Effect of Ethanolic Caesalpinia Sappan Fraction on in Vitro Antiviral Activity Against Porcine Reproductive and Respiratory Syndrome Virus

veterinary sciences

Article Effect of Ethanolic Caesalpinia sappan Fraction on In Vitro Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Chaiwat Arjin 1 , Surat Hongsibsong 2 , Kidsadagon Pringproa 3, Mintra Seel-audom 1, Warintorn Ruksiriwanich 4,5 , Kunrunya Sutan 2, Sarana Rose Sommano 4,6 and Korawan Sringarm 1,4,*

1 Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] (C.A.); [email protected] (M.S.-a.) 2 School of Health Science Research, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] (S.H.); [email protected] (K.S.) 3 Department of Veterinary Bioscience and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand; [email protected] 4 Cluster of Research and Development of Pharmaceutical and Natural Products Innovation for Human or Animal, Chiang Mai University, Chiang Mai 50200, Thailand; [email protected] (W.R.); [email protected] (S.R.S.) 5 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand 6 Department of Plant and Soil Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand * Correspondence: [email protected]

Citation: Arjin, C.; Hongsibsong, S.; Pringproa, K.; Seel-audom, M.; Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major epidemic in Ruksiriwanich, W.; Sutan, K.; pig production, leading to economic losses in the pig industry worldwide. The use of medicinal Sommano, S.R.; Sringarm, K. Effect of plants with antiviral properties might be useful help to prevent and control PRRSV outbreaks. Ethanolic Caesalpinia sappan Fraction Caesalpinia sappan (CS) heartwood is an important herbal ingredient used in Thai folk medicine, on In Vitro Antiviral Activity against possessing various biological activities, including antiviral activity. The present study focuses Porcine Reproductive and on the in vitro antiviral activity against PRRSV of a semi-purified fraction of ethanolic CS crude Respiratory Syndrome Virus. Vet. Sci. extract using preparative high-performance liquid chromatography. Qualification of the fractions 2021, 8, 106. https://doi.org/ illustrating positive antiviral activity was carried out with liquid chromatography–quadrupole time- 10.3390/vetsci8060106 of-flight mass spectrometry. The preparative chromatography separated the crude extract into six consecutive fractions, among which the first fraction showed potential antiviral activity by inhibiting Academic Editor: Kwonil Jung PRRSV replication in a MARC-145 monolayer (virus titer 2.75 median tissue culture infective dose (TCID )/mL (log ) vs. 9.50 median log10 TCID /mL of the control) at 72 h post-infection, and this Received: 29 April 2021 50 10 50 Accepted: 8 June 2021 fraction included byakangelicin, brazilin, naringenin, and brazilein. These results provide useful Published: 9 June 2021 information for further study to effectively develop the CS bioactive antiviral compounds against PRRSV as a feed additive or veterinary drug in the pig industry.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: Caesalpinia sappan; porcine reproductive and respiratory syndrome virus (PRRSV); antivi- published maps and institutional afﬁl- ral activity; fractionation; LC–QTOF-MS iations.

1. Introduction Copyright: © 2021 by the authors. Porcine reproductive and respiratory syndrome (PRRS) is an important major infec- Licensee MDPI, Basel, Switzerland. tious disease that has adversely impacted the global pig industry. Porcine reproductive This article is an open access article and respiratory syndrome virus (PRRSV), the causative agent of PRRS, is an enveloped distributed under the terms and single-stranded positive-sense RNA virus that belongs to the Arteriviridae (order Nidovirales) conditions of the Creative Commons family [1,2]. PRRSV induces reproductive failure in pregnant sows and respiratory distress Attribution (CC BY) license (https:// in pigs of all ages [3] as well as predisposing pigs to infection by bacteria and other viral creativecommons.org/licenses/by/ pathogens [4,5]. Even though vaccinations have been used to control this disease, some 4.0/).

Vet. Sci. 2021, 8, 106. https://doi.org/10.3390/vetsci8060106 https://www.mdpi.com/journal/vetsci Vet. Sci. 2021, 8, 106 2 of 14

vaccine types, including modified live vaccines, have not been successful in eradicating the virus and do not provide complete immunity from heterologous infections [6,7]. Moreover, there are currently no specific drug treatments in clinical use available against PRRSV infection. The use of antibiotics might help in controlling secondary infections during PRRSV infections [8]. However, the use of antibiotics could promote antibiotic resistance, which has an adverse impact on animal health and is consequently associated with resistant infections in humans [9]. Therefore, attempts have been made to search for medicinal plants with antiviral potential to enable the development of a novel antiviral drug to prevent PRRSV [6,10,11]. Our previous studies discovered that the Caesalpinia sappan (CS) crude extract had antiviral activity against PRRSV by inhibiting PRRSV replication in vitro in the MARC-145 cell line [12]. CS is a well-known medicinal plant belonging to the Leguminosae family [13], distributed and cultivated in tropical Asian regions such as Southern China, India, Myanmar, Vietnam, Sri Lanka, and Thailand. The dried heartwood of CS has been used for a long time in oriental folk medicines such as in Indian Ayurveda and Traditional Chinese Medicine [14]. It is also the source of a natural red dye that can be used as a coloring agent in food, beverage, and cosmetics [15]. A decoction made from its heartwood is used in Namya-Uthai as a cardiotonic and to quench thirst [16]. In Northern Thailand, especially in the Chiang Mai, Nan, and Lampang provinces, a CS heartwood decoction is used as an anti-inflammatory agent for the treatment of traumatic disease and arthritis [13]. Many biological properties of CS have been reported such as antioxidant [12,13,17,18], antibacterial [15,16,19], anti-inflammatory [17,20], hypoglycemic [21,22], and hepatoprotec- tive [23] activities. In addition, the constituents of CS extract exhibited high activity against the influenza virus (H3N2) [24]. Bunluepuech and Tewtrakul [25] also reported that CS ethanolic extract could exhibit HIV-1 inhibitory activity. However, defining the exact antiviral agent had been difficult due to the complex phytocomposition of the ethanolic extract. Fractionation is, therefore, very crucial to further characterize bioactive compound groups with antiviral properties. Preparative high-performance liquid chromatography (HPLC) is a classical chromatographic method extensively used in the separation, purification, and upscale of natural products of interest [26]. No data are yet available on the HPLC fractionation of CS extract, which conveys antiviral activity against PRRSV. Therefore, the objectives of this study were separate and collect semi-purified fraction of CS extract by preparative HPLC, to determine each semi-purified fraction with in vitro antiviral activity against PRRSV, and to characterize the responsible bioactive compounds.

2. Materials and Methods 2.1. Experimental Plan This study was designed to identify the CS extract fraction with antiviral activity against PRRSV (Figure1). A crude extract of CS heartwood was obtained with 95% ethanol. Analytical high-performance liquid chromatography (HPLC) was use to optimize the analysis conditions and chromatogram fingerprint for CS crude extract. Next, preparative HPLC was performed to separate and collect each semi-purified fraction of CS. All semi-purified fractions of CS underwent a cell cytotoxicity test to define the optimum concentration for testing their antiviral activity in cells (expressed as 50% cytotoxic concentration (CC50). The antiviral activity for the inhibition of virus infection and replication was determined as a function of the CC50. The immunoperoxidase monolayer assay (IPMA) was used to assess virus titration. Lastly, the effective antiviral bioactive compounds in the semi-purified fractions were characterized using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–QTOF-MS). Vet. Sci. 2021, 8, x FOR PEER REVIEW 3 of 14 Vet. Sci. 2021, 8, 106 3 of 14

FigureFigure 1. 1.TheThe experimental experimental procedure procedure toto determinedetermine the the CS CS fraction fraction with within vitro in vitroantiviral antiviral activity activity againstagainst PRRSV. PRRSV. 2.2. Sample Collection and Extraction 2.2. Sample Collection and Extraction The heartwood of CS was collected from Samoeng District, Chiang Mai Province, Thailand.The heartwood The heartwood of CS was was chopped collected and from dried atSamoeng 60 ◦C in aDistrict, hot-air ovenChiang for 48 Mai h. Then, Province, Thailand.it was ground The heartwood to powder was and sievedchopped through and dried a 1 mm at mesh. 60 °C Thein a finehot-air powder oven (500 for g) 48 was h. Then, it wasmacerated ground with to powder 95% ethanol and (3sieved× 28 L)through for 72 h a at 1 roommm mesh. temperature, The fine and powder the extraction (500 g) was maceratedwas repeated with twice. 95% ethanol The obtained (3 × 28 ethanolic L) for 72 crude h at extracts room temperature, were combined and and the filtered extraction wasthrough repeated Whatman twice. No.The 1obtained filter paper. ethanolic The solvent crude was extracts completely were evaporatedcombined usingand filtered a rotary evaporator at 40 ◦C. The extracts were stored at −20 ◦C until further use. through Whatman No. 1 filter paper. The solvent was completely evaporated using a ro- tary2.3. evaporator Analytical High-Performanceat 40 °C. The extracts Liquid Chromatographywere stored at (HPLC) −20 °C until further use. The ethanolic CS crude extract was dissolved in 10% methanol to a final concentration 2.3.of Analytical 1 mg/mL. High-Performance It was then semi-purified Liquid Chromatography before identifying (HPLC) fingerprints of the peak using analyticalThe ethanolic HPLC. TheCS crude analytical extract HPLC was was dissolved performed in using10% methanol an Agilent to 1220 a final Infinity concentra- II tionLC of (Agilent 1 mg/mL. Technologies, It was then Santa semi-purified Clara, CA, USA). before Reverse-phase identifying columnfingerprints chromatography of the peak us- × µ ingwas analytical performed HPLC. using The a YMC analytical Triart C-18HPLC (250 was performed4.6 mm, 5.5 usingm) (YMC, an Agilent Kyoto, 1220 Japan). Infinity The mobile phase consisted of 1% acetic acid in distilled water (A) and 1% acetic acid in II LC (Agilent Technologies, California, USA). Reverse-phase column chromatography methanol (B). The gradient elution started at a flow rate of 1 mL/min with 95% A and wasdecreased performed to 80% using A ina YMC 5 min, Triart 70% A C-18 in 5 min,(250 65%× 4.6 A mm, in 5 min,5.5 μ 55%m) (YMC, A in 5 min,Kyoto, 25% Japan). A in The mobile5 min, phase and 5% consisted A in 5 min of before1% acetic holding acid for in 10distilled min and water returning (A) and to 95% 1% Aacetic in 5 min. acid Thein meth- anoltotal (B). run The time gradient was 45 elution min per started sample. at The a flow photodiode rate of 1 array mL/min detection with wavelengths 95% A and weredecreased to 80%set to A 280 in and5 min, 330 70% nm. A in 5 min, 65% A in 5 min, 55% A in 5 min, 25% A in 5 min, and 5% A in 5 min before holding for 10 min and returning to 95% A in 5 min. The total run 2.4. Semi-Purified Fractionation of CS Extract by Preparative HPLC time was 45 min per sample. The photodiode array detection wavelengths were set to 280 and 330The nm. ethanolic CS crude extract was prepared by dissolving it in 10% methanol to a final concentration of 10 mg/mL before passing through 0.45 and 0.22 µm filters. The filtrated CS crude extract was fractionated using a YMC LC-Forte/R recycling preparative 2.4. Semi-Purified Fractionation of CS Extract by Preparative HPLC HPLC (YMC, Kyoto, Japan) with a photodiode array detector (YMC-YUV-3400). It was operatedThe ethanolic at 18 mL/min CS crude using extract 1% acetic was acid prepared in distilled by waterdissolving (solvent it in A) 10% and 1%methanol acetic to a finalacid concentration in methanol (solvent of 10 mg/mL B). The before linear gradient passing used through was as 0.45 follows: and 0.22 0 min, μm 5% filters. B; 5 min, The fil- trated20% CS B; 10 crude min, 30%extract B; 15 was min, fractionated 35% B; 20 min, using 45% B;a YMC 25 min, LC-Forte/R 75% B; 30 min, recycling 95% B; 35preparative min, HPLC95% (YMC, B; and 45Kyoto, min, 5%Japan) B. The with preparative a photodiode HPLC array column dete wasctor a YMC-Actus (YMC-YUV-3400). Triart C18 It was × µ operated(250 mm at 1820 mL/min mm, 5 m, using pore size1% acetic 12 nm) acid (YMC, in Kyoto,distilled Japan). water The (solvent detection A) wavelength and 1% acetic was set to 280 and 330 nm. Each injection volume was equivalent to 100 mg/load. Each acid in methanol (solvent B). The linear gradient used was as follows: 0 min, 5% B; 5 min, 20% B; 10 min, 30% B; 15 min, 35% B; 20 min, 45% B; 25 min, 75% B; 30 min, 95% B; 35 min, 95% B; and 45 min, 5% B. The preparative HPLC column was a YMC-Actus Triart C18 (250 mm × 20 mm, 5 μm, pore size 12 nm) (YMC, Kyoto, Japan). The detection wavelength was set to 280 and 330 nm. Each injection volume was equivalent to 100 mg/load. Each semi-purified CS extract fraction was collected and evaporated using a rotary evaporator

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semi-puriﬁed CS extract fraction was collected and evaporated using a rotary evaporator before drying the sample with nitrogen blown through needles onto the samples in tubes. Next, the samples were weighed and kept at −20 ◦C until they were used for the antiviral bioassay and phytocomposition determination by LC-QTOF-MS.

2.5. Cells and Virus MARC-145 cells (CRL-12231, American Tissue Collection Center (ATCC), Virginia, USA) were grown in Dulbecco’s modiﬁed Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin and incubated at 37 ◦C in 5% CO2 in a humidiﬁed incubator. PRRSV (VR2332 North American genotype) was propagated in MARC-145 cells, and the virus cells were titrated using IPMA and then ◦ stored at −80 C. Virus titer was determined and expressed as TCID50 according to the Reed-Muench method [27].

2.6. Cell Cytotoxicity The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used to determine the effect of the CS extract fractions on MARC-145 cell viability. Brieﬂy, Marc-145 cells were seeded into 96-well plates at a density of 5000 cells/well and ◦ incubated in a 5% CO2 atmosphere at 37 C for 24 h. When cells achieved at least 90% conﬂuence, the medium was removed and replaced with a medium containing twofold serial dilutions of the CS extract fraction. The medium without plant extract was used as a ◦ control. The plates were incubated at 37 C under a 5% CO2 atmosphere for 72 h. Thereafter, the medium was removed, and 20 µL of freshly prepared 5 mg/mL MTT solution was added to each well, before incubating again at 37 ◦C for 4 h. Then, the medium was removed and replaced with 150 µL of DMSO to dissolve the crystals, and the plates were shaken for 5 min to dissolve any air bubbles before measuring the MTT signal absorbance at 550 nm. The results were represented as CC50, describing the extract concentration that reduced cell viability by 50% when compared to untreated controls [28].

2.7. Inhibition of Viral Infection Assay The inhibition of viral infection assay was performed as previously described [11]. Brieﬂy, the PRRSV at a multiplicity of infection (MOI) of 1 was mixed with media containing CS fractions at the cytotoxicity test concentration and two lower concentrations in twofold dilutions before incubating at 37 ◦C for 1 h. PRRSV mixed with 1% DMSO served as the positive control. Thereafter, the MARC-145 cells at 5000 cells/well in 96-well plates were inoculated with the mixture of PRRSV and CS fraction or control before incubating at 37 ◦C for 1 h. Next, the medium was removed and replaced with fresh 2% FBS medium. The supernatant was collected for virus titration 24 h later.

2.8. Inhibition of Viral Replication Assay The inhibition of the viral replication assay was performed as previously described [11]. Brieﬂy, MARC-145 cells were seeded into 96-well plates at a density of 5000 cells/well and infected with PRRSV at an MOI of 1 at 37 ◦C for 1 h. Then, PRRSV was removed from each well and replaced with media containing diluted CS extract fractions at the concentration from the cytotoxicity test and at two lower concentrations in twofold dilutions. Medium containing 1% DMSO served as the positive control. The plates were cultured under ◦ standard conditions (5% CO2 atmosphere at 37 C). The supernatants were collected at 24, 48, and 72 hpi to quantify virus titer.

2.9. Virus Titer The IPMA was used to assess virus titration as previously described [11]. Briefly, cells were fixed with 100 µL of 4% cold formalin for 15 min at room temperature (RT). The fixed cells were washed once with 100 µL of phosphate-buffered saline (PBS) and twice with 100 µL of 0.5% PBS Tween-20 (PBST), before blocking with 100 µL of 1% bovine serum Vet. Sci. 2021, 8, 106 5 of 14

albumin (BSA) in 0.5% PBST for 30 min at RT. Then, the cells were washed. Next, 70 µL of anti-PRRSV NC protein monoclonal antibody (Median Diagnostics, Gangwondo, Korea) was diluted at a ratio of 1:400 and added to cells at RT for 60 min, before incubating again with 50 µL of peroxidase-conjugated AfﬁniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, USA) at a ratio of 1:600 for 60 min at RT. The cells were then washed and counterstained with the 1,5-diaminopentane (DAP) substrate for 5 min before they were washed with distilled water and examined under a microscope. Virus titer was determined using the Reed–Muench method [27], expressed as TCID50, describing the virus concentration required to infect 50% of the given cell culture [29].

2.10. Characterization of Semi-Purified Fraction by LC-QTOF-MS LC-MS evaluation was performed using a quadrupole time-of-flight mass spectrome- ter (QTOF-MS) according to the adopted method of Chellappan et al. [30]. The samples were dissolved in 0.01% formic acid and ethanol (1:1, v/v) to obtain a final concentration of 1 mg/mL and then cleaned up using QuEChERS dispersive SPE kit, fat + pigments (Agilent Technology, Santa Clara, CA, USA) before passing through a 0.22 µm membrane filter. The characterization was performed using an Agilent 1290 Infinity II series, coupled to a 6546 LC/Q-TOF instrument (Agilent Tech., Santa Clara, CA, USA), consisting of a degasser, binary pump, column oven, UV–Vis detector, and thermostat autosampler. The instrument settings were optimized as follows: LC conditions, UV at 330 nm; 0.2 mL/min flow rate; injection volume of 10 µL; and a gradient mobile system starting with 5% ACN and 95% water (1% formic acid), decreasing to 20% ACN in 5 min, 30% ACN in 5 min, 35% ACN in 5 min, 45% ACN in 5 min, 75% ACN in 5 min, and 95% ACN until the run ended. The chromatographic separation was accomplished using a ZORBAX Eclipse Plus C18 (2.1 × 150 mm, 1.8 µm). The MS conditions involved an electrospray ionization (ESI) probe in positive mode. The nebulizer was operated at 20 psi with 7 L/min N2 flow. The capillary temperature was kept at 300 ◦C, while the sample flow rate was set at 8 µL/min. The m/z range was 50–1000, the capillary voltage was 4500 V, and the dry heater temperature was 280 ◦C. The chemical structures and other parameters for compounds were determined using an online database (www.chemspider.com, accessed on 1 February 2021).

2.11. Statistical Analysis The cell viability was calculated using a regression analysis of dose–response curves for the MTT assay expressed as CC50. Comparisons of the mean of differences in antiviral activities of CS extract fractions were analyzed using a one-way analysis of variance and Tukey’s post hoc test. All statistical analyses were performed using the SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). A p-value < 0.05 was considered statistically signiﬁcant.

3. Results 3.1. Extraction Yield of Ethanolic CS Heartwood Crude Extract The ethanolic CS heartwood crude extract yield is shown in Table1. It was found that a yield as high as 7.020% was achieved from the raw material of CS heartwood (500.323 g) in ethanol.

Table 1. The ethanolic crude CS heartwood extraction yield.

Sample Weight (g) Yield (g) % Yield CS heartwood 500.323 35.124 7.020

3.2. Analytical HPLC and Preparative HPLC Semi-Purification of CS Extract Figure2 shows that six semi-purified peaks were marked for preparative HPLC. At 280 nm, the ethanolic CS crude extracts were separated into five dominant semi-purified peaks (A). The yields of semi-purified CS extract fractions are given in Table2. The CS extract fraction yields ranged from 33.75 to 51.94 mg/g extract. Fraction 1 was isolated Vet. Sci. 2021, 8, x FOR PEER REVIEW 6 of 14

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extract fraction yields ranged from 33.75 to 51.94 mg/g extract. Fraction 1 was isolated with a mobile phase of 30:70 A/B at an Rt of 12.76 min, resulting in 39.54 mg/g extract. with a mobile phase of 30:70 A/B at an Rt of 12.76 min, resulting in 39.54 mg/g extract. Fractions 2–5 wereFractions isolated 2–5 with were a isolatedmobile phase with a mobileof 35:65 phase A/B ofat 35:65Rts of A/B 15.32, at Rts 17.14, of 15.32, 18.85, 17.14, 18.85, and 19.18 min resultingand 19.18 in min 51.94, resulting 42.32, in33.75, 51.94, and 42.32, 45.75 33.75, mg/g and extract, 45.75 mg/g respectively. extract, respectively. The The results show that resultsF2 was show isolated that F2in wasthe highes isolatedt yield, in the highestwhereas yield, F4 led whereas to the F4 lowest led to theyield. lowest yield. Moreover, a singleMoreover, dominant a single peak dominant of 330 nm peak was of apparent 330 nm was at apparent an Rt of at 26.99 an Rt min of 26.99 (B). min Frac- (B). Fraction tion 6 was isolated6 waswith isolated a mobile with phase a mobile of 75:25 phase A/B, of 75:25 resulting A/B,resulting in 37.43 inmg/g 37.43 extract. mg/g extract.

FigureFigure 2. Representative2. Representative chromatograms chromatograms of semi-puriﬁed of semi-purified CS extract CS onextract HPLC on UV HPLC at (a) UV 280 at nm (a and) 280 (b nm) 330 and nm. (b) 330 nm. Table 2. The preparative HPLC fraction yields of ethanolic CS crude extract. Table 2. The preparative HPLC fraction yields of ethanolic CS crude extract. Fractions Retention Time (min) Yield (mg/g Extract) Fractions F1Retention Time (min) 12.76 Yield (mg/g Extract) 39.54 ± 5.85 F1 F2 12.76 15.32 39.54 ± 5.85 51.94 ± 5.12 ± F2 F3 15.32 17.14 51.94 ± 5.12 42.32 5.46 F4 18.85 33.75 ± 4.60 F3 F5 17.14 19.84 42.32 ± 5.46 45.75 ± 3.22 F4 F6 18.85 26.99 33.75 ± 4.60 37.43 ± 4.46 F5 19.84 45.75 ± 3.22 F6 3.3. Cell Cytotoxicity 26.99 37.43 ± 4.46 Prior to the inhibition of viral replication assay, MARC-145 cell viability was deter- 3.3. Cell Cytotoxicitymined. In this study, we expressed the cell viability as 50% cytotoxicity concentration Prior to the inhibition(CC50), as shown of viral in Figurereplication3. The resultassay, shows MARC-145 that F4 had cell the viability highest CCwas50 deter-of 601.18 µg/mL mined. In this study,toward we MARC-145 expressed cells, the followed cell viability by F1 withas 50% a CC cytotoxicity50 of 535.91 µ g/mL.concentration On the other hand, we found that F6 had the lowest CC of 12.83 µg/mL, while F2, F3, and F5 had a CC of (CC50), as shown in Figure 3. The result shows that50 F4 had the highest CC50 of 601.18 50 29.24, 155.47, and 261.62 µg/mL, respectively. μg/mL toward MARC-145 cells, followed by F1 with a CC50 of 535.91 μg/mL. On the other hand, we found that F6 had the lowest CC50 of 12.83 μg/mL, while F2, F3, and F5 had a CC50 of 29.24, 155.47, and 261.62 μg/mL, respectively.

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700 700 601.18601.18 535.91535.91 600600

500500

400400 (µg/ml) (µg/ml) 261.62261.62

50 300 50 300 CC 200CC 200 155.47155.47 12.83 100100 12.83 29.2429.24 0 0 F1F1 F2 F2 F3 F3 F4 F4 F5 F5 F6 F6 Fractions Fractions FigureFigure 3. 3.Cytotoxicity CytotoxicityCytotoxicity of of the of the theCS CS extract CS extract extract fractions fractions fractions towa towa towardrdrd MARC-145 MARC-145 MARC-145 cells cells cellsdeterm determ determinedinedined by by the the by 3-(4,5- 3-(4,5- the 3- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MARC-145 cells were incu- (4,5-dimethylthiazol-2-yl)-2,5-diphenyldimethylthiazol-2-yl)-2,5-diphenyl tetrazolium tetrazolium bromide bromide (MTT) (MTT) assay. assay. MARC MARC-145-145 cells were cells wereincu- bated with various concentrations of CS extract fractions or control (medium without CS extract incubatedbated with with various various concentrations concentrations of ofCS CS extract extract fractions fractions or or control control (medium (medium without without CS extractextract fractions)fractions) for for 72 72 h hprior prior to to the the MTT MTT assay. assay. F1, F1, fraction fraction 1; 1;F2, F2, fraction fraction 2; 2;F3, F3, fr actionfraction 3; 3;F4, F4, fraction fraction 4; 4; fractions) for 72 h prior to the MTT assay. F1, fraction 1; F2, fraction 2; F3, fraction 3; F4, fraction 4; F5, F5,F5, fraction fraction 5; 5;and and F6, F6, fraction fraction 6. 6.CC CC50 50mean mean 50% 50% cytotoxic cytotoxic concentration. concentration. fraction 5; and F6, fraction 6. CC50 mean 50% cytotoxic concentration. 3.4.3.4. Inhibition Inhibition of ofPRRSV PRRSV Infection Infection Different semi-purified CS fraction concentrations according to the CC50 were tested Different semi-purifiedsemi-purified CSCS fractionfraction concentrationsconcentrations accordingaccording toto thethe CCCC5050 werewere testedtested forforfor the thethe inhibition inhibitioninhibition of ofof in inin vitro vitrovitro viral viralviral infection. infection.infection. Our Our results results in in Figure Figure 44 4show show show that thatthat the thethe semi- semi-semi- purifiedpurifiedpurified CS CS fractions fractions fractions did did did not not not significantly significantly significantly inhibit inhibit inhibit PRRSV PRRSV PRRSV infection infection infection in in MARC-145 MARC-145 in MARC-145 cells cells ( cellsp ( p> > 0.05).(0.05).p > 0.05).

Figure 4. Virus titer describing the inhibition of PRRSV infectivity of six CS extract fractions at 24 h FigureFigure 4. 4.Virus Virus titer titer describing describing the the inhibition inhibition of of PRRSV PRRSV in infectivityfectivity of of six six CS CS extract extract fractions fractions at at24 24 h h post-infection (hpi). No signiﬁcant difference for different concentrations of the CS extract fractions. post-infectionpost-infection (hpi). (hpi). No No significan significant differencet difference for for different different concentratio concentrationsns of of the the CS CS extract extract fractions. fractions. (p > 0.05). (p( >p 0.05).> 0.05).

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3.5. Inhibition of PRRSV Replication 3.5. InhibitionIn this study, of PRRSV we treated Replication PRRSV with various concentrations of semi-purified CS extract Infractions this study, according we treated to their PRRSV CC50 values with various as well concentrations as two lower concentrations of semi-purified as CSthe extractrange of fractions 12.83–535.91 according μg/mL to not their affecting CC50 values the proliferative as well as activity two lower of MARC-145 concentrations cells asat thethree range time ofintervals 12.83–535.91 (24, 48,µ andg/mL 72 hpi). not affecting The results the obtained proliferative from activitythe antiviral of MARC-145 test iden- cellstified at F1 three as having timeintervals significant (24, potential 48, and for 72 hpi).anti-PRRSV The results replication obtained activity from in the a antiviraldose-de- testpendent identified manner F1 as(Figure having 5). significant F1 at 535.91 potential μg/mL for showed anti-PRRSV a stronger replication inhibitory activity activity in a µ dose-dependentagainst PRRAV replication manner (Figure at a virus5). F1 titer at 535.91of 2.75 TCIDg/mL50/mL showed (log10 a) at stronger 72 hpi inhibitorycompared activitywith the againstcontrol PRRAV(p < 0.05). replication As shown atin aFigure virus 6 titer (bro ofwn 2.75 staining TCID of50 /mLthe cells), (log10 the) at immun- 72 hpi comparedoperoxidase with monolayer the control assay (p < 0.05).(IPMA) As shownindicated in Figurethat F16 (brown blocked staining PRRSV of replication the cells), the in immunoperoxidaseMARC-145 cells. Interestingly, monolayer assay F4 at (IPMA) 155.47 indicatedμg/mL could that F1inhibit blocked PRRSV PRRSV replication replication in in MARC-145 cells. Interestingly, F4 at 155.47 µg/mL could inhibit PRRSV replication in MARC-145 cells at a virus titer of 4.25 TCID50/mL (log10). MARC-145 cells at a virus titer of 4.25 TCID50/mL (log10).

Figure 5. Virus titer describing the inhibition of PRRSV replication of six CS extract fractions at 24, 48, and 72 h post infection (hpi);Figurea,b,c 5. pVirus< 0.05 titer compared describing with the different inhibition concentrations of PRRSV ofreplication the CS extract of six fractions. CS extract fractions at 24, 48, and 72 h post infection (hpi); a,b,c p < 0.05 compared with different concentrations of the CS extract fractions.

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Figure 6. Immunoperoxidase monolayer assay (IPMA) showing the inhibitioninhibition of PRRSV replicat replicationion in MARC-145 cells by F1. Scale bar in the ﬁgure:figure: 200 µμm.

3.6. Characterization by LC–QTOF-MS 3.6. Characterization by LC–QTOF-MS According to its antiviral activity against PRRSV, the most effective fraction was F1. According to its antiviral activity against PRRSV, the most effective fraction was F1. Thus, it was subjected to the identification of bioactive compounds using LC–QTOF-MS Thus, it was subjected to the identification of bioactive compounds using LC–QTOF-MS analysis. The spectrum showing specific peaks and their corresponding compounds is analysis. The spectrum showing specific peaks and their corresponding compounds is displayed in Figure 7. The active compounds of F1 revealed from LC–QTOF-MS analysis displayed in Figure7. The active compounds of F1 revealed from LC–QTOF-MS analysis are listed in Table 3. Fraction 1 had 17 prominent peaks, but only 11 peaks produced a are listed in Table3. Fraction 1 had 17 prominent peaks, but only 11 peaks produced a matching score > 90%. Among those, six compounds produced a matching score > 99%, matching score > 90%. Among those, six compounds produced a matching score > 99%, which were identified as byakangelicin, brazilin, naringenin, brazilein, tricin, and which were identified as byakangelicin, brazilin, naringenin, brazilein, tricin, and wogonin, wogonin, corresponding to (M − H)– ions at m/z 333.0979, 285.0768, 271.0612, 283.0613, corresponding to (M − H)– ions at m/z 333.0979, 285.0768, 271.0612, 283.0613, 329.0665, 329.0665, and 283.0612, respectively. and 283.0612, respectively.

Table 3. Compounds identified from F1 of ethanolic crude CS extract according to LC–QTOF-MS. Table 3. Compounds identiﬁed from F1 of ethanolic crude CS extract according to LC–QTOF-MS. No. Compound Name Structure Rt Matching Score (%) m/z * (M − H)− Mass Mass Diff (Tgt/ppm) Compound Matching Mass Diff 1 No. Catechin C15 HStructure14 O6 4.466 Rt 98.52 289.0658m/z *289.0718 (M − H) 290.079− Mass −0.10 Name Score (%) (Tgt/ppm) 2 Isorhamnetin C16 H12 O7 5.750 95.57 315.0509 315.0510 316.0583 −0.16 − 3 1Kaempferol Catechin C15 CH1510 OH614 O5.7506 4.466 93.02 98.52 285.0402 289.0658 285.0425 289.0718 286.0476 290.079 −0.36 0.10 2 Isorhamnetin C H O 5.750 95.57 315.0509 315.0510 316.0583 −0.16 4 (+)–Epicatechin C15 H1614 O612 6.5177 97.80 349.0928 289.0718 290.079 −0.13 3 Kaempferol C15 H10 O6 5.750 93.02 285.0402 285.0425 286.0476 −0.36 5 Brazilein C16 H12 O5 6.600 99.64 283.0613 283.0612 284.0685 0.16 4 (+)–Epicatechin C15 H14 O6 6.517 97.80 349.0928 289.0718 290.079 −0.13 6 Kaempferide C16 H12 O6 7.051 98.79 299.056 299.0561 300.0633 −0.16 5 Brazilein C16 H12 O5 6.600 99.64 283.0613 283.0612 284.0685 0.16 7 Brazilin C16 H14 O5 7.684 99.83 331.0822 285.0768 286.084 −0.41 6 Kaempferide C16 H12 O6 7.051 98.79 299.056 299.0561 300.0633 −0.16 15 12 5 8 7Naringenin Brazilin C CH16 OH14 O8.9185 7.684 99.80 99.83 317.0666 331.0822 271.0612 285.0768 272.0684 286.084 −0.34− 0.41 9 8Byakangelicin Naringenin C17 CH1518 OH712 O9.1855 8.918 99.97 99.80 333.0979 317.0666 333.0980 271.0612 334.1052 272.0684 −0.27− 0.34 10 9Tricin Byakangelicin C17 CH1714 OH718 O9.9357 9.185 99.10 99.97 329.0665 333.0979 329.0667 333.0980 330.0738 334.1052 −0.44− 0.27 11 10Wogonin Tricin C16 CH1712 OH514 O9.9357 9.935 99.10 99.10 329.0665 329.0665 283.0612 329.0667 284.0683 330.0738 −0.50− 0.44 − 11* The m/z of Wogonin (+)-epicatechin Cis16 shownH12 O as5 M + CH9.9353COO− (compound 99.10 mass 329.0665 + 59.0440), while 283.0612 that of brazilin, 284.0683 naringenin, and0.50 − − *wogonin The m/z ofis (+)-epicatechinshown as M + isHCOO shown as(compound M + CH3COO mass(compound + 45.0174). mass + 59.0440), while that of brazilin, naringenin, and wogonin is shown as M + HCOO− (compound mass + 45.0174).

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Figure 7.7. LC–QTOF-MS analysisanalysis ofof F1, F1, along along with with the the top top three three compounds compounds in in terms terms of matchingof matching scores: scores: (A )( LC–QTOF-MSA) LC–QTOF- chromatogramMS chromatogram of F1; of (F1;B) ( massB) mass spectrum spectrum of byakangelicinof byakangelicin ((M ((M− H)− H)− −= = 333.0979); 333.0979);( C(C)) singlesingle MSMS spectrumspectrum ofof brazilinbrazilin ((M((M ++ HCOO)− == 331.0822); 331.0822); and and (D (D) )MS MS spectrum spectrum of of naringenin naringenin ((M ((M + + HCOO) HCOO)−− = =317.0666). 317.0666).

4. Discussion Medicinal plantsplants representrepresent an an alternative alternative source source of antiviralof antiviral agents agents that that can potentiallycan poten- tiallybe used be insteadused instead of antibiotics. of antibiotics. The bioactive The bioactive ingredients ingredients of medicinal of medicinal plants illustrateplants illustrate a wide arange wide ofrange biological of biological properties prop sucherties assuch antibacterial, as antibacterial, antioxidant, antioxidant, anti-inflammatory, anti-inflammatory, and andantiviral antiviral activities. activities. As the As mostthe most commonly commonly identified identified active active compound compound group, group, flavonoids flavo- noidsare the are major the major class illustrating class illustrating anti-viral anti-viral properties properties [31]. Previous [31]. Previous studies studies have reportedhave re- portedthat flavonoids, that flavonoids, including including genistein genistein and catechins, and catechins, could reduce could thereduce infectivity the infectivity of various of

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viruses affecting humans and animals, including adenovirus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), PRRSV, and rotavirus [32,33]. Many studies also reported the potential of medicinal plants against PRRSV. Kaewprom et al. [34] presented that Thymus vulgaris and Nepeta cataria hydrosols blocked the viral attachment, adsorption, replication, and release of PRRSV load in MARC-145 cells. Anantikulchai et al. [35] showed that a turmeric extract could inhibit PRRSV replication in vitro. Pringproa et al. [11] found that the crude extract of Cynodon dactylon potentially inactivated PRRSV and inhibited replication of PRRSV in vitro. Furthermore, an ethanolic crude CS extract exhibited effective antiviral activity against PRRSV [12]. However, the specific bioactive compound groups in CS crude extract illustrating antiviral properties were not elucidated. Therefore, we separated a semi-purified CS crude extract before identifying the effective compound groups with antiviral activity against PRRSV. Prior to antiviral activity determination, the cell cytotoxicity of fractions was established to avoid cell damage. The fractions with the lowest cell cytotoxicity were F4 and F1 with CC50 of 601.18 and 535.91 µg/mL, respectively. Adnan et al. [36] explained that antiviral compounds should be highly effective while showing minimal toxicity to normal cells and tissues. In this study, the ethanolic CS crude extract was fractioned before testing antiviral activity at concentrations according to the CC50. All CS fractions showed no effect against PRRSV in terms of inhibiting viral infection. This is in line with the results previously reported for CS crude extract [12]. Although the CS fractions did not prevent viral infection, they effectively inhibited PRRSV replication, especially F1. F1 was evaluated for its bioactive ingredients by LC–QTOF-MS, revealing that the main bioactive components of F1 were flavonoids such as brazilin, naringenin, brazilein, and catechin. Table3 lists the compounds with a matching score > 90%. The most dominant compound found in F1 was byakangelicin. Byakangelicin is a major natural coumarin compound found in Angelica dahurica, which is a folk medicine in which the roots are used to treat cold and fever in East Asian countries. It also possesses various pharmacological properties, such as antibacterial, antiasthmatic, hypotensive, anti-inflammatory, and antioxidant activities [37]. Likewise, it has been reported that byakangelicin exhibits effective anti-HIV activity [38]. Moreover, other identified flavonoid compounds in F1 were brazilin, naringenin, and brazilein. Brazilin is the main homoisoflavonoid constituent in CS heartwood [39]. Brazilin and brazilein exhibit various pharmacological properties such as anti-inflammatory [40,41], antioxidant [17], and antibacterial activities [42,43]. Laksmiani et al. [44] reported favor- able in silico molecular docking studies of brazilin and brazilein from CS heartwood with angiotensin-converting enzyme 2 (ACE2). In addition, they were able to act as en- try inhibitors of SARS-Cov-2 by inhibiting ACE2 and transmembrane protease serine 2 (TMPRSS2). Moreover, CS heartwood also inhibited RNA-dependent RNA polymerase (RdRp), which plays a role in RNA replication in the host cell. Nevertheless, until now, no report was available on the antiviral activity of brazilin against PRRSV. We assume that it might play a part in the inhibition of PRRSV replication. To this end, we anticipate that the mechanism of brazilin needs to be investigated in the future in a similar manner to Laksimiani et al. [44]. Naringenin is a flavanone considered one of the most important flavonoids due to its potential biological properties such as antioxidant, anti-inflammatory, and antiviral activities [45,46]. Naringenin is typically found in citrus fruits [45]. Several reports have shown that naringenin exhibits high potential as an antiviral agent. Frabasile et al. [47] found that the administration of naringenin to Huh 7.5 cells could inhibit Dengue virus replication. It is possible that naringenin might inhibit proteases during the virus replication process. Khaerunnisa et al. [48] reported that flavonoids such as naringenin, kaempferol, quercetin, and apigenin are the most recommended compounds that may act as potential inhibitors of the SARS-CoV-2 main protease. Our study, however, classified the complex composition of the CS fraction with the highest potential antiviral activity. Further purification steps of this fraction, accompanied by antiviral activity testing of the purified compounds, may confirm the effectiveness of C. sappan heartwood as an anti-PRRSV agent. We report, for the first time, the potential antiviral activity of the detected compounds (in- Vet. Sci. 2021, 8, 106 12 of 14

cluding byakangelicin, brazilin, naringenin, and brazilein) toward PRRSV. Future studies can aim to determine the mechanism of action underlying the antiviral activity of these compounds toward PRRSV.

5. Conclusions CS extract has potential anti-PRRSV activity in vitro. The ethanolic CS crude extract was separated into six semi-purified fractions by preparative HPLC. This study showed that only the initial fraction detected at 280 nm illustrated effectiveness against PRRSV in terms of inhibiting viral replication. LC–QTOF-MS analysis revealed the coumarin compound byakangelicin and flavonoids including brazilin, naringenin, and brazilein as the main bioactive compounds in F1, which could play a role as anti-PRRSV agents. These compounds merit further evaluation through purification and structure elucidation.

Author Contributions: Conceptualization, K.S. (Korawan Sringarm), S.H., W.R. and K.P.; methodol- ogy, C.A., K.S. (Kunrunya Sutan) and K.S. (Korawan Sringarm); validation, S.H., M.S.-a. and K.S. (Korawan Sringarm); data curation, S.H., M.S.-a. and W.R.; formal analysis, C.A.; investigation, S.H., S.R.S., and K.S. (Korawan Sringarm); project administration, K.S. (Korawan Sringarm); resources, S.H., K.P. and K.S. (Korawan Sringarm); writing—original draft, C.A.; writing—review and editing, C.A., S.H., W.R., S.R.S. and K.S. (Korawan Sringarm). All authors have read and agreed to the published version of the manuscript. Funding: This research was supported financially by a Ph.D. scholarship of the Research and Researcher for Industries Projects (RRi), Thailand Science Research and Innovation (TSRI), under contract no. PHD61I0042. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Institutional Review Board Statement: All experimental protocols were reviewed and approved by the Chiang Mai University Institutional Biosafety Committee (Ref. No. CMUIBC0663001). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The research project is supported by RRi projects, TSRI. This research work was partially supported by Chiang Mai University. Conflicts of Interest: The authors declare that they have no conflict of interest.

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