Chemical Constituents from Chirita longgangensis var . hongyao with Inhibitory Activity against Porcine Respiratory and Reproductive Syndrome Virus

Dois novos quinenóides chiritalona A and B, e uma nova neolignina 7’E-4,9-dihidroxi3,3’,5’-trimethoxi-8,4’-oxineolign-7’-en-9’-al, além dos conhecidos (-)-8-hidroxi-α-dunniona, digiferruginol, 2,5-dimetoxi-1,4-benzoquinona e hederagenina, foram isolados do caule de Chirita longgangensis var. hongyao. As estruturas dos novos compostos foram elucidadas por análise detalhada de dados obtidos pela técnicas de NMR (ressonância magnética nuclear) e MS (espectrometria de massas), e a configuração absoluta de chiritalona A foi determinada por análise de difração de raios X de monocristal utilizando o parâmetro Flack. A atividade inibitória dos compostos com relação ao vírus da síndrome respiratória e reprodutiva suína (PRRSV) foi medida pelo método do efeito citopático (CPE). Digiferruginol e hederagenina apresentaram efeito fraco com relação ao PRRSV com valor IC50 de 80,5 ± 16,9 μmol L -1 (SI = 19,9) e 43,2 ± 7,4 μmol L (SI = 13,1), respectivamente.


Introduction
Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is characterized by respiratory disorders in young pigs and reproductive failure in sows.It is widespread in most major pig-producing areas throughout the world and is one of the most important causes of economic loss to the swine industry.In China, in 2006 only, PRRS spread to more than 10 provinces (or autonomous regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. 1 Although previous studies provided a basis for development of pharmacological agents to inhibit PRRSV replication, so far there are no effective drugs to overcome this problem, 2 and many vaccine strategies developed to control the disease are not yet completely successful. 3,4uinones are a widespread group of oxygen-substituted aromatic compounds, and some of them are considered as inhibitory agents against both RNA and DNA virus. 5lants of Gesneriaceae such as Streptocarpus dunnii Mast., Chirita eburnea Hance, Sinningia aggregata (Ker-Gawl.)Wiehler, and Didymocarpus hedyotideus Chun.][8][9][10] Chirita longgangensis var.hongyao, a species of Gesneriaceae, is distributed in Guangxi of China, 11 and is used in the treatment of bone fractures, wounds and pains. 12][15][16][17] Our group conducted a phytochemical research on the stems of the plant, which led to the isolation of seven compounds (1-7) including two new quinones (1 and 2) and the new neolignan (3) (Figure 1).All of the isolates were evaluated for their inhibitory activity against PRRSV.The structure elucidation of the new constituents and the bioassay results are reported.
Compound 3 was isolated as an oil, and its molecular formula established as C 21 H 24 O 8 from the HRESIMS quasi-molecular ion [M -H] -at m/z 403.1390 (calcd.403.1392).The IR absorption bands of 3 were indicative of OH presence of OH (3440 cm -1 ), CHO (1659 cm -1 ) and    Ph (1583, 1502 and 1425 cm -1 ) groups.The 1 H and 13 C NMR data of 3 showed high similarity with those of the known neolignan 8 isolated from Eucommia ulmoides Oliver (Figure S18 in the Supplementary Information (SI) section), 18 which displays a negative optical rotation [[α] D 25 -15.5 (c 0.04, MeOH)].Since the optical rotation of 3 was positive, its 7R,8S configuration was excluded.Nevertheless, due to the lack of available sample material, the CD spectrum of 3 could not be recorded, and the absolute configuration could not be assigned.The structure of 3 was further supported by HMBCs as shown in Figure 2. The coupling constant of 5.0 Hz between H-7 and H-8 suggested the two H-atoms were in erythro-configuration. 19ccordingly, 3 was determined as 7'E-4,9-dihydroxy-3,3',5'-trimethoxy-8,4'-oxyneolign-7'-en-9'-al.
The inhibitory activity of compounds 1-7 against PRRSV was measured by the cytopathic effect (CPE) method. 24igiferruginol (5) and scutellaric acid (7) showed weak effect on PRRSV with an IC 50 (concentration of compound required for 50% inhibition) value of 80.5 ± 16.9 μmol L -1 (selectivity index, SI > 19.9) and 43.2 ± 7.4 μmol L -1 (SI = 13.1),respectively, compared with tilmicosin phosphate (IC 50 = 225.1 ± 27.4 μmol L -1 , SI = 3.8) (Table 2).No significant inhibitory effects were observed for other compounds at a concentration of 200 μmol L -1 .Among the quinones (1, 2 and 4-6), only the active compound (5) belongs to the anthraquinone type.These results suggest that the carbon skeleton of anthraquinone might be necessary for the PRRSV-inhibitory activity.][27] The inhibitory activity of the oleanane-type triterpene 7 against protein tyrosine phosphatase 1B (PTP1B) has been evaluated, but it was found to be inactive. 28The antiviral activity of compounds 5 and 7 against PRRSV is here reported for the first time.
Furthermore, by the real-time fluorescent quantitative reverse transcription-polymerase chain reaction (FQ RT-PCR), [29][30][31] the relative expression ratio of non-strach polysaccharides NSP9 and open reading frame ORF7 genes of PRRSV was tested.As shown in Table 3 and Figure 5, NSP9 mRNA relative expression level was significantly reduced by compounds 5 and 7 at the concentrations of 50 μmol L -1 or more (P < 0.001) in a dose-dependent manner.Also, ORF7 mRNA relative expression level was significantly reduced by compounds 5 and 7 at the concentration of 200 μmol L -1 (P < 0.001).The viral RNA-dependent RNA polymerase (RdRp; nsp9), a key enzyme for the RNA synthesis of PRRSV, is enc oded by the NSP9 gene. 324][35] The down regulation of NSP9 and ORF7 mRNA expression implied that the replication of PRRSV RNA and the assembly of the virons might be inhibited by the compounds.

General experimental procedures
Melting points were determined using an X-4 melting point apparatus (Yingyu Yuhua Apparatus Factory, Gongyi, P. R. China), and were not corrected.Bruker SMART APEX-II and Bruker APEX DUO diffractometers using  graphite-monochromated Cu K α radiations were employed for the intensity data collection, and the structures of compounds were solved by direct methods (SHELXS97). 36ptical rotations were determined on a JASCO DIP-370 automatic digital polarimeter.UV spectra were recorded on a Shimadzu double-beam 210A spectrometer.IR spectra were recorded on a Bio-Rad FTS-135 infrared spectrophotometer.Circular dichroism (CD) spectra were obtained using a JASCO J-715 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) in CD

Plant material
The stems of Chirita longgangensis var.hongyao were collected from Jingxi County of Guangxi Zhuang Autonomous Region, P. R. China, in June 2010.The plant material was identified by Prof. Chun-Lin Long, and a voucher specimen (No.JX1001) was deposited at the Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and isolation
The air-dried stems of Chirita longgangensis var.hongyao (4.5 kg) were exhaustively extracted with MeOH (4, 3 and 3 h, successively) at 70 ºC, and the solvent evaporated under reduced pressure.2), largest difference peak and hole = 0.332 and -0.437 e Å -3 .The intensity data for 1 were collected on a Bruker APEX DUO diffractometer using graphite-monochromated Cu K α radiation.The structure of 1 was solved by direct methods (SHELXS97), 36 expanded using difference Fourier techniques, and refined by the program and full-matrix least-squares calculations.
The nonhydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions.Crystallographic data for the structure of 1 were deposited in the Cambridge Crystallographic Data Centre (deposition No. CCDC 852437).for 1.5 h.The number of surviving cells was measured with a Bio-Tek ELx 800.ELISA microplate reader could show the detection wavelength of 450 nm (L 1 ) and the reference wavelength of 650 nm (L 2 ).The 50% cytotoxic concentration (CC 50 ) was obtained by nonlinear regression analysis of logistic curves (the value of L 1 -L 2 to different concentrations of compounds).

Cytopathic effect inhibition assay
YN-1 strain of PRRSV was isolated from local pigs in Yunnan Province, P. R. China. 39The antiviral activity of tested compounds against viruses was measured by the CPE inhibition assay. 24Tissue culture medium infective dose (TCID 50 ) of 500 viral particles with twofold serial dilutions of the compounds were added to each test well, and the plates were reincubated for 4 days to allow development of a CPE if any.A non-infection control was made in the absence of natural products and tilmicosin phosphate was used for drug control.The concentration reducing CPE by 50% with respect to virus control was estimated from graphic plots and was defined IC 50 .The selectivity index was calculated from the ratio CC 50 /IC 50 .

Statistical analyses
All experiments were performed in three replications.Continuous variables, expressed as mean ± standard deviation, were compared using one-way ANOVA (analysis of variance).Statistical analyses were conducted with SPSS 17.0 Statistics software.

Conclusions
In this research, seven compounds were isolated from the stems of C. longgangensis var.hongyao.Compounds 1 and 2 are new quinonoids, and 3 is a new neolignan.The inhibitory activity of all compounds against PRRSV was measured by the CPE method.Among the quinones (1, 2 and 4-6), only the active compound 5 belongs to the anthraquinone type.The results suggest that the carbon skeleton of anthraquinone might be necessary for the PRRSV-inhibitory activity.

a 1 H
NMR of 1 recorded in CDCl 3 at 400 MHz; b 13 C NMR of 1 recorded in CDCl 3 at 100 MHz; c 1 H NMR of 2 recorded in DMSO-d 6 at 600 MHz; d 13 C NMR of 2 recorded in DMSO-d 6 at 150 MHz; e 1 H NMR of 2 recorded in CD 3 OD at 100 MHz; f 13 C NMR of 2 recorded in CD 3 OD at 100 MHz.

Figure 3 .
Figure 3. ORTEP drawing of the asymmetric unit of compound 1.

of 1 except that the signal for one ketone group in 1 was replaced by the signal for the O-bearing CH group (d C 72.4 ppm) in 2. This implied that one of the ketone groups was reduced to an O-bearing CH group in 2. The group was located at C-4 by the HMBCs correlations Figure 1. Chemical structures of compounds 1-7. Vol
.23, No. 10, 2012

Table 1 .
NMR data of compounds 1 and 2 (d in ppm, J in Hz)

Table 2 .
Inhibitory effects of 5 and 7 on PRRSV in Marc-145 cells 50 : the median cytotoxic concentration; SI: selectivity index.