4.1 HERBOMINERAL FORMULATIONS WITH SPECIAL REFERENCE …shodhganga.inflibnet.ac.in/bitstream/10603/38973/8/08_chapter 4.pdf · 4.1 HERBOMINERAL FORMULATIONS WITH SPECIAL REFERENCE

Chapter-4 Preparation and Physicochemical Evaluation 107

4.1 HERBOMINERAL FORMULATIONS WITH SPECIAL REFERENCE

TO RASA

“Rasa Shastra” although basically means the “science of mercury”

it also refers to the science of making minerals suitable for the body so

that they can be used as medicines (Kulkarni, 1982; Shastry, 1999).

Minerals such as mercury and arsenic are as such considered toxic

(Saper, 2004) but by proper shodhana (detoxification) process, they can

be made into wonderful medicines. When mercury is properly prepared,

it balances all three doshas (humors) of the body, has a soothing effect,

and prevents disease and aging process. It nourishes all vital body parts

and increases the strength of the eyes (Ghanekar, 1981). In India, the

Ayurvedic physician uses 20 % pure herbal preparations, 30 % pure

mineral preparations and 50 % herbomineral preparations. This

percentage of usage illustrates that there is much value to using mineral

preparations (Sharma, 1983).

In Vedas, gold and silver had a ritualistic use. Rasa Shastra is

believed to have come out in the 6th and 7th century. The Buddhist sage,

Nagarjuna, is considered the first to use mercury and is believed to have

done exhaustive work in the creation and establishment of Rasa Shastra.

As per Ayurveda mercury is a vrisya (aphrodisiac), balya (tonic), snigdha

(anointing), rasayana (rejuvenative), vrana sodhana and ropana (wound

cleaner and healer), and krimighna (Anthelmintic and Antimicrobial).

Mercury is said to give a firm physique, a stable mind, and considered to

be the destroyer of diseases. When compounded with herb the mercury

heightens the medicinal properties of the concerned herb.

Traditional herbomineral formulations have been widely used for

thousands of years in many countries. Metals have been used in disease

treatment since time immemorial. Role of these herbomineral

preparations for curing skin diseases such as psoriasis, eczema,

alopecia, allergy, diabetic ulcer, warts and leprosy is well studied

[Joseph, 2008]. Most of the medicines are mixture of compounds and

because of their synergistic action toxicity is diminished, and

bioavailability through the cells of the body is increased. Treating the


minerals with herbal juices and further trituration may lead to reduction

in particulate size up to nano or near nano scale (less than 100 nm)

enabling their increased potency. These drugs are known to be effective

even in low concentrations.

4.2 COLLECTION, IDENTIFICATION AND AUTHENTICATION OF HERBS

The dried fruits of Piper longum, Piper nigrum, roots of Aconitum

ferox, and rhizomes of Zingiber officinale were obtained from Amruth

Kesari herbs and chemicals, Bangalore (Karnataka). The herbs were

authenticated by Prof. K. Prabhu, Botanist, S.C.S. College of Pharmacy,

Harapanahalli (Karnataka) as Herbarium Voucher Specimens no.

scscop/phcog/herb 452, 517, 691 and 584 respectively. After procuring

the herbs were properly cleaned and dried. The dried drugs were stored

in well closed labeled containers.

4.3 COLLECTION AND IDENTIFICATION TEST FOR METALS AND

NON-METALS

The metals as parada (mercury), manahsila (arsenic form of

arsenic disulphide) and non-metals as gandhaka (sulphur), and tankana

(borax) were obtained from Amruth Kesari herbs and chemicals,

Bangalore (Karnataka). The identification test for metals and non-metals

were performed according to procedure (Chatwal, 2009) as below.

Table 4.1 Identification of parada (mercury)

S.No Test Inference

1 Sample being examined was placed on well scraped copper foil. A dark grey stain was produced which on

rubbing became shining. When dried copper foil was heated in a test tube, spot disappeared.

+

2 Sample being examined, when potassium iodide solution was added, a red precipitate was formed. The

ppt was soluble in an excess of potassium iodide

+

3 To the sample, 2M sodium hydroxide solution was added until it became strongly alkaline. A dense yellow

precipitate was produced.

+

4 Sample being examined, 6M hydrochloric acid was

added, white precipitate was produced which was blackened by adding dilute ammonia.

+

(+) Indicates presence of (parada) mercury


Table 4.2 Identification of manahsila (arsenic disulphide)

S.No Test Inference

1 Sample melted at 320 °C and burnt with a bluish flame

releasing fumes of arsenic and sulfur.

+

2 5 ml of the sample solution was heated on a water-bath

with an equal volume of hypophosphorus reagent,

brown precipitate was obtained.

+

(+) Indicates presence of manahsila (arsenic disulphide)

Table 4.3 Identification of gandhaka (sulphur)

S. No Test Inference

1 Sample melted at about 115 ºC to yield yellow mobile

liquid which became dark and viscid on further heating

at about 160 ºC.

+

2 When sample was viewed under microscope, it consists

of grouped amorphous subglobular particles without

any admixture of crystals.

+

(+) Indicates presence of gandhaka (sulphur)

Table 4.4 Identification of tankana (borax)

S. No Test Inference

1 Dissolved 0.1 gm of sample by gently warming with

5 ml of methanol to which a few drops of sulphuric

acid was added. When solution was ignited,

produced a green colour flame.

+

(+) Indicates presence of tankana (borax)

4.4 SHODHANA / DETOXIFICATION PROCESS OF HERB, METAL

AND NON-METAL INGREDIENTS

In Rasa Shastra almost all the drugs are advised to be processed

with specific shodhana process before their internal use. It has been

observed that if metals / minerals are used in their impure form, these

are likely to produce some harmful effects or may cause various diseases

in the body (Prajapati, 2009). Thus shodhana processes are prescribed

to each metal / mineral to remove physical and chemical impurities, as


well as to convert their mineral forms into some suitable forms for their

further treatment/processing. Shodhana is not only a process of

chemical purification but it is a specific process of addition and

separation which causes physical, chemical and biological changes in the

metals. These changes depend on the structure, constituents /

constitution, impurity and properties of particular substance.

Shodhana process of so-called heavy metals as mercury and

arsenic form of arsenic disulphide and non-metals as sulphur and borax

and herbs such as Aconitum ferox was carried out as per prescribed

procedure (Sharma, 1979).

4.4.1 Shodhana of vatsanabha (A. ferox)

Method: 50 gm of vatsanabha roots were cut into small pieces to which

100 ml of cow urine was added and it was kept overnight and dried in

sunlight. Process was repeated for three days and during each day

similar quantity of fresh cow urine was used. Finally after drying the

detoxified vatsanabha was powdered and stored in a glass jar.

Observation: Colour of cow urine changed from yellow to brownish.

Percentage yield: 98.41 % of shuddha (detoxified) vatsanabha.

4.4.2 Shodhana of parada (mercury)

Method: 50 gm of parada and 50 gm of calcium carbonate were taken in

a porcelain mortar and triturated for 24 hrs. Parada was then separated

from mixture and washed with hot water. To collected parada, 50 gm of

garlic and 25 gm of rock salt powder were added and then mixture was

triturated for 48 hrs till black colour precipitate was formed. Precipitate

was washed with hot water to obtain detoxified parada was dried,

weighed and packed in a glass jar.

Observation: After trituration with calcium carbonate, almost all parada

was mixed with it and colour of the mixture turned to gray. Parada was

scattered throughout the mixture. Pale garlic and rock salt was changed

to black colour within two hours of trituration.

Percentage yield: 61.72 % of shuddha (detoxified) parada.


Precaution: Trituration process was completed carefully to avoiding the

splitting of parada from porcelain mortar and pestle.

4.4.3 Shodhana of manahsila (arsenic disulphide)

Method: Manahsila was detoxified by giving bhavana of adrak (ginger)

juice, the adrak juice was prepared by mixture of 50 gm fresh adrak

(ginger) and 100 ml of distilled water. 50 ml of the fresh adrak juice was

added to 50 gm of manahsila powder in glass beaker and closer was

properly covered by cloth and stored overnight. Manahsila settled at

bottom of beaker and unwanted adrak juice was decanted off. The

detoxified manahsila was collected dried in shade, triturated the final

powder, and stored in a glass jar. The process was repeated for seven

times for 7 days during each time (each day) fresh adrak juice was added

for shodhana.

Observation: Brick red manahsila powder turned dull red after

shodhana. Slight weight loss of manahsila in the form of fine particles

floating on adraka juice was seen during decantation.

Percentage yield: 98.53 % of shuddha (detoxified) manahsila.

4.4.4 Shodhana of gandhaka (sulphur)

Method: 50 gm of powdered gandhaka was taken in glass beaker and

melted with 1 ml of cow ghee, liquefied sulphur was passed (sieved) into

another glass beaker containing 100 ml of milk through cloth tied over

the mouth of glass beaker. Solidified form of gandhaka settled at bottom

of beaker was taken out and washed with hot water to obtain

pure/detoxified gandhaka. By this process, the stony substances present

in gandhaka remained on the cloth which was separated and finally

giving purified gandhaka. After drying it was powdered, weighed and kept

in a glass jar. The process was repeated for seven times and during each

process milk was changed in glass beaker i.e. fresh milk was taken for

each process.

Observation: Average time taken to melt the gandhaka was three

minutes, crystalline dark yellow gandhaka turned granular and dull

yellow after shodhana. The poisonous substances in the gandhaka were


floated on the milk mixed with ghee which was decanted. The purified

gandhaka remained at bottom of the beaker in the solidified form.

Percentage yield: 97.15 % of shuddha (detoxified) gandhaka.

Precaution: Powder form of gandhaka was heated over mandagni. Cloth

was slightly smeared with ghee. After each quenching, gandhaka was

thoroughly washed with hot water.

4.4.5 Shodhana of tankana (borax)

Method: Weighed 100 gm of tankana placed in a stainless steel pan and

heated to remove its water content. Obtained pure tankana was dried,

triturated and packed in glass jar.

Observation: On heating tankana was spread over the stainless steel

pan i.e. bulkiness was increased.

Percentage yield: 58.38 % of shuddha (detoxified) tankana.

4.5 PREPARATION OF SHWASKUTHAR RASA

Shwaskuthar Rasa - a herbomineral formulation of Ayurveda was

prepared according to Ayurvedic practice (AFI, 2000).

Table 4.5 Formula of Shwaskuthar Rasa

S. No Ingredient Quantity

1 Rasa - suddha (Parada) – Mercury 4 gm

2 Gandha – suddha (Gandhaka) - Sulphur 4 gm

3 Manahsila - suddha (Arsenic disulphide) 4 gm

4 Tankana – suddha (Borax) 4 gm

5 Visa - suddha (Vatsanabha ) – A.ferox 4 gm

6 Maricha (Piper nigrum ) 36 gm

7 Sunthi (Zingiber officinale) 4 gm

8 Pippali (Piper longum) 4 gm

Note: Ingredients as mercury, sulphur, arsenic disulphide, borax and A. ferox were detoxified.

4.5.1 Method of preparation

Preparation of Kajjali: Initially equal quantities of shuddha parada and

shuddha gandhaka were taken (1:1) in a porcelain mortar in reference

amount and mixture was triturated continuously for 40 hrs till it

attained the required blackish appearance (Kajjalabha) and parada


attained lusterless (Nishchandra) state i.e. till the shining of parada was

lost. This intermediate form of preparation comprising mercury and

sulphur is called Kajjali (mercuric sulfide) of non-metallic components.

Preparation of formulation: Prepared kajjali (mercuric sulfide) was

triturated with the reference amount of powders of manahsila,

vastanabha, tankana and trikatu (A preparation containing powders of

equal parts of maricha, pippali and adrak) for 6 hrs by using porcelain

mortar and pestle and the remaining reference quantity of maricha was

added to mixture which was triturated for 72 hrs to obtained fineness

powder of Shwaskuthar Rasa - a herbomineral formulation and it was

allowed for drying and stored in glass jar for further studies.

4.5.2 Confirmation of formulation completion test for Shwaskuthar

Rasa

Confirmation tests for fineness and completion process of prepared

Shwaskuthar Rasa were performed following Ayurvedic procedure. As per

Ayurvedic text, the Rasa preparation process is considered final only

when the preparation passes through and clears certain tests

(Rasatarangini, 1979).

a) The preparation should not show any luster of mercury i.e. mercury

should loose its completely luster indicating that no mercury exists in

metal form and 100 % metallic mercury has been absorbed and

assimilated in the formulation by interacting with other ingredients.

b) The preparation should be fine enough to be filled between fine

markings of fingers.

c) Particle size of preparation should be so small that it when sprinkled

in a glass filled with water, the formulation particles should float on

the water surface only and should not submerge or sediment in water.

d) When small amount of preparation is kept on tongue the preparation

should not impart any taste.

e) Ingestion of small amount of equivalent to pinch the formulation

should not cause / produce the nausea or vomiting.


f) The ingredients in general and metals in particular should not regain

their original state when heated with mixture (seeds of Abrus

precatorius, honey, ghee, borax and jaggery).

g) When certain quantity of final preparation is heated with weighed

amount of silver foil, the weight of silver foil should not increase. This

test probably confirms that all the ingredients of the preparation have

been interacted and assimilated within themselves and no part /

portion of any of the ingredient was free to interact with silver.

When passed through all these test as per Ayurvedic text can only

be considered complete and final formulation and fit for human

consumption as medicine. When looked and viewed in the light of

modern science, after passing these tests the finding can be interpreted

and concluded as below.

I. Metals no more remains in form as metals they completely loose their

metallic state / form.

II. All ingredients of the formulation are integrated in their whole

someness with each other that their unsaturation is completely lost

and thus they became completely inactive or in a way chemically inert

form /taste.

III. As such neither the formulation impart any metallic taste nor exhibit

any sensation of nausea or vomiting thus indicating / proving that

the preparation is completely and totally acceptable to the system /

body.

IV. And finally on the basis of these tests it can be intended and assumed

that particle size of the final formulation reaches to nano or near

nanoscale. Which on one hand increases the surface area enormously

for its faster release / absorption and assimilation enabling its higher

bio-availability and enhanced therapeutic efficacy. On the other hand

the particle size of formulation reaches to such a small level that the

drug particle can enter the cell and can come out from the cell

environment after performing its therapeutic role /effect.


Table 4.6 Confirmation of formulation completion test for prepared

Shwaskuthar Rasa

S.No Test Observation Result

1 Nischandratva: Sample of

prepared formulation was taken

in a petri dish and observed for

any luster in daylight through

magnifying glass.

No luster was found Completion

of process

2 Rekhapurnatvam: A pinch of

prepared formulation was taken

in rubbed gradually and slowly

between the thumb and index

finger.

Formulation entered

the lines of the finger

and did not easily

removed or washed-

out from the cleavage

of the finger lines.

Fineness of

prepared

formulation

3 Varitaratavam: A small amount

of the prepared sample was

sprinkled over the water in a

beaker.

Particles floated over

the surface of the

water.

Fineness of

prepared

formulation

4 Nisvadutvam: When a small

amount of sample was kept on

the tongue

Tasteless was found Completion

of process

5 Avami: Ingestion of 5-10 mg of

the sample.

Did not produce any

nausea / vomiting.

Completion

of process

6 Apunarbhavata: Sample when

mixed with equal quantity of

mitrapanchaka (seeds of Abrus

precatorius, honey, ghee, borax

and jaggery), sealed in earthen

pot thereafter heated and

allowed self cooling.

Ingredients didnot

regain their original

state.

Completion

of process

7 Niruttha: Sample was mixed

with fixed weight of silver leaf,

kept in earthen pot and heat

was applied and self cooling.

Weight of silver was

not increase.

Completion

of process


4.6 EVALUATION OF PREAPRED SHWASKUTHAR RASA

4.6.1 Organoleptic evaluation

A small amount of Shwaskuthar Rasa herbomineral formulation

was spread over a watch glass and it was physically examined for general

appearance i.e sparsh, rupa, rasa, shabda and gandha.

Table 4.7 Organoleptic characters of prepared Shwaskuthar Rasa

4.6.2 Physicochemical characters of formulation

4.6.2.1 Loss on drying

Accurately weighed about 1 gm of air dried Shwaskuthar Rasa

formulation was transferred in a previously weighed weighing bottle. The

bottle was stopered loosely and kept in an oven at 105 0C for 30 min. The

bottle was then cooled to room temperature in desiccator and weighed till

a constant weight was achieved. The loss on drying was calculated with

reference to air-dried formulation sample (Table 4.11).

4.6.2.2 Ash value

Accurately weighed about 1 gm of Shwaskuthar Rasa formulation

was evenly distributed in the crucible. Previously heated to redness for

30 min and allowed to cool in a desiccator and weighed. The material in

crucible was dried at 105 C for one hour and ignited to constant weight

in muffle furnace gradually increasing the temperature. The crucible was

allowed to cool in desiccator after each ignition. After prolonged ignition a

carbon free ash could not be obtained. The percentage of ash on the

dried formulation basis was then calculated and recorded (Table 4.11).

S.No Parameters Observation

1 Sparsha (Touch & texture) No coarse particles

2 Rupa (Colour) Black colour

3 Rasa (Taste) Tasteless

4 Shabda (sound) No metallic sound

5 Gandha (Odour) Unspecific


4.6.2.3 Acid insoluble ash

The ash obtained was boiled with 25 ml of 2M hydrochloric acid for

5 min and the insoluble matter was collected in a gooch crucible. It was

then washed with hot water, ignited, cooled in a desiccator and weighed.

The percentage of acid-insoluble ash on the dried formulation basis was

calculated (Table 4.11).

4.6.2.4 Measurement of particle size and surface characteristics

The average particle size of Shwaskuthar Rasa was measured in 10

mM NaCl by dynamic light scattering with a Zetasizer from Malvern

Instruments (MAL1023461). Surface analysis of particles was done using

scanning electron microscope (SEM) (JEOL JSM6100).

SEM is a widely used technique employed to image the surface of

sample. It provides outstanding image resolution, unique image contrast

and a large depth of field. An SEM forms image by rastering a highly

focused electron beam, typically with energies of 1 to 20 keV, across a

sample and detecting the secondary or backscattered electrons ejected.

The secondary electrons originate from the top 5-15 nm of the sample

and provide information on the topography (Russell and Daghlian,

1985; Barnes et al., 2002). A portion of Shwaskuthar Rasa was

sprinkled onto a double side carbon tape and mounted on aluminium

stubs, in order to get a higher quality secondary electron image for SEM

(Photograph 4.1).

4.6.2.5 Phase identification of minerals

X-ray powder diffraction (XRD) is one of the most powerful

techniques for qualitative and quantitative analysis of crystalline

compounds or crystalline phases. The information obtained includes

types and nature of crystalline phases present, degree of crystallinity,

and amount of amorphous content and orientation of crystallites. XRD is

an instrumental technique that is used to identify minerals, as well as

other crystalline materials. The method has been traditionally used for

phase identification, quantitative analysis and the determination of


structure imperfections. XRD is particularly useful for identifying fine

grained minerals and mixtures. If the sample is a mixture, XRD data can

be analyzed to determine the proportion of the different minerals present.

Other information obtained can include the degree of crystallinity of

minerals present, possible deviations of the minerals from their ideal

compositions and the structural state of the minerals (John and Shelby,

1995).

When a material is irradiated with a parallel beam of

monochromatic X-rays, the atomic lattice of the material acts as a three

dimensional diffraction grating causing the X-ray beam to be diffracted to

specific angles. The diffraction pattern, that includes position (angles)

and intensities of the diffracted beam, provides information about the

material. Angles are used to calculate the interplanar atomic spacings (d-

spacings). Because every crystalline material gives a characteristic

diffraction pattern and can act as a unique fingerprint, the position (d)

and intensity (I) information is used to identify the type of material by

comparing them with patterns for over 80,000 data entries in the

International Powder Diffraction File (PDF) database, complied by the

Joint Committee for Powder Diffraction Standards (JCPDS). By this

method, identification of any crystalline phase, even in a complex

sample, can be made. Compounds/phases are identified by comparing

diffraction data against a database of known materials. The position (d)

of diffracted peaks also provides information about how the atoms are

arranged within the crystalline compound. The intensity information is

used to assess the type and nature of atoms. Determination of lattice

parameter helps understand extent of solid solution in a sample. Width

of the diffracted peaks is used to determine crystallite size and micro-

strain in the sample.

The powder XRD patterns of the prepared Shwaskuthar Rasa were

recorded using X‟pert pro Panalytical X-ray diffractometer with CuKα

radiation (λ=1.5406 A°) operating at 45 KV and 40 mA for the angle (2θ)

ranging from 5-50 degree at a scanning rate of 3 degree/second.


Fig 4.1 Standard XRD pattern of mercuric sulphide (HgS)


Fig 4.2 Standard XRD pattern of mercuric oxide (HgO)


Fig 4.3 Standard XRD pattern of sulphur (S)


Table 4.8 XRD analysis of prepared Kajjali (mercuric sulphide)

Pos.

[°2Th.]

FWHM

[°2Th.]

d-spacing

[Å]

Rel. Int.

[%]

Area

[cts*°2Th.]

Mineral phase

identification

16.4345 0.1840 5.39393 15.25 45.76 HgS

26.4590 0.2342 3.36872 100.00 306.17 HgS

27.7947 0.1673 3.20979 11.70 25.60 HgS

28.0822 0.1506 3.17757 10.20 25.56 S

30.6646 0.1840 2.91562 32.73 101.04 HgO

43.8034 0.2175 2.06677 28.26 103.09 HgS

Pos.[°2Th.] - Diffraction angle for the peak FWHM [°2Th.] - Full width of diffraction peak at half maxima d-spacing [Å] - The distance between adjacent planes of atoms Rel. Int. [%] - The intensity of the diffraction maximum Area [cts*°2Th.]- Peak intensity

Table 4.9 XRD analysis of prepared Shwaskuthar Rasa

Pos.

[°2Th.]

FWHM

[°2Th.]

d-spacing

[Å]

Rel. Int.

[%]

Area

[cts*°2Th.]

Mineral phase

identification

16.4126 0.1673 5.40107 12.30 31.75 HgS

20.2896 0.1506 4.37693 9.76 22.68 HgO

23.1078 0.1673 3.84910 22.33 57.65 S

26.4088 0.2175 3.37500 100.00 335.68 HgS

27.7600 0.1840 3.21372 12.35 35.08 HgS

28.1028 0.1506 3.17530 8.87 20.62 S

30.4977 0.1673 3.02824 36.69 94.74 HgO

31.8976 0.1673 2.92184 33.03 85.30 HgO

36.1022 0.2007 2.48798 3.46 10.71 HgS

43.7219 0.2844 2.07043 27.57 121.01 HgS

45.3820 0.2613 1.97655 18.49 58.93 HgO

49.1298 0.2448 1.85291 3.52 17.97 HgS

Pos.[°2Th.] - Diffraction angle for the peak FWHM [°2Th.] - Full width of diffraction peak at half maxima d-spacing [Å] - The distance between adjacent planes of atoms Rel. Int. [%] - The intensity of the diffraction maximum Area [cts*°2Th.]- Peak intensity


Fig 4.4 (A) XRD pattern of prepared Kajjali (B) XRD pattern of prepared Shwaskuthar Rasa

4.6.2.6 Elemental analysis

EDAX (Energy dispersive X-ray analysis) makes use of the X-ray

spectrum emitted by a solid sample bombarded with a focused beam of

electrons to obtain a localized chemical analysis. Qualitative analysis

involves the identification of the lines in the spectrum and is fairly

straight forward owing to the simplicity of X-ray spectra. Quantitative

analysis (determination of concentrations of the elements present) entails

measuring line intensities for each element in the sample. By scanning

the beam in a television-like raster and displaying the intensity of a

selected X-ray line, element distribution images or 'maps' can be

produced. Also, images produced by electrons collected from the sample

reveal surface topography. The scanning electron microscope (SEM) is

closely related to the electron probe, is designed primarily for producing

electron images, but can also be used for element mapping, and even

point analysis, if an X-ray spectrometer is added. EDAX systems are

Inte

nsit

y (C

ounts

) In

tensit

y (C

ounts

)

Angle (Two theata)


attachments to SEM where the imaging capability of the microscope is

used to identify the specimen of interest.

A representative portion of prepared Shwaskuthar Rasa was placed

in an alumina crucible and the temperature was varied from 40-400 C.

EDAX (EDAX Inc. Mahwah, NJ, USA) attached to SEM for the detection

of various elements in Shwaskuthar Rasa (Table 4.11).

4.6.2.7 Determination of heavy metals

An ICP-MS (Inductively Coupled Plasma – Mass spectroscopy) is an

instrument capable of determining the concentrations of trace elements

in materials. The material is introduced into the plasma, where it is

vaporized, atomized, and ionized then passed through a magnetic

quadrupole to detector. The instrument is capable of ultra low detection

limits of parts per million for elements. For quantitative detection of so-

called heavy metals in parts per million (ppm) in Shwaskuthar Rasa

formulations an inductively coupled plasma-mass spectrometer (ICP-MS,

Perkin-Elmer ELAN-6000) was used (Table 4.11).

4.6.2.8 Study of chemical bonding / organic molecules

An infrared spectrum represents a fingerprint of a sample with

absorption peaks which correspond to the frequencies of vibrations

between the bonds of the atoms making up the material. Because each

different material is a unique combination of atoms, no two compounds

produce the exact same infrared spectrum. Therefore, infrared

spectroscopy can result in a positive identification (qualitative analysis)

of every different kind of material. The mid-IR (400-4000 cm-1) is the

most commonly used region for analysis as all molecules possess

characteristic absorbance frequencies and primary molecular vibrations

in this range (Davis and Mauer, 2010). Mid-infrared spectroscopy

methods are based on studying the interaction of infrared radiation with

samples. As IR radiation is passed through a sample, specific

wavelengths are absorbed causing the chemical bonds in the material to

undergo vibrations such as stretching, contracting, and bending.


Functional groups present in a molecule tend to absorb IR radiation in

the same wave number range regardless of other structures in the

molecule, and spectral peaks are derived from the absorption of bond

vibrational energy changes in the IR region. Thus there is a correlation

between IR band positions and chemical structures in the molecule. It

also provides qualitative information about functional groups (Smith,

1996).

The infrared spectrum in the low frequency region (50-400 cm-1)

was recorded on a Bruker IFS 66 V/S vacuum Fourier transform

interferometer, where as the spectra from 400-4000 cm-1 region were

recorded using FTIR spectrophotometer (Spectrum RXI, Perkin Elmer).

For IR spectra, Shwaskuthar Rasa formulation was mixed in potassium

bromide (KBr) to make translucent pellet and spectrum was recorded

(Table 4.10 and Fig 4.5, 4.6).

Table 4.10 FT-IR spectrum of prepared Shwaskuthar Rasa

S.No Frequency range

Mode of vibration Inference / Remark

1 3363.6 O-H stretching in intermolecular hydrogen bonding

Alcohol

2 2930.2 C-H stretching in methyl Methyl group

3 1634.6 C=O stretching Carbonyl group

4 1440.7 C-H bending in methyl Methyl group

5 1346.7 O-H bending Alcohol

6 1253.1 C-O-C stretching Ethers

7 1131.6 C-O stretching Alcohol

8 1080.1 C-N stretching Amine group

9 708.0 C-S stretching Carbon sulphide


Fig 4.5 FIR spectrum of prepared Shwaskuthar Rasa

Fig 4.6 FT-IR spectrum of prepared Shwaskuthar Rasa


Table 4.11 Physicochemical characters of prepared Shwaskuthar

Rasa formulation

4.7 RESULTS AND DISCUSSION

The herbs were identified and authenticated by botanist along with

citing specimen number for individual herbs. Metals such as mercury

and arsenic, non-metals as sulphur and borax were identified with

specific inorganic radicals test which showed positive results with

respected metals and non-metals (Table 4.1, 4.2, 4.3, 4.4).

It is noteworthy that very specific shodhana (detoxification and

purification) processes and techniques were carried out as per Ayurvedic

text individually for mercury, arsenic disulphide, sulphur, borax and

roots of A. ferox which convert the toxic metals / minerals / herbs into a

suitable therapeutic form. Percentage yield of shodhana process of roots

of A. ferox (98.41 %), mercury (61.72 %), arsenic disulphide (98.53 %),

sulphur (97.15 %) and borax (58.38 %) was found.

S.No Parameters Shwaskuthar Rasa

1 Loss on drying (%) 0.32

2 Ash value (%) 41.70

3 Acid insoluble ash (%) 15.53

4 Particles size (µm) 1.22

5 Phase identification HgS and HgO

6 Elemental content (Wt %) C – 31.24, S - 0.89,

N - 12.40, O - 42.63,

Na - 2.37, Ca -1.62,

Cl – 3.46, H - 4.65.

7 Heavy metal content (ppm) Hg – 0.94

As – 8.78

8 Organic macromolecules 9 sharp peaks


The Shwaskuthar Rasa prepared as per Ayurvedic text (AFI, 2000)

using the required ingredients (Table 4.5) was subjected to all test

prescribed for the finished herbomineral formulation as per literature for

quality test (Rasatarangini, 1979) for its final fitness for human

consumption. It was tested for fineness of formulations with

Rekhapurnatvam and Varitaratavam although completion test of

prepared formulation was performed with Nischandratva, Nisvadutvam,

Avami, Apunarbhavata and Niruttha (Table 4.6) as per Ayurvedic quality

control test gives fineness and completion of prepared formulation.

Evaluated organoleptic characters of Shwaskuthar Rasa showed

non-metallic sound, absence of coarse particles, black colour, and

absence of taste (Table 4.7). The physicochemical characters were found

as loss on drying (0.32 %), ash value (41.70 %) and acid insoluble ash

values (15.53 %) (Table 4.11).

Characterization of Shwaskuthar Rasa using modern analytical

techniques was necessary to determine the effect of the process. The

average particle size from Zetasizer and particle shape and surface

characteristics from the SEM photograph, Shwaskuthar Rasa showed

spongy structure with irregular particles size lying in the submicron

range (Photograph 4.1). The reason is the use of the organic materials

from herbal source in the preparation of formulation. From the image it

is clear that nanosize crystallites are agglomerated giving rise to micro

sized particles with loss of grain boundaries. These studies confirm that

Shwaskuthar Rasa is nanocrystallite with submicron size particle (1.22

µ). The particle size recorded can be characterized as the desired

specification of the final Shwaskuthar Rasa.

XRD pattern of Kajjali shows peaks due to mercuric sulfide,

mercuric oxide and free sulfur (Fig 4.1 JCPDS File number-02-461, Fig

4.2 01-0896, Fig 4.3 20-1227 respectively) while the XRD pattern of

prepared Shwaskuthar Rasa shows peaks due to major presence of

mercuric sulfide (Fig 4.1 JCPDS File number-02-461) and mercuric


oxide (Fig 4.2 JCPDS File number-01-0896) and low intensity of sulfur

(Fig 4.3 JCPDS File number-20-1227). No extra diffraction peaks were

observed in the case of Shwaskuthar Rasa confirming that while in the

initial stages of the processing of the formulation, free sulfur is present

in significant amount, whereas after through trituration major mercuric

sulfide (HgS) and mercuric oxide (HgO) which were observed in the

preparation. Not only the diffraction peaks in the XRD pattern of

Shwaskuthar Rasa corresponding to mercuric sulfide became sharper

and intense in final preparation compared to Kajjali sample but some

new peaks also appeared due to mercuric sulfide, which were not present

in the Kajjali sample (Table 4.8, 4.9 and Fig 4.4 A, B). This observation

confirms that the trituration of Kajjali helps in the formation of mercuric

sulfide and increases the crystallinity in the formulations. The crystallite

size was calculated from XRD pattern following the Scherrer formula Dp

= λ × 0.94/ (B1/2 × Cos θ). Where, „Dp‟ is the crystallite size for (h k l)

plane, λ is the wavelength of the incident X-radiation [CuKα (λ=1.5406

A°)], β is the full width at half maximum (FWHM) in radians and θ is the

diffraction angle for (h k l) plane. It is notable here that the FWHM in

case of Kajjali is high in comparison to the finally prepared Shwaskuthar

Rasa confirms that the size of the crystallite increases. It is obviously

due to trituration process of the Kajjali sample. Thus the XRD study

confirms the presence of nanocrystalline structure of the formulation.

In addition to the metal Hg and As used in the formulation, other

metals were also expected in the formulation which enters in it during its

detoxification and trituration process. EDAX has been used to

detect/estimate the elements present in a considerable amount where as

the ICP-MS method was used to detect/estimate (Hg and As) elements

present in trace amount. Chemical composition of Shwaskuthar Rasa

using EDAX and trace metal composition of Shwaskuthar Rasa using

ICP-MS has been listed (Table 4.11). Abundance of C (31.24 %), N (12.40

%) and O (42.63 %) in the formulation was observed which is obviously

from the herbs used in the preparation of the formulation. Ca (1.62 %)


conducive to healthy metabolism and prevention for stomach lesions,

was also found to be present in the final Shwaskuthar Rasa product. Na

(2.37 %) needed for maintaining normal fluid balance was also present in

the final product (Table 4.11). These elements (Ca, Na, S) act as

additional supplement improving the curative properties of the

formulations. Other elements such as H (4.65 %) and Cl (3.46 %) were

also found in the formulations. Presence of heavy metals was tested in

Shwaskuthar Rasa as Hg (0.94 ppm) and As (8.78 ppm). Their

concentration was found to be well within the safe limits in Shwaskuthar

Rasa as recommended by WHO. The additional elements present in the

formulation are clearly due to herbal ingredients used and so may be

called as bioavailable. It is notable that the proportion of mercury in

Shwaskuthar Rasa does not seem to follow a consistent trend, as part of

it is certainly lost during the preparation through direct trituration

process. This aspect raises the safety concerns regarding the process

used and warrant for evolving the procedure to minimize the loss of

mercury in process.

FIR spectrum of Shwaskuthar Rasa in the region from 50-400 cm-1

was studied (Fig 4.5). Crystalline mercuric sulfide (HgS) is known to

have absorption at 83, 92 and 100 cm-1 and its presence in the same

absorption range in the present FIR spectra indicate that Shwaskuthar

Rasa is essentially mercuric sulfide. This observation supports the XRD

analysis. FT-IR spectrum of Shwaskuthar Rasa in the region from 400-

4,000 cm-1 is shown (Table 4.10 and Fig 4.6). There are fairly sharp

peaks at 708, 1080, 1131, 1253, 1346, 1440, 1634, 2930 and 3363 cm-1

which indicate the presence of the organic compounds in the

formulation. Which arise from the usage of herbs. The presence of

appreciable concentration of C, H, O, and N (Table 4.11) also suggests

the presence of organic molecules in the preparation again from herbs

used. It is presumed that the organic molecules present in herbal

ingredient of the preparation play vital therapeutic role in such

preparations.


Particle size of the preparation may be attributed to the trituration of

detoxified metals, non-metals and herbs for a long duration which

causes the change in the chemical nature of ingredients. The FT-IR

analysis shows the possibility of presence of organic matter in the

formulations. This could be due to the formation of organometallic

complexes in the Shwaskuthar Rasa formulation. The observation made

and the studies discussed here are quite promising. Several significant

possibilities and future prospects of the prepared formulation could be

debated with these results. The particle size of the prepared formulation

matches well with the colloidal size and this suggest the possibility that

these colloidal particles may get attached to the human intestine and

providing a large surface area and thereby increasing the absorption of

other nutrients and ingredients, which are added to it during the process

of preparation or prescribed to the patient along with the treatment

tenure. Thus, these drugs may act as the absorbent. Further, metal

compound may act as the carrier for the organic matter derived from the

herbal ingredient used during the pharmaceutical processing. In short,

metals as a carrier for the organic contents from A. ferox, P. nigrum,

P. longum, and Z. officinale, are known to be useful in treatment of

asthma, allergy, cough and inflammation etc. From XRD studies of

Shwaskuthar Rasa it was concluded that mercuric sulfide (HgS) in

nanocrystalline range (31-66 nm), associated with organic molecules

probably plays an important role in making it biocompatible and non-

toxic at therapeutic doses. Other essential elements present in

Shwaskuthar Rasa may act as additional supplement and help in

increasing the over-all efficacy of the formulation.

Documents

4.1 HERBOMINERAL FORMULATIONS WITH SPECIAL REFERENCE …shodhganga.inflibnet.ac.in/bitstream/10603/38973/8/08_chapter 4.pdf · 4.1 HERBOMINERAL FORMULATIONS WITH SPECIAL REFERENCE