Anti-Cancer Activity of Cayratia Auriculata Ethanolic Extracts Against Cancer Cell Line A549-An In Vitro Analysis

Lung cancer is the world's leading cause of death and it has been noted that 7.6 million deaths were recorded in 2008. Death rates have been forecast to rise by more than 11 million in 20301. Usually, 80 percent of cases of histologically non-small cell lung cancer are a big health issue2. Due to the high incidence rate, rapid development and poor prognosis, lung cancer-related deaths have been the leading cause3. With an average survival rate of around 15 percent, it has become a burden worldwide4. There are more medications available today to manage lung cancer. Chemotherapy is the routine treatment for lung cancer, but it does have several side effects that also affect the quality of life of patients5. It is still deficient, considering the numerous treatment methods available to avoid these side effects6,7. Therefore, new drugs or innovative ways to cure or avoid this global warming disease are being pursued. Several medicinal plants have been used globally for the treatment of different forms of cancer 8. Conventional medicine with its low toxicity and side effects has the ability to treat anticancer activity. According to the WHO study, in contrast to modern medicine, 80 % of the world 's population relies on conventional medicine because of its less side effects, cost effectiveness, protection, and effective prognosis9. Even today, several tribal groups are practicing herbal medicine to cure various diseases10. The medicinal plant belonging to the Vitaceae family is Cayratia auriculata. Cayratia auriculata (Roxb.) gamble is a synonym for Cyphostemma auriculatum (Roxb), a climber well cultivated in dry evergreen to dry deciduous forests and well distributed in Andhra Pradesh, Tamilnadu, and Maharashtra11,12. For several diseases, cayratia auriculata has been used as herbal medicine. The leaf is the most important part of the plant as it has potent ethno-medicinal properties. It is used in the treatment of snake bite, dog bite, abscess, rheumatism, cardiovascular disease, tumours, and also in the treatment of cold, cough, worm infestation13. The genus is also used for bloody dysentery and diarrhoea in veterinary medicine. Based on previous studies, a study on the cytotoxic activity of C. auriculata extract is still limited to lung cancer. The present study was therefore conducted to determine the anticancer and cytotoxicity potential of Cayratia auriculata unrefined ethanolic leaf extract.

Lung cancer is the world's leading cause of death and it has been noted that 7.6 million deaths were recorded in 2008. Death rates have been forecast to rise by more than 11 million in 2030 1 . Usually, 80 percent of cases of histologically non-small cell lung cancer are a big health issue 2 . Due to the high incidence rate, rapid development and poor prognosis, lung cancer-related deaths have been the leading cause 3 . With an average survival rate of around 15 percent, it has become a burden worldwide 4 . There are more medications available today to manage lung cancer. Chemotherapy is the routine treatment for lung cancer, but it does have several side effects that also affect the quality of life of patients 5 . It is still deficient, considering the numerous treatment methods available to avoid these side effects 6,7 . Therefore, new drugs or innovative ways to cure or avoid this global warming disease are being pursued. Several medicinal plants have been used globally for the treatment of different forms of cancer 8 . Conventional medicine with its low toxicity and side effects has the ability to treat anticancer activity. According to the WHO study, in contrast to modern medicine, 80 % of the world 's population relies on conventional medicine because of its less side effects, cost effectiveness, protection, and effective prognosis 9 . Even today, several tribal groups are practicing herbal medicine to cure various diseases 10 . The medicinal plant belonging to the Vitaceae family is Cayratia auriculata. Cayratia auriculata (Roxb.) gamble is a synonym for Cyphostemma auriculatum (Roxb), a climber well cultivated in dry evergreen to dry deciduous forests and well distributed in Andhra Pradesh, Tamilnadu, and Maharashtra 11,12 . For several diseases, cayratia auriculata has been used as herbal medicine. The leaf is the most important part of the plant as it has potent ethno-medicinal properties. It is used in the treatment of snake bite, dog bite, abscess, rheumatism, cardiovascular disease, tumours, and also in the treatment of cold, cough, worm infestation 13 . The genus is also used for bloody dysentery and diarrhoea in veterinary medicine. Based on previous studies, a study on the cytotoxic activity of C. auriculata extract is still limited to lung cancer. The present study was therefore conducted to determine the anticancer and cytotoxicity potential of Cayratia auriculata unrefined ethanolic leaf extract.

Plant content processing and authentication
Cayratia auriculata was collected in November 2018 from the botanical garden of Andhra University, Visakhapatnam, Andhra Pradesh. The plant content has been identified and approved by Dr. S.B. Padal, Associate Professor, Department of Botany, Visakhapatnam, Andhra University.

Preparation of extracts for plants
Fresh leaves of Cayratia auriculata were washed in flowing tap water, dried in the shade and powdered. Approximately 75 g of coarsely powdered plant materials (75g/250mL) were extracted for 10 to 12 hours in a soxhlet extractor. The desired compounds were condensed in the distillation flask after several cycles, the extracts were finally collected, then condensed and finally dried to a constant weight. Before usage, dried extracts were stored at 20 ° C. In an in vitro trial, this extract was used.

Procedure of MTT assay proceedings
In lung cancer, the effect of the drug was tested against the cell line (A549) by MTT assay according to the standard procedure 15 . The cells were seeded in 96-well micro plates (1x106 cells / well) and incubated in a 5 % CO2 incubator at 37 ° C for 24 hours and allowed 80% confluence to expand. The medium was then substituted and the cells were treated with drugs at varying concentrations, such as 12.5, 25, 50, 100, 150, 200 μg / mL and were incubated for 24 hours. The cells were then washed and applied to each well with phosphate-buffer saline (PBS, pH-7.4) and 20 μL (MTT) solution (5 mg / mL in PBS). The plates were then placed for an additional 2-4 h at 37oC in the dark. In 100μL DMSO the formazan crystals were dissolved and the absorbance at 570 nm was spectrometrically read. The morphological changes of the untreated (control) and treated cells were observed and photographed after 24 h under a bright field microscope. The concentration required for 50% inhibition (IC50) was calculated and graphically determined. The cell viability percentage was expressed as a formula. Graphs are plotted using the X-axis sample concentration and the percentage of Y-axis cell viability. In each experiment, sample control and cell control were used to compare the full assessments of cell viability. To understand the biological pharmacological characteristics of a chemotherapeutic agent, the determination of a half maximum inhibitory concentration (IC50) is essential 16 .

STATISTICAL ANALYSIS
Cell viability data were analysed using one-way ANOVA followed by multiple comparison tests by Dennett with the same sample size using SPSS 17.0.0. The difference was significant when P < 0.005 was assessed. Table 1 tabulates the test concentration and the corresponding percentage of cell growth inhibition. C. Compared with control, auriculata exhibited a cytotoxic effect. At 150 and 200μg / ml concentrations, with 61 percent and 73.7 percent, respectively, the highest cytotoxicity was observed. The findings show that cytotoxicity is directly proportionate to the concentration of the extract. Price IC50 of methanolic extract C. auriculata was found to be 102.9μg / ml versus the A549 cell line. Morphological differences were observed in the untreated (control) and treated cells after 24 h under a bright field microscope ( Figure 1).

DISCUSSION
The key origins of medicines are plant-based herbal extracts. Many herbal extracts and distilled flavonoids have been developed to become medicinal drugs in clinical practise and are used therapeutically in medicine. To evaluate the cytotoxic activity of C. auriculata ethanol extracts, the MTT (3-(4, 5-dimethylthiazole-2-il)-2,5diphenyltetrazolium bromide) assay method was used. This technique has been widely used to assess the extract's cytotoxic activity at various concentrations 17 . The basic concept of the method is that every living cell has mitochondrial activity, so the greater the activity, the greater the number of living cells 18 . A cellular reductase enzyme exists in mitochondria that can transform the tetrazolium compound in the MTT reagent into the crystal of purple formazan. The effect of the extract's cytotoxic activity is apparent when the tetrazolium compound 's original yellow colour does not change to purple, suggesting low mitochondrial activity. The lower the activity of the mitochondria, the lower the volume of living cells. Usage of the ELISA reader to read the absorption of the sample at 595 nm 17 . The MTT assay uses the IC50value as a parameter for the interpretation of cytotoxic activity. IC50 is the amount of concentration of the extract that can suppress the cancer cells' 50 percent activity. The relationship between cytotoxic activity and the IC50 value is inversely proportional, so the higher cytotoxic activity is indicated by the lower IC50 value.
In present study, we have found that value of methanol extract of C. auriculata showed IC50 around 102 μg/mL range, which means that C. auriculata extracts are weakly active in inhibiting A549 lung cancer cells activity. Subsequently, C. auriculata extracts, as a natural anticancer agent, may be further developed. As for anti-tumor medicine, it can be a new source and can be effectively used as an immunological antimalignant compound. The preliminary phytochemical screening using chemical methods conducted on the ethanolic extract revealed the presence of various phytochemicals like flavonoids, phenols, coumarin, saponins, tannins, terpenoids, steroids and glycosides. The existence of flavonoids can be responsible for the anticancer property.

CONCLUSION
In the current research, C.auriculata ethanolic extract was shown to be a strong suppressant for cell division and proliferation. This anticancer activity may be due to the presence of glycosides and flavonoids. Further studies are required to conclude the actual phytoconstituents and underlying mechanisms responsible for the anticancer property.

ACKNOWLEDGEMENT
We are thankful to management of SRIHER for all the encouragement and support.