Comparative stagewise mapping of trace elements using ICP-OES in five Ayurvedic Marine Drugs highlights their posologic and clinical implications

Rasashastra , an Ayurvedic pharmaceutical branch, scientifically converts natural resources into palatable therapeutic dosage forms. Ayurvedic Marine Drugs (AMD) are mainly calcium carbonate/ calcite, but in Ayurvedic treatment modality they are uniquely used for several ailments. Moreover, their detailed microchemical compositions have seldom been reported. In this study, five raw materials, conch, oyster shell, cowry, coral, and pearl, their transitions during the manufacturing process, and the final drugs were analyzed for identification and quantitation of minor elements using a validated inductively coupled plasma optical emission spectrophotometry method. All the five raw materials contained traces of Al, Ag, B, Ba, Cu, Co, Cr, Fe, Hg, Li, K, Mg, Mn, Na, Ni, Pb, S, Sr, Tl, and Zn. Interestingly, B, Co, Cr, Li, Mn, and Ni were absent in the finished products, whereas Pb and Hg exhibited a considerable decrement during the drug formation. The first three components of principal component analysis together explained 72.09% of variance. Raw AMD indicated positive factor scores, whereas their respective purified and incinerated forms exhibited negative scores. Cluster analysis derived a dendrogram typically forming incinerated AMD group but not for the raw forms. Coral with Mg as highest content was a remarkable outlier in both these analyses as compared to Na in others. In discriminant analysis of the presence/absence of elements and periodic table-based classifications, coral and pearl showed higher variation indicating as discriminants when equated with others. Periodic table-based classification was a better model for discrimination. Correlation analysis in both classifications revealed a strong correlation of conch with oyster shells and vice versa, cowry and pearl with oyster shell and a weak correlation of coral with pearl. Coral was a good predictor followed by conch, pearl, oyster shell, and cowry, sequentially, and can be considered a moderately sensitive but highly specific model. Thus, ICP-OES technique is highly precise and accurate for assessing the micro-composition of raw, in-process, and finished products to ensure their transitions, quality, and genuineness. Such characterized traditional medicines should be further investigated in depth for their mechanistic aspects.


Introduction
India shares an integral geography with distinct marine eco-zones with over 8000 km of coastline and thereby rich marine biodiversity (Serajuddin et al. 2018).Since time immemorial, along with fishing, marine mollusks such as oysters, bivalves, cephalopods, etc. have been harvested as food commodities (Mohamed and Venkatesan 2017).The shells of marine organisms have been also explored as marine drugs for centuries in Indian Systems of Medicine like Ayurveda, Unani, and Siddha (Gopal et al. 2008).Ayurvedic medicines composed of natural resources such as herbs, metals, minerals, animal products, and marine products are used either singly or in combination for their acclaimed health benefits.
One of the sub-classes of Ayurvedic Marine Drugs (AMD), the calcium-containing group (Sudha Varga), is extensively used in several ailments like hyperacidity, osteoporosis, peptic ulcers, fever, cardiovascular diseases, hemorrhagic disorders, eye diseases, diabetes, bronchitis, asthma, etc. (Mallic et al. 2013).Although categorized in one calcium group, these medicines have been attributed to unique therapeutic uses, thereby implying their diverse nature.Therefore, the five most commonly used AMD, Shankha Bhasma (incinerated conch), Shukti Bhasma (incinerated oyster shell), Kapardik Bhasma (incinerated cowry), Praval Bhasma (incinerated coral), and Mouktik Bhasma (incinerated pearl) are included in the present study.The specialty of these drugs lies in their origin from marine creatures that deposit calcium carbonate (Marin et al. 2012).These are mollusks, bivalves, and corals formed in a specific marine atmosphere, where several elements other than calcium are deposited in the CaCO 3 architecture (Furuhashi et al. 2009).
Several Ayurvedic pharmaceutical manufacturers routinely produce incinerated conch (Chavan et al. 2018), oyster shell (Sharma et al. 2016;Dubey et al. 2009a;Biradar et al. 2017), cowry (Devanathan et al. 2010;Kulshrestha et al. 2013), coral (Lata and Biradar 2015), and pearl (Dubey et al. 2009b) using different traditional methods of purification of raw material in liquid medium (Shodhana) followed by incineration (Puta) (Anonymous 2003).Moreover, the availability of several grades of raw materials in their various natural resources may lead to variable-quality drugs coming into the market.The classical texts of Ayurveda describe the preferred forms of human consumption.However, to date, neither raw materials nor finished product monographs enlisting their complete characterization are published in the Ayurvedic Pharmacopoeia of India.
Furthermore, the unique and varied therapeutic effects of AMD have been elaborated in several authoritative books and reviews of pharmacological actions (Sharma 1979).All these AMD are good appetizers (deepana/agnivardhak) while most of them are coolants,

Graphical abstract
sweet in taste, strengthening, detoxifying, complexion enhancers, and aphrodisiacs.Specific properties such as alkaline and adsorbent of Shankha Bhasma; unctuous and taste enhancer of Shukti Bhasma; hotness of Kapardik Bhasma; digestive and antiseptic of Praval Bhasma; and longevity enhancer, cardiac tonic, cognition enhancer, growth promoter, intellect enhancer, eye tonic, osteo-promoter, and dentition promoter of Mouktik Bhasma are also described (Supplementary File 1 Table S1).Similarly, at least two of these marine drugs are indicated for irritable bowel syndrome, duodenal ulcer, asthma, eye diseases, cough, and general debility conditions.Specific diseases indicated are hyperacidity and acne for Shankha Bhasma; heart diseases, spleen diseases, abdominal diseases, and urinary calculi for Shukti Bhasma; ear discharge and boils for Kapardik Bhasma; hemorrhagic disorders, and excessive sweating for Praval Bhasma; and burning sensations, chronic fever, Diabetes Mellitus, fever during dentition, and osteoporosis for Mouktik Bhasma (Supplementary File 1 Table S2), thus suggesting some general and specific diversity in these drugs.
This background information clearly sets the objectives of the present study.A comprehensive elemental assay is required for the genuine raw materials and all the finished products to manufacture unified and highquality drugs, establish their limits in the final medicine, and ensure batch-to-batch consistency.Furthermore, it will be scientifically challenging and valuable to study the transition of elemental status from raw materials to intermediates to the final products.This stagewise analysis may differentiate all five AMD types based on temporal/ spatial factors responsible for their diverse nature.It might be possible that some of their properties are common.However, several other actions are more drugspecific because of the presence, concentration, and speciation of such fortified minerals, as suggested by a few previous reports (Sur et al. 2013;Sreejith 2012).Standardization, elemental profiling, and thorough characterization of AMD have therefore become mandatory for authenticity and a better understanding of their mode of action in wide-ranging diseases.
Previous attempts have analyzed AMD by titration, atomic absorption spectrometry, inductively coupled plasma optical emission spectrophotometry (ICP-OES), X-ray fluorescence spectrometer, etc.They reported the presence of elements such as Ca, Mg, Na, Al, B, Mg, Fe, Cr, Co, Cu, Fe, K, Mn, Zn, Zr, Ni, Sr, Ti, P, Cl, S, Si, Sn, Sb, As, and Mo in incinerated conch (Chavan et al. 2018;Sur et al. 2013); incinerated oyster shell (Sharma et al. 2016); incinerated cowry (Devanathan et al. 2010;Vedhagiri et al. 2012); incinerated coral (Lata and Biradar 2015) and pearl (Joshi et al. 2015).Although data on incinerated pearls are limited to only heavy metals, freshwater cultured and natural pearls have been reported for the presence of Sr, Mn, Ba, Ni, Cr, Pb, Al, Cu, and Na (Zhang et al. 2014).However, the repeatability of these elemental data, as well as the analytical methods used to characterize these medicines, is inadequate.In contrast, spectrophotometric methods have gained tremendous accuracy, sensitivity, and specificity leading to their applications in all biological fields in recent years.It has been further advocated for application in traditional medicines, especially in the estimation of heavy metal contamination (Li et al. 2020).
In the present study, all five AMD samples were analyzed specifically by ICP-OES for elemental composition in raw, purified, and finished product samples digested in a microwave-assisted system.The effects of purification and incineration processes were assessed for trace elements and heavy metal residues.Such comprehensive trace element analysis of Ayurvedic medicines during their manufacturing process by ICPO-OES detailing the sample preparation method, experimental setup of the instrument, and data analysis has not been reported.This application of analytical science through ICP-OES in the investigation of Ayurvedic medicines is a stepping milestone for the Ayurvedic pharmaceutical industries and clinical studies in the future.

Materials and methods
Two raw material samples of five marine-origin materials were analyzed.Each raw material, divided into three batches, was cleaned with de-ionized water (post-wash).They were then cleaned by an Ayurvedic purification process using specific liquid media and incinerated in the presence of trituration media to obtain the finished products.Additionally, two market brands of each marine drug were also assessed.A schematic presentation of the study is provided in Supplementary File 2 Fig. S1.The contribution of elements through the Aloe barbadensis (AB) leaf pulp used for trituration before incineration was also tested.The results were subjected to multivariate, cluster, and discriminant analyses.The derived elemental concentrations were used for the theoretical calculation of alkaline earth element proportions and per dose elemental composition in individual drugs, as well as in Kamdudha Vati (an Ayurvedic medicine containing all these drugs in equal proportion along with starch of Tinospora cordifolia and ghee roasted red ochre powder).

Procurement of study materials
Raw conches, oyster shells, and cowries were purchased from S.V. Ayurvedic Bhandar, Agriculture Produce Market Committee, Navi Mumbai, India; coral chips from Agasti Agroved, Pune, India; while cultured pearls from Hari Parshuram Aushadhalaya, Pune, India.They were deposited in the institutional repository for future reference (conch: V-M&M/08 and 22, oyster shell: V-M&M/14 and 23, cowry: V-M&M/09 and 24, coral: V-M&M/06 and 25, and pearl: V-M&M/04 and 26).AB and cow's buttermilk were procured from institutional agriculture and dairy farms, respectively, while lemons were purchased from the local market.The AB leaves and lemons were authenticated at the Indian Drug Research and Analytical Laboratory, Pune, India.One set of finished product batches, in triplicate, was prepared in the Bhasma Section of Atharva Nature Healthcare Pvt. Ltd., Pune, India, while two sets of other marketed samples were purchased from Manakarnika Aushadhalaya, Pune, India.The details are provided in Supplementary File 1 Table S3.

Instruments, chemicals, and accessories
ICP-OES from Thermo-Fisher Scientific, Waltham, USA (iCap7200DUO), accompanied by Chiller (Thermoflex 900) were used for the elemental assay.Certified ultrahigh purity grade Ar gas of 99.999% purity was used for the generation of plasma flame while moisture and oxy trap (PCI Analytics, Mumbai, India) purifiers were used during Ar gas flow.Ethos Easy Microwave Digestion System from Milestone Srl, Sorisole BG, Italy, with 100-mL polytetrafluoroethylene digestion vessels withstanding maximum temperature up to 300 °C and pressure up to 100 bars was used for microwave-assisted sample digestion.Acid HNO 3 (Primer Plus Trace Analysis Grade > 68%, Fisher Chemical, Fisher Scientific UK Ltd., Loughborough, UK) and oxidizing agent H 2 O 2 (Ultrapure 30-32%, JT Baker, Avantor Performance Materials, PA, USA) were used to digest of all the samples.De-ionized water (pH 7.02) prepared on the WaterPro BT System (Labconco, MO, USA) was used for all dilutions while potable water (pH 6.85) was used for the washing and purification processes.Multi-element (Ag, Al, B, Ba, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Sr, Tl, Zn) and As standards from Merck (Certipur, Darmstadt, Hesse, Germany) while Hg and S from Sigma-Aldrich (TraceCERT, St. Louis, MO, USA) of 1000 ppm (mg/L) concentration, each, were used.The values of trace element impurities in HNO 3 , H 2 O 2 , de-ionized water, and potable water used during analysis are displayed in Supplementary File 1 Table S4.

Drug preparation, sample processing, and basic physicochemical analysis
The raw materials were soaked in hot water for 16 h, rinsed properly, and dried at 105 °C for 3 h (post-wash samples).Conch, oyster shell, and cowry were purified in dilute lemon juice (1:3 v/v) while coral and pearl in cow's buttermilk by boiling for 3 h.Further, they were triturated for 3 h with AB leaf pulp, except for pearl triturated with rose water, and then incinerated in the traditional way between 650 and 750 °C (Sharma 1979).All raw, post-wash, and purified materials were powdered, sieved through mesh #200, and dried at 105 ⁰C for 3 h before further analysis.Coding was performed as RM 1 and 2 for raw materials of Batches 1 and 2, respectively, P for purified, while Bhasma (B) or incinerated (I) for the finished product.All samples were noted for organoleptic characters, the effect of HCl, pH, and Ca percentage (Anonymous 1987).

Inductively coupled plasma optical emission spectrophotometry (ICP-OES) analysis
Powdered samples weighing 100 mg and AB leaf pulp 0.5 mL were placed in digestion vessels, and 8 mL HNO 3 was added slowly followed by 1 mL H 2 O 2 .The mixture was allowed to react for 15 min.The vessels were tightly closed and placed in the rotor of the microwave digestion system.The system was run at a power of 1500 microwave Watts for 15 min to reach 200 °C, stabilized for 15 min at 200 °C, and then cooled to room temperature for 20 min.The vessels were cleaned with 2% HNO 3 , washed with de-ionized water for 15 min, and dried in an oven at 80 ⁰C for each usage.The digested samples were made up to 50 mL with de-ionized water, introduced through the aspiration loop via a peristaltic pump (50 rpm), and spray nebulized into Ar plasma.The ICP-OES instrument parameters and wavelengths of the elements selected for analysis are displayed in Supplementary File 1 Table S5.The wavelength exhibiting the highest expected intensity was used for calculations.

Validation
Validation studies were conducted according to the standard guidelines (Anonymous 2005).The precision for Ag, Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Na, Ni, Pb, S, Sr, Tl, and Zn was checked for interday and intra-day systems as well as method precision by injecting six replicates of reference standards and samples, respectively (expressed by SD and %RSD).Reference standards with 250 ppb (µg/L) strength for each of these elements were aspirated for system precision and concentration measurements.Six samples (100 mg each) of incinerated cowry (KAP-B/3) were digested, diluted to 50 mL with de-ionized water, and aspirated against linearity (1-1000 ppb) of each element under test for method precision.The limit of detection (LOD) and limit of quantification (LOQ) were estimated for Sr and K in incinerated cowry.The concentration selected for the LOD was 10% of the elemental content in the samples (150 ppm for Sr and 100 ppm for K), while for the LOQ, three times the LOD concentration was studied.Furthermore, to validate the LOQ parameters,50,100,150%,i.e.,225,450,and 675 ppm of Sr and 150,300, and 450 ppm of K concentrations, were tested.These concentrations were spiked in the sample (100 mg), digested, and aspirated for recovery.Linearity was assessed for these elements by preparing nine concentrations (1-1000 ppb).

Multivariate data analysis
Multivariate data analysis was conducted using principal component analysis (PCA) and agglomerative hierarchical cluster analysis (AHCA) to assess the relationship between the trace elements detected in AMD through a correlation matrix.Statistical analysis was performed using XLSTAT (2020XLSTAT ( .4.1.1023Trial version, https:// www.xlstat.com/ en/) for Windows 10 (Addinsoft Inc., NY, USA) with a maximum of 5 filter factors and n-type standardization.The results obtained for elemental concentrations in the raw, purified, and incinerated samples were compared by pattern recognition of the data.AHCA and PCA were applied to the generated data matrix (15 × 22), with the samples organized in rows (15) and element concentrations (variables) in columns ( 22) to achieve the pattern, and the distance biplot was retrieved.The data were initially auto-scaled, wherein data were centered according to their mean value, and then, each was divided by the standard deviation such that all the variables were given the same importance (Barbosa et al. 2019).A heat map for the elemental concentrations of 18 trace elements and four heavy metals was generated using Multiple Experiment Viewer (Version: MeV_4_9_0_r2731_win, https:// osdn.net).

Discriminant analysis
IBM SPSS Inc. (version 27.0, https:// www.ibm.com) was used to classify the finished products based on readings of all the 22 elements studied wherein each classified group had n > 5.The analysis was carried out with two possible classifications: firstly, grouped as either detected in all the five AMD (Ag, Al, Fe, K, Mg, Na, Sr, S, Tl, Zn) or not detected in all of the drugs (As, B, Ba, Cd, Co, Cr, Cu, Hg, Li, Mn, Ni, Pb), and secondly, categorizing these elements based on the elemental periodic table as Group 1 (I-IIA: Li, Na, K, Mg, Sr, Ba), Group 2 (I-VIIIB: Cr, Mn, Fe, Co, Ni, Cu, Ag, Zn, Cd, Hg) and Group 3 (III-VIA: B, Al, Tl, Pb, As, S).

Calculation of alkaline earth metals proportion and per dose elemental concentration
The average values of Ca, Ba, Mg, and Sr obtained from the experimental batches were converted to g/g of raw materials and finished products.Further, moles and millimoles, for each, were deduced using their respective molar masses to obtain the proportion as mmol/mol.The probable concentrations of only those elements detected and calculated as percentages were used for estimation per element per dose in mg/mg and mg/g, as well as ppm per dose or per day.Similarly, their probable contribution in the formulation Kamdudha Vati was deduced.

Physicochemical analysis
Representative photographs of raw and purified materials, as well as finished product samples of each AMD, are shown in Supplementary File 2 Fig. S2.The color of the experimental batches did not vary considerably while the marketed sample had white, grayish white, off-white, and creamy white shades to pink.Similarly, experimental batches exhibited no precipitate in acidic solution while blackish brown to gelatinous white precipitate was present in market samples, except for conch.All the incinerated experimental batches had Ca content between 38 and 42%w/w and pH ranging from 9.75 to 10.75 (moderately alkaline), while it ranged between 32.5 and 43.06%w/w and pH from 9.17 to 11.94 (strongly alkaline), respectively, for market samples.The individual readings are provided in Supplementary File 1 Table S6.

Validation studies
The %RSD was less than 5% for each element in inter-and intra-day system precision, except for Na at 184.67 nm (5.54%), and less than 10% for Cr, K, Na, and Sr and more than 10% for Al, As, B, Ba, Co, Li, S, and Zn in inter-and intra-day method precision.However, a %RSD of less than 10% was noted in Ag and Cu for intra-day only and in Fe, Mg, Mn, and Ni for inter-day only.The calculated values for percent recovery for all three selected levels of LOQ were between 75 and 120%,i.e.,111 ± 2.7,94 ± 11.8 and 114 ± 3.2 for Sr and 104 ± 4.3,99 ± 3.6 88 ± 7.7 for K after 50, 100 and 150% of spiking, respectively.All the elements tested for linearity from 1 to 1000 ppb of concentration showed R 2 values not less than 0.999 on correlation coefficient analysis using Qtegra Software Version 2.6 (Thermo-Fisher Scientific, Waltham, MA, USA).Please refer to Supplementary File 3 for further details.

ICP-OES analysis of raw, purified, and incinerated forms of experimental batches
The trace elements in percentage and heavy metals (As, Cd, Hg and Pb) in ppm ± standard deviation calculated from the raw data are enumerated in Tables 1, 2, and 3 for raw, purified, and incinerated samples, respectively.ICP-OES testing of AMD in these forms revealed the presence of several elements in minor amounts in the CaCO 3 matrix.The impact or contribution of trace elements impurities from HNO 3 , H 2 O 2 , de-ionized water, and potable water was considered negligible in deducing the final concentrations of studied elements in all the AMD samples (Supplementary File 1 Table S4).The raw data for all duplicate samples, their duplicate readings, the final concentration deduced, regression equations, and calculations are provided in Supplementary File 4.
It was noticed that minor elements, viz.Al, Fe, K, Mg, Na, S, Sr, and Zn, were detected in larger quantities (ranging from 0.01 to 4%) and were essentially present in all the stages of the manufacturing.Cu and Mn were also present in lower quantity.B, Co, Cr, Li, and Ni were present in the raw form only but not in the purified and incinerated forms.Ag was only present in raw conch and its successive stages while in other drugs it was absent in raw forms but was present in successive stages.Ba was present in both, raw and incinerated forms of oyster shell, coral, and pearl while only present in raw forms of conch and cowry.Tl was detected in the raw and incinerated forms but not in the purified form, for all the samples.The heavy metal concentrations in all the raw materials were very high although there was significant variation in both batches.The effects of manufacturing steps on the major elements studied in the experimental batches are shown in Fig. 1.

Effect of the manufacturing process on heavy metal content
The content of Pb in raw materials (n = 2) and the effects of washing with hot water, purification with dilute lemon juice, and incineration (all, n = 3) on its content are shown in Fig. 2.There was a substantial reduction in Pb content during all pharmaceutical processes in all AMD.All incinerated samples of the experimental batches showed Pb concentrations less than 10 ppm.The other three heavy metals (As, Cd, and Hg) did not reveal any substantial changes during any of the pharmaceutical processes for all the samples and were less than 3, 0.3, and 1 ppm, respectively.

Principal component and agglomerative hierarchical cluster analysis
The distribution of elements present in the samples as active variables and separation of different stages of five AMD in PCA for the F1 vs F2 axes are displayed in Fig. 3.The score plot of matrices showed the distribution of the samples, while the loading matrix highlighted the importance of the variables.Thus, PCA resulted in three-dimensional graphs of the scores or loadings.The first (F1), second (F2), and third (F3) principal components accounted for 42.89%, 17.14%, and 11.99%, respectively, together explaining 72.02% of the total data variance based on the eigenvalues.All raw AMD samples showed positive factor scores, while their purified and incinerated counterparts exhibited negative factor scores.Details of PCA are given in Supplementary File 5 while the Scree plot and biplot of axes F1 vs F3 components are given in Supplementary File 2 Fig. S3 and S4, respectively.AHCA was applied by employing Ward's linkage method with Euclidean distances to calculate the inter-point distances and similarities between the samples.Dendrograms and level chart bars obtained from the data matrix generated using elemental concentrations are presented in Supplementary File 2 Fig. S5.However, a vivid heat map is illustrated in Fig. 4. The dendrogram depicted a trend toward the formation of incinerated material group and to some extent the purified material group.However, the raw AMD did not form a distinct group based on the concentration of studied minor elements.Interestingly, coral in all forms was an outlier which corroborated the results of PCA.

Discriminant analysis
The trend of elements detected in the finished products was as conch (Na > Sr > K > Mg > Fe > S > Al > Zn > Tl > C u > Ag), oyster shell (Na > Sr > K > Mg > S > Mn > Al > T l, Zn > Cu > Ag), cowry (Na, Sr, K, Mg, S, Al, Fe, Tl, Zn, Ag) coral (Mg, Na, S, Sr, K, Fe, Al, Mn, Zn, Cu, Tl, Ag), and pearl (Na, Sr, S, Mg, Mn, Al, Fe, K, Tl, Zn, Cu, Ag).However, the discriminant analysis, as detailed in Supplementary File 6, disclosed some important findings.For elements present in all the drugs, conch, oyster shell, and cowry did not show much variation in mean and standard deviation values, but coral and pearl had higher variation.In contrast, for elements absent in all the drugs, only coral showed higher variation.Similar findings were recognized in analysis based on periodic and 2 of the periodic table may be considered a better model for discrimination.Similarly, the eigenvalue of the presence/ absence method was less for 100% variance but higher for F1 in the periodic table method with 96.5% variance.The correlation analysis using pooled withingroup matrices in both classifications revealed that conch had the strongest correlation with oyster shells and vice versa, followed by cowry and pearl.Cowry had the strongest correlation with oyster shell followed by conch and pearl while pearl correlated closely with oyster shell followed by conch and cowry, based on standardized canonical discriminant function coefficients.Coral had the weakest correlation with pearl followed by conch, cowry, and oyster shell.Hence, coral was a good predictor followed by conch, pearl, oyster shell, and cowry, sequentially.Overall, the discriminant analysis showed that the elemental assay and its analysis by both methods was a moderately sensitive but highly specific model of prediction.In summary, 68.2% originally grouped cases and 59.1% cross-validated group cases were correctly classified in the presence/ absence method while 63.6% and 45.5%, respectively, by the periodic table-based classification method.

ICP-OES analysis of market samples
The duplicate readings and regression analysis of all the elements studied in marketed samples are listed in Supplementary File 4 and the final concentrations in Supplementary File 1 Table S7.In all the marketed samples, K, Na, S, and Sr were detected while B, Co, Cr, Cu, Li, Mn, Ni, and Tl were not detected.Ba was also detected in all the samples, except in one sample of cowry, while Ag and Fe were recorded only in incinerated conch.Al was not detected in conch and one sample of oyster shell while Zn was detected only in conch and one sample of oyster shell.The heavy metals were present in high quantities in marketed samples (0.4-12 ppm of As, 76 ppm of Hg, and up to 316 ppm of Pb).Out of the ten marketed samples tested, five had As less than 3 ppm; two showed Hg less than 1 ppm; and seven had Pb less than 10 ppm while all the samples had Cd less than 0.3 ppm.

ICP-OES analysis of Aloe barbadensis leaf pulp
The samples of AB leaves showed the presence of elements in minor quantities, and the raw data are given in Supplementary File 1 Table S8.Table 4 shows the readings and percentages of elements present in leaf pulp Fig. 1 Elemental concentration change in experimental batches during manufacturing, viz.raw, purified, and incinerated forms.Aluminum, iron, magnesium, potassium, sodium, strontium, sulfur, and zinc were studied.An increasing trend of aluminum content during successive steps of pharmaceutical processes was observed (a) while a decreasing trend was seen for iron content (b).Conch, cowry, coral and pearl denoted initial decrease and then increased concentration of magnesium (Right vertical axis denotes values for coral).However, Mg increased throughout in oyster shell (c).Potassium levels remained almost unchanged after purification but increased after incineration in all the samples, except cowry, which decreased sharply after incineration (d).On the contrary, sodium levels illustrated initial decrease after purification and then increase after incineration (1e).Strontium content remained unchanged in conch but increased in oyster shell and coral after incineration.However, cowry and pearl revealed initial decrease and then increased levels of strontium after incineration (f).An increase in sulfur content after purification and decrease after incineration were noted, except for pearl, which depicted clear decreasing trend from raw to incinerated stage.However, for coral it remained quite higher even after incineration as compared to others (g).Zinc showed extensive decrease at the purified stage and remained low at the incineration stage except for conch, which clearly revealed the opposite trend (h) along with the possible percentages that might have contributed to the finished products after incineration.Substantial quantities of Ca, Al, Fe, S, Na, Mg, and K were present in AB leaf pulp without considering the abiotic factors like watering and soil conditions, age, and season of AB.It also revealed that the concentration of K was higher, followed by Ca and other elements.

Calculation of alkaline earth elements proportion and per dose elements in AMD
The calculation details are depicted in Supplementary File 1 Table S9.The Mg/Ca proportion for conch, oyster shell, and cowry finished products was found to be doubled as compared to their raw counterparts.However, coral and pearl did not exhibit substantial change.The Sr/ Ca ratio did not display any change in raw materials and finished products for conch and cowry, but it increased in finished products of oyster shell (~ 2 times) and coral (~ 6 times) when compared to their respective raw forms.However, Sr/Ca proportion decreased (~ 5 times) in the case of pearl.The Ba/Ca proportion was found to be decreased (oyster shell and coral) or absent (conch and cowry) in the finished products when compared with their respective raw forms.However, the proportion was found to be 10 times increased in pearl.The probable concentration of elements per dose and day for individual AMD as well as in the formulation Kamdudha Vati are detailed in Supplementary File 1 Table S10.

Discussion
Marine natural resources are used for nutrition, medicine, cosmetics, jewelry, games, religious, etc. purposes and possess exorbitant economic importance (Ekin and Sesen 2018;Santhiya et al. 2013).Conch/ conch shell (Xancus pyrum), cowry/ cowrie/ porcelaneous shell (Cypraea moneta), oyster/ oyster shell/ common oyster cell/ bivalve shell/ pearl oysters (Ostrea edulis), pearl (Pictada margaritifera), and coral (Corallium rubrum) (Anonymous 2008) analyzed in the present study are marine drugs mentioned in Ayurveda, classified as calcium group (Sudha varga) and water-source drugs (Jalaj dravya) (Mallic et al. 2013).Additionally, pearls and coral are also included in the gems group (Ratna varga) (Sharma 1979).Medicinally desirable types of conches (white), cowries (yellow), and oyster shells (producing pearl) are readily available in the Fig. 2 Effect of pharmaceutical processes on lead content in experimental batches of AMD during manufacturing stages.Raw materials, post-wash samples, i.e., washed with hot water, purified samples, i.e., boiled in dilute lemon juice or cow's butter milk and incinerated/ Bhasma samples, are depicted here Indian market, but pearl and coral are sold as natural/ cultured or synthetic.All these commodities were easily available a few years ago from natural resources, but commercialization, industrialization, and conservation policies (Sathianandan 2013) have urged for aquaculture and synthetic production to cater to the increasing demands.However, the traditional therapeutic claims attributed to these drugs are for natural products which may not be inculcated completely in cultured and synthetic forms due to environmental setup (Zhang et al. 2014).Along with improper raw materials, inappropriate processing methods may also lead to variation.Nevertheless, spurious finished drugs may also be marketed, such as synthesized calcium carbonate.The present study was therefore initiated to identify and correlate the origin of raw materials and examine the changes during the processing of these materials until their conversion into a palatable dosage form.

Stagewise mapping of micro-elements highlights raw material quality assurance, their qualitative and quantitative changes leading to AMD-specific blends and AMD quality control
The main substrate of these shell buildings is calcium carbonate in aragonite or calcite form, but during calcification their extra pallial space is pooled with precursor ions Ca 2+ and HCO 3¯ along with other major ions (Na ) as well as minor ions (Fe, Sr, Cu, Mn, Al, Cr, Pb, Ti, Ag, Sn and Ba) (Saha et al. 2000;Misogianes and Chasteen 1979).These act as macromolecular components and get incorporated into the shells during their architectural development.The elements studied in these AMD could be categorized as essentially present in high amounts, essentially present in low amounts, absent, and ought to be within permissible limits.It was noted that in the finished products, Sr, Fe, K, Mg, Na, S, and Al were essentially present in high amounts (0.01-4%); as well as between Hg-Cu, Al-K, and Ba-Mn (r > + 0.80).Similarly, Ag-Ba-Cd-Tl, Al-Cr, Cr-K, Mn-Na-Sr, Ba-S, and S-Tl revealed negative correlation (r between − 0.5 and − 0.7) Fig. 4 Heatmap showing complete linkages of elements detected in AMD during their manufacturing stages.Comparison is done between raw (R), purified (P), and incinerated (I) forms Table 4 Elements detected in Aloe barbadensis (AB) leaf pulp used for trituration before the incineration process 1a, 1b and 2a, 2b represent 2 samples each from two different AB cultivated fields, respectively. 15.6 kg of AB leaf pulp was used to process 8 kg of material in each batch. 2 87.5% of average yield is considered after incineration Ag, Ba, Cu, Tl, and Zn were essentially present in lower amounts (< 0.01%); B, Co, Cr, Li, Mn, and Ni were not detected while As, Cd, Hg, and Pb were within permissible limits.
Though several liquid media are mentioned in classical texts for purification, a single and easily available acidic medium was used (Sharma 1979) considering the general rule of Amlam Shodhan Jaranam (Rasavagbhat. Rasa Ratna Samuchchaya. 1962), meaning acidic media cleanses and digests.The boiling process in acidic media highly decreased the heavy metals from raw materials, probably due to the influence of the shifted S (Fig. 1g) and decreased Fe content (Fig. 1b) leading to leaching action.The second step was trituration with herbal media and then incineration to get calcium carbonate (Chavan et al. 2018;Sharma et al. 2016;Dubey et al. 2009aDubey et al. , 2009b;;Biradar et al. 2017;Devanathan et al. 2010;Kulshrestha et al. 2013;Lata and Biradar 2015).Calcination of seashells (without any trituration) has revealed CaCO 3 to CaO conversion between 700 and 900 °C with the presence of other elements like Na, Ti, Fe, Cu, Sr, Co, Ba, Cr, Al, etc. (Nordin et al. 2015).Similarly, these AMD also contained several other trace elements in the calcite matrix.A mild to moderate increase of Sr (all samples) and K (except, pearl) in the finished products can be explained due to the volumetric loss of organic matrix (10-20%) after incineration (Chavan et al. 2018).Na and Mg mass losses in all the samples during purification may be attributed to the dissolution of the outer nacreous layer, which probably contains these in larger amounts.However, the final increment after incineration may be due to the contribution from AB juice used for trituration.The Mg level in coral was according to previous reports and confirmed its natural origin (Meibom et al. 2004;Vielzeuf et al. 2013).The trace impurities in ultrapure nitric acid, hydrogen peroxide, de-ionized water, and reverse osmosis water used for washing were in extremely low percentages or absent (Supplementary File 1 Table S4) as compared to the quantified elements in raw and processed AMD (Tables 1,2, and 3).Hence, the interference from solvent, diluent, and washing media was meager.All these possibilities can be studied prospectively in liquid media and in-process samples to understand the mechanisms by simultaneously using advanced techniques like electron probe micro-analysis, time-to-flight secondary ion mass spectrometry, energy dispersive spectrometry (Farre et al. 2011), X-ray absorption near edge structure spectroscopy, etc. (Dauphin et al. 2012;Cuif et al. 2008;Bellaaj-Zouari et al. 2011).
Multivariate data analysis was done to understand the association between the stages of manufacturing and trace elements in these AMD as reported for mollusk species (Barbosa et al. 2019).Both PCA and AHCA showed discrete groupings of purified and incinerated forms of these drugs as compared to their raw counterparts (Figs. 3 and 4).PCA showed a clear association with Sr, Mg, Na, Al, K and Tl in incinerated forms while S and Ag in purified forms indicating the usefulness of this study to establish a positive correlation in these AMD.Other elements showed a negative correlation and considerable variation in their raw forms.Though other analytical techniques have been reported to study market samples (Dhamal et al. 2013), a noteworthy variation was noticed in the market samples analyzed in this study wherein several trace elements were not detected.This signifies the importance of raw material standardization and the meticulous performance of all the manufacturing steps under good manufacturing practices.
Elemental assays of these AMD when compared with previous reports, irrespective of raw material, method of incineration, repeatability of the manufacturing method and analytical instrument used, clearly indicate the essential presence of several trace elements in variable concentrations.The comparative data are compiled in Supplementary File 1 Table S11.Out of these reports, only oyster shell and pearl were analyzed as raw, purified, and incinerated forms while others were studied at raw and/ or incinerated stages only.Notably, Al, Fe, K, Mg, Na, and Zn in conch (Chavan et al. 2018;Sur et al. 2013); Al, Cu, Fe, K, Mg and Zn in oyster shell (Sharma et al. 2016); Mg and Sr in cowry (Vedhagiri et al. 2012); Ag, Cu, Fe, K, Mg, Mn, Na, S, Sr, Zn in coral (Lata and Biradar 2015) and Al, Co, Cu, Fe, K, Mg, S, and Zn in pearl (Joshi et al. 2015) are commonly reported.Interestingly, S, Sr in conch (Chavan et al. 2018;Sur et al. 2013); Na, Sr in oyster shell (Sharma et al. 2016); K, Na, S in cowry (Vedhagiri et al. 2012); and Na, Sr in pearl (Joshi et al. 2015) were not reported in previous studies (Joshi et al. 2015).On the contrary, Mo, Ti, and Zr in coral (Lata and Biradar 2015); P in pearl (Joshi et al. 2015) and Sb in oyster shell (Sharma et al. 2016) were additionally reported.
The theoretical calculation for alkaline earth element proportions was an attempt to derive putative values for future studies.Alkaline earth metal proportion is a well-established parameter for assessing the effect of sea surface temperature on marine carbonates (Schrag 1999;Deng et al. 2010;Cantarero et al. 2017).Since these marine drugs are inevitably used in Ayurveda, this parameter has been epitomized here to contemplate their probable impact on identity, safety, and efficacy.However, experiments using ICP-mass spectrometry and laser ablation-ICP-OES can be used to confirm the proportions.Moreover, elements per dose in individual AMD as well as compound formulations like Kamdudha Vati (containing all five AMD) may be useful to plan mechanistic studies for their reported therapeutic activities like gastric ulcer, vertigo, hyperacidity, etc. (Chandra et al. 2010).

Unique micro-elemental compositions of AMD may be responsible for genome-wide signaling/activating metabolic networks supporting the holistic healing approach of Ayurveda
The secondary objective of the present study was to highlight the importance of the analysis of trace elements from a clinical perspective.Elements play dual roles as life essentials as well as having medicinal value, i.e., essential (E) and non-essential (NE).A study of the mineral composition of femoral bones assessed in young and elderly females depicted that bone essentially contains Ca (22.6, 24.4%) and P (10.6, 10.7%), along with other minerals like Mg (3.3, 3.1), K (0.4, 0.7), Fe (2.4, 0.2) and Na (25.3, 27.9) in mg/g while Sr (54.6,89.3)and Ba (< 5.0, 9.0) both in µg/g (Milovanovic et al. 2011).Ca (E) shares similar biochemistry with Mg (E).Free Ca 2+ regulates skeletal and cardiac muscle contraction, bone and tooth mineralization as well as the resorption of regulatory mechanisms for Ca 2+ , Mg 2+ , and O 4 P −3 .Even the Na + exchanger is responsible for controlling Ca 2+ overload in the cytoplasm by extruding Ca 2+ ions into extracellular space.Mg is distributed in bones, skeletal muscles, soft tissues, and extracellular fluids and is an enzyme cofactor in the mechanisms of genomic information.It also stabilizes lipid membranes, nucleic acids, and ribosomes and plays a crucial role in metabolic signals and signaling cascades by participating in regulating enzyme activity and directing macromolecules.Na and K (both E) are crucial in cellular homeostasis where specific protein pumps for Na + and K + in cell membranes generate nervous impulses with a mechanism based on a sodium-potassium pump (Chellan and Sadler 2015).Sr (NE) is naturally present in foodstuffs like grains, leafy vegetables, dairy products, corn, cabbage, onions, and lettuce.It is also present in seawater (up to 13 ppm) and exceeds any of the other cations except the most common, Na, Mg, Ca and K (Anupama and Lavanya 2016).Nowadays, Sr is widely accepted in several therapeutic forms like acetate, citrate, ranelate, or chloride and is prescribed orally for sensitive teeth, osteoporosis, and osteoarthritis as well as intravenously for bone pain related to cancer (Anonymous Strontium 2020). 89SrCl has shown promising relief in bone metastatic lung, breast and prostate cancer patients (Ye et al. 2018) while Sr-90 stops pain signals within bones and is also useful to treat a variety of eye disorders.Sr in minute amounts in the body can increase the retention of calcium by the body, help to prevent bone loss due to osteoporosis, prevent re-absorption of bone, build stronger teeth, act as a cavity preventer, and promote overall absorption of other minerals too (Anupama and Lavanya 2016;Wasserman 1998;Cabrera et al. 1999;Nielson 2004;Wang and Yeung 2017;Querido et al. 2016).Sr is also included in marketed sea salt blended with I, Mo, In, Rb, and W to boost the absorption and effectiveness of other ingredients for providing their specific health benefits (Anupama and Lavanya 2016).Hence, Sr is strongly associated with Ca metabolism (Huixu et al. 2018;Vezzoli et al. 1998;Jimenez et al. 2019) which agreeably highlights the use of these AMD having naturally fortified Sr in their incinerated/Bhasma form.
Similarly, S (E) protects protein integrity, reduces oxidative stress, participates in enzymatic reactions, regulates inflammation, etc. Fe (E) occupies human genome code for over 500 Fe-containing proteins, and Fe-S proteins are universal in cells having roles like electron transfer, catalysis, and iron regulation.The transition metal Cu (E) is involved in respiration, angiogenesis, and neuromodulation.Zn (E) has a role in protein structure and enzymatic catalysis as well as regulating the mammalian circadian clock by stabilizing period proteins.Ag (NE) is potentially used as an antimicrobial, anti-ulcer, wound healer, antiviral, and drug delivery agent.Although Al (NE) is considered detrimental due to oxidative stress caused by its deposition in the brain, it is extensively used in several cosmetics and as an adjuvant in vaccines.Tl (NE), even if considered toxic, is used as medicine for the treatment of external genital warts and added to the standard chemotherapy regimen for preventing bone marrow toxicity.Moreover, intravenous Tl was found to be anti-inflammatory and anti-apoptotic (Chellan and Sadler 2015).Here, the role of S, Fe, Cu, Zn, Ag, Al, and Tl in these AMD needs to be thoroughly investigated through in vivo studies.
The presence of several essential and non-essential elements indicates that these Bhasma are fortified calcium carbonate minerals.This probably highlights the mention of these marine drugs in several diseases like acid peptic disease, irritable bowel syndrome, ulcers, osteoporosis, cardiac tonic, eye disease, and so on (Thakur et al. 2016).Several in vivo and clinical studies have shown promising results in gastroesophageal reflux disease by conch (Ranade and Chary 2013); peptic ulcer by oyster shell (Chouhan et al. 2010); acid peptic syndrome (Ravte et al. 2015), fever, wound healing, and infections by cowry (Immanuel et al. 2012); as well as bone mineralization (Reddy et al. 2003) and ulcers (Dubey et al. 2009b) by coral.The discriminant analysis classified conch, oyster shell, and cowry together while coral and pearl have distinct microchemical compositions.The specific activities (Sharma 1979) of coral in cough, hemorrhagic disorders, excessive sweating, and those of pearl in burning sensation, chronic fever, Diabetes Mellitus, fever during dentition, and osteoporosis may be attributed to their microchemical-based discrimination.Similarly, the properties (Sharma 1979) of coral like being easily digestible, digestive, and antiseptic, and those of pearl like longevity enhancer, cardiac tonic, cognition enhancer, growth promotor, intellect enhancer, eye tonic, osteogenic and dentition promotor may also be due to this microchemical specificity.
Recent studies have focused on biological chemistry along with the influence of environmental factors on the selection of elements by animals as well as the speciation of elements responsible for these functions (Williams et al. 2012).Moreover, it is also important to consider the synergistic actions of elements, like Cu and Fe, Cu and Zn, Fe and S, etc. (Chellan and Sadler 2015).Though current research is engrossed in targeted actions, the holistic approach found in Ayurveda and the Chinese system of medicines for utilizing natural resources needs to be elicited.Marine drugs prepared using traditional methods can be considered one such class of natural products that may be attributed to holistically attained benefits.In the last 50 years, application of the ICP-OES technique in food, microplastics, materials, dietary supplements, human tissue, and body fluid analysis has gained popularity and urged experts for its valuable contribution to trace element research (Douvris et al. 2023).Thus, the use of ICP-OES is an emerging technique for the analysis of traditional medicines, especially marine materials (Yu et al. 2017) which are planned for further pre-clinical/ clinical studies.
On this ground, these five AMD have been classically described with specific therapeutic actions and used as individual drugs in several diseases along with specific herbs.Few attempts have been reported to distinguish these marine drugs (Gawalkar and Jadar 2017) though all are in calcium carbonate form.Further, these marine drugs are also used as ingredients, either singly, e.g., in Svarnamalini Vasant Rasa (only pearl) for chronic fever, or in permutations like four together, e.g., in Trailokyachintamani Rasa (except, oyster shell) for phthisis (Shah et al. 1958) or all five together e.g., in Kamdudha Vati (Chandra et al. 2010) for hyperacidity.Clinical trials are investigating the role of Kamdudha Vati in reducing the side effects of radiotherapy and chemotherapy in several types of cancer patients and Trailokyachintamani Rasa as an anticancer in breast cancer patients along with other drugs.The efficacy observed in such trials may be attributed to these AMD drugs along with their fortified trace/ minor elemental composition (CTRI 2007 CTRI/2017/01/007684; 007687; 007689; 007690 and CTRI/2018/01/011644).

Conclusions
In conclusion, the present study reports a method of validated ICP-OES analysis for Quality Control and Quality Assurance.Several trace elements were observed to be present in all the AMD containing calcium (in carbonate form) as a substrate.The study revealed that the Ayurvedic methods of manufacturing these drugs eliminated the traces of heavy metals up to acceptable limits for human use.Certain groups of essential elements remained unchanged while others revealed noticeable changes throughout the manufacturing process.However, a considerable variation in elemental composition was recorded in market samples as compared to the experimental batches.Multivariate analysis showed typical separation and grouping of elements at different stages of manufacturing.Discriminant analysis classified the drugs as conch, oyster shell, and cowry together while pearl and coral were distinct based on elemental concentration.The presence of trace or minor elements in these AMD and their position in the periodic table were satisfactorily usable for discrimination.The triturating medium might be a contributing factor to the fortification of already present minor elements.
A familiar analytical technique like ICP-OES is convenient for quantifying a wide range of elements simultaneously.However, elemental speciation can be assessed further for a better understanding of medicinal chemistry.The elemental screening and quantification of these AMD will aid in developing detailed monographs.It will also be advantageous to assess the genuineness of the natural raw materials available as by-products for the development of therapeutic agents.Understanding the natural bio-mineralization pattern of these different marine species may explain their different acclaimed therapeutic actions.Several in vitro, in vivo, and clinical

Fig. 3
Fig. 3Principal component analysis of the elements detected in AMD during different stages of manufacturing.It shows distribution of active variables (elements) overlapping with materials at different stages against axes F1 and F2.The Pearson correlation co-efficient (r) expressed in correlation matrix at 0.05 significance level suggested stronger positive correlations between As, B, Co, Cr, Fe, Ni, Pb, and Zn (r > + 0.75) as well as between Hg-Cu, Al-K, and Ba-Mn (r > + 0.80).Similarly, Ag-Ba-Cd-Tl, Al-Cr, Cr-K, Mn-Na-Sr, Ba-S, and S-Tl revealed negative correlation (r between − 0.5 and − 0.7)

Table 1
Minor elements (%) and heavy metals (ppm) detected in raw Ayurvedic Marine Drugs (AMD)

table -
equal (p < 0.0001) by rejecting the null hypothesis.The Wilk's Lambda was 0.769 (p = 0.468) for the presence/ absence method and was 0.463 (F1) and 0.962 (F2) with p = 0.219 and 0.956, respectively, in the periodic tablebased method.Hence, classification based on Groups 1

Table 2
Minor elements (%) and heavy metals (ppm) detected in purified form of five AMD

Table 3
Minor elements (%) and heavy metals (ppm) detected in incinerated AMD