Extraction and Crystal Structure of Physalin B

YANG Yn-Jun SHA Cong-Wei CHEN Mei-Guo HU Ling



Extraction and Crystal Structure of Physalin B

YANG Yan-Juna②SHA Cong-WeibCHEN Mei-GuoaHU Linga

a(510520)b(510440)

The title compound of physalin B (C28H32O9), a main active physalin ofL, was isolated from the whole plant ofL, andcharacterized by X-ray diffraction analysis. It crystallizes in the monoclinic system, space group21with C28H32O9,= 12.4996(2),= 14.35620(10),= 14.75190(10) Å,= 2607.97(5) Å3,= 4,D= 1.382 mg/cm3,M= 542.56,(000) = 1152, and= 0.870 mm-1. The final= 0.0389 and=0.1037 for 47670 observed reflections (> 2()). The rigid molecule consists of eight fused rings involving two lactones. There are two C28H32O9molecules in ansymmetric unit, and the title compound is stacked into a 3D layer structure through hydrogenbonds. In the 5～20 μmol/L range, physalin B can significantly inhibit the secretion of inflammatory cytokines TNF-and IL-6 on RAW264.7 cells. The results suggest that physalin B has anti-inflammatory activity in vitro.

physalin B, crystal structure,L, physalin

1 INTRODUCTION

Physalin B, possessing a novel 13,14-seco-16,24- cycloergostane skeleton, was initially isolated fromthevar.[1].In the vivo anti-inflammatory experiment, physalin B appeared to be mostly due to the activation of glucocorticoid receptors, so it may represent novel therapeutic options for the treatment of inflammatory diseases[2]. physalin B has the potential to be developed as an effective chemotherapeutic lead compound for the treatment of malignant melanoma[3]. Itshows strong cytotoxicity against multiple tumor cell lines, including KB, A431,HCT-8, PC-3, and ZR751, with EC50values less than 4 μg/mL[4].The antimitotic activity of physalin B wasthe most active, withIC50values of 12.5 mM, for the first cleavage[5].Physalin B may be considered responsible for the antimicro- bial activity. It (200 μg/mL) can inhibite S. aureus ATCC 6538P by ±85%[6]; physalins B exhibits a minimum inhibitoryconcentration value (MIC) againstH37Rv strain of 128 g/mL[7].

Physalin B did not afford crystals suitable for crystallograpohy before[8]. As part of our ongoing research into bioactive constituents from medicinal plants, we isolated physalin B from fresh plants of, locally known as “”, collec- ted from Jiedong (Guangdong, China), and got the well-grown prismatic crystals of physalin B which could be subjected to crystallographic analysis. We describe here the crystal structure determination of physalin B by X-ray diffraction (Fig. 1).

Fig. 1. Chemical structure of physalin B

2 EXPERIMENTAL

2. 1 Extraction and crystallization

The air-dried and milled whole plants of(8 kg) were extracted with ethanol (85%) three times (3 × 30 L) under immersion for 1 week each. After filtration and evaporation of the solvent under reduced pressure, the combined crude ethano- lic extract (1067 g) was mashed and then dissolved successively with petroleum ether and EtoAc,-BuOH to afford dried petroleum ether-soluble (101 g), EtoAc-soluble (49 g) and-BuOH-soluble (48 g), respectively. Accordingly, the EtoAc-soluble extract was subjected to medium pressure column chromatography over silica gel (LC60A 40-63 micron) and eluted using a step gradient of a petroleum ether and EtoAc solvent system (100:0, 100:1, 80:1, 50:1, 25:1, 10:1, 5:1, 3:1, 2:1,1:1) at a flow rate of 50 mL min-1, pressure 20 bar, to obtain ten fractions (F1-F10) based on TLC profile. Each fraction was concentratedVFurther puri- fication of subfraction F7 (petroleum ether and EtoAc solvent system: 5:1) by repeated column chromatography over silica gel (LC60A 40-63 mi- cron) with petroleum ether-EtoAc (100:0 to 1:1) followed by thin-layer chromatography gave trans- parent crystals physalin B suitable for X-ray dif- fraction analysis (80.0 mg).

2. 2 Physical and chemical properties

Melting point for the colorless prismatic crystals wasdetermined on an YANACO MP-500P meltingpoint apparatus from Yanaco New Science Inc. With- out correction. m.p.: 253～254 ℃ (petroleum ether-.EtoAc). NMRstudies were carried out with a BrukerDRX-400instrument (400MHz) with (CD3)2CO as the solvent.1HNMR,13C-NMR, DEPT,1H-1HCOSY, HMQC and HMBC were detected. The details are summarized in Table 1. Mass spectra were recorded on a Thermo MAT 95XP (Thermo- Finnigan) equipment HR-EI:/510.1883 (C28H30O9, Calcd. for 510.1884).

Table 1. NMR Spectral Data for Physalin B (in Acetone-d6)

2. 3 Crystal structure determination

A transparent crystal cultured from ether-EtoAc with dimensions of 0.43mm × 0.41mm × 0.36mm was selected for X-ray diffraction analysis. The de- termination of unit cell and data collection were performed with Cu-radiation (= 1.54178 Å) on an Xcalibur, Atlas, Gemini ultra diffractometer (Agilent Technologies, santa clara, USA)equipped with a graphite-monochromator. A total of 47670 reflections were collected in the range of 3.04≤≤67.05° by using an-scan mode at 150(2) K, in which 9280 were independent withint= 0.0348 and 8908 observed reflections with> 2() were used in the structure determination and refinements. The structure was solved by direct methods and expan- ded by difference Fourier techniques with SHELXL- 97 program[10-11]. The hydrogen atoms were deter- mined with theoretical calculations and refined iso- tropically. Non-hydrogen atoms were refinedaniso- tropically to= 0.0407,= 0.1056 (= 1/[2(F2) + (0.0664)2+ 0.5452], where= (F2+ 2F2)/3),= 1.058, (Δ/)max= 0.001, (Δ)max= 0.297 and (Δ)min= –0.276 e·Å3.

2. 4 Establishment of inflammatory cell model and physalin B concentration

The establishment of cell models: The cell density was set as 1 × 105/mL, 100 μL/well in 96-well plates, including the control group and groups with diffe- rent LPS (Lipopolysaccharide) concentration. LPS groups: LPS was added to the cultured cells at the final concentration of 10, 100 and 1 μg/mL; Normal control groups: while stimulators were added to other groups, volumes of the normal control groups were supplemented with complete culture. After LPS groups were incubated for 8 h, the supernatant was collected. The levels of pro-inflammatory cytokines IL-6, TNFwere measured by ELISA to determine the dose of LPS stimulation.

The establishment of drug concentration: RAW- 264.7 cells were seeded in 96-well plates, including the control group, LPS groups, and test groups with different concentration. After the cells were adhered, each test group was added and mixed gently with different concentration of physalin B. After the groups, expect for the control group, were incubated at 37 ℃ for 2 h, they were added with LPS solutions and cultured for 8 h. Afterwards, the supernatant was collected, and the pro-inflammatory cytokines IL-6, TNF-levels were measured by ELISA.

2. 5 Effects of physalin B on RAW264.7 cells TNF-α and IL-6 levels

RAW264.7 cells were seeded in 24-well plates, including the control group, LPS group, the positive drug test (dexamethasone) and test groups with different concentration. After the cells were adhered, dexamethasone was added to the positive group. Each test group was added with different concentra- tion of physalin B. After 2 h, each group, except for the control group, was added and mixed gently with LPS solution of a final concentration of 1 μg/mL. After the groups were incubated at 37 ℃ for 8 h, the supernatant was collected, and the TNF-α, IL-6 protein were measured by ELISA according to the ELISA’s instructions.

3 RESULTS AND DISCUSSION

3. 1 Molecular structure and selected bond lengths and bond angles of physalin B

The molecular structure of physalin B isshown in Fig. 2. Theselected bond lengths and bond angles are listed inTable 2.

3. 2 Creation of a model of inflammatory cells

After the mouse macrophages were simulated with different LPS concentration, the TNF-α and IL-6 levels secreted by mouse macrophages RAW- 264.7 were observed. The result showed that 1 ug, 100 ng and 10 ng/mL LPS could significantly ele- vate the TNF-α and IL-6 levels with a concentra- tion-dependent relationship (Fig. 3).

3. 3 Physalin B dose screening

As shown in Fig. 4, while control group cells had low TNF-α and IL-6 levels, the TNF-α and IL-6 levels secreted in RAW264.7cells increased signi- ficantly after the RAW264.7cells were simulated by LPS. The difference between two groups was statistically significant (P < 0.05); Physalin B sample (between 20 and 5 μmol/L) could significantly inhibit the secretion of TNF-α and IL-6 (Fig. 4). When compared with the LPS stimulation group, the difference between the groups was statistically significant (P < 0.05).

Table 2. Selected Bond Lengths (Å) and Bond Angles (°) for the Title Compound

Fig. 2. Molecular structure and atomic numbering scheme of the title compound

3. 4 Impact of Physalin B on the IL-6, TNF-α expression in RAW264.7 cells

As shown in Fig. 5, while the TNF-α and IL-6 levels in the control group cells are low, they increa- sed significantly after the cells were stimulated by LPS. Positive drug dexamethasone (1 μmol/L) could significantly inhibit the secretion of proinflamma- tory cytokines TNF-α and IL-6. Physalin B sample (in 20, 10 and 5 μmol/L concentration) could signi- ficantly inhibit the secretion of inflammatory cyto- kines TNF-α and IL-6 (Fig. 5).

Fig. 5. Impact of physalin B on the TNF-α and IL-6 secretion in RAW264.7 cells (± s, n = 3) ΔP < 0.05, VS control group. *P < 0.05, VS LPS group

In the symmetric unit, there are two molecular C29H30O10. Fig. 2 is the molecular structure with atomic numbering scheme for these two molecules. Physalin B is a highly oxygenated steroidal lactone having eight fused rings. The six-membered ring A (C(1)～C(5)/C(10)) is in a half-chair conformation. Ring B (C(5)～C(10)) is trans-fused to ring A and adopts a chair conformation. The spiro-fused five- membered ring D (O(5)/C(14)～C(17)) assumes a vertical conformation, and lactone E (O(4)/C(18)/ C(13)/C(17)/C(20)) shows a planar five-membered ring. Ring F (C(16)/C(17)/C(20)/C(22)～C(24)) is in a boat-chair conformation, allowing the C(24)– C(16)–C(17), C(16)–C(17)–C(20), C(22)–C(23)–C(24) and C(20)–C(22)–C(23) bond angles to widen to 113.97(17), 114.48(17), 109.62(17) and 114.85(18)º, respectively. Ring H (O(6)/C(22)～C(26)) adopts a half-chair conformation. The two epoxy seven- and eight-membered rings (O(9)C(14)～C(16)C(24)C(25)C(27) and C(8)C(9)C(11)～C(14)C(17)O(5)) are in half-chair and envelope conformations, re- spectively. The3-3bond distances lie in the range of 1.508(3)～1.566(2) Å. The bond angles involving the spiro atom C(17) range from 102.53(15) to 115.20(16)°. The methanol molecules of solvation participate in two hydrogen bonds. A number of O(11)–H(11)×××O(1) and O(2)–H(2A)×××O(10) hydrogen bonds are observed in the molecular and crystal structures. The molecules are linked together by these interactions to form a two-di- mensional molecular network. The detailed infor- mation could be seen in Table 3.

Table 3. Hydrogen Bonds for Physalin B (Å, °)

Symmetry transformations used to generate the equivalent atoms: i) –,+1/2, –

The activity results show that the ant-inflammtory activity of physalin B (10μmol/L)is similar to that of dexamethasone (1μmol/L). The impact of its action can not be suppressed by the glucocorticoid receptor (GR) antagonist Ru486, but the positive drug dexamethasone can be suppressed by Ru486. The results suggest that physalin B has anti- inflammatory pharmacological activity in vitro, but its mechanism is not showing the glucocorticoid receptor (GR) related. The anti-inflammatory me- chanism needs further study.

(1) Matsuura, T.; Kawai, M.; Nakashima, R.; Butsugan, Y. Structures of physalin A and physalin B, 13,14-seco-16,24-cyclo-steroids fromvar.1970, 664–670.

(2) Vieira, A. T.; Pinho, V.; Lepsch, L. B.; Scavone, C.; Ribeiro, I. M.; Tomassini, T.; Ribeiro-dos-Santos, R.; Soares, M. B. P.; Teixeira, M. M; Souza, D. G. Mechanisms of the anti-inflammatory effects of the natural secosteroids physalins in a model of intestinal ischaemia and reperfusion injury.2005, 146, 244–251.

(3) Hsu, C. C.; Wu, Y. C.; Farh, L.; Du, Y. C.; Tseng, W. K.; Wu, C. C.; Chang, F. R.Physalin B from physalis angulata triggers the NOXA-related apoptosis pathway of human melanoma A375. 2012, 50, 619–624.

(4) Kuo, P. C.; Kuo, T. H.; Damu, A. G.; Su, C. R.; Lee, E. J.; Wu, T. S.; Shu, R. X.; Chen, C. M.; Bastow, K. F.; Chen, T. H.; Lee, K. H. Physanolide A, a novel skeleton steroid, and other cytotoxic principlesfrom physalis angulata..2006, 14, 2953–2956.

(5) Magalhães, H.I.F.; Veras, M.L.; Pessoa, O.D.L.; Silveira, E.R.; Moraes, M.O.; Pessoa, C.; Costa-Lotufo, L.V.Preliminary investigation of structure-activity relationship of cytotoxic physalins.2006, 3, 9–13.

(6) Silva, M. T. G.; Simas, S. M.; Batista, T. G. F. M.; Cardarelli, P.; Tomassini, T. C. B. Studies on antimicrobial activity, in vitro, ofL. (Solanaceae) fraction and physalin B bringing out the importance of assay determination.,Rio de Janeiro. 2005, 100, 779–782.

(7) Januario, A. H.; Rodrigues, E.; Pietro, R. C. L. R.; Kashima, S.; Sato, D. N.; Franca, S. C. Antimycobacterial physalins fromL. (Solanaceae).2002,16, 445–448.

(8) Masao, K.; Bunsho, M.; Tooru, T.; Yoshihisa, M.; Tatsuo ,Y.; Tsunehiro, F.; Hatsuo, Y.; Yasuo, B.; Kiyoshi, O.; Mitsuo, H. Crystal structures of 5,6-epoxy and 2,3-dihydro derivatives of physalin B, a 13,14-seco-16,24-cyclosteroid, and their1H NMR spectral analysis.. 1994, 67, 222–226.

(9). CrysAlis171.NET, Agilent Technologies, Version 1.171.35.19 2011.

(10) Sheldrick, G. M.. University of Göttingen, Germany 1997.

(11) Bruker..; Madison, Wisconsin, USA 2005, 10.

25 July 2013;

14 March 2014 (CCDC 908233)

the Guangdong Provincial Science and Technology Projects (No. 2011B031700072), and Guangdong Natural Science Foundation (No. 9151063201000036,S2013010013484), Guangdong Provincial Administration of traditional Chinese medicine project (No. 2008435)

. Tel: 020-28854995, E-mail: ncyyj@163.com