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 transmission 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:
[email protected] 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 particles 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.
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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 preparation 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 necessary for its therapeutic application is also not known. However, at present, attempts at establishing a complete physicochemical composition—complete elemental balance, atom balance, surface composition, detailed particle size distribution, and phase composition—have been made. A complete physiochemical characterization of any one particular Bhasma has not been reported. Chemical evaluation of different Ayurvedic preparations 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 spectroscopy. 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 characterization 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 physicochemical properties including bulk and surface composition elemental analysis, crystalline phase analysis, 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
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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 preparation 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 supernatant 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, Dandenong, Australia) with a standard ICP torch and peristaltic pump. The operating conditions for ICP-OES were as follows:
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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; integration 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 composition. 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 composition 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 operating 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 coefficients (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 hemispherical 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
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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 A ). 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 suspension 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-fractionated 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 electron 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
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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 (wt%)
Std. dev (ICP) (wt%)
a
O
SEM-EDAX (wt%)
Std. dev (EDAX) (wt%)
SEM-EDAX (atom%)
Std. dev (EDAX) (atom%)
11.70
1.27
32.63
2.48
Na
0.140
0.060
a
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
0.100
0.023
0.36
0.10
0.25
0.07
0.547
78.86
1.91
54.07
3.02
Cu Zn a
78.82
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 interesting 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 A (2h = 36.25°), d = 2.81 A ´˚ (2h = 31.75°), and d = 1.62 A (2h = 56.58°) confirm 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 ˚´ (2h = 43.23°). In the appearing at d = 2.09 A Jasada Bhasma sample the characteristic peak of ´˚ crystalline metallic zinc at d = 2.09 A was absent. This result confirms the absence of any crystalline zinc metal in the final product of the Jasada Bhasma.
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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
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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 vacancies on the surface of the sample. Zn2p3/2 peak analysis of Jasada Bhasma also shows (Fig. 2c) considerably reduced peak area of the fully coordinated Zn peak at 1022.0 eV, strongly suggesting a substantial oxygen deficiency on the surface of the material.
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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
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
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.
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). Henceforth, these fractionated particles in 30 nm size range are referred as nanoparticulate fraction in all the subsequent studies.
Particle size analysis: evidence for nanoparticles
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.
Dynamic light scattering (DLS) Particle size analysis from DLS instrument is shown in Fig. 3. The result shows size of the particles
Transmission electron microscopy (TEM)
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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 fraction 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, estimated 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
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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.
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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 preparation 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 amorphous 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 crystalline zinc metal is also confirmed by XRD analysis. Therefore, ICP and EDAX analysis results, combining 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 predominantly 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 conserved 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 oxidative 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.
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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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