The Cytotoxic Activity and Metabolic Profiling of Hyptis rhomboidea Mart. et Gal

Many naturally occurring chemical metabolites with significant cytotoxic activities have been isolated from medicinal plants and have become the leading hotspot of anti-cancer research in recent years. Hyptis rhomboidea Mart. et Gal is used as a folk medicine in South China to treat or assist in the treatment of liver disease, ulcers, and edema. But its chemical constituents have not been fully investigated yet. This study aimed to assess the cytotoxicity of H. rhomboidea, which was chemically characterized by chromatography–mass spectrometry methods. The results showed that the 95% ethanol extract of H. rhomboidea has marked inhibitory effects on five human cancer cell lines (HL-60, A549, SMMC-7721, MDA-MB-231, and SW480), with IC50 values ranging from 15.8 to 40.0 μg/mL. A total of 64 compounds were identified by ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and gas chromatograph–mass spectroscopy (GC-MS) analysis of H. rhomboidea crude extract. Among them, kaempferol, quercetin, rosmarinic acid, squalene, and campesterol were found to be abundant and might be the major metabolites involved to its bioactivity. The cytotoxic characterization and metabolite profiling of H. rhomboidea displayed in this research provides scientific evidence to support its use as medicinal properties.


Introduction
Cancer is one of the most dangerous diseases, posing a significant threat to human health worldwide.According to GLOBOCAN 2020 statistics on cancer incidence and mortality, there were approximately 19.3 million new cancer cases and nearly 10 million cancer deaths in 2020 [1].Among them, breast cancer (2.3 million), lung cancer (2.24 million), colorectal cancer (1.97 million), prostate cancer (1.44 million), and stomach cancer (1.1 million) are still high-incidence diseases, with lung cancer causing the highest number of deaths annually.Despite substantial advancements in cancer biology, oncology, and surgical techniques in recent years, the overall survival rate for cancer has only slightly improved over the past decades, and this disease remains one of the leading causes of death [2][3][4] Currently, the main treatment methods for cancer include radiotherapy, chemotherapy, and surgery [5].While these treatments can be effective, the toxicity of anti-cancer drugs and the development of drug resistance in cancer cells often diminish their efficacy and result in significant side effects, such as nausea and loss of appetite [6][7][8].Research has discovered that some natural plant compounds possess significant cytotoxicity against cancer cells lines, and they are abundantly available and have few toxicity side effects [9].Therefore, natural plant compounds are important raw ingredients for exploring novel anti-cancer drugs.Combining natural anti-cancer compounds with conventional chemotherapy drugs can help to jointly inhibit cancer cell growth, prevent the development of drug resistance in Among them, 75 small-molecule drugs (41% of the total) originated from natural products and their derivatives [11].Therefore, natural products play significant role in new drug discovery and development.
Hyptis rhomboidea Mart.et Gal (Figure 1), also called H. decurrens, Pycnanthemum decurrens, is a medicinal plant belonging to the Lamiaceae family, native to tropical and subtropical regions of North America and widely distributed in the coastal areas of South China [12,13].It is used as Chinese folk medicine to assist in the treatment of hepatitis, ulcers, and edema [14].Phytochemical and pharmacological studies revealed that H. rhomboidea mainly contains phenylpropanoids, terpenoids, and phenolic acid.For example, hyprhombin C and epi-hyprhombin B (first identified in H. rhomboidea) demonstrate excellent anti-xanthine oxidase activity [15].Caryophyllene oxide can be used to relieve pain without inducing psychoactive side effects, while ursolic acid is effective in treating autoimmune arthritis [16,17].However, there are no reports about the cytotoxic activity of H. rhomboidea in human cancer cells.
In this study, the cytotoxic activities of H. rhomboidea ethanol extract against five human cancer cell lines including leukemia (HL-60), hepatoma (SMMC-7721), lung cancer (A-549), breast cancer (MDA-MB-231), and colon cancer (SW480) were assessed by the MTS method [18].Based on the activity screening results, the metabolic profiling of H. rhomboidea extract was further analyzed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and gas chromatography-mass spectrometry (GC-MS).This analysis helped to explore the potential cytotoxicity of natural products from the medicinal plant H. rhomboidea against human cancer cell lines.In this study, the cytotoxic activities of H. rhomboidea ethanol extract against five human cancer cell lines including leukemia (HL-60), hepatoma (SMMC-7721), lung cancer (A-549), breast cancer (MDA-MB-231), and colon cancer (SW480) were assessed by the MTS method [18].Based on the activity screening results, the metabolic profiling of H. rhomboidea extract was further analyzed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and gas chromatography-mass spectrometry (GC-MS).This analysis helped to explore the potential cytotoxicity of natural products from the medicinal plant H. rhomboidea against human cancer cell lines.

Cytotoxic Activity Evaluation of H. rhomboidea Crude Extracts
To investigate the cytotoxic activity of ethanol extracts from H. rhomboidea, five types of human cancer cells (HL-60, A549, SMMC-7721, MDA-MB-231, and SW480) were incubated with 100 µg/mL of the extracts.The cell viability of the cultured cancer cells was measured using MTS assays, evaluated the cytotoxicity of 95% and 50% ethanol extracts of H. rhomboidea against the five human cancer cell lines.
The 95% ethanol extract (HR-95) exhibited significant cytotoxic activity against the growth of the five types of cancer cells in vitro.Compared with normal human cells (BEAS-2B), HR-95 exhibited a significantly higher inhibitory effect against the five human tumor cell types (Figure 2).Notably, the extract was particularly effective in inhibiting the growth of leukemia (HL-60) and breast cancer (MDA-MB-231) cells, with rates of 96.10% and 90.54%, respectively.The inhibition rates against hepatoma (A549), lung cancer (SMMC-7721), and colon cancer (SW480) cell growth were 78.24%, 79.43%, and 85.64%, respectively.In contrast, the 50% ethanol extract (HR-50) had low inhibitory effect on cell viability and promoted HL-60 cell growth by 41.52%.For the negative control, both extracts exhibited no cytotoxic activity against the growth of human normal lung epithelial cells (BEAS-2B), with inhibition rates of only 14.20% and 1.57%, respectively (Table 1).measured using MTS assays, evaluated the cytotoxicity of 95% and 50% ethanol extracts of H. rhomboidea against the five human cancer cell lines.
The 95% ethanol extract (HR-95) exhibited significant cytotoxic activity against the growth of the five types of cancer cells in vitro.Compared with normal human cells (BEAS-2B), HR-95 exhibited a significantly higher inhibitory effect against the five human tumor cell types (Figure 2).Notably, the extract was particularly effective in inhibiting the growth of leukemia (HL-60) and breast cancer (MDA-MB-231) cells, with rates of 96.10% and 90.54%, respectively.The inhibition rates against hepatoma (A549), lung cancer (SMMC-7721), and colon cancer (SW480) cell growth were 78.24%, 79.43%, and 85.64%, respectively.In contrast, the 50% ethanol extract (HR-50) had low inhibitory effect on cell viability and promoted HL-60 cell growth by 41.52%.For the negative control, both extracts exhibited no cytotoxic activity against the growth of human normal lung epithelial cells (BEAS-2B), with inhibition rates of only 14.20% and 1.57%, respectively (Table 1).

Screening of the 95% Ethanol Extract for Cytotoxic Activity against Human Cancer Cell Lines
After assessing the cytotoxic activity of H. rhomboidea (HR-95), it was observed that HR-95 exhibits obviously inhibitory activity, with half-maximal effectiveness against the growth of the five human cancer cell lines in vitro.Therefore, the cell viability of cancer cells was determined using the MTS method at different concentrations of HR-95 (0, 0.8, 4, 20, 100 µg/mL).Positive control compounds included different concentrations of cisplatin and Taxol.
After 48 h of incubation, cell viability assays were performed to evaluate the cytotoxic effects of HR-95 on the five human cancer cell lines.HR-95 and the positive control cisplatin exhibited poor inhibitory effects on the proliferation of human normal lung epithelial cells (BEAS-2B), and HR-95 possessed satisfactory cytotoxic activity against the five human cancer cell lines compared with the positive control cisplatin, with particularly noteworthy inhibitory effects on breast cancer (MDA-MB-231) and colon cancer cells (SW480) (Figure 3).Based on IC 50 calculations, HR-95 exhibited distinct half-maximal inhibitory concentrations against the following five cancer cell lines: HL-60 (36.44 ± 1.82 µg/mL), A549 (40.02 ± 0.58 µg/mL), SMMC-7721 (26.20 ± 0.77 µg/mL), MDA-MB-231 (22.95 ± 1.59 µg/mL), and SW480 (15.81 ± 0.63 µg/mL) (Table 2).The results indicate that HR-95 contains compounds with potential cytotoxic activity against human cancer cell lines and warrants further isolation and identification for characterization.

Metabolic Profiling of H. rhomboidea
The results of the MTS assay showed that the 95% ethanol extract of the whole H. rhomboidea (HR-95) plant had great cytotoxic activity, while 50% extract had no inhibitory activity.To elucidate the chemical constituents (HR-95) with cytotoxic activity, LC-MS and GC-MS analysis were performed to characterize the metabolite profiles.

Non-Volatile Chemical Compound Analysis
To identify the non-volatile compositions of HR-95, UPLC-QTOF-MS conditions were optimized to establish the suitable elution gradient, ratio of formic acid in the mobile phase, operating voltage, and other parameters and achieve optimal chromatographic peak intension and separation.The identification of metabolites was performed by combining the positive and negative ion modes to obtain base peak chromatograms.The results were retained at the initial 10 min, and chromatographic peaks found in blank samples were excluded to enhance the accuracy of the analysis (Figure 4).Different types of metabolites were detected between positive and negative ion modes, with the total peak intensity notably higher in positive ion mode than in negative ion mode.Based on the retention time, high-resolution mass spectra molecular ions and t fragment ions of peaks were obtained from the low-energy and ramp collision energy MS E data.According to the rules of ion fragmentation, compared with open-source ma spectral libraries and the literature on the Hyptis genus, 25 compounds with relatively hig contents including eleven flavonoids, six terpenoids, two coumarins, three aliphatic d rivatives, two aromatic derivatives, and one saccharide were tentatively identified fro HR-95 (Table 3).Based on the retention time, high-resolution mass spectra molecular ions and the fragment ions of peaks were obtained from the low-energy and ramp collision energy of MS E data.According to the rules of ion fragmentation, compared with open-source mass spectral libraries and the literature on the Hyptis genus, 25 compounds with relatively high contents including eleven flavonoids, six terpenoids, two coumarins, three aliphatic derivatives, two aromatic derivatives, and one saccharide were tentatively identified from HR-95 (Table 3).
Among the identified compounds, three types of flavonoids were categorized as follows: flavonols, flavones, and flavanes.These flavonoids exhibited strong molecular or quasi-molecular ion peaks using the ESI ion source.3 and not described here.The possible identifications of the other 12 peaks are summarized in Table 3 and not described here.

Discussion
H. rhomboidea, a traditional herbal medicine used to treat hepatitis, has been investigated for its chemical constituents, antimicrobial activity, and cytotoxicity activity against the murine tumor cell line in few studies [14,38].We evaluated the cytotoxic activity of H. rhomboidea ethanol extract on five human cancer cell lines for the first time.The results indicated that the 95% ethanol extract of H. rhomboidea (HR-95) exhibits pronounced inhibitory activity, while the 50% extract (HR-50) shows no significant effect.However, HR-50 significantly inhibits NO production in murine monocyte-macrophages, indicating its excellent anti-inflammatory effects.At a concentration of 50 µg/mL, HR-50 achieved an inhibition rate of 97.23% on NO production.Nevertheless, it also exhibited cytotoxicity (Table S1).In addition, both of the extracts (HR-95 and HR-50) showed no cytotoxic against non-cancer cells (normal human lung epithelial cells), which demonstrated that the H. rhomboidea extracts have selectivity against human cancer cells.This finding indicates that the potential cytotoxicity of the ethanol extract from H. rhomboidea are relative to the polarity of its metabolites, and the big polar compounds from the 50% ethanol extract may associate with their anti-inflammatory activity.These results support the medicinal value of H. rhomboidea.
The MTS assay revealed that the 95% ethanol extract of H. rhomboidea exhibited lower cytotoxic activity against the five human cancer cell lines compared with the positive controls (cisplatin and Taxol), yet it still demonstrated a moderate level (the extract showed over half inhibitory rate at 100 µg/mL) of an inhibitory effect, with IC 50 values ranging from 15.8 µg/mL to 40.0 µg/mL.This may be attributed to the low concentration of the bioactive metabolites and the interaction of multiple compounds.The separation and purification of bioactive compounds can enhance their cytotoxicity against cancer cells.Consequently, the chemical composition of HR-95 was investigated using LC-MS and GC-MS.
In this study, we employed LC-MS and GC-MS methods to analyze and characterize the chemical composition of HR-95, resulting in the identification of various potential biologically active compounds.Among them, flavonoids were the main components in H. rhomboidea crude extract.Previous studies have reported that flavonoids possess extensively cytotoxicity against cancer cells [39,40].Studies have found that 3 ′ ,4 ′ ,5 ′ -trimethoxyflavonol induces apoptosis in TRAMP C2 prostate cancer cells [41].Quercetin not only triggers apoptosis in human colon cancer cells by inhibiting the nuclear factor-kappa B pathway but also suppresses proliferation and invasion in breast cancer cells through the up-regulation of miR-146a.It further induces autophagy-associated cell death in HL-60 cells via the CaMKKβ/AMPK/mTOR signaling pathway and exhibits other anti-tumor effects as well [42][43][44][45].Kaempferol induces the cell cycle at the G2/M phase in MDA-MB-453 breast cancer cells and reverses the EMT process in gastric, ovarian, and breast cancer cells by modulating key markers such as E-cadherin, Smad2/4, TGF-β, N-cadherin, vimentin, and Snai1 [46,47].Astragalin has been shown to effectively inhibit the proliferation of leukemia (HL-60), hepatocellular (HepG2), skin (HaCaT), and lung (A549) cancer cells [48].Meanwhile, genkwanin demonstrates strong efficacy in inhibiting the proliferation and metastasis of A549 and H69AR cancer cells and shows potential in treating colon cancer and B16F10 melanoma [49].The effective inhibition of human tumor cells by the H. rhomboidea extract may be attributed to these compounds.
Previous studies have found that the main components of H. rhomboidea are phenolic acids and glycosides, which have inhibitory effects on plant pathogens such as Fusarium graminearum and Exserohilum turcicum [38].The ethanol extract can improve the levels of serum TNF-α and IL-2 in mice, boost SOD activity, and decrease MDA content; thus, it can against the growth of digestive tumor cells with an inhibition activity of 68.84% [14].However, there are few reports on the chemical composition of H. rhomboidea and their cytotoxicity against human cancer cells.In this study, we observed satisfactory activity of the ethanol extract of H. rhomboidea on human cancer cells for the first time.Subsequently, using metabolomics approaches with UPLC-QTOF-MS and GC-MS, we analyzed the chemical composition of the extract and identified potential active compounds associated with cytotoxicity, providing new insights into the isolation and purification of bioactive compounds.Moreover, merging bioactivity with metabolomic techniques can provide an effective strategy to discover bioactive compounds from medicinal plants [18], thereby addressing the limitations of traditional methods in isolating bioactive compounds.

Plant Materials
The H. rhomboidea plant was collected from Ledong county, Hainan province, China.Voucher specimens were verified by Dr. Haijun Yang (South China Agricultural University) and stored at −40 • C in the laboratory of chemical ecology until extraction.

Perparation of Crude Extract
The aerial part of H. rhomboidea was naturally air-dried and ground into a powder.The dried powder was weighed to 200 g accurately and evenly divided into 2 portions (100 g each).Subsequently, samples are extracted with 50% ethanol and 95% ethanol for 48 h and then sonicated at 20 • C for 30 min.Each sample underwent two extractions using the same method.The filtrate was concentrated with a vacuum rotary evaporator to produce dry residues.The 50% ethanol crude extract was 328 mg, and the 95% ethanol crude extract was 169 mg.The extracts were stored at −20 • C until use.

UPLC-QTOF-MS Analysis of Non-Volatile Compounds
The 95% ethanol crude extract of H. rhomboidea was dissolved in methanol to a final concentration of 1 mg/mL and filtered through a 0.22 µm PTFE filter for analysis [61].The prepared samples were analyzed using an ACQUITY UPLC system connected to a Xevo G2-XS QTOF spectrometer (Waters Milford, Milford, MA, USA).Separation was achieved on an ACQUITY UPLC ® BEH C18 column (2.1 mm × 50 mm, 1.7 µm; Waters) maintained at 40 • C, with an injection amount of 2 µL.The elution utilized a two-solvent system as follows: solvent A was water with 0.1% formic acid, and solvent B was acetonitrile with 0.1% formic acid.The gradient program was as follows: 0-1.0 min, 10% B; 1.0-2.0min, 10~30% B; 2.0-8.0 min, 30-98% B; 8.0-10.0min, 98% B; 10.0-10.2min, 98~10% B; 10.2-13.0min, and 10% B. The flow rate was set to 0.3 mL/min, and the autosampler temperature was maintained at 16 • C. High-resolution mass spectrometry was detected in both negative and positive ion sensitivity mode with an electrospray ionization source.The essential parameters of the Xevo G2-XS QTOF-MS included a capillary voltage of +2.50 kV and −1.5 kV, source temperature of 120 • C, desolvation temperature of 350 • C, cone gas flow rate of 50 L/h, desolvation gas flow rate of 800 L/h, scan time of 0.5 s, mass range of 100-1000 m/z, low energy of 5 V, and collision energy of 20~50 V.All compounds were detected using MS E mode to obtain fragmentation data in a single analysis.

GC-MS Analysis of Volatile Compounds
The 95% ethanol crude extract of H. rhomboidea was dissolved with methanol (with 5 mg/L ethyl caprate) to a concentration of 1 mg/L.Subsequently, the extracts were sonicated for 30 min in a 15 • C water bath, followed by drying over anhydrous sodium sulfate and stored at −4 • C for 12 h.Then, 1 mL of extraction solution was aspirated and filtered through a 0.22 µm PTFE into a 2 mL sample vial for GC-MS analysis.
The GC-MS analysis was conducted using a SHIMADZU TQ8040 GC-MS instrument, which featured a triple quadrupole configuration and a DB-5MS column (30 m × 0.25 mm × 0.25 µm, Agilent Technologies, Santa Clara, CA, USA).The GC parameters were as follows: helium was used as the carrier gas at a flow rate of 1 mL/min, the injection temperature was set to 230 • C, the detector temperature was 250 • C, the sample volume was 1 µL, and the split ratio was 3: 1.The oven temperature was increased using the following procedure: starting at 60 • C for 2min, ramped at 6 • C/min to 250 • C and kept for 15 min, then maintained 250 • C for 10 min.The MS utilized an EI ionization source operating at 70 eV, with a scan range of 50-500 m/z.The ion source temperature was set at 200 • C, and the GC-MS interface temperature was maintained at 250 • C. The raw compound data files were converted from QGD format to CDF format using SHIMADZU GSMSsolution (Ver.4.52) for subsequent analysis.

Data Analysis
The raw data acquired from UPLC-QTOF-MS and GC-MS analyses were initially converted to ABF format files by ABF converter (Ver.1.3).Subsequently, compound relative content, mass spectra information, relative retention index (RI), and relative retention time (RT) were obtained using MSDAIL (Ver.4.9) for compound identification.The MSDAIL parameters were set to default.The non-volatile compounds of UPLC-MS were identified using the Massbank library, MSDAL standard compound library, METVARE self-built library, and published mass spectra.Meanwhile, the volatile compounds of GS-MS were identified using the NIST 2020 mass spectral library and published mass spectra, and the compounds with matching ≥ 800 were retained.

Conclusions
In this study, we first discovered that the 95% ethanol extract of H. rhomboidea exhibited moderate cytotoxic activities against five human cancer cell lines (HL-60, A549, SMMC7721, MDA-MB-231, and SW480), with IC 50 values ranging from 15.8 to 40.0 µg/mL.Based on metabolomic analysis using UPLC-QTOF-MS and GC-MS methods, a total of 25 nonvolatile and 39 volatile compounds from the active crude extract were identified, and several compounds had the potential cytotoxicity need further isolation and verification.These findings highlight the cytotoxic potential of H. rhomboidea and provide valuable insights for its further exploration and application in cancer research and therapeutic development.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/molecules29174216/s1:Table S1.The inhibitory rate of nitric oxide (NO) generation for two H. rhomboidea ethanol extracts and the positive control.
Author Contributions: W.G., J.Z. and R.Z. prepared the crude extracts and conducted the chemical analysis of the samples; W.G. and J.Z. performed the HPLC-QTOF-MS/MS analysis; R.Z. and J.Z. carried out the GC-MS analysis; J.Z., W.G. and K.Y. conducted partial cytotoxicity and anti-inflammatory cell experiments, with technical assistance provided by the Kunming Institute of Botany; J.Z., W.G. and P.L. analyzed the results; J.Z. wrote this manuscript; I.J., P.L. and J.Y. proofread this manuscript; J.Y. and P.L. were responsible for project management.All authors have read and agreed to the published version of the manuscript.

Molecules 2024 ,Figure 4 .
Figure 4. Base peak chromatograms of the 95% ethanol extract of H. rhomboidea (HR-95).(a) Ba peak chromatograms of the positive ion mode.(b) Base peak chromatograms of the negative i mode.Compound peaks are ranked by number.

Figure 4 .
Figure 4. Base peak chromatograms of the 95% ethanol extract of H. rhomboidea (HR-95).(a) Base peak chromatograms of the positive ion mode.(b) Base peak chromatograms of the negative ion mode.Compound peaks are ranked by number.

Figure 6 .
Figure 6.Relative content of identified volatile compounds.(a) Relative content of compounds classified by functional groups.(b) Relative content of different terpenoid types.

Figure 6 .
Figure 6.Relative content of identified volatile compounds.(a) Relative content of compounds classified by functional groups.(b) Relative content of different terpenoid types.

Table 1 .
Inhibitory rate (%) of two H. rhomboidea ethanol extracts on five types of human cancer cells.

Table 2 .
The IC 50 value (µg/mL) of H. rhomboidea crude extract on five human cancer cell lines.
HR-95: 95% ethanol extract of H. rhomboidea.Cisplatin and Taxol were used as positive controls.The values are expressed as mean ± SD of IC 50 (n = 3).

Table 4 .
Essential compounds in the 95% ethanol extracts of Hyptis rhomboidea extracted using GC-MS.
RT: retention time.RI: retention index.Library No. includes the CAS ID and NIST ID of compounds.Relative content is calculated by peak area/total peak area of compounds.

Table 4 .
Essential compounds in the 95% ethanol extracts of Hyptis rhomboidea extracted using GC-MS.