INTRODUCTION:

Nature has served mankind by providing most of its needs, like food, shelter and natural remedies to maintain health. One of the important applications is using plants as medicine to treat diseases and various illnesses. According to WHO, about 80% of the world’s population still depends on natural (herbal) medicines for its primary healthcare needs.¹

Cancer is a disease caused by loss of cell cycle control, associated with abnormal and uncontrolled cell growth.

In the past, an individual’s genes, environment, and diet were considered to be the cause of cancer and the underlying mechanisms were unclear. At present advancements in scientific research and the availability of the latest technology has enabled researchers to characterize and understand the mechanisms causing cancer and also to develop new methods to treat or prevent cancer.

Plants serve as a source of food, medicine and many useful articles in day-to-day life. Various plants are used for their medicinal properties in the traditional systems of medicine, based on the traditional uses these plants are being investigated to confirm their medicinal properties by modern scientific methods and also explore possibilities to develop new medicines which can be accommodated in the modern system of medicine.

Lantana camara Linn. is a flowering ornamental plant belonging to the family Verbenaceae; which has been used in the traditional systems of medicine to treat different ailments such as mouth ulcers, eczema, inflammation of skin and respiratory infections etc.

Native to tropical America, known by several common names viz. black sage, cuasquito, angel lip, flowered sage, shrub verbena, white sage and wild sage all over the world. It is a significant weed of which there are some 650 varieties in over 60 countries or island groups.²

It was probably introduced in India before the nineteenth century. Lantana camara is distributed throughout India in moderate to high rainfall and well-drained regions; considered a weed, it has been found to have fundamental medicinal properties.

In India, the ash of the entire plant is used externally for chronic ulcers.³

The decoction of the leaves and the stem is used externally as an excellent wash for eczema or chronic inflammation of the skin.⁴

Powdered fresh leaves are used for sprains. The flowering tops in decoction are used for coughs, colds, fever, jaundice, and chest diseases.⁴

The decoction of the root is recommended for use against infections of the respiratory tract, mumps, and gargles and is a good mouthwash for ulcers of the mouth.⁴

Leaves extract of Lantana camara is reported to possess anticancer activity⁵^,⁶^,⁷^,⁸, active against murine tumors⁹, extracts of leaves and roots reported to possess antibacterial activity^10,11, anti-microbial,¹² hypoglycemic¹³ and wound healing^14,15 activity.

MTT assay is a cell viability method used to access anticancer activity. Tim Mosmann first described it. The colourimetric method utilizes the reduction of yellow tetrazolium salt (3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide) to measure cell metabolic activity. Viable cells contain NAD (P) H- dependent oxidoreductase enzymes, which reduce the reagent to formazan, an insoluble crystalline product with a deep purple colour. Formazan crystals are then dissolved using the solubilizing solution and absorbance is measured at 500-600 nm wavelength using a plate reader.

MATERIALS AND METHODS:

Collection of Plant material:

The plant specimen for the proposed study was collected from the Naubad Forest range in Bidar District, Karnataka.

Care was taken to select healthy plants and normal organs.

Authentication:

The plant under study was authenticated by Prof B.S. Sajjan, Head Dept. of Botany B.V. Bhoomreddy College Bidar.

Chemicals:

DMEM (Dulbecco's Modified Eagle Medium), Dimethyl sulfoxide (DMSO), Ethyl acetate, Hexane, Methanol, MTT and Cisplatin were procured from Sigma Aldrich and Hi Media Mumbai.

Preparation of Extract:

The collected roots were washed in free flowing water to clear it from soil and other adhered material and finally rinsed with distilled water, cleaned roots were cut into small pieces and left to dry at room temperature in the clean laboratory environment free from dust and pollutants. The dried roots of Lantana camara were subjected to further size reduction in a mechanical grinding mill and extracted using soxhlet apparatus with different solvents with increasing polarity. Soxhlet extractor was set up properly, then 146.5g of sample was loaded into the thimble and extracted successively with approximately 750 ml of Hexane, followed with Ethyl Acetate 1L for 24hrs and Methanol 1L for 48 hrs, the recovered extracts were concentrated using a rotary evaporator to recover the solvent at 40ͦ℃ and the concentrate obtained was dried in a desiccator at room temperature. The Ethyl Acetate and Methanol extracts were dissolved in distilled water to obtain suspensions of selected test concentrations to evaluate anti-cancer activity by MTT assay.¹⁶

Procedure^17–21

MCF-7 Cells, named after the Michigan Cancer Foundation (MCF) is the most studied human breast cancer cell line worldwide, were selected to evaluate the anti-cancer activity by MTT assay.

1. The cells were trypsinised and aspirated into a 15ml centrifuge tube. The cell pellet was obtained by centrifugation at 300 x g. The cell count was adjusted, using DMEM medium, such that 200μl of suspension contained approximately 10,000 cells.

2. To each well of the 96-well microtitre plate, 200μl of the cell suspension was added, and the plate was incubated at 37^oC and 5% CO₂ atmosphere for 24 h.

3. After 24 h, the spent medium was aspirated. 200μl of different test concentrations (100, 200, 300, 400, and 500 μg/ml from stock) of test drugs were added to the respective wells. The plate was incubated at 37^oC and 5% CO₂ atmosphere for 24 h.

4. The plate was removed from the incubator, and the drug-containing media was aspirated. 100μl of medium containing 10% MTT reagent was added to each well to get a final concentration of 0.5mg/ml, and the plate was incubated at 37^oC and 5% CO₂ atmosphere for 3 h.

5. The culture medium was removed completely without disturbing the crystals formed. Then 100μl of solubilization solution (DMSO) was added, and the plate was gently shaken in a rotary shaker to solubilize the formed formazan.

6. The absorbance was measured using a microplate reader at a wavelength of 570 nm and also at 630 nm. The percentage growth inhibition was calculated after subtracting the background and the blank. The concentration of the test drug needed to inhibit cell growth by 50% (IC₅₀) was generated from the dose-response curve for the cell line.

Ethics and Consent:

The present study is invitro and was carried out on cancer cell lines cultured in laboratory. Ethical approval is not required.

Table 1. MTT Assay, MCF-7 Cell line Vs Sample Ethyl Acetate Extract (EA) Test concentrations of Ethyl Acetate extract (in µg/ml)

	Blank	Untreated	Cisplatin 15 µg/ml	100	200	300	400	500
Reading 1	0.057	1.363	0.471	1.189	1.105	0.997	0.93	0.829
Reading 2	0.052	1.391	0.496	1.202	1.153	1.047	0.887	0.842
Mean OD	0.0545	1.377	0.4835	1.1955	1.129	1.022	0.9085	0.8355
Mean OD-Mean Blank		1.3225	0.429	1.141	1.0745	0.9675	0.854	0.781
Standard deviation		0.01979899	0.01767767	0.009192388	0.033941125	0.035355339	0.030405592	0.009192388
Standard error		0.014	0.0125	0.0065	0.024	0.025	0.0215	0.0065
% Standard error		1.058601134	0.945179584	0.491493384	1.814744802	1.890359168	1.625708885	0.491493384
% Viability		100	32.43856333	86.27599244	81.24763705	73.15689981	64.57466919	59.05482042

Reading 1: Absorbance measured at 570nm

Reading 2: Absorbance measured at 630nm

Table 2: MTT Assay, MCF-7 Cell line Vs Sample Methanol Extract (ME) Test concentrations of Methanol extract (in µg/ml)

	Blank	Untreated	Cisplatin 15 µg/ml	100	200	300	400	500
Reading 1	0.057	1.363	0.471	1.003	0.891	0.802	0.803	0.632
Reading 2	0.052	1.391	0.496	0.996	0.879	0.832	0.796	0.682
Mean OD	0.0545	1.377	0.4835	0.9995	0.885	0.817	0.7995	0.657
Mean OD-Mean Blank		1.3225	0.429	0.945	0.8305	0.7625	0.745	0.6025
Standard deviation		0.01979899	0.01767767	0.004949747	0.008485281	0.021213203	0.004949747	0.035355339
Standard error		0.014	0.0125	0.0035	0.006	0.015	0.0035	0.025
% Standard error		1.058601134	0.945179584	0.264650284	0.4536862	1.134215501	0.250284	1.890359168
% Viability		100	32.43856333	86.27599244	81.24763705	73.15689981	64.57466919	59.05482042

Reading 1: Absorbance measured at 570nm

Reading 2: Absorbance measured at 630nm

Figure: 3: MCF-7 Cell line Vs Sample Methanol Extract (ME)

Figure4: MCF-7 Cell line Vs Sample Methanol Extract (ME)

Figure 5: Images of MTT Microtitre plates

Slide at Reading 1 (570 nm)

Slide at Reading 2(630 nm)

Untreated

Cisplatin

15µg/ml

100µg/ml

200µg/ml

300µg/ml

400µg/ml

500µg/ml

100µg/ml

200µg/ml

300µg/ml

400µg/ml

500µg/ml

EA: Ethyl Acetate Extract, ME: Methanol Extract

The IC₅₀ values of the test compounds for MCF-7 cell-line 24-hour treatment were found to be:

Table 3: IC₅₀ values

Sample name	MCF-7 cell line IC₅₀ (in µg/ml) 24hr
EA	>500µg/ml
ME	450.05µg/ml

DISCUSSION:

Natural products or their derivatives have been exhibited to have significant anticancer potentials due to their ability to inhibit tumour growth, angiogenesis, and metastasis without many side effects.²²

In order to prepare anticancer medicines from natural resources like plants, testing cytotoxic compounds and screening raw extracts of plants is necessary. Therefore, availability of natural products with higher effectiveness and lower side effects is desired. Medicinal herbs are important for cancer treatment due to their multiple chemical compounds for discovering new active materials against cancer.²³

Upon review of literature, pharmacological activities on various parts of Lantana camara plant have been reported.

Leaf extracts of Lantana camara were reported for antiprolifirative activity against HEP-2 (laryngeal cancer) and Ncl- H 292 (lung cancer) cell lines.⁵

Leaves of Lantana camara were reported to exhibit cytotoxic effect on vero cell line.^6,24

Methanolic extracts of different parts of Lantana camara exhibited significant cytotoxic activity against five different human cancer cell lines.⁷

Methanolic extracts of Lantana camara leaves show cytotoxic effect against MCF-7 cell line.⁸

Oleanonic acid isolated from Lantana camara was screened for anti-cancer activity against murine tumour and three other human cancer cell lines. The extracts have shown good cytotoxic activity against A375 (Malignant skin Melanoma) cancer cells.⁹

Lantadene A isolated from leaves of Lantana camara was effective against LNCap (prostate cancer) cells without effecting normal prostate cells.²⁵

Ethanolic extracts of Lantana camara induced apoptosis in MCF-7 (Breast cancer) cell line, which was regulated by Bcl-2 family.²⁶

Alkaloids from Methanolic extract of Lantana camara leaves were tested for antiproliferative effect against MCF-7, HCT 116 and HeLa cancer cell lines having significant effect on MCF-7 (Breast cancer) and HCT 116 (Colon cancer) cell lines and weak effect of HeLa (Cervical cancer) cell lines.²⁷

Lantana camara stem extracts with different solvents evaluated for anticancer activity against HL-60 Leukemia cell lines.²⁸

Ethanolic extracts of Lantana camara leaves and roots have been reported to possess anti-bacterial activity.²⁹

Methanolic extracts of different parts of Lantana camara have shown anti-bacterial activity against Gram positive Bacillus cereus and Gram negative Salmonella typhi.¹¹

Antihyperglycemic activity of methanol extracts of Lantana camara leaves was reported in alloxan induced diabetic rats.¹³

Hypoglycaemic activity of methanolic extracts of Lantana camara fruits was reported in streptozocin induced diabetic rats.³⁰

Aqueous extracts of Lantana camara was reported for anti-inflammatory activity in albino rats.³¹

Wound healing property of ethanolic leaf extracts of Lantana camara was reported in adult male Wister rats.³²

Oral administration of aqueous extracts of Lantana camara leaves remarkably decreased the number and duration of epileptic seizures as compared to sodium valproate in kainite treated mice.³³

Anti-cancer activity of different extracts of Lantana camara roots was investigated using (3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide) assay on MCF-7 (Breast Cancer) cell lines. A mitochondrial enzyme in the living cells succinate dehydrogenase cleaves the tetrazolium ring and converts MTT into an insoluble purple formazan and the amount of formazan produced is directly proportional to the number of viable cells.³⁴

Given the complications of the modern therapies currently considered for cancer treatment, the high costs of conventional therapies, and the growing incidence of cancer in both developed and developing countries, it is necessary to develop more novel approaches with higher efficiency and lower toxicity to normal cells and side effects.³⁵

In the present study, an attempt has been made to study the cytotoxic activity of Ethyl acetate and Methanol extracts of Lantana camara root against MCF-7 cell lines by including an untreated sample as control and a sample treated with Cisplatin as the reference standard. The results of the MTT assay are mentioned in Tables 1 and 2. Both the extracts have shown significant cytotoxicity at concentrations of 500µg/ml as compared to that of Cisplatin at concentration of 15µg/ml. The effectiveness of the extracts is estimated by plotting the results as a graph of Percentage viability versus concentration of the extracts to obtain the IC₅₀values in Figures 1. and 3. which are >500µg/ml for Ethyl acetate extract and 450.05µg/ml for Methanol extract. The photographs of microtitre plates are tabulated in Figure 5.

Recent publications showed the effect of many herbal plants in the treatment of a wide range of illnesses. Also, several naturally produced herbal formulations are currently available for cancer patients. Most of the chemotherapeutic agents were cytotoxic to normal cells and build up drug resistance. Therefore scientific consideration and testing of traditionally used herbs for the treatment of various malignancies could also be considered a very important source for new chemotherapeutic drugs.^36,37

Limitations of the study:

As described the selected plant is considered a weed and grows in wild and was collected from different places in vast region, the root material was subjected to soxhlet extraction and the crude extracts thus obtained were taken for pharmacological investigations on one type of cancer cell line. Plant material obtained from different geographic and cliamatic regions may vary in composition and concentration of active constituents and may give varied response on the selected or different cancer cell lines.

CONCLUSION:

Lantana camara, once considered an obnoxious weed, has been used to treat different ailments in the traditional systems of medicine. Based on this, it has been scientifically investigated for most of its medicinal properties, and different phytoconstituents have been isolated and linked adequately to its pharmacological activity. In this study the anticancer potential of the root extracts was evaluated using a well-established invitro method MTT assay on the MCF7 cell line, the ethyl acetate and methanol extracts have shown significant anti-tumour potential, and the results found in this study reveal there are potent active constituents in the Lantana camara root extracts which have been effective against the selected cell lines.

This gives rise to a need for further detailed investigation to purify and pinpoint the active constituent and to evaluate its mechanism of action leading to the development of new molecules which may proceed to further pharmacological investigations.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 28.02.2023 Modified on 11.07.2023

Research J. Pharm. and Tech 2024; 17(3):1166-1172.

DOI: 10.52711/0974-360X.2024.00181