GC/MS analysis, free radical scavenging, anticancer and β-glucuro-nidase inhibitory activities of Trillium govanianum rhizome

Abstract

The current study aims to investigate the phytochemical constituents, antioxi-dant, β-glucuronidase inhibitory and anticancer activities of Trillium govanianum rhizome. Phytochemical screening revealed the presence of steroidal glycosides, saponins, sterols, flavonoids and carbohydrates. GC/MS analyses of n-hexane fraction identified 12 compounds, including 70% unsaturated and 30% saturated fatty acids. Higher free radical scavenging capacity was observed in n-hexane and chloroform fraction compared with the other fractions. Based on IC₅₀ (μg/mL) values, antiproliferative activity on HeLa cells was observed for chloroform (0.8 ± 0.2), ethyl acetate (1.4 ± 0.1) and butanol (1.6 ± 0.3) fractions by comparison to anticancer drug doxorubi-cin (0.3 ± 0.0). Similarly, all fractions exhibited cytotoxicity on PC-3 cells. Moreover, the methanol extract (IC₅₀: 140.8 ± 3.8) and butanol fraction (196.2 ± 1.9) exhibited a moderate level of β-glucuronidase inhibitory activity. These findings may validate the folkloric uses of T. govanianum rhizome in cancer management, and can be a promising candidate as an anticancer agent.

Introduction

Medicinal plants are capitalized in cure of different ailments since time immemorial. This can be attributed to the presence of secondary metabolites, arising from primary metabolism by different biosynthetic path-ways. Important classes of secondary metabolites include alkaloids, glycosides, tannins, steroids, terpe-noids, saponins and phenolic compounds. These are biologically active molecules and potential activities of any medicinal plant are primarily due to these meta-bolites (Okigbo et al., 2009). After isolation of wide varieties of active compounds and exploration of their efficacies and safety profiles, it is proved that medicinal plants have a value in the modern synthetic era. Numerous drugs have entered the international phar-macopoeias through the study of ethnopharmacology and traditional medicine (Patwardhan et al., 2005).

Keeping in view the importance and scope of medicinal plants, the present study was aimed to investigate the phytochemical constituents and biological activities of Trillium govanianum Wall. ex Royle, used in the traditional systems of medicine. T. govanianum belongs to the family Trilliaceae and is mainly distributed in South Asia, from Pakistan to Bhutan, at an altitude of 2700-3800 m (Muhammad, 2011). The genus Trillium includes long lived herbaceous flowering plants and different species are widely distributed throughout the world; the species reported from Pakistan is T. govania-num (Flora of Pakistan). The Asian species of Trillium produce secondary metabolites that are mainly exploit ted in cancer treatments (Huang et al., 2011). North American species of Trillium are known to have anti-fungal and antibacterial properties (Akihito et al., 2008). In folk medicine, T. govanianum is used to cure dysen-tery, in wound healing, inflammation, sepsis, menstrual and sexual disorders (Mahmood et al., 2013; Pant et al., 2010; Savita et al., 2013). It has been reported that the powdered plant is used as body and sexual tonic (Khan et al., 2013). Due to the folkloric knowledge of this plant, it is relevant to provide scientific evidence to its traditional uses, as well as to screen this valuable herb for any possible potential biological activity that will provide a base for isolation of targeted lead com-pounds. In this study, to the best of our knowledge we have explored for the first time, the antioxidant, anti-cancer and β-glucuronidase inhibitory activities of T. govanianum rhizome, its phytochemical constituents and proximate fatty acids composition.

Materials and Methods

Plant material

Plants of T. govanianum were collected from Dir Upper, Kohistan Valley (34° 54' and 35° 52' North latitudes and 72° 43' and 73° 57' East longitudes, at an altitude of 2700 -3800 m) in August, 2013. The plant was identified by Mr. Ghulam Jelani (Curator) of the Department of Botany, University of Peshawar and a voucher speci-men [No. Bot. 20092 (PUP)] was deposited in the herbarium of the same department.

Extraction and fractionation

The shade dried rhizomes of T. govanianum (7 kg) were grounded and extracted with methanol (40 L) at room temperature for a period of 14 days (3 X 40 L). The combined methanolic extract was evaporated to dry-ness to yield a brownish gummy residue (512 g), which was further fractionated (solid-liquid partition) into n-hexane (81 g), chloroform (94 g), ethyl acetate ( 85 g) and n-butanol (115 g) fractions.

Phytochemical screening

Different qualitative chemical tests of the n-hexane, chloroform, ethyl acetate, n-butanol and methanol fractions were performed for the determination of plant metabolites like alkaloids, tannins, phenolic com-pounds, flavonoids, saponins, sterols and carbohydra-tes, according to the recommended standard protocols (Shome and Joshi, 1984).

Gas chromatography/mass spectrometry (GC/MS)

GC/MS analysis was carried out on a 6890N Agilent gas chromatograph coupled with a JMS 600 H JEOL mass spectrometer. The compound mixture was sepa-rated on a fused silica capillary SPBI column, 30 m × 0.32 mm, 0.25 µm film thicknesses, in a temperature program from 50 to 256°C with a rate of 4°C/min with 2 min hold. The injector was at 260°C and the flow rate of the carrier gas (helium) was 1 mL/min. The EI mode of JMS 600 H JEOL mass spectrometer has ionization volt of 70 eV, electron emission of 100 µA, ion source temperature of 250°C and analyzer temperature of 250°C. Sample was injected manually in split mode. Total elution time was 90 min. MS scanning was performed from m/z 85 to m/z 390 (Faizi et al., 2014).

Derivitization

The n-hexane fraction was esterified to the more volatile methyl esters by methanol boron trifluoride method. Hexane solution (5 mL) obtained from the extraction was treated with boron trifluoride methanol (10%) solution and refluxed for 2 min on a water bath, and then 5 mL n-hexane was added, after an another 1 min of reflux, the solution was treated with 15 mL saturated NaCl solution under vigorous stirring. The organic layer was then separated and dried over anhydrous CaCl₂ (Botinestean et al., 2012).

GC/MS identification of components

Identification of proximate fatty acid components of the non-polar fraction (n-hexane) was based on the com-puter evaluation of mass spectra of sample through NIST-based AMDIS (automated mass spectral deconvo-lution and identification software), direct comparison of peaks and retention times with those for the standard compounds as well as by following the characteristic fragmentation patterns of the mass spectra of particular classes of compounds.

DPPH free radical scavenging assay

The in vitro antioxidant activity was evaluated by DPPH free radical scavenging assay according to method described elsewhere (Lue et al., 2010) with slight modifications. Two milliliters of 0.1 mM DPPH free radical solution in methanol were added to 1 mL of different concentrations (1, 10, 30, 50, 100, 200 μg/mL) of the fractions or standards in methanol. The solutions were shaken thoroughly on a vortex (Gyromixer, Pakland Scientific Production, Pakistan) and incubated in the dark at ambient temperature for 30 min. Absorbance was then measured at 517 nm using UV/Visible spectrophotometer (Lambda 25, PerkinElmer, USA) against control which consisted of 0.1 mM DPPH free radical solution without extracts or standards. Blank consisted of methanol alone. Ascorbic acid and BHT were used as standards. The percent DPPH free radical scavenging was calculated using the following formula: DPPH (%) = [(A_I-A_II/A₁) × 100], where A_I is the absorbance of the control reaction and A_II is the absorbance in the presence of the sample.

Anticancer assay

The cytotoxic activity of samples was determined by the MTT assay, according to Baydoun and Mosmann with slight modification (Baydoun et al., 2013; Mosmann, 1983), on two cancer cell lines, i.e. HeLa (cervical cancer cells) and PC-3 (prostate cancer cells). For the assay, cells were grown in DMEM (Dulbecco’s modified Eagle medium) and MEM (modified Eagle’s medium) containing 10% FBS and 2% antibiotic (penicillin and streptomycin) and maintained at 37°C, in 5% CO₂, for 24 hours, in a flask. Cells were plated (1 × 10⁵ cell/mL) in 96-well flat bottom plates and incubated for 24 hours for cell attachment. Various concentrations of test sample/fractions ranging from 1.25-20 µM were added into the well and incubated for 48 h. A 50 µL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; 0.5 mg/mL] aliquot was added to each well 4 hours before the end of incubation. Medium and reagents were aspirated and 100 µL DMSO was added and mixed thoroughly for 15 min to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader (Spectra Max 340; Molecular Devices, CA, USA). Finally, IC₅₀ values were calculated. Doxorubicin was used as the positive control (Baydoun et al., 2013).

β-Glucuronidase assay

The β-glucuronidase assay was performed according to the method described previously, using p-nitrophenyl β-D-glucuronide as substrate (Collins et al., 1997). The enzyme mixture (total volume 0.25 mL) contained 50 mL of p-nitrophenyl glucuronide, 190 mL of acetate buffer, 5 mL enzyme and 5 mL of inhibitor. The assay mixture was incubated at 37ºC for 40 min, the reaction was stopped by the addition of 50 mL of 0.2M Na₂CO₃, and the absorbance was measured at 405 nm. D-saccharic acid-1,4-lactone was used as a standard inhi-bitor. The percent inhibitory activity (%) was calculated using the formula; [(E-S)/EX100], where ‘‘E’’ is the activity of enzyme without test material and ‘‘S’’ is the activity of enzyme with test material.

Chemicals and reagents

Boron trifluoride solution in methanol (10%) was purchased from Fluka Chemie (Buchs, Switzerland). Sodium hydroxide solution (methanolic; 0.5N) and sodium chloride (analytical grade) were obtained from Merck (Darmstadt, Germany), while methanol (HPLC grade) and n-hexane (HPLC grade) were from Fischer Scientific (Leicestershire, UK). Helium gas (purity 99.9999%) was procured from Pak Gas (United Arab Emirates). Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT) were from Sigma-Aldrich (Germany).

Results

Phytochemical screening

The preliminary (qualitative) phytochemical tests on T. govanianum rhizome revealed the presence of secondary metabolites in methanol extract and its fractions, such as glycosides, steroidal saponins, tannins, sterols and flavonoids (Table I). Noteworthy, samples were very rich in steroids, steroidal glycosides and saponins.

Gas chromatography/mass spectrometry (GC/MS)

The proximate fatty acid composition of n-hexane frac-tion was carried out by GC/MS analysis. Twelve compounds were identified by comparison of n-hexane GC/MS spectra with the mass library search (NIST based AMDIS), as shown in Table II. Unsaturated fatty acids (70%) were more abundant than saturated fatty acids (30%). Among the unsaturated fatty acids, high (C₁₉H₃₀O₃), 9-hexadecenoic acid methyl ester (C₁₇H₃₂O₂) and cis-13-eicosenoic acid (C₂₀H₃₈O₂) were detected, where as 2-methyl hexadecanoic acid methyl ester (C₁₈H₃₆O₂) and ethyl 13-methyl tetradecanoate (C₁₇H₃₄O₂) represented the saturated fatty acids present at higher concentra-tions.

DPPH free radical scavenging assay

Antioxidants from natural sources are effective in reducing the toxic effects in human due to xenobiotic exposure (Wong et al., 2006). DPPH free radical scavenging assay is considered as a standard method for the assessment of the antioxidant activity of pure compounds, natural extracts and fractions (Loo et al., 2007; Lue et al., 2010; Rufino et al., 2009). Our results indicated that a higher scavenging capacity was measured in Hex-Fr and CHL-Fr (Figure 1 and 2) compared with the other fractions. This can be attributed to the presence of fatty acids, i.e. 9,12-octadecadienoic acid and hexadecanoic acid, in Hex-Fr, and glycosides, saponins and flavonoids in CHL-Fr, as previously reported on diverse plant species (Table II) (Bodoprost et al., 2007; Ha YL et al., 1990; Naga et al., 2012; Ying et al., 2014).

Anticancer assay

Figure 1: Percent of DPPH free radical scavenging activity of extract/fractions or standards (ascorbic acid and BHT). Results are mean ± SEM of three different experiments

Figure 2: DPPH free radical scavenging activity of extracts/fractions or standards (ascorbic acid and BHT). Results are mean ± SEM of three different experiments

The cytotoxic activity of methanol extract and fractions against two cancer cell lines, HeLa (cervical cancer cells) and PC-3 (prostate cancer cells), was evaluated by MTT assay. All fractions exhibited cytotoxicity against both cancer cell lines (Table III). In particular, anticancer activity of chloroform fraction on HeLa cells was slightly lower than that of doxorubicin, with IC₅₀ of 0.8 ± 0.2 and 0.3 ± 0.0, respectively. Similarly, this fraction was also most effective against PC-3 cells (IC₅₀ = 2.7 ± 0.3), though to a lesser extent than doxorubicin (IC₅₀ = 1.4 ± 0.2). Moreover, the butanol fraction, although possessed moderate cytotoxic effect against the HeLa cells (IC₅₀ = 1.6 ± 0.3) but was less effective in inhibiting the PC-3 cells (IC₅₀ = 4.0 ± 0.3).

β-Glucuronidase inhibitory assay

Based on the IC₅₀ values (µg/mL) the methanol extract (140.8 ± 3.8) and butanol fraction (196.2 ± 1.9) exhibited a moderate level of enzyme inhibitory activity in comparison to the standard D-saccharic acid-1,4-lactone (Table IV).

Discussion

Almost 70% of breast cancer patients are estrogen receptor alpha (ERα) positive and estrogen-dependent. Estrogen receptors play a crucial role in the development and progression of breast cancer which function as a transcription factor to influence cell differentiation, proliferation, and apoptosis (Anderson et al., 2002). Endocrine therapy is a treatment of breast cancer by preventing estrogen from binding estrogen receptors. Tamoxifen is the first generation of estrogen receptor modulators in clinical practice for the treatment of metastatic breast cancer and has achieved great therapeutic effects (Renoir et al., 2013). The estrogen receptor has become a promising target for synthesizing low-toxic and highly effective estrogen receptor inhibitors (Ariazi and Jordan, 2006).

Naphthoquinones are a kind of common natural compounds, which have bactericidal, anti-oxidant and anti-viral effects (Zhivetyeva et al., 2016; Novais et al., 2018). Some studies have shown that naphthoquinone derivatives have anti-tumor and apoptosis-inducing effects (Li et al., 2017; Liu et al., 2018). In this study, we found a compound which can selectively inhibit MCF-7 cells (estrogen receptor positive) but has lower cytotoxicity against MDA-MB-231(estrogen receptor negative). Also, we found that MCF-7 cells were more toxic to com-pound 34 when estrogen in MCF-7 cells was deprived for 24 hours (supplement). On the other hand, phenol red in culture media significantly attenuated the inhibition of MCF-7 cells (supplement). The reason may be that phenol red can simulate the effects of estrogen (Berthois et al., 1986; Welshons and Jordan, 1987) and result in the competitive binding with estrogen receptors between compound 34 and estrogens. Previous studies have demonstrated that raloxifene and tamoxifen could be metabolized by both rat or human liver microsomes to electrophilic diquinone methide and o-quinones and the classical electrophilic quinone methide might contribute to the potential toxicity of raloxifene and tamoxifen (Yu et al., 2004; Liu et al., 2005; Dowers et al., 2006). From the literature reports and our MTT experimental data, we hypothesized compound 34 was likely to be an estrogen receptor inhibitor. We continued to explore its mechanism of inhibiting MCF-7. Hoechst 33342 is a common dyeing solution for detecting apoptosis. Fluorescent photographs showed that the MCF-7 cells treated with compound 34 were densely stained and the cells of control were natural blue, which revealed that compound 34 could induce apoptosis in MCF-7 cells. Next, we detected the changes of mitochondrial membrane potential by JC-1 staining, because the decrease in mitochondrial membrane potential is a sign of early cell apoptosis. The results showed that the cells in the control group were red, and gradually turned green as the compound concentration increased, indicating that the mitochondrial membrane potential was decreasing and was dose-dependent. It has been reported in the literature that quinones are highly redox active molecules which can redox cycle with their semiquinone radical anions leading to the formation of reactive oxygen species (ROS) (Bolton and Dunlap, 2017) and ROS accumulation leads to a membrane potential decrease in cellular mitochondria and activation of intrinsic apoptotic pathways (Skulachev, 2006; Yee et al., 2014). Therefore, we performed intracellular ROS assay and found that ROS was significantly accumulated in MCF-7 cells treated with compound 34 compared to the control. However, NAC did not reverse the cytotoxicity against MCF-7 cells. At the same time, Western blotting showed that the intrinsic pathway marker protein of cytochrome c was increased and the expression level of procaspase-3 was down-regulated. Computer simulation improves the efficiency of drug development (Zhong and MacKerell, 2007), we conducted molecular target docking through the online molecular docking network. The results showed that the compound could dock with the estrogen receptor and the docking score was 4.7, better affinity than tamoxifen. Metastasis is a multi-step process that involves the movement and invasion of cancer cells, which is a key problem for cancer treatment (Deryugina and Quigley, 2006). Therefore, inhibition of metastasis is essential for effective cancer treatment. Scratch test results showed that compound 34 could inhibit cell migration, it may also be a promising migration inhibitor.

Conclusion

To the best of our knowledge, this is the first report on the phytochemical analysis, antioxidant, β- glucuro-nidase inhibitory and anti cancer activities of this plant species. These findings indicated that T. govanianum rhizome possesses a promising potential as anticancer medicinal plant, even if further studies, now in progress, are necessary to isolate active constituents responsible for the observed efficacy.

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