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EVALUATION OF ANTIOXIDANT EFFECT AND ANTICANCER ACTIVITY AGAINST OVARIAN CANCER (OVCAR-3) CELL LINE OF BRASSICA OLERACEA AND HERBOMINERAL COMPLEX


S Patil* and A Ansari
Department of Pharmacognosy, 23 Jote Joy Building, Rambhau Salgaonkar Marg, Cuffe Parade, Colaba, Mumbai, INDIA.

Abstract

Botanical and nutritional compounds have been used for the treatment of cancer throughout history. These compounds also may be useful in the prevention of cancer. Population studies suggest that a reduced risk of cancer is associated with high consumption of vegetables and fruits. Dietary botanicals, including cruciferous vegetables such as cabbage and broccoli, Allium vegetables such as garlic, onion, green tea, Citrus fruits, soybeans, tomatoes, and berries are in preclinical or clinical trials for cancer. Indole-3-carbinol (I3C), naturally found in high concentration in cruciferous vegetables, has been shows promising activities in preventing development and growth of breast cancer.

The main aim of the study was to screen the hydro alcoholic extracts from of Brassica oleracea. f rubra and Brassica oleracea. f alb leaves, herbomineral complex and Indole-3-carbinol for their in vitro anti-oxidant and anticancer activity against OVCAR-3 cell lines. In vitro antioxidant activity of extracts was determined by DPPH Radical assay, Inhibition of lipid peroxidation. Cytotoxic effect of extracts and herbomineral was evaluated by performing Sulphorhodamine B (SRB) assay on OVCAR-3 cell lines.Extracts and herbomineral complex were found to possess significant antioxidant activity.

In SRB colorimetric assay, Indole-3-carbinol, Brassica oleracea. f rubra() and Brassica oleracea. f alba().Extract and their herbomineral complex with zinc was evaluated and compared to anticancer activity. Extract of white cabbage shows a more potent activity herbomineral complex of red cabbage and white cabbage showed anticancer activity. Herbomineral complex of white cabbage has showed more potent activity then red cabbage.The observed anticancer activity of extract and complex may be due to its antioxidant potential

Key words: Indole-3-carbinol, breast cancer, Antioxidant, Anticancer.

INTRODUCTION

Cancer has been a scourge on the human population for many years. Although numerous advances have been made in prevention, diagnosis and treatment of the disease, it still continues to torment mankind. As is widely believed, cancer is the result of many genetic and epigenetic changes in a population of cells as well as in the surrounding stroma and blood vessels. These genetic alterations disrupt several molecular pathways to the cell and lead to self-sufficiency in growth signals, insensitivity to growth control signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, invasion and metastasis1.

Cancer chemoprevention has developed as a major field of scientific investigation. Cancer chemoprevention is defined as pharmacological intervention in synthetic or naturally occurring compounds that may prevent, inhibit, or reverse carcinogenesis, or prevent the development of invasive cancer. Dietary consumption of foods and herbal medicines is a convenient method of administering potentially chemo preventive phytochemicals in a cost-effective manner. Numerous reports suggest a protective role of a diet rich in fruits and vegetables.Overall, diets high in vegetables and fruits (_400 g/d) may prevent at least 20% of all cancers. Some of the most convincing evidence for the health benefits of fruit and vegetable consumption relates to the reduced risk of gastrointestinal cancers, such as those associated with the mouth, pharynx, esophagus, stomach, colon, and rectum. Vegetables are more effective than fruits.

5 The mechanismsby which vegetables and fruits reduce cancer are multipleand complex. Various stages of carcinogenesis may be inhibited, and various in vitro or in vivo systems may be used to model these inhibitory effects in preclinical studies. It is logical to acquire compellingin vitro data prior to performing tests of animal models, and it is generally necessary to isolate and characterize active chemical principles before moving on to the animal model and clinical studies2.

Ovarian cancer is the second most common gynecological malignancy following uterine corpus cancer and it is the fifth leading cause of cancer death in women. There are important differences in their incidence across the world. In Europe in 2008, estimated incidence was 66,734 cases with an estimated mortality of 41,929 women. In United States, ovarian cancer was diagnosed in 21,880 with 13,850 cancer deaths last year. Both incidence and mortality are declining in USA and Europe.

Higher incidence rates are observed in North America and European countries exceeding 10 per 100.000 inhabitants. Lower rates are observed in South America (7,7 per 100.000) and Southern Asia (7,5 per 100.000). (Parkin et al, 2005) Such geographical variations are due to differences in oral contraceptive use practices, pregnancy history, breast-feeding and other hormonal factors. (Permuth –Wey & Sellers, 2009) The relative risk for developing ovarian cancer is 1.39% (lifetime risk). It affects 12.9 per 100,000 women per year. Incidence rate of ovarian cancer increases with aging, being more prevalent in the eighth decade of life. At diagnosis, mean age is 63 years, and 62% of patients have advanced disease. Inherited ovarian cancer presents at younger age.

(www.Seer.gov,Ferlay et al 2010) Five year overall survival is 93.5% for localized disease, 73.4% for loco regional disease (regional lymph node involvement) and 27.6% for distant disease. Genetic studies on ovarian cancer indicate that most of the cases are sporadic while 5 to 10 percent are inherited, generally due to germline mutations. Three histological subgroups have been described: epithelial tumors, stromal tumors and germ-cell tumours. Ninety percent of cases are epithelial tumours arising from the ovarian surface epithelium or Mullerianderivatives. These tumours are typical in postmenopausal women. The World Health Organization classification defines six more histotypes: serous, mucinous, endometrioid, clear cell and squamous cell carcinomas. According to their architectural features like glandular or papillary components, carcinomas have been classified into three histological grades, well differentiated, moderately differentiated, poorly or undifferentiated. Malignant germ cell tumour affects younger women. (De Vita et al, 2009) Despite the high incidence, ovarian cancer etiology is still poorly understood3.

Cruciferous or Brassica vegetables come from plants in the family known to botanists as Cruciferae or alternatively, Brassicaceae. Plants in the Cruciferae family have flowers with four equal-sized petals in the shape of a ‘crucifer’ cross. “Brassica” is the Latin term for cabbage. Many commonly consumed cruciferous vegetables come from the Brassica genus, including broccoli, Brussels sprouts, cabbage, cauliflower, collard greens, kale, kohlrabi, mustard, rutabaga, turnips, bok choy and Chinese cabbage. Although not in the Brassica genus, arugula, horseradish, radish, wasabi and watercress are also cruciferous vegetables.

Like other vegetables, cruciferous vegetables contain a number of nutrients and phytochemicals with cancer chemopreventive properties, including folate, fiber, carotenoidsand chlorophyll. However, cruciferous vegetables are unique in that they are rich sources of glucosinolates, sulfur-containing compounds that are responsible for their pungent aromas andspicy (some say bitter) taste . The hydrolysis of glucosinolates by the plant enzyme myrosinase results in the formation of biologically active compounds, including indoles and isothiocyanates (Fig. 1) 4. More than 100 glucosinolates with unique hydrolysis products have been identified in plants. For example, broccoli is a good source of glucoraphanin, the glucosinolate precursor of sulforaphane (SFN), and glucobrassicin, the precursor of indole-3-carbinol (I3C) 5.

Figure: 1 Breakdown of glucosinolates  

Herbomineral Complex: Process for formation of herbomineral complex is known as chelation. Chelation is the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central atom.

Phenolic compounds constitute a diversified group of plant secondary metabolites in terms of structure, molecular weight and physicochemical and biological properties. They exhibit strong antioxidant properties. Antioxidant properties can result from their free radical scavenging activity but major drawback of phenolic compound is their poor bioavailability. Literature resources reveled that formation of complex of these phenolic compound with mineral will increase bioavailability and free radical scavenging activity.

Their ability to chelate transition metal ions, especially Fe (II) and Cu (II), also plays an important role. Metal ions can generate highly reactive oxygen free radicals by Fenton or Haber-Weiss chemistry. In the Fenton reaction the hydroxyl radical (HO) is produced from hydrogen peroxide. In the iron-catalyzed Haber-Weiss reaction the superoxide radical (O2•−) reduces ferric to ferrous ions, which then are again involved in generating of hydroxyl radical. Extremely reactive hydroxyl radicals can interact with many biological macro- and small molecules and therefore lead to lipid peroxidation, DNA damage and polymerization or denaturation of proteins. The binding of transition metal ions by phenolic compounds can stabilize prooxidative activity of those ions6, 7.

Objectives of the Present Research Work

The aim of the present research project was:

  • To explore Anticancer activity of leaves of Brassica oleracea.f rubra and Brassica oleracea. f alba
  • Comparison of Anticancer activity of Brassica oleracea.f rubra and Brassica oleracea. f alba

The main objectives of the present study were:-

  • Collection, Authentication and processing of the plant material.
  • Extraction of the leaves of Brassica oleracea f. alba (White cabbage) , B. oleracea f. rubra (Red cabbage), family : Brassicaceae
  • Preparation of Herbomineral complex of Brassica oleracea leaf extract with Zinc metal and its characterization
  • In vitro Evaluation and comparison of antioxidant potential of the standard Indole-3-Carbinol and Ethanolic extract of Brassica oleracea.
  • Evaluation and comparison of cytotoxic activities of Ethanolic extract of Brassica oleracea, Indole-3-Carbinol and the Herbomineral complex.

Material and Methods

Plant material

Leaves of Brassica oleracea. f rubra and Brassica oleracea. f alba were collected from Colaba market, Mumbai. The plant (specimen#: 155503 and 155519) was authenticated at Guru Nanak Khalsa College, Department of Botany, Mumbai

Preparation of plant extracts and herbomineral complex 8,9

Freshly collected leaves of Brassica oleracea. f rubra and Brassica oleracea . f alba were screened for presence of foreign matter and any unwanted plant parts like flower buds or stem. The leaves were then macerated and subjected to extraction using Ethanol and Water as solvent (9:1) at temperature not exceeding 60°C in an ultra sonicator. The extracts thus obtained were concentrated in evaporating dish on the water bath to obtain dry extract. The dry extract was weighed and extractive value was calculated. The Extract obtained was further subjected to phytochemical evaluation by using test reagents to determine the presence of various phytoconstituents viz; flavonoids, alkaloids, glycosides, saponins, Tannins, phenols, sugars and proteins.

HCl buffer (0.1M) is prepared with addition of 0.1M KCl at PH 5.0. ZnCl2 was dissolved in the same buffer at a concentration of 8mM. Brassica oleracea. f rubra and Brassica oleracea extract solution was prepared in methanol i.e. (10μg/ml). 1ml of Brassica oleracea. f rubra and Brassica oleracea . f alba extract was mixed with 3ml salt solution and kept aside for few hours. After few hours precipitates were formed which were settle down at the bottom of the test tube. Herbomineral complex of Brassica oleracea. f rubra and Brassica oleracea . f alba extract with zinc was formed in the form of precipitates, solvent was evaporated in evaporating dish and dried precipitates were collected.

In vitro antioxidant assay

DPPH assay- 

The scavenging activity was determined by DPPH assay previously reported by Blois (2009).7.9mg of DPPH was accurately weighed and dissolved in 100ml methanol to obtain 200µM solution of DPPH. Aliquots of different concentrations of extracts (10µg/ml,20µg/ml,30µg/ml,40µg/ml,50µg/ml) and fractions (5µg/ml,10µg/ml,50µg/ml,100µg/ml,150µg/ml,200µg/ml) were added to 1ml of DPPH solution. After incubating test tubes for 30min in dark, absorbance was measured at 517nm against blank10. Extracts and fractions were compared with standard Quercetin.The radical scavenging activity was calculated from the equation:-

% radical scavenging activity = (Abs control-Abs sample) / Abs control× 100  

Abs- Absorbance.

Inhibition of Lipid peroxidation

Freshly excised rat liver was processed to get 10% homogenate in cold phosphate buffer, pH 7.4 Degree of lipid peroxidation was assayed by estimating the TBARS formed. The reaction mixture contains 0.1 ml of different concentrations of extracts (10µg/ml,20µg/ml,30µg/ml,40 µg/ml,50µg/ml) and fractions (5µg/ml,10µg/ml,50µg/ml,100µg/ml,150µg/ml,200µg/ml) were added to the brain homogenate. 0.2 ml SDS (8.1%), 1.5 ml of acetic acid (20%) solution and 1.5 ml of aqueous solution of TBA (0.8%) were added to the homogenate mixture the mixture was finally made up to 4.0 ml with distilled water, and heated at 95°C for 60 min. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of mixture of n-butanol and pyridine (5: 1) were added, and the mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer (upper layer) was measured at 532 nm 12.

The radical scavenging activity was calculated from the equation:-

% radical scavenging activity = (Abs control-Abs sample)/Abs control× 100

In vitro cytotoxicity evaluation:

The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine. For present screening experiment, cells were inoculated into 96 well microtiter plates in 100 μL at plating densities as shown in the study details above, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs.

After 24 h, one 96 well plate containing 5*103cells/well was fixed in situ with TCA, to represent a measurement of the cell population at the time of drug addition (Tz). Experimental drugs were initially solubilized in dimethyl sulfoxide at 100mg/ml and diluted to 1mg/ml using water and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate (1mg/ml) was thawed and diluted to 100 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml with complete medium containing test article. Aliquots of 10 μl of these different drug dilutions were added to the appropriate microtiter wells already containing 90 μl of medium, resulting in the required final drug concentrations i.e.10 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml 12.

Sulphorhodamine B assay:

Sulforhodamine B (SRB) assay was developed by Skehan and colleagues to measure drug-induced cytotoxicity and cell proliferation for large-scale drug-screening applications. Its principle is based on the ability of the protein dye sulforhodamine B to bind electrostatically and pH dependent on protein basic amino acid residues of trichloroacetic acid-fixed cells. Under mild acidic conditions it binds to and under mild basic conditions it can be extracted from cells and solubilized for measurement. After incubation for 48 hours assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of cold 30 % (w/v) TCA and incubated for 60 minutes at 4°C. The supernatant was discarded; the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (50 µl) at 0.4 % (w/v) in 1 % acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing five times with 1 % acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on a plate reader at a wavelength of 540 nm with 690 nm reference wavelength 12 

Results

DPPH assay

White cabbage andRed cabbageshowed promising free radical scavenging effect on DPPH radical in a concentration dependent manner. The IC50 values of white cabbage and red cabbage were found to be 18.03µg/ml and 69.89µg/ml respectively (Figure 2, 3). The extracts were compared with standard Quercetin having IC50 value of 35.04μg/ml.

Inhibition of Lipid peroxidation assay

White cabbage and Red cabbage elicited concentration dependent inhibition of FeSO4 induced lipid peroxidation in rat brain homogenate. The IC50 values of white cabbage and red cabbage were found to be 47.04μg/ml and 21.05μg/ml respectively (Figure 4, 5). IC50 value of Quercetin was found to be 22.93/ml

Fig 2: DPPH free radical scavenging activity of white cabbage.

Fig 3: DPPH free radical scavenging activity of red cabbage.

Fig: 4 In vitro inhibition of Lipid peroxidation activity of white cabbage

Fig: 5 In vitro inhibition of Lipid peroxidation activity of red cabbage

Sulphorhodamine B assay

The GI50 value of EWC and ERC was found to be 15.0μg/ml and 28.5μg/ml respectively when compared with standard anticancer drug ADR having GI50 value of less than 10µg/ml (Figure 6) EWC and ERC did not show any significant cytotoxic activity.

Table.6 Reports of invitro testing for anticancer activity

Drug concentrations (µg/ml) calculated from graph
OVCAR-3 LC50 TGI GI50*
Std I3C >80 74.0 21.9
EWC 59.7 37.3 15.0
ERC >80 64.3 28.5
HMWC >80 67.7 30.0
HMRC >80 >80 46.6
ADR <10 <10 <10

These results indicate that EWC extract was found to be more potent than ERC showing less GI50 value. HMWCand HMRCwas found to be 30.0μg/ml and 46.6μg/ml respectively when compared with standard anticancer drug ADR having GI50 value of less than 10µg/ml (Figure 6) EWC and ERC did not show any significant cytotoxic activity. These results indicate that HMWC was found to be more potent than HMRC showing less GI50. (Figure 7)

Figure.7 Growth curve of human ovarian cell line

Discussion

In the present study, the antioxidant potential of EWC and ERC was evaluated with the help of in-vitro antioxidant models like DPPH free radical scavenging activity and Lipid peroxidation,

Free radical scavenging activity of the extracts was evaluated based on their ability to scavenge DPPH. Bleaching of DPPH absorption is a representative of the capacity of the test drug to scavenge free radicals independently. Here, EWC and ERC presented good scavenging of DPPH free radical which might be due to their ability to transfer electrons to the DPPH radical thereby showing reduction in absorbance.

In the current method effectiveness, of EWC and ERC is evaluated as inhibitor of lipid peroxidation. Extracts reduced the initiating perferryl radical with the formation of ubisemiquinone and H2O2. Additionally, it is possible that they might have also eliminated lipid peroxyl radicals. Extracts ERC were found to be exerting more potent antioxidant effect in preventing lipid peroxidation than standard Quercetin.

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