Bioassay-Guided Isolation and HPLC Quantification of Antiproliferative Metabolites from Stahlianthus thorelii

In folk medicine, Stahlianthus thorelii Gagnep. has been used to treat diseases related to inflammation, ulcers, and cancer. There are no reports concerning the chemical components and bioactivities of S. thorelii; thus, this study aims to explore the phytochemicals, quantify the main compounds, and test the anticancer activity of isolates from S. thorelii. Dried rhizomes were extracted with 95% ethanol and, then, partitioned, fractionated, and isolated. On the basis of the result of the antiproliferative activity of the fractions, seven isolates were yielded and were identified by spectroscopic analyses. The inhibition of cancer proliferation was determined by an MTT assay and the deployed IC50 to value their efficacy. Seven compounds containing one new C-benzylated dihydrochalcone derivative, thorechalcone A (1) and 2–7 were isolated from S. thorelii. In terms of the bioactivity, compounds 1 and 3 displayed promising antiproliferative activity (WiDr, A549, and HepG2), with IC50 values <40 µM. The HPLC-UV method of quantification of two major compounds (3 and 4) was also validated. This study presented the isolations of antiproliferative potentials of new chalcone and known flavonoid derivatives from S. thorelii. The validated simple, accurate, and rapid HPLC method could be deployed for the quality control of herbal drugs.


Introduction
Zingiberaceae is commonly known as a zinger family consisting of perennial herbs used in traditional medicines. This family is widely distributed in Asia, Africa, and America, comprising more than 1300 species and around 52 genera. Many species in this family have already been studied for their phytochemicals and bioactivities, which display significant anticancer, antimicrobial, anti-inflammation bioactivities [1]. The potential of anticancer activities of the species in the Zingiberaceae family have In this way, the bioactivity-guided fractionation of S. thorelii extracts led to isolating one new compound, thorechalcone A (1), together with six known compounds, 2-7 ( Figure 1). Molecules 2020, 25, x FOR PEER REVIEW 3 of 14
The 13 C-NMR, 1 H-NMR and ESIMS data of 1-7 are shown in the supplementary section as Figures S1-S22.

Evaluation of the Anticancer Potential of Isolated Compounds
In the present study, an MTT cell proliferation assay was deployed for assessing the cytotoxicity of the fractions and isolated compounds. The high level of MTT reduction correlated to the high proliferation of cancer cells. The number of viable cells led to a large amount of formazan, which was created in a culture. Mitomycin c, which is a positive control, was used as a reference compound for all the cytotoxic assays in this study.
As shown in Table 3, the cytotoxicity of compounds 1-6 was tested on four cancer cell lines (A549, WiDr, HepG2, and MCF-7) by the MTT assay. The compounds 1 and 3 exhibited stronger cytotoxic activities on cell lines A549, WiDr, and HepG2, due to their possessing a chalcone structure. Interestingly, compound 1 had more potent cytotoxicity against the three cell lines than that of 3, based on the additional o-hydroxybenzyl moiety in structure 1. Although the remaining compounds 4-6 displayed moderate activities on different cell lines, this study was the first to report antiproliferation on four tumor cell lines for 4 and 5. In previous reports, 4 ((+)-crotepoxide) showed antimutagenicity in a Vitotox assay [17] and 1,6-deoxytingtanoxide (5) displayed mild cytotoxicity against human pancreatic cancer cell lines [21]. The bioassay data showed that compound 6 (O-methoxybenzoyl benzoate) did not have significant cytotoxicity on MCF-7; this result correlated with a previous study [22].

Quantitative Analysis of Compounds 3 and 4
The validated method was applied to the quantitative analysis of bioactive compound 3 and major yielded compound 4 from the S. thorelii rhizome. It was considered that 210 nm could be best used to analyze the profile of the compounds after comparing the record of the chromatograms of the extract solution running at wavelengths within 200 to 550 nm. Good linearity was obtained for each of constituents (R 2 > 0.995). The titled plant sample, Stahlianthus thorelii, was collected from three different areas in December 2018 (shown in Table 4). The quantitation was determined with three preparative samples and was analyzed three times. The concentrations were detected based on a linear regression and the average contents were obtained (p < 0.01). The contents of compounds 3 and 4 in the S. thorelii rhizome were 0.106% and 0.013%, respectively.

Validation of the HPLC-UV Analysis Method
To evaluate the quality of S. thorelii, two major compounds, 4 and anticancer 3, were retained for their quantification in the plant material. The HPLC-UV method was validated in terms of the analysis of the system suitability, specificity, precision, accuracy, linearity, and limits of detection and quantification. The validation summary is found in Table 5. According to ICH guidelines, system suitability testing is based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such. Testing is checked by calculating the retention time (t R ), peak area (A), theory Molecules 2020, 25, 551 8 of 14 plate (N), and resolution (Rs) factors. A residual sum of the squares of all the calculated parameters that is less or more than 2%, is within the acceptable limits, indicates good selectivity of the method, and ensures system performance. The linearity of this method was confirmed by a linear regression function. The calculations were based on seven different concentrations (n = 7). The regression equations were calculated using y = 32638x (R 2 = 0.9956) and y = 57440x (R 2 = 0.9969) for compounds 4 and 3, respectively, as illustrated in Table 5.
The limit of detection (LOD) and limit of quantification (LOQ) were determined by calculating them as three and 10 times the intensity of the baseline noise, respectively. The LOD and LOQ of compound 4 were 0.05 and 0.17 µg/mL, respectively, and the LOD and LOQ of compound 3 were 0.025 and 0.08 µg/mL, respectively. The specificity of the method was investigated. Specificity represents the ability to assess an analyte unequivocally in the presence of components which may be expected to be present by comparing the retention time and the UV spectra with the standard. As can be seen from Figure 2, the retention times of the compound 4 peak (24.8 min) and the compound 3 peak (30.0 min) in the sample were the same as the peaks in the standard. In addition, the peaks in the sample separated absolutely, and the peak area went up when adding the standards into the sample. Whereas, in the blank sample, there was no peak in the retention time of compound 4 and compound 3. This result indicated that the method could be used to analyze compound 4 and compound 3. Furthermore, the solvent did not influence the major compound peaks, and PAD purity studies confirmed the purity of the investigated peaks.
The repeatability was determined using six samples (n = 6) by calculating the retention time and the peak area. The RSDs of all the calculated parameters were less than or equal to 2% (retention time) and 5.3% (peak area). The accuracy was calculated based on the recovery concentration in three levels. The amount of the standard was put into the powdered rhizome at a ratio of 80%, 100%, and 120% of the content of compounds 4 and 3 in the sample. According to the calculated concentrations of compounds 4 and 3, the percentage of recovery of these compounds could be determined and the recovery parameters were less than or equal to 5.3%.

Plant Material
The plant material of S. thorelii was collected in An Giang Province, in the south of Vietnam, in 2017. The material was identified by the Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, where the voucher specimen (STV-20170224) was deposited. The rhizomes were dried and ground.

Extraction and Isolation
Air-dried rhizoma of S. thorelii (15 kg) were extracted three times using 95% ethanol (40 L) at 40 • C for 24 h each time. The extract was evaporated under reduced pressure to get the crude extract (500 g). Next, the extracted ethanol of S. thorelii was suspended and dissolved in 500 mL of H 2 O and partitioned continually by ethyl acetate and n-butanol (3 × 800 mL). After evaporation in a vacuum, fractions were prepared sequentially by ethyl acetate fraction (SEA) (120 g, 24%), n-butanol fraction (SBU) (40.5 g, 8.1%), and aqueous fraction (SW) (320 g, 64%) from the ethanol extracts.
Thorechalcone  were used for the analysis. The data were solved using the direct method, and the structure was refined by full-matrix least-squares on F 2 values. All non-hydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atom positions were idealized geometrically and allowed to ride on their parent atoms. The final indices were R1 = 0.0593, wR2 = 0.1337 with goodness-of-fit = 1.056. The final X-ray model is shown in Figure 3. The crystallographic data of compound 1 were deposited in the Cambridge Crystallographic Data Centre (CCDC), and the CCDC deposition number is CCDC 1948088. with I > 2σ (I) were used for the analysis. The data were solved using the direct method, and the structure was refined by full-matrix least-squares on F 2 values. All non-hydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atom positions were idealized geometrically and allowed to ride on their parent atoms. The final indices were R1 = 0.0405, wR2 = 0.0853 with goodness-of-fit = 1.062. The final X-ray model is shown in Figure 3. The crystallographic data of compound 2 were deposited in the Cambridge Crystallographic Data Centre (CCDC), and the CCDC deposition number is CCDC 1949802.

Colorimetric MTT In Vitro Assay
An MTT assay was employed to evaluate the proliferation of a number of cancer cell lines. Human lung carcinoma (A549), human breast adenocarcinoma (MCF-7), human colon adenocarcinoma (WiDr), and human hepatocellular carcinoma (Hep G2) were cultured in 75-T flasks with MEM supplemented with 5% FBS and a 1% penicillin-streptomycin antibiotics suspension. The cell suspensions after trypsinizing were seeded in 96-well plates at densities of 3000 cells per well. The 96-well plates were then incubated at 37 • C/5% CO 2 to adhere the cell to the surface of the plate. After reaching the confluence (80%), the cells were treated with 200 µL medium containing 20 to 80 µg/mL of the extract(s) and the fraction(s), and 1 to 40 µg/mL of the pure compound(s), and incubated for 72 h before reading the results. Later, the medium was removed by vacuum suctioning and 200 µL of 0.2 mg/mL MTT was added for 4 h at 37 • C. At the end of the incubation, the MTT was discharged, and 200 µL of dimethyl sulfoxide (DMSO) was added into 96-well plates. The absorbance of the solution was measured at 570 nm by a microplate reader (Dynatech, MR 7000) (Dynatech Labs, Chantilly, VA, USA). The same procedure was conducted with mitomycin c (positive control) at a concentration of 0.4, 0.2, and 0.1 µg/mL. The mean IC 50 was the inhibitor concentration, which reduced the proliferation of cells by 50%; this value was determined under the experimental conditions and calculated by the average of at least three independent tested results.
The cell viability was measured using this equation:

HPLC-SPD Separation Profile for Analysis of Seven Isolates from S. thorelii
The HPLC-SPD analysis was performed using a 250 × 4.6 nm i.d., 5 µM, Cosmosil C 18 column with an LC-20AT pump and an SPD-10A diode array detector (Shimazu). (Shimadzu, Kyoto, Japan). The mobile phase flow rate and the injection volume were 0.8 mL/min and10 µL, respectively. The wavelengths were set at 203 and 254 nm. The mobile phase was comprised of H 2 O (A) and ACN (B) and used a gradient program of separation condition as: 255 to 35% B from 0 to 15 min, 35% to 40% B from 15 to 20 min, 40% to 45% B from 20 to 30 min, 45% to 50% B from 30 to 35 min, 50% to 60% B from 35 to 45 min, 60% to 65% B from 45 to 55 min, 65% to 75% B from 55 to 85 min, and 75% to 100% B from 85 to 100 min ( Figure 4).

Quantitation of Bioactive Compound 3 and Major Yielded Compound 4
Concentrated solutions of two standards (3 and 4) were prepared in the ACN, each at 1000 mg/L. After diluting the stock solutions with ACN to obtain the developing solutions, seven concentration levels were found in the range of 2.5-500 mg/L. The worked solutions were filtered by a PVDF filter (0.45 µM, Millipore) before HPLC injection. Linear regression analysis was applied to achieve linearity by the integrated peak areas (Y) vs. the concentration of each standard (X, mg/L) at five different concentrations.

Validation of the HPLC-UV Method
According to the ICH harmonized tripartite guidelines, validation of the analytical procedure included system suitability, accuracy, repeatability, specificity, detection limit, quantitation limit, and linearity. Samples, calibration, and validation standards were prepared separately. mobile phase flow rate and the injection volume were 0.8 mL/min and10 μL, respectively. The wavelengths were set at 203 and 254 nm. The mobile phase was comprised of H2O (A) and ACN (B) and used a gradient program of separation condition as: 255 to 35% B from 0 to 15 min, 35% to 40% B from 15 to 20 min, 40% to 45% B from 20 to 30 min, 45% to 50% B from 30 to 35 min, 50% to 60% B from 35 to 45 min, 60% to 65% B from 45 to 55 min, 65% to 75% B from 55 to 85 min, and 75% to 100% B from 85 to 100 min (Figure 4).

Quantitation of Bioactive Compound 3 and Major Yielded Compound 4
Concentrated solutions of two standards (3 and 4) were prepared in the ACN, each at 1000 mg/L. After diluting the stock solutions with ACN to obtain the developing solutions, seven concentration levels were found in the range of 2.5-500 mg/L. The worked solutions were filtered by a PVDF filter (0.45 μM, Millipore) before HPLC injection. Linear regression analysis was applied to achieve linearity by the integrated peak areas (Y) vs. the concentration of each standard (X, mg/L) at five different concentrations.

Preparation of Calibration Standards
This study measured 10.0 mg of compounds 3 and 4, respectively, into a volumetric flask and diluted them to 10.0 mL with MeOH. Next, 250 µL of each solution were placed into a volumetric flask and diluted to 10.0 mL with MeOH. The final concentrations for compound 4 and compound 3 were 250 µg/mL and 55 µg/mL, respectively. Finally, 2 mL of this solution was filtered through a PVDF filter (0.45 µM, Millipore) for HPLC analysis.

Preparation of Validated Extracted Samples
This study measured 2.0 g of the powdered S. thorelii rhizome into a 50.0 mL glass Erlenmeyer flask, to which 20.0 mL of MeOH was added. The solution was then ultrasonicated three times for 30 min before being filtered and evaporated to about 5.0 mL of a concentrated extract. The concentrated extract was put into a 20.0 mL volumetric flask, rinsed two times with 5.0 mL of MeOH, ultrasonicated, and diluted to 10.0 mL with MeOH. Finally, 2.0 mL of this solution was filtered through a PVDF filter (0.45 µm, Millipore) for HPLC analysis.

Statistical Analysis
The results obtained were displayed as mean ± standard errors (SD). The experiments were conducted in triplicate on three different occasions. Statistical analyses were performed using SPSS software (IBM SPSS®software 20) (SPSS Inc., Chicago, IL, USA). The difference between two or more groups based on one-way of ANOVA multiple comparisons, where p < 0.01 indicates statistical significance. Inhibitory concentrations at (IC 50 ) were attained from the Sigma plot (12.5) using the nonlinear regression equation log (concentration) versus response-variable slope.

Conclusions
Bioassay-guided isolation yielded seven compounds from 95% EtOH extract of S. thorelii, compound 1 was a new C-benzylated dihydrochalcone derivative, compound 3 [(2E)-1-(2,4-dihydroxy-6-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-2-propen-1-one] was isolated from a plant for the first time, and this was the first time for the other compounds to be reported in this plant. The effective antiproliferative extracts and compounds yielded from S. thorelii through our studies provide scientific evidence to support this plant serving as a folk medicine to reduce tumor size. The bioassay results also revealed that dihydrochalcone derivatives 1 and 3 had promising cytotoxic effects against the HepG2 and WiDr cell lines.
Similar to the first report, the contents of the major compounds in S. thorelii were determined simultaneously by a simple, accurate, and rapid HPLC-UV data. Compound 3, the most potent anticancer isolated compound, together with the major yielded isolate, compound 4 [(+)-Crotepoxide], were chosen as analytical markers of this species. The quantification data of 4 and 3, as shown in Figure 5 and Table 5, concluded that the content of these analytical markers was suitable for comparing the titled plants collected in several areas. It is suggested that this validated method could be applied for the quality control of this herbal medicine in the future.

Conflicts of Interest:
The authors declare no conflict of interest.

Conflicts of Interest:
The authors declare no conflict of interest.