1. INTRODUCTION:

Cancer is one of the most non-communicable diseases in humans. According to the American Cancer Society, there were annually increasing about 2%–3% of deaths caused by cancer reported worldwide. Thus, around 3.5 million people all over the world were killed by cancer. Several chemically synthetic antitumor agents are used to treat cancer, but the wide range of side effects encourages many cancer patients to seek alternative or complementary methods of treatment.¹Natural product based medicine becomes one option to treat cancer.

Nowadays natural product or herbal-based medicine represented about 60%–80% of all drugs in use by 1990. Moreover, 70%–95% of world populations applied herbal-based medicine for their primary care, particularly in developing countries.² Biological activity investigations related to antimicrobial and cytotoxic properties have been associated with the quinolone alkaloids content. Quinoline alkaloids from L. amara can inhibit Mycobacterium tuberculosis H37Rv. Moreover, one of quinoline alkaloids, namely, lunacridine was reported as DNA topoisomerase II inhibitor compound.³ The type of this mechanism will lead to antimicrobial and cytotoxic activities based on the inhibition on cell proliferation. In our effort to develop a standardized herbal-based medicine from Indonesia medicinal plants, we previously conducted the physicochemical standardization of the wood extract of L. amara collected in South Sulawesi.⁴ In this recent paper, further phytochemical standardization including the determination of total alkaloid and the concentration of lunacrine as a marker compound were performed to ensure the extract purity and quality which is responsible for affecting biological activity. Cytotoxic activity of wood extract was also performed to prove the potential bioactivity of standardized extract. The use of plants as Medicines is as old as human civilization itself and out of about 258, 650 species of higher plants reported from the world; more than 10% are used to cure ailing communities. Many of the existing medicinal system such as Ayurveda, Unani, Homeopathy, Naturopathy, Siddha and other alternative medicinal system have been utilizing plants as effective medicines to cure many harmful diseases.⁵ The world health organization (WHO) has estimated that 80% of the earth’s inhabitant relied on traditional medicine for their primary health care needs and most of these therapies involved the use of plant extract or their active compounds (Bruneton, 1995). Justicia adhatoda (L.) Nees (family Acanthaceae) is a shrub widespread throughout the tropical regions of Southeast Asia.⁶ The name J. adhatoda (L.) Nees and Adhatoda zeylanica Medic are used synonymously. It is commonly known as Vasaka or Malabar nut. It is a perennial, evergreen and highly branched shrub (1.0 m to 2.5 mm height) with unpleasant smell and bitter taste. This study was therefore undertaken to evaluate the effect of methanolic extract of the leaf of Adhatoda vasaka on different cancer cell lines like MCF-7, MDA-MB-231, Hela, HepG2 cell lines.

2. MATERIALS AND METHODS:

2.1 PREPARATION OF EXTRACT:

The collected materials (leaves) were chopped into small pieces separately, shade-dried, and coarsely powdered (sieve no. 40) using a pulverizor The coarse powders were subjected to extraction with organic solvents 80% of methanolic extract (distilled water + methanol) and ethanol by maceration method. The extract was collected and distilled off on a water bath and the solvents Removed by vaccum. The resulted extracts were used for phytochemical screening.⁷

2.2 Preliminary Phytochemical Screening:

All the extracts were subjected to preliminary phytochemical tests as per standard procedure.

2.3 Physico-Chemical Analysis:

The powdered plant materials were morphologically and organoleptically screened and subjected to physicochemical analysis in accordance with the WHO Guidelines (WHO, 1998). The various parameters considered were

1. Determination of Ash Values:

Ash values of a crude drug is the inorganic residue remaining after incineration, which simply represents inorganic salts, naturally occurring in drug or adhering to it or deliberately added to it as a form of adulteration. Hence, the ash values are helpful in determining the quality and purity of a crude drug in the powdered form

2. Determination of Total Ash:

Weighed 2 to 3g of the air-dried crude drug in a tared platinum or silica dish and incinerated at a temperature not exceeding 450°C until free from carbon, cooled and weighed. Calculated the % of total ash with reference to the air-dried drug.

3. Determination of Water Soluble Ash:

Boiled the ash, (obtained in ash value determination), for 5 minutes with 25 ml of water; collected the insoluble matter in a Gooch crucible or on an ashless filter paper, washed with hot water, and ignited for 15 minutes at a temperature not exceeding 4500C. Subtracted the weight of the insoluble matter from the weight of the ash; the difference in weight represented the water-soluble ash. Calculated the % of water-soluble ash with reference to the air-dried drug.

4. Determination of Acid Insoluble Ash:

Boiled the ash, (obtained in ash value determination), with 25ml of 2M hydrochloric acid for 5 minutes, collected the insoluble matter in a Gooch crucible or on an ashless filter paper, washed with hot water, ignited, cooled in a desiccator and weighed. Calculated the % of acid-insoluble ash with reference to the air-dried drug.

5. Determination of Sulphated Ash:

Heated a silica or platinum crucible to redness for 10 minutes, allowed to cool in a desiccator and weighed. Unless otherwise specified in the individual monograph, transferred to the crucible 1g of the substance being examined and weighed the crucible and the contents accurately. Conducted the ignition in a place protected from air currents. Allowed the crucible to cool, added a few drops of sulphuric acid and heated. Ignited as before, allowed to cool and weighed. Repeated the operation until two successive weighings did not differ by more than 0.5mg.

6. Determination of Loss on Drying:

The loss on drying test is designed to measure the amount of water and volatile matters in a sample, when the sample is dried under specific conditions. If the substance is in the form of large crystals, reduce the size by rapid crushing to a powder. The test should be carried out on a well mixed sample of the substances. Weighed a glass stoppered, shallow weighing bottle that has been dried under the same conditions to be employed in the determination. Transferred to the bottle the quantity of the sample specified in the related monograph, covered it and accurately weighed the bottle and the contents. Distributed the sample as evenly as practicable by gentle sidewise shaking to a depth not exceeding 10 mm. Placed the loaded bottle in the drying chamber (oven or desiccator) as directed in the monograph, removed the stopper and left it also in the chamber. After drying was completed, opened the drying chamber, closed the bottle promptly and allow it to cool at room temperature (where applicable) in a desiccator before weighing. Weighed the bottle and the contents.

2.4 In-vitro Studies of Cytotoxic activity of Different Cancer Cell Lines:

1. Tumor Cell Line and Culture Conditions^8,9:

Human cancer cell lines, MCF-7, MDA MB-231, HeLa, HepG2, were procured from the National Centre for Cell Sciences (NCCS, Pune, India) and maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin (100 μg /mL at 37°C in 5% CO2 environment according to standard recommended protocols.

2. In-vitro Assay:

The cellular toxicity of both methanolic and ethanolic extracts of the compounds were investigated against different human cancer cell lines using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Sigma) assay. Pale yellow MTT was converted to purple formazan crystals by mitochondrial enzymes in viable cells. Cells at a density of (5x10⁴ cells per well) were seeded in clear 96-well plates and incubated for 24h. After 24h treatment the medium was replaced with fresh medium along with 10µl of the MTT solution (5 mg ml^-1in PBS) was added to each well together with 200µl of DMEM growth medium. The plates were further incubated for 4h at 37^◦C. Then the medium was removed and the resultant formazan crystals were dissolved in 200ml of dimethyl sulfoxide.

3. RESULTS:

3.1 Evaluation of Phytochemical and Physicochemical Studies of Adathoda vasica:

The phytochemical investigation of the ethanolic extract of Adathoda vasica was carried out with standard protocol. The extraction was carried out using by cold maceration. From the above table it is evident that the both methanolic and ethanolic extract of the Adathoda vasica extract consists of carbohydrates, steroids, Alkaloids, Tannins, Flavanoids, Saponins, gums and mucilage.

3.2 Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of HeLa cells:

The in vitro cytotoxicity of treated and non-treatedcells was investigated using MTT cell viability assay against HeLa cell line. The cytotoxicity of a treated ethanolic and methanolic extracts with increasing concentrations were analysed. The killing effect of the methanolic extract was more pronounced at the low concentrationsand exhibited higher cytotoxicity compared to ethanolic extract. However, the difference was not significant at the higherµg/ml concentrations. When the experimental cells were exposed to the highest concentration 7 for the period of 24h, 65% and 60.% of cells were killed by both methanolic and ethanolic extractsrespectively (Figure 1). The higher cytotoxic effect of both extracts could be attributed to the competent celluar uptake and intracellular distribution of extract compounds.

Fig 1: Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of HeLa cells

In vitro cytotoxicity of HeLa cells treated with different concentration of both methanolic and ethanolic Extracts after 24h incubation. All experiments were repeated thrice and found highly reproducible

3.3 Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of HepG2 Cells:

The In vitro cytotoxicity of treated and non-treated cells were investigated using MTT cell viability assay against HepG2 cell line. The cytotoxicity of a treated ethanolic and methanolic extracts with increasing concentrations were analysed. The killing effect of the methanolic extract was more pronounced at the low concentrations (5µg/ml) and exhibited higher cytotoxicity when compared to ethanolic extract. However, the difference was still significant even at the higher concentrations (40 µg/ml). When the experimental cells were exposed to the highest concentration 40 µg/ ml for the period of 24h, 90% and 60% of cells were killed by both methanolic and ethanolic extracts respectively (Figure 2). The higher cytotoxic effect of methanolic extracts could be attributed to the competent celluar uptake and intracellular distributionthan ethanolic compounds.

Fig 2: Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of HepG2 Cells

In vitro cytotoxicity of HepG2 cells treated with different concentration of both methanolic and ethanolic Extracts after 24h incubation. All experiments were repeated thrice and found highly reproducible.

3.4 Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of Mcf-7 Cells:

The In vitro cytotoxicity of treated and non-treated cells were investigated using MTT cell viability assay against MCF-7 cell line. The cytotoxicity of a treated ethanolic and methanolic extracts with increasing concentrations were analysed. The killing effect of the ethanolic extract was less pronounced at the low concentrations (5µg/ml) and exhibited higher cytotoxicity at (15µg/ml) when compared to methanolic extract. However, the difference was still significant even at the higher concentrations (45 µg/ml). When the experimental cells were exposed to the highest concentration 50µg/ml for the period of 24h, 90% and 85% of cells were killed by both ethanolic and methanolic extracts respectively (Figure 3). The higher cytotoxic effect of ethanolic extracts could be attributed to the competent cellular uptake and intracellular distribution than methanolic compounds.

Fig 3: Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of Mcf-7 Cells

In vitro cytotoxicity of MCF-7 cells treated with different concentration of both methanolic and ethanolic Extracts after 24h incubation. All experiments were repeated thrice and found highly reproducible.

3.5 Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of MDA MB-231 Cells:

The In vitro cytotoxicity of treated and non-treated cells were investigated using MTT cell viability assay against MDA MB-231 cell line. The cytotoxicity of a treated ethanolic and methanolic extracts with increasing concentrations were analysed. The killing effect of the methanolic extract was more pronounced at the low concentrations (1µg/ml) and exhibited higher cytotoxicity at (10µg/ml) when compared to ethanolic extract. However, the difference was still significant even at the higher concentrations (10µg/ml). When the experimental cells were exposed to the highest concentration 10µg/ml for the period of 24 h, 80% and 65% of cells were killed by both methanolic and ethanolic extracts respectively (Figure 4). The higher cytotoxic effect of methanolic extracts could be attributed to the competent cellular uptake and intracellular distribution than methanolic compounds.

Fig 4: Effect of Ethanolic Extract of Adathoda vasica in In-vitro Cytotoxicity of MDA MB-231 Cells

In vitro cytotoxicity of MDA MB-231 cells treated with different concentration of both methanolic and ethanolic Extracts after 24h incubation. All experiments were repeated thrice and found highly reproducible.

4: DISCUSSION:

Adhatoda vasica has an tropical distribution and is found throughout South-East Asia. In various parts of the world, decoctions of the leaves, fruits, bark or wood of Adhatoda vasica are employed as a febrifuge. Externally the fresh, dried or powdered leaves are applied as a poultice, apparently for their astringent properties, to treat wounds, swellings, burns and to ripen boils, sores and ulcers.

In the present study an attempt has been made for the pharmacognostical study, which helps to identify the plant for futher investigation and phytochemical study for investigation of chemical constituents of the plant and free radical scavenging activity by in vitro method and anti cancer activity to prove its efficacy on support of folklore uses.

Qualitative phytochemical analysis of leaf extract showed the presence of terpenoid, steroid, flavonoids, tannin and saponins. Ethno botanicol information and thorough literature review of plant made us to investigate anti inflammatory, anticancer of leaf under investigation. Besides earlier reports, present phytochemical investigation revealed the presence of phytoconstituents such as flavonoids, tannins, steroids and terpenoids also supports our present pharmacological investigation. Hence anti-cancer activity was carried only for leaf extract of the plant and found to be effective from the results observed in the current investigation, it may be concluded that the Methanolic extract of Adhatoda vasica. Linn., leaf at the dose of 200mg/kg and 400mg/kg body weight displays a significant anti-cancer activity in MCF-7, MDA MB-231, HeLa, HepG2.

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Received on 06.07.2019 Modified on 14.08.2019

Res. J. Pharmacognosy and Phytochem. 2019; 11(4):212-216.

DOI: 10.5958/0975-4385.2019.00036.0