LC-MS/MS-Based Metabolomic Profiling of Constituents from Glochidion velutinum and Its Activity against Cancer Cell Lines

This study aimed to establish the phytochemical profile of Glochidion velutinum and its cytotoxic activity against prostate cancer (PC-3) and breast cancer (MCF-7) cell lines. The phytochemical composition of G. velutinum leaf extract and its fractions was established with the help of total phenolic and flavonoid contents and LC-MS/MS-based metabolomics analysis. The crude methanolic extract and its fractions were studied for pharmacological activity against PC-3 and MCF-7 cell lines using the MTT assay. The total phenolic content of the crude extract and its fractions ranged from 44 to 859 µg GAE/mg of sample whereas total flavonoid contents ranged from 20 to 315 µg QE/mg of sample. A total of forty-eight compounds were tentatively dereplicated in the extract and its fractions. These phytochemicals included benzoic acid derivatives, flavans, flavones, O-methylated flavonoids, flavonoid O- and C-glycosides, pyranocoumarins, hydrolysable tannins, carbohydrate conjugates, fatty acids, coumarin glycosides, monoterpenoids, diterpenoids, and terpene glycosides. The crude extract (IC50 = 89 µg/mL), the chloroform fraction (IC50 = 27 µg/mL), and the water fraction (IC50 = 36 µg/mL) were found to be active against the PC-3 cell line. However, the crude extract (IC50 = 431 µg/mL), the chloroform fraction (IC50 = 222 µg/mL), and the ethyl acetate fraction (IC50 = 226 µg/mL) have shown prominent activity against breast cancer cells. Moreover, G. velutinum extract and its fractions presented negligible toxicity to normal macrophages at the maximum tested dose (600 µg/mL). Among the compounds identified through LC-MS/MS-based metabolomics analysis, epigallocatechin gallate, ellagic acid, isovitexin, and rutin were reported to have anticancer activity against both prostate and breast cancer cell lines and might be responsible for the cytotoxic activities of G. velutinum extract and its bioactive fractions.


Introduction
After cardiovascular diseases, cancer is the major cause of death [1]. Though the modern age has advanced pharmaceuticals for cancer treatment, we are still deprived of the radical cure and patients often undergo miserable adverse effects while receiving treatments [2]. Cancer treatments include surgical resection of the cancerous mass, radiotherapy, and chemotherapy [3]. Common problems with the available therapeutic options are a high risk of adverse reactions, resistance, ineffectiveness, and high cost. Cancer diagnoses 2. Results and Discussion 2.1. Phytochemical Analysis 2.1. 1

. Total Phenolic Contents
Based on the results obtained, the crude extract and fractions of G. velutinum showed a significant amount of total phenolic content, as shown in Table 1. The highest phenolic content was shown by the ethyl acetate fraction (859 ± 1.3 µg GAE/mg) followed by crude extract (588 ± 3 µg GAE/mg), chloroform (266 ± 1 µg GAE/mg), and aqueous (77 ± 2.3 µg GAE/mg) fractions. Polyphenols are well known for their anti-inflammatory, antioxidant, and anticancer properties [16]. In this study, a significant amount of phenolic content was estimated in the crude extract and fractions of G. velutinum. Therefore, the presence of a remarkable number of polyphenols depicts the pharmacological value of G. velutinum.

Total Flavonoid Contents
The obtained results from total flavonoid contents (TFC) showed that the extract and its fractions contain a considerable quantity of TFCs, as shown in Table 2. The ethyl acetate fraction showed the maximum quantity of flavonoid contents (315 ± 1 µg QE/mg) followed by crude extract (118 ± 2 µg QE/mg) and the chloroform (79 ± 1.3 µg QE/mg) fraction. Flavonoids are plant bioactive constituents of great interest due to their remarkable antioxidant, anti-inflammatory, antibacterial, antifungal, and anticancer properties [17]. Hence, the presence of a remarkable number of flavonoids in the crude extract and its fractions also signifies the importance of G. velutinum for medicinal use, especially in inflammation and cancer.

LC-MS/MS-Based Chemical Profiling
Based on the anti-prostate and anti-breast cancer activities of the crude extract of G. velutinum leaves, it was investigated to establish the detailed phytochemical profile, using the LC-MS/MS (tandem mass) and GNPS-based metabolomics platform. Further, the fractionlevel biological activity against the prostate cancer cell line (PC-3) and the breast cancer cell line (MCF-7) was determined for the polarity-based fractions obtained through solventsolvent extraction from the G. velutinum crude extract. The fractions were also analyzed through tandem mass spectrometry and the GNPS molecular networking platform. Based on previous literature, none of the species from the genus Glochidion were studied for detailed phytochemical profiling using LC-MS/MS-based modern metabolomics approaches.

Anticancer Activity
In this study, the anticancer potential of the crude extract and fractions of G. velutinum was evaluated using the MTT assay. The treatment of PC-3 cells with G. velutinum extract and its fractions for 24 h resulted in the reduction of cell viability in comparison to DMSO (control) treated cells (no cell death). From Figure 4, it can be observed that the n-hexane fraction presented the least cytotoxic effect (IC 50 = 325 µg/mL) on PC-3 cells, whereas ethyl acetate and n-butanol fractions showed a moderate effect with respective IC 50 values of 196 µg/mL and 123 µg/mL, respectively. The stronger effects were observed for the chloroform fraction (IC 50 = 27 µg/mL), followed by the aqueous fraction (IC 50 = 36 µg/mL) and G. velutinum extract (IC 50 = 89 µg/mL). demonstrated a good safety margin of the tested samples. Based on earlier reports [9,36], selectively targeting cancer cells with minimum toxicity to normal cells prevents damaging effects on body organs [9]. Therefore, G. velutinum extract can be considered safe for the isolation of anticancer constituents.  Similarly, the treatment of MCF-7 cells with G. velutinum extract and its fractions showed a decrease in cell viability in comparison to DMSO-treated cells. From Figure 5, among the tested samples, the n-butanol fraction presented the least cytotoxic effect (24% cell growth inhibition) on MCF-7 cells, whereas the aqueous and n-hexane fractions showed a moderate effect with IC 50 values of 522 µg/mL and 523 µg/mL, respectively. The stronger effects were observed for the chloroform fraction (IC 50 = 222 µg/mL), followed by the ethyl acetate fraction (IC 50 = 226 µg/mL) and G. velutinum extract (IC 50 = 431 µg/mL). The IC 50 values of G. velutinum extract and its fractions for the MCF-7 and PC-3 cell lines have also been given in Table 5.    Plant-derived natural products remain the major contributors to pharmacotherapy, especially for cancer and infectious diseases [24]. Among these phytoconstituents, polyphenols have been extensively explored for the treatment of cancer [25][26][27]. In this work, phenolics, flavonoids, fatty acids, terpenoids, coumarins, and sugars were identified as major constituents of G. velutinum extract and its fractions. Based on literature surveys of the identified compounds in the crude extract and its fractions, epicatechin gallates were previously reported for anticancer activity for prostate cancer cell lines [28]. Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation [29]. Isovitexin, ellagic acid, trehalose, and rutin were also identified as possessing activity against prostate cancer cell lines [30]. On the other hand, it is also noteworthy that the above-mentioned constituents, i.e., epigallocatechin-3-gallate (EGCG), isovitexin, isokaempferide, quinic acid, rhoifolin gallic acid, luteolin, ellagic acid, and rutin have also been shown to possess activity against breast cancer cell lines [31][32][33][34][35]. Hence, the presence of all these compounds may be responsible for the activity of the crude extract and its fractions against prostate and breast cancer cell lines. These results indicate that the fractions of G. velutinum extract may also be useful for the isolation of anticancer constituents, especially against prostate and breast cancer.
Moreover, the G. velutinum extract and its fractions were tested against peritoneal macrophages (at a maximum tested concentration of 600 µg/mL), and it was observed that the G. velutinum extract and its fractions induced negligible toxicity to normal macrophages in comparison to the control that is presented in Figure 6. Safety is the major concern for the development of novel therapeutic agents [9]. In this study, the low cytotoxic effect of the G. velutinum extract and its fractions against normal macrophages demonstrated a good safety margin of the tested samples. Based on earlier reports [9,36], selectively targeting cancer cells with minimum toxicity to normal cells prevents damaging effects on body organs [9]. Therefore, G. velutinum extract can be considered safe for the isolation of anticancer constituents.

Collection and Identification of the Selected Plant
The leaves of G. velutinum Wight were collected from Batrasi Reserved Forest, District Mansehra, Pakistan, in June. The plant specimen was authenticated by Dr. Abdul Majid, Assistant Professor, Department of Botany, Hazara University, Pakistan. The voucher specimen (GV-ZH-02/18) was deposited to the Department of Environmental Sciences Herbarium, COMSATS University Islamabad, Abbottabad Campus for future records.

Processing of Plant Material and Extraction
The collected plant material was shade-dried at ambient temperature (24-26 °C ). The dried material was powdered (5 Kg) and subjected to extraction using methanol (15 L) with occasional shaking. The extraction was performed for 14, 7, and 3 days, respectively to completely exhaust the plant material. The extracted material was filtered through muslin cloth followed by Whatman filter paper. A vacuum rotary evaporator (Büchi Rotavapor ® R-300 Flawil, Switzerland) was used for the concentration of filtrates to get the crude extract [37]. The yield of G. velutinum crude extract was 410 g (8.2%).

Collection and Identification of the Selected Plant
The leaves of G. velutinum Wight were collected from Batrasi Reserved Forest, District Mansehra, Pakistan, in June. The plant specimen was authenticated by Dr. Abdul Majid, Assistant Professor, Department of Botany, Hazara University, Pakistan. The voucher specimen (GV-ZH-02/18) was deposited to the Department of Environmental Sciences Herbarium, COMSATS University Islamabad, Abbottabad Campus for future records.

Processing of Plant Material and Extraction
The collected plant material was shade-dried at ambient temperature (24-26 • C). The dried material was powdered (5 Kg) and subjected to extraction using methanol (15 L) with occasional shaking. The extraction was performed for 14, 7, and 3 days, respectively to completely exhaust the plant material. The extracted material was filtered through muslin cloth followed by Whatman filter paper. A vacuum rotary evaporator (Büchi Rotavapor ® R-300 Flawil, Switzerland) was used for the concentration of filtrates to get the crude extract [37]. The yield of G. velutinum crude extract was 410 g (8.2%).

Fractionation of Crude Extract
The crude extract (340 g) was suspended in distilled water and extracted with various organic solvents (n-hexane, chloroform, ethyl acetate, and n-butanol) in increasing order of polarity using established protocols [38,39]. The organic layers and final residual aqueous layer after solvent-solvent extraction were dried with the help of a rotary evaporator to obtain crude fractions. The crude extract of G. velutinum yielded various fractions including n-hexane (20 g), chloroform (180 g), ethyl acetate (15 g), n-butanol (35 g), and an aqueous fraction (40 g).

Total Phenolic Contents (TPC)
The TPC of the G. velutinum extract and its fractions was determined using the Folin-Ciocalteu reagent (FCR) method according to the procedure reported by John et al., 2014 [40]. Gallic acid was used as the standard polyphenolic compound to construct a calibration curve for the measurement of the contents of the samples. The samples of gallic acid, plant extract, and fractions were prepared in methanol (0.5 mL) and mixed with FCR (1.5 mL). The mixture was incubated at room temperature for 5 min. Then, 4 mL of Na 2 CO 3 solution (7.5%) was added to the above mixture and the volume was made up to 25 mL with distilled water. After a 30 min incubation, absorbance was measured at 765 nm with a UV/VIS spectrophotometer (Model UVD-3000, Labomed, Inc., Los Angeles, CA, USA) against distilled water as a blank. Results were expressed as micrograms of gallic acid equivalents (GAE)/mg of dry extract.

Total Flavonoid Contents (TFC)
The TFC of the G. velutinum extract and its fractions was determined using the aluminum chloride colorimetric method according to the procedure reported by Vyas et al. (2015) with slight alteration [41]. The calibration curve for the determination of contents in the samples was constructed using quercetin as the standard compound. The samples of quercetin, plant extract, and fractions were dissolved in 0.5 mL methanol. In test tubes, the measured volumes (500 µL) of quercetin, crude extract, and fractions solution were placed. Each test tube was filled with distilled water (3 mL) and 0.3 mL sodium nitrite solution. After 5 min, 0.3 mL of aluminum chloride (10% w/v) and sodium hydroxide (1 M) solutions were added to the test tubes. Finally, distilled water was used to adjust the volume (up to 10 mL). The absorbance was measured at 415 nm using a UV-visible spectrophotometer (Model UVD-3000, Labomed, Inc., Los Angeles, CA, USA). The calibration curve for quercetin was generated by plotting the absorbance against different concentrations of the extract and its fractions. The results were expressed as micrograms of quercetin equivalents (QE)/mg of dry extract.

LC-MS/MS-based Metabolomic Profiling
The LC-MS/MS analysis of the extract and various fractions was performed according to the procedure mentioned in Bashir et al. (2021) with modifications where required using negative ion mode [21]. Briefly, samples of methanolic extract of G. velutinum and fractions were prepared by dissolving in HPLC grade methanol at a concentration of 1 mg/mL. The samples were vortexed, sonicated, and then filtered through a 0.45 µm membrane filter and transferred to Orbitrap HPLC vials. The Dionex Ultimate 3000 UHPLC system coupled with Velos Pro Orbitrap Mass spectrometer was employed. A reverse-phase (C-18) analytical column (50 mm, 2.1 mm, 1.6 µm) was used as the stationary phase while acetonitrile and water both acidified with 0.1% formic acid was employed as the mobile phase in the gradient elution HPLC program. An amount of 10 µL of each sample was injected into the column and a flow rate of 0.5 mL/min was maintained. The LC-MS/MS analysis was carried out using the electrospray ionization (ESI) technique in negative ion mode. The Global Natural Product Social (GNPS) molecular networking platform (https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp, accessed on 30 October 2022) and its various tools were used for the dereplication and tentative identification of the various phytochemical constituents of the crude extract and its fractions [22].

Evaluation of the Anticancer Activity
The MTT assay was performed to evaluate the anticancer properties of the extract and its fractions [9,42].

Cell Culture
For cancer cells, cell lines (PC-3, and MCF-7) were purchased from American Type Culture Collection (ATCC) and grown in a Dulbecco's Modified Eagles Medium (DMEM) with the addition of 10% fetal bovine serum (FBS), L-glutamine, and antibiotics. The cells were kept at 37 • C in a humified environment of 5% CO 2 and 95% oxygen. Cancer cells were cultured in culture flasks at specific concentrations, such as 1 × 10 5 in 10 mL of complete culture media for a 25 cm 2 flask. From these culture flasks, cells were used for further analysis.
For normal cells, macrophages were isolated from the peritoneum of mice. The cells were washed and seeded in cell culture flasks of 25 cm 2 supplemented with Dulbecco's Modified Eagle Medium (DMEM) media with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotics. Then, culture flask cells were seeded in a 96-well plate and were treated with the crude extract and fractions of G. velutinum at a concentration of 600 µg/mL [43].

MTT Assay
For the MTT assay, the cultured cells were trypsinized and plated individually in 96-well plates (Costar ® , Corning Inc., Corning, NY, USA) at 10,000-per-well density. After 24 h, cells were incubated with various concentrations (75-600 µg/mL) of the crude extract and its fractions for 24 h. Similarly, DMSO, a control (<1%), was mixed with media and added to the control wells. Then, the media was aspirated, and fresh media (100 µL) containing the MTT reagent (BioShop Inc., Burlington, ON, Canada) was added to each well. Plates were incubated for an additional 4 h. For the solubilization of formazan crystals, 100 µL of dimethyl sulfoxide (DMSO) was added to each well. The plates were scanned at 492 nm using a plate reader (Model VEGA 500, Easy Access International Co., Ltd., Shanghai, China). Experiments were conducted in triplicate and percent cell viability was calculated by the following formula: % Viability = Absorbance of sample Absorbance of negative control × 100

Statistical Analysis
The results were statistically analyzed through a two-tailed Student's t-test by applying GraphPad Prism (version 5.0). The data were presented as MEAN ± standard deviation with a confidence interval of 95%.

Conclusions
In conclusion, G. velutinum extract and its fractions have a remarkable number of polyphenols. Moreover, the present work has established the detailed phytochemical profile of G. velutinum, which indicated 46 compounds in the crude extract and its fractions comprising mostly phenolics, flavonoids, fatty acids, terpenoids, coumarins, and sugars. In addition, G. velutinum extract and its fractions presented promising cytotoxic effects against prostate and breast cancer cells. Furthermore, the traditional use of this plant as an anticancer treatment was confirmed and strengthened by these results. This study suggested that G. velutinum has anticancer potential and may be used for the isolation and development of relatively safer anticancer drugs.