INTRODUCTION:

According to WHO, about 70% of world population extensively use traditional and alternative medicines for the healthcare¹. “Ayurveda” which exactly means “knowledge of life”. This traditional form of medicine is thought to be thousands of years old, Ayurveda remained as applicable as ever then and now—and it is given great importance every day as individuals globally use its timeless knowledge in their everyday lives.

Bhasma which is an ancient medicinal trend is a beautiful gift of Ayurveda given to the humankind. The bhasma is the incinerated state of metals which is prepared taking a lot of care to remove the metallic qualities of the original metal and transform it into nano size particles, which makes it to adopt medicinal qualities and used for treatment of human beings^2,3,4,5,6.

According to the experts of these shastras, the process of preparation should be strictly followed to get the exact component. It means all these metals with plant and herbal extracts added to it, should be prepared with the same lengthy process propounded for it and incinerated in the same manner as mentioned in the shastras. This result in the metals to lose its metallic qualities and toxicity and change into such compounds which have medicinal and healing qualities of very high grade which when taken as medicines by human beings is absolutely safe and works as a curative drug without any side effects.

In Ayurveda, Yashada bhasma has great medicinal value, and had been in use since hundreds of years for various medicinal purposes like eye disorder, diabetes, blood disorder etc. It is very useful in treatment of Leucorrhoea, eye disorder, Eczema, anemia, respiratory disorder, wound healing etc⁷. But, validation of this biological formula has not been introduced, due to which the quality control of this product has not been taken care of. It is believed that standardization is the capacity for confirming the quality and is used to describe all methods, which are taken during the manufacturing process and quality control leading to a reproducible quality. Thus, standardization method should be introduced to ensure its qualities and also to describe all its measures^8. The quality control and manufacturing process of this product needs a great attention to make it consumable for all humankind ^9,10.

The present study has been carried out to analyze and compare the formulated and marketed samples of Yashada bhasma through some physico chemical and modern standardization techniques like XRD, DLS, Zeta Potential, TGA, SEM, EDAX and ICP MS. The opinion propounded was that either the bhasma preparation did not follow the proper process as mentioned in the text or else a shortcut method may have been followed due to certain reasons which raise a question on their safety and efficacy^11,12 and render it unacceptable as approved drugs on international level^13,14. Thus, with an objective to find out more regarding the same and to look towards the standardization of the bhasma, the present study has been carried out.

MATERIAL AND METHODS:

Preparation of Yashada bhasma:

Yashada bhasma was prepared according to the methodology laid down in Ayurvedic texts ^15-18 in the analytical lab of National Institute of Ayurveda, Jaipur, Rajasthan.

Yashada bhasma was prepared by three step process i.e. Shodhana (purification), Jarana (roasting) and Marana (calcination). Shodhana was carried out by heating and quenching of Yashada (Zinc) in five different liquid medias i.e. Tila Taila (sesame oil), Kanji (fermented ayurvedic preparation), Takra (buttermilk), Kulattha Kwatha (decoction of horse gram), and Gomutra (cow urine) for seven times. For Vishesha Shodhana same process was repeated in Nirgundi herbal extract (Vitex negundo) for three times¹⁹. Shodhita metal is converted into powdered form. For Jarana process, shuddha Yashada and apamarga churna (Achyranthes aspera) was taken in an iron pan and was allowed to melt at high temp, metal was converted in to powder form and thus, no part of the metal remained in metallic form. This powder is further processed for Marana, triturated with kumari swarasa (aloe vera juice) to form chakrikas and subjected for calcination process in muffle furnace at temperature 500⁰.The same procedure was repeated using fresh aloe vera juice every time until the bhasma, which passed the classical test characterization was obtained. The other samples of marketed Yashada bhasma were procured from local market of Jaipur and their documentation regarding their batch no, date and manufacturing was done²⁰.

Characterization with Comparative Assessment of Formulated and Marketed Yashada Bhasma:

Details of formulated Yashada bhasma (prepared in National institute of Ayurveda, Jaipur) and marketed Yashada bhasma (procured from Jaipur market) samples were given specific codes of BYB1, BYB2 and CYB whereas the formulated bhasma was coded as OMM. These formulations were characterized through classical methods of analysis as mentioned in the text, at National Institute of Ayurveda, Jaipur. Physical characterization was carried out at Ayushraj Institute, Jaipur and Manipal University Jaipur. Modern characterization techniques like XRD, TGA, DLS, Zeta potential and ICPMS was done at Sastra University, Thanjavur and SEM and EDAX analysis at MNIT, Jaipur.

Classical characterization²¹and Physical characterization of Bhasma:

The qualitative evaluation of bhasma particles forms the basic analytical validation technique, where different bhasma samples are examined for physical properties like color, taste, lusterness, fineness and floating properties etc. The quantitative analysis of bhasma includes physical and chemical properties of bhasma samples^{22, 23}. This is carried out by finding Total Ash value, Acid insoluble Ash, Water soluble Ash, Loss on drying and pH value.

Modern Characterization of Bhasma:

The modern techniques of characterization of bhasma give a clear picture on the safety and efficacy of the bhasma samples^24,
25.

XRD Analysis^{26, 27}: XRD of formulated and purchased samples were carried out for detection of crystalline or amorphous nature of compounds and phases of Zinc metal. Crystallite size, structural identification, and purity of samples were also determined by X-ray diffractometer (Bruker D8 focus) using Cu K_α radiation of wavelength 𝜆=1.5418 A^o. The 2ϴ value ranged from 20^o to 80^o.

DLS and Zeta potential analysis: Particle size and Zeta potential analyzer determined Particle size, surface charge, polydisperse index (PDI) and stability of all Yashada bhasma samples. Nano-ZS series 633 nm laser (Malvern Instruments Limited, UK) was used for nano particle size distribution.

TGA Analysis: Thermo gravimetric is a technique that measures the variation of mass of a sample during thermal treatment. An SDT Q600 TGA/DSC of TA instrument is used to record the loss in weight of samples with temperature range 0⁰- 800⁰.

Table 1: Ancient and Physical characterization of BYB1, BYB2, CYB, OMM

Sample	Varna (Color)	Niswadu (Taste	Nishchandrata (Lusterless)	Varitara (Lightness)	Nirdhuma (Fumes)	Rekhapurnata (Fineness)
BYB1	Yellow	Tasteless	No luster	Positive	No fumes	Positive
BYB2	Yellow	Tasteless	No luster	Positive	No fumes	Positive
CYB	Yellow	Pungent	No luster	Positive	Mild fumes	Positive
OMM	Creamish	Tasteless	No luster	Positive	No fumes	Positive
Samples	Total Ash Value	Acid Insoluble Ash	Water Soluble Ash	Loss on Drying	pH value
BYB1	95.20%	62.33%	9.55%	0.78%	9.23
BYB2	96.13%	66.21%	11.78%	0.80%	10.00
CYB	85%	67%	15%	2.5%	11.00
OMM	99.76%	46.10%	1.66%	0.01%	8.58

SEM Analysis²⁸: SEM analysis is useful for determining the three-dimensional configuration and surface morphology of all formulations. Small quantity of samples was sprinkled on to a double-sided carbon tape and mounted on aluminum stubs, to get electron image for SEM and EDAX analysis. Morphology of particles, size of particles and elemental composition of Yashada bhasma samples were carried by using SEM-EDAX instrument, NOVA NANOSEM 450 (XT Microscope server, FEI Netherlands).

ICPMS Analysis: ICPMS is carried for determination of heavy metals present in bhasma samples. Thermo Series-II, NWR 213 (Thermo Fisher Scientific, Germany) was used for detection of heavy metals present in samples. The ICP source converts the atoms of the elements in the sample to ions. These ions are then separated and detected by the mass spectroscopy.

RESULTS:

Ancient and Physical characterization:

Ancient characterization like Varna, Taste, Nishchandrata, Varitara, Nirdhuma and Rekhapurnata of all samples and Physical characterization of bhasma samples like Total Ash value, Acid Insoluble ash, Water soluble ash, Loss on drying and pH value of all samples are shown in Table 1.

Modern characterization of Bhasma:

X-ray Diffraction:

XRD result of formulated Yashada bhasma and marketed Yashada bhasma samples are illustrated in Fig.1. BYB 1 sample shows the diffraction peaks at angle 2θ= 31.70^◦, 34.36^◦, 36.18^◦, 47.47^◦, 56.51^◦, 62.79^◦, 66.29^◦, 67.87^◦, 69.00^◦, 72.36^◦ ,76.88^◦, and 81.27^◦, corresponding to the hkl value (1 0 0), (0 0 2),(1 0 1), (1 0 2),(1 1 0),(1 0 3), (2 0 0),(1 1 2),(2 0 1),(0 0 4),(2 0 2) and ( 1 0 4) with reference to the JCPDS File No (89-1397). It confirms the presence of ZnO (zinc oxide) in hexagonal p6₃mc symmetry. The XRD pattern of BYB2 is similar to that of BYB1 and again this result confirms the presence of ZnO (zinc oxide) with JCPDS File No (89-1397). In CYB sample XRD peaks at angle 2θ= 31.72^◦, 34.39^◦, 36.25^◦, 47.49^◦, 56.53^◦, 62.82^◦, 66.31^◦, 67.89^◦,69.02^◦,76.90 corresponding to the hkl value (1 0 0), (0 0 2),(1 0 1), (1 0 2),(1 1 0),(1 0 3), (2 0 0),(1 1 2),(2 0 1),(2 0 2) matches with JCPDS File No (89-1397) confirming the presence of zinc oxide(ZnO). Some peaks of CYB sample are at angle 2θ= 20.98^◦, 25.41^◦, 31.32^◦, 38.60^◦, 40.79^◦, 43.23, 48.64^◦, 52.22^◦, 55.69^◦, 65.42^◦, corresponding to value (0 1 1), (2 0 0), (1 0 2), (0 2 2), (1 2 2), (3 1 1),(3 0 2),(0 4 0), (3 2 2),(0 2 4) confirms the presence of CaSO₄as it matches with JCPDS File No (72-0916).

Fig. 1: XRD spectra of BYB1, BYB2, CYB and OMM

XRD analysis of OMM pattern shows hexagonal ZnO (zincite phase) crystalline phase, with JCPDS card 79-0208 and P63mc space group, located at 2θ= 31.42^◦, 34.09^◦, 35.91^◦, 47.22^◦, 56.28^◦, 62.58^◦, 66.09^◦, 67.67^◦, 68.80^◦, 72.36^◦ and 76.71^◦_. Crystallite size of all Yashada bhasma samples were calculated by the prominent peak of corresponding XRD pattern by using Debye Scherrer’s formula (D=0.89𝜆/𝛽cos𝜃), Where D is the crystallite diameter, λ is the x-ray wavelength (0.15418 nm), β is the full width at half maximum intensity (FWHM) of the diffraction peak, and θ is the diffraction angle of the peak pattern of the of bhasma sample. Crystallite sizes of all the samples are as shown in Table 2.

Table 2: Crystallite size of BYB1, BYB2, CYB, OMM

Sample	2θ value	FWHM	Crystallite size(nm)
BYB 1	36.18	0.185	44.65
BYB 2	36.18	0.157	53.40
CYB	36.19	0.174	47.48
OMM	35.91	0.252	32

DLS and ZETA Potential:

Particle size of all bhasma samples as calculated by DLS method are shown in Fig. 2. BYB1 and BYB2 show mean particle diameter of 1397 and 1046 nm, CYB shows 858 nm while OMM shows very less particle size of diameter 339 nm. Zeta potential values of BYB 2, CYB and OMM are approximately same i.e., -18.3, -16.5, -20 but for BYB 1, zeta potential value is slightly higher (-8.4) than other samples as shown in Fig. 3, (a-d).

Fig. 2: Particle size of BYB1, BYB2, CYB and OMM

TGA:

TGA analysis curves of all bhasma samples are presented in Fig. 4. Weight loss of sample BYB1, BYB2, and OMM is found to be 0%, while sample CYB shows 2.78% weight loss above 800⁰C temperature.

Fig. 4: TGA Spectra of BYB1, BYB2, CYB and OMM

SEM and EDAX analysis:

SEM micrographs and EDAX analysis of all bhasma samples are presented in Fig. 5 and Table 3 respectively.

Fig. 5 SEM results of (a) BYB1, (b) BYB2, (c) CYB and (d) OMM

SEM image of BYB1, BYB2, CYB and OMM shows difference in particle size, agglomeration and morphology of particles as shown in Fig. 5. BYB1 shows spongy and globular particle with size ranges between 400nm-1.2µm, BYB2 shows spongy, non-symmetrical and non-specified particles with size ranging 250nm-1.2µm, in CYB particles are in rod shaped, non-porous and symmetrical structure with size ranging from 650nm-2.2 µm, while OMM shows globular, porous and symmetrical particles with size ranging from 130- 700nm.EDAX results shown in Table 3 represents different concentration of elements in each sample. OMM shows higher concentration of zinc and oxygen as compare to BYB1, BYB2, and CYB.

Table 3: Elemental analysis of BYB1, BYB2, CYB AND OMM

Elements	BYB1	BYB2	CYB	OMM
	Concentration (%w/w)
Zn	49.28	38.58	35.50	65.18
O	20.92	20.72	18.50	30.45
C	28.97	39.49	40.30	-
Al	0.15	0.51	-	-
Ca	-	-	5.7	0.49
P	-	-		2.72
K	-	-		0.70
Fe	-	-		0.40

ICPMS Analysis:

ICPMS results show the presence of heavy metals in all bhasma samples, but their concentration in each sample is different. In BYB1, BYB2 and CYB the concentration of lead is 6442 ppm, 7738 ppm and 3226 ppm while in formulated sample OMM, the concentration of Pb is only 36 ppm.

DISCUSSION:

Zinc is a very important element for all physiological processes in human body, as it plays vital role in cell functioning, boosting immune system, promoting wound healing, control diabetes and regulates many more functions in human body²⁹. Imbalance of zinc in human system causes appetite loss, eye and skin lesions, and diabetes and weight loss. Also, zinc deficiency causes anemia, low insulin level, as it is slowly absorbed by body. This deficiency of zinc cannot be fulfilled by taking zinc metal directly, as their direct consumption might create harmful side effects in human body. It may be noted that for its suitable consumption, they are formulated by proper incineration process as mentioned in classical texts. Different steps (Shodhana, Jarana, and Marana) of preparation of bhasma not only remove the impurities present in the metal but integrate much medicinal property which renders them suitable for treatment of many ailments.

Yashada bhasma may be prepared using various methods and raw materials as per texts laid down in many literatures. These may vary from one manufacturer to another as described in the Ayurvedic textbooks³⁰. It is needed to deduce the chemical composition, check upon the physical properties and structure of these bhasmas which after its complete preparation eliminates the toxicity present in the metal resulting in giving excellent medicinal effect to the human body.

Firstly, all bhasma samples (formulated and marketed samples) were subjected to ancient tests. Color of OMM sample was found to be different from other marketed samples as color of bhasma sample depends upon the material used in Marana process. In OMM, Kumari swarasa was used as bhavana dravya, so that OMM has creamish color while BYB1, BYB2, CYB have yellow color. Varitara, Rekhapurnata are done to check the lightness and fineness of bhasma as fineness and lightness will enhance the absorption and curing property of bhasma. Similarly, Nishchandrata and Nirdhuma show the absence of metallic part in all bhasma samples as there was no luster and no fumes were found emerging out from bhasma samples except CYB as it shows mild fumes may have some metallic part remaining. Total ash content shows proper incineration of BYB1, BYB2 and OMM as they have ash value >95%, while in CYB, ash content value is 85% shows that there may be some moisture and organic impurity present in bhasma sample. Greater acid insoluble value of all bhasma samples revealed that some inorganic matter is present in all bhasma samples which was insoluble in hydrochloric acid used as a solvent in this test. Water soluble ash value of OMM is very less (1.66%) as compared to BYB1, BYB2, CYB which shows that inorganic matter present in OMM is insoluble in water, comparatively high-water soluble ash value of BYB1, BYB2, and CYB confirms the presence of water-soluble matter in samples. Loss on drying measures the water content and volatile matter present in bhasma samples. Less value of LOD (<1%) in OMM, BYB1, BYB2 shows absence of water in bhasma samples. Increased value of LOD in CYB sample confirms the presence of water in it which may have been inserted by the use of bhavana dravya during Marana process. The pH value represents acid or basic nature of samples. All samples were found to be basic in nature.

Instrumental analysis using XRD confirms the presence of zinc oxide in all bhasma samples as their major portion. All bhasma samples have sharp peaks with different intensities reveals the crystallite nature of samples. OMM, BYB1, BYB2 have only ZnO crystallite peaks in it, and no peaks of zinc metal revealed that all zinc had been converted into its oxide form³¹. While XRD peaks of CYB shows some peaks of CaSO₄matches with JCPDS File No (72-0916). Thus, Calcium may have been present in zinc metal already or may have come from some herbal juice and plant extract that get converted into CaSO₄during preparation process. The average crystallite size of all bhasma samples is found to be less than 100 nm from Scherer’s formula which confirms proper incineration of zinc metal and properly incinerated nano size bhasma particles can be easily absorbed by targeted site.

Particle size of samples BYB1, BYB2, and CYB is higher (1397, 1046, 858 d nm) as compared to size of OMM (339d nm) sample. The greater particle size of all samples obtained from DLS method as compared to XRD result because in DLS method, was attributed to the fact that when particles are dispersed in aqueous media, these particles colloid together and form suspension of negatively charged hydrophobic particles. This may be due to the repeated incineration method during Marana process³², although OMM shows decreased particle size as compared to marketed samples. This may be due to proper incineration of bhasma sample. TGA result confirms the stability of all bhasma samples at high temperature.

Zeta potential value of all samples shows their stable condition. SEM result shows the average particle size of BYB1, BYB2, CYB and OMM bhasma samples are in micrometer range, whereas particle size of OMM bhasma sample is in nanometer range. ICPMS result shows the higher concentration of lead in all marketed samples as compared to formulated sample that may causes harmful effects in human body. SEM results are in good agreement with particle size obtained from DLS analysis. Less particle size of OMM may be attributed due to repetitive burning in Marana process. EDAX confirms the presence of zinc and oxygen as major components in all bhasma samples but the concentration is different in all samples. BYB1, BYB2, CYB had decreased amount of zinc and oxygen as compared to OMM which reveals that major part of zinc has converted into zinc oxide in OMM sample. Some trace elements like phosphorus, potassium and calcium present in OMM sample, may be attributed from some herbal juice or plant extracts (Kumari swarasa is rich with Ca, Na, Mg) during preparation method³³. Fe might be introduced from iron pan used in Jarana process at high temperature.

CONCLUSION:

Variation in the results of the Marketed samples BYB1, BYB2, CYB clearly indicates that they have not been prepared strictly as per literature mentioned in Ayurvedic texts. Even there had been variation in the results of BYB1and BYB2 with the same brands but different batches. These raise a serious concern over not only safety of these formulations but their efficacy also. Thus, there is an urgent need of some standard operating protocol being made for the preparations of these bhasmas and strict regulations should be made that these ayurvedic medicines should be prepared strictly using the formulation methods indicated in the Ayurvedic texts only then it will adopt the quality which shall be useful and devoid of side effects for human consumption.

SOURCES OF FUNDING:

Seed money project under endowment fund (EF/2016/05-05) from Manipal University Jaipur, Jaipur, Rajasthan.

CONFLICT OF INTEREST:

None.

ACKNOWLEDGEMENTS:

The authors are thankful to Manipal University Jaipur for providing funds in the form of a seed money project and Prof. K. Shankar Rao and Prof. P. Suresh from National Institute of Ayurveda, Jaipur for providing the research and lab facilities related to preparation of Yashada Bhasma. The authors would also like to extend their heartfelt thanks to Prof. Brindha from Shastra University, Tanjavore and MNIT, Jaipur for providing the research facilities related to characterization of Bhasma samples.

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Received on 07.09.2020 Modified on 30.12.2020

Research J. Pharm.and Tech 2021; 14(12):6392-6398.

DOI: 10.52711/0974-360X.2021.01105