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The effects of Nigella sativa Ns Anthemis hyalina Ah and Citrus sinensis Cs extracts on the replication of coronavirus and the expression of TRP genes family Mustafa Ulasli Serdar A Gurses Recep Bayraktar Onder Yumrutas Serdar Oztuzcu Mehri Igci Yusuf Ziya Igci Ecir Ali Cakmak Ahmet Arslan Received 25 September 2013 Accepted 2 January 2014 Published online 12 January 2014 C211 The Author s 2014 This article is published with open access at S Abstract Extracts of Anthemis hyalina Ah Nigella sativa Ns and peels of Citrus sinensis Cs have been used as folk medicine to fight antimicrobial diseases To eval uate the effect of extracts of Ah Ns and Cs on the repli cation of coronavirus CoV and on the expression of TRP genes during coronavirus infection HeLa CEACAM1a HeLa epithelial carcinoembryonic antigen related cell adhesion molecule 1a cells were inoculated with MHV A59 mouse hepatitis virus A59 at moi of 30 1 50 dilu tion of the extracts was found to be the safe active dose ELISA kits were used to detect the human IL 8 levels Total RNA was isolated from the infected cells and cDNA was synthesized Fluidigm Dynamic Array nanofluidic chip 96 96 was used to analyze the mRNA expression of 21 TRP genes and two control genes Data was analyzed using the BioMark digital array software Determinations of relative gene expression values were carried out by using the 2 DDCt method normalized threshold cycle Ct value of sample minus normalized Ct value of control TCID 50 ml tissue culture infectious dose that will produce cytopathic effect in 50 of the inoculated tissue culture cells was found for treatments to determine the viral loads The inflammatory cytokine IL 8 level was found to increase for both 24 and 48 h time points following Ns extract treatment TRPA1 TRPC4 TRPM6 TRPM7 TRPM8 and TRPV4 were the genes which expression levels changed significantly after Ah Ns or Cs extract treatments The virus load decreased when any of the Ah Ns or Cs extracts was added to the CoV infected cells with Ah extract treatment leading to undetectable virus load for both 6 and 8hpi Although all the extract treatments had an effect on IL 8 secretion TRP gene expression and virus load after CoV infection it was the Ah extract treatment that showed the biggest difference in virus load Therefore Ah extract is the best candidate in our hands that contains potential treatment molecule s Keywords Coronavirus CoV C1 TRP channels C1 Anthemis hyalina C1 Nigella sativa C1 Citrus sinensis Introduction Coronaviruses CoV members of the Coronaviridae family are enveloped viruses that contain non segmented positive stranded genomic RNA 1 6 Ultrastructural analysis of CoV have revealed that they form pleomorphic particles that are roughly spherical but show variations in size 80 120 nm in diameter and shape 7 10 The entire CoV replication cycle take places in the cytoplasm In general enveloped viruses are able to use a variety of cellular membranes at different steps of their life cycles In fact almost any membranous subcellular compartment appears to be used by particular viruses to support their replication complex and assembly process 10 11 Electronic supplementary material The online version of this article doi 10 1007 s11033 014 3019 7 contains supplementary material which is available to authorized users M Ulasli therefore viruses may be targeting cellular mechanisms that regulate this concentration One of the possible targets of viruses to change the intracellular calcium concentration are the ion channels The transient receptor potential proteins TRPs is group of ion channels family that are responsible for a wide range of cellular functions including adjusting intracellular Ca 2 concentration 21 The TRP superfamily can be divided into seven families TRPC canonical TRPV vanilloid TRPM melastatin TRPN and TRPA ankyrin TRPML mucolipin and TRPP polycystin 22 TRP channels are triggered by a many different stimuli such as growth factors chemicals temperature depletion of intracellular calcium level and etc Triggering mechanism of channels and selectivity can be different between the TRPs 23 The expression levels of TRPM and TRPV families have been associated with tumor growth and progression 23 25 Herbal remedies have been used as folk medicine to treat many diseases Some herb extracts were shown to inhibit virus replication 26 27 One of these herbs is a chamomile plant called Anthemis hyalina Ah A hyalina belongs to plant family of Asreraceae genus Anthemis species A hyalina DC Chamomile is regularly used in tea making and to relieve stomachache and digestive system problems Chamomile was shown to have anti inflammatory effects and to decrease wound healing time 28 Antimicrobial activity of different species in the genus of Anthemis have been documented 29 Twenty four different components were characterized in Ah extract The major components were carvacrol 38 4 and a pinene 30 9 30 Citrus sinensis Cs belongs to plant family of Rutaceae genus citrus species Citrus sinensis L Osbeck Another herb used for various treatments is Cs known as sweet orange In Turkey people consume Cs by making jam and eating especially during flu seasons in order to get more vitamin C which is believed to help recover from flu Cs peels extract contain high amount of flavonoids limonene and linalool 31 Cs peel extract has antioxidant and antimicro bial activities 31 32 Limonene is an insecticidal compound with low oral or contact toxicity to mammals 33 Flavonoids have also been shown to have antiviral activity 34 35 Nigella Sativa belongs to the plant family of Ranun culaceae genus Nigella species Nigella sativa L Ns seeds that are known as black cumin is another plant that is used for various treatments In Turkey Ns seeds are commonly used on breads and cookies In addition some people mix Ns seeds and honey to use for upper respiratory tract infections and some stomach diseases Ns extract has antiviral antitumor and antimicrobial activities 36 38 Ns extract oil contains thymoquinone 27 8 57 0 q simen 7 1 15 5 karvakrol 5 8 11 6 t anetol 0 25 2 3 4 terpineol 2 0 6 6 and longifoline 1 0 8 0 39 40 It has been revealed that thymoqui none ve dithymoquinone are active components in Ns extract 41 In this study we set to find the effects of Ah Cs and Ns extracts on the replication of CoV and the possible involvement of TRP genes in the replication of CoV Materials and methods Cells virus and time course analysis of MHV infection HeLa CEACAM1a the epithelial carcinoembryonic anti gen related cell adhesion molecule 1 and murine fibroblast LR7 cells 42 that were used to propagate and titrate MHV A59 mouse hepatitis virus A59 were maintained in Dulbecco s Modified Eagle Medium DMEM Sigma St Louis MO containing 10 fetal calf serum Thermo Waltham MA 100 IU of penicillin ml and 100 lg ml of streptomycin both from Life Technologies Rochester NY HeLa CEACAM1a cells were inoculated with MHV A59 at a moi of 30 43 44 After 30 min the infected cells were washed and maintained in complete medium Sub sequently the infected cells and culture supernatants were 1704 Mol Biol Rep 2014 41 1703 1711 123 collected for analysis at 0 6 8 h p i When plant extracts were added to the HeLa CEACAM1a cells they were added after viral infection and were washed away 1 h later Preparation of extracts Air dried plant powdered seeds of Ns flowers and buds of Ah and peels of Cs were used for extraction 100 g of material of each separately All extraction experiments were performed by 200 ml ethanol addition onto powders and incubation at room temperature for a 24 h The extracts were filtered using Whatman filter paper and ethanol was eliminated by rotary vacuum evaporator at 55 C176C The plant extracts were dissolved in 10 ml distilled water Quantitative analysis of IL 8 by enzyme linked immunosorbent assay ELISA ELISA kits were used to detect the human IL 8 according to the user s manual Quantikine ELISA Human CXCL8 IL 8 Immunoassay DY208 R D Systems Minneapolis MN HeLa CEACAM1 cells were treated with 1 50 and 1 100 diluted concentrations of Ns Ah and Cs extracts Additionally HeLa CEACAM1 cells were infected with MHV A59 Infected cells were treated with 1 50 and 1 100 diluted concentrations of Ns Ah and Cs extracts Subse quently culture supernatants were collected at 0 6 and 8 h post infection to measure IL 8 levels by using enzyme linked immunosorbent assay ELISA Monitoring the extracellular release of the virus Infected HeLa CEACAM1a supernatants were collected and end point dilutions were made for TCID 50 analysis at 6 and 8 h post infection The amount of virus present in the culture supernatants were evaluated by using LR7 cells and TCID 50 values were calculated Isolation of total RNA and TaqMan analysis Total RNA was isolated from the infected cells using RNeasy mini kit Qiagen Hilden Germany according to the manufacturer s instructions with subsequent DNaseI treatment on the column The amount of RNA concentra tion was determined by spectrometry using a Nanodrop 100 Thermo Scientific DE USA cDNA synthesis Complementary DNA cDNA was synthesized by using Qiagen miScript Reverse Transcription Kit Qiagen Sample and Assay Technologies Hilden Germany according to manufacturer s instructions Mixture of 59 miScript RT buffer and miScript Reverse Transcription mix was prepared at given ratios and 1 25 ll of it was distributed for each reaction 0 2 lg of RNA samples was added on reaction mixtures and they were incubated at 37 C176C for 60 min then at 95 C176C for 5 min Synthesized cDNAs were then diluted 1 5 with low EDTA TE buffer and subsequently transferred on ice block and kept at 20 C176C till use Fluidigm real time PCR quantifications Fluidigm Dynamic Array nanofluidic chip 96 96 was used for mRNA expression assay Fluidigm South San Fran cisco CA USA mRNA expression levels of 23 genes were analyzed by BioMark TM HD System real time PCR Fluidigm Table 1 Scrutinizing of the data and counting of the generated signals in reaction chambers was achieved by fluidigm real time PCR Analysis software Fluidigm Intracellular calcium level determination using atomic absorption spectrometer HeLa CEACAM1a cells were infected with MHV A59 cells After infection HeLa CEACAM1 cells were incubated with Ns Ah and Cs extracts for 6 and 8 h Infected cells were collected for quantification of total calcium ions Concentra tion of calcium ions lg l was determined by using an AAS 400 Perkin Elmer USA atomic absorption spectrometer FAAS in an air acetylene flame according to the user s manual provided by the manufacturer A calcium hallow cathode lamp was used as the radiation source at 423 nm Statistical analysis Data was analyzed using the BioMark digital array software and the numbers of positive chambers were corrected to estimate the true number of copies This number was used to determine the number of copies in the original sample Determinations of relative gene expression were carried out by using the 2 DDCt method comparative Ct method where Ct values of the samples of interest are compared with a control uninfected cells after the Ct values of the control and the samples are normalized to an endogenous house keeping gene Expression levels were shown relative to the uninfected cells GAPDH was used as the housekeeping gene for normalization of the expressions Results and discussion Determination of maximum non toxic doses In order to assess any possible toxic effects of Ns Ah and Cs extracts on HeLa CEACAM1a cells serial dilutions Mol Biol Rep 2014 41 1703 1711 1705 123 1 10 1 50 1 100 1 1000 1 10 000 of Ns Ah and Cs extracts were incubated with HeLa CEACAM1a cells for 24 and 48 h The cell viability was assessed by the tetrazolium salt 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide MTT assay These three plant extracts showed toxic effect at 1 10 dilution however toxicity of the extracts clearly decreased at 1 50 dilution for 24 and 48 h time points Figs 1 2 These results suggest that the cytotoxic effect of Table 1 The list of genes whose expression levels were measured by BioMark TM HD System real time PCR TRPA1 TRPC4 TRPC7 TRPM3 TRPM6 TRPV1 TRPV4 GAPDH TRPC1 TRPC5 TRPM1 TRPM4 TRPM7 TRPV2 TRPV5 TRPC3 TRPC6 TRPM2 TRPM5 TRPM8 TRPV3 TRPV6 Fig 1 Effects of Ns Ah and Cs extracts on the survival of HeLa CEACAM1a cells HeLa CEACAM1a cells were incubated with the diluted extracts for 24 h and cell toxicity was measured by MTT assay The bars show the averages from 2 independent experiments with 4 repeats for each treatment Error bars show standard error C negative control Fig 2 Effects of Ns Ah and Cs extracts on the survival of HeLa CEACAM1a cells HeLa CEACAM1a cells were incubated with the extracts for 48 h and cell toxicity was measured by MTT assay The bars show the averages from two independent experiments with four repeats for each treatment Error bars show standard error C negative control 1706 Mol Biol Rep 2014 41 1703 1711 123 these plants extracts were concentration dependent but not time dependent in that less cells were viable at 1 10 dilution compared to 1 50 dilution Based on these results the plant extract dilution of 1 50 was accepted as an active dose IL 8 levels following extract treatments of CoV infected cells The levels of the inflammatory cytokine IL 8 in the supernatants of cells that were treated with the plant extracts were determined by ELISA Fig 3 IL 8 was chosen for its possible role in preventing DC cells from priming T cells during CoV infection and inducing calcium signaling that might be related to TRP genes 45 IL 8 levels increased following treatment by Ns Ah and Cs extracts for both 1 50 and 1 100 dilutions at 24 h However IL 8 levels had decreased at 48 h for Ah and Cs treated cells This suggests that Ns extract was better at stimulating IL 8 secretion from infected cells to lead to a bigger inflammatory response When IL 8 levels were measured following plant extract treatment of infected cells 6 and 8 h post infection it was found that IL 8 levels were below the lowest standard and very low Supplementary figure Therefore we cannot comment on the effect of plant extracts on IL 8 secretion from CoV infected cells Expressions of TRP genes following extract treatment of CoV infected cells To show effects of the Ns Ah and Cs extracts on expres sion of TRP genes during CoV infection active doses of the Ns Ah and Cs extracts were added to HeLa CEA CAM1a cells one hour before CoV infection Subse quently the cells were infected with CoV for 6 h post infection and 8 h mRNA expression levels for TRP channel families were investigated by using fluidigm RT PCR GAPDH expression was used for normalization of gene expression Uninfected cells were used as a negative control The infected but not plant extract exposed cells were used as positive control The expressions of TRP genes were evaluated 6 and 8 h post infection These time points were chosen because CoV shows its cytopathic effects by 8 h post infection 43 The changes in the expression levels were determined by calculating fold changes 2 DDCt for the TRP genes and normalizing them by dividing with the fold change for the control gene GAPDH Fold change values greater than 1 indicate an up regu lation and fold change values less than 1 indicate down regulation However fold change values more than 2 and less than 0 5 are considered significant Based on the TRP genes expression analysis results the expression levels were mainly down Fig 4 The TRP channels affected by extracts were TRPA1 TRPC4 TRPM6 TRPM7 TRPM8 and TRPV4 Treatment with Ns Ah and Cs extracts during CoV infection resulted in the down regulation of TRPM6 and TRPA1 for both 6 and 8 h time points TRPC4 was down regulated following Ns and Ah treatments for both 6 and 8 h time points TRPM7 was down regulated only after Ns treatment for the two time points Ah treatment resulted in down regulation of TRPV4 for the two time points Cs treatment resulted in down regulation of TRPM8 for the two time points Monitoring the extracellular release of the virus In order to determine the number of viruses following extracts treatment and viral infection TCID50 ml was determined for the conditions and it was found that com pared to the control group extract treatments lowered the Fig 3 Effects of Ns Ah and Cs extracts on the secretion of IL 8 from HeLa CEACAM1a cells after 24 and 48 h The bars show the averages from two independent experiments with four repeats for each treatment Error bars show standard error C negative control Mol Biol Rep 2014 41 1703 1711 1707 123 virus loads Fig 5 In the case of Ah treatments there was no detectable virus Following Ns treatment the number of viruses was very low at 6 h post infections and it was 1 10th of the control amount by 8 h post infections Cs treatment resulted in the same number of viruses at 6 and 8 h post infections which were 1 10th of the positive control numbers by 8 h Intracellular calcium levels Following the determination of the expression of TRP genes we sought to find the intracellular calcium levels Fig 6 Intracellular calcium levels did not change after addition of plant extracts to uninfected cells However addition of plant extracts to CoV infected cells increased intracellular calcium levels Fig 4 TRP gene expression levels following Ns Ah and Cs extract treatment of infected HeLa CEACAM1a cells after 6 a and 8 h post infections b compared to uninfected cells The bars show the averages from two independent experiments Error bars show standard error TCID50 ml 0 20000 40000 60000 80000 100000 120000 Ah Ns Cs Control 6hpi 8hpi Fig 5 Virus loads following Ns Ah and Cs extract treatment of infected HeLa CEACAM1a cells after 6 and 8 h post infections The amount of virus present in the culture supernatants were evaluated by using LR7 cells and TCID 50 values were calculated The bars show the averages from two independent experiments with two repeats 1708 Mol Biol Rep 2014 41 1703 1711 123 Conclusion The results presented here suggest that treatment of cells with Ns Ah or Cs extracts prior to infection with CoV decreases the replication of the virus IL 8 secretion was increased at 24 h for Ns and Ah extract treatments but Cs treatment did not show a significant increase in IL 8 secretion at 24 h However IL 8 secretion was very low at early time points after infection Fig 3 Therefore it is not known if IL 8 levels are related to a decreased virus load Following gene expression analysis the TRP genes with changed expression levels were identified as follows TRPM6 and TRPA1 were the two genes that were down regulated for both 6 and 8 h time points in CoV infected and extract treated cells TRPC4 gene was down regulated following Ns and Ah treatments for both 6 and 8 h time points TRPM7 was down regulated only after Ns treatment for the two time points TRPV4 gene was down regulated following Ah treatment for the two time points and TRPM8 was down regulated following Cs treatment for the two time points A general down regulation trend for TRP genes was observed in the CoV infected and extract treated cells Together with the data showing that virus loads were decreased upon extract treatments we predicted that TRP genes might be involved in CoV survival inside cells Considering that replication of other viruses were related to intracellular calcium concentration and TRPs modulate ion concentrations including calcium we measured intracel lular calcium concentrations and found that they increased following plant extract treatments of infected cells The controversial TRP gene expression and calcium concen tration results suggest that they may not be related to decreased viral loads in plant extract treated infected cells or decreased TRP gene expression and or increased cal cium concentration decrease viral loads through unknown mechanisms Undetectable levels of the virus after Ah treatment can mean a specific factor being involved fol lowing that treatment and this factor can b