4.1 HERBOMINERAL FORMULATIONS WITH SPECIAL REFERENCE...
Transcript of 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
Chapter-4 Preparation and Physicochemical Evaluation 108
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
Chapter-4 Preparation and Physicochemical Evaluation 109
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
Chapter-4 Preparation and Physicochemical Evaluation 110
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.
Chapter-4 Preparation and Physicochemical Evaluation 111
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
Chapter-4 Preparation and Physicochemical Evaluation 112
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
Chapter-4 Preparation and Physicochemical Evaluation 113
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.
Chapter-4 Preparation and Physicochemical Evaluation 114
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.
Chapter-4 Preparation and Physicochemical Evaluation 115
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
Chapter-4 Preparation and Physicochemical Evaluation 116
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
Chapter-4 Preparation and Physicochemical Evaluation 117
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
Chapter-4 Preparation and Physicochemical Evaluation 118
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.
Chapter-4 Preparation and Physicochemical Evaluation 119
Fig 4.1 Standard XRD pattern of mercuric sulphide (HgS)
Chapter-4 Preparation and Physicochemical Evaluation 120
Fig 4.2 Standard XRD pattern of mercuric oxide (HgO)
Chapter-4 Preparation and Physicochemical Evaluation 121
Fig 4.3 Standard XRD pattern of sulphur (S)
Chapter-4 Preparation and Physicochemical Evaluation 122
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
Chapter-4 Preparation and Physicochemical Evaluation 123
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)
Chapter-4 Preparation and Physicochemical Evaluation 124
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.
Chapter-4 Preparation and Physicochemical Evaluation 125
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
Chapter-4 Preparation and Physicochemical Evaluation 126
Fig 4.5 FIR spectrum of prepared Shwaskuthar Rasa
Fig 4.6 FT-IR spectrum of prepared Shwaskuthar Rasa
Chapter-4 Preparation and Physicochemical Evaluation 127
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
Chapter-4 Preparation and Physicochemical Evaluation 128
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
Chapter-4 Preparation and Physicochemical Evaluation 129
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 %)
Chapter-4 Preparation and Physicochemical Evaluation 130
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.
Chapter-4 Preparation and Physicochemical Evaluation 131
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.