INTRODUCTION:

Curcuma Longa Linn (turmeric) is the most important Indian spice and medicinal herb belongs to the Zingiberaceae family which is extensively used in Ayurveda, Unani andSiddha medicine and as polyphenols, responsible for the yellow colour of this rhizome^1,2. Curcumin activates the DDR (DNA damage response), providing an opportunity and rationale for the clinicalapplication of these nutraceuticals in the chemoprevention of prostate cancer³curcumin was having anticancer activities⁴.

Curcumin has also shown anti-proliferative effect in multiple cancers, and is an inhibitor of the transcription factor NF-B and downstream gene products (including c- myc, Bcl-2, COX- 2, NOS, Cyclin D1, TNF-a, interleukins and MMP-9). In addition, curcumin affects a variety of growth factor receptors and cell adhesionmolecules involved in tumor growth, metastasis ⁵. Curcuminis the most important component of the rhizomes of Curcuma longa L. (turmeric) and was extracted from turmeric plant in a pure crystalline form for the first time in 1870. Aqueous extract of Curcuma longa rhizome showed the antifertility effects in male mice by significantly decreasing sperm counts, sperm motility and seminal pH and cause significantly higher mortality of spermatozoa⁶.Curcumin and itsderivatives have received immense attention in the past two decades due to their bio functional properties such as anti-tumor, antioxidant, and anti-inflammatory activity. Camelia Sinensis (green tea) is widely recognized to have beneficial effects on human health. Epigallocatechingallate (EGCG) is the most abundant polyphenol in green tea and believed to be mostly responsible for its various biologicaleffects. Flavonoids and polyphenol compounds have been reported by to have favorable properties such as anticarcinogenic, antimutagenic, antimicrobial and anti-oxidant properties⁷. Flavonoids and polyphenols of Camellia sinensis also prevent cancer cell survival^8,9.

MATERIALS AND METHODS:

Rawmaterial (Curcumin) was collected from the dried rhizome of Curcumalonga and the sample of green tea (Camellia Sinensis) was collected from the dried leaves of 100% organic green tea from Hindustan Herbals Limited.

Preparation of Extract:

The process of extraction requires the raw materials in the form of paste orpowder andit is rinsed with a suitable solvent that selectively extracts a colouring matter. Crushed powder of Curcuma longa rhizome and leavesofCameliasinensis (1:1 ratio) was mixed with ethyl acetate solvent and extracted with microwave extractor for 60s. The extraction was finished when the sample reached 60ºC and filtered with Whatman filter paper. Thefiltrate was allowed to dry and stored at 4ºC.

Sample

Solvent

Yield

Curcuma longa and

Cameliasinensis (1:1 ratio)

Ethyl acetate

0.141 g

Phytochemical Estimation:

Test for phenols:

Lead acetate test: The extract was taken; 3ml of 10% lead acetate solution was added. Presence of phenolic compounds was determined by the formation of bulky white precipitate.

Determination of Total Phenolic Content:

The test sample was mixed with 0.5ml of water and 0.2 ml of Folin- Ciocalteu's phenol reagent (1:1). After 5 min, 1ml of saturated sodium carbonate solution (8% w/v in water) was added to the mixture and the volume was made up to 5ml with distilled water

The reaction was kept in the dark for 30 mins and the absorbance of blue color from different sample was measured by UV-VIS Spectrophotometer at 765nm. The total phenolic content was calculated as Gallic acid equivalents GAE/g of dry plant material on the basis of a standard curve of Gallic acid.

In vitro Anti-Cancer activity:

MTT Assay:

By counting viable cells after staining with a vital dye the presence of unknown compounds was determined. . MTT, is a water-soluble tetrazolium salt forming a yellowish solution when prepared in media or salt solutions lacking phenol red. By mitochondrial dehydrogenase enzymes of viable cells dissolved MTT was converted to an insoluble purple formazan. Formazan can be solubilized using acidified isopropanol, DMSO, or other solvents (Pure propanol or ethanol). The resulting purple solution was spectrophotometrically measured. Degree of cytotoxicity was determined by increase or decrease in cell number results in a change in the amount of formazan formed.

MTT reagent (The solution is filtered through a 0.2µm filter and stored at 2-8ºC for frequent use or frozen for extended periods)

1. DMSO

2. CO2incubator

3. Micro Plate reader

4. Inverted microscope

5. Refrigeratedcentrifuge

Cell lines and Culture Medium:

A549 (Human lung adenocarcinoma) cell line was procured from the National Centre for Cell Science, Pune, India (NCCS). Stock cell was cultured in medium supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100μg/ml) in an humidified atmosphere of 5% CO2 at 37oC until confluent. The cell wasdissociated with Trypsin Phosphate Versene Glucose (TPVG) solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). Viability of the cells was determined and centrifuged. Again 50,000cells/well was seeded in a 96 well plate and incubated for 24 hrs. at 37^oC, 5% CO2 incubator.

Source of reagents:

DMEM, FBS, Pen strip, Trypsin procured from Himedia.

Procedure:

The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 105 cells/ml using respective media containing 10% FBS. To the 96 well microtiterplate100μl of the diluted cell suspension (50,000cells/well) was added to each well. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100μl of different concentrations of test drugswere added on to the microtiter plates. The plate was the nincubatedat 37^oC for 24h in 5% CO2 atmosphere. After incubation the test solutionsin the wells were discarded and 100μl of MTT (1mg/1ml of MTT in PBS) was added to each well. The plate was incubated for 4h at 37^oC in 5% CO2 atmosphere. The supernatant have removed and 100μl of DMSO is added and the plate shakengently to solubilize the formed Formazan. At a wave length of 570nm, the absorbance was measured using a microplate reader. Using the following formula, the percentage of cell viability can befound.

Sample abs

Cell viability (%) = ------------------------- X 100

Control abs

Preparation of the gel:

Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarosegels are prepared using a w/v percentage solution. The concentration of agarose in the gel will depend on the size of the DNA fragments, with most gels ranging from between 0.5% - 2%. Bath capacity should not be more than 1/3 of the flask volume. Add the active ingredient to a flask containing agarose. Swirl to mix. The most effective gel baths are TAE (40mM Tris-acetate, 1mM EDTA) and TBE (45 mM Tris-borate, 1mMEDTA).

Melt the agarose/buffer mixture. This is usually done by heating in a micro wave oven, but can also be done over the Bunsen flame. In the 30s, remove the flask and scroll through the contents to blend in well. Repeat until the agarose is completely dissolved. Add ethidium bromide (EtBr) to a mixture of 0.5μg/ml. Alternatively, the gel may also be stained after electrophoresis using a bath containing 0.5μg/ml EtBr for 15-30 minutes.

DNA Fragmentation Assay:

Figure: 2 DNA Fragmentation Assay

RESULTS:

Total Phenolic content:

Phenolic compounds are important plant constituents with redox properties responsible for antioxidant activity. The hydroxyl groups in plant extracts are responsible for facilitating free radicalscavenging.

Total phenol content in the plant extract (CLCS) using the calibration curve, was found to be 1137.45mg of Gallic acid equivalents/g dry weight of extract.

Table: 1 Quantitative phytochemical analysis - Total phenoliccontent

Sample	Concentration	Singlet	Duplicate	Triplicate	Mean	TPC
Gallic acid	0 µg	0	0	0	0
	2 µg	0.058	0.054	0.052	0.054667
	4 µg	0.084	0.088	0.085	0.085667
	8 µg	0.156	0.153	0.157	0.155333
	16 µg	0.277	0.274	0.278	0.276333
	32 µg	0.642	0.646	0.643	0.643667
	64 µg	1.136	1.139	1.135	1.136667
Sample	Concentration	Singlet	Duplicate	Triplicate	Mean	TPC
CLCS	500 µg	1.951	1.945	1.947	1.947667	1137.45

Figure 4

Anti-cancer activity:

The anticancer activity was carried out with lung cancer cell line (A-549) by MTTassay method. When cancer cells were incubated with combination of curcuma longa and camellia sinensis extracts, they induced cytotoxicity in a significant time which implicit the damage to the membrane integrity of cell.

Cisplatin:

Cisplatin is a simple inorganic compound which has proved to be a very effective anticancer drug, especially against testicular tumors. The compound has therapeutic limitations based primarily on toxic side effects and acquired resistance. The cisplatin method conducts extensive research to understand the chemical basis of theselimitations and to develop logically. The cisplatin action is under intense study in order to under stand the chemical basis for the selimitations and to develop in rational manner a new generation of platinum-based antitumor drugs. Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered to be a cytotoxic drug that kills cancer cells by damaging DNA andpreventing DNA synthesis. Mannered on the cell disintegration, when the plant extracts were incubated with lung cancer cells and cell death were calculated (Table: 2 and 3, Fig: 5 and 6). The IC50 value of the given test sample of curcuma longa and camellia sinensis and the standard Cisplatin was found to be 22.72µg and 3.89µg . This is clearly stated that the selection of A-549 cellline might provide a good evidence for the treatment of lungcancer.

Table:2 Anti Cancer Activity of CLCS extract on Lung cancer cell (A-549)

Sample	Concentration	% of Viability	IC50
CLCS	3.125	97.30566739
	6.25	93.80613193
	12.5	63.02260762	22.72
	25	50.23227005
	50	22.01920099
	100	13.00712295

Fig: 5 Anti Cancer Activity of CLCS extract on Lung Cancer Cell (A-549)

Table: 3 Anti-Cancer Activity of Cisplatin on Lung Cancer Cell (A-549)

Sample	Concentration	% of Viability	IC50
Cisplatin	3.125	67.17249923
	6.25	38.40198204
	12.5	15.91824094	3.89
	25	9.321771446
	50	4.707339734
	100	1.827191081

Fig: 6 Anti Cancer Activity of Cisplatin on Lung Cancer Cell (A-549)

DISCUSSION:

Several extraction techniques and solvents are used for obtaining anticancer extracts from plant sources. Extraction techniques include solvent extraction (SE) microwave assisted extraction (MAE), Soxhlet extraction and supercritical fluid extraction¹⁰. Among these, MAE is a relatively newmethod used for the extraction of natural products¹¹. The microwave- assisted extraction system for biologically active compounds has many advantages over other conventional extraction methods. Microwave- assisted extraction methods required shorter time, less solvents, provide higher extraction rates and better products with lower costs¹². MAE was more effective than conventional extraction methods in the extraction of tea polyphenols and tea caffeine. MAE also usedto optimize the extraction of phenolic compounds from grapeseeds13. Antioxidant properties of black tea extract was obtained from Camellia sinensis extract¹⁴.V. Curcumin was extracted by various techniques¹⁵. Plant phenolic is an important component that contributes to quality, color, and flavor function and plays an important role in both individual air purifiers and free scavengers, which helps reduce cell damage. It indicated that a higher TPCin ethanolic extracts is present compared to that in aqueous extracts. In turmeric the TPC ranged from 4.52% to 7.68% in aqueous for extraction and from 6.15% to 16.07% in ethanolic extracts. According to the Database 60% of the ingredients of Camellia sinensis extract isolated to the polyphenols.The effects of polyphenols extracts Camellia sinensis on A431, HaCaT and DU145 cell lines (human epidermoid, keratinocyte, prostate carcinoma cells, respectively) and reported the induction of apoptosis in these cells¹⁶. In a similar study conducted. Camellia sinensis leaf extracts were affected on the PC-3 (human metastatic prostate cancer) cell line in an in vitro study and showed efficacy of Camellia sinensis extracts on the PC-3 cell line. Recent studies have indicated that inhibition of tumor cell growth is associated with the promotion of apoptosis and decrease in telomerase expression in a dose dependentmanner.

CONCLUSION:

In conclusion, it was observed that the combinations of plants Curcuma longa and Camellia sinensis contains phenolic compounds that hold strong anticancer capacity based on the experiments performed which add scientific evidence to carry out additional studies to probe the lead compounds present in the herbalplants and to evaluate its anticancer potential on in vivo clinical trials to measure the efficacy on human beings.

According to the results of the current studies, Curcuma longa Camellia sinensishas potential anticancer influence, leaves of Camellia sinensis areused to produce green tea evaluated anticancer effects in cell culture. To the destructive and predictable side effects of conservative anticancer treatments, it is essential to utilize an efficient, cost-effective and anti contradictory treatment. So, this study aimed to evaluate the anticancereffects of Curcuma longa Camellia sinensis via an in vitro investigation. The results of this study showed that Cisplatin was the most effective among all concentrations of Camellia sinensis and proved the inhibition of tumor cells, and its anticancer effects that are innovative and more effective.

ACKNOWLEDGMENT:

The authors are thankful to Dr. R. Suresh Greensmed lab for helping in successfully completing the project.

CONFLICT OF INTEREST:

All the authors declare that there is no conflict of interest.

REFERENCES:

1. Sri Vasavi Reddy A, J. Suresh, Hemant K.S. Yadav, Apurva Singh. A Review on Curcuma longa. Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 158-165.doi:10.5958/0974-360x

2. Acharya YT (1994) CharakaSamhitha of Agnivesh with the Ayurveda Dipika commentary. (4thedn), Chaukambha Sanskrit Samstha, Varanasi, India 32-33.

3. Sastry JLN (2005) Illustrated DravyagunaVijnana. (2ndedn), ChaukhambhaOrientalia, Varanasi, India 513-518

4. Horie S (2012) Chemoprevention of Prostate Cancer: SoyIsoflavonesandCurcumin. Korean JUrol53: 665-672.doi:10.4111/kju.2012.53.10.665

5. Wilken R (2011)Curcumin:A review of anti-cancer properties and therapeutic activity inheadandneck squamous cell carcinoma. MolCancer 10: 12.doi:10.1186/1476-4598-10-12

6. Anita Raj Hembrom, AartiVerma, V.N. Singh. Antifertility Effects of Rhizome of Curcuma longa on Seminal Parameters of Swiss Albino Male Mice. Research J. Pharm. and Tech. 8(4): April, 2015; Page 404-406.doi:10.5958/0974-360x

7. Fan D-m, Fan K, Yu C-p, Lu Y-t, Wang X-c (2017). Tea polyphenols dominate the short- term tea (Camellia sinensis) leaf litter decomposition. J Zhejiang UnivSci, 18, 99-108.doi:10.1631/jzus.B160004

8. Yildirim A, Mavi A, Oktay M, Kara AA, Algur OF, Bilaloglu V (2000). Comparison of antioxidant and antimicrobial activities of tilia (TiliaargenteaDesf. Ex. D.C.) sage (Salvia triloba L.) and black tea (Camellia sinensis L.) extracts. J. Agr. Food Chem. 48(10): 5030- 5034.doi:10.1021/jf000590k

9. Ann Beltz L, Kay Bayer D, Lynn Moss A, Mitchell Simet I (2006). Mechanisms of cancer prevention by green and black tea polyphenols. Anti-Cancer Agents Med Chem, 6, 389-406. (PDF) Evaluation of Anticancer Activity of Camellia Sinensis in the Caco-2 Colorectal Cancer Cell Line.doi:10.2174/187152006778226468.

10. Chen LG, Ding L, Zhang HR, Li J, Wang YT, Wang XP, Qu CL, Zhang HQ (2006). Dynamic microwave-assisted extraction coupled with online spectrophotometric determination of safflower yellow in Floscarthami. Anal. Chim. Acta 580: 75–82.doi:10.1016/j.aca.2006.07.040

11. Bicchi C, Liberto E, Cordero CEI, Sgorbini B, Rubiolo P. 2009. Stir bar sorptive extraction (SBSE) and headspace sorptive extraction (HSSE): an overview. LCGC North America 27: 376–391.doi:10.1016/j.chroma.2005.07.099.

12. Ganzler, K.; Salgo, A.; Valko, K. J. Chromatogr. 1986, 371.doi:10.1016/s0021-9673(01)94714

13. Pan et al., X. Pan, G. Niu, H. Liu Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves Chem. Eng. Process., 42 (2003), pp. 129-133.doi.org/10.1016/S0255-2701(02)00037-5

14. Dhrubajyoti Sarkar, SubhashisDebnath, Sekhar Kumar Bose. Phytochemical analysis and assessment of Antioxidant properties of black tea extract obtained from Camellia sinensis. Research J. Pharm. and Tech. 2020; 13(10):4539-4544.doi:10.5958/0974-360x

15. V. Vyshali, N. Lalitha Jyotsna, VasudhaBakshi, Syed Ismail, DibyalochanMohanty, T. Kavitha, M. Manoj. Optimization of Extraction of Curcuminoids from Turmeric Powder (Curcuma longa). Research Journal of Pharmacy and Technology. 2021; 14(9):4615-0.doi:10.5958/0974-360x

16. Pedro AC, Granato D, Rosso ND (2016). Extraction of anthocyanins and polyphenols from black rice (Oryza sativa L.) by modeling and assessing their reversibility and stability. Food Chem, 191, 12-20.doi:10.1016/j.foodchem.2015.02.045

Received on 15.02.2022 Modified on 12.07.2022

Research J. Pharm. and Tech 2023; 16(5):2192-2196.

DOI: 10.52711/0974-360X.2023.00360