Introduction

Cancer is the second leading origin of death, after cardiovascular ailments. Amongst the non-communicable illnesses, morbidity and mortality of cancer are high in worldwide (Hoyert et al., 2003; Lopez, Mathers, Ezzati, Jamison, & Murray, 2006; Mathers & Loncar, 2006). Cancer is an abnormal cell mutation and multiplication, considered as an only most extensive lethal disease, which holds numerous health risks around the world with 6 million mortalities every year (Pandey, Sharma, & Dudhe, 2012; Shaikh, Pund, Dawane, & Iliyas, 2014; Solowey et al., 2014). It has been assessed, in 2050, 24 million people will be affected by cancer newly (Schwartsmann et al., 2002; United Nations Development Programme (UNDP), 2000). Presently existing beneficial herbal drugs are cytotoxic & aside from distressing cancer cell growth, active phytoconstituents too detrimentally influences the prompt multiplying ordinary cells, together with entities limited in the digestive pathway, bone marrow, hair consequently provoking tummy side and adverse effects such as biliousness, alopecia, vomiting & myelosuppression. Rao MRP and his research team members in 2007, described the efficiency of tumour medications is repeatedly restricted through their insolvability in addition to uncertainty, less amount on the tissue and cells engrosses them, also malignant tumour drug conflict.

Anti-neoplastic remedies always have also been accompanying with an improvement of a secondary tumour. Many chemotherapeutic agents are symptomatic relief only, not able to stop the total mechanism of a tumour, that's why in need of better tolerated efficacious drugs. The traditional system of medicines has a long use from gold old days, enhanced patient forbearance and public recognition in the management of malignancy (Cragg & Newman, 2001; Newman, Cragg, & Snader, 2000), moreover the attention in mother flora in place of foundation of prospective chemotherapy-induced agents (Schwartsmann et al., 2002). The recent natural products research and development initialled towards the innovation of novel anti-proliferative agents have been sustained by upgrading the science and technology in drug discovery of anti-tumour. The traditional system of medicines plays an essential role in principal health care structure surrounded by rural inhabitants since allopathic anti-cancer remedies are away from the reach of the public for the reason of expensive. Medicinal plants consume active, potential part in hindrance plus management of malignance which achieves healing outcome through constraining cancer triggering hormones, enzymes, exciting DNA restoration mechanism, upholding the manufacturing anti-oxidant defensive enzymes, persuading then improving the immunity (Thakore, Mani, & Kavitha, 2012).

Herbal remedy delivers a noble base used for systematic assessment of effective anti-tumor therapeutics. P. murex (Pedalium murex Linn) generally well-known to the world as “Large Caltrops” and is belonging to the family Pedaliaceae. This medicinal plant is rich in glycosides, proteins, polyphenolics (flavonoids and phenolics). Due to high polyphenolics present in this plant, it is duly necessary to investigate its anti-cancer activity. The main objective of this paper was the evaluation of potent pharmacological agents from P. murex leaves. For that purpose, processes of extraction were performed, in vitro anti-oxidant and anti-cancer activities were also evaluated.

Experimental Methods

Collection of P. murex leaves

The plant leaf of P. murex was collected in the district of Namakkal by the side of Vattamalai, Kumarapalayam Town, Tamil Nadu.

Authentication of P. murex leaves

Leaves were identified by Prof & Dr Jayaraman in Chennai (Institute of Herbal Botany). Authentication reference: No.PARC/2014/3070.

Extracts preparation

The collected and cleaned P. murex leaves were desiccated under darkness and then thoroughly crushed into granular materials using hands. The uniform size of granular powder was allowed by way of mesh number 40. Stored, packed in a sealed bottle for the intended use. The desiccated grainy leaves (200 gram) were defatted using the non-polar solvent (60-80⁰C) petroleum ether to eliminate the waxy substances, chlorophyll and foreign matters, which commonly affect the isolation of plant phytoconstituents. The defatted marc of non-polar solvent was dried in the shade and then extracted by 95 per cent v/v ethanol in soxhlet apparatus (72 hrs). Distil the solvent, and the resultant semisolid material was dried in a vacuum evaporator.

Preliminary phytochemical analysis

The two solvent extracts of P. murex were exposed towards qualitative phytochemical investigation for the credentials of several herbal active components like carbohydrates, flavonoids, saponins, alkaloids, proteins & amino acids, steroids, tannins, glycosides, terpenoids (Chatwal & Anand, 2003; Harborne, 2005; Krishnaswamy, 2003).

In vitro free radical scavenging activities

Estimation of total anti-oxidant capacity

The anti-oxidant potential of P. murex polar and non-polar extracts was screened depends upon the thiocyanate technique (Mitsuda, Yasumoto, & Iwami, 1966). To make a stock solution, 20 milligrams of P. murex extracts were solubilized with 20 ml water. Five different concentration of P. murex leaf extracts (12.5, 25, 50, 100 and 200 mg) or standard sample of 0.04 M potassium phosphate buffer (2.5 millilitres, pH 7.0) was mixed with emulsion linoleic acid (2.5 ml) in buffer 0.04 M potassium phosphate (pH 7.0). Individual sample, the standard solution was incubated by 37^°C in shady. Throughout incubation, at intervals, every solution was agitated for three minutes. 0.1 millilitre of incubated solution, 0.1 ml (ferric chloride & thiocyanate) were transferred to ethanol (4.7 ml) carried test tube. At that time, 5 min incubated the mixture. The peroxide value was estimated finally through measuring the absorbance on 500 nanometers in a spectrophotometer (Bio-Crom Gmbh,8500 II, Zurich, Switzerland).

During the oxidation of linoleic acid, peroxides were formed & oxidized ferrous to ferric ions. Fe³⁺ ions made complex with SCN- & showed a more absorbance at 500 nanometers. From this time greater value of absorbance designated the presence of more oxidation of linoleic acid. Blank solutions were prepared without adding P. murex plant extracts or standard drugs. 5 ml emulsion of linoleic acid made up of Tween-20 (17.5 gm), linoleic acid 15.5 ml and 0.04 M K₃PO₄ buffer (pH 7.0). The other side, control solution (5 ml) was mixed with 2.5 millilitres of the linoleic acid mixture & 2.5 ml of 0.04 M potassium phosphate buffer (pH 7.0). The average of all the obtained data about total anti-oxidant estimation was a duplicate investigation. A subsequent equation calculated the percentage of lipid peroxidation inhibition:

% Cell inhibition = [A₀ - A₁/A₀] × 100, A₀ = Absorbance of control, A₁= absorbance of the sample.

Estimation of DPPH free radical scavenging capacity

One millimole methanol dissolved DPPH radical solution was made and then various concentrations of leaf extracts of P. murex prepared from one millilitre of stock solution. This solution was mixed vigorously and keep it aside in place of 30 minutes on dim light at average room temperature, measure absorbance with a spectrophotometer (517 nm). The obtained values were subjected to formula (Blois, 1958; Sánchez-Moreno, Larrauri, & Saura-Calixto, 1999; Zhu, Hackman, Ensunsa, Holt, & Keen, 2002).

% DPPH free radical scavenging capacity = Absorbance (Control – Extract) / Absorbance (Control) x 100. One ml of 1 mmol solution of DPPH radical was mixed with one ml of methanol solution for control.

In vitro Anti-cancer Activity

The ethanol (polar solvent) extract of P. murex was exposed to in vitro anti-cancer activity in two different cell lines such as human colorectal adenocarcinoma (HCT116) & human liver cancer (HepG2) by MTT assay methodology. These two tumour cell lines were collected from National Centre for Cell Science (NCCS), Pune). HCT116 cells were grown-up in Dulbecco’s modified eagle’s medium (DMEM), HepG2 cells were developed in EMEM (Eagles minimum essential medium) having 10 per cent FBS (fetal bovine serum). These cell lines were preserved at 95% air, 37^oC, 5% Carbon dioxide & 100% relative humidity. These cell cultures weekly maintained; the culture medium was altered once every two weeks. The above cell lines were treated by doses of EEPM (62.5, 125,250 & 500 µg/ml) respectively. Further, the percentage of cell inhibition was calculated (Monks et al., 1991; Mosmann, 1983).

Results and Discussion

The powdered plant leaves of P. murex [Pedaliaceae] were extracted effectively with petroleum ether. Then the solvent was filtered, removed defatted marc was exposed to constant extraction consuming 95 per cent v/v ethanol by soxhlet apparatus. These two extracts of Pedalium murex Linn leaf were exposed to preliminary phytochemical investigation exhibited the existence of carbohydrates, flavonoids, glycosides, saponins, alkaloids, tannin and steroids, which is stated in Table 1.

Endless generation of free radicals occurs in the living system and its origins of extensive damages of cells, tissues and organs, which may lead to numerous ailment states, specifically degenerative disarrays and inflammatory sicknesses. Anti-oxidants from herbal plants battle against the diseased cells and avert the syndromes through scavenging the free radical molecules and its formation, to inhibit the lipid peroxidations and many other oxidative mechanisms in the human body. The results of the total anti-oxidant ability of EEPM was more significant (65.23% at 200 µg/ml) which was compared to petroleum ether extract (64.34 %) (Table 2 & Figure 1).

One of nitrogen centred stable free radical (DPPH) is conservatively used to determine and find the free radical foraging accomplishments of anti-oxidants of traditional herb extracts otherwise synthesized compounds (El-Maati, Mahgoub, Labib, Al-Gaby, & Ramadan, 2016; Kalaivani & Mathew, 2010). Decreased level of absorbance (517 nm) to reduce the capability of radical DPPH by anti-oxidants. The 2,2-diphenyl-1-picrylhydrazyl radical pursuing activity of P. murex leaf extracts was assessed by the standard drug ascorbic acid (positive control) (Table 3 & Figure 2).

DPPH reduction is always directly proportional to the quantity of anti-oxidant content present in the particular extract. If the extract has greater anti-oxidant phytoconstituents reduces the higher amount of DPPH radicals. Ethanol extract of this plant revealed the extreme DPPH scavenging action in 200 μg/ml (69.23%), had IC₅₀ value - 96.77 μg/ml, and it exhibited the significant anti-oxidant potential. Based on extractive values, preliminary qualitative analysis and in vitro radical scavenging and suppressing activities, EEPM was carefully chosen for in vitro anti-cancer activity.

The assessment of the anti-malignancy activity of traditional herbal extracts is essential and ultimately safe for treatment and its protocols. It empowers to ascertain the intrinsic plant toxicity (Padmaja et al., 2002; Rahman, Akhtar, Sahabjada, & Arshad, 2016) and possessions of severe overdose. MTT assay method is applied in screen and evaluates the crude herbal extracts and isolated compounds to measure the toxicity. This method of assessments provides a sign of potential cytotoxic activities of the established vegetal extracts. In this assay method, mitochondrial dehydrogenase reducing the MTT and yields the formazan product in purple colour. This one is frequently used in vitro methods to cytotoxic activity measurement of diverse of herbal extracts, isolated active constituents and toxic materials against cell lines of cancer (Morshed et al., 2011).

The results of anti-cancer studies of EEPM produced a significant action against HCT116, HepG2 cell lines by in vitro method. The obtained data of percentage inhibition of cell by EEPM at different concentrations were presented in Table 4 & Table 5. The difference in cell inhibition of EEPM was represented in Figure 3 & Figure 4. The cell inhibition results indicated that EEPM exhibited significant inhibition in liver cancer cell lines when linked to colon cancer cell lines. HepG2 cell line showed more significant anti-cancer activity.

Herbal plant products are considered as foundations of potent innovative drugs with less adverse and side effects and have aided as dynamic resources of cancer treatment (Ezhilarasan et al., 2017). The anti-neoplastic effects of secondary metabolites like flavonoids have been described to induces the inhibition of cell growth and a variety of cancer cell mechanism apoptosis (Carlo, Mascolo, Izzo, & Capasso, 1999). Flavonoid aglycones, flavonoid glycosides and phenolic acids as anti-oxidants are commonly acknowledged for their beneficial effects on human well-being and their anti-tumour properties. Li, Zhang, and Wang (2017) reported 5, 7-Dimethoxy flavone, a natural flavonoid showed potential cancer-protective effect against (HepG2) liver cancer cell line. This preliminary investigation result designates EEPM exhibits significant anti-malignance effect against HepG2 cell lines in MTT assay method. EEPM have anti-tumour activity might be due to the existence of phyto flavonoids and glycosides, phenolic active constituents.

Conclusion

From the results, we conclude, medicinal herb P. murex hold a vast number of potential secondary active metabolites which embrace potent anti-oxidant (inhibit the oxidation or free radical formation) ability established on experimentations accomplished which enhance the scientific proof to demeanour advanced level of studies, to scrutinize the potent lead moieties existing in this herb and assess the plant anti-neoplastic potential on rodent models and initiate an endeavour to implement clinical studies on human beings. P. murex showed significant anti-cancer activity against these cancer cell line owing to the existence of potent anti-oxidant phytoactive constituents like flavonoids & phenolic compounds of extract. Advanced studies are required to assess the in vivo potential of potent lead constituents in other animal research, and novel enigmatic pharmacological actions also need to be discovered.