In vitro Efficacy of Some Plant Extracts Against Plant ...

Middle-East Journal of Scientific Research 24 (8): 2450-2459, 2016 ISSN 1990-9233 © IDOSI Publications, 2016 DOI: 10.5829/idosi.mejsr.2016.24.08.102173

In vitro Efficacy of Some Plant Extracts Against Plant Tumour and MCF-7 Breast Cancer Cell Line Paras Jain, H.P. Sharma and Fauziya Basari Laboratory of Plant Physiology and Biotechnology, University Department of Botany, Ranchi University, Ranchi, India Abstract: The aim of present study was to evaluate the anticancer activity of some plant extracts on plant tumour and MCF-7 Breast Cancer cell line. Five medicinal plants were selected for scientific validation of ethnomedicinal knowledge collected from local people of Jharkhand. The cytotoxicity of four different concentrations of plant extracts on MCF-7 was evaluated by the SRB assay method. The efficacy of plant extracts on plant tumour was screened by Crown Gall Potato Tumour Assay. At the concentration of 80 µg/ml all the plants showed more than 50% inhibition of MCF-7 cell line growth with a maximum of 99.5 % shown by Semecarpus anacardium followed by Phyllanthus amarus (73.7%)>, Gloriosa superba (68.7%)>, Bacopa monnieri (66.6%) and Plumbago zeylanica (56.5%). Similar result were also found in the case of plant tumour at the concentration of 1000µg/Disc Semecarpus anacardium exhibited a striking 85% inhibition followed by 79% by Phyllanthus amarus, 72% by Gloriosa superba, 66% of Plumbago zeylanica and 60% by Bacopa monnieri. After getting encouraging result, Semecarpus anacardium was subjected to gas chromatography and mass spectroscopy. The GC-MS study summarized the presence of a total 32 phytochemical constituents in methanolic extracts of Semecarpus anacardium. These constituents may be responsible for pharmacological activities. Key words: Cancer

SRB Assay

Crown Gall Tumour Assay

INTRODUCTION Cancer is among one of the biggest health problems, the world is facing today. A significant part of drug discovery in the last forty years has been focused on agents to prevent or treat cancer. This is not surprising because, in most developed countries and to an increasing extent, in developing countries, cancer is amongst the three most common causes of death and morbidity [1]. The process of cell renewal and cell death is delicately balanced and various type of mature cells in the body have a given life span; as these cells die new cells are generated by the proliferation and differentiation of various types of stem cells. Sometimes cell do not respond to normal growth control mechanisms. These cells give rise to clones of cells that can expand to a considerable size, producing a tumour or neoplasm [1].

MCF-7

Medicinal Plants

GC-MS Analysis

Treatments for cancer may involve surgery, radiotherapy and chemotherapy and often a combination of two or all three is employed. Due to the toxic and adverse side effects of synthetic drugs as well as conventional treatments are being failed to fulfill their objectives (tumour control), for these consequence herbal medicine has made a comeback to improve the fulfillment of our present and future health needs [2]. Medicinal plants are rich in phytochemicals which are used for curing of various human diseases and also play an important role in healing. Large number of plants and their isolated constituents has been shown to possess potential anticancer activity. As they are valuable source of novel cytotoxic agents and are still in performance better role in health concern [3]. Out of huge number of ethnomedicinal plants screening for their anticancer activities is prerequisite for any advance study. Therefore different bioassays offer vast advantages for screening of

Corresponding Author: Paras Jain, Laboratory of Plant Physiology and Biotechnology, University Department of Botany, Ranchi University, Ranchi, India. Tel: +91-9693329694.

2450

Middle-East J. Sci. Res., 24 (8): 2450-2459, 2016

medicinal plant extracts for different purposes, i.e. antitumour, antibacterial, antioxidant, phytotoxic properties. Potato disc bioassay is one of the methods which is based on Agrobacterium tumefaciens infection on potato disc, where tumour inhibition with plant extract is one of the positive signs for plants to be studied for their anticancer activities. Crown-gall is a neoplastic disease of plants caused by A. tumefaciens following by the transfer and expression of its special type of DNA segment (TDNA) in the plant genome through type IV secretion system (T4SS) [4]. The inhibition of crown-gall tumour initiation on potato discs showed good agreement with plant extracts and its anticancer drug. The number of similarity between crown gall and animal tumour has been proved and documented by various scientists. Tumour can form in both plant and animal kingdom. There is a very close relation of carcinogenic process in both plant and animals. In plants tumour growth is known as” GALL” and the causative agent is the species of Agrobacterium. Malignancy in plants similar to that of animals has been reported as long back as in 1920 [5]. Ullrich and Aloni [6] has nicely explained the similarity of plant tumour with animal tumour, i.e., The genetic/epigenetic alteration of a cell or group of cells leading to loss of cell cycle control, subsequent unchecked cell proliferation and the production of a macroscopic, generally undifferentiated tumour and diversion/development of vasculature to feed the tumour structure through angiogenesis (in animals) or vascularization (in plants). The sulforhodamine B (SRB) assay, which was developed in 1990, remains one of the most widely used methods for in vitro cytotoxicity screening [7]. This assay has been widely used for drug-toxicity testing against different types of cancerous and non-cancerous cell lines [8]. The SRB assay provided a rapid and sensitive method for measuring the drug-induced cytotoxicity in both attached and suspension cultures in 96-well microtiter plates. SRB is a bright pink Aminoxanthine dye with two sulfonic groups. Under mild acidic conditions, SRB binds dye to basic amino acid residues in TCA (Trichloro acetic acid) fixed cells to provide a sensitive index of cellular protein content that is linear over a cell density range of visible at least two order of magnitude [7, 9]. If the effective plant extract would be find out for the inhibition of tumour forming mechanism

and cancer cell line growth inhibition, it would be used in drug developmental research for tumour treatment in human. In present study we had selected Five Plant for the scientific authentication and validation of ethnomedicinal knowledge collected by local people of Jharkhand. Bacopa monnieri L. (family: Scrophulariaceae) locally known as Brahmi is a reputed drug of Ayurveda. It is widely used in traditional medicine to treat various nervous disorders, cardio tonic, digestive aid, memory enhancer. The main principal bioactive compound of this plant is Bacosides [10]. Phyllanthus amarus (Family: Euphorbiaceae) is a herb locally known as Bhumyamala. It is an annual herb (weed) which grows in the wild after first showers of monsoon. Maharshi Charak, Indian Mythology has considered this herb to be most effective in the treatment of jaundice, asthma, increasing appetite, improving digestion, stimulating liver and producing laxative effects. In the Unani System of medicine this herb is good for sores and chronic dysentery. Its seeds are used in the treatment of ulcers, wounds, scabies and ringworms. The main principal bioactive compound of this plant is lignin [11]. Plumbago zeylanica Linn (Family: Plumbaginaceae) is shrub which is locally known as Chitrak and Doctorbush. The literature reveals its wide application in traditional system of medicines against various diseases, as anti-inflammatory, anti-malarial, anti-fertility, antimicrobial, anti-oxidant, blood coagulation, wound healing, memory enhancer and anti-cancer. The main principal bioactive compound of this plant is plumbagin [12]. Gloriosa superba Linn (Family: Liliaceae) is an endangered plant locally known as Kalikari or Agnisikha. The plant grows in sandy-loam soil in the mixed deciduous forests in sunny positions. Traditionally it is used for the treatment of ulcers, leprosy, piles, inflammations, intestinal worm infestations, thirst, bruises, skin problems and snakebite. It is poisonous, toxic enough to cause human and animal fatalities if ingested. Gloriosa species contain the most important alkaloid “colchicines” [13]. Semecarpus anacardium L. (family Anacardiaceae) is a tree commonly known as “Bhilawa”. It is found in moist tropical forests. It is a plant well-known for its medicinal value in ayurvedic and siddha system of medicine. Several experiments have proved its antiatherogenic, anti-inflammatory, antioxidant, antimicrobial,

2451


anti-reproductive, CNS stimulant, hypoglycemic, anticarcinogenic and hair growth promoter activities. The most significantcomponent of the S. anacardium Linn. is bhilwanols [14]. The aim of present investigation was to scientific determination of anticancer efficacy of some plant extracts on plant tumour and MCF-7 Breast Cancer cell line.

(Known anticancer drug) was used as positive control replacing test extracts. The potato discs were incubated for 20 days at 30°C incubator, after which Lugol’s solution (10% KI, 5% I2) was added, the tumour counts were done under a stereo microscope and compared with negative controls (DMSO in the place of extract). Percent inhibition of tumour was calculated as described [16]. More than 20% tumour inhibition is considered significant [17].

MATERIALS AND METHODS Human Cell Lines: Human breast cancer cell lines namely MCF-7 were grown in T-75 flasks containing 50 mL of RPMI-1640 medium with glutamine, bicarbonate and 5% fetal calf serum. Medium was changed at 48-hour intervals. The cell lines were maintained at 37°C in a 5% CO2 atmosphere with 95% humidity. Cells were dissociated with 0.25% trypsin and 3 mW 1, 2cyclohexane diamine tetra acetic acid in NKT buffer (137 nuW NaCl, 5.4 mAf KC1 and 10 mAf Tris; pH 7.4). Experimental cultures were plated in microtiter plates (Costar, Cambridge, MA) containing 0.2 mL of growth medium per well at densities of 1, 000-200, 000 cells per well.

Collection of Plant Materials: Roots of Plumbago zeylanica, nut of Semecarpus anacardium, rhizome of Gloriosa superba, whole plant of Phyllanthus amarus and Bacopa monnieri were collected from the Botanical Garden, University Department of Botany, Ranchi University. The roots were washed thoroughly 2-3 times with running tap water and kept in shade to dry after final washing with autoclaved water. Solvent Extraction: The methanolic extracts were prepared. Sohxlet extraction was done for Semecarpus anacardium and remaining four plant cold extraction were performed. The dried powders of plant mix with desirable amount of methanol (1:10 ratio) were placed in shaker incubator for 48 hours. The extract was filtered; the filtrate obtained was dried and dried extract was re dissolved in Dimethylsulphoxide (DMSO) to form stock solutions, which were filter sterilized (0.2µm) before testing.

In vitro Assay for Cytotoxic Activity: In vitro cytotoxicity of five plants against MCF-7 human breast cancer cell lines was determined using sulforhodamine B assay (SRB) as described previously. The cell growth was measured using ELISA reader after staining with Sulforhodamine B dye (SRB) which binds to basic amino acid residues in the trichloroacetic acid (TCA) fixed cells.

Collection and Maintenance of A. tumefaciens Strain: Two Agrobacterium tumefaciens strains (NCIM 2939 and NCIM 2145) were collected from National Collection of Industrial Microorganism (NCIM), National Chemical Laboratory, Pune and maintained in YEB medium for further use. Antitumour Potato Disc Bioassay: Antitumour assay of plant extracts was performed according to standard potato disc bioassay [15]. Fresh, disease-free potatoes were obtained from a local market. With the help of cork borer potato discs cut into 0.5 cm diameter in size. Disk were surface sterilized by immersion in HgCl2 0.1% for 5 min, washed 3-4 times with autoclaved distilled water. 5 disks were placed in each Petri plates containing 1 percent autoclaved distilled water Agar medium. Disks were immersed with 600 µl plant extract + 150 µl sterilized distilled water (SDW) and 750 µl A. tumefaciens in PBS suspension for assay. Adriamycin

Preparation of Cell Suspension for Assay Cell Fixation: Different concentration (10, 20, 40 and 80 µl) of test sample with DMSO (vehicle control) and positive control were added in culture medium to the wells containing the cells. Only DMSO was added in control wells. Washing cultures with buffer prior to fixation to remove serum protein commonly causes cell detachment and loss. To avoid this potential problem, cultures were fixed with TCA before washing. Cells attached to the plastic substratum were fixed by gently layering 50 µl of cold 50% TCA (4°C) on top of the growth medium in each well to produce a final TCA concentration of 10%. The cultures were incubated at 4 °C for 1 hour and then washed five times with distilled water to remove TCA, growth medium and low-molecular-weight metabolites and

2452


serum protein. Plates were air dried and then stored until use. Background optical densities were measured in wells incubated with growth medium without cells. Cells in suspension were allowed to settle out of solution. When these cells were physically resting on the bottom of the wells, 50 µl of cold 80% TCA (4 °C) was gently layered on top of the overlying growth medium. The cultures were left undisturbed for 5 minutes and then refrigerated at 4°C for an additional hour of fixation. This procedure led to the attachment of single cell suspensions to the plastic substratum, provided that cells were in contact with it when the fixative was applied.

set at 270 and 280°C and split ratio is 20 throughout the experiment periods. The ionization mass spectroscopic analysis was done with 70 eV. Mass spectra were recorded across the range of 40 to 1000 m/z for the duration of 35 min. Identification of components was based on comparison of their mass spectra with those of Wiley and NIST libraries and those described by Adams [18] as well as on comparison of their retention indices with literature [19, 20]. RESULTS AND DISCUSSION

SRB Assay: TCA-fixed cells were stained for 30 minutes with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. At the end of the staining period, SRB was removed and cultures were quickly rinsed four times with 1% acetic acid to remove unbound dye. The acetic acid was poured directly into the culture wells from a beaker. Residual washed solution was removed by sharply flicking plates over a sink, which ensured the complete removal of rinsing solution. Because of the strong capillary action in 96-well plates, draining by gravity alone often failed to remove the rinse solution when plates were simply inverted. After being rinsed, the cultures were air dried until no standing moisture was visible. Bound dye was solubilized with 10 mM unbuffered Tris base (pH 10.5) for 5 minutes on a gyratory shaker and OD was measured at 510 nm. Calculations: Cell viability and growth in presence of test material was calculated as follows:

Percent growth inhibition in presence of test material was calculated as under: 100- Percent growth in presence of test material GC-MS Analysis: The GCMS analysis of Plant extracts was performed using GC-MS SHIMADZU MS 2010 instrument equipped with AB innowax column (60 × 0.25 mm id, film thickness 0.25 µm). Initially, oven temperature was maintained at 50×C for 3 min and temperature was gradually increased up to 280°C at 30 min and 0.2 µl of sample was injected for analysis. Helium was the carrier gas. The flow rate of helium gas was 1.2 ml/min. The sample injector and mass transfer line temperature were

Crown Gall Potato Tumour Assay: The potato disc assay is an inexpensive and reliable bioassay that provides useful indication of antitumour activity of test samples by the inhibition of the development of crown gall tumour on the disc of potato tubers. In fact, the inhibition of Agrobacterium tumefaciens induced tumours (or Crown Gall) in potato disc is an assay based on antimitotic activity and can detect a broad range of known and novel antitumour effects [21]. This assay is based on the hypothesis that antitumour agents might inhibit the initiation and growth of tumours in both plant and animal systems, because certain tumorigenic mechanisms are similar in plants and animals [15]. The Methanolic extracts of selected plants were assessed for antitumour activity using the Crown gall potato tumour assay as described in materials and methods, the result of which have been presented in Table 1. Phyllanthus amarus and Gloriosa superba showed more than 50% inhibition at a concentration of 100µg/Disc, Simultaneously Semecarpus anacardium and Plumbago zeylanica also exhibited significant inhibition as shown in Table 1. At a higher dose of 1000 µg/Disc Semecarpus anacardium exhibited a striking 85% inhibition followed by 79% by Phyllanthus amarus, 72% by Gloriosa superba, 66% by Plumbago zeylanica and 60% by Bacopa monnieri. All extracts showed considerable level of antitumour activity. The extracts exhibited dosedependent inhibition of the growth of gall tumour caused by Agrobacterium tumefaciens. Significant tumour inhibition was observed at 100µg/Disc and 1000µg/Disc concentrations, but not at 10µg/Disc. The results were compared with negative control (without treating plant extract; only Agrobacterium tumefaciens strains) and the percent of tumour inhibition was calculated.

2453

Middle-East J. Sci. Res., 24 (8): 2450-2459, 2016 Table 1: Effect of different plant extracts on tumour inhibition percentage

Plant name

Tumour inhibition percentage ----------------------------------------------------------------------------------------------------------------------10µg/Disc 100µg/Disc 1000µg/Disc

Semecarpus anacardium L. Plumbago zeylanica L. Gloriosa superba L. Phyllanthus amarus L. Bacopa monnieri L.

26 24 28 30 21

48 39 54 62 42

85 66 72 79 60

Table 2: Effect of different plant extracts on MCF-7 Breast Cancer Cell Line Human Breast Cancer Cell Line MCF7 % Control Growth Drug Concentrations (µg/ml)

Pz Bm Sa Gs Pn ADR

Experiment 1 -------------------------------10 20 40 80

Experiment 2 ------------------------------10 20 40 80

Experiment 3 ---------------------------------10 20 40 80

Average Values --------------------------------------------------------------------10 20 40 80

90.8 89.4 32.9 21.4 22.2 -9.1

87.5 99.7 73.6 59.4 52.9 15.5

86.3 85.2 61.6 39.5 37.9 10.1

88.2 ±2.33 91.4 ±7.46 56.0 ±20.9 40.1 ±19.35 37.7 ±15.35 5.5 ±12.92

83.4 79.6 26.9 21.3 19.9 -12.5

66.0 50.1 12.9 20.2 18.4 -31.5

41.9 23.2 -14.0 19.4 11.2 -59.8

87.0 90.4 71.3 51.0 49.6 7.3

71.4 79.7 54.8 46.8 43.3 -9.7

44.9 37.6 9.9 42.2 38.7 -41.9

83.1 82.4 52.2 38.8 37.5 8.5

67.0 63.5 37.1 37.2 33.6 -16.9

43.7 39.5 5.6 32.3 29.0 -51.1

84.5 ±2.17 84.1 ±5.60 50.1 ±22.27 37.0 ±14.92 35.7 ±14.93 1.1 ±11.79

68.1 ±2.87 64.4 ±14.82 34.9 ±21.03 34.7 ±13.47 31.8 ±12.55 -19.3 ±11.10

43.5 ±1.50 33.4 ±8.91 0.5 ±12.74 31.3 ±11.43 26.3 ±13.94 -50.9 ±8.95

Pz = Plumbago zeylanica; Bm= Bacopa monnieri; Sa= Semecarpus anacardium; Gs= Gloriosa superba; Pn= Phyllanthus amarus; ADR= Adriamycin (Doxorubicin; Known drug)

Phyllanthus amarus(73.7%)>, Gloriosa superba(68.7%)>, Bacopa monnieri (66.6%) and Plumbago zeylanica(56.5%).

The results were also compared with positive control (Camptothecin- an established anticancer drug), which completely inhibited the growth of gall tumour on potato discs. This result may be attributed to its DNA damaging activities. Camptothecin is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I (topo I) and is isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree) [22]. SRB Assay: SRB assay was performed by using various concentrations (10, 20, 40 and 80 µg/ml) of plant extract. The experiment was repeated for three time and results were summarized in Table 2 which shows anti-proliferative activity of the above plants. A dose dependent response for cell growth inhibition was shown by various extracts (Table 2). However, considerable variation was seen in magnitude of inhibition and amount of extract. P. amarus and Gloriosa superba showed more than 50% inhibition (62.3 & 59.9 % respectively) for MCF-7 cell proliferation even at a small dose of 10 µg where as Semecarpus anacardium, Plumbago zeylanica and Bacopa monnieri exhibited 44, 11.8 & 8.6 % growth inhibition respectively at the same concentration. At the concentration of 80 µg/ml all the plants showed more than 50% inhibition with a maximum of 99.5 % shown by Semecarpus anacardium followed by

GC-MS Analysis: After getting encouraging result of nut extract of Semecarpus anacardium plant against MCF-7 breast cancer cell line and plant tumor, we had done phytochemical analysis of this plant through gas chromatography and mass spectroscopy. In the GC-MS analyses, total 32 compounds were identified in the Methanolic extract of Semecarpus anacardium. The identification of phytochemicals is based on the peak area (which represents the percentage of that compound), molecular weight and molecular formula. The chromatogram are present in Figure 1. 2-(1Oxohexadecyl)-5-methylpyrrole(33.13%), Bi-1-cycloocten1-yl (12.18%), Oleic Acid (11.20%), p-Menth-3-en-9-ol (10.95%), n-Hexadecanoic acid (5.72%), Asarone (4.45%), are the major components whereas some other minor components are also present (Table 3, Fig. 3). This study highlighted the presence of many secondary metabolites in the nut parts of Semecarpus anacardium, provide an overview of the different classes of molecules present that have led to their pharmacological activities. This study confirmed that the plant extract could be used for the treatment of various diseases. The GC-MS analysis of extracts showed the

2454


Fig. 1: Effect of plant extracts on MCF-7 breast cancer cell line growth curve Pz = Plumbago zeylanica; Bm= Bacopa monnieri; Sa= Semecarpus anacardium; Gs= Gloriosa superba; Pn= Phyllanthus amarus; ADR= Adriamycin (Doxorubicin; Known drug)

Fig. 2: Chromatogram (GC/MS) of the methanolic extract of S. anacardium 2455

Middle-East J. Sci. Res., 24 (8): 2450-2459, 2016 Table 3: Chemical constituents present in the methanolic extract of S. anacardium Peak

R. Time

Area

Area% Name of compounds

Molecular formula Mol. Weight

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

5.75 8.714 11.298 11.659 12.456 13.185 13.656 14.699 14.891 15.092 15.145 15.277 16.595 16.812 16.98 17.157 17.437 18.304 19.133 19.938 20.192 20.699 20.975 21.042 21.441 21.498 21.609 22.326 22.393 23.125 23.233 30.982

225746 435583 388010 7311618 417481 433704 1231333 2508392 997313 1206716 1104158 9392959 2580728 258808 18414139 3413901 1005915 1028264 1214529 20021871 6937587 881280 229446 154758 54451705 18003775 5103803 397830 382821 370266 2246812 1599780

0.14 0.27 0.24 4.45 0.25 0.26 0.75 1.53 0.61 0.73 0.67 5.72 1.57 0.16 11.2 2.08 0.61 0.63 0.74 12.18 4.22 0.54 0.14 0.09 33.13 10.95 3.11 0.24 0.23 0.23 1.37 0.97

DODECANE Tetradecane Tetradecane Asarone alpha.-Bisabolol Tetradecanoic acid 9-Octadecenoic acid (Z)-, methyl ester 9-Hexadecenoic acid, methyl ester, (Z)Hexadecanoic acid, methyl ester cis-9-Hexadecenoic acid 9, 12-OCTADECADIENOIC ACID (Z, Z)n-Hexadecanoic acid 9-Octadecenoic acid, methyl ester, (E)Octadecanoic acid, methyl ester Oleic Acid Octadecanoic acid Eicosane Eicosane HEXACOSANE Bi-1-cycloocten-1-yl Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester Tetracontane 2-CYCLOHEXYLIDENE-1, 3-DIMETHYLIMIDAZOLIDINE Eicosanoic acid, 2-[(1-oxohexadecyl)oxy]-1-[[(1-oxohexadecyl)oxy]methyl]ethy 2-(1-Oxohexadecyl)-5-methylpyrrole p-Menth-3-en-9-ol

C12H26 C14H30 C14H30 C12H16O3 C15H26O C14H28O2 C19H36O2 C17H 32O2 C17H34O2 C16H30O2 C18H32O2 C16H32O2 C19H36O2 C19H38O2 C18H34O2 C18H36O2 C20H42 C20H42 C26H54 C16H26 C 19H 38O 4 C40H82 C11H20N2 C 55H 106O 6 C21H 37NO C10H 18O

170 198 198 208 222 228 296 268 270 254 280 256 296 298 282 284 282 282 366 218 330 562 180 862 319 154

Tetratriacontane Z-6, 17-Octadecadien-1-ol acetate p-Menth-3-en-9-ol 3, 4, 4-TRIMETHYL-3-(3-OXO-BUT-1-ENYL)-BICYCLO[4.1.0]HEPTAN-2STIGMAST-5-EN-3-OL, (3.BETA.)-

C34H70 C20H 36O2 C10H18O C14H 20O 2 C29H50O

478 308 154 220 414

presence of various types of anticancer compounds like 9-octadecenoic acid (Z) - methyl ester (C19H36O2) having anti-carcinogenic activity. Cancer is a disease recognised by seven hallmarks: unlimited growth of abnormal cells, self sufficiency in growth signals, insensitivity to growth inhibitors, evasion of apoptosis, sustained angiogenesis, inflammatory microenvironment and eventually tissue invasion and metastasis [23, 24, 25]. From the year 1981–2002 reports showed that approximately 60% of anticancer agents are derived from natural products. Herbal drugs do not only serve as drugs but also provide a rich source of novel structures that may be developed into novel anticancer agents [26]. Plant-based compounds are well known for the development of several clinically useful anti-cancer drugs i.e. including taxol, vinblas-tine, vincristine, the camptothecin derivatives, topotecan and irinotecan and etoposide derived from epipodophyllotoxin [27]. These molecules might act as cancer-blocking agents, preventing initiation of the carcinogenic process and as

cancer-suppressing agents, inhibiting cancer promotion and progression [28]. In addition a number of other mechanisms are also involved in the process. Kumar and Pandey [29] have described the mechanisms by which flavonoids can exert their anticancer activity. The DNA replication may be due to inhibition of DNA topoisomerase II, a key enzyme in DNA replication. The arrest the cell growth in cell cycle, as they reduces the rate of cell division by preventing the entry of cell into the prophase and subsequent phases which was concluded from the antimitotic and antiproliferative results that may be considered as an alternate mechanism of action of extracts in cell growth inhibition. The activity might be also depended upon the morphology of cell lines and mechanism of action of the plant extract [30]. The antioxidants are also known to play a key role in reducing cancer cell proliferation and tannins are known as strong lipid peroxidation inhibitors [31, 32]. Hence, due to the presence of tannins and related compounds as the major compounds in plant extracts may play anti-cancer activities.

2456


Fig. 3: Chemical structure of some major compounds 2457


Further experiments are also needed, both in vitro and in vivo to understand mechanism of action. CONCLUSIONS From the results of the present study it could be concluded that Semecarpus anacardium, Phyllanthus amarus and Gloriosa superba have high potential activity against cell proliferation in case of both Plant and animal system. These plants could be used as sources of potent agents for anticancer drug development. As such these plants could be further investigated for the individual components of the methanolic extract is recommended to try to determine the ingredient(s) that may be responsible for its antineoplastic effects and the mechanism of growth inhibition, which may be used as natural and low cost drugs to fight cancer with minimal or no side effects. ACKNOWLEDGEMENTS Authors are heartily thankful to Dr. A. K. Srivastava, Head of Botany Department, Ranchi University for his support. We are greatly thankful to ACTREC, Tata Memorial Centre, Mumbai and AIRF, JNU, New Delhi for providing all necessary facilities to carry out cell line work. We also acknowledge UGC for their financial supports which make enabled us to carry out investigations. REFERENCES 1. Al Diab, A., S. Qureshi, K.A. Al Saleh, A.H. Al Qahtani, A. Aleem and M.A. Algamdi, 2013. Review on breast cancer in the Kingdom of Saudi Arabia. Middle-East J. Sci. Res., 14(4): 532-543. 2. Harun-ur-Rashid, M.D., M.A. Gafur, G.M. Sadik and M.A.A. Rahman, 2002. Biological activities of a new acrylamide derivative from Ipomoea turpithum. Pakistan J. Biol. Sci., 5(9): 968-969. 3. Malaviya, T. and P.K. Sharma, 2014. Phytochemical, Pharmacological Profile and Commercial Utility of Tropically Distributed Plant Bauhinia variegata. Global Journal of Pharmacology, 8(2): 196-205. 4. Zupan, J., T.R. Muth, O. Draper and P. Zambryski, 2000. The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. The Plant Journal, 23(1): 11-28. 5. Levin, I. and M. Levine, 1920. Malignancy of the crown-gall and its analogy to animal cancer. The Journal of Cancer Research, 5(3): 243-260.

6. Ullrich, C.I. and R. Aloni, 2000. Vascularization is a general requirement for growth of plant and animal tumours. Journal of Experimental Botany, 51(353): 1951-1960. 7. Skehan, P., R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H. Bokesch, S. Kenney and M.R. Boyd, 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. Journal of the National Cancer Institute, 82(13): 1107-1112. 8. Monks, A., D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff and M. Gray-Goodrich, 1991. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. Journal of the National Cancer Institute, 83(11): 757-766. 9. Skehan, P., R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H. Bokesch and S. Kenney, 1989. Evaluation of colorimetric protein and biomass stains for assaying drug effects upon human tumor cell lines. In The Proceedings of the American Association for Cancer Research, 30(612): 11. 10. Al-Snafi, A.E., 2013. The pharmacology of Bacopa monniera. A review. International Journal of Pharma Sciences and Research, 4(12): 154-159. 11. Ogunjobi, A.A. and M.A. Abiala 2013. Antimicrobial Activity of Senna alata and Phyllanthus amarus. Global Journal of Pharmacology, 7(2): 198-202. 12. Jain, P., H.P. Sharma, F. Basri, B. Baraik, S. Kumari and C. Pathak, 2014. Pharmacological Profiles of EthnoMedicinal Plant: Plumbago Zeylanica l. A Review. Int. J. Pharm. Sci. Rev. Res, 24(1): 157-163. 13. Kavina, J., R. Gopi and R. Panneerselvam, 2012. Difenoconozole and Propiconazole’s Effects on Antioxidant Potentials of Gloriosa superba Linn. World Journal of Agricultural Sciences, 8(3): 247-252. 14. Jain, P. and H.P. Sharma, 2013. A Potential Ethno medicinal Plant: Semecarpus anacardium Linn.-A Review. International journal of Research in Pharmacy and Chemistry, 3(3): 564-572. 15. Galsky, A.G., J.P. Wilsey and R.G. Powell, 1980. Crown Gall Tumor Disc Bioassay a possible aid in the detection of compounds with antitumor activity. Plant Physiology, 65(2): 184-185. 16. Mclaughlin, J.L., L.L. Rogers and J.E. Anderson, 1998. The use of biological assays to evaluate botanicals. Drug Information Journal, 32(2): 513-524.

2458


17. Ferrigni, N.R., J.E. Putnam, B. Anderson, L.B. Jacobsen, D.E. Nichols, D.S. Moore, J.L. McLaughlin, R.G. Powell and Jr C.R. Smith 1982. Modification and evaluation of the potato disc assay and antitumor screening of Euphorbiaceae seeds. Journal of Natural Products, 45(6): 679-686. 18. Adams, R.P., 1995. Identification of essential oil components by Gas Chromatograph Mass Spectroscopy. Allured, Carol Stream, IL, USA. 19. Vanden Dool, H. and P.D. Kratz, 1963. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. Journal of Chromatography A, 11: 463-471. 20. Ghosh, M. and B.N. Sinha, 2010. "GC-MS studies on the bark extracts of Litsea polyantha Juss." MiddleEast J. Sci. Res., 5(6): 441-444. 21. Mclaughlin, J.L., 1991. Crown gall tumours on potato discs and brine shrimp lethality: two simple bioassays for higher plant screening and fractionation. Methods in Plant Biochemistry, 6: 1-32. 22. Wall, M.E., M.C. Wani, C.E. Cook, K.H. Palmer, A.A. McPhail and G.A. Sim, 1966. Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from camptotheca acuminata1, 2. Journal of the American Chemical Society, 88(16): 3888-3890. 23. Yeong, Ha, L. Nancy, K. Grimm and M.W. Pariza, 1989. "Newly recognized anticarcinogenic fatty acids: identification and quantification in natural and processed cheeses." Journal of Agricultural and Food Chemistry 37(1): 75-81. 24. Russo, G.L., 2007. Ins and outs of dietary phytochemicals in cancer chemoprevention. Biochemical Pharmacology, 74(4): 533-544. 25. Mohamed, G. Layaly, S. Hussein, S. Salwa, S. Reda ElSayed and El. G. Abdel-Wahab, 2014. "Targeting Apollon via siRNA Induces Apoptosis in Different Human Malignancies. Academic Journal of Cancer Research, 7(4): 233-238.

26. Reddy, A.S., S.N.A. Malek, H. Ibrahim and K.S. Sim, 2013. Cytotoxic effect of Alpinia scabra (Blume) Náves extracts on human breast and ovarian cancer cells. BMC complementary and alternative medicine, 13(1): 1. 27. Shoeb, M., 2006. Anticancer agents from medicinal plants. Bangladesh Journal of Pharmacology, 1(2): 35-41. 28. Maurya, D.K., N. Nandakumar and T.P.A. Devasagayam, 2010. Anticancer property of gallic acid in A549, a human lung adenocarcinoma cell line and possible mechanisms. Journal of Clinical Biochemistry and Nutrition, 48(1): 85-90. 29. Kumar, S. and A.K. Pandey, 2013. Chemistry and biological activities of flavonoids: an overview. The Scientific World Journal, Article ID 162750: pp: 1-16. 30. Sivashanmugam, M.U.A. and P. Jagannath, 2011. Induction of apoptosis and cytotoxic activities of Apium graveolens Linn. using in vitro models. Middle East J. Sci. Res., 9: 90-4. 31. Chinery, R., R.D. Beauchamp, Y. Shyr, S.C. Kirkland, R.J Coffey and J.D. Morrow, 1998. Antioxidants reduce cyclooxygenase-2 expression, prostaglandin production and proliferation in colorectal cancer cells. Cancer Research, 58(11): 2323-2327. 32. Okuda, T., Y. Kimura, T. Yoshida, T. Hatano, H. Okuda and S. Arclchi, 1983. Studies on the activities of tannins and related compounds from medicinal plants and drugs. I. Inhibitory effects on lipid peroxidation in mitochondria and microsomes of liver. Chemical and Pharmaceutical Bulletin, 31(5): 1625-1631.

2459