Characterization and anticancer potential of Withania somnifera fruit bioactives (a native species to Pakistan) using gas chromatography-mass spectrometer, nuclear magnetic resonance and liquid chromatography-mass spectrometry-electrospray ionization

Abstract

INTRODUCTION:

Withania somnifera (W. somnifera) is a plant with remarkable pharmacological properties. The plant has an impressive profile of medicinal uses in the folk medicine system of several civilizations.

AIM:

This comprehensive study is aimed to characterize phytochemicals in fruit of W. somnifera and tested for in vitro anticancer potential to find out active candidate in disease prevention and treatment.

METHODS:

The bioactive components from W. somn-ifera fruit were extracted with polar and non-polar solvents. Anticancer potential of the isolated bioactive was assessed against different cancer cell lines through MTT assay and Incucytes imaging analysis. The extracts were characterized for secondary metabolites using GC-MS (gas chromatography-mass spectrometer), LCMS (liquid chromatography-mass spectrometry)-ESI (electrospray Ionization) and 1H-NMR (electrospray Ionization) techniques.

RESULTS:

Both freeze-dried and rotary evaporator con-densed extracts exhibited anticancer potential against MDA-MB-231, MCF7- SKOV3 and SKBR3 cell lines. The tested extracts have cell growth inhibition potential ag-ainst mammalian cancer cell line. Hexacosanedioic acid purified from n-hexane extract through HPLC was inves-tigated for its cytotoxicity against breast cancer cell line SKBR3 by using Incucytes imaging analysis. Conclusion: We found that a variety of bioactive compounds existed in this plant. One identified compound that was not investigated for cytotoxicity in previous studies was purified and its application showed cytotoxicity on breast cancer cell lines. A number of bioactive identified from W. somnifera fruit may have an effective potential for development into chemotherapy drugs.

1. INTRODUCTION

Withania somnifera (W. somnifera) is known as the king of medicinal plants in various traditional medicinal systems including Ayurveda. It belongs to the family Solanaceae or “night shade” of the genus Withania, with around 60 species reported. This plant grows in a slightly hot climate and is widely distributed in Pakistan, India, Iran Mediterranean regions of Asia and Africa. The plant is known by different common names such as, “auksin” in Pakistan, “winter cherry” in English, “ashwagandha” in India and “kansuensis kuang” in China.¹ The root and leaves of W. somnifera are commonly used in Iran, China, India and Israel for the treatment of different ailments due to wide spectrum of pharmacological activities such as anti-aging, anti-inflammatory, anti-arthritis, antitumor and hepatoprotective roles² Phytochemically, 40 withanolides, 12 alkaloids and a number of lactones, glycosides and saponins have been reported from different parts of this plant.³

The extraction of chemical constituents and their immunomodulatory activity mainly depend on the choice of different extraction solvents and the origin of plant.⁴ Although a great deal of attention has been given to the phytochemicals profiling of this plant worldwide, however, W. somnifera from Pakistani flora have not yet been characterized for their secondary metabolites and anticancer activity against mammalian cancer cell lines.

Soon Valley (District Khushab of Punjab province) in Pakistan is famous for its rich flora of medicinal plants. This valley is characterized by specific agro-climatic conditions (relatively low temperature and rain fed agro-ecological zones) which distinguished this area from adjoining regions.⁵

Among many other medicinal plants in Soon valley, W. somnifera is very much popular among local community for its multiple medicinal uses.³ However, rarely reports are available on the phytochemicals profiling of W. somnifera fruit harvested from Soon valley of Pakistan. In this research, we, for the first time comprehensively characterized and explored the secondary metabolites profile of W. somnifera fruit collected from wildly grown plants in the Soon valley. Moreover, cytotoxicity of the bioactive extracts using in vitro cytotoxicity assays with human cancer cell lines was evaluated.

2. MATERIALS AND METHODS

2.1. Fruit collection and processing

The fruit of Withania somnifera was collected in 2017 from the Punjab province (Soon valley, District Khushab) of Pakistan. The fruits (supplemental Figure 1) were dried under shade at room temperature before they were ground and stored for future use in sealed polyethylene bags.⁵

Figure 1. Cell viability of four cancer cell lines against treated extracts, positive control drugs and negative control media with corresponding 1% DMSO concentration. Values are reported as mean ± SD (n = 3) of three separate experiments.

A: SKBR3; B: MDA-MB-231; C: MCF7; D: SKOV3.

2.2. In vitro cytotoxicity assays

In vitro cytotoxic evaluation of the plant crude extracts was made against three mammalian breast cancer cell lines MDA-MB-231, MCF7, and SKBR3, and one ovarian cancer cell line SKOV3.

In addition to the crude extracts, the cancer cells were also incubated with different concentrations of purified compounds and monitored using the Incucyte^® microscope in same way as Tabassam et al ⁶ performed. The anticancer activity results were calculated using the Incucyte^® ZOOM integrated analysis software (v2016A).

2.3. Bioactives profile: ¹H-NMR analysis

10 mg samples were dissolved in 700 µL of DMSO-d₆ and analyzed on a Bruker Advance III (400 MHz) NMR spectrometer. NMR spectra were processed with the Topspin Bruker software.⁷

2.4. LCMS-ESI

Extract samples were analyzed using a Thermoscientific TSQ Quantum Access Max (Triple Quadrupole) with a 600 Acela pump mass spray equipped with an electrospray ionization source (N₂) in positive full scan mode 50-1400 m/z. The instrument parameters were: spray voltage 3,500 V, vaporizer temperature 350°C, sheath gas pressure 60psi, capillary temperature 380°C and tube lens offset 109. The column used for separation was a Zorbax Eclipse plus C18 (3.0 × 150 mm) 3.5-micron (Agilent USA). Sample of crude extract (1mg) was dissolved in 1 mL methanol to prepare a stock solution. For LC, a gradient mobile phase was used comprising mobile phase A (2 mM NH₄OAc) while mobile phase B was MeCN. The injection volume was 10μL and flow rate was set as 0.3 mL/min.⁸

2.5. Volatile composition

The volatile composition of the extract samples was investigated using a modified method reported by¹⁹ Wang et al (2016). Each sample was analysed on a Perkin Elmer Clarus 680 GC with an automated liquid injection mode and coupled with a Clarus SQ 8 C quadrupole mass system having an EI ionization range of 4-1000 m/z. column used with specifications of (30 m × 2.5 µm) and an injection volume was 1 µL.

2.6. Separation/Purification/identification

The n-hexane extract was analysed on a Shimadzu Nexra HPLC system having a multichromatic PDA detector and a D₂ lamp, using an Agilent Eclipse C18 (4.6 × 250mm) column with 5 μm particle size by the method of Seal et al.¹⁰ The injection volume was 50 µL and the flow rate was maintained at 0.5 mL/min. The results were analyzed at three different wavelengths (λ = 272, 280, and 310 nm). A gradient program was run for 60 min total run time. Mobile phase A was 1% v/v AcOH in H₂O and mobile phase B was MeCN. The pump B percentage gradually increased to 10% from 0-10 min, then 10-28 min to 40%, 28-39 min to 60 % and then 39-55 min it remained at 60%. For the last 5 min the initial gradient of 10% mobile phase B was used. Fractions were collected manually and identified using a Bruker Solarix 2xR 7T APCI with high resolution power from (450 000 @1 Hz and 400 m/z, 20 000 000) through a PDA detector with a direct infusion method.

2.7. Statistical analysis

Statistical analysis was performed by using GraphPad prism 19. Values were taken as in triplicate. The mean ± standard deviation values between various groups were measured where statistically significance of data considered as P < 0.05.

3. RESULTS AND DISCUSSION

3.1. Extract yield and anticancer potential

In the present work, the W. somnifera plant fruit extract was explored for its anticancer potential against mammalian cancer cell lines through the MTT assay. In the present study, we used different polarity solvents (H₂O > MeOH > CH₂Cl₂ > n-hexane) to produce extracts. The maximum yield was obtained with CH₂Cl₂: H₂O extract (75%), followed by the MeOH extract (60%) and the minimum yield was obtained with the n-hexane extract (50%) that are in line with earlier literature reports.¹¹

We found that non-polar solvent (n-hexane) extract from W. somnifera fruit had the highest anticancer activity. Anticancer potential of different solvent extracts, in terms of cell viability, along with positive control (Cisplatin and MMAE) and negative control (DMSO) are depicted in Figure 1.

The extracts were used within the concentration range of 31.25-500 μg/mL with five dilutions. The effect of cellular viability was observed in a dose dependent manner. “Our” studies are consistent with those of Khang et al ¹² who showed that the plant extract exhibits anticancer activity. Aqueous CH₂Cl₂ extract was found to be effective at 500μg/mL with promising cell growth inhibition against SKOV3 and MCF7 cell line. While other extracts concentration (250, 125, 31.25 μg/mL) also showed considerable reductions in cell growth. The anticancer activity of the tested extracts can be in due part to the presence of anticancer compounds.

The n-hexane extract showed promising anticancer effects towards SKBR3 cells. Herein, we can presume that this extract is rich in biologically active compounds. Previously work reveals that this plant family (The nightshade family) contains lactones and alkaloids such as withaferin-A, that is reported to inhibit cancer cell growth and played a role in neuromodulation. They have been shown to cause G (2)/M cell cycle arrest and modulation of cyclin B1, p34 (cdc2), and PCNA levels. They also decreased levels of STAT3 expression and its phosphorylation on two-point Tyr (705) and Ser (727), leading to the modification of expression levels of p53-mediated apoptotic markers-Bcl₂, Bax, caspase-3, and cleaved PARP, consequently they played a significant role in inhibition of tumor growth.¹³

The MeOH extract showed less activity amongst others against SKBR3 cells. Moreover, this trend was also observed in the other cancer cell lines tested. An earlier study reported that MeOH root extract of Withania somnifera are highly active against breast cancer cell lines even at very low concentration 10 μg/mL.¹⁴

3.2. GCMS analysis of active fraction

The n-hexane extract of Withania somnifera fruit was subjected to a methanolysis reaction for derivatization as previously performed whereby volatile composition was analyzed by method¹⁵ Soares et al. (2019). The GC-MS analysis with NIST library search showed a list of compounds (1-10) Phthalic acid) 1, 1,3-bis (1',1'-dimethyl ethyl) benzene 2, Hexadecane 3, 2-methyl- dodecane 4, n-Hexadecanoic acid (Palmitic acid) 5, Ecosane 6, Methyl palmitic ester 7, Methyl (9-trans, 12-trans)-nonadeca-9,12-dienoate (Methyl linolic ester) 8, (9-cis, 12-cis)-Nonadecane-9,12-dioic acid (linoleic acid) 9, Tetracosanoic acid 10 as revealed in (supplemental Table 1) along with their structure and reported activities from different plants see spectrum in (supplemental Figure 2).

Table 3.

Hexacosanedioic acid mass and various confirmatory parameters

Actual m/z (amu)	Ion formula	Score	Compound	Calculated m/z (amu)	Mean mass Err (mmu)	Mean error (ppm)	M Sigma	Relative double bond		eConf	N-Rule	XR Score
427.37864	C26H51O4	100	Hexacosane-dioic acid	427.3782	-4	-1.4	89.1		2	even	ok	n.a

Figure 2. Ten compounds.

In the present study, the spectrum is analyzed with the NIST library and it showed ten compounds in comparison with mass of already known compounds. Four different classes of compounds are identified, which are aromatic/acyclic carboxylic acids / fatty acids, alkane (hydrocarbons), alkaloids and lipids. Compared to all phytochemical groups, the tested extract was noted to be rich in fatty acid constituents. Some fatty acids, like linoleic acid 9 and palmitic acid 5, are reported in the literature with their anticancer activities against breast cancer cell lines. It is likely that the presence of these phytochemical similar views like (Xu et al; ²² Mericli et al;¹⁹ Nahid et al;²⁰ Vimalavady et al;¹⁷ Krishnan et al ¹⁶) are partly responsible for the cytotoxicity of these extracts.^{16,17,18,19,20,21,22}

3.3. Atmospheric pressure chemical ionization (APCI) mass spectrometry

Withania plant is known to have a variety of phenol, saponin, alkaloid, terpenoids and steroidal constituents. To check the presence of such high molecular mass compounds in non-polar solvent (such as n-hexane) extracts, the highly sensitive APCI-MS was used. Data analysis through Scifinder and Metlin databases showed presence of the following ten compounds shown in spectrum (Figure 2, Supplemental Table 2 and Figure 3).

Figure 3. Anticancer activity of Hexacosanedioic acid towards breast cancer cell line.

The palmitic acid 5 and aconitine alkaloid 11 were found in higher quantities in the n-hexane fruit extract. Our results are in line with.²⁶ who investigated Withania fruits from India and found that palmitic acid 5 as the metabolite with highest abundance. In our analysis, we report the presence of withangulatin-A for the first time in fruits of Withania. It was previously reported in fruits of Physali angulate, a phylogenetic neighbor of Withania somnifera.²⁷ We also identified withafastuosin-D diacetate 17 that has been previously found in the roots of Withania plant, displaying cytotoxic activity.²⁸ Vamonolide 16, isolated from the leaves and fruit part of medicinal plants as reported in Physalis angulate plant of the Solanaceae family, is known to show antiparasitic activity Phytosphingosine 18 showed anti-inflammatory and antimicrobial activities against acne vulgaris.²⁹ Alkaloids, such as aconitine 11 and withangulatin-A 12, have been found to have anticancer activity.

Most of the alkaloids approved clinically to be used as an active drug against cancer such as taxol, vinca alkaloids (vincristine and vinflunine). The polyphenolic compounds such as epicatechin-3-O-gallate 13 have been found to be effective for prevention of oxidative damage in plasma, and have shown anti-proliferative activity against colon cancer cell lines.^30,31

3.4. ¹H-NMR

The partially purified active fraction from n-hexane extract of Withania somnifera fruit was further confirmed through the appearance of signals in NMR spectrum (Supplemental Figure 4). The similar group of compounds was identified as with MS and GC. The processed data analysis of spectrum presented in (Supplemental Figure 5) showed that a few diagnostic signals are associated with aliphatic compounds. The structure of a couple of compounds was confirmed by looking at the chemical shifts (δ in ppm) and multiplicity of signals in the following crude spectrum.

Figure 4. cell experiment result.

A: % Confluency after 3 days treatment; B: negative control; C: indicating with drug 25 μg/mL conc; Sample highest concentration 50 μg/mL.

The presence of palmitic acid 5 was confirmed by ¹H-NMR spectrum peaks after their identification through GC and APCI. A triplet peak at 0.88 ppm corresponded to methyl (C-17) protons. A pentet at 1.85 ppm and a triplet at 2.30 ppm of methylenic protons appeared for C-3 and C-4 respectively. A broad singlet of carboxylic proton (-COOH) was observed at 10.99 ppm. A multiplet of many protons appeared at 0.80 ppm due to the alkyl chain (C-4 to C-16). Similarly, the emergence of a broad singlet, a triplet, a pentat and a broad singlet [for terminal carboxylic ¹Hs (C-1 to C-26), methylenic ¹Hs (C-2 to C-25), methylenic ¹Hs (C-3 to C-24) and the ¹Hs of alkyl residual chain (C-4 to C-26)] at 10.90, 2.30, 1.85, 0.80 ppm, respectively concluded the structure of hexaco-sanedioic acid.

3.5. LC-MS-ESI

We tried various methods of different gradients, mobile phases reported by³² Trivedi et al. (2017) work on similar plant; however, we were unable to predict compounds from mixture (poor separation) and could only find compound hexacosanedioic acid with Chemical C library.

Our findings are in agreement with Piasecka et al,³³ who reported limitations of analytical technique and revealed the same complication linked to the separation process of LC-MS instrument with crude mixture of 1 mg/mL concentration. Nevertheless, this is a good approach to confirm the mass of a pure compound with known standards or evidence to the whole mass of single compound which is already identified through other techniques because it gives trustworthy explanation of elemental ratio of protonated molecule by its sensitive mass analyzer.

3.6. Purification / identification

The n-hexane extract was subjected to analytical HPLC for the separation and purification. All fraction collected manually through fraction collector. The UV active fraction was purified. The single peak was collected after various run (Supplemental Figure 6). The purified compound was analyzed by HRMS and structures were identified after analysis with Solarix 2R software. The purified compound was hexacosanedioic acid (Supple-mental Figure 7) as parented in Table 3 with all specification.

3.7. Biological assay through incucyte zoom

Growth inhibition of breast cancer cell line was checked by using three different concentrations (ranging from 12.5, 25, 50 μg/mL) of hexacosanedioic acid.

After 3 days incubation, we got the result through incucyte software and noticed that our sample significantly inhibited cell growth in comparison to negative control with 50 μg/mL concentration.

Maximum 98% confluency is with control while sample is effective in 50 μg/mL concentration with 45% live cell confluency. Drug is more active even with 25 μg/mL concentration showing 20% average confluency as presented in Figure 3. Values are results of three independent experiment with P value less than 0.05.

The % living cell confluency was observed with negative control: drug and our sample. The highest confluency rate almost 98% observed with negative control, while drug 25 μg/mL showed lowest confluency 20%. Our sample highest concentration regarded as IC₅₀ value 50 μg/mL kills most of the cells and decreased 45% confluency in comparison to negative control which indicates promising results towards SKBR3 cell lines as mentioned in Figure 4A-C.

4. CONCLUSIONS

The extract from Withania somnifera fruit from Pakistan was investigated for its phytochemical and pharmacological potential. This is the first com-prehensive study to highlight this plant’s potential with three different solvent extracts based on polarity. We have tested these extracts for anticancer activity against various mammalian cell lines. We found that the extracts were cytotoxic, but a particular extract obtained with n-hexane displayed a higher cytotoxic potential against the breast cancer cell line SKBR3.

Several analytical techniques (GCMS, LCMS, APCI, HPLC and HRMS) were used to identify and characterize secondary metabolites in the subject plant extracts. We found that LCMS-ESI was a less effective approach for crude extract profiling due to inefficiency with separation of compound mixture. The GC-MS was found to be effective for compounds having molecular weight of 500 Da only. The APCI proved to be an effective tool while profiling metabolites from non-polar solvent of crude mixture. It gives rare fragmentation and we easily pick up the whole m/z value to know about the nature of compounds in comparison with already known compounds. The ¹H-NMR confirmed the presence of identified metabolites through MS results. Therefore, APCI and NMR together provided credible information about the chemical makeup of plant.

Using HPLC, we purified one compound, hexaco-sanedioic acid and confirmed its structure and through high resolution MS. Incucyte analysis is a more accurate way of analyzing cells proliferation rather than MTT assay. We can clearly observe differences between tested compounds and control so we measured the cytotoxic activity of our compound through this assay.

The most promising compound hexacosanedioic acid was isolated from n-hexane extract. It presented cytotoxicity against SKBR3 cells; however, its efficacy was not as high as standard drug MMAE. Future study will be made via cell migration assay against other types of cancer cell lines so as to predict full spectrum anticancer potential.

5. ACKNOWLEDGMENTS

QT acknowledges the IRSIP Scholarship Program of Higher Education commission of Pakistan for funding. We are thankful to Dr Nick Proschogo in the School of Chemistry at The University of Sydney for his help with Mass Spectroscopy experiments.

Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this article.