Hibiscus sabdariffa L. and Its Bioactive Constituents Exhibit Antiviral Activity against HSV-2 and Anti-Enzymatic Properties against Urease by an ESI-MS Based Assay

For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS) and its bioactive constituent protocatechuic acid (PCA), have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS)-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC50 values of 0.92 and 1.43 µg∙mL−1, respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC50 value of 82.4 µg∙mL−1. This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.


Introduction
Herpes simplex virus (HSV) infections are quite common in humans, affecting about 90% of the world population. HSV is a member of Herpesviridae, a wide family of enveloped-DNA viruses that cause several clinically significant syndromes in both adults and neonates [1]. HSV-2 is mainly connected with genital infection, and has been recorded as a risk factor of HIV infection in humans [2]. Currently, treatment of HSV-2 infection mainly relies on the use of acyclovir and related nucleoside analogues that target viral DNA polymerase. Unfortunately, although several strategies have shown high efficacy results, HSV-2 infection treatment fails in about 30-45% of infected adults [3,4]. This is due to the extensive use of acyclovir and related nucleoside analogues, which has created drug resistance, associated with other adverse effects, alongside with the establishment of viral latency and reactivation that occurs in the presence of humoral-and cell-mediated immunity [5]. To date, no prophylactic HSV vaccine has been found to be entirely effective in the prevention of HSV infections [6,7]. Therefore, it is crucial to find alternative strategies to combat this viral resistance, and increase treatment efficacy results.

Determinations of Concentration of PCA and Total Polyphenols Content in Plant Material
The results demonstrated that the concentration of PCA in AEHS was found to be 94.1 µg/g dry weight of Hibiscus calyces ( Figure 1) and total polyphenols content calculated as mg of gallic acid equivalent was 106.0 mg/g of dry extract of Hibiscus calyces.

Anti-HSV-2 Activity and Cytotoxicity
AEHS and PCA were evaluated with respect to their inhibitory effect on HSV-2 replication. Before performing the antiherpetic assay, we assessed the cytotoxicity of each sample in Vero cells by the neutral red dye-uptake method. The CC 50 values for PCA and acyclovir were found to be higher than 200 µg·mL −1 (Table 1). Antiherpetic activity was determined by the titer reduction assay in infected Vero cells using quantitative real-time reverse transcription PCR. Ten acyclovir-sensitive strains of HSV-2 (clinical isolates) were used and typed by quantitative real-time reverse transcription PCR using primers pairs H 2 M 40 5 -GTACAGACCTTCGGAGG-3 and H 2 P 4 5 -CGCTTCATCATGG GC-3 for identification. AEHS was not active against HSV-2. This could be related to the low concentrations of antiherpetic compounds in the crude extract. PCA showed potent anti-HSV-2 activity compared with that of acyclovir with EC 50 values of 0.92 and 1.43 µg·mL −1 , respectively, and selectivity indices >217 and >140, respectively. PCA exhibited cytotoxic effect on Vero cells at concentration higher than its EC 50 . The selectivity index (SI) is fundamental to determine any possible toxic effect of any compound on the cells that could be confused with an antiviral activity. Based on our results, PCA demonstrated anti-HSV-2 activity with SI > 217. 4 higher than acyclovir (>140). Thus, the SI verifies the safety index of PCA.
Molecules 2017, 22,722 3 of 12 selectivity indices > 217 and > 140, respectively. PCA exhibited cytotoxic effect on Vero cells at concentration higher than its EC50. The selectivity index (SI) is fundamental to determine any possible toxic effect of any compound on the cells that could be confused with an antiviral activity. Based on our results, PCA demonstrated anti-HSV-2 activity with SI > 217. 4 higher than acyclovir (> 140). Thus, the SI verifies the safety index of PCA.

Anti-urease Inhibitory Properties by ESI-MS-based Assay
Urease inhibitors play an essential role as alternative antibiotics in the treatment of gastrointestinal and urinary tract infections caused by urease-producing bacteria [8]. Enzyme activity and inhibition studies were determined by an ESI-MS based method. The method is based on the detection of urea depletion in the absence and presence of inhibitors by monitoring catalytic reaction through simultaneous detection of the concentration changes of urea.

Determination of Urease Kinetic Parameters Km and Vmax
It is known that the essential function of enzymes is to enhance the rate of biochemical reactions. Therefore, to understand enzyme function, it is very crucial to study the kinetic description of their activity. To determine kinetic parameters of the urease-catalyzed reaction including the Michaelis constant (Km) and the maximal reaction rate (Vmax), we monitored the enzymatic reaction through decreasing of concentration of urea at five concentrations (137.0, 208.2, 274.0, 416.4 and 694.1 µmol•L −1 ) and constant concentration of urease (60.0 µg•mL −1 , Figure 2). The initial rate of the reaction was determined for each concentration from the rate constant and initial substrate concentration. A plot of 1/initial velocity versus 1/(urea) generates a Lineweaver-Burk plot and a linear regression fit to this data was used to determine the Km and Vmax (Figure 3). The good linearity of the Lineweaver-Burk plot (R 2 = 0.99) for urea shown in Figure 3 ensures the accuracy of steady-state kinetics in this

Anti-Urease Inhibitory Properties by ESI-MS-Based Assay
Urease inhibitors play an essential role as alternative antibiotics in the treatment of gastrointestinal and urinary tract infections caused by urease-producing bacteria [8]. Enzyme activity and inhibition studies were determined by an ESI-MS based method. The method is based on the detection of urea depletion in the absence and presence of inhibitors by monitoring catalytic reaction through simultaneous detection of the concentration changes of urea.

Determination of Urease Kinetic Parameters K m and V max
It is known that the essential function of enzymes is to enhance the rate of biochemical reactions. Therefore, to understand enzyme function, it is very crucial to study the kinetic description of their activity. To determine kinetic parameters of the urease-catalyzed reaction including the Michaelis constant (K m ) and the maximal reaction rate (V max ), we monitored the enzymatic reaction through decreasing of concentration of urea at five concentrations (137.0, 208.2, 274.0, 416.4 and 694.1 µmol·L −1 ) and constant concentration of urease (60.0 µg·mL −1 , Figure 2). The initial rate of the reaction was determined for each concentration from the rate constant and initial substrate concentration. A plot of 1/initial velocity versus 1/(urea) generates a Lineweaver-Burk plot and a linear regression fit to this data was used to determine the K m and V max (Figure 3). The good linearity of the Lineweaver-Burk plot (R 2 = 0.99) for urea shown in Figure 3 ensures the accuracy of steady-state kinetics in this method.          For inhibitor screening, the IC 50 value is an apparent measure of the potency of an inhibitor at specific experimental conditions. Figure 4 shows the reaction progression slope for the depletion of urea in the presence and absence of inhibitors following the first-order kinetics conditions per equation C t = C 0 × e −kt , where C t is the concentration at time (t), C 0 is the initial concentration and k is the reaction rate constant [26]. From Figure 4, the reaction rate constant in the presence of inhibition (k) is lower than the reaction rate constant in the absence of inhibition (k 0 ), where k in the presence of acetohydroxamic acid (AHA; used as a reference urease inhibitor) and AEHS (k = 0.0477 and 0.0975 min −1 , respectively) and (k 0 = 0.1934 min −1 ) at concentrations of 274.0 µmol·L −1 for urea and 60.0 µg·mL −1 for urease, and concentrations of inhibitors (AHA = 13.2 µmol·L −1 and AEHS = 81.0 µg·mL −1 ). It has been reported that the percent inhibition is defined as percent reduction of the reaction rate constant (k) as compared with the rate constant in the absence of an inhibitor (k 0 ). Thus, % activity is usually determined as k/k 0 and % Inhibition is 1 -% activity. Therefore, to determine IC 50 value of an inhibitor the following equation % activity = k/k 0 =1 − [I]/([I] + IC 50 ) = IC 50 /([I] + IC 50 ), where [I] is the concentration of the inhibitor, was used as previously described [26]. IC 50 for AHA was determined to be 4.3 µmol·L −1 and for AEHS to be 82.4 µg·mL −1 . The used method proved to be robust and determined IC 50 value for AHA compared favourably with previous data reported in literature (IC 50 for AHA = 5 µM) [27]. For inhibitor screening, the IC50 value is an apparent measure of the potency of an inhibitor at specific experimental conditions. Figure 4 shows the reaction progression slope for the depletion of urea in the presence and absence of inhibitors following the first-order kinetics conditions per equation Ct = C0 × e −kt , where Ct is the concentration at time (t), C0 is the initial concentration and k is the reaction rate constant [26]. From Figure 4, the reaction rate constant in the presence of inhibition It has been reported that the percent inhibition is defined as percent reduction of the reaction rate constant (k) as compared with the rate constant in the absence of an inhibitor (k0). Thus, % activity is usually determined as k/k0 and % Inhibition is 1 -% activity. Therefore, to determine IC50 value of an inhibitor the following equation % activity = k/k0 =1 − [I]/([I] + IC50) = IC50/([I] + IC50), where [I] is the concentration of the inhibitor, was used as previously described [26]. IC50 for AHA was determined to be 4.3 µmol•L −1 and for AEHS to be 82.4 µg•mL −1 . The used method proved to be robust and determined IC50 value for AHA compared favourably with previous data reported in literature (IC50 for AHA = 5 µM) [27].  For inhibitor screening, the IC50 value is an apparent measure of the potency of an inhibitor at ecific experimental conditions. Figure 4 shows the reaction progression slope for the depletion of rea in the presence and absence of inhibitors following the first-order kinetics conditions per uation Ct = C0 × e −kt , where Ct is the concentration at time (t), C0 is the initial concentration and k is e reaction rate constant [26]. From Figure 4, the reaction rate constant in the presence of inhibition ) is lower than the reaction rate constant in the absence of inhibition (k0), where k in the presence acetohydroxamic acid (AHA; used as a reference urease inhibitor) and AEHS (k = 0.0477 and 0.0975 in −1 , respectively) and (k0 = 0.1934 min −1 ) at concentrations of 274.0 µmol•L −1 for urea and 60.0 g•mL −1 for urease, and concentrations of inhibitors (AHA = 13.2 µmol•L −1 and AEHS = 81.0 µg•mL −1 ). has been reported that the percent inhibition is defined as percent reduction of the reaction rate nstant (k) as compared with the rate constant in the absence of an inhibitor (k0). Thus, % activity is sually determined as k/k0 and % Inhibition is 1 -% activity. Therefore, to determine IC50 value of an hibitor the following equation % activity = k/k0 =1 − [I]/([I] + IC50) = IC50/([I] + IC50), where [I] is the ncentration of the inhibitor, was used as previously described [26]. IC50 for AHA was determined be 4.3 µmol•L −1 and for AEHS to be 82.4 µg•mL −1 . The used method proved to be robust and etermined IC50 value for AHA compared favourably with previous data reported in literature (IC50 r AHA = 5 µM) [27].   found to be 7.5%. Although current practices in stability studies rely on the addition of an internal standard to assure that short-term and long-term signal fluctuations do not influence quantitative analyses, we avoided the use of an internal standard which could interfere with enzymatic reaction or exact determination of substrate concentration. Therefore, we measured a calibration curve after each experiment to correct instability of MS signal. found to be 7.5%. Although current practices in stability studies rely on the addition of an internal standard to assure that short-term and long-term signal fluctuations do not influence quantitative analyses, we avoided the use of an internal standard which could interfere with enzymatic reaction or exact determination of substrate concentration. Therefore, we measured a calibration curve after each experiment to correct instability of MS signal.

Discussion
For decades, treatment of infectious diseases has been a focus of interest for both researchers and healthcare providers, as the arising issue of drug resistant strains has become a serious problem worldwide. This study adds to the growing body of knowledge on the antiviral activity of polyphenol-enriched extracts derived from plants. We show for the first time that PCA, an active compound of H. sabdariffa L. exerts potent antiviral activity against clinical isolates of HSV-2, the mechanism of which involves the inhibition of viral replication. Plant-derived phytochemicals, such as polyphenols, phenolics, terpenes, alkaloids and other substances have been reported to possess inhibitory properties on HSV replication [7,28]. For instance, polyphenols have been found to interfere with the early phases of the HSV replicative cycle and/or with viral particles directly [29]. In this study, PCA showed excellent ability to inhibit HSV-2 replication and hence might open new gates for the development of anti-HSV-2 drugs. In addition, PCA is known to exhibit cytotoxic effect on cancer cells with various mechanisms of action [30,31]. Therefore, it should be taken into consideration the non-toxic doses, especially with topical preparations containing PCA. Considering the significant global incidence, morbidity, and mortality rates of viral sexually transmitted infections (STIs), the development of new, safe, topically applied antiviral agents for their prevention is of high priority [32]. Therefore, in recent years, a new approach has been under focus of interest to maximize the treatment efficacy by combining natural products with nucleoside analogues, resulting in reduction of cytotoxicity [33]. Thus, this approach will reduce the cytotoxicity of PCA. However, further studies should be carried out to determine its safety as well as the possibility of adverse effects in vivo.

Discussion
For decades, treatment of infectious diseases has been a focus of interest for both researchers and healthcare providers, as the arising issue of drug resistant strains has become a serious problem worldwide. This study adds to the growing body of knowledge on the antiviral activity of polyphenol-enriched extracts derived from plants. We show for the first time that PCA, an active compound of H. sabdariffa L. exerts potent antiviral activity against clinical isolates of HSV-2, the mechanism of which involves the inhibition of viral replication. Plant-derived phytochemicals, such as polyphenols, phenolics, terpenes, alkaloids and other substances have been reported to possess inhibitory properties on HSV replication [7,28]. For instance, polyphenols have been found to interfere with the early phases of the HSV replicative cycle and/or with viral particles directly [29]. In this study, PCA showed excellent ability to inhibit HSV-2 replication and hence might open new gates for the development of anti-HSV-2 drugs. In addition, PCA is known to exhibit cytotoxic effect on cancer cells with various mechanisms of action [30,31]. Therefore, it should be taken into consideration the non-toxic doses, especially with topical preparations containing PCA. Considering the significant global incidence, morbidity, and mortality rates of viral sexually transmitted infections (STIs), the development of new, safe, topically applied antiviral agents for their prevention is of high priority [32]. Therefore, in recent years, a new approach has been under focus of interest to maximize the treatment efficacy by combining natural products with nucleoside analogues, resulting in reduction of cytotoxicity [33]. Thus, this approach will reduce the cytotoxicity of PCA. However, further studies should be carried out to determine its safety as well as the possibility of adverse effects in vivo.
Urease inhibitors are considered promising therapeutic agents for the treatment of ureolytic bacterial infections [34]. Over the past two decades, a large number of plant-derived products were found to possess in vitro inhibitory activities against urease and intensive efforts were then made to evaluate the efficacy of these inhibitors in vivo [35]. Unfortunately, most of these investigations failed to prove the efficacy of those studied drugs in vivo due to problems of hydrolytic instability, toxicity and adverse side effects [36]. In this study, we report for the first time the anti-urease activity of AEHS by a developed ESI-MS based assay. Since H. sabdariffa L. is commonly used in traditional folk medicine in the form of a herbal tea or as a dietary supplement [37]. This indicates that AEHS could be used safely as natural urease inhibitor for the treatment of diseases caused by urease-producing bacteria. Additionally, the use of AEHS could overcome the problem of preventing the use of urease inhibitors in vivo and in clinical trials; due to their possible toxicity, instability and undesirable side effects. It has been reported that polyphenols and phenolic compounds have been shown to possess potent anti-urease activity. Moreover, crude plant extracts containing polyphenols have been shown an excellent ability to interact with wide range of enzymes [8,12]. Therefore, we may suggest that anti-urease activity of AEHS could be related to its rich content of polyphenols. Identification of bioactive molecules from AEHS and confirmation of the key components contributing to anti-urease activity should be studied in further investigations.

Plant Collection and Extraction Procedure
Hibiscus sabdariffa L. was collected from the northern part of Aswan, Egypt in June 2014, and identified by Sherif T. S. Hassan in the Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences (Brno, Czech Republic). A voucher specimen of the plant was deposited with the number EGHS5 at the herbarium of the department. One-gram air dried Hibiscus calyces were extracted in 10 mL of boiled water by sonication extraction for 30 min. The extract was stored at 4 • C for further use. A 1 mL of aliquot of crude extract was evaporated to dryness and then weighted to yield a final amount of 0.0243 g of dry extract.

Determination of Concentration of PCA in Plant Material
Air dried Hibiscus calyces (0.51 g) were extracted in 10 mL of boiled water by sonication extraction for 30 min. HPLC-MS (Agilent 1200 HPLC system, Böblingen, Germany) coupled with a mass spectrometer (Sciex-3200QTRAP-hybrid triple quadrupole/linear ion trap, Toronto, ON, Canada) fitted with Electrospray Ionization (ESI) were used for the analysis. For the quantification, an external calibration method with standard PCA (Sigma Aldrich, Prague, Czech Republic) was used as previously described [38].

Determination of Total Polyphenols by Folin-Ciocalteu Method
The total polyphenols content was determined per the Folin-Ciocalteu method as previously described by ISO 14502-1 [39].

Viral Strains, Cultures, Cell Lines and Reagents
For antiviral activity, Vero cells (ATCC: CCL 81, UK; were obtained from the Motol University Hospital, Prague, Czech Republic) were grown in Eagle's minimum essential medium (MEM; Cultilab, Campinas, UK) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, UK), 100 U/mL penicillin G, 100 µg·mL −1 streptomycin and 25 µg·mL −1 amphotericin B (Sigma-Aldrich, Berlin, Germany) and maintained at 37 • C in 5% CO 2 atmosphere. Ten clinical isolates of HSV-2 (isolated from patients with HSV-2 infection) were kindly obtained from the Motol University Hospital, Prague, Czech Republic). All clinical isolates were typed by quantitative real-time reverse transcription PCR using primers pairs H 2 M 40 5 -GTACAGACCTTCGGAGG-3 and H 2 P 4 5 -CGCTTC ATCATGGGC-3 for identification as previously described [40] and then were propagated in Vero cells. The cytopathic end-point assay was used to determine the titers which were expressed as 50% tissue culture infective dose (TCID 50 /mL) as previously described [41]. Viral stocks were stored at −80 • C.

Determination of Cytotoxicity
Cytotoxicity was evaluated by the neutral red dye-uptake method as previously described [42]. Briefly, AEHS, protocatechuic acid (PCA; Sigma Aldrich, Prague, Czech Republic) and acyclovir (positive control; Sigma-Aldrich, Berlin, Germany) were prepared in 0.1% dimethyl sulfoxide (DMSO). Stock solutions were prepared in deionized water at concentration of 400 µg·mL −1 and sterilized. Vero cell monolayers were cultivated in 96-well microtiter plates with two-fold serial dilutions of AEHS, PCA and acyclovir, and incubated for 48 h at 37 • C in 5% CO 2 atmosphere. After incubation, the morphological alterations of the treated cells were determined using an inverted optical microscope (Leitz, Berlin, Germany) and the maximum non-toxic concentrations (MNTC) were determined. The 50% cytotoxic concentrations (CC 50 ) of AEHS, PCA and acyclovir were calculated as the concentration that reduces cell viability by 50%, when compared to the untreated controls as priviously described [43].

Anti-HSV-2 Activity
Antiherpetic activity was determined by the titer reduction assay as previously described [44]. Briefly, acyclovir was used as a positive control and Vero cell monolayers were treated with AEHS, PCA and acyclovir at concentrations at which no change was observed in cell morphology, and 80% cell viability were determined. 100 TCID 50 /mL of HSV-2 acyclovir-sensitive suspensions were added to treated and untreated cell cultures and incubated at 37 • C for 48 h in a 5% CO 2 atmosphere. After incubation, the virus titers in treated and untreated cells, were determined. Anti-HSV-2 activity was evaluated as percentage inhibition (PI) using antilogarithmic TCID 50 values as follows: PI = [1 − (antilogarithmic test value/antilogarithmic control value)] × 100. The dose response curve was determined from the MNTC, and the 50% effective concentration (EC 50 ) was determined as the concentration required for 50% protection against virus-induced cytopathic effects. Selectivity index (SI) value was calculated as the ratio of CC 50 /EC 50 .

Statistical Analysis
Experiments were generated in duplicate in at least three independent experiments. PRISM software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis (one-way ANOVA test) and to calculate EC 50 and CC 50 parameters.

Enzyme, Substrate, Inhibitors and Reagents
All chemical reagents were obtained from commercial suppliers and used without further purification. Urease (Type III, from Canavalia ensiformis), urea, acetohydroxamic acid (AHA; standard urease inhibitor) were purchased from (Sigma Aldrich, Prague, Czech Republic).

Instrumentation
Urease inhibitory activity was evaluated using a system pump-injector (Agilent 1200) coupled with a Sciex-3200QTRAP-hybrid triple quadrupole/linear ion trap mass spectrometer fitted with Electrospray Ionization (ESI). The system runs in flow injection analysis (FIA) mode without a HPLC column. The operational parameter settings were as follows: curtain gas (CUR), 25 psi; nebulizer gas (GS1), 50; auxiliary gas (GS2), 40; declustering Potential (DP), 15 V; ion spray voltage, −4000 V; turbo temperature (TEM), 450 • C. MS in positive ion mode was used in multiple reaction monitoring (MRM) analysis for detection and quantitation of urea with transition m/z 61→44 ( Figure 6). 0.1% HCOOH and 1 mM HCOONH 4 were used as mobile phases with the flow rate set at 0.5 mL·min −1 . The injection volume was 10 µL. (MRM) analysis for detection and quantitation of urea with transition m/z 61→44 ( Figure 6). 0.1% HCOOH and 1 mM HCOONH4 were used as mobile phases with the flow rate set at 0.5 mL•min −1 . The injection volume was 10 µL.

Anti-urease Activity by ESI-MS-based Assay
Several important experimental conditions were investigated and taken into consideration to optimize the efficacy of the ESI-MS-based assay analysis, such as the buffer concentration, the buffer pH, and the type of sample vials. Under the optimized experimental conditions, experiments were employed and aimed at evaluating urease assay conditions that would be compatible with ESI-MS. The enzymatic reaction took place in 1 mM HCOONH4 buffer, which was found to be suitable (pH 7.5) containing 60.0 µg•mL −1 of urease, over a substrate (urea) concentrations ranging from 137.0 to 694.1 µmol•L −1 at room temperature (25 °C). Urea was chosen as a natural substrate for the enzymatic reaction. Urea concentrations were maintained at concentrations below 694.1 µmol•L −1 to avoid excess substrate inhibition. The concentrations of inhibitors (AHA = 13.2 µmol•L −1 and AEHS = 81 µg•mL −1 ) were used and were within the range of their effectiveness to inhibit urease activity. Enzymatic reaction was carried out by pre-incubating urease in 1 mM HCOONH4 buffer with each inhibitor during 180 min to reach binding equilibrium followed by adding urea. The solutions were directly injected automatically into FIA system and the concentration changes of urea were monitored. For the analysis of the kinetics of substrate depletion by ESI-MS, areas (total counts) under peaks for substrate in the FIA record were integrated. Each measured sequence consists of five measurements. Briefly, to determine the repeatability of measurements, we performed multiple measurements of enzymatic reaction of the same sample. The precision of time course analysis was calculated as RSD (%) of multiple measured slopes (lower than 10%). The slopes represent rate constants, which are used for determination of enzyme activity and inhibition studies. For clarity of figures, multiple measurements have not been presented in Figures 2 and 4. As shown in Figure 5, multiple measurements and evaluation of slopes are presented.
For measuring the rate constants for determining Km and Vmax, we set to each experiment different substrate concentrations. Furthermore, for screening inhibitors, we set to each experiment

Anti-urease Activity by ESI-MS-Based Assay
Several important experimental conditions were investigated and taken into consideration to optimize the efficacy of the ESI-MS-based assay analysis, such as the buffer concentration, the buffer pH, and the type of sample vials. Under the optimized experimental conditions, experiments were employed and aimed at evaluating urease assay conditions that would be compatible with ESI-MS. The enzymatic reaction took place in 1 mM HCOONH 4 buffer, which was found to be suitable (pH 7.5) containing 60.0 µg·mL −1 of urease, over a substrate (urea) concentrations ranging from 137.0 to 694.1 µmol·L −1 at room temperature (25 • C). Urea was chosen as a natural substrate for the enzymatic reaction. Urea concentrations were maintained at concentrations below 694.1 µmol·L −1 to avoid excess substrate inhibition. The concentrations of inhibitors (AHA = 13.2 µmol·L −1 and AEHS = 81 µg·mL −1 ) were used and were within the range of their effectiveness to inhibit urease activity. Enzymatic reaction was carried out by pre-incubating urease in 1 mM HCOONH 4 buffer with each inhibitor during 180 min to reach binding equilibrium followed by adding urea. The solutions were directly injected automatically into FIA system and the concentration changes of urea were monitored. For the analysis of the kinetics of substrate depletion by ESI-MS, areas (total counts) under peaks for substrate in the FIA record were integrated. Each measured sequence consists of five measurements. Briefly, to determine the repeatability of measurements, we performed multiple measurements of enzymatic reaction of the same sample. The precision of time course analysis was calculated as RSD (%) of multiple measured slopes (lower than 10%). The slopes represent rate constants, which are used for determination of enzyme activity and inhibition studies. For clarity of figures, multiple measurements have not been presented in Figures 2 and 4. As shown in Figure 5, multiple measurements and evaluation of slopes are presented.
For measuring the rate constants for determining K m and V max , we set to each experiment different substrate concentrations. Furthermore, for screening inhibitors, we set to each experiment different inhibitor concentrations or different types of inhibitor. A calibration curve after each experiment was measured to correct instability of MS signal.

Conclusions
Plant-derived phytochemicals provide an excellent option for the treatment of infectious diseases. The present findings indicated that PCA exhibited notable inhibitory properties on HSV-2 replication and thus has promising application in the development of anti-HSV-2 drugs. However, further studies should be carried out to evaluate its safety, validate the activity in vivo, and to eliminate the possible adverse effects of this compound by using improved delivery techniques prior its possible practical application. In addition, our results indicate that AEHS exerted anti-urease activity according to a developed ESI-MS-based assay. However, further studies must be performed to identify the active compounds in AEHS that contribute to the anti-urease activity, and evaluate their toxicity and structure-activity relationships.