INTRODUCTION:

Phytomedicines are important component derived from various parts of the plants with various applications in pharmaceutical and herbal industry. Development of herbal remedies is more popular now a days due to less side effects and easy availabilities of medicinal plants (Nishaa et al., 2013; Udayaprakasha et al., 2015). Presences of bioactive secondary metabolites in the medicinal plants are more responsible to cure several diseases in mankind (Rafiqul et al., 2015). The practice of medicinal plants as herbal remedies in the treatment of various ailments, has been recorded way back since the ancient history. These plants remarkably serve as strong antioxidants and free radical scavenging agents, which ultimately results in prevention of autoimmune diseases and cancer (Ghate et al., 2014; Ghagane et al., 2017).

Cancer is a major public health problem affecting both developing and developed nations, but whereas 50% of cancer patients in developed countries die of the disease, in developing countries 80% of cancer victims are diagnosed with late-stage incurable tumors (Stewart and Wild, 2015; Senthilraja and Kathiresan, 2015). Herbal medicines are being practice at large scale for primary health care in developing countries (Rackley et al. 2006). Chemotherapy is one of the potential treatments for prolonging the patient’s life. According to World Health Organi zation (WHO) about 80% of world population relies on such systems of medicine (Ankad et al. 2014). About 2000 drugs of plant origin are derived from the evidences of different traditional and folktale practices as reported by Indian Materia Medica (Muralidharan and Narasimhan, 2012). Almost 60% of anticancer drugs are of natural origin, such as plants (i.e., vincristine, irinotecan, camptothecines) and microorganisms (i.e., doxorubicin, dactinomicines, mitomycin and bleomycin) (Grever, 2005; Senthilraja and Kathiresan, 2015).

Sapindaceae family is known for its traditional medicinal uses as a diuretic, stimulant, expectorant, natural surfactant, sedative, vermifuge and against stomachache and dermatitis in many parts of the world. Chemical investigations of this family have led to the isolation of saponins, diterpenes and flavonoids, among other secondary metabolites. Several saponin and acyclic sesquiterpene and diterpene oligoglycosides have been isolated as main secondary metabolites of several Sapindaceae species used in traditional oriental medicine (Cavalcanti et al., 2001). Ethnomedical information exposed that extracts from members of the family Sapindaceae are normally used for the treatment of boils, ulcers, pain, dermatological troubles, wound healing, diarrhoea and dysentery (Sofidiya et al., 2007). Taking into consideration the above facts, an attempt has been made to evaluate the anticancer activity of the medicinal plant Majidea zanquebarica J.Krik. ex Oliv. (Sapindaceae) syn. Harpullia zanquebarica used in the Indian traditional medicine system.

MATERIALS AND METHODS:

Collection and identification of the plant:

The source of material is Majidea zanquebarica J. Krikex Oliv. (Sapindaceae) was collected from in and around Coimbatore District, Tamil Nadu, India and authenticated by the Botanical Survey of India (No. BSI/SRC/5/23/10-11/tech-671).

Direct extraction with different solvents:

All the shade dried bark powder of M. zanquebarica was alone mixed with hexane separately at 1:10 ratio (w/v) in a clean flask and kept overnight under shaken condition. The process was repeated three times, but using fresh solvent at every extraction. The resultant extracts were filtered through Whatman No.1 filter paper. The solvents were removed by rotary evaporator, under reduced pressure (Buchi, Switzerland) at 40°C to yield thick syrupy extracts and used for further studies. The same residue was re extracted with methanol, filtered, evaporated, concentrated and also used for further studies.

In vitro cytotoxicity:

Cell culture:

Human cancer cell lines, such as PC-3 (Prostate carcinoma), A-431 (Epidermoid carcinoma), HeLa (Human cervical carcinoma) and MCF (Breast cancer) were obtained from National Centre for Cell Science (NCSS), Pune. The cell lines were cultured in 1.1 mixture of Dulbecco’s modified Eagles’s Medium/Hams F12 (GIBCO) supplemented with nutrients, amino acids and vitamins. 10% heat inactivated fetal bovine serum (FBS) and antibiotics such as penicillin and streptomycin were added and the culture was maintained at 37°C in an atmosphere of 5% CO₂ and 95% humidity. Cells were passaged weekly and the culture medium was changed every 3-4 days. Cultures were allowed to reach 80% confluence before experiments were performed.

In vitro MTT Assay:

Materials required:

DMEM/F-12 (GIBCO) medium preparation, fetal bovine serum (10%), 10ml of FBS was made up to 100ml with DMEM medium. MTT 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide), PBS, Trypsin, Penicillin (100units/ml), Streptomycin (100 µg/ml), DMSO (Sigma)

Procedure:

The hexane and 70% methanol extract were added as 2X concentration to the cell in
100μl volumes and the concentration range from 100, 10, 1, 0.1, 0.01μg/ml. The plates were further incubated for 48hrs in the CO₂ incubator. Cell viability was then determined using the MTT assay. Briefly, 50μl of MTT (5mg/ml in PBS) was added to each well and incubated for 2 h, and then the plate was centrifuged at 1500rpm for 5 min at 4°C. The MTT solution was removed from the wells by aspiration. The absorbance was recorded at a wavelength of 570nm on a Synergy H4 Multimode Microplate Reader (BioTek). Each concentration was performed in quadruplicate and cumulative variation was maintained less than 20% between the data points. The growth inhibitory (GI) effect of the hexane and 70% methanol was determined by comparing the optical density of the treated samples against the optical density of the control. Vehicle-treated cells were taken as 100% viable. From the optical densities, the percentage growth was calculated using the following formula:

If T is greater than or equal to T₀, then the

Percentage growth = 100× [(T-T₀)/(C-T₀)] and

If T is less than T₀, then the

Percentage growth = 100× [(T-T₀)/T₀)], where

T is the optical density of test,

C is the optical density of control,

T₀ is the optical density at time zero.

From the percentage growth, a dose response curve was constructed and GI-50 value was interpolated from the growth curve.

RESULTS AND DISCUSSION:

Interest in the pharmacological effects of bioactive compounds on cancer treatments and prevention has increased dramatically over the past twenty years. It has been shown to possess numerous anti-cancer activities in various cancer cells through different forms of cytotoxic effects without exhibiting considerable damage to normal cells (Katiyar et al., 2009; Mantena et al., 2006a; Senthilraja and Kathiresan, 2015). The results of the cytotoxicity of hexane and 70% methanol extract of M. zanquebarica was evaluated in 5 logarithmic concentrations (100, 10, 1, 0.1, and 0.01µg and DMSO as vehicle control) in four cancer cell lines, such as PC-3 (Prostate carcinoma), A-431 (Epidermoid carcinoma), HeLa (Human cervical carcinoma) and MCF (Breast cancer), using MTT assay are summarized in (Figs.1 and 2). Among the extracts, hexane bark extract had shown more percentage of inhibition on A431 cell lines. Out of the different concentrations used, the hexane extract showed IC₅₀ value of 100 µg/ml concentration in all the cell lines.

Figure 1: Percentage growth against Bark methanol extract of M. zanquebarica

Figure 2: Percentage growth against Bark Hexane extract of M. zanquebarica

Recently, a plant derived bioactive substance that is capable of selectively arresting cell growth in tumour cells has received considerable attention in cancer chemopreventive approaches (Galati et al., 2000; Jang et al., 2005; Selvaraj and Agastian et al., 2017). Understanding the occurrence of phytochemicals in medicinal plants is advantageous and presently, the discovery of new drug compounds or lead molecules from plants is mainly based on the systematic examination of different plant extracts or plant based products. Also, this preliminary knowledge can decipher a new source for economically valued chemical compounds (Gezahegn et al., 2015; Mohanty et al., 2014). Current anti-estrogen medicine, tamoxifen, is widely used in the prevention and treatment of estrogen receptor positive breast cancer (Lazarus et al., 2009). Synergistic effects of various components of plant extracts are believed to contribute to effectiveness of herbal preparations used in traditional medicine. Synergy research in phytomedicine is really needed to address this issue. Many chemical substances derived from medicinal plants are known to be effective and versatile chemo-preventive agents in a number of experimental models of carcinogenesis. This finding indicates the significance of the selected plant as a potential resource for the discovery of novel leads for maneuvering them as effective anticancer agents.

CONCLUSION:

It can be summarized that hop- 22(23) en- 3β- ol triterpenoid saponins isolated from bark extract of M. zanquebarica have significant anticancer activities against A431, MCF-7, HeLa and PC-3. The findings suggest that the plant could be regarded as a promising alternative for the development of efficient and effective anticancer drug from natural sources. Nevertheless, the research data of the present findings may serve as a guideline for the standardization and validation of natural drugs containing the selected medicinal plants as ingredients.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 21.05.2020 Modified on 16.06.2020

Research J. Science and Tech. 2020; 12(3):173-176.

DOI: 10.5958/2349-2988.2020.00023.6