In vitro Evaluation of Anticancer Activity of Karanthai Legium (Kl) –
A Siddha Medicine for Cervical Carcinoma against Hela Cell Line By MTT Assay
1PG Scholar, Department of Gunapadam, National Institute of Siddha, Chennai - 47.
2Associate Professor, Department of Gunapadam, National Institute of Siddha, Chennai - 47.
3Director, National Institute of Siddha, Chennai - 47.
*Corresponding Author E-mail: carolinpaul97@gmail.com
ABSTRACT:
Cervical cancer become a major global burden and of course considered one of the leading causes of cancer-related deaths in women, particularly in developing countries. Viral induction of human papilloma viruses (HPV) can disturb the basic cellular mechanism of growth control. Currently medicinal plants have become the source of drug discovery in research for treating cancer. Siddha medicine is an ancient medical system of India which classifies diseases into 4448 types. In Siddha literature, Cancer is mentioned as Vippuruthinoipadalam (Carcinoma like illness), Kiranthi noi, Mega Katti, Pilavai and Putru noi (tumour). Literary evidences have a classic collection of herbal, mineral, herbo- mineral medicines for Cancer. Many anti-cancerous drugs are now in practice in Siddha medicine. Among them, Karanthai Legium (KL) a classical herbo mineral drug having anticancer potential is mentioned in Siddha Literature. The main objective of this study is to evaluate anti- cancer activity against HeLa cell using MTT assay. Present study revealed that, Karanthailegium (KL) at high as 200µg/ml showed high cytotoxicity activity as high as 72.44% against HeLa cell lines. IC50 of the tested KL against HeLa cells was calculated as104.90µg/ml. It was concluded from the result of the present study that the formulation KL possesses promising anti-cancer activity.
KEYWORDS: Anti-cancer activity, Karanthai legium, HeLa cell lines, MTT assays, Siddha medicine.
INTRODUCTION:
Cancer has become a leading cause of death that could reduce the life expectancy throughout the world1. According to estimates from the World Health Organization (WHO) in 2019, 2 cancer ranks third or fourth in 23 countries and it is the first or second leading cause of female death in 112 countries before 70 years of age. Among them, cervical cancer is the second most common cancer in women worldwide. Most deaths occur among women living in low- and middle-income countries due to poor access to screening and treatment services.
Cervical cancer was highlighted in the “Political Declaration of the High-level Meeting of the General Assembly on the Prevention and Control of Non-communicable Diseases” and in the “comprehensive global monitoring framework” under development. It includes key indicators of non-communicable diseases and a set of global targets for its prevention and control 2.
Cervical intraepithelial neoplasia (CIN) is a premalignant lesion with three stages: CIN1, CIN2, or CIN3.2. When untreated, CIN2 or CIN3 (collectively referred to as CIN2+) can develop into cervical cancer. Approximately 1–2% of women have CIN2+ every year with a higher ie,10% prevalence rate is reported in HIV-positive women3. Cancer causing factors linked to either increasing average socioeconomic levels or a diminishing risk of persistent infection with high-risk HPV, resulting from improvements in genital hygiene, reduced parity, and a diminishing prevalence of sexually transmitted disease4. Currently, the use of herbal medicines in cancer treatment has undergo a thoughtful attention due to its various phyto-metabolic contents with several biological activities. Hence, it is a need to develop safe and effective treatment method for the prevention and treatment of cervical cancer5.
In Siddha system of medicine contain 64 therapies were divided into 32 internal and 32 external therapies. Among them Legium is one of the internal medicine, which composed of drug or drug powder, decoction with added ghee,sugar (or) jaggery. Karathai legium (KL) is one of the herbomineral drug indicates for kiranthi, karappan, soolai, kannaputru, lingaputru, yoniputru, pilavaiputru, kunmam. It is one of the simple herbo mineral preparations for cancer management. The study was carried out to validate anticancer activity of Karanthai legium (KL) on HeLa cells through MTT assay.
The raw drug was purchased in Ramasamy Chettiyar, Paris, Chennai and authenticated by botanist (NISMB5352022) and from Department of Gunapadam ,National Institute of Siddha ,Chennai.
Thirikadugu (Zingiber officinalis, Piper nigrum, Piper longum), Kadukkai (Terminalia chebula), Thandrikai (Terminalia bellirica), Vasambu (Acorus calamus), Indhuppu (Rock salt), Valuluvai (Celastruspaniculatum), Kostam (Saussurealappa), Karunjeeragam (Nigella sativa), Omam (Trachyspermumammi), Arasu (Ficusreligiosa), Elam (Elettaria cardamom), Manjal (Curcuma longa), Karbogaarisi (Psoraleacorylifolia), Seviyam (Piper nigrum– black peper root), Vaividangam (Embeliaribes), Anaithipilli (Scindapsus officinalis), Jathikai (Myristica fragrans), Shenbaga poo (Micheliachampaca), Thalisam (Abiesspectabilis), Lavangam (Syzygium aromaticum), Athimathuram (Glycyrrhizag labra), Jathipat hiri (Myristica fragrans) - eachĽ palamand Root of Karanthai (Spaeranthusamaranthoides) obtained.
Except for root of karanthai, all of the aforementioned raw drugs were refined, ground, and converted into a fine powder. Karanthai root was washed, dried, and ground into a powder. The same amounts of powdered karanthai root to the net weight of the aforementioned raw drugs is taken and mixed well. This powder was added slowly and thoroughly combined with melted ghee. Finally, stirred with honey6.
Serial dilutions of test formulation (10, 50, 100 and 200 μg/ml) were prepared using Double-distilled water.
The subculture of HeLa cell lines done in Dulbecco’s Modified Eagle’s Medium (DMEM), after discarding the culture medium, it was trypsinized separately, taken in a flask. To the disintegrated cells 25mL of DMEM with 10% FCS was added. By gently passing a pipette the cells suspended in the medium were homogenized.
One ml of the homogenized cell suspension was added to each well of a 96 well culture plate along with different concentration of KL sample (0 to 200µg/mL) and incubated at 37°C in a humidified CO2 incubator with 5% CO2. The cells were incubated for 48 hrsand then observed under an inverted tissue culture microscope. With 80% confluence of cells cytotoxic assay was carried out.
For cytotoxicity assay (3‐(4, 5‐dimethyl thiazol‐2yl)‐2, 5‐diphenyl tetrazolium bromide (MTT) were used. Mitochondrial Succinate dehydrogenase and reductase of viable cells, cleaved MTT yielding purple product formazan that could be measured. The production of formazan is directly proportional to the number of viable cells and inversely proportional to the degree of cytotoxicity. After incubation for 48hrs MTT was added to the wells and left for 3 h in room temperature. Then the content of all the wells were removedusing pipette and 100µl SDS in DMSO was added to dissolve the formazan crystals. The absorbances were read using Lark LIPR9608 micro plate reader at 540 nm 7. The % cell viability was calculated using the following formula:
% Cell viability = A540 of treated cells X 100 / A540 of control cells
Graphs are plotted using the Concentration of the sample as X-axis and % of Cell Viability at Y-axis.To compare the full cell viability assessments (Figure 1) in each assay cell control and sample control were included.
The half maximal inhibitory concentration (IC50) quantitatively measures the effectiveness of a compound in inhibiting a biological or biochemical function. It indicates the quantity of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process by half. To determineIC50 of a drug a dose-response curve was constructed and the effect of different concentrations of antagonist on reversing agonist activity was examined. The concentration that could be needed to inhibit half of the maximum biological response of the agonist denotes IC50. The nonlinear regression analysis (curve fit) based on sigmoid dose response curve gives IC50 values for cytotoxicity tests.
The in-vitro cytotoxicity activity of the drug KL against HeLa cells were inhibited significantly; however the increase of sample concentrations showed a high increment of cytotoxicity and it was clearly observed in the results (Table 1). The samples tested at a maximum dose of 200µg/ml showed high cytotoxicity activity of about 72.44% against HeLa cell lines. It was proven that the high cytotoxicity effect of the test sample showed cell disintegration after 48h of treatment against the selected tested cell lines at higher concentrations (figure 2). The IC50 of the tested Karanthai legium against HeLa cells was calculated as 104.90µg/ml . The anticancer activity of Karanthai legium was plotted in (figure 1). The images of HeLa cell lines treated with various concentration of Karanthai legium (KL) are shown in (figure 2).
Table 1: In vitro cytotoxicity effect of drug KL against HeLa cells lines
|
Sample Conc.(µg/mL) |
% Cell Viability KL |
|
0.0 |
100.00 |
|
25.0 |
75.89 |
|
50.0 |
55.52 |
|
100.0 |
40.25 |
|
200.0 |
27.56 |
|
IC50 |
104.90 µg/ml |
Figure 1: Anticancer activity of Karanthai legium(KL) against the HeLa Cell lines.
Cytotoxicity effect of KL-H against HeLa Cell lines
Control Concentration 25 µg/Ml
Concentration 50 µg/mL Concentration 100 µg/Ml
Concentration 200 µg/mL
Figure 2: Cytotoxicity effect of Karanthai legium (KL) against HeLa Cell lines
According to WHO, Cervical Cancer is the fourth most common cancer among women population. Cervical cancer (more than 95%) is due to human papilloma virus (HPV).It can be cured, if diagnosed at early stage8. Cervical cancer is common due to lack of awareness and preventive measures for HPV among women. HPV vaccinations have an important role in prevention of cervical cancer but very few are aware to this and very few numbers of women gets vaccinated. Since ancient times, plant-based formulations have been practiced as remedies against diverse ailments9. Over the past two decades, interest in traditional medicines has increased considerably in many parts of the world10.
In Siddha system medicines were prepared using herbal, minerals, metals and animal products. Many higher order medicines have been mentioned in Siddha literatures for the treatment and management of cancer. In that Karanthai legium is one of the herbo- mineral drugs which is indicates for cervical cancer. The each ingredient in preparation has their unique anti-cancer and antioxidant properties. As karanthai is one of the kaya karpam (rejunivation) tha acts as an antioxidant. They prevent from ageing, Skin wrinkling, greying and promote longevity. As drug KL is a herbal drug which has safety and efficacy for cervical cancer. Present study reveals that, samples tested (Karanthai legium) at high as 200µg/ml showed high cytotoxicity activity as high as 72.44% against HeLa cell lines.
The half maximal inhibitory concentration (IC50) is an important predicating factor to measure the potency or active entity of the formulation in inhibiting biochemical or metabolic function11. Normal quantification of IC50 of a drug determined by regular dose-response curve, that the concentration required for the inhibiting the 50 % of the cell population. Lower the value higher will be the potency12. The Sphaeranthus amaranthoides has the presence of steroids, alkaloids, sugars, phenolics, flavonoids, saponins, tannins. From the results of the present study, it was observed that the IC50 of the tested Karanthai legium against HeLa cells was calculated as104.90µg/ml.
As much of occurrence of cervical cancers is due to HPV, the number of screenings should be increased more among the rural population. The result obtained from the in vitro studies reveals that the Siddha Herbo Mineral formulation Karanthai legium(KL) has potent anti cancer activity and it can be used in the management of cervical cancer among women population.
Further detailed studies have to be conducted to validate the medicine for global acceptance.
The author has stated no conflict of interest.
The Author would like to thank the guide Dr. Mariappan for guiding and supporting for this manuscript. The author also likes to thank Head of the Department (i/c) Department of Gunapadam, The Director, National Institute of Siddha, for the support extended.
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Received on 22.05.2023 Modified on 10.08.2023
Accepted on 12.10.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(2):807-810.
DOI: 10.52711/0974-360X.2024.00125