Screening of Selected Ethno-Medicinal Plants for Anti-Cancer Activity

Cancer is an epidemic worldwide. As per the report of the National Centre for Health Statistics in 2019, approximately 606,880 patients were died due to cancer and 1,762,450 new cases were detected. To overcome this scenario, worldwide research is going on to combat this disease. Among these, treatment of cancer through plant phytochemical has attended much prominence in the last few decades. Phytochemicals are produced by plants for their defense mechanism. However, these can also be used to treat many deadly diseases, among them cancer is most common. In the present study, we selected three Ethno-medicinal plants namely Careya arborea (leaf), Ficus religiosa (leaf) and Amorphophallus campanulatus (tuber) to evaluate their efficacy against A549 human lung cancer cells. MTT assay is widely used to assess cell viability. Human lung adenocarcinoma cell lines were purchased from NCCS, Pune and maintained in the laboratory under all standard conditions. Cells were grown, using ready to use media containing 4.5 grams of glucose, L-glutamine and sodium pyruvate. Growth factors were made available to the cells through the fetal bovine serum. We treated the cells with different concentrations of methanol and distilled water extracts of the selected plants and investigate the cell viability using a statistical test. We found varied significant differences between control cells and the cells treated with plant extracts.


Introduction
Lung cancer is most common in men and the third most common cancer in women. In low and middle income countries 50% deaths are due to lung cancer [1]. Today, despite worldwide cancer eradication and awareness programmes, cancer established itself a killer disease around the globe.
When normal cells lose their regulatory mechanisms and halt apoptosis leads to abnormal growth of cells and tissue, called as cancer. These mechanisms make cell resistant to chemotherapeutic drugs [2]. Indeed, it took more than five decades to establish a systematic drug discovery on cancer what we have today. However, chemotherapeutics as a treatment in cancer do not devoid their intrinsic problems. Use of these agents for cancer treatment often creates more secondary complications in patient. For example, use of a chemotherapeutic agent, called as 5-fluorouracil is known to cause myelotoxicity, cardiotoxicity and often thought to act as a vasospastic agent in few documented cases [3,4]. Drug doxorubicin has been reported to cause renal toxicity, cardiac toxicity and myelotoxicity. Another widely used drug named bleomycin is known to cause pulmonary toxicity and cutaneous toxicity. Similarly, cyclophosphamide drug is used to treat many malignant conditions, has been reported for bladder toxicity in the 448 Screening of Selected Ethno-Medicinal Plants for Anti-Cancer Activity form of alopecia, immune suppression and hemorrhagic cystitis. Meaning, modern and advanced chemotherapeutic drugs are failed to fulfil the lung cancer treatment [4][5][6][7][8].
Therefore, there is regular and constant demand to develop new therapeutics against cancer to meet the safety and costing strategies. Hence development of new anti-cancer therapeutics using traditional natural products and plants has attracted much attention of scientific communities in the last 3 decades [9,10]. At present, about 60% anticancer drugs are derived from the plants due to its affordability and lower side effects (except few plants). Plants like Vinca alkaloids, Taxus diterpenes, Podophyllum lignans and Camptotheca alkaloids have been found most effective against cancer. Moreover, flavopiridol isolates from the Indian tree Dysoxylum binectariferum and compound like meisoindigo isolated from the Chinese plant Indigo feratinctoria have been reported for their potential anti-cancer activities than conventional drugs [2][3][4]. Approximately more than 3000 plant varieties worldwide have been known and used to treat cancer. In Asia, about 50% people use these plants and its derived compounds to treat cancer [1]. India, bestowed with a large biodiversity. From ancient time Indians have been using plant derived compounds to treat various ailments. Indeed, plants produces different kind of phytochemicals for their protection and development in different habitats, perhaps, hence selection of a same plant from different habitat and region may give different results [11]. Therefore, in present study we selected aforesaid three ethno-medicinal plants to check their efficacy against anti-cancer activities.

Plant Selection and Authentication
The selected plants are collected from Umred forest during July to November 2018. Plant authentication was done with the help of herbariums at the department of Botany, Dr Ambedkar College, Deekshabhoomi Nagpur.

Plant Extract Preparation
Selected plants were dried in a shed and then subjected to Soxhlet apparatus and Rotary evaporator for their methanol and distilled water extracts. Suitable concentrations of the extracts were prepared using 1% Dimethyl Sulphoxide.

Cell Line
Human lung adenocarcinoma A549 cells were purchased from the National Centre for Cell Science (NCCS) Pune and maintained in Ham's F12 nutrient mixture (Himedia), with 10% Fetal Bovine Serum (FBS) (Hyclone) and 1% streptomycin and penicilin antibiotic (Hyclone) at 37 °C at 5% CO 2 . Experiments were done on fourth passaged cells.

Cell Viability Assay
Cell viability was assessed using MTT assay. 2×10 4 cells were seeded in required wells of 96 well plate. Cells with media were incubated at 37°C in CO 2 incubator (Sartorious) for 24 hours with and without plant extracts. After 24 hours, media was removed and MTT (Himedia) was added at a concentration 5 mg/ml and again incubated for 4 hours. After 4 hours MTT was removed and purple coloured farmazon crystals dissolved in 100µl DMSO solution. Absorbance was measured at 570 nm using ELISA plate reader (Bio-Rad). Results were expressed in percentage using following formula:

Statistical Analysis
Statistical analyses were done using Medcalc statistical software. Student "t' test assuming unequal variance was used to assess the significant differences between studied groups. Data was presented in Mean±SEM. P<0.05 was considered as a significant level.

Results
We found varied significant differences when selected plants with different extracts at different concentrations compared with control. For Careya arborea, we found significant decreased cell viability for 10 µg/ml (p<0.0001) and 20 µg/ml (p<0.001) methanol extract. However, we found non-significant difference for 40 µg/ml methanol extract when compared with control. However, for distilled water extract, we found significantly increased cell viability for 20 µg/ml (p<0.05) and 40 µg/ml (p<0.01) concentrations. A non-significant difference was found when 10 µg/ml extract compared with control (Table 1; Figure 1: a and b). For Ficus religiosa, we found significant increased cell viability for 10 µg/ml (p<0.0001) and 40 µg/ml (p<0.001) methanol extract.

Discussion
Screening of plants for anti-cancer activities urge to search for more reliable and potent drugs. Worldwide research is going to derive natural product-based therapeutics not only for cancer but also to treat other ailments. So, being one of the greatest and diverse biodiversity, India should lead the world. Hence through this work, we drive to focus the plants of central India for deriving such novel therapeutics. Prodigious work done on plants has already explored publications on plant-derived compounds as novel drug molecules.
Moreover, to determine active biological compounds from the plants we need to explore our traditional knowledge about plants and natural product that we have gathered since ancient time against various diseases; by providing a scientific shred of evidence. Plants have various phytochemicals which may lead to in-vitro cell survival or cell death. By isolating such bio-active compound from plants we may treat various diseases [12,13]. Cancer is a condition where there is continuous cell growth with a lost apoptosis mechanism. This may lead to tumour formation resulting disrupt cellular mechanism and death. To suppress tumour growth numerous medications and the treatments are available, but a question is always posed for their reliability and safety [14]. Therapeutics derived from natural products may oppose this question and hence plant derived bioactive compounds have attended much importance in the field of cancer research [15]. Therefore, the present study aimed to screen three ethno-medicinal plants for anti-lung cancer activity using A549 human adenocarcinoma cells. In the present study, among all three selected ethno medicinal plants methanolic extract of Careya arborea exhibited the decreased cell viability as compared to the control. Result showed that this plant may have a property to treat human lung cancer. Methanolic extract of Careya arborea at the concentration of 10 ug/ml and 20 ug/ml exhibited the decreased cell viability, however with a concentration at 40ug/ml showed a non-significant difference. This is possibly due to the fact that, we used a crude methanolic extract of the plant, which contains numerous useful and unusual bioactive compounds [16]. Distilled water extract of the plant showed increased cell viability, suggesting its non-potential to treat A549 cells. Methanolic and distilled water extracts of Ficus religiosa and Amorphophallus campanulatus also showed significant increased cell viability suggesting their non-anti-cancerous activities. Of note, the possible reason for the non-linear relationship between dose and effects for all studies plants could be the crude extracts. Further, the phytochemicals or plants secondary metabolites synthesised by plants may promote cell growth and differentiation. Cancer is modulated by a variety of cellular signalling pathways, albeit, to inhibit cancer via modulating such signalling pathways plant must possess special kind of biochemical compounds that can modulate such cancer leading mechanisms. Although Ficus religiosa and Amorphophallus campanulatus did not show anti-cancer activities, their phytochemicals might use for other ailments; as each plant may synthesise different phytochemicals at different climatic conditions. Thus, one can predict that Careya arborea from different geographical locations and habitat conditions may exert different results.
Worldwide research on medicinal plants suggested their complete phytochemical and biochemical analysis, required to derive their best possible biological outcomes. Tinospora cardiofolia also is known as giloya in Hindi and heartleaf plant in English is a smooth climbing shrub often abundant in India, Myanmar, China and Shri Lanka. In Ayurveda, this plant is considered to be "amrita" for its longevity, youthfulness and vitality. This plant is also well known for its anti-inflammatory, anti-arthritis and anti-allergic properties often use against general debility, dyspepsia, fever, urinary diseases and jaundice. Stem extract found to be active against various skin diseases. This plant is also reported for high anti-oxidant content and immune-modulatory activities. Methanolic extract of Tinospora cardiofola contains alkaloid like choline, isocolumbin, tinosporin, columbine, palmatine, magnoflorine and tetrahydropalmatine as active components. Dose dependant increased concentration of methanolic extract of this plant found effective against HeLa cancer cell lines. However, it's anti-cancer activity also found in rats where it reported for rapid tumour suppression ata dose of 50 mg/Kg [4,[17][18][19][20][21][22][23][24].
Another example is related to Ziziphus nummularia, also known as beri in Hindi and jujube in English, is a shrub, with purplish stem, found abundant in India, Pakistan, Afganistan, Egypt etc. Different plant parts like root, bark, stem, flowers and seeds have been reported for different biological activities. The bark and the stem of this plant found rich in two phytochemicals called betulin and betulinic acid. Both compounds exhibited anti-cancer activities. Betulinic acid has been reported to induce apoptosis in cancer cells by increasing reactive oxygen species concentration, topoisomerase inhibition, mitogen-activated protein kinase (AMP kinase) cascade activation, and angiogenesis inhibition and by modulating pro-growth transcriptional activators. Moreover, betulinic acid has been reported to induce apoptosis by p53 and CD95 independent mechanism. It has been revealed that the combined treatment of betulnilic acid and other anti-cancer drugs induces loss of mitochondrial membrane potential and releases cytochrome-c and thereby induce apoptosis [4,[25][26][27][28][29][30][31][32].
From above cited examples it has been assumed that the selected plants of this study might possess properties against not only for cancer but also for other ailments and hence, demand in detail study to elucidated their phytochemicals rather active biochemical structures, to open a new window for researchers in central India.

Conclusions
Among selected three ethno-medicinal plants crude methanolic extract of Careya arborea might have a potential to treat human lung adenocarcinoma. Other two plants may have different types of potentials to treat varied diseases and ailments. Although this is a basic study providing first superficial inference; finding bioactive compounds using high-end techniques like high-performance liquid chromatography (HPLC) and Gas chromatography-mass spectrophotometry (GC-MS) analysis are required. Further, the bioactive guided assay may use as a track to elucidate enormous biological functions of these plants.