Physico-chemical and Toxicological

standardization of a Siddha medicine, ‘Sarva noi

Linga chenduram’.

R. Radhakrishnan1, H. Vetha Merlin Kumari 2, H. NaliniSofia3, K. Manickavasakam4

Abstract:

Siddha, a time tested, safe and effective system ofTraditional Medicine is being practiced widely in Tamilspeaking parts of our country and abroad. The specialtylies in the ability of the Siddha Physicians to preparetheir own medicines. ‘Sarva noi linga chenduram’ is one amongthe medicines used in Siddha for treating ‘Azhal Keel Vayu’which may be correlated with Osteo arthritis having thesymptoms of Pain and swelling in knee joints etc. So far,no scientific standardization has been carried out pre-clinically. So in the present study standardization of thetest drug was done through Physico-chemical analysis, Acuteand long term toxicity studies as per WHO guidelines fortraditional medicine.

Oral toxicity studies ensured the safety usage of thedrug to animals up to a maximum dose of 23.4 mg/animal.Physico- chemical analysis showed the presence ofconstituents like Calcium, Iron and Sulphur. The minimumparticle size (3 µ) unveiled in the Particle per Million

1 P.G. Scholar, National Institute of Siddha, Chennai, Tamilnadu.

2 & 3 Lecturers, Dept. of Medicine (Maruthuvam), National Institute of Siddha, Chennai, Tamilnadu. dr.sofiacourtdurai@gmail.com 3

4 Director, National Institute of Siddha (NIS), Chennai, Tamilnadu. dr.kmvm@gmail.com

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analysis shows the existence of the drug in micro particlesize which contributes to its therapeutic effect by theincreased bio availability.

Key words: Siddha Medicine, Toxicology, Physico-chemicalstandardization

BACKGROUND

Increasing use of natural products as a result ofpatients seeking for Siddha medicines to prescribed drugshas provoked a great interest in research in Siddha. InSiddha system of medicine the use of metals, minerals andchemical products are predominant. For a medicine to beeffective, the inorganic substances have to be brought totheir atomic form. The siddhars developed the knowledge ofbringing inorganic substances into atomic and ionic formwhich can be easily absorbed in the system when ground withherbal juice and put in the fire with a calculated number ofcow dung cakes.

The special feature of the Siddha medicine is that mostof the preparations are in compound formulation, and becauseof its synergistic action, toxicity is being diminished,thereby increasing bioavailability through the cells of thebody. Siddha system has its own well developed chemistry andsiddhars tireless striving in the direction of developmentresulted in genesis of thousands of mineral and metallicpreparations.

Siddhars are the pioneers in the use of metals andminerals in the treatment of diseases.Siddhars hasclassified the chemicals into metals of 11 varieties, saltsof 25 varieties, pashanam of 64 varieties and uparasas of120 varieties. All the metals and minerals used in Siddhasystem are completely in a detoxified state as per themethod known “suddhi muraigal”. During this process, themetal molecules are rendered non toxic.

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Sarva noi linga chenduram is a Siddha formulation. Ithas the ingredients of venkaram (borax) and lingam(cinnabar). Cinnabar has been used in traditional medicinesfor thousands of years for various ailments especiallysoolai (throbbing pain) and vatha diseases. Among themercurial compounds HgS is found to be the least toxic andless absorbed than any other form of mercury.Standardization of this formulation is must according to GCPbefore introducing in clinical practice. Modern analyticaltechiniques of scanning electron microscopy (SEM) andinductively coupled plasma optical emission spectrometry(ICP-OES), is used to determine physicochemical fingerprint.

In Siddha literature, Azhal keel vayu is describedunder vatha diseases. Azhal keel vayu is a chronicdegenerative joint disease characterized by pain andphysical disability. Osteo arthritis is a major contributorto morbidity and utilization of health care resourcesworldwide. In India it is a second most prevalent diseaseafter diabetes (according to the survey titled, TNS AROGYA2006-2007, the health monitor).

MATERIAL AND METHODS

1. Physico- chemical analysis

Inductively coupled plasma optical emissionspectrometry (ICP-OES), is an analytical technique used forthe detection of trace metals. The intensity of thisemission is indicative of the concentration of the elementwithin the sample. A Perkin-Elmer Optima ICP spectrometer isused for routine ICP-OES analysis done at SAIF, IIT Madras,Chennai-36

SEM analysis is done at SAIF, IIT Madras, Chennai-36 todetermine particle size distributions. The SEM is carriedout by using FEI-Quanta FEG 200-High Resolution Instrument.

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2. Toxicity studies

Preparation of test substance

SARVA NOI LINGA CHENDURAM is brick red in colour .Thetest substance was insoluble in water. In order to obtainand ensure the uniformity in drug distribution, the drug wasdissolved by Normal solution (10%).

Test Animals for acute toxicity study

Swiss albino mice (20-25 gm) of either sex bred in thelaboratory animal facility of the King institute, Chennaiand stocked at Animal house, National institute of Siddha,Chennai. The animals were housed in polypropylene cagesprovided with bedding of husk and allowed to acclimatize forone week under standard conditions of temperature(27±2ºc )with a 12:12 h natural light : dark cycle beforecommencement of the experiment. The animals were maintainedfreely on standard pellets (Sai meera foods pvt.ltd,Bangalore) and water. All animal experiments were incompliance with International Guidelines for handlingexperimental animals. Animal studies were conducted withprior permission obtained from the Institutional AnimalEthics Committee, National Institute of Siddha, Chennai,with protocol ethical clearance number1248/ac/09/CPCSEA/December/IAEC 2011

Test Animals for sub acute toxicity study

Male and Female Wister albino rats (150-200 gms) ofeither sex bred in the laboratory animal facility of theKing institute, Chennai and stocked at Animal house,National institute of Siddha, Chennai. The animals werehoused in polypropylene cages provided with bedding of huskand allowed to acclimatize for four week under standard

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conditions of temperature(27±2ºc ) with a 12:12 h naturallight : dark cycle before commencement of the experiment.The animals were maintained freely on standard pellets (Saimeera foods pvt.ltd, Bangalore) and water. All animalexperiments were in compliance with International Guidelinesfor handling experimental animals. Animal studies wereconducted with prior permission obtained from theInstitutional Animal Ethics Committee, National Institute ofSiddha, Chennai, with protocol ethical clearance number1248/ac/09/CPCSEA/December/IAEC 2011

Acute toxicity study

Swiss albino mice of both sex were randomly grouped(n=10) to orally receive 2.34 mg single dose of Sarva noilinga Chenduram. The control groups received distilled water(5ml/kg). The dose level was calculated according to bodyweight, and surface area. Since the clinical dose was 130mg\day, it was converted to animal dose (2.34 mg) and thenadministered. The animals were observed continuously forfirst 4 hrs, then periodically during the first 24 hrs tocheck any physiological and behavioral responses. Animalswere observed for body weight and mortality for 14 days. Ifanimals dye during the period of study, the animals weresacrificed. At the end of the 14th day all animals weresacrificed and necroscopy was done.

Sub acute toxicity study

Adult Wister albino rats of both sexes were randomlygrouped (n=10) to orally receive 4.68, 23.4, 46.8 mg/kg ofSNLC, Control group received distilled water (5ml/kg).Treatment was given once a day for 30 days. On day 31animals fasted overnight and all the animals were sacrificedon the 31st day and assessed for sub acute toxic effect. Allthe animals were weighed and examined for clinical signs atthe start and end of the treatment.

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Histopathological studies

Tissue samples of organs from control and treatedanimals were preserved in 10% formalin for preparation ofsections using microtome. The organs included liver,kidneys, heart, lungs and stomach of the animals werepreserved and they were subjected to histopathologicalexamination.

The organ pieces (3-5 micron ) were fixed in 10%formalin for 24 hours and washed in running water for 24hours .Samples were dehydrated in tissue processor and thencleaned in benzene to remove absolute alcohol .Embeddingwas done by passing the cleared sample through three cupscontaining molten paraffin at 50 degree c and then a cubicalblock of paraffin made by the L moulds it was followed bymicrotome and the slides were stained with haematoxylin–eosin stain .Stained sections of each organ were examinedunder light microscope at high (40X) power magnification.All the histo pathological slides were prepared at Dept .of.Pathology,Vels university,pallavaram Chennai.

Results

Preliminary Qualitative Phyto chemical analysis showedthat the drug possess Calcium, Carbonate, Iron, Phosphate,Aluminium, Magnesium, Mercury, Silicate, Sodium, andunsaturated compunds.

Physicochemical properties of Sarva Noi Linga Chenduram.

Table-1 Colour characters of Sarva Noi Linga Chenduram.

S No Solvent used Under ordinarylight

Under ultraviolet light

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1 Powderedmaterial

Dark Brown Dark Brown

Table -2 Physicochemical properties of Sarva Noi LingaChenduram.

S No. Parameters Valuesobtained(%w/w)

Heavy/ toxicmetals

1 Total ash value 9.15 Lead BDL2 Acid insoluble ash 0.77 Cadmiu

mBDL

3 Water soluble ash 5.63 Mercury

4.251mg/L

4 Moisture content 10.24 Arsenic

BDL

Table-3 Colour, nature and percent yields of extracts ofSarva Noi Linga Chenduram

S.no. ExtractSolvents

Colour

Nature

%Yield(w/w)

SEM-Micrographparticalsize rangein micron

pH

1 Water Darkbrown

Solid 49 1 – 2.5micron

8.5 –8.7

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Particle Size Of Sarva Noi Linga Chenduram

Results of Acute toxicity study.

Sarva noi linga Chenduram at the dose 2.34mg/animaldid not exhibit any mortality in mice. No behavior changeswere noted for the first 4 hours and for the next 24 hoursand throughout the study period of 14 days. No weightreduction was noted before and after the acute studyduration. Reflexes were found to be normal before and afterthe study. All other observations were found to be normalbefore and after the study. In Necropsy, the organs of theanimal such as Liver, Heart, Lungs, Pancreas, Spleen,Stomach, Intestine, Kidney, Urinary bladder, Uterus allappeared normal.

Results of Sub acute toxicity study

No weight loss, abnormal animal behaviours, metabolicfunctions [urination, lacrimation, defaecation etc.,] andmortality were noted. In necropsy of the animal organs

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showed normal appearance and weight. All Haematological andbiochemical parameters were within normal limits. Thestastical report of the Haematological and Biochemical datasdid not show any significant difference, between the controland test groups. In Histopathological studies, No abnormalfindings were observed in the organs such as Heart, Liver,Lungs, Kidney and Stomach in 1X (4.68 mg/kg), 5X (23.4 mg/kg)and 10X (46.8 mg/kg) compared with control group.

Figure1. Sub acute effect of SNLC on rat heart Control group

1x(4.68 mg/kg)

showing normal shows normal cardiacmuscle bundles Myocardial fibers in a longitudinal section

5x (23.4mg /kg) 10x(46.8mg/kg)

shows normal Myocardial fibers in a longitudinal section shows

hypertrophic cardiacmuscle bundles

Figure2. Sub acute effect of SNLC on rat lung

Control group 1x (4.68mg/kg)

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shows normal bronchioles shows normal bronchiolesand alveoli and alveoli

5x (23.4mg /kg) 10x (46.8mg/kg)

shows normal bronchioles and alveoli shows normalbronchioles alveoli

and focal collections oflymphocytesFigure3. Sub acute effect of SNLC on rat liver

Control group 1x(4.68 mg/kg)

Liver of control animal showing liver shows almostnormal hepatocytes parenchyma with central vein and portal traid

5x (23.4mg /kg) 10x (46.8mg/kg)

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Shows dilated central veins Shows lymphocyticinfiltration in the portal triad

Figure4 . Sub acute effect of SNLC on rat kidney

Control group 1x(4.68mg/kg)

kidney of control showing normal renal shows normalrenal tissue withparenchyma with glomeruli (G)and glomeruli andtubulestubules (T)

5x (23.4mg /kg) 10x (46.8mg/kg)

shows renal parenchyma with normally Shows normalrenal tissue few tubulesappearing glomeruli. Show tubular casts inthe lumen.. Figure .5. Sub acute effect of SNLC on rat stomach Control group 1x(4.68mg/kg)

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shows normal mucosal glands. shows normal mucosalglands. 5x (23.4mg /kg) 10x(46.8mg/kg)

shows near normal mucosal gland Shows normal Intestinalmucosal lining with mild exudates. with mild exudates Conclusion

Oral toxicity studies conducted ensured the safetyusage of the drug to animals up to a maximum dose of 23.4mg/animal. Physico chemical analysis showed the presence ofconstituents like Calcium, Iron, Sulphur which played a rolein repairing and preventing the joint damage in the disease.Preparation of Bhasmas and chenduram in Siddha medicinegives much importance to particle size. It is mentioned inthe texts, that the process of marana (incineration) shouldbe repeated to get smaller particle size. The minimumparticle size (3 µ) unveiled in the (Particle Per Millionsize) PPM analysis shows the existence of the drug in microparticle size which contributes its therapeutic effect bythe increased bio availability.

Reference:

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1. Dr. R. Thiagarajan, LIM, ‘Gunapadam – Thathu Jeevavaguppu’, sixth edition, 2006, published by Department ofIndian Medicine & Homeopathy, Chennai.

2. Nayanar.M 1926. Theraiyar karisal.Buvaneshwari press.,Madras page 84.

3. Hardy et.al. A report on the composition of mercurialsused in traditional medicines in Oman. Journal ofEthnopharmacology.49., 17-22

4. Anupoga vaithiya Navaneetham, part IV- Hackim Pa.Mu. Abdullahsaibu

5. Compendium of Indian medicinal plants, vol II- B.N mehrotra,

Ram.P rastogi.

6. Siddha materia medica (Mineral and vegetable kingdom) - Dr.

Anaivari Anandan & Dr. M.S. Thulasimani

7. Siddha Maruthuvam Pothu – Dr. Ka. Na Kuppusamy Muthaliyar

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