characterization of makaradhwaja by …...kajjali). confirmatory test was done for the quality...

33

CHARACTERIZATION OF MAKARADHWAJA BYINSTRUMENTAL, PHYSICAL AND TOXICOLOGICAL

METHODS

Research Article

Lopamudra Bhattacharyya*, Pratip Kumar Debnath, Joyram Hajra,Sachchida Nand Upadhyay

National Research Institute of Ayurveda and Drug Development, Kolkata, West Bengal, India. *Corresponding author. e-mail: [email protected]

ABSTRACT: Ancient Ayurvedic formulation Makaradhwaja (a compound of Gold, Mercury andSulphur) has been characterized in the present study adopting latest instrumental methods such as X-Ray Diffraction analysis (XRD), Fourier Transform Infrared Spectroscopy (FTIR analysis) andScanning electron microscopy (SEM). The XRD data gives the size of the drug particle ranging from28.62 to 29.08 nm. In FTIR graph broad peaks were arise at 3474 cm-1and 1637 cm-1 due to presenceof the Makaradhwaja in the sample. The molecule seems to be spherical shaped in SEM micrograph(images obtain from 500 µm – 1 µm range). Evaluation of Physical properties has been performed sideby side for quality control of the drug. Two weeks study for acute oral toxicity resulted in no mortalitiesor evidence of adverse effects, implying that the formulation is nontoxic. Throughout 14 days of thetreatment no changes in behavioural pattern, clinical sign and body weight of rat in both control andtreatment groups. Also there were no any significant elevations observed in the biochemical analysisof the blood serum. The overall results suggest that Makaradhwaja is safe and it can be administeredas pharmaceutical formulation.

Key words: Makaradhwaja, Traditional medicine, Characterization.

INTRODUCTIONMercury in its purified form is being used

for the preparation of different formulationsfrom thousands of years in the field oftraditional system of medicine (Debnath 2006).Makaradhwaja is one of the most popularmercurial preparation of Ayueveda havingpotent action against a range of ailments (bothsomatic and psychological). The process ofpreparation and therapeutic benefits of this

formulation has been depicted in ancientAyurvedic literatures (Mishra 2006). Gold,Mercury and Sulphur are used for thepreparation of Makaradhawaja in the ratio of1:8:16. After procurement all the ingredientsi.e. Mercury, Sulphur and Gold were purifiedseparately through a range of scientific methodsas per ancient text. Plant, animal and mineralproducts were used in these processes (Reddy2007). Then purified basic ingredients of

Explor Anim Med Res,Vol.5, Issue - 1, 2015, p. 33-43

34

Exploratory Animal and Medical Research, Vol.5, Issue 1, June, 2015

Makaradhwaja (Mercury, Gold and Sulphur)were mixed with each other (in mortar pastel)maintaining the chronological order. At firstMercury and Gold were triturated and mixedwith each other followed by the addition ofSulphur into the mixture. Constant trituratingin mortar pastel transformed the mixer into ahomogenous black compound (known asKajjali). Confirmatory test was done for thequality assurance of Kajjali and finally it wassubjected to the process of sublimation. Foroptimum yield of the finished producttemperature was maintained 6 hours each in120-200 oC, 200-400 oC and 400-600 oC rangeduring the preparation (Mishra 2006, Khedekaret al., 2011, Kulkarni 1998). Bright redcoloured Makaradhwaja was obtain by thisprocess. The yielded material was trituratedthoroughly to get fine powder of the drug andstored in a closed container. Table .1 representsthe schematic chart of Makaradhwajapreparation. Figure 1a and 1b represents thephotograph of ancient and modern techniquesof Makaradhwaja preparation. This drug isexclusively used for good health and longevity(Shastri Ambikadatta 2005, Shastri 2004). It isrejuvenator as well as immunomodulator(Prajapati et al., 1997). In Geriatric problemsit acts as vitalizer and general tonic(Mahadihassan 1985). Apart from these benefitsMakaradhwaja is used for degenerative changesand inflammatory conditions (Sinyorita et al.,2011). It is an excellent drug for anxiety, stressand insomnia. The branch of Ayurveda dealswith metallic and mineral preparations is knownas Rasashastra. These preparations are highlypotent and have specific pharmacologicalactions (Debnath 2006). It acts as catalyst invarious metabolic processes take place inhuman body. In spite of their unique therapeutic

benefits, the application of metallic preparationsin healthcare is restricted. This is due tounavailability of scientific and rationalguidelines for standardization of theseformulations. The characterization ofMakaradhwaja as well as the quality assuranceis the main aim and objective of the presentstudy. Starting from easy Physical and Chemicaltests for standardization the drug has beenstudied with the help of different sophisticatedmethods of analysis (such as X-Ray Diffraction,Fourier Transform Infrared Spectroscopy andScanning Electron Microscopy). Finally theacute oral toxicity study has been conducted toevaluate the toxic effect of the drug. Theanimals of control and treated groups wereobserved for any changes in behavioral pattern,clinical sign, body weight and bloodbiochemical parameters (Subramanion et al.,2011, Halim et al., 2011).

MATERIALS AND METHODSSource of the Drug: The Makaradhwaja

used in this study has been kindly supplied byDr. P.K. Prajapati of Department of Rasashastra& Bhaishajya Kalpana, Gujrat AyurvedUniversity, Jamnagar.

Study of Physical properties: This includesthe colour, odor, pH, solubility, loss on drying,loss on ignition, total ash value of the drug. Themoisture content has been calculated throughthe loss on drying test. The loss on ignition andtotal ash value were also calculated. Table.2represents all data obtained from this study.

Study by Instrumental methodsA. X-Ray Diffraction (XRD) analysis of the

powder Makaradhwaja (by using RigakuUltima III with a Bragg – Brentano geometryand a Cu-k∝ radiation, λ= 0.154). This method

35

is useful to determine the presence of differentmolecules of a drug sample. The size ofmolecules can also be calculated from thevalues of XRD (Bhati-Kushwaha et al., 2014).

B. Scanning electron microscopy (SEM)analysis was done by using JEOL, JSM (Modelno: 6360). SEM analysis gives the clear idearegarding the inner structure of the drugmolecule.

C. Fourier transform infrared spectroscopy(FTIR analysis) of the powder Makaradhwajawas taken by using Schimadzu (Model: prestige22) FTIR spectrophotometer. The peaks foundin FTIR spectrum authenticate the results ofXRD.

Acute Oral Toxicity Study 24 healthy adult Wistar albino rats of either

Table.1: The process of Makaradhwajapreparation.

Table: 2 Physical properties of powderMakaradhwaja.

Table. 3 Physical appearance and behavioural observation record of animals in four groups.

Characterization of Makaradhwaja by Instrumental, physical and toxicological methods.

36


sex weighing about 160 to 200 gm and agedapproximately 10 weeks were taken for thestudy and distributed into four groups. Thestudy related to the animals was authorized bythe institutional animal Ethical committee(Proposal No. IAEC/2012/08). One weekacclimatization to the laboratory conditions wasperformed before the study. Drinking water andfood were provided to the animals throughoutthe experiment. Only food supply was

discontinued for a period of 12 hours prior totreatment (though the drinking water was stillin free) as reported by Halim et al., 2011. Foodsupply was started again one hour aftertreatment. From the literature study it has beenfound that human therapeutic dose ofMakaradhwaja is 2 mg/ kg body weight per day(Sinyorita et al., 2011). The suitable dose forrat had been calculated for the toxicologicalstudy. The animals of Group A have been treated

Table. 4 Blood biochemical parameters of animals in control and treatment group. Values are givenas the Mean± S.E.; p values less than 5% were considered statistically significant (p < 0.05).

Figure.1a Ancient technique of Makaradhwajapreparation.

Figure.1b modern technique of Makaradhwajapreparation.

37

with ten times of human therapeutic dose ofthe drug converted to rat dose. The animals ofGroup B have been treated with five times ofhuman therapeutic dose of the drug convertedto rat dose. The animals of Group C have beentreated with human therapeutic dose of the drugconverted to rat dose. The animals of Group Dhave been treated as control group. Carefulobservation of animals was done continuouslyfor the first 3 hours after dosing for toxicsymptoms. The number of survivors was noted

after 24 hours and daily observation wasmaintained up to 14th day. Animals wereweighed and visual observations for mortality,behavioral pattern, changes in physicalappearance and signs of illness were conducteddaily during this period (Subramanion et al.,2011, Halim et al., 2011). At the end of 14days blood samples from the animals of GroupA and Group D was collected for bloodbiochemical parameters such as blood glucose,serum cholesterol, total bilirubin, serum ureaand serum creatinine.

Figure: 2 The X-Ray Diffraction (XRD) graph of powder Makaradhwaja; the average mean size ofdrug particle is ranging from 28.62 – 29.08nm.


38


Figure: 3.a Presence of some particle ofdifferent size and shape (500µm).

Figure.3.b Crystals of different size with a darkbackground (100µm).

Figure.3.c Molecules are scattered in a mannerof snow fall over a wavy ground (10µm).

Figure.3.d Prominent view of space betweentwo crystals (5µm).

Figure.3.e Spherical shaped drug moleculespread over a wavy surface (5µm).

Figure.3.f Prominent view of the unevensurface and its layer (5µm).

39

Figure.3.g Sponge ball like shaped drugmolecules (1µm).

Figure.3.h Spheres of drug particle (1µm).

Figure: 4 FTIR spectrum of Makaradhwaja.


40


RESULTS AND DISCUSSIONStudy of Physical properties: This study

reveals that the Makaradhwaja is fine powderand bright red in colour. It has no specific odoror rather it can be said to be odorless. The pH ofthe drug is 5.2. It is heavier than water. It nearlyinsoluble in water or ether or alcohol. It issoluble in aqua regia. Table.2 represents thedifferent physical properties of the drug.

Study by Instrumental methodsX-Ray Diffraction analysis of the drug has

been done to determine the presence ofMercury, Sulphur and Gold in the sample(Figure 2). All the samples show the presenceof those molecules. No free molecule of Hg orS was present in the sample and they are all inthe form of HgS. Side by side the presence ofGold molecule in the sample has also beendetected. In the literature it is mentioned thatin traditional method of Makaradhwaja

Figure.5 The percentage of increase in body weight o f animals in acute oral toxicity study.

preparation the gold does not combines with thefinal product collected in the neck of thecontainer. It is found to be precipitated at thebottom of the container (Kulkarni 1998, Shastri2005, Shastri 2004). So the present study is thescientific proof for validation of the ancientmethod of Makaradhwaja preparation. The sizeof the particles of Makaradhwaja has also beendetermined from the data obtain from XRDgraph with the help of Scherrer’s equation:

D = 0.94 λ / (β cos θ)Where D= the average crystallite domain size

perpendicular to the reflecting planes; λ =X-ray wavelength; β =Full width at half maximum(FWHM); θ = the diffraction angle. The averagemean size powder of Makaradhwaja is rangingfrom 28.62 – 29.08nm (Bhati-Kushwaha et al.,2014, Barbe et al., 2004 and Oshal et al., 2010).

Fourier transform infrared spectroscopy(FTIR analysis) of Makaradhwaja has beenrepresented by Figure.4. From the literature

41

review it has been found that broad peaks at3400 cm-1 and 1600 cm-1 arise in the FTIRspectra of Makaradhwaja due to characteristicOH stretching (γ-OH) H OH bending (δ OH)vibrational band due to adsorbed water in thesample (Austin 2012). In the present studybroad peaks at 3474 cm-1and 1637 cm-1 werefound due to the same reasons. According tothe study of Physical property it has beenestimated that the L.O.D. of the sample is0.55%. Broad FTIR peaks at 3474cm-1 indicatesthe presence of phenolic OH group and at1637cm-1 indicates the presence keto group(Austin 2012 and Barbe et al., 2004).

Scanning Electron Microscopy (SEM) studywas performed in micro meter scale rangingfrom 500 µm to 1 µm. The image of the drugreveals its spongy structure. The molecules arespherical shaped scattered over a wavy surface.Application of intense heat during thepreparation of drug may be the cause offormation of this uneven surface. The dataobtain from SEM study have been presented inFigure. 3. a, b, c, d, e, f, g and h.

Acute Oral Toxicity StudyThe acute oral toxicity study has been

conducted for evaluation of the toxic effect ofthe drug. A two weeks study on rat withmultiplied therapeutic dose reveals that thegrowth of the animals was normal in controland treatment groups.

The percentage of increase in body weightof in Group.1 on Day 7 and Day 14 were foundto be 3.24% and 5.82%, respectively. Thepercentage increase in body weight of rats inGroup.2 on Day 7 and Day 14were found to be2.68% and 5.27%, respectively. The percentageincrease in body weight of rats in Group.3 onDay 7 and Day 14 were found to be 2.86% and

5.72%, respectively. The percentage increasein body weight of rats in Group.4 on Day 7 andDay 14 were found to be 2.5% and 5.36%,respectively. The results signify that there weregradual increases in body weight of treatmentand control rats and the rate of weight gain isalmost the same in both groups (Figure 5). Thefood and water consumption of the treatmentrats were also not significantly different ascompared to the control rats measuredthroughout the study.

The animals in all groups are observed andno visual abnormality was detected in any oneof the animals. In physical observation of thetreated rats throughout the study, none of theanimals showed signs of toxic effect such aschanges on skin and fur, eyes, mucusmembrane, heart beat, behavioural pattern,tremors, salivation, diarrhoea, lethargy, sleepand coma (Table.3). No mortality has beenobserved throughout the 14 days study period.The clinical biochemistry values (showed inTable.4) of blood glucose, serum cholesterol,total bilirubin, serum urea and serum creatinineof the treatment rats were not significantlydifferent as compared to the control rats(Subramanion et al., 2011 and Halim et al.,2011).

CONCLUSIONMakaradhwaja, a well known mercurial

preparation has been characterized by easy andcost effective methods. Latest scientifictechniques have been adopted forcharacterization of the drug. The XRD analysisreveals the presence of Mercury, Gold andSulphur in the formulation. The size of the drugmolecules has also been calculated from thedata obtained in XRD analysis. The molecularstructural of the drug has been observed in SEM


42


analysis. The spongy spherical shaped drugmolecules scattered over a wavy surface havebeen identified in SEM study. FTIR spectrumof the powder Makaradhwaja has also donewhich supports the result of XRD. A two weeksstudy on laboratory animals did not produce anytoxic effect. All the animals were healthy aftercompletion of study. There were no significantdifference in body weight, physical signs andblood biochemical parameters of treatment andcontrol group.

ACKNOWLEDGEMENTThe first author is very much thankful to the

institutional S.O.P. Laboratory for co-operationin the present study. She extends gratitudetowards Dr. M.K.Mitra, Professor, Departmentof Metallurgical and Material Engineering,Jadavpur University, for his help and supports.She is also very much grateful to Dr. P.K.Prajapati of Department of Rasashastra &Bhaishajya Kalpana, Gujrat AyurvedUniversity, Jamnagar for his kind help.

REFERENCES

Austin A (2012) Chemical Characterization of aGold and Mercury based Siddha Sasthric Preparation–Poorna chandrayam, Am J Drug Discov Dev 2 (3):110 – 123.

Bhati-Kushwaha, Malik CP (2014) Biosynthesisof silver nanoparticles using fresh extracts of Tridax

procumbens Linn. Ind J Exp Biol 52: 359-368.

Barbe C, Bartlett J, Kong L, Kim F, Qiang LH,Larkin M, Calleja S, Bush A, Colleja G (2004)Silica particles: A novel drug delivery system,Advanced Materials. 16: 1959 - 1966.

Debnath PK (2006) Molecules of metals andminerals in Ayurveda: the interior science for healthand diseases. Papers on the Ayurvedic Studies, Ed.Brahmananda Gupta, The Asiatic Society, Kolkata.124-140.

Halim SZ, Abdullah NR, Afzan A, Rashid BA,Abdul Jantan I, Ismail Z ( 2011) Acute toxicity studyof Carica papaya leaf extract in Sprague Dawleyrats. J Med Plants Res 5: 1867-1872.

Khedekar S, Patgiri BJ, Ravishankar B, PrajapatiPK (2011) Standard Manufacturing Process ofMakaradhwaja prepared by Swarna Patra - Varkhaand Bhasma. Ayu J 32 (1) 109 - 115.

Kulkarni DA (1998) Rasratna Samuchchaya (byVagbhatta) Hindi commentary. MeharchandLaxmandas publication, New Delhi. 3 (20) 45.

Mishra SN (2006) Rasendra Chintamani (byDhundhukanath), Hindi Commentary. ChaukhambaOrientallia, Varanasi. 8/20-28, 112-113.

Mahadihassan S (1985) Cinnabar as the bestalchemical drug of longevity, called Makaradhwajain India. Am J Clin Med. 13: 93 - 108.

Oshal F, Mossalayi H (2010) Effect of matriceson size and morphology of HgS nanoparticles.Scholars Research Library. Der Pharma Chemica 2(5): 33-37.

Prajapati PK, Joshi D, Dube GP (1997) AnExperimental Study on Makaradhwaja. AncientScience of Life. Vol no XVI: 302-306.

Reddy D, Ramchandra K (2007) Text Book ofRasa Shastra. Chaukhambha Sanskrit Bhawan,1 st. Edn, Varanasi. 5: 231-234.

43

Shastri Ambikadatta (2005) Bhaishajya Ratnabali(by Govinda Das Sen) - Vidyotini Vyakhya,Vajikarana Rogadhikar, Chaukhamba SanskritSansthan, Varanasi. 74/114 - 123: 1126.

Shastri KN (2004) Rasa Tarangini (by S.N.Sharma) Hindi commentary. Motilal Banarasi Das.Delhi. 15/ 59 – 61: 374.

*Cite this article as: Bhattacharyya L, Debnath PK, Hajra J, Upadhyay SN (2015)Characterization of Makaradhwaja by instrumental, physical and toxicological methods. ExplorAnim Med Res 5(1): 33-43.

Sinyorita S, Ghosh CK, Chakrabarti A, AuddyB, Ghosh R, Debnath PK (2011) Effect of AyurvedicMercury Preparation Makaradhwaja on Geriatriccanine - A Preliminary Study. Ind J Exp Biol 49(7): 534-539.

Subramanion LJ, Zuraini Zakaria , Yeng Chen ,Yee Ling Lau, Lachimanan Yoga Latha, SreenivasanS (2011) Acute Oral Toxicity of Methanolic SeedExtract of Cassia fistula in Mice. Molecules 16:5268-5282.


characterization of makaradhwaja by …...kajjali). confirmatory test was done for the quality...

Documents