Tahira Aziz Mughal  ( Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan )
Fazilat Aslam  ( Department of Botany, Lahore College for Women University, Lahore, Pakistan )
Zubaida Yousaf  ( Department of Botany, Lahore College for Women University, Lahore, Pakistan )
Numrah Nisar  ( Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan )
Ping Chung Leung  ( Institute of Chines Medicine, The Chines University of Hong Kong, Hong Kong )

Objective: To evaluate the cytotoxicity of crude aqueous and ethanol extracts of Zaleya pentandra against oestrogen receptor-positive breast cancer cell line Michigan Cancer Foundation-7.

Method: The study was conducted at the Institute of Chinese Medicine, the Chinese University of Hong Kong, Hong Kong, from March to September 2017, and comprised Zaleya pentandra herbaceous perennial plant collected from Pakistani cities of Shakargarh, Lahore and Sialkot. Both aqueous and ethanol extracts were prepared in solvents following Soxhlet extraction technique. Rate of reduction in viability of cancer cell line was studied through MTT (dye compound 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay on the basis of time of incubation and the dose of extract. Analysis was performed using High Performance Liquid Chromatography with Diode Array Detector in order to found the compounds responsible for the death of cancer cells.

Results: Cell viability was observed to be dose-dependent (range: 16.7-37.4%) for aqueous extracts. Minimum inhibition concentration was 16.65% at 200μg/ml after 24 hours of incubation, whereas maximum inhibition was 37.39% at 3200μg/ml. Ethanol extracts showed less inhibition, with maximum inhibition being 25.29% at 1000μg/ml and minimum 13.57% at 62.5μg/ml. Certain polar compounds, like Hydroxytrosol and Tyrosol, could be obtained from the aqueous extracts only.

Conclusion: Zaleya pentandra aqueous extract was found to have potential benefit towards cytotoxicity of breast cancer cells.

Keywords: HPLC-DAD, Zaleya pentandra, ZP, Ethanol extracts, Cytotoxicity. (JPMA 70: 35; 2020). https://doi.org/10.5455/JPMA.299690

Introduction

Cancer, a disease as old as the humankind, despite all the advancements in medical sciences, is still a major health issue worldwide.1 There have been 28 types of cancers reported in 184 countries of the world. In 2008, 12.7 million new cancer cases and 7.6 million cancer related deaths took place, while almost 14.1 million new cancer cases and 8.2 million cancer-related deaths were recorded in 2012, indicating an alarming increase. 2 It was also estimated that the number of new cases would rise to 19.3 million per year by 2025. More than half of all cancers (56.8%) and cancer-related deaths (64.9%) in 2012 occurred in the less developed world. 2 Breast cancer is the leading cause of cancer deaths among females, and it is the most frequently diagnosed cancer, accounting for 23% of the total and 14% of related deaths. 3 In recent years it has become clear that breast cancer collectively consists of many molecularly-distinct tumours arising from the epithelial cells of the breast and does not represent a single disease. 4 In cancer research done using in vitro models, cell lines seem to be a key component for the molecular diagnosis in breast cancer cases as they can be widely used in numerous features of laboratory research. 5 Among all the breast cancer cell lines, Michigan Cancer Foundation-7 (MCF- 7) cells represent an important candidate as they are used universally in research for oestrogen receptor (ER)- positive breast cancer cell experiments and many of its sub-clones with varying nuclear receptor expression levels. 6MCF-7 cell line has been propagated for many years by multiple groups and is the most regularly used line in laboratory research worldwide as it is usually known to have low metastatic potential, and is a poorly-aggressive and non-invasive cell line. 7-9Clonal variations have been found in MCF-7 cells as extensive aneuploidy with important variations in chromosome numbers ranging 60-140 having been observed. Additional cytogenetic variances have also been reported about the presence or absence of specific marker chromosomes. 10 Literature suggests a raised level of genetic instability in MCF-7 cells. Due to different culture conditions, karyotype differences could reflect changes in selective pressure. Besides, MCF-7 cells comprise a division of stem cells able to create clonal va riability, and, as a model for breast tumour heterogeneity, this was proposed as an explanation for the heterogeneity of this cell line. Both the genomic and ribonucleic acid (RNA) expression levels of different MCF- 7 variants undergo divergence.10 MCF-7 cells have been used universally in research for oestrogen receptor (ER)-positive breast cancer cells. MCF- 7 cells are compatible for anti-hormone therapy resistance studies since they are easily cultured and, when treated with such targeted-therapy, they hold ER expression. Populations of MCF-7 cells modified to various anti-hormone environments have been produced in order to explore the properties of acquired antihormone resistant breast cancer cells. The only breast cancer cell line which has produced more data ever of practical knowledge for patient care than any other is the MCF-7 cell line. 6 Traditionally, herbal drugs have played a vital role in the cure of different diseases. In recent years, plant-based compounds have been preferred. Quinoline isolated from Quinine plant has been used in many synthetic medicines. 11 Zaleya Pentandra (Linn.) (AP) belongs to the genus Zaleya (Family: Aiozaceae). It is a prostrate and branched herb species found in Pakistan, especially in southern Punjab, Azad Kashmir, Cholistan and Nara deserts of Balochistan. 12,13Six Zaleya species are reportedly used in traditional medicines to treat and manage cancer, bronchial disease, fever, constipation, urinary tract infection as well as kidney and bladder stones etc. 14 ZP has anti-ulcerogenic activity through some unknown mechanism and it has been used in the area by traditional healers for a vast variety of ailments. Besides, it is also used as fodder for cattle in some desert areas. 15-17The plant reportedly contains antioxidant activity as well. 18 Pentandraon, a novel compound which is chemically a steroidal hormone, was isolated from nethanolic extract of ZP in the form of amorphous solid. 12 Another steroidal compound, named pentandraol, was isolated as amorphous solid from the methanolic extract of ZP which is probably responsible for the antibacterial activity against Bacillus (B.) spizizenii and Staphylococcus (S.) aureus bacterial strains, and the methanolic extract also showed antioxidant activity. 13 Another compound , pentandradiol, was isolated from the methanolic extract of ZP. The dichloromethane extract of ZP showed significant anti-diabetic activity against alpha ()- glucosidase. 19 The current study was planned to evaluate the cytotoxicity of crude aqueous and ethanol extracts of ZP against ER-positive breast cancer cell line Michigan Cancer Foundation-7 (MCF-7).

 

Materials and Methods

The study was conducted at the Institute of Chinese Medicine, the Chinese University of Hong Kong, Hong Kong, from March to September 2017, and comprised ZP herbaceous perennial plant collected from Pakistani cities of Shakargarh, Lahore and Sialkot. Fresh plant material, including root, stem and leaves, was processed by washing with distilled water to remove the foreign matter, like dust, dung, insects. They were then shade-dried and crushed with pestle and mortar into fine powder particles and stored for further experiments. The plant specimens were identified by Prof. Dr. Zaheeruddin Khan, a well-known taxonomist at The Government College University, Lahore, (www.gcu.edu.pk) and voucher specimen was deposited (PM # 667) in Prem Madan Herbarium, Lahore College for Women University, Lahore (www.lcwu.edu.pk). Water and ethanol were used as solvents; one is polar and the other non-polar. ZP extraction were carried out by Soxhlet extrac tion method. 20 Ethanol and water extraction were carried out at 60°C and 100OC respectively. Ethanol was evaporated under vacuum by rotary evaporator to yield crude ethanol extract. The water extract was first centrifuged and then stored in Freeze-dried mode. All solvents used were of analytical grade (Merck). ZP aqueous extract 32mg/ml was used to dissolve in the full Roswell Park Memorial Institute (RPMI) medium 1640 (Invitrogen GIBCO, NY, USA) containing 10% Foetal Bovine serum (FBS; Invitrogen GIBCO) and 1% penicillin, 1% streptomycin (PS; Invitrogen GIBCO) to prepare the stock solution while the highest concentration was 3.2mg/ml. The ethanol extract 200mg/ml was dissolved in dimethyl sulfoxide (DMSO; Sigma) first as stock solution and then in full RPMI medium to get the highest concentration 1mg/ml. The extracts were completely homogenised using ultra homogeniser (ultrasonic cleaner Branson 5510-sigma Aldrich). The 0.22m syringe filter was used to sterilise and filter the aqueous and ethanol stock solutions. Further dilutions were made with the full culture medium RPMI to the defined concentrations.21 Human breast cancer cell line MCF-7(American Type Culture Collection [ATCC], MD, USA). These cells originated from a metastatic adenocarcinoma which expressed ER. The cell lines were maintained in a humidified incubator at 37°C in 5% carbon dioxide (CO2) atmosphere. RPMI medium 1640, supplemented with 10% FBS (Invitrogen GIBCO), 100 units / ml penicillin and 100g/ml streptomycin (Invitrogen GIBCO). During MCF-7 harvesting, trypsin was used as the detaching agent for cells. Culture flasks containing RPMI medium with supplements were then transferred into new culture flasks for further growth and activation of cell lines. The cell lines were transferred into new culture flasks more than 4 times so that all the cells could get completely activated. Haemocytometer was used to count the harvested cells while the trypan blue exclusion test was used to determine cell viability. The 96-well flat-bottom Costar culture plates (Corning Inc., MA, USA) were used to seed the MCF-7 cells while keeping cell density 5x104 cells seeded per well in 100l of culture medium RPMI 1640 supplemented with 10% FBS, 1% penicillin and 1% streptomycin. The defined concentrations of the aqueous and ethanol ZP extracts were prepared in the culture medium containing MCF-7 cells, and 100l of solution was added to each well. Control wells were added with 100l of plain culture medium with MCF-7 cells. And 96 well plates containing cultured MCF-7 cells along with the ZP were then incubated for a defined time of 16 hours, 24 hours and 48 hours. ZP extract treated MCF- 7 cells. Proliferative response and cell death were determined using MTT cytotoxicity assay and cell viability assay. MTT is the abbreviation for the dye compound 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. This dye is helpful measuring the functionality (Cytotoxicity) of animal and human cells by calculating the activity of enzymes that reduce MTT. That is why it is called MTT assay. 21 After incubation of MCF-7 cells with the ZP extract for 16, 24 and 48 hours in 96-well plates, they were taken out of the incubator and 30l of  5mg/ml MT T in phosphate -buffered saline (PBS; Invitrogen GIBCO) was added to each well and the plates were incubated again at 37OC for 3-4 hours. They were then taken out and the entire liquid medium was pipetted out of the wells. One hundred microliter of DMSO (Sigma) was then added to each well. Absorbance of the dissolved solution was detected at 540nm by a Benchmark microtiter plate reader (Bio-Rad Laboratories, CA, USA). The absorbance of untreated cultured cells was considered 100%.22



To evaluate the effect of the ZP extract on cell viability, cells were exposed to the ZP extract at concentrations producing 50% growth inhibition (IC50) for the tumour cell lines, as determined by MTT assay, for 24 and 48 h. The viable cells were counted by trypan blue exclusion test. The percentage of dead cells was calculated from the ratio of dead cells to total number of cells x 100%.21 Both the aqueous and ethanolic extracts were subjected to HPLC analysis (Waters 600 Controller System) with the separation achieved at thermo-scientific NX 5MC18 column (250×4.6 mm) at 25OC. The detector photodiode detector (Waters 432 Controller System) was set at the wavelength of 235nm. Samples were sonicated briefly and passed through 0.45m membrane filter paper (Whattman, GE Healthcare) before analysis. Solvent phase was methanol (solvent A), and 0.1% formic acid in HPLC grade water (solvent B). Gradient programme commenced at 5% of A and 95% of B at 0 min reaching to linear gradient of 100% A and 0% B over 65 min and held at for 10 min in that composition at 1ml/min flow rate. The nature of the compounds was determined by comparing it with the literature data.^23,24,25 Further confirmation and concentration was achieved through Vicenin-2 as reference standard, due to its availability, following the protocol as suggested by Gouvea et al., 2017. Vicenin-2 is a flavonoid exhibit anti-inflammatory and anti-cancer activity and found in hydro alcoholic plant extracts. 26 The reference compound was dissolved in water and ethanol in similar manner asthe samples. The experiments were performed using complete randomised design in triplicate manner with six replicates  for each tested dilution / concentration of all the crude extracts. Data was expressed as mean% MTT absorbance (ratio of absorbance in extract-treated well to that of control well x 100%) ± standard deviation (SD) of three independent experiments with six wells each for in vitro studies. Data was entered in the form of spreadsheet in Microsoft excel and statistical analysis was done using one-way analysis of variance (ANOVA) in SPSS 25.0. P<0.05) was considered statistically significant.

 

Results

All the treatments with ethanol extract for 16 hours and 24 hours showed ER-positive MCF-7 breast cancer cells inhibition and the results were statistically significant (p<0.05) for 250, 500 and 1000μg/ml. The treatment at 48 hours with ethanol extract did not show the significant inhibition for cancer cells (p>0.05). All the treatments with ZP aqueous extracts showed apoptosis of cancer cells. The 200 and 400μg/ml aqueous extract treatment showed significant inhibition (p<0.05), while the 800, 1600 and 3200μg/ml aqueous extract treatment showed significant inhibition of ER-positive MCF-7 breast cancer cells (p<0.005) for 16 and 24 hours treatment. At 48 hours, no significant inhibition was observed (p>0.05) (Figures 1-2).





HPLC-DAD analysis revealed certain important polar and partially polar compounds from the extracts (Table).



Chromatograms showed certain compounds from aqueous extracts with Retention Time 24, 27 and 35 minutes (Figure 3).



Discussion

Extracts of ZP active in crude form were found to have a dose-dependent efficacy just like some studies have found in the case of Morinda lucida and Catharanthus roseus, reporting that activity increased with increase in concentration. 27-30The aqueous extract of ZP proved to be highly effective against apoptotic induction of cell death compared to the ethanol extract. Results revealed that higher the concentration, maximum would be the apoptotic cell cycle arrest and cell death. In case of aqueous extract,the maximum inhibition was observed in the highest concentration of 3200μg/ml treatment with (p <0.005), while in ethanol extract, maximum inhibition of ERpositive MCF-7 breast cancer cells was observed in the highest concentration of 1000μg/ml treatment. Maximum activity in aqueous fraction proved that compounds responsible for activity were polar in nature. Increase in activity while moving towards polar solvent indicated the presence of compounds in different fractions, but their quantity might be increased in aqueous fraction due to ability of water to dissolve more compounds than the other solvents. Water decoction is more effective than other non-polar extracts.31 The polarity trend was also seen in fractions of Aesculus indica and Aspidosperma tomentosum. 32 ZP is herbaceous perennial plant that has not been fully explored in laboratory to know its chemical profile and bioactive ingredients. The results of the current study, among other things, provide evidence of time - dependent efficacy of ZP extracts. Vicenin-2 is a flavonoid exhibit anti-inflammatory and anti-cancer activity and found in hydro alcoholic plant extracts. The presence of vicenin-2 in both extracts as evident from the HPLC analysis, revealed the secret behind the anti-cancer property of ZP extracts.26 ER-positive MCF-7 cells proliferation of cancer cells might be due to the presence of steroidal hormone-like compounds, such as pentandraon, pentandraol and pentandradiol, in the crude extract of ZP as has been reported in literature.12,13,19 Future studies on animal models are recommended in order to better understand the mechanism of ZP action and to move towards low-cost anti-cancer herbal drugs.

Conclusion

Zaleya pentandra aqueous extract was found to have potential benefit towards cytotoxicity of breast cancer cells.

Disclaimer: The text is part of a PhD thesis.

Conflict of Interest: None.

Source of Funding: The Higher Education Commission of Pakistan.

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