). The IR spectrum showed the presence of hydroxyl (3431 cm À1 ), carbonyl (1730 cm À1 ) and olenic (1629 cm À1 ) groups. The NMR data of aglycone part for 5 (Tables 1 and 2) were similar to those of actaeaepoxide-3-O-a-Dxylopyranoside. 25 The main differences were that a methine (C-22) at d C 86.6 was absent, instead, there's another methylene (d C 42.1), and the upeld shis of C-23, C-24, and C-25 by 3.1 ppm, 5.4 ppm and 8.6 ppm, respectively. These changes could be explained as that, in 5, a methylene replaced a methine at C-22, and two hydroxy groups instead of the ternary epoxy ring at C-23 and C-24. These deductions were further conrmed by the HMBC coupling of H-20 (d H 4.81)/C-22 (d C 42.1). The sugar unit was connected to C-3 and identied as L-arabinose using same approaches as those of 1. In addition, the orientations of core structure and the conguration of C-24 of 5 were determined on the basis of the ROESY associations as those of 4. Therefore, the structure of 5 was determined as 12b-acetoxy-23,24-dihydroxy-7(8)-en-15-deoxy-isodahurinyl-3-O-a-L-arabinopyranoside.
On the basis of the  (Table 1), indicating 6 is a CTs glycoside with an acetoxy group and a double bond. The sugar unit was connected to C-3 and deduced as L-arabinose by using same approaches as those of 1. Comparison of NMR data of 6 and hydroxyshengmanol-7 (8) compounds (S, J # 2 Hz; R, J z 6). 15,26 The molecular formula C 30 H 44 O 7 of 7 was deduce from its HRTOF-ESIMS at m/z 539.2988 [M + Na] + (calcd 539.2985). The spectroscopic features of 7 were identical to 6 except for a carbonyl group and a hydroxy group at C-3 and C-12, respectively. HMBC correlation of CH 3 -29 (d H 1.11) with the carbonyl carbon (d C 214.6) and the upeld shi of C-12 by 4.8 ppm further conrmed these elucidations. Same orientations of H-8, H-12, H-17, and H-23, as well as the conguration of C-24 between 7 and 6 were determined on the basis of the ROESY associations and comparison of coupling constant of H-24 with known compounds. Thus, the structure of 6 and 7 were determined as 12b-acetoxy-7 (8) 25 The main differences were that no double bond at C-7 and C-8, and the absence of an acetoxy group at C-12 in 8.  (Fig S100 †). Therefore, the structure of 8 was determined as actaeaepol.
The molecular formula of compound 9 was determined as C 32 H 48 O 9 from HRTOF-ESIMS at m/z 599.3188 [M + Na] + (calcd 599.3196). In the 1 H NMR spectrum (Table 1), signals due to an extremely downeld shied cyclopropane methylene at d H 1.02 (1H, d, J ¼ 3.4 Hz) and 2.01 (1H, d, J ¼ 3.9 Hz), an anomeric proton at d H 4.82 (d, J ¼ 7.0 Hz), an olenic hydrogen atom at d H 5.21 (brd, J ¼ 6.3 Hz), four tertiary methyl groups at d H 1.12-1.60, and a secondary methyl signal at d H 0.90 (d, J ¼ 5.9 Hz), were observed. The 1 H-1 H COSY spectrum indicated that 9 had part structure of -CHCH(CH 3 )CH 2 -(for C -17, C-20 to C-22). Aforementioned data together with HMBC associations of H-22 (2H, d H 2.43 and 2.51) with the carbonyl carbon (d C 211.2) and the oxygenated carbon at d C 82. 3 (C-24, d), and H-17 (d H 2.25) with the oxygenated carbon at d C 81.9 (C-16, s) and C-24, exhibited that 9 was a foetidonol-type CTs glycoside. The sugar unit was connected to C-3 and identied as L-arabinose using same ways as those of 1. Compound 9 had a similar structure as that of foetidonol-3-O-b-D-xylopyranoside, 27 except for a double bond at C-7 and C-8, and a hydroxy group at C-11. The 1 H-1 H COSY coupling of H-6 and the olenic proton at d H 5.21,) and C-9, as well as the molecule weight further conrmed these elucidations. Therefore, the structure of 9 was determined as 11bhydroxy-7(8)-en-foetidonol-3-O-a-L-arabinopyranoside.
The HREIMS of 10 exhibited a molecular ion at m/z 488.3508 [M] (d,J ¼ 6.9 Hz). These data indicated that 10 possessed one more tertiary methyl group in the skeleton than a usual CTs. In 13 C NMR spectrum, a pair of tetrasubstituted olenic carbons at d C 134.9 (C-8, s) and 136.0 (C-9, s), and the characteristic ketal signal for C-16 of cimigenol-type CTs at d C 112.7 (s) were observed. HMBC associations of H-7/C-8 (d C 134.9, s), H-11/C-9 (d C 136.0, s), H-1/CH 3 -19 (d C 19.6, q), and CH 3 -19/C-9 and C-10, located the double bond at C-8 and C-9, and the CH 3 -19 at C-10, respectively. The rest of NMR resonances of 10 were identical to those of cimigenol. 28 Therefore, the structure of 10 was determined as 19b-methyl-8(9)-encimigenol.

Alteration of morphology and phenotype on PMAinduced THP-1 cells
The normal THP-1 cells are ball-shaped without adhering to the surfaces of the plastic culture plates. 11 Cultured by adding 100 nM PMA for 24 h, the cells became at and amoeboid in shape, and adhered to the dish bottom ( Fig. S97A and B †). Moreover, the differentiation of monocyte to macrophage was determined on the basis of 97% of the PMA-induced THP-1 cells were CD68 positive and 33% of these cells were CD11b positive with ow cytometry analysis ( Fig. S97C and D †).

Cytotoxic activities of compounds 1-12 on PMA-induced THP-1 cells
Before conducting further bioassays, the cytotoxicities of compounds 1-12 on PMA-induced macrophages were tested by MTT assay. As shown in Fig S98, † compounds 1, 2, 6, 10, and 12 indicated notable cytotoxic effect (25% to 75% inhibition) on the cell viability from the concentration of 25 mM. Thus, these molecules were discarded for further studies. Conversely, compounds 3-5, 7-9 and 11 were chose for successive investigations due to their negligible cytotoxicity even at 50 mM (about 10-15% reduction on the cell viability) (Fig. S98 †). In addition, the experimental concentration range was set as10 to 50 mM in the present study.

Inhibition of PMA-induced THP-1 cells migration by compound 7
The enhanced migration or invasion of peripheral macrophages is a characteristic feature in pathological process of atherosclerosis. 4,5 Because of compound 7 showed signicant inhibition on the mRNA expression of CD147 and MMPs, the regulatory effect of this molecule on the migration of PMAinduced THP-1 cells was further studied by scratch wound assay. As a result, 7 (aer 24 h incubation) potently and doserelated decreased the number of migrated cells with an IC 50 value of 5.87 AE 0.27 mM (Fig. 3F and S99 †).

Conclusion
As mentioned in the introduction, substances, which inhibit CD147 and MMPs expression may hold great potentials to prevent the development of atherosclerosis. Indeed, antiatherogenic drugs, such as uvastatin, attenuating the MMPs and EMMPRIN productions partially contribute to their clinical effects. [30][31][32] Natural products (NPs) are important resources of active molecules for modern drug development. 33 Previously, two undescribed CTs (YG and HC) from C. foetida, with notable inhibitions on CD147 and MMPs mRNA expression, were identied. Successive investigations on the aerial parts of C. yunnanensis led to characterize eleven new CTs, including nine glycosides (1- 6, 9, and 11-12) and two aglycones (7 and 8), along with one undescribed lanostane-type triterpene (10). Compounds 7 and 8, two aglycones, showed noticeable inhibitory effects on the mRNA expression of CD147 and MMPs, as well as migration ability of the induced THP-1 cells. By contrast, all tested glycosides were inactive. It is worth noting that 7 is the most potent molecule among these four active CTs. Given the critical roles of CD147 and MMPs in stabilizing atherosclerotic plaques, 7 may have a promising effect in retarding the development of the vulnerability of the plaque, deserve to conduct more sophisticated animal studies in future.
In summary, our studies show that CTs, specially aglycones, are potential resources of anti-atherosclerosis bioactive agents and deserve further extensive exploration. DMEM/F12 with different concentrations of compounds 1- 12 (0, 5, 10, 25, 50, 75, and 100 mM) were added to instead of the original medium, and cultured for another 48 h. Cell viability was evaluated by MTT assay: each well was added with 20 mL of MTT to a nal concentration of 0.5 g L À1 for 4 h before using 150 mL DMSO to solubilize the reactive dye. Each well was recorded by a Bio-Rad microplate reader to absorbance value of 570 nm. All the experiments were repeated in triplicate.

Isolation of total RNA and RT-PCR
RevertAid™ First Strand cDNA Synthesis Kit was applied to extract total RNA of differentiated THP-1 cells treated with compounds 3-5, 9 and 11 (10 mM), along with 7 and 8 (1, 3, 10, 25, and 50 mM) for 24 h (5 Â 10 5 cells per mL) following the manufacturer's instructions. cDNA was synthesized from the isolated total RNA based on the instruction of the PrimeScript RT reagent Kit. Briey, PrimeScript RT Enzyme Mix 1 (0.5 mL), 5 Â PrimeScriptTM Buffer (2 mL), oligo dt Primer (0.5 mL) and Random 6 mers (0.5 mL) were gently mixed with 1 mg RNA from each sample to a reaction volume of 10 mL with RNase free water, then incubated for 15 minutes at 37 C to activate the reverse transcriptase enzyme. Finally, the reaction was stopped by 85 C for 5 seconds.
Aer reverse transcription, cDNA was used to carry out realtime quantitative RT-PCR on ProFlex™ PCR system by SYBR Premix Ex Taq (Takara). The nal volume of TR-PCR reaction is 25 mL, containing 12.5 mL SYBR green master mix, 1 mL cDNA, 0.5 mL each forward and reverse primer, and 10.5 mL nucleasefree water. For information of primers see Table S1. † Thermal cycling conditions for all genes were as follows: template predenaturation (10 min at 95 C), denaturation (15 seconds at 95 C), annealing and extension (30 seconds at 60 C) for 40 cycles. Internal reference is GAPDH mRNA, and fold changes of mRNA expression for each target relative to GAPDH were calculated by the 2 ÀDDCt method. Expression of mRNA is determined as the change in mRNA copy numbers relative to negative control cells (undifferentiated THP-1 cells). All the experiments were carried out in triplicate.

Wound-healing migration assay
The differentiated THP-1 cells were seeded and grown into full conuence in 6 well plates. 2% FBS DMEM/F12 media was used to inactivated cell proliferation for 12 h, then wounded with pipette tips. Fresh DMEM/F12 medium with or without 1, 3, 10, 25, and 50 mM of 7 was added to the scratched monolayers. Images were took aer 24 hours using a Nickon inverted microscope (magnication, 10Â). The migration cell number of PMA-induced THP-1 cell group was dened as control. All the experiments were performed in triplicate.