Antioxidant, Antiproliferative, Pro-apoptotic and cell cycle arrest properties of crude extract and biofractions of Hybanthus enneaspermus Linn. to combat breast cancer

 

Liesl Maria Fernandese Mendonca¹*, Arun Bhimrao Joshi²,

Anant Bhandarkar², Himanshu Joshi³

¹Department of Pharmacology, Goa College of Pharmacy, 18th June Road, Panaji, Goa 403001 India.

²Department of Pharmacognosy and Phytochemistry, Goa College of Pharmacy,

18th June Road, Panaji, Goa, 403001 India.

³Department of Pharmacology, College of Pharmacy, Graphic Era Hill University,

Bhimtal campus, Uttarakhand 263156 India.

 

ABSTRACT:

Objective: According to the World Health Organisation, breast cancer is presently the most common cancer diagnosed in women globally. Polyphenolic compounds act as antioxidants, improve health, and reduce risk and proliferation of various types of cancers. Hybanthus enneaspermus Linn. is a beneficial medicinal plant, reported to possess antimicrobial, antioxidant, anti-inflammatory, neuroprotective, cardioprotective, and nephroprotective properties etc. Methods: The current study involved the evaluation the antioxidant, antiproliferative, apoptotic and cell cycle arrest potential of the ethanolic leaf extract of Hybathus enneaspermus Linn. (EEHE), its toluene soluble, toluene insoluble, ethyl acetate and methanol soluble biofractions viz. TFHE, ITHE, EFHE, and MFHE to combat breast cancer. In vitro antioxidant activities were evaluated using DPPH, Hydrogen peroxide, Nitric oxide and ABTS free radical scavenging assays. In vitro antiproliferative activity against MCF-7 cells was assayed using the Sulforhodamine method, while apoptosis and cell cycle assays were analysed by flow cytometry. Results: MFHE exhibited significant in vitro antioxidant activity with IC₅₀ values of 21.10±0.39 μg/mL and 25.99±4.66μg/mL, when compared against standard ascorbic acid with IC₅₀ values of 11.19±1.09 μg/mL and  9.30±0.26μg/mL in DPPH and nitric oxide assays respectively.  EFHE displayed substantial antioxidant potential in ABTS and hydrogen peroxide assays with IC₅₀ values of 40.38±0.88μg/mL and 99.11± 13.59μg/mL, while ITHE showed considerable activity with IC₅₀ < 100μg/mL in DPPH, nitric oxide and ABTS assays. TFHE demonstrated significant antiproliferative activity by sulforhodamine assay, with GI₅₀ value of 10.22 6.72µg/mL, while EEHE and ITHE showed substantial activity with GI₅₀ values of 41.42±3.74µg/mL and 64.37±7.07µg/mL respectively, as against the standard drug Adriamycin (GI₅₀ < 10µg/mL) used. In the apoptosis assay, ITHE showed 11.31±0.82% cells in late apoptosis and 34.48±1.57 % cells in necrosis as compared to standard Adriamycin indicated 13.67±1.02 % cells in late apoptosis and 8.58±0.65 % cells in necrosis. In cell cycle analysis, ITHE displayed significant apoptotic activity with 20.15±1.37 % cells in SubG₁ phase and 13.99±1.65 % cells arrested in G₂-M phase as compared to the control. Conclusion: The study thus revealed that MFHE, EFHE and ITHE biofractions showed significant antioxidant activities, while EEHE, TFHE and ITHE exhibited substantial antiproliferative activity against mammary cancer cells. Additionally, ITHE induced remarkable apoptotic activity and cell cycle arrest in the MCF-7 cells. The therapeutic benefits may be credited to the bioactive constituents present in the ITHE fraction viz. polyphenolics, flavonoids etc.; however, the molecular mechanisms may need to be evaluated further. 

 

 

 

1. INTRODUCTION: 

Breast cancer is currently the most common type of cancer diagnosed worldwide and is reported to be the foremost cause of cancer-related mortalities in women of developed and underdeveloped countries^1,2. Tremendous research is being carried out to combat, alleviate symptoms, and cure patients suffering from this debilitating disease. However, current treatment approaches like chemotherapy or radiation therapy account for innumerable adverse effects, drug resistance, and increased financial burden to the patient community. Naturally occurring plant metabolites are comparatively safe, have minimal side effects, cost-effective, and show good bioavailability³.

 

Plants have been used in traditional medicine systems for health and therapeutic benefits. The World Health Organisation (WHO) states that herbal medicines still play a key role in medical treatment in developing nations.

 

Plants and dietary foods rich in polyphenolics like flavonoids, tannins, gallocatechin, etc., act as natural antioxidants that can improve health and reduce the risk of cancers⁴. As reactive free radical scavengers, plant antioxidants help minimize oxidative stress, preserve cellular integrity, and maintain physiological functions and homeostasis in the human immune system⁵. Phytochemicals have demonstrated anticancer activity by inhibiting tumor cell proliferation, inducing apoptosis, suppressing cell cycle, metastasis, angiogenesis, etc⁶. Several studies indicate that combining conventional therapies with natural products provided better efficacy, additive or synergistic anticancer benefits with reduced side effects, and resistance to chemotherapy and radiotherapy⁷.

 

Hybanthus enneaspermus (L.) F. Muell, synonym Ionidium suffruticosum (L.) Ging, belonging to the family Violaceae, grows as a small, diffuse, perennial herb in tropical and subtropical countries⁸. Traditionally the plant is used to treat urinary calculi, strangury, painful dysentery, vomiting, wandering of mind, asthma, epilepsy, cough etc. and provide tone to the breasts⁹. Pharmacologically, the plant is reported to possess antibacterial, antifungal, antiviral, antioxidant, anti-inflammatory, antidiabetic, antiarthritic, anti-nociceptive, antiallergic, anticonvulsant, antihyperlipidemic, neuroprotective, cardioprotective, nephroprotective, antiplasmodial and anticancer activities.

 

It is reported to contain a variety of phytoconstituents, viz. flavonoids, steroids, terpenoids, phenols, tannins, alkaloids, saponins, cardiac glycosides, cyanogenic glycosides, anthraquinone glycosides, dipeptides, sugars, amino acids, etc. that contribute to the pharmacological activities of the plant^10-13.

 

Based on the pharmacological profile of the plant and its folklore claims, the present research was undertaken to evaluate the therapeutic efficacy of the ethanolic extract of the whole plant of H. enneaspermus and its biofractions against MCF-7 breast cancer cell lines. The extract and biofractions showing considerable antioxidant and antiproliferative activities were subjected to apoptosis and cell cycle analysis to study the probable mechanism of action.

 

2. MATERIALS AND METHODS:

Chemicals and reagents:

Propidium iodide (PI), DPPH and ABTS were obtained from Sigma Aldrich (USA). Annexin V-FITC Kit was obtained from Beckman Coulter (Indianapolis, USA).  Dulbecco’s Modified Eagle Media with low glucose and Fetal bovine serum were procured from Gibco, Invitrogen (USA). Antibiotic – Antimycotic 100X solution and hydrogen peroxide solution (Qualigens) were obtained from Thermofisher Scientific (USA). Phosphate-buffered saline (PBS) was obtained from HiMedia (USA) and ascorbic acid, sulphanilamide, sodium nitroprusside and potassium persulfate were obtained from HiMedia (India). High purity analytical grade solvents and chemicals were used for the study. Reference standards were procured from Sigma-Aldrich (St. Louis, Mo., USA).

 

Cell cultures:

The MCF-7 breast cancer cell lines were bought from National Centre for Cell Science, Pune, India and stock cells were cultured in RPMI 1640 media, enhanced with 10% fetal bovine serum and 2mM L-glutamine. DMEM was added along with inactivated FBS (10%), penicillin (100 IU/mL), and streptomycin (100μg/mL) in 5% CO₂ at 37°C until confluent. TPVG solution was used to separate the cells.  

 

2.1. Plant collection and preparation of experimental samples:

Fresh whole plants of H. enneaspermus were collected from Mangalore in August 2017 and authenticated by Dr. Sunita Garg, Emeritus Scientist CSIR-NISCAIR, New Delhi (NISCAIR/RHMD/Consult/2017/3101-50).  The plants were washed to remove extraneous matter, shade dried, cut into small pieces, and powdered using a mechanical grinder. 3Kg of the powdered drug was macerated with absolute ethanol over a period of 4 days with occasional shaking to yield the ethanolic extract of H. enneaspermus (EEHE). The process was repeated with the fresh solvent that was further filtered using the Whatmann filter paper (No.1), distilled off using a rotary evaporator and then evaporated to dryness to yield the extract. 250g of EEHE was refluxed with toluene successively thrice for one and half hours, filtered and the solvent evaporated to dryness to yield toluene soluble, toluene insoluble fractions of the ethanolic leaf extract of H. enneaspermus viz., TFHE, ITHE. Similarly, 15g of ITHE was refluxed successively with ethyl acetate and methanol, to yield ethyl acetate and methanol soluble fractions (EFHE and MFHE), which were stored at 4℃ until further used.

 

2.2. Qualitative phytochemical screening:

The experimental samples (EEHE, TFHE, ITHE, EFHE and MFHE) and standard ascorbic acid were screened to identify the phytoconstituents present by standard procedures^14-16.

 

2.3. In vitro antioxidant activity:

The in vitro antioxidant activity of the experimental samples was measured using the DPPH, hydrogen peroxide, nitric oxide and ABTS radical scavenging methods.

 

2.3.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay:

The antioxidant activity of the experimental samples and standard was studied using DPPH free radicals with slight modifications of the technique portrayed by Chanda S et al., Ghagane S et al., Chen Z et al^17-19.

 

2.3.2. Hydrogen peroxide radical scavenging assay:

Hydrogen peroxide free radicals were used to estimate the scavenging activity of the experimental samples and standard by adopting the assays performed by Raju D et al. with slight modifications^20,21.

 

2.3.3. Nitric oxide radical scavenging (NO) assay:

The assay evaluated the ability of the experimental samples to scavenge the nitric oxide free radicals generated by sodium nitroprusside and measured by the Griess reaction against the standard Ascorbic acid. This assay was performed in accordance with the procedure described by Green et al. with slight modifications^17,22-25.

 

2.3.4. ABTS radical scavenging (ABTS) assay:

The ABTS free radical was produced using ABTS radical cation decolorization assay, by treating 10mg of ABTS with 2mg potassium persulfate in water, and the ability of the experimental samples and standard to scavenge these free radicals were determined using the methods described by Seena H et al., Prior R et al. and Salama Z et al. with slight modifications^5,26,27.

 

2.4. In vitro anticancer activity:

2.4.1. Cell proliferation assessment:

The antiproliferative activity of the experimental samples was evaluated by Sulforhodamine (SRB) assay in accordance with the procedure described by Mendonça L et al^28-30.            

 

2.4.2. Apoptosis assay:

The experimental samples showing substantial antiproliferative activity were subjected to apoptosis assay and analysed by flow cytometry, as per the technique employed by Pozarowski P et al. and Tilekar K et al^31,32.  

 

2.4.3. Cell cycle assay:

The potential of the experimental samples to induce cell cycle arrest was studied using cell cycle assay and analysed by flow cytometry as per the method described by Tilekar K et al³².

 

Statistical analysis:

All analyses were performed in triplicate and recorded as mean ± standard deviation (SD). Statistical analysis was done using IBM© SPSS version 23.0 statistical software (IBM Corporation, New York, USA) and GraphPad Prism version 7.3. One-way Analysis of Variance (ANOVA) along with Duncan's multiple range test was used to calculate statistical significance.

 

3. RESULTS:

3.1. Extraction and fractionation yield:

The yield of the EEHE was reported to be 268.37g, while that of its toluene soluble fraction (TFHE) and toluene insoluble fraction (ITHE), were 149.28g and 53.42g, respectively. Ethyl acetate (EFHE) and methanol (MFHE) soluble fractions yielded 3.16g and 7.81g, respectively. 

 

3.2. Preliminary phytochemical screening:

The preliminary phytochemical analysis of H. enneaspermus revealed the presence of various active phytoconstituents. EEHE confirmed the presence of alkaloids, flavonoids, glycosides, saponins, tannins, phenolics, steroids, triterpenoids, sugars and amino acids.  While TFHE showed significant quantities of steroids and triterpenoids; ITHE, EFHE and MFHE showed substantial amounts of flavonoids, tannins and phenolics. 

 

3.3. In vitro free radical scavenging activity:

Four in vitro free radical scavenging assays were used to assess the antioxidant activity of the ethanolic extract of H. enneaspermus and its biofractions. IC₅₀ value indicates the concentration of the sample, at which 50% of the free radicals are scavenged. The results of the in vitro free radical scavenging activity performed using DPPH, hydrogen peroxide, nitric oxide and ABTS methods are depicted in Figure 1. Amongst the selected experimental samples, MFHE exhibited significant antioxidant potential with IC₅₀ values of 21.10±0.39 μg/mL and 25.99±4.66μg/mL as compared to the standard ascorbic acid with IC₅₀ values of 11.19±1.09 μg/mL and 9.30±0.26μg/mL in DPPH and nitric oxide free radical scavenging assays, respectively.  EFHE displayed substantial in vitro antioxidant potential in ABTS and hydrogen peroxide assays with IC₅₀ values of 40.38±0.88μg/mL and 99.11±13.59μg/mL respectively, while ITHE showed considerable activity with IC₅₀ < 100μg/mL in DPPH, NO and ABTS assays, as against the standard and other experimental samples tested.

 

 

Figure 1: Antioxidant activities of EEHE and its biofractions by DPPH,    

 

Hydrogen peroxide, Nitric oxide and ABTS free radical scavenging assays.

Each value is stated as the mean±SD (n = 3). Within each assay, the means with different superscript letters are statistically significant (ANOVA, p < 0.05, and subsequent post hoc multiple comparisons with Duncan's test).

 

3.4. In vitro anticancer activity:

The growth control of MCF-7 human breast cancer cell lines by sulforhodamine B (SRB) assay is demonstrated in Figure 2. After treatment with 80µg/mL of samples, EEHE, TFHE and ITHE showed a reduction in MCF-7 cell size, as the cells underwent significant changes in morphology. Treatment with TFHE, caused the cells to become rounder and compact, comparable to the treatment with standard Adriamycin, while EFHE and MFHE did not show significant changes in cell morphology (Figure 3). TFHE depicted significant growth inhibition of MCF-7 cells at GI₅₀ value of 10.22 ±6.72µg/mL, while EEHE and ITHE showed antiproliferative activity with GI₅₀ values of 41.42±3.74 µg/mL and 64.37±7.07µg/mL respectively, while EFHE and MFHE showed GI₅₀  > 80µg/mL, as against the standard drug Adriamycin (GI₅₀ < 10µg/mL). Hence, EEHE, TFHE and ITHE were further subjected to apoptosis and cell cycle analysis at their respective GI₅₀ concentrations. In the apoptosis assay, ITHE showed 11.31±0.82 % cells in late apoptosis and 34.48±1.57 % cells in necrosis as compared to standard Adriamycin that showed 13.67±1.02 % cells in late apoptosis and 8.58±0.65 % cells in necrosis (Figure 4 and 5). In cell cycle analysis, ITHE showed significant apoptotic activity with 20.15±1.37% cells in SubG₁ phase and 13.99±1.65 % cells arrested in G₂-M phase as compared to control (Figure 6 and 7).

 

 

Figure 2: Antiproliferative activity of EEHE and its biofractions against MCF-7 cell lines.

 

Each value is stated as the mean ± SD (n = 3). For each concentration, the means with different superscript letters are statistically significant (ANOVA, p < 0.001, and subsequent post hoc multiple comparisons with Duncan's test).

 

Figure 3: MCF-7 cells morphology of (a) control, (b) standard drug Adriamycin, (c-g) EEHE and its biofractions.

 

Figure 4: Apoptosis analysis of EEHE and its biofractions on MCF-7 cell lines.

Each value is stated as the mean±SD (n = 3). Within each stage, the means with different superscript letters are statistically significant (ANOVA, p<0.001, and subsequent post hoc multiple comparisons with Duncan's test).

 

Figure 5: Apoptosis analysis of (a) control, (b) standard drug Adriamycin, (c-e) EEHE and its biofractions (TFHE, ITHE) against MCF-7 cells.

 

 

Figure 6: Cell cycle analysis of EEHE and its biofractions on MCF-7 cells.

Each value is stated as the mean ± SD (n = 3). Within each stage, the means with different superscript letters are statistically significant (ANOVA, p < 0.001, and subsequent post hoc multiple comparisons with Duncan's test).

 

Figure 7. Cell cycle analysis of (a) control, (b) standard drug Adriamycin, (c-e) EEHE and its biofractions (TFHE, ITHE) against MCF-7 cells.

 

4. DISCUSSION:

Cancer is one of the prominent causes of morbidity and mortality worldwide, with current treatment regimens being very expensive, producing intolerable side effects, triggering drug resistance, and ultimately dramatically reducing the quality of life³³. Cancer cells are considered immortal as they possess an infinite potential for replication, growth, and proliferation by disturbing the normal cell cycle and apoptotic mechanisms³⁴. Apoptosis is important for normal physiological functions of the body and is also identified as one of the important targets for cancer prevention and treatment³⁵. Defects in apoptosis or dysregulation of the apoptotic program results in cancer, autoimmune diseases etc³⁶. Dysfunction of the cell cycle checkpoints that usually sense DNA damage and ensure repair results in genomic instability, accumulation of damaged DNA, uncontrolled cell proliferation and carcinogenesis³⁷. Oxidative stress also contributes to chronic diseases like autoimmune disorders, neurodegenerative diseases, cardiovascular disorders, cancer etc³⁸. Consumption of fruits, vegetables, and certain beverages (tea and wine rich in polyphenols) are rich sources of antioxidants and reduce the risk of these diseases. Plants are a promising source of therapeutic entities, e.g., Vincristine, etoposide, irinotecan, taxanes, curcumin and camptothecins are plant-derived anticancer compounds that have worked wonders in the prevention and treatment of cancer^33,39. Natural compounds are preferred therapeutic agents owing to their low toxicity levels and fewer side effects as compared to their synthetic counterparts⁴⁰. H. enneaspermus Linn is reported to have significant antioxidant activity, that may be primarily attributed to its high phenolic content^8-13. The results of our study illustrated the efficacy of the various biofractions of the ethanolic extract of H. enneaspermus Linn (EEHE) viz. TFHE, ITHE, EFHE, MFHE to scavenge the DPPH, hydrogen peroxide, nitric oxide and ABTS free radicals, wherein ITHE, MFHE and EFHE advocated the antioxidant activity (Figure 1) of the plant as previously claimed in the literature.

 

Based on the mechanisms at molecular level, breast cancer can be classified into three main subtypes i) Hormone receptor-positive [estrogen receptor (ER) ± or progesterone receptor (PR) ±]; ii) human epidermal growth factor receptor (EGFR) 2 (HER2) - positive; and iii) triple negative^30,41. Over three-quarters of breast cancers are ER (±) and can be treated with hormone therapies, e.g. tamoxifen and aromatase inhibitors. However, after five years of treatment, ER (±) patients become resistant to hormonal medication and need to be supplemented with adjuvant therapies.  Studies demonstrated that phytoconstituents like phenolics, flavonoids, tannins, nitrogen-containing compounds and triterpenes have good anticancer potential and can act as adjuvant therapies. Isoflavones like daidzein, genistein etc., inhibit proliferation and induce apoptosis of breast cancer cells; epigallocatechin gallate, a polyphenolic catechin suppresses growth and angiogenesis while increasing apoptosis; flavonols (quercetin, kaempferol etc.) and phytochemicals like resveratrol, lignans, curcumin possess antiproliferative activity against breast cancer cells⁴¹. Amongst the samples screened for anticancer activity, the toluene biofraction significantly inhibited the growth of MCF-7 human breast cancer cells, while the crude ethanolic extract EEHE and its insoluble toluene biofraction (ITHE) also depicted considerable antiproliferative activity by SRB assay, hence these samples were evaluated for their potential to induce apoptosis and cell cycle arrest.

 

Most chemotherapeutic agents exhibit anticancer action by activating a cascade of events, e.g. activation of caspases, inter-nucleosomal DNA fragmentation etc, resulting in apoptosis or producing cell cycle arrest ⁴². It was interesting to note that amongst all the experimental samples tested, ITHE induced significant apoptosis of the mammary cancer cells that was comparable to the standard Adriamycin, exhibiting prominent cell cycle arrest in G₂-M/SubG₁ phase, thereby affirming its anticancer activity by apoptotic and cell cycle arrest mechanisms.

 

The presence of secondary metabolites like polyphenolics, flavonoids etc. in ITHE may be responsible for its potential antioxidant, antiproliferative and pro-apoptotic anticancer activities ^{3,26,35,43-46}. Thus, ITHE can be effectively used as a potential candidate or adjuvant to chemotherapeutic treatment of breast cancer; however, further studies will help to validate its claim.

 

5. CONCLUSION:

On the basis of our observations, we can conclude that the methanolic, ethyl acetate and insoluble toluene biofraction of the ethanolic extract of H. enneaspermus Linn (EEHE) have significant antioxidant potential. While

 

TFHE, ITHE and EEHE depicted considerable antiproliferative activity against MCF-7 breast cancer cells, ITHE induced significant apoptotic activity of the mammary cancer cells along with cell cycle arrest. Thus, ITHE can be considered a promising agent for mammary tumour therapy with the capacity to combat oxidative stress and improve the quality of life. The future prospects of this study may involve the isolation of active compounds and the exploration of their mode of action against breast cancer using in vivo experimental models.

 

6. ACKNOWLEDGMENT:

The contributors are thankful to the authorities and Principal of Goa College of Pharmacy, Government of Goa, for providing the facilities to perform the research activities; Central research laboratory, Maratha Mandal’s NGH Institute of dental sciences and research centre, Belagavi, and Anticancer drug screening facility (ACDSF), Advanced Centre for Treatment, Research & Education in Cancer, (ACTREC) at Tata Memorial Centre, Navi Mumbai, India, for facilitating the anticancer research activities.

 

7. CONFLICT OF INTERESTS:

All authors have no conflict of interests.

 

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Accepted on 18.11.2022          © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(9):4127-4134.