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Physicochemical characterization of an Indian traditional medicine, Jasada

Bhasma: Detection of nanoparticles containing non-stoichiometric zinc oxide

Article  in  Journal of Nanoparticle Research · April 2009

DOI: 10.1007/s11051-008-9414-z

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J Nanopart Res
DOI 10.1007/s11051-008-9414-z
RESEARCH PAPER
Physicochemical characterization of an Indian traditional
medicine, Jasada Bhasma: detection of nanoparticles
containing non-stoichiometric zinc oxide
Tridib Kumar Bhowmick Æ Akkihebbal K. Suresh Æ
Shantaram G. Kane Æ Ajit C. Joshi Æ Jayesh R. Bellare
Received: 9 February 2008 / Accepted: 18 May 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Herbs and minerals are the integral parts
of traditional systems of medicine in many countries.
Herbo-Mineral medicinal preparations called Bhasma
are unique to the Ayurvedic and Siddha systems of
Indian Traditional Medicine. These preparations have
been used since long and are claimed to be the very
effective and potent dosage form. However, there is
dearth of scientific analytical studies carried out on
these products, and even the existing ones suffer from
incomplete analysis. Jasada Bhasma is a unique
preparation of zinc belonging to this class. This
particular preparation has been successfully used by
traditional practitioners for the treatment of diabetes
and age-related eye diseases. This work presents a
first comprehensive physicochemical characterization
of Jasada Bhasma using modern state-of-the-art
techniques such as X-ray photoelectron spectroscopy
(XPS), inductively coupled plasma (ICP), elemental
analysis with energy dispersive X-ray analysis
(EDAX), dynamic light scattering (DLS), and trans-
mission electron microscopy (TEM). Our analysis
A. C. Joshi: Private Practitioner (Vaidya).
T. K. Bhowmick
A. K. Suresh
S. G. Kane

J. R. Bellare (&)
Department of Chemical Engineering, Indian Institute
of Technology, Powai, Mumbai 400076, India
e-mail: jb@iitb.ac.in
A. C. Joshi
22, Shukrawar Peth, Pune, India
shows that the Jasada Bhasma particles are in oxygen
deficient state and a clearly identifiable fraction of
particles are in the nanometer size range. These
properties like oxygen deficiency and nanosize par-
ticles in Jasada Bhasma might impart the therapeutic
property of this particular type of medicine.
Keywords Electron microscopy

Nanoparticle
X-ray diffraction (XRD)

X-ray photoelectron spectroscopy (XPS)

Dynamic light scattering (DLS)
Nanomedicine
Introduction
Ayurveda is an intricate system of healing that
originated in India thousands of years ago. Siddha is
also an ancient Indian system of medicine in which
attention is given to minerals and metals in addition to
plant constituents. Medicinal preparations called
Bhasma are unique to the Ayurvedic and Siddha
systems of medicine. These are made from a variety
of base materials, e.g. Jasada, Tamra, and Louha
Bhasma are made from zinc (Zn), copper (Cu), and
iron (Fe), respectively. These medicines are taken
orally and the dose of a Bhasma is very small,
commonly a small heap of powder the size of a grain
of rice. Some Bhasmas have been pasted with honey,
butter, or ghee, and the paste has been taken orally
(Suoboda 1998). However, the mechanism of action of
these unique preparations is not clearly understood yet.
123

A complex and elaborate procedure for the preparation
of Bhasma was described by Nagarjuna around 800
AD (Sharma 1978), and is strictly followed till today.
The rigidity of the particular preparation method for a
particular Bhasma makes it a defined, unique prepara-
tion of the metal, but its exact chemical composition
has not yet been well characterized. In addition,
whether the particular preparative method imparts
any unique physiochemical property to Bhasma nec-
essary for its therapeutic application is also not known.
However, at present, attempts at establishing a
complete physicochemical composition—complete
elemental balance, atom balance, surface composi-
tion, detailed particle size distribution, and phase
composition—have been made. A complete physio-
chemical characterization of any one particular
Bhasma has not been reported.
Chemical evaluation of different Ayurvedic prep-
arations of iron has been done by Pandit et al. (1999)
with the help of atomic absorption spectroscopy, but
no effort was made to close the material balance on
the sample. Evaluation of chemical constituents of
Swarna Bhasma (gold ash) was studied by Mitra et al.
(2002), with the help of atomic absorption spectros-
copy. Their report accounts for only for 38.87% of
total mass by the different cations including 20.34%
gold (Au).Very few publications for the characteriza-
tion of Jasada Bhasma have been reported. The
preparation process and structural characterization of
Jasada Bhasma were studied by Bhgawat et al.
(2003). It was reported that hexagonal ZnO was the
major phase present in the Bhasma and six cationic
elements other than Zinc were present in smaller
amounts. No attempts to characterize the surface and
bulk composition separately have been reported.
Our objectives in the present work have been to
characterize Jasada Bhasma in terms of its physico-
chemical properties including bulk and surface
composition elemental analysis, crystalline phase ana-
lysis, particle size analysis, and surface composition.
Materials and methods
Material
In our experiments, the primary material used was
Jasada Bhasma. This Jasada Bhasma was prepared
by an authentic traditional practitioner, in whose
123
J Nanopart Res
family these Bhasmas have been synthesized for a
few generations. Jasada Bhasma was prepared by
following the method described in Ayurvedic texts
(Shastri 1988). Details of the preparation procedure
are described in following section.
Jasada Bhasma preparation
The traditional method for Jasada Bhasma prepara-
tion is long and involves several steps. Jasada
Bhasma, which has been used in our study, was
prepared by the following method. Traditional names
of each step have been mentioned in parentheses.
(i) In the first step, raw zinc was melted and quenched
into slaked lime water (ratio of 1:200) for over 70 min.
The procedure (Shodhan) was repeated seven times. (ii)
In the second step, the product from the previous step
was melted, while adding equal weight of turmeric
powder slowly. The process (Jarana) of heating was
continued until the smoke stops. (iii) In the third step, the
product from the previous step was added to water in a
container, allowed to settle overnight, and the superna-
tant was poured off and the process (Dhavana) repeated
seven times. (iv) In the fourth step, the product from the
third step was mixed with turmeric quath (a decoction of
turmeric in water) in a mortar and ground with pestle
continuously. This process (Bhavana) continues until all
the water goes off. (v) In the fifth step, the ground
material from the Dhavana step was made into discs and
placed inside an earthenware pot, which was covered
with an inverted earthenware pot. The entire assembly
was placed in a pit with dry cow-dung. The dung was set
on fire. The process (Puta) continues for 4–5 h. (vi)
After the Puta, step material was allowed to cool
overnight. Each Puta step was followed by Bhavana
step and this combination was repeated 13 times before
completion of Jasada Bhasma preparation. Final Jasada
Bhasma was collected in fine powder form after
grinding the material from the previous step.
Elemental analysis with inductively coupled
plasma (ICP)
The elemental composition was measured by ICP-AES
(Plasmalab, 8440, GBC Scientific Equipment, Dand-
enong, Australia) with a standard ICP torch and peri-
staltic pump. The operating conditions for ICP-OES
were as follows:
J Nanopart Res
Frequency: 27.12 MHz; power RF: 1.2 kW;
plasma gas flow rate: 18 L/min; auxiliary gas flow
rate: 6 L/min; sample uptake: 2.5 mL/min; integra-
tion time: 5.0 s; Nebulizer: GMK nebulizer; Spray
Chamber: cyclonic chamber.
Sample preparation for ICP
Because of the difficulty of dissolving Bhasma in
solvents, a novel two-step method was developed for
sample preparation. The first step, commonly used in
such investigations, involves treating the Bhasma
sample with a (1:1) mixture of conc. HNO3 and
HClO4. The bulk of the sample dissolves in such a
solution. However, a fair amount of residue was left
behind after filtration through a filter paper (Millipore,
USA). The elements in the dissolved sample are
quantitatively estimated using ICP. In order to achieve
total analysis, the residue from the first step was
weighed and fused with LiBO2 after heating for 1 h at
1,000 °C. This second step was sufficient to fully
solubilize the residue in dilute HNO3. This solution
from second step was also analyzed as before and the
two estimates were combined to get the total compo-
sition. This modified two step sample preparation
procedure that leaves no residue was used in this
investigation to achieve a complete mass balance, and
it is an important aspect of this work as compared to
others previously reported in the literature.
Elemental analysis with energy dispersive X-ray
analysis (EDAX)
Quantitative determination of bulk elemental composi-
tion in the Bhasma sample was carried out by EDAX
(EDAX Inc., Mahwah, NJ, USA) which was attached
with Environmental Scanning Electron Microscope
(ESEM) (Quanta 200, FEI, Hillsboro, Oregon, USA).
For EDAX analysis, the Bhasma sample was packed
into a hole in an aluminum stub (9 mm diameter, 9 mm
depth). The operating parameters were: 30 keV,
count-rate 1,500 ± 500 counts/s, working distance
10 mm, chamber pressure set to\2.2 9 10-4 torr, tilt
angle 0°, and accumulation time 50 s.
A calibration sample of both copper and aluminum
was used for the calibration of the EDAX. A piece of
copper grid was pasted on an aluminum stub and
placed in the microscope chamber and the sample
adjusted in such a way that the field of view is
roughly two-thirds copper (so that the Cu Ka and the
Al Ka peaks are close to the same height). The
spectrum appeared with the Al Ka peak (1.486 kev)
and Cu Ka peak (8.04 kev). Microscope parameters
during calibration were kept the same as the operat-
ing parameters, which were mentioned above. The
calibration of EDAX was rechecked by analyzing
different analytical reagent grade salts. To maximize
the quality of the result, under the same microscope
operating parameters standard less element coeffi-
cients (SEC) factors were adjusted for each analyzed
element.
A gaseous secondary electron detector (GSED)
was used for the image formation. The relative
elemental compositions of the Bhasma particles were
computed directly with EDAX software, using
‘‘ZAF’’ (atomic number, absorption, fluorescence)
correction. Analyses were performed on 13 different
points for each sample. The elements measured were
O, Mg, Al, Si, S, K, Ca, Mn, Fe, Cu, Zn, and Sn.
Surface analysis with X-ray photo electron
spectroscopy (XPS)
The surface composition of Jasada Bhasma particles
was analyzed by XPS (Thermo VG Scientific,
Multilab-2000). The instrument is equipped with
Mg Ka X-ray source (1253.60 ev) and a hemispher-
ical detector (Clam-4). The monochromatic Mg Ka
radiation source was operated at 15 kV and 150 W.
The base pressure of the XPS analysis chamber was
approximately 5 9 10-10 milli-bar. The spectra were
recorded with X-ray spot size of 650 lm, dwell time
50 ms and step size of 0.05 eV. Spectra were handled
with Avantage V-2.26 software. Calibration of the
instrument was done with the gold (Au4f7/2) peak at a
position of 83.8 eV. For XPS analysis, the powder
samples were made into pellets with the help of a
hydraulic press. For comparative study, ZnO (AR
Grade salt) particles were also analyzed with XPS.
Crystalline phase identification with X-ray
diffraction (XRD)
To determine the different crystalline phases present
in the Jasada Bhasma XRD studies was done. For
comparison, hexagonal ZnO (AR grade) was also
analyzed with XRD. XRD patterns were obtained
using an X’Pert Pro (Phillips) X-ray powder
123

diffractometer. Powdered samples were studied by
placing a thin layer in conventional cavity mounts.
The samples were scanned from (10–90°) 2h. The Cu
anode X-ray was operated at 40 kV and 30 mA to
´
give monochromatic Cu Ka X-rays (k = 1.54056 Å).
Mean crystallite size of Jasada Bhasma was
calculated from XRD graph using the Debye–Scherrer
formula.
Dh;k;l ¼ ðk  kÞ=ðbD  cos hB Þ
where Dh,k,l, mean effective size of crystal; k = 0.9
(constant); k, X-ray wavelength; bD, full width half
maxima (FWHM) of peak, hB, Bragg scattering
angle. The mean crystallite size was calculated after
averaging the crystal size value from seven most
intense reflection peaks of the XRD graph.
Size fractionation of Bhasma
Particle size distribution in the bulk powder has been
analyzed, and a nanoparticulate fraction was extracted
from the sample. The nanoparticulate fraction of
Bhasma particles was isolated by filtration. A sus-
pension of 1 mg/mL Jasada Bhasma was made in
Milli Q water. Suspension pH was adjusted to 3.0 with
0.1 M acetic acid for better dispersion of the particles
in aqueous media. Sample was filtered through the 0.5
micron filter (Millipore, USA). Both the un-fraction-
ated Bhasma as well as nanoparticulate fraction were
subjected to size analysis by DLS and TEM.
Particle size analysis
Two independent methods, dynamic light scattering
(DLS) (Brookhaven-Zeta Plus) and transmission elec-
tron microscopy (TEM) (FEI, Technai 12, 120 kV),
have been used for size analysis of the particles.
Size determination with DLS
DLS was performed using Brookhaven-Zeta plus
(Holtsville, NY, USA) instrument to determine the
mean particle size of the Jasada Bhasma samples.
The instrument is powered with an argon laser
source, which produces 660 nm laser light for
analysis. For the analysis of the sample, a suspension
of 10 mg/mL concentration of Jasada Bhasma has
been made in MilliQ water. Sample pH was adjusted
to 3.0 with 0.1 M acetic acid for better dispersion of
123
J Nanopart Res
the sample in the aqueous media. The suspension was
sonicated for 10 min before analysis. The instrument
(Brookhaven-Zeta plus) measures the change of
intensity of the laser light with time at a particular
angle after interaction with spherical shape particles
suspended in aqueous media. From the computed
auto-correlation function, the hydrodynamic diameter
of the spheres was measured.
Size determination with TEM
TEM (Tecnai-G2, FEI, Hillsboro, USA) was done for
measuring the size and shape of un-fractionated and
nanoparticulate fractionated Jasada Bhasma sample.
The sample preparation for TEM analysis was as
follows.
The Jasada Bhasma sample was first dispersed in
Milli-Q water for making 1 mg/mL suspension in a
glass beaker and the beaker sonicated for 10 min. For
bulk sample analysis, one drop of sample suspension
was taken on a carbon-coated grid (Ladd research,
USA) and dried at room temperature as well as under
IR laser lamp for complete drying before analysis
under TEM.
Results
Chemical composition: analysis by ICP and
EDAX
A complete analysis of the Jasada Bhasma was
possible by ICP and EDAX. This analysis was made
possible by the two-step solubilization procedure
developed in this work, which ensured complete
dissolution of the entire powder sample. Average
elemental content of three repeated ICP analysis of
Jasada Bhasma is shown in Table 1. Result shows
that Jasada Bhasma contains twelve elements (Na,
Mg, Al, Si, S, K, Ca, Mn, Fe, Cu, Zn, and Sn), where
Zn is the major element (78.82 wt%).
EDAX analysis also shows the presence of twelve
elements (O, Mg, Al, Si, S, K, Ca, Mn, Fe, Cu, Zn,
and Sn) in the Jasada Bhasma, where zinc
(78.86 wt%) is present as major element followed
by oxygen (11.7 wt%). As SEM coupled EDAX,
microprobe analysis is semi-quantitative, analysis of
several spots was carried out, and finally an average
value is considered. The average of 13-point analysis
J Nanopart Res

Table 1 Comparison of elemental composition of Jasada Bhasma detected by ICP and EDAX method
Elements ICP Std. dev SEM-EDAX Std. dev SEM-EDAX Std. dev (EDAX)
(wt%) (ICP) (wt%) (wt%) (EDAX) (wt%) (atom%) (atom%)
a
O 11.70 1.27 32.63 2.48
a a
Na 0.140 0.060
Mg 1.472 0.161 0.68 0.09 1.25 0.14
Al 0.636 0.106 0.56 0.11 0.93 0.18
Si 4.110 0.694 4.92 0.63 7.82 0.80
S 0.151 0.038 0.23 0.12 0.32 0.17
K 0.736 0.042 1.14 0.13 1.31 0.15
Sn 0.515 0.280 0.21 0.07 0.08 0.03
Ca 1.470 0.248 0.83 0.10 0.93 0.11
Mn 0.018 0.001 0.01 0.00 0.01 0.00
Fe 0.583 0.063 0.50 0.07 0.40 0.06
Cu 0.100 0.023 0.36 0.10 0.25 0.07
Zn 78.82 0.547 78.86 1.91 54.07 3.02
a
Method not able to detect the particular element

of the sample is shown in Table 1. The result shows
(Table 1) the presence of 78.86% (wt) zinc and
11.7% (wt) oxygen in the sample. Table 1 also shows
that when oxygen detected by EDAX (11.70%) is
added to the elemental composition (88.32%)
detected by ICP analysis, a 100% accountability of
the total sample contents is achieved. Thus, EDAX
and ICP results are internally consistent and have
been given 100% material balance on elements.
Atom balance has revealed (Table 1) some inter-
esting features: The total oxygen needed for fulfilling
stoichiometry of all cationic elements is *67 atom%.
However, from the EDAX analysis the amount of
oxygen present in the sample is only *33 atom%. The
maximum possible amount of oxygen bound with other
cationic elements except zinc is 13 atom%. This leaves
the net available oxygen for zinc (54 atom%) to be
20 atom%. Thus the atom ratio of zinc to available
oxygen is nearly 2:1. This strongly suggests that only
half of the total Zn is bound with oxygen.
Crystal structure determination with XRD
XRD technique was used to identify presence of the
crystalline phases in the sample. XRD pattern of
Jasada Bhasma was shown in Fig. 1a. Sample
identification was done by matching d spacing with
the standard JCPDS database (Fig. 1c). Result shows
ZnO is the major crystalline phase present in Jasada
Bhasma. Standard hexagonal ZnO also was analyzed
with XRD and diffraction pattern is shown in Fig. 1b.
´ ´
Peaks at d = 2.47 Å (2h = 36.25°), d = 2.81 Å
´
(2h = 31.75°), and d = 1.62 Å (2h = 56.58°) con-
firm the presence of ZnO (hexagonal) as the major
crystalline phase in the sample. Mean crystal size of
the Jasada Bhasma particles was reported after
averaging the crystal size calculated from seven most
intense reflection peaks [(1 0 1), (1 0 0), (0 0 2), (1 1 0),
(1 0 2), (1 0 3), (1 0 1)] of the graph. The mean
crystal size of Jasada Bhasma is found to be 52.7 nm
(Fig. 1c) whereas the mean crystal size of standard
hexagonal ZnO is 47.9 nm (Fig. 1c).
Comparative analysis of XRD results between Jasada
Bhasma and standard hexagonal ZnO shows the major
reflection peaks of both the samples are at identical
positions. This result indicates an identical crystal
structure of Jasada Bhasma with standard hexagonal
ZnO but Jasada Bhasma particles have *10% higher
crystal size compared to standard hexagonal ZnO.
The starting material in the preparation of Jasada
Bhasma was crystalline metallic zinc sheet with the
characteristic (data not shown) peak of zinc metal
´
appearing at d = 2.09 Å (2h = 43.23°). In the
Jasada Bhasma sample the characteristic peak of
´
crystalline metallic zinc at d = 2.09 Å was absent.
This result confirms the absence of any crystalline
zinc metal in the final product of the Jasada Bhasma.

123

Fig. 1 (a) XRD spectra of Jasada Bhasma, (b) XRD spectra
of hexagonal standard ZnO, (c) d spacing of most three intense
peaks of Jasada Bhasma and standard hexagonal ZnO. Mean
crystal size of Jasada Bhasma and ZnO. XRD spectrum of
Jasada Bhasma is similar with the hexagonal ZnO
Surface analysis with XPS
The binding energy of Zn2p peaks was calibrated by
taking Au4f7/2 peak at 83.8 eV as the standard peak
(Database 2001; Powell et al. 2001). Peak positions
123
J Nanopart Res
of Zn2p3/2 and Zn2p1/2 XPS spectra of Jasada
Bhasma and ZnO are shown in Fig. 2a, d.
In the case of ZnO (Fig. 2a), the binding energy of
Zn2p3/2 and Zn2p1/2 appeared at 1022.0 and
1045.1 eV, respectively, and these values agree well
with the standard XPS database (database, 2001).
Jasada Bhasma peak positions of Zn2p3/2 and Zn2p1/2
are at 1021.9 and 1045.0 eV, respectively. The result
shows the binding energy of Zn2p3/2 peak in the case
of Jasada Bhasma to have shifted to a lower energy
level compared to Zn2p3/2 peak of ZnO.
After deconvolution of Zn2p3/2 peak of ZnO
(Fig. 2b), we could observe the appearance of three
peaks at 1023.0, 1022.0, and 1019.3 eV. Appearance
of peaks at 1023.0 eV is described as the basic
‘independent of crystal size’ peak and is observed in
all spectra by Tay et al. (2006).
After deconvolution of Jasada Bhasma Zn2p3/2
peak (Fig. 2c), in addition to all three peaks (1023.0,
1022.0, 1019.3 eV) of ZnO, two extra peaks appeared
at 1021.0 and 1020.8 eV. The presence of the extra
peaks suggests a change in the binding state of zinc,
which can physically be described by a loss in
number of oxygen atoms in Bhasma ZnO particle.
In the hexagonal ZnO crystal, zinc cation is
coordinated with four oxygen atoms tetrahedrally
and shows a peak at 1022.0 eV. In the case of Zn2p3/2
peak of ZnO this peak at 1022.0 eV is the dominant
peak. This is expected when there is no oxygen
deficiency in the tetrahedrally coordinated Zn atom.
However, in the tetrahedrally coordinated ZnO
structure, an oxygen deficient atom produces two
new binding energy peaks at 1021.0 and 1020.8 eV,
respectively (Tay et al. 2006).
Deconvolution of Zn2p3/2 peak of Jasada Bhasma
shows appearance of two new peaks, which were
shifted 0.8 and 1.2 eV at lower binding energy
direction compared to Zn2p3/2 peak of ZnO
(1022.0 eV). This feature is associated with the
variation of number of broken bonds per zinc atom
with oxygen atom in a crystal. Thus, appearance of
new peaks and shift of 0.8 and 1.2 eV at lower
binding energy direction confirm the oxygen vacan-
cies on the surface of the sample. Zn2p3/2 peak
analysis of Jasada Bhasma also shows (Fig. 2c)
considerably reduced peak area of the fully coordi-
nated Zn peak at 1022.0 eV, strongly suggesting a
substantial oxygen deficiency on the surface of the
material.
J Nanopart Res
Fig. 2 (a) Zn2p peak of Jasada Bhasma and ZnO analyzed by
XPS. (b) Zn2p3/2 peak of ZnO after deconvolution analyzed by
XPS. (c) Zn2p3/2 peak of Jasada Bhasma after deconvolution
analyzed by XPS. (d) Peak positions of Zn2p3/2 peak of ZnO
XPS spectra for O1s of both samples have also
been obtained. By curve fitting and estimating the
area under the curve for Zn and O, the O/Zn atom
ratio is estimated to be 0.98 and 0.59 for synthetic
ZnO and Jasada Bhasma, respectively (Fig. 2d) (Xu
et al. 2004). This is in agreement with the details of
XPS above. This result is also in general agreement
with the oxygen deficiency brought out clearly by
the atom balance in bulk material analysis with
EDAX.
Particle size analysis: evidence for nanoparticles
Dynamic light scattering (DLS)
Particle size analysis from DLS instrument is shown
in Fig. 3. The result shows size of the particles
and Jasada Bhasma after deconvolution. Appearance of two
new peaks at 1020.8 and 1021.2 ev and decreased intensity of
1022.0 ev peak compared to ZnO indicate oxygen deficient
nature of Jasada Bhasma
present in un-fractionated part of Jasada Bhasma is
0.85–1.35 lm with the mean size 1.2 lm (Fig. 3a).
Analysis of the fractionated part of Jasada Bhasma
shows the presence of 25–50 nm particles with the
mean size 10–25 nm in the sample (Fig. 3b). Hence-
forth, these fractionated particles in 30 nm size range
are referred as nanoparticulate fraction in all the
subsequent studies.
Transmission electron microscopy (TEM)
Particle size analysis of Jasada Bhasma sample also
had been done with TEM and the result is shown in
Fig. 3. The photomicrograph of bulk particle
(Fig. 3c) shows a wide distribution of size in the
sample. We also observe that the particles are
irregular in shape and present in aggregated form.
123
 J Nanopart Res

Fig. 3 Particle size

distribution result of Jasada
Bhasma by DLS. (a) Bulk
particle size distribution;
(b) particle size distribution
of Jasada Bhasma after
filtration through 0.5 lm
filter. Particle size analysis
with transmission electron
microscope; (c) TEM
photograph of bulk particles
of Jasada Bhasma;
(d) TEM photograph of
Jasada Bhasma particles
present in the filtrate after
filtration through 0.5 lm
filter. Mean particle size of
Jasada Bhasma is showing
*1 lm. After filtration
through 0.5 lm filter we
can observe significant
number of small particles of
10–25 nm size present in
the sample

The TEM photomicrograph of nanoparticulate frac-
tion of Jasada Bhasma particles (Fig. 3d) shows the
appearance of 15–25 nm particles in the sample, and
a much narrower distribution of sizes. The particles
are spherical in nature. This result is in good
agreement with the DLS result and confirms the
presence of significant amount of nanometer size
particles in the sample. Point to be noted here is that
the filtration of 1,000 ppm (:788 ppm Zn) Bhasma
allows the passage of nanoparticulate particles of
Bhasma, which contain only 11.5 ppm zinc, esti-
mated by ICP.
Both DLS and TEM results are in accord to show
the presence of significant amount of nanoparticles in
the Jasada Bhasma sample, which may be one of the
potential active ingredients.
Discussion
Preparation procedure of Bhasma is very elaborate.
However, this process has been followed strictly till
today for maintaining the quality and efficacy of the
product. By the process of Shodhana surface of the
metals was cleaned and prepared for the next
processing step. In the next processing (Jarana) step
zinc metal was melted and a mixture of turmeric and
molten zinc metal was formed. In the third step
(Dhavana) water wash was useful to exclude the
excess unabsorbed turmeric from product. In the
fourth step (Bhavna) of the process material was
converted into powder form while grinding them in
mortar and pestle. Repeated last step (Puta) of the
process helps to produce very fine quality powder,
which certainly improves the quality of the final
Bhasma.
Physicochemical analysis of Jasada Bhasma has
shown some unique properties in this material.
Elemental analysis with ICP and EDAX shows
several other elements other than zinc and oxygen,
such as Na, Mg, Al, Si, S, K, Ca, Mn, Fe, Cu, and Sn
are present in this material as trace amount. The
specific roles of these trace elements present in
Jasada Bhasma are not yet clear.

123
J Nanopart Res
An interesting feature of these results is the atom%
for Zn (54.07%) and oxygen (32.63%) as per the
EDAX analysis. Considering definite amount of
oxygen would be bound with the other elements,
the net oxygen available for binding with the Zn
would even be less than the 32.63 atom%. Thus, the
atom ratio of this available oxygen is nearly 2:1. This
strongly suggests that only half of the total Zn is
bound with oxygen.
The combination of ICP and EDAX analysis
results suggest the possibility of different types of
structures in Jasada Bhasma as described below.
(i) Considering the details of the method of prep-
aration of Jasada Bhasma and allowing for the
possibility of oxidation on surface of Zn particle,
there can be a core and shell morphology, with a
layer of ZnO surrounding a Zn core.
(ii) Jasada Bhasma may have a mixture of amor-
phous Zn and crystalline ZnO particles
(iii) ZnO may exist in a highly oxygen deficient
form.
We have attempted to distinguish between these
possibilities as the following.
XRD analysis has shown that Jasada Bhasma
particles contain crystalline hexagonal ZnO, ruling
out the firtst possibility. Absence of any free crystal-
line zinc metal is also confirmed by XRD analysis.
Therefore, ICP and EDAX analysis results, combin-
ing with the XRD result, have strongly suggested the
possibility of the oxygen deficient structure inferred
from the elemental analysis.
Our XPS analysis study has shown that the surface
as well as the bulk Jasada Bhasma particles are in
oxygen deficient state and have different structures
compared to the stoichiometric ZnO, shown by XPS
analysis.
Thus, taking into account the ICP, EDAX, XRD,
and XPS results, it can be said that the ZnO
contained in Jasada Bhasma is present predomi-
nantly in the hexagonal crystalline form but with a
considerable amount of oxygen deficiency. Results
also ruled out the first two possibilities of Jasada
Bhasma structure inferred from ICP and EDAX
result. This leaves out only one possible structure of
Jasada Bhasma, which has to be a crystalline ZnO in
high oxygen deficient form. In order to obtain high
catalytic activity and good electronic characteristics
the non-stoichiometric ZnO materials are now in
major research focus (Pan et al. 2005). So far, no
reports on medicinal uses of non-stoichiometric ZnO
have been found. This highly oxygen deficient ZnO
in a medicinal preparation like Jasada Bhasma raises
the possibility that oxygen deficient property can be
one of attributing factors imparting the medicinal
value in it.
Our physicochemical analysis show that particles
fractionated by filtration of 1,000 ppm (:788 ppm Zn)
Jasada Bhasma suspension has the size in 10–25 nm
range as revealed in DLS and TEM analysis, hence
termed as the nanoparticulate fraction. Analysis
revealed that it contains only 11.5 ppm zinc estimated
by ICP (data not shown). Nanosize particles are
attached with the cell surface and can diffuse readily
inside the cells (Limbach et al. 2005). Thus, the size of
the particle is able to influence the efficacy as shown by
others studies (Alexiou et al. 2000; Chithrani et al.
2006). Due to extremely small sizes (10–25 nm) these
particles can impart contribution in the biological
effect of Jasada Bhasma. It would be interesting to test
the effect of this nanoparticulate fraction and compare
it with that of the un-fractionated Bhasma on the
biological system from various perspectives. It can be
possible that small particles (10–25 nm size range) are
able to enter the cells by permeation, whereas larger
size particles ([100 nm) cannot permeate inside the
cells easily.
The unique physicochemical properties brought
out by this work make it interesting to investigate the
specific roles of Jasada Bhasma and nanosize Jasada
Bhasma particles on the biological system. It will be
also important to study which step(s) of the elaborate
preparation process, which has been strictly con-
served over the years, imparts these unique properties
in the Bhasma. Another interesting study would be
the investigation of the effect of different zinc
compound including ZnO on the biological system,
to understand more precisely the importance of
oxygen deficiency in crystalline ZnO, which is
present in Jasada Bhasma. Although, very little
attention has been paid to the therapeutic application
of the oxygen vacant materials, recent medical
findings have shown importance of oxygen vacancy
in different diseases by controlling the lethal oxida-
tive stress (Schubert et al. 2006; Contreras et al.
2007; Korsvik et al. 2007). These studies are being
done by us and are being reported concurrently
elsewhere.
123

Conclusion
Although Bhasmas are complex materials, we have
shown that a physicochemical analysis is possible
using modern techniques to unravel their mode of
action. From the studies described here, we have shown
that in Jasada Bhasma, zinc is the major element
present, next to oxygen. It contains hexagonal ZnO as
crystalline phase. This material is highly oxygen
deficient in nature. In a crystal, only half of the total
zinc atoms bound to the oxygen atom. A significant
number of particles present in the material within the
nanometer size range of 10–25 nm, and it is possible
to fractionate the sample by size. Altogether, these
characterization techniques will be most attractive
techniques for the standardization of herbomineral
medicines.
Acknowledgments This work was supported by the Department
of Biotechnology, Govt. of India, (Nanobiotechnology fund),
Ministry of Human Resource Department (MHRD), Govt. of
India. The authors are grateful to Regional Sophisticated
Instrumentation Center (RSIC), Bombay, India, for the
technical assistance in ICP analysis, and to XRD laboratory,
Department of Material Science, Indian Institute of Technology,
Bombay, India, for the technical assistance in XRD analysis.
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