world journal of pharmaceutical research · vaiththiya viththuvan mani s.kannuchamipillai,...

www.wjpr.net Vol 8, Issue 8, 2019.

Abinaya et al. World Journal of Pharmaceutical Research

851

ANTICANCER POTENTIAL OF A NOVEL SIDDHA METALLO-

MINERAL FORMULATION “KAALAMEGA NARAYANA

CHENDHOORAM” AS MENTIONED IN “ATHMARAKSHA MIRTHAM

ENNUM VAITHIYA SAARA SANGERAHAM” THROUGH A A549 CELL

LINE STUDY FOR LUNG CANCER

Dr. R. Abinaya*1, Dr. R. Vijaya Nirmala

1, Dr. R. Karolin Daisy Rani

2 and Dr. M. D.

Saravana Devi3

1Post Graduate, Department of Gunapadam (Pharmacology), Government Siddha Medical

College, Arumbakkam, Chennai, Tamil Nadu, India.

2Lecturer, Department of Gunapadam (Pharmacology), Government Siddha Medical College,

Arumbakkam, Chennai, Tamil Nadu, India.

3Head of the Department, Department of Gunapadam (Pharmacology), Government Siddha

Medical College, Arumbakkam, Chennai, Tamil Nadu, India.

ABSTRACT

Aim and Objective: The aim of the present study is to validate the

Anti Lung cancer potentials of a novel siddha metallo-mineral

formulation Kaalamega Narayana Chendhooram as mentioned in

Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham through a

A549 cell line study. Methods: Cancer is a dangerous disease with

increasing its prevalence day by day. There is no such medications

which have a complete cure for cancer without any adverse effect. The

super natural scientists gave a boon of siddha system of medicine

which have the solutions for various diseases including Cancer without

any adverse effect. Kaalamega Narayana Chendhooram as mentioned

in Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham, a novel

siddha metallo-mineral formulation which has a anticancer potential

was underwent globally accepted technique for screening anticancer potentials through MTT

assay for KMNC was done on A549 cell lines with various concentration of the drug as 6.25

µg/ml, 12.5 µg/ml, 25 µG/ml, 50 µg/ml, 100 µg/ml. Raising the modern medical science to

support the effect of heavy metals usage in aspect of cancer treatment. Results: At the end of

World Journal of Pharmaceutical Research SJIF Impact Factor 8.074

Volume 8, Issue 8, 851-864. Research Article ISSN 2277– 7105

Article Received on

15 May 2019,

Revised on 05 June 2019,

Accepted on 25 June 2019,

DOI: 10.20959/wjpr20198-15356

*Corresponding Author

Dr. R. Abinaya

Post Graduate, Department

of Gunapadam

(Pharmacology),

Government Siddha Medical

College, Arumbakkam,

Chennai, Tamil Nadu, India.



852

this preliminary research study, that the trail drug of a novel siddha metallo-mineral

formulation Kaalamega Narayana Chendhooram as mentioned in Athmaraksha Mirtham

Ennum Vaithiya Saara Sangeraham through a A549 cell line study was found to be a potent

anticancer medicine on human lung cancer cells. It showed (IC 50) 50% inhibitory

concentration at 50µg/mL. There has been showed that there was a decrease in a number of

cell with increasing the concentration of the drug as 6.25 µg/ml, 12.5 µg/ml, 25 µG/ml, 50

µg/ml, 100 µg/ml showed the percentage of viability as 93.73%, 83.56%, 61.28%, 40.95%,

23.96.% Thus the Further studies with the trail medicine KMNC will pave the right path

towards the safe and potent as anticancer medicine. Conclusion: The present preliminary in-

vitro study of a potent siddha metallo-mineral formulation Kaalamega Narayana

Chendhooram as mentioned in Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham

through a A549 cell line study was found to be a potent anticancer medicine on human lung

cancer cells.

KEYWORDS: Kaalamega Narayana Chendhooram, KMNC, Chendhooram, Siddha,

metallo-mineral formulation, A549 cell line, MTT assay, Lung cancer, Cancer.

INTRODUCTION

Cancer is the disease of the cell, which is the one of the leading cause of death World wide. It

is a generic term for a group of more than 100 diseases that can affect any part of the body.[1]

Cancer is the second leading cause of clinical mortality in developed countries. Cancer and

tumors are the result of altered, excessive and invasive cell reproduction in other nearby

healthy tissues, their development can be originated from diverse causes, like genetics, to the

consumption of potentially toxic and harmful foods. In India, tumors (neoplasms) are the

third cause of mortality.[2]

Cancer is a disease of the cell with progressive, persistant, purposeless and uncontrolled

growth of cell. If it occurs in lungs is called lung cancer. It is a malignant lung tumor.[3]

Worldwide, lung cancer is the most-common cancer among men in terms of

both incidence and mortality, and among women has the third-highest incidence, and is

second after breast cancer in mortality. In 2012, there were 1.82 million new cases

worldwide, and 1.56 million deaths due to lung cancer, representing 19.4% of all deaths from

cancer.[4]

https://en.wikipedia.org/wiki/Lung_tumor

https://en.wikipedia.org/wiki/Incidence_(epidemiology)

https://en.wikipedia.org/wiki/Mortality_rate



853

Across the developed world, 90% of lung cancer deaths in men and 70% of those in women

during the year 2000 were attributed to smoking.[5]

The main causes of cancer are smoking, tobacco chewing, poor oral health, malnutrition,

alcohol, dietary imbalances, HPV infections, obesity, betel quid, hormones and chronic

infections leading to chronic inflammation.[6]

Siddha system of medicine plays an important role in delivering huge line of treatment for

various life threatening diseases including cancer. The term Cancer was mentioned in the

ancient siddha classical literature in the name of Putru (undetermined growth), Arpudham

(spectacular tumors), and Vanmeegam (precarious tumors).[7]

Pathology of Cancer in Siddha system of medicine

The pathology of Siddha System of medicine explained about the three humors such as

Vaadham called Wind, Pitham called Fire, and Kapham called Phlegm which maintains the

human body in a steady state without any diseased conditions. The normal proportion of three

humours were in the ratio of 1:1/2:1/4. The change in the ratio of this humors will affect the

normal physiology of the body and results in causing diseases.

1. Pitham is responsible for various metabolic functions in cells.

2. In the pathology of cancer the humour called Pitham which agni or fire gets gradual

decrease.

3. As a result of decreased Pitham there is progressive increase of Kapham which as phlegm

or water and Vatham which is Wind.

4. Increase of Kapham results in excessive tissue growth and Vatham results in anabolic

growth.

5. The inversely proportional of Kapham and Vatham to Pitham results in cancer.

6. The functions of Vatham, Pitham, Kapham were action, protection and destruction.

7. In cancer the protective function of Pitham loses its power due to the dominant of

destructive powers of Kapham and action Vatham.

8. Thus Kapha Vatham results in cancer.[8]

Nanomedicine of Siddha in dealing with Cancer

The miraculous of Siddha system of medicines, the higher order formulations like Parpam,

Chendhooram, Kattu, Kazhangu, Chunnam, Padhangam were considered as nano medicine

due to ultra fine particles around 1 – 100 nm in their particle. Due to the nano nature of



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medicine with greater surface area, they acts at the cellular and nuclear level, prolonged

duration of action, with increased bioavailability, required theraupetic response with less

toxic effects. With all the characteristic nature of nano medicine, thus KMNC plays an

important role in treating cancer cells.[9]

MATERIALS AND METHODS

THE DIFFERENT TYPES OF KMNC PREPARATIONS WERE AVAILABLE IN

DIFFEREN CLASSICAL SIDDHA LITERATURES.THEY ARE LISTED AS BELOW

Vaiththiya Viththuvan Mani S.Kannuchamipillai, Chikichcha Raththina Theepamennum

Vaithya Nool, Page No: 247, B.Rathna Nayaagar & Sons, Thirumakal Vilasa

Achchakam, Chennai 79.

Kandhasamy Mudhaliyaar, Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham,

First edition 1931, Page No : 496, B.Rathna Nayaagar & Sons, Thirumakal Vilasa

Achchakam, Chennai 79.

Vaiththiya Viththuvan Mani S.Kannuchamipillai, Kannusamy Paramparai Vaithiyam,

Page No: 327, B.Rathna Nayaagar & Sons, Thirumakal Vilasa Achchakam, Chennai 79.

Vaiththiya Viththuvan Mani S.Kannuchamipillai, Kannusamiyam, Page No: 120,

B.Rathna Nayaagar & Sons, Thirumakal Vilasa Achchakam, Chennai 79.

Vaiththiya Viththuvan Mani S.Kannuchamipillai, Kannusamy Paramparai Vaithiyam,


All the above mentioned the classical siddha text books shows the same ingredients and the

same indications of KMNC but all the above preparations follows different medicinal

preparation methods.

The current research derived the medicinal preparation the siddha text, Kandhasamy

Mudhaliyaar, Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham, First edition 1931,


SELECTION OF THE DRUG

For this present study, the metello-mineral formulation “KAALAMEGA NARAYANA

CHENDHOORAM” was taken as the compound drug preparation for oral cancer mentioned

in the classical Siddha literature “Athmarakshamirtham Ennum Vaithiya Saara Sangeraham”

written by Kandhasamy Mudhaliyaar, pg no:493, First Edition 1931.



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Ingredients of the drug

1. Purified Vediuppu [Potassium nitrate] – 840 gm

2. Purified Thurusu [Cupric sulphate] – 210 gm

3. Purified Padikaaram [Aluminium potassium sulphate (Alum)] – 840 gm

4. Purified Vengaram [Sodium bicarbonate (Borax)] – 210 gm

5. Purified Navacharam [Ammonium Chloride]-210gm

6. Purified Pooneeru [Impure Sodium Carbonate (Fullers Earth)] – 105 gm

7. Purified Jaathilingam [Red sulphate of mercury]-525gm

8. Purified Gandhagam [Sulphur] – 420 gm

9. Purified Kalluppu [Sodium chloride]- 210 gm

10. Purified Rasam [Hydragyrum] – 1050 gm

11. Purified Aritharam [Tri sulphate of Arsenic (Yellow Orpiment)]- 350 gm

12. Purified Manosilai [Di suphate of Mercury (Red Orpiment)]- 140gm[10]

Kandhasamy Mudhalaiyaar, Kaalamega Narayana Chendhooram, Athmaraksha

Mirtham Ennum Vaithiya Saara Sangeraham First Edition 1931, P.no.493,94.

Collection of the raw materials

All the raw materials were purchased from R.N. Rajan country drug store, Parrys corner,

Chennai.

Identification and Authentication of the drug

The raw materials were identified and authenticated by the experts of Gunapadam,

Government Siddha Medical College, Arumbakkam, Chennai- 106.

The specimen sample of each raw material has been kept in the PG Gunapadam department

individually for future reference.

Procedure

840 gm of 8th

solution of Vediuppu [Potassium nitrate] and Padigaram [Aluminium

potassium sulphate (Alum)] were taken.

Along with that, 210 gm of Thurusu [Cupric sulphate], Vengaram [Sodium bicarbonate

(Borax)], Navacharam [Ammonium Chloride], Kalluppu [Sodium chloride Impura] were

taken and then mixed with 105 gm of Pooneeru [Impure Sodium Carbonate (Fullers

Earth)].



856

Above ingredients were ground into fine powder and divided into 3 parts.

First part of the powder was underwent distillation process, the end product was mixed

with 2nd

part of powder and dried.

Second part of the powder was underwent distillation process, the end product was mixed

with 3rd

part of powder and dried.

Third part of the powder was undergoes distillation process, the final end product was

taken and kept in a sealed bottle.

The Jaathilingam [Red sulphate of mercury]-525 gm, Aritharam [Tri sulphate of Arsenic

(Yellow orpiment)]-350 gm, Gandhagam [Sulphur] 420 gm, Rasam (Mercury)- 1050 gm,

Manosilai [Di sulphate of mercury (Red Orpiment)] 140 gm wereground, along with the

end product of distillation for 12 hours (4 saamam) and made into fine powder and dried.

Dried powder was kept in a mud pot which was sealed with 7 mud pasted plaster.

Another mud pot with small quantity of sand was taken and above preparation was kept

into it and sealed the lid with mud pasted plaster.

The mud pot was ignited by using Aavarai stick for 30 hours (10 saamam), after 30 hours

“Chendhooram” was obtained

Drug profile

Drug name Kaalamega Narayana Chendhooram

Dosage 244 mg of Chendhooram [1/2 Panavedai ]

Route Enteral (oral)

Adjuvant Thipili chooranam with honey (bd for 48 days – 1 mandalam)

Indications Kannaputru [Oral Cancer], Elaippu [Tuberculosis], Kuttam 18 [Hansen’s

Disease]

Reference “AthmarakshaMirutham Ennum Vaithiya Saara Sangeeraham”.[13]

Fig No: 1: Final Product of Kmnc Chendhooram.



857

INVITRO ANTICANCER EFFECT DETERMINATION BY MTT ASSAY

The study was conducted in Biogenix Research Centre, Trivandrum., Kerala, India. A549

(Lung cancer) cells was initially procured from National Centre for Cell Sciences (NCCS),

Pune, India and maintained Dulbecco’s modified Eagles medium, DMEM (Sigma aldrich,

USA).

The cell line was cultured in 25 cm2 tissue culture flask with DMEM supplemented with 10%

FBS, L-glutamine, sodium bicarbonate (Merck, Germany) and antibiotic solution containing:

Penicillin (100U/ml), Streptomycin (100µg/ml), and Amphoteracin B (2.5µg/ml). Cultured

cell lines were kept at 37ºC in a humidified 5% CO2 incubator (NBS Eppendorf, Germany).

The viability of cells were evaluated by direct observation of cells by Inverted phase contrast

microscope and followed by MTT assay method.

Cells seeding in 96 well plate

Two days old confluent monolayer of cells were trypsinized and the cells were suspended in

10% growth medium, 100µl cell suspension (5x104 cells/well) was

seeded in 96 well tissue

culture plate and incubated at 37ºC in a humidified 5% CO2 incubator.

Preparation of compound stock

1mg of sample was weighed and dissolved in 1mL DMEM using a cyclomixer. The sample

solution was filtered through 0.22 µm Millipore syringe filter to ensure the sterility.

Anticancer Evaluation

After 24 hours the growth medium was removed, freshly prepared each compounds in 5%

DMEM were five times serially diluted by two fold dilution (100µg, 50µg, 25µg, 12.5µg,

6.25µg in 500µl of 5% DMEM) and each concentration of 100µl were added in triplicates to

the respective wells and incubated at 37ºC in a humidified 5% CO2 incubator. Non treated

control cells were also maintained.

Anticancer Assay by Direct Microscopic observation

Entire plate was observed after 24 hours of treatment in an inverted phase contrast tissue

culture microscope (Olympus CKX41 with Optika Pro5 CCD camera) and microscopic

observation were recorded as images. Any detectable changes in the morphology of the cells,

such as rounding or shrinking of cells, granulation and vacuolization in the cytoplasm of the

cells were considered as indicators of cytotoxicity.



858

Anticancer Assay by MTT Method

Fifteen mg of MTT (Sigma, M-5655) was reconstituted in 3 ml PBS until completely

dissolved and sterilized by filter sterilization.

After 24 hours of incubation period, the sample content in wells were removed and 30µl of

reconstituted MTT solution was added to all test and cell control wells, the plate was gently

shaken well, then incubated at 37ºC in a humidified 5% CO2 incubator for 4 hours. After the

incubation period, the supernatant was removed and 100µl of MTT Solubilization Solution

(Dimethyl sulphoxide, DMSO, Sigma Aldrich, USA) was added and the wells were mixed

gently by pipetting up and down in order to solubilize the formazan crystals. The absorbance

values were measured by using microplate reader at a wavelength of 540 nm (Laura B.

Talarico et al., 2004).

The percentage of growth inhibition was calculated using the formula:

Mean OD Samples x 100

Mean OD of control group

APPENDIX

Instruments and reagents used

DMEM media -Sigma Aldrich, USA D5648

Fetal Bovine Serum -Gibco, US orgin-

0.25% Trypsin - Invitrogen, USA 25200-056

Micropipettes - F1 Thermoscientific USA

CO2 Incubator - Eppendorf, GERMANY

Phase Contrast Microscope - Olympus, JAPAN with Optika Pro 5 Camera

MTT - Sigma Aldrich M5655

ELISA Reader - ERBA, GERMANY

Culture Plates and Flasks - NUNC, Thermoscientific USA

Image Magnification - 10X.[11]

% of viability =



859

Fig No: 2: Micro Plate Reader.

Fig No: 3: Phase Contrast Microscope.

Fig No: 4: CO2 Incubator.



860

RESULTS AND DISCUSSIONS

Table No. 1: Effect of Kmnc In A549 Lung Cancer Cell Lines: LC50 Value: TC-

53.7163µg/mL (Calculated using ED50 PLUS V1.0 Software).

Sample Concentration

(µg/mL) OD value I OD value II OD value III Average OD

Percentage

Viability %

Control 0.6885 0.6946 0.7119 0.6983 100.00

Sample code: KMNC

6.25 0.6738 0.6547 0.6351 0.6545 93.73

12.5 0.5542 0.5863 0.6100 0.5835 83.56

25 0.4697 0.3973 0.4168 0.4279 61.28

50 0.2954 0.2746 0.2878 0.2859 40.95

100 0.1616 0.1761 0.1643 0.1673 23.96

Fig No: 5: Graphical Representation of A549 Cell Line.

Fig No: 6: Cell Line Images.



861

Control group

6.25 µg/mL

12.5 µg/mL

25 µg/mL



862

50 µg/mL

100 µg/mL

DISSCUSSION

Cytotoxicity Assay by MTT

MTT colorimetric method, is a method for detecting cell survival and growth methods. This

assay is based on the metabolic reduction of 3- (4,5- dimethylthiazol-2-yl) -2,5-difeniltetrazol

(MTT) by mitochondrial enzyme succinate dehydrogenase in a colored compound blue

(formazan), allowing to determine the functionality of the mitochondrial treated cells. This

method has been widely used to measure survival and cell proliferation. The amount of living

cells is proportional to the amount of formazan produced. Cell lines derived from NCCS,

Pune were free from any kind of bacterial and fungal contamination.



863

Table-1 and Graph -1 shows the drug dose and % of Inhibition of A549 Cell line after

treating with KMNC. It can be observed by the result of MTT assay that the IC dose of

KMNC is 50μg/ml. As the dose increases the A549 cell viability decreases. It was found that

the % of growth inhibition increasing with increasing concentration of KMNC steadily from

6.25 μg/ml to 100 μg/ml on A549 line (Table-1) and (Graph -1) that IC value on A549 cell

line was 50 and R value was found to be 0.6983.

KMNC at different doses (6.25-100 μg in100 μl of 5% DMEM) was administered for 24 hrs.

It was found that the number of cells decreases as the dose increases and at approximately 50

μg/ml dose of extract, 50% of the cells (A549 cells) were less as compared to normal control

as shown in graph no (1). The percentage of cells viability was determined by calculating the

O.D of treated against the control. Reading optical density (OD) is performed in a

spectrophotometer at a wavelength of 540 nm. Comparison values are made on a basis of

50% inhibition of growth (IC50) in treated cells with specific agents. Results are tabulated in

Table (1) and Graph (1). It showed that (IC 50) 50% inhibitory concentration at 50µg/mL.

There has been showed that there was a decrease in a number of cell with increasing the

concentration of the drug as 6.25 µg/ml, 12.5 µg/ml, 25 µG/ml, 50 µg/ml, 100 µg/ml showed

the percentage of viability as 93.73%, 83.56%, 61.28%, 40.95%, 23.96.% Thus the Further

studies with the trail medicine KMNC will pave the right path towards the safe and potent as

anticancer medicine.

CONCLUSION

Cancer stands as a one of the leading cause of death World wide. There is no such complete

cure for cancer without any adverse effect. Thus there is a need for evaluating new medicines

for cancer without any adverse effect. Siddha system of medicine has a rich collections of

medicines for treating various diseases including acute and chronic diseases. With the

treasure of Siddhars and Siddha system of medicine I explored the anticancer effect of KMNC

in A549 cell lines, it gives the better results in cancer cell lines. Thus the traditional medicinal

preparation KMNC with its nano particle size will provide a magical remedy in the field of

cancer management.

ACKNOWLEDGEMENT

First and foremost I would like to thank the Almighty for his showers, grace, strength and

caliber for doing various research. In the name of Siddhars who has given me power and

courage to accomplish this work, I bow my head on thanks and gratitude to Siddhars for their



864

blessings. Finally, I would like to acknowledge the person who mean world to me, My

mother Mrs. A.Pushpavalli Rajendran for her lovable support and encouragement towards my

various research work.

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