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Article

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

by
Syed Luqman Shah
1,
Kashif Bashir
2,
Hafiz Majid Rasheed
1,
Jamil Ur Rahman
1,
Muhammad Ikram
1,
Abdul Jabbar Shah
1,
Kamlah Ali Majrashi
3,
Sulaiman Mohammed Alnasser
4,
Farid Menaa
5,* and
Taous Khan
1,*
1
Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan
2
Department of Pharmaceutical Sciences, Pak-Austria Fachhochschule, Institute of Applied Sciences and Technology, Mang, Khanpur Road, Haripur 22650, Pakistan
3
Biological Sciences Department, College of Science and Arts, King Abdulaziz University, Rabigh 21911, Saudi Arabia
4
Department of Pharmacology and Toxicology, Unaizah College of Pharmacy, Qassim University, Buraydah 52571, Saudi Arabia
5
Department of Oncology and Nanomedicine, California Innovations Corporation, San Diego, CA 92037, USA
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(24), 9012; https://doi.org/10.3390/molecules27249012
Submission received: 5 November 2022 / Revised: 14 December 2022 / Accepted: 14 December 2022 / Published: 17 December 2022
(This article belongs to the Special Issue Analysis of Phytochemical Components)
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Abstract

:
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.

1. 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 are constantly increasing day by day, and it is expected that the incidence will increase in the future. Based on incidence rates, female breast cancer is the most commonly diagnosed cancer followed by lung, colorectal, prostate, and stomach cancer. Based on mortality, lung cancer is at the top, followed by colorectal, liver, stomach, and breast cancer. The global cancer burden is expected to be about 28.4 million cases in 2040 [4].
Prostate cancer is considered one of the most frequently occurring cancers in the male population [5,6]. The accumulation of somatic mutations in the prostate epithelial cell genome during a patient’s lifetime is thought to be a significant link between prostate cancer and the disease. Genes that control cell growth, cell proliferation, and cell death are the most common targets for mutations [5]. Metastasis to other organs results in more complications and death. Resistance of the tumor cells is also a major problem in the treatment of prostate cancer while treating it with cytotoxic agents. Docetaxel was introduced and approved by the FDA in 2004 for the treatment of prostate cancer, but later on, resistance was developed against docetaxel. Other new chemical entities were also approved by the FDA, but questions were raised regarding their safety, efficacy, and cost. During the past decade, treatment strategies for patients with advanced prostate cancer relating to various stages after treatment with curative intent, as well as castration-resistant prostate cancer, have extensively evolved with the introduction and approval of several new agents including sipuleucel-T, radium-223, abiraterone, enzalutamide, and cabazitaxel [7].
Breast cancer is the most commonly diagnosed cancer, worldwide. The prevalence of breast cancer has increased over the past several decades [4]. There are 2.3 million new cases of breast cancer in both sexes combined today [4]. Breast cancer typically begins as ductal hyper-proliferation and progresses to benign tumors or even metastatic carcinomas when they are repeatedly stimulated by numerous carcinogenic stimuli [8]. Both oncogene and anti-oncogene mutations and abnormal amplification play critical roles in the development and progression of breast cancer [8]. The combination of pertuzumab, trastuzumab, and docetaxel has been approved for the treatment of breast cancer [8]. All of these agents have shown a significant improvement in overall survival, but these are associated with severe side effects such as non-selectivity, resistance, low bioavailability, and efficacy [9]. Such issues reduce the motivation of researchers focusing to treat cancer patients [7].
Traditionally, plants have been used in all communities for healing different diseases, and experimental results highlight the potential of plants as sources of anticancer compounds [10,11]. In several cases, phytochemical constituents have been used directly or chemically modified in the development of anticancer medications. More than 60% of drugs used for the treatment of cancer are derived from natural resources, according to the Food and Drug Administration (FDA) [7].
Glochidion velutinum Wight belongs to the family Euphorbiaceae [12]. It is mostly found in Pakistan, Burma, Nepal, China, Vietnam, and India. It is commonly known as Mattachar, Kaalikaath, Velvety Melon Feather foil, and Downy Melon Feather foil [13,14]. It has traditionally been used to treat cancer, diabetes, inflammation, wounds, coughs, and diarrhea. Flavonoids, glycosides, saponins, and alkaloids are known to be the constituents of G. velutinum. The reported pharmacological activities of G. velutinum include antibacterial, antioxidant, cytotoxic, antidiabetic, anti-inflammatory, and antiurolithiatic activity. G. velutinum has been traditionally used in cancer treatment [15]; however, according to the literature, its extract, fractions, and constituents have not been pharmacologically evaluated for anticancer potential against various cell lines including prostate cancer. Furthermore, it has not been investigated for detailed metabolite profiling, isolation, and characterization of its various constituents. Therefore, the current study focused on the phytochemical profiling of G. velutinum extract and its fractions, and their evaluation against prostate (PC-3) and breast cancer (MCF-7) cell lines.

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.

2.1.2. 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.

2.1.3. 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 fraction-level 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 solvent–solvent 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.
HPLC-MSn is routinely employed in the separation and tentative identification of complex mixtures of natural product origin [18,19]. Therefore, this technique was used to carry out a comprehensive and detailed phytochemical analysis of the crude extract and fractions of G. velutinum leaves. A total of 46 compounds were tentatively identified in the crude extract and subsequent polarity-based fractions of G. velutinum leaves. Based on the phytochemical analysis results, the crude extract was found to contain flavonoid glycosides, coumarins, fatty acids, and sugars. The chloroform fraction was found to be most bioactive against prostate cancer and breast cancer. It was found to contain flavonoid glycosides, flavans, pyranocoumarins, hydrolysable tannins, monoterpenoids, carbohydrate conjugates, and fatty acids, as shown in Figure 1 and Figure 2. Ethyl acetate, being another bioactive fraction against both types of cancer, was found to contain benzoic acid derivatives, coumarin glycosides, flavans, flavones, flavonoid glycosides, diterpenoids, terpene glycosides, pyranocoumarins, O-methylated flavonoids, and linoleic acid derivatives, as shown in Figure 3. The various constituents observed in the crude extract and fractions from G. velutinum leaves are listed in Table 3 along with their m/z values in negative ion mode, MS/MS fragmentation patterns, and retention time.
The reported chemical constituents that were isolated from the plant include D-mannitol, β-amyrin, stigmasterol, glochidonol, glochidone, glochidiol, betulin, β-daucosterol, glochidioside, glochidioside N, glochidioside Q, and epimachaerinic acid [11,20]. The major phenolic compounds identified in the G. velutinum extract and its fractions include ellagic acid (9), gallic acid (32), 2-hydroxycinnamic acid (HCA) (34), epigallocatechin gallate (EGCG) (26), gallocatechol (3), trans-piceid (24), and 1,3,6-tri-O-galloylglucose (37). The flavonoid glycosides include isoquercitrin (28), rutin (10), rhoifolin (41), kaempferol 3-O-glucoside (39), homoorientin (38), vitexin-2-O-rhamnoside (27), vicenin-2 (25), isovitexin (5) and myricetin 3-galactopyranoside (6), hyperoside (7), and luteolin 4’-O-glucoside, (42) whereas isokaempferide (35), 5,6,2′-trimethoxy flavone (15), catechin (33), and luteolin (40) were identified as simple flavonoids. Trehalose (2), arabinose (44), and maltotriose (29) were observed as sugars. Quinic acid (1) was identified as a cyclic polyol. Digalactosylmonoglycerol (DGMG) (19) and monogalactosylmonoglycerol (MGMG) (31) were identified as glycosyl glycerol. 9-Hydroxy-10E,12Z,15Z-octadecatrienoic acid (20), FA 18:4+2O (18), FA 18:1+3O (12), stearidonate (18:4(n−3)) (13), methyl (2E,4E,8E)-7,13-dihydroxy-4,8,12-trimethyltetradeca-2,4,8-trienoate (16), and (10E,15E)-9,12,13-trihydroxyoctadeca-10,15-dienoic acid (11) were identified as fatty acids (Table 3 and Table 4). Roseoside (36) was identified as a fatty acyl glycoside.
Some typical fragmentation patterns were distinctive for the tentative identification of flavonoid glycoside showing galactose (162), rhamnose (146), and glucose (162) for O-glycosides [21]. Moieties such as 60, 90, and 120 were characteristic of C-glycosides [22]. Gallic acid and HCA fragmented with the loss of CO2 moiety [23].

2.1.4. 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 (IC50 = 325 µg/mL) on PC-3 cells, whereas ethyl acetate and n-butanol fractions showed a moderate effect with respective IC50 values of 196 µg/mL and 123 µg/mL, respectively. The stronger effects were observed for the chloroform fraction (IC50 = 27 µg/mL), followed by the aqueous fraction (IC50 = 36 µg/mL) and G. velutinum extract (IC50 = 89 µg/mL).
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 IC50 values of 522 µg/mL and 523 µg/mL, respectively. The stronger effects were observed for the chloroform fraction (IC50 = 222 µg/mL), followed by the ethyl acetate fraction (IC50 = 226 µg/mL) and G. velutinum extract (IC50 = 431 µg/mL). The IC50 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.

3. Materials and Methods

3.1. 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.

3.2. 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%).

3.3. 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).

3.4. Phytochemical Evaluation

3.4.1. 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 Na2CO3 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.

3.4.2. 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.

3.4.3. 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].

3.5. Evaluation of the Anticancer Activity

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

3.5.1. 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% CO2 and 95% oxygen. Cancer cells were cultured in culture flasks at specific concentrations, such as 1 × 105 in 10 mL of complete culture media for a 25 cm2 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 cm2 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].

3.5.2. 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

3.6. 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%.

4. 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.

Author Contributions

Conceptualization, S.L.S. and T.K.; methodology, S.L.S., K.B., H.M.R., M.I. and F.M.; software, S.L.S., K.B., H.M.R., M.I. and F.M.; validation, A.J.S., T.K., M.I. and F.M.; formal analysis, S.L.S., K.B., H.M.R., M.I., J.U.R. and F.M.; investigation, S.L.S., K.B. and H.M.R.; resources and data curation, T.K., K.A.M., S.M.A. and F.M.; writing—First draft, S.L.S., K.B., H.M.R., A.J.S., K.A.M., S.M.A., F.M. and T.K.; writing—revisions and editing, S.L.S., K.B., H.M.R., A.J.S., K.A.M., S.M.A., F.M. and T.K.; visualization, K.A.M., S.M.A., F.M. and T.K.; supervision, T.K., M.I. and F.M.; project administration, T.K., M.I. and F.M.; funding acquisition, S.M.A., K.A.M. and F.M. Submission: M.I. and F.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

Upon request to the corresponding authors.

Acknowledgments

All the authors are thankful to Higher Education Commission (HEC), Pakistan, for funding this project under Scholarship Phase-II batch-V, Indigenous Ph.D. Fellowships Program for 5000 Scholars. We are also thankful to Phillip Crews, Distinguished Research Professor of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA for his help in LC-MS/MS analysis.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not Applicable.

References

  1. Otto, T.; Sicinski, P. Cell cycle proteins as promising targets in cancer therapy. Nat. Rev. Cancer 2017, 17, 93–115. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Sun, G.; Rong, D.; Li, Z.; Sun, G.; Wu, F.; Li, X.; Cao, H.; Cheng, Y.; Tang, W.; Sun, Y. Role of small molecule targeted compounds in cancer: Progress, opportunities, and challenges. Front. Cell Dev. Biol. 2021, 9, 694363. [Google Scholar] [CrossRef] [PubMed]
  3. Debela, D.T.; Muzazu, S.G.Y.; Heraro, K.D.; Ndalama, M.T.; Mesele, B.W.; Haile, D.C.; Kitui, S.K.; Manyazewal, T. New approaches and procedures for cancer treatment: Current perspectives. SAGE Open Med. 2021, 9, 20503121211034366. [Google Scholar] [CrossRef] [PubMed]
  4. Sung, H.; Ferlay, J.; Seigel, R.; Lavarsanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2021, 71, 209–249. [Google Scholar] [CrossRef]
  5. Rebello, R.J.; Oing, C.; Knudsen, K.E.; Loeb, S.; Johnson, D.C.; Reiter, R.E. Prostate cancer. Nat. Rev. Dis. Primers 2021, 7, 2021. [Google Scholar] [CrossRef]
  6. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin. 2019, 69, 7–34. [Google Scholar] [CrossRef] [Green Version]
  7. Komura, K.; Sweeney, C.J.; Inamoto, T.; Ibuki, N.; Azuma, H.; Kantoff, P.W. Current treatment strategies for advanced prostate cancer. Int. J. Urol. 2018, 25, 220–231. [Google Scholar] [CrossRef] [Green Version]
  8. Arnold, M.; Morgan, E.; Rumgay, H.; Mafra, A.; Singh, D.; Laversanne, M.; Vignat, J.; Gralow, J.R.; Cardoso, F.; Siesling, S.; et al. Current and future burden of breast cancer: Global statistics for 2020 and 2040. Breast 2022, 66, 15–23. [Google Scholar] [CrossRef]
  9. Rasheed, H.M.; Wahid, F.; Ikram, M.; Qaisar, M.; Shah, A.J.; Khan, T. Chemical profiling and anti-breast cancer potential of hexane fraction of Sphaeranthus indicus flowers. Trop. J. Pharm. Res. 2021, 20, 1931–1939. [Google Scholar] [CrossRef]
  10. Oliveira, P.G.; Otero, P.; Pereira, A.G.; Chamorro, F.; Carpena, M.; Echave, J.; Corral, M.F.; Gandara, J.S.; Prieto, M.A. Status and challenges of plant-anticancer compounds in cancer treatment. Pharmaceuticals 2021, 14, 157. [Google Scholar] [CrossRef]
  11. Babaei, G.; Aliarab, A.; Abroon, S.; Rasmi, Y.; Aziz, S.G.G. Application of sesquiterpene lactone: A new promising way for cancer therapy based on anticancer activity. Biomed. Pharmacother. 2018, 106, 239–246. [Google Scholar] [CrossRef] [PubMed]
  12. Benny, S.; Krishnakumar, K.; Sandhya, A.S. Glochidion velutinum: An overview. J. Bio Innov. 2019, 8, 419–430. [Google Scholar]
  13. Deb, J.; Saha, S.; Deb, N.K. Review on Glochidion velutinum Wight, (Euphorbiaceae): A medicinal plant. Int. J. Herb. Med. 2019, 7, 1–3. [Google Scholar]
  14. Chaitnya, R.; Sandhya, S.; Banji, D. HRBC membrane stabilizing property of root, stem and leaf of Glochidion velutinum. Int. J. Res. Pharm. Biomed. Sci. 2011, 2, 256–259. [Google Scholar]
  15. Sandhya, S.; Chaitanya, R.; Vinod, K.R.; Rao, K.N.V.; Banji, D.; Sudhakar, K.; Swetha, R. An updated review on the genus Glochidion plant. Arch. Appl. Sci. Res. 2010, 2, 309–322. [Google Scholar]
  16. Huang, W.Y.; Cai, Y.Z.; Zhang, Y. Natural phenolic compounds from medicinal herbs and dietary plants: Potential use for cancer prevention. Nutr. Cancer 2010, 62, 1–20. [Google Scholar] [CrossRef]
  17. Fernandez, J.; Silvan, B.; Entrialgo-Cadierno, R.; Villar, C.J.; Capasso, R.; Uranga, J.A.; Lombo, F.; Abalo, R. Antiproliferative and palliative activity of flavonoids in colorectal cancer. Biomed. Pharmacother. 2021, 143, 112241. [Google Scholar] [CrossRef]
  18. Allard, P.-M.; Péresse, T.; Bisson, J.; Gindro, K.; Marcourt, L.; Pham, V.C.; Roussi, F.; Litaudon, M.; Wolfender, J.-L. Integration of molecular networking and in-silico ms/ms fragmentation for natural products dereplication. Anal. Chem. 2016, 88, 3317–3323. [Google Scholar] [CrossRef]
  19. Olivon, F.; Grelier, G.; Roussi, F.; Litaudon, M.; Touboul, D. MZmine 2 data-preprocessing to enhance molecular networking reliability. Anal. Chem. 2017, 89, 7836–7840. [Google Scholar] [CrossRef]
  20. Srivastava, R.; Kulshreshtha, D.K. Triterpenoids from Glochidion heyneanum. Phytochemistry 1988, 27, 3575–3578. [Google Scholar] [CrossRef]
  21. Saeed, A.; Bashir, K.; Shah, A.J.; Qayyum, R.; Khan, T. Antihypertensive activity in high salt-induced hypertensive rats and lc-ms/ms-based phytochemical profiling of Melia azedarach L. (Meliaceae) leaves. BioMed Res. Int. 2022, 2022, 2791874. [Google Scholar] [CrossRef] [PubMed]
  22. Bashir, K.; Naz, S.; Rasheed, H.M.; Farooq, U.; Shah, A.J.; McCauley, E.P.; Crews, P.; Khan, T. Tandem high resolution mass spectrometry based phytochemical composition of Sauromatum guttatum tubers and its enzyme inhibitory potential with molecular docking. J. Chromatogr. A 2022, 1672, 463055. [Google Scholar] [CrossRef]
  23. Bashir, K.; Naz, S.; Farooq, U.; Wahid, F.; Shah, A.J.; McCauley, E.P.; Crews, P.; Khan, T. Assessing the ethnobotanical potential of Carissa opaca berries by merging outcomes from metabolomics profiling, enzyme assays, and in silico docking studies. Food Chem. 2021, 363, 130259. [Google Scholar] [CrossRef] [PubMed]
  24. Atanasov, A.G.; Zotchev, S.B.; Dirsch, V.M.; Supuran, C.T. Natural products in drug discovery: Advances and opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216. [Google Scholar] [CrossRef] [PubMed]
  25. Niedzwiecki, A.; Roomi, M.W.; Kalinovsky, T.; Rath, M. Anticancer efficacy of polyphenols and their combinations. Nutrients 2016, 8, 552. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Oyenihi, A.B.; Smith, C. Are polyphenol antioxidants at the root of medicinal plant anti-cancer success? J. Ethnopharmacol. 2019, 229, 54–72. [Google Scholar] [CrossRef]
  27. Roleira, F.M.F.; Tavares-da-Silva, E.J.; Varela, C.L.; Costa, S.C.; Silva, T.; Garrido, J.; Borges, F. Plant derived and dietary phenolic antioxidants: Anticancer properties. Food Chem. 2015, 183, 235–258. [Google Scholar] [CrossRef]
  28. Johnson, J.J.; Bailey, H.H.; Mukhtar, H. Green tea polyphenols for prostate cancer chemoprevention: A translational perspective. Phytomedicine 2010, 17, 3–13. [Google Scholar] [CrossRef] [Green Version]
  29. Albrecht, D.S.; Clubbs, E.A.; Ferruzzi, M.; Bomser, J.A. Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation. Chem.-Biol. Interact. 2008, 171, 89–95. [Google Scholar] [CrossRef]
  30. Salehi, B.; Fokou, P.V.T.; Yamthe, L.R.T.; Tali, B.T.; Adetunji, C.O.; Rahavian, A.; Mudau, F.N.; Martorell, M.; Setzer, W.N.; Rodrigues, R.M.; et al. Phytochemicals in prostate cancer: From bioactive molecules to upcoming therapeutic agents. Nutrients 2019, 11, 1483. [Google Scholar] [CrossRef] [Green Version]
  31. Samimi, S.; Ardestani, M.S.; Dorkoosh, F.A. Preparation of carbon quantum dots-quinic acid for drug delivery of gemcitabine to breast cancer cells. J. Drug Deliv. Sci. Technol. 2021, 61, 102287. [Google Scholar] [CrossRef]
  32. Rezaei-Seresht, H.; Cheshomi, H.; Falanji, F.; Movahedi-Motlagh, F.; Hashemian, M.; Mireskandari, E. Cytotoxic activity of caffeic acid and gallic acid against MCF-7 human breast cancer cells: An in silico and in vitro study. Avicenna J. Phytomed. 2019, 9, 574–586. [Google Scholar] [PubMed]
  33. Ahmed, S.; Khan, H.; Fratantonio, D.; Hasan, M.M.; Sharifi, S.; Fathi, N.; Ullah, H.; Rastrelli, L. Apoptosis induced by luteolin in breast cancer: Mechanistic and therapeutic perspectives. Phytomedicine 2019, 59, 152883. [Google Scholar] [CrossRef] [PubMed]
  34. Castro e Silva, J.H.; Ferreira, R.S.; Pereira, E.P.; Braga-de-Souza, S.; Alves de Almeida, M.M.; Creusa dos Santos, C.; Butt, A.M.; Caiazzo, E.; Capasso, R.; Amaral da Silva, V.D.; et al. Amburana cearensis: Pharmacological and neuroprotective effects of its compounds. Molecules 2020, 25, 3394. [Google Scholar] [CrossRef]
  35. Xiong, L.; Lu, H.; Hu, Y.; Wang, W.; Liu, R.; Wan, X.; Fu, J. In vitro anti-motile effects of rhoifolin, a flavonoid extracted from Callicarpa nudiflora on breast cancer cells via downregulating podocalyxin-ezrin interaction during epithelial mesenchymal transition. Phytomedicine 2021, 93, 153486. [Google Scholar] [CrossRef]
  36. Rahman, S.A.S.N.; Abdul Wahab, N.; Abd Malek, S.N. In vitro morphological assessment of apoptosis induced by antiproliferative constituents from the rhizomes of Curcuma zedoaria. Evid. Based Complement. Altern. Med. 2013, 2013, 257108. [Google Scholar]
  37. Said, A.; Naeem, N.; Wahid, F.; Javed, A.; Rasheed, H.M.; Khan, T.; Sajjad, W.; Shah, K.; Siraj, S. Mechanisms underlying the wound healing and tissue regeneration properties of Chenopodium album. 3 Biotech 2020, 10, 452. [Google Scholar] [CrossRef]
  38. Khan, T.; Ali, S.; Qayyum, R.; Hussain, I.; Wahid, F.; Shah, A.J. Intestinal and vascular smooth muscle relaxant effect of Viscum album explains its medicinal use in hyperactive gut disorders and hypertension. BMC Complement. Altern. Med. 2016, 16, 251. [Google Scholar] [CrossRef] [Green Version]
  39. Khan, S.; Khan, T.; Shah, A.J. Total phenolic and flavonoid contents and antihypertensive effect of the crude extract and fractions of Calamintha vulgaris. Phytomedicine 2018, 47, 174–183. [Google Scholar] [CrossRef]
  40. John, B.; Sulaiman, C.T.; George, S.; Reddy, V.R.K. Total phenolics and flavonoids in selected medicinal plants from Kerala. Int. J. Pharm. Pharm. Sci. 2014, 6, 406–408. [Google Scholar]
  41. Vyas, S.; Kachhwaha, S.; Kothari, S.L. Comparative analysis of phenolic contents and total antioxidant capacity of Moringa oleifera Lam. Pharmacogn. J. 2015, 7, 44–51. [Google Scholar]
  42. Kamiloglu, S.; Sari, G.; Ozdal, T.; Capanoglu, E. Guidelines for cell viability assays. Food Front. 2020, 2020, 332–349. [Google Scholar]
  43. Khan, Z.U.; Khan, T.; Mannan, A.; Ali, A.; Ni, J. In Vitro and Ex Vivo Evaluation of Mangifera indica L. Extract-Loaded Green Nanoparticles in Topical Emulsion against Oxidative Stress and Aging. Biomedicines 2022, 10, 2266. [Google Scholar] [CrossRef] [PubMed]
Molecules 27 09012 g001 550
Figure 1. Phytochemical classes of bioactive chloroform fraction from G. velutinum leaves using LC-MS/MS-based molecular networking analysis.
Figure 1. Phytochemical classes of bioactive chloroform fraction from G. velutinum leaves using LC-MS/MS-based molecular networking analysis.
Molecules 27 09012 g001
Molecules 27 09012 g002 550
Figure 2. Chemical constituents observed in the chloroform fraction of G. velutinum leaves using LC-MS/MS and GNPS classical molecular networking.
Figure 2. Chemical constituents observed in the chloroform fraction of G. velutinum leaves using LC-MS/MS and GNPS classical molecular networking.
Molecules 27 09012 g002
Molecules 27 09012 g003 550
Figure 3. Classes of chemical constituents were identified using GNPS molecular networking and MolnetEnhancer technique to identify the chemical space present within ethyl acetate fraction of G. velutinum leaves.
Figure 3. Classes of chemical constituents were identified using GNPS molecular networking and MolnetEnhancer technique to identify the chemical space present within ethyl acetate fraction of G. velutinum leaves.
Molecules 27 09012 g003
Molecules 27 09012 g004 550
Figure 4. Presentation of growth inhibitory effects of G. velutinum extract and its fractions on PC-3 cells. The results are expressed as a percentage of the negative control. Data are means ± SD of three independent experiments. *** represent p ≤ 0.001.
Figure 4. Presentation of growth inhibitory effects of G. velutinum extract and its fractions on PC-3 cells. The results are expressed as a percentage of the negative control. Data are means ± SD of three independent experiments. *** represent p ≤ 0.001.
Molecules 27 09012 g004
Molecules 27 09012 g005 550
Figure 5. Presentation of growth inhibitory effects of G. velutinum extract and its fractions on MCF-7 cells. The results are expressed as a percentage of the negative control. Data are means ± SD of three independent experiments. * and *** represent p ≤ 0.05 and p ≤ 0.001, respectively.
Figure 5. Presentation of growth inhibitory effects of G. velutinum extract and its fractions on MCF-7 cells. The results are expressed as a percentage of the negative control. Data are means ± SD of three independent experiments. * and *** represent p ≤ 0.05 and p ≤ 0.001, respectively.
Molecules 27 09012 g005
Molecules 27 09012 g006 550
Figure 6. Presentation of growth inhibitory effects of G. velutinum extract and its fractions (at maximum tested concentration) against normal cells.
Figure 6. Presentation of growth inhibitory effects of G. velutinum extract and its fractions (at maximum tested concentration) against normal cells.
Molecules 27 09012 g006
Table
Table 1. Total phenolic contents of crude extract and fractions of G. velutinum.
Table 1. Total phenolic contents of crude extract and fractions of G. velutinum.
Entry No.ExtractTotal Phenol
(µg GAE/mg)
1Crude588 ± 3.0
2n-Hexane54 ± 1.2
3Chloroform266 ± 1.0
4Ethyl acetate859 ± 1.0
5n-Butanol44 ± 2.1
6Aqueous77 ± 2.3
Table
Table 2. Total flavonoid contents of crude extract and fractions of G. velutinum.
Table 2. Total flavonoid contents of crude extract and fractions of G. velutinum.
Entry No.ExtractTotal Flavonoid
(µg QE/mg)
1Crude118 ± 2.0
2n-Hexane20 ± 1.1
3Chloroform79 ± 1.3
4Ethyl acetate315 ± 1.0
5n-Butanol24 ± 1.5
6Aqueous29 ± 1.4
Table
Table 3. The phytochemical profile of G. velutinum leaves was established with the help of LC-MS/MS-based molecular networking.
Table 3. The phytochemical profile of G. velutinum leaves was established with the help of LC-MS/MS-based molecular networking.
Crude Extract of G. velutinum
Entry #Compound NameRTm/zMS2 Fragmentation PatternMolecular FormulaExact Mass
1Quinic acid0.43191.0568111.1887 (100), 173.3876, 85.1423, 127.066, 146.063, 93.184C7H12O6192.0633
2Trehalose0.55341.1099179.3955 (100), 161.3627, 143.3026, 119.2360, 113.2109C12H22O11342.1162
3Gallocatechol1.14305.0642221.1006 (100), 179.1527, 273.0852, 261.1541C15H14O7306.0739
4Citric acid1.71191.0566147.3730 (100), 111.1887, 85.1423C6H8O7192.0270
5Isovitexin2.96431.0991311.1170 (100), 341.1700, 269.1172, 367.1852C21H20O10432.1056
6Myricetin, 3-Galactopyranoside3.71479.1910316.6702 (100), 193.4596, 271.5861C21H20O13480.0903
7Hyperoside 4.02463.5582301.5882 (100)C21H20O12464.0954
8CGHTM, ME4.05447.0950285.5274 (100), 327.6831, 313.7282C17H26O11406.1475
9Ellagic acid6.07300.9967257.0632 (100), 229.0459, 185.1000, 271.0589, 151.1032C14H6O8302.0062
10Rutin6.38609.0831463.1980 (100), 301.1688C27H30O16610.1533
119,12,13,TriHODE6.76327.2881229.2324 (100), 291.2702, 211.2155, 171.2333C18H32O5 328.2249
12FA 18:1+3O6.84329.1445229.2349 (100), 171.1871, 211.1918, 293.2625C18H34O5 330.2406
13Stearidonate (18:4n3)7.35277.206233.6909 (100), 205.7536, 179.5107C18H27O2-275.2011
148,8-DDPCM8.19339.8762183.4248 (100), 299.6149, 197.4348C19H18O6342.1103
155,6,2′-Trimethoxyflavone8.77311.166183.1256 (100), 247.3156, 198.1505C18H16O5312.0997
16MDTT9.06311.1662183.373 (100), 149.344, 271.601, 247.682C18H30O4310.2144
n-Hexane fraction
17Azelaic acid6.24187.2562125.1266 (100), 143.2794, 97.4454, 159.1357C9H16O4188.1048
12FA 18:1+3O6.84329.1445229.2349 (100), 171.1871, 211.1918, 293.2625C18H34O5 330.2406
18FA 18:4+2O7.15307.180097.0354 (100), 267.0800C18H28O4308.1987
19DGMG 18:37.41721.3603675.5287 (100), 397.2563, 415.2704C33H56O14676.3670
209-HOA7.73275.2000231.2642 (100), 177.2366, 203.3373, 255.2677, 239.4132C18H30O3294.2194
2113-HODE7.92295.3241195.2709 (100), 171.2408, 179.2919, 251.3765, 181.2437C18H32O3296.2351
22Docosanol8.97325.2862183.1292 (100)C22H46O 326.3548
Chloroform fraction
23p-Mentha-1-ene-6-one3.59216.5700171.0872 (100), 144.1471, 125.9763,
168.9540		C10H16O 152.1201
9Ellagic acid6.07300.9967257.0632 (100), 229.0459, 185.1000
271.0589, 151.1032		C14H6O8302.0062
24trans-piceid5.70433.2105387.2457 (100), 225.1868, 193.112, 313.1065C20H22O8 390.1314
25Vicenin-25.70593.2812473.1857 (100), 353.1384, 503.181, 383.1578, 413.2072C27H30O15594.1584
26EGCG5.85457.0732331.1129 (100), 305.1384, 169.0689, 413.1637, 193.0952C22H18O11 458.0849
27Vitexin-2-O-rhamnoside6.01577.1510311.1990 (100), 413.2145, 341.1857, 293.2155, 395.136, 283.16C27H30O14578.1635
28Isoquercetin6.01463.3657301.1005 (100), 316.0813, 343.0984C21H20O12 464.0954
10Rutin 6.38609.0831463.1980 (100), 301.1688C27H30O16610.1533
119,12,13,TriHODE 6.76327.2881229.2324 (100), 291.2702, 211.2155, 171.2333C18H32O5 328.2249
29Maltotriose7.50449.7303502.4152 (100), 503.2143, 418.2113, 491.4700, 523.2600, 371.3013C18H32O16504.1690
309,10-DiHOME7.52313.2362201.1504 (100), 183.1968, 293.0565, 277.2592, 171.1771, 195.1924C18H34O4 314.2457
155,6,2′-Trimethoxyflavone8.77311.166183.1256 (100), 247.3156, 198.1505C18H16O5312.0997
31MGMG 18:37.72559.308331.4297 (100), 305.10051, 169.0092C27H46O9514.3141
19DGMG 18:37.41721.3603675.5287 (100), 397.2563, 415.2704C33H56O14676.3670
148,8-DDPCM8.19339.8762183.4248 (100), 299.6149, 197.4348C19H18O6342.1103
Ethyl acetate fraction
32Gallic acid0.42169.0149125.2083 (100), 141.1750, 81.4432, 69.1584 C7H6O5170.0215
3Gallocatechol1.14305.0642221.1006 (100), 179.1527, 273.0852, 261.1541C15H14O7306.0739
33Catechin2.20289.1863245.1277 (100), 205.1055, 179.1031C15H14O6 290.0790
5Isovitexin2.96431.0991311.1170 (100), 341.1700, 269.1172, 367.1852C21H20O10432.1056
342-HCA3.50163.0392119.0751(100), 135.1361C9H8O3 164.0473
35Isokaempferide 4.67301.5982257.5770 (100), 229.4959, 272.5718 C16H12O6300.0633
36Roseoside 5.38387.2834207.0929 (100), 163.1177, 225.0903C19H30O8 386.1940
371,3,6-tri-O-galloylglucose5.80635.0832465.1552 (100), 483.1615, 313.1343, 423.1522, 591.1775, 221.1012C27H24O18 636.0962
26EGCG5.85457.0732331.1129 (100), 305.1384, 169.0689, 413.1637, 193.0952C22H18O11 458.0849
38Homoorientin 5.89447.0904327.0962 (100), 357.0830, 285.0861, 313.0800 C21H20O11 448.1005
28Isoquercetin6.01463.3657301.1005 (100), 316.0813, 343.0984C21H20O12 464.0954
39Kaempferol 3-O-glucoside6.13447.0922285.0800 (100), 301.0750, 307.1052
327.1223		C21H20O11 448.1005
40Luteolin6.59285.0380241.1324 (100), 175.0822, 199.1310, 217.0913, 151.0380C15H10O6 286.0477
41Rhoifolin6.63577.1302269.1225 (100), 431.1875, 413.1603, 307.1234, 327.1476C27H30O14 578.1635
119,12,13-TriHODE 6.76327.2881229.2324 (100), 291.2702, 211.2155, 171.2333C18H32O5 328.2249
12FA 18:1+3O6.84329.1445229.2349 (100), 171.1871, 211.1918, 293.2625C18H34O5 330.2406
22Docosanol8.97325.2862183.1292 (100)C22H46O 326.3548
148,8-DDPCM8.19339.8762183.4248 (100), 299.6149, 197.4348C19H18O6342.1103
n-Butanol fraction
1Quinic acid0.43191.057111.1887 (100), 85.1423, 127.2440, 146.063, 93.184C7H12O6192.0633
2Trehalose0.50341.1099179.3955 (100), 161.3627, 143.3026, 119.2360, 113.2109C12H22O11342.1162
5Isovitexin2.96431.0991311.1170 (100), 341.1700, 269.1172, 367.1852C21H20O10432.1056
7Hyperoside 4.02463.0932301.5882 (100)C21H20O12464.0954
42Luteolin 4’-O-glucoside4.16447.0980284.582 (100), 327.7294, 315.7570, 255.572C21H20O11448.1005
Aqueous fraction
2Trehalose0.55341.1099179.3955 (100), 161.3627, 143.3026, 119.2360, 113.2109C12H22O11342.1162
436,7-Dimethyl-4-hydroxycoumarin0.67188.3989131.3810 (100), 147.4211, 85.2340C11H10O3190.0629
4Citric acid1.71191.0566147.3730 (100), 111.1887, 85.1423C6H8O7 192.0270
44Arabinose3.36174.9568131.2210 (100), 147.2940, 155.4096 C5H10O5150.0528
35Isokaempferide 4.67301.5982257.5770 (100), 229.4959, 272.5718 C16H12O6300.0633
148,8-DDPCM8.19339.8762183.4248 (100), 299.6149, 197.4348C19H18O6342.1103
451-THONONE8.44311.175183.5197 (100), 149.3188C15H20O7312.1209
467-Methoxyflavonol8.49266.669097.0762 (100), 245.4699C16H12O4268.0735
Table
Table 4. List of some identified compounds abbreviated in Table 3.
Table 4. List of some identified compounds abbreviated in Table 3.
Serial No.AbbreviationCompound Name
1CGHTM, MECyclopenta(c)pyran-4-carboxylic acid, 1-(beta-D-glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-4a,7-dihydroxy-7-methyl-, methyl ester
28,8-DDPCM(8,8-dimethyl-2,10-dioxo-9H-pyrano[2,3-f]chromen-9-yl) (Z)-2-methylbut-2-enoate
3MDTTMethyl (2E,4E,8E)-7,13-dihydroxy-4,8,12-trimethyltetradeca-2,4,8-trienoate
49,12,13,TriHODE(10E,15E)-9,12,13-trihydroxyoctadeca-10,15-dienoic acid
5EGCGEpigallocatechin gallate
6FA 18:1+3O9,12,13-Trihydroxy-10-octadecenoic acid
7FA 18:4+2O(10E,12E,14E)-16-hydroxy-9-oxooctadeca-10,12,14-trienoic acid
89-HOA9-Hydroxy-10E,12Z,15Z-octadecatrienoic acid
913-HODE13-hydroxy-9,11-octadecadienoic acid
109,10-DiHOME(12Z)-9,10-Dihydroxyoctadec-12-enoic acid
112-HCA2-Hydroxycinnamic acid
121-THONONE1-[2-methyl-6-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]ethanone
Table
Table 5. IC50 values of G. velutinum extract and fractions for MCF-7 and PC-3 cell lines.
Table 5. IC50 values of G. velutinum extract and fractions for MCF-7 and PC-3 cell lines.
Entry No.Sample NameMCF-7 Cell Line IC50 (µg/mL)PC-3 Cell Line IC50 (µg/mL)
1Crude431.7889.02
2n-Hexane fraction523.63325.87
3Chloroform fraction222.2727.63
4Ethyl acetate fraction226.35196.83
5n-Butanol fraction≤50% (Not applicable)123.36
6Aqueous fraction522.4136.95
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Shah, S.L.; Bashir, K.; Rasheed, H.M.; Rahman, J.U.; Ikram, M.; Shah, A.J.; Majrashi, K.A.; Alnasser, S.M.; Menaa, F.; Khan, T. LC-MS/MS-Based Metabolomic Profiling of Constituents from Glochidion velutinum and Its Activity against Cancer Cell Lines. Molecules 2022, 27, 9012. https://doi.org/10.3390/molecules27249012

AMA Style

Shah SL, Bashir K, Rasheed HM, Rahman JU, Ikram M, Shah AJ, Majrashi KA, Alnasser SM, Menaa F, Khan T. LC-MS/MS-Based Metabolomic Profiling of Constituents from Glochidion velutinum and Its Activity against Cancer Cell Lines. Molecules. 2022; 27(24):9012. https://doi.org/10.3390/molecules27249012

Chicago/Turabian Style

Shah, Syed Luqman, Kashif Bashir, Hafiz Majid Rasheed, Jamil Ur Rahman, Muhammad Ikram, Abdul Jabbar Shah, Kamlah Ali Majrashi, Sulaiman Mohammed Alnasser, Farid Menaa, and Taous Khan. 2022. "LC-MS/MS-Based Metabolomic Profiling of Constituents from Glochidion velutinum and Its Activity against Cancer Cell Lines" Molecules 27, no. 24: 9012. https://doi.org/10.3390/molecules27249012

APA Style

Shah, S. L., Bashir, K., Rasheed, H. M., Rahman, J. U., Ikram, M., Shah, A. J., Majrashi, K. A., Alnasser, S. M., Menaa, F., & Khan, T. (2022). LC-MS/MS-Based Metabolomic Profiling of Constituents from Glochidion velutinum and Its Activity against Cancer Cell Lines. Molecules, 27(24), 9012. https://doi.org/10.3390/molecules27249012

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