Characterization and potential novel applications of zinc-based traditional medicine, Yashad Bhasma

Abstract

Background

Yashad Bhasma (YB), the incinerated metal ash of zinc, has been used for centuries in Ayurveda to address a variety of conditions, including eye diseases, diabetes mellitus, anemia, respiratory illnesses, etc.

Objective

This research aimed to synthesize and characterize YB and to evaluate its potential antimicrobial, antioxidant, and anti-angiogenic activities.

Materials and methods

In this study, YB is synthesized by optimizing the traditional method. Morphological and physicochemical characterization are performed using XRD, XPS, SEM, TEM, EDAX, DLS, TGA-DSC, and FTIR. The antimicrobial activity of YB is assessed using the well diffusion technique against the gram-positive bacterium Staphylococcus aureus (S. aureus) and the gram-negative bacterium Escherichia coli (E. coli). The antioxidant potential is evaluated using the 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) radical scavenging assay and Nitric oxide (NO) radical scavenging assay. A chick chorioallantoic membrane (CAM) assay is performed on fertilized chick eggs to study the anti-angiogenesis potential of YB.

Results

The XRD patterns of YB showed the presence of cubic and hexagonal phases of ZnS having average crystallite size of 32.66 nm. XPS data supports the formation of ZnS phase of YB. SEM and TEM data confirmed the size of YB NPs in a range of 250–350 nm. The EDAX analysis confirmed the presence of Zn (37.2 %) and S (21.18 %). The mean particle diameter was 361 nm in DLS. TGA-DSC findings verified that the synthesized material is stable up to 435.80 °C. The FTIR confirms the presence of organic moieties in YB along with ZnS phase. YB effectively inhibited the growth of S. aureus and E. coli. The ability of YB to scavenge DPPH and NO radicals is found to be concentration dependent (50–250 μg/mL). The study also demonstrated that YB has notable antioxidant activity. The disappearance of blood vessels beneath the sample-loaded disk after 7 days indicated the effective anti-angiogenic properties of YB.

Conclusion

Altogether, YB exhibited significant antimicrobial, noteworthy antioxidant, and anti-angiogenic activities, indicating its potential as a promising therapeutic agent.

1. Introduction

Nutritionally essential trace metal elements like Zn, Cu, and Fe have been made available in their medicinal forms as Bhasmas (incinerated metallic ash). These have been used for medicinal purposes in Ayurveda for centuries. Ayurveda, the traditional Indian health system, utilizes various metals and minerals for their therapeutic properties. Ancient scholars conducted numerous experiments to render metals and minerals biocompatible and bioavailable for safe internal use. The transformation of metals into biocompatible and safe medicinal forms is well established in Ayurveda through a process called Bhasmikarana. In this process, metals are converted into an ash form with particle sizes ranging from nanometers to micrometers. The ancient texts of Ayurveda extensively describe the process of converting metals into their Bhasma form, with various methods of Bhasmikarana documented chronologically in subsequent literature [1]. Additionally, Bhasmas are distinct chemical entities that do not share similar physicochemical properties with engineered nanoparticles (NPs). Understanding the specific chemical nature of Bhasma is essential to achieve the desired effects while avoiding the toxic effects that are commonly associated with most engineered metal nanoparticles (MNPs) [1].

The essential role of zinc in human health was first established in the year 1961. It is necessary for proper growth and development in human beings. Its catalytic, structural, and regulatory functions ascertain its significant role in human physiology. Its deficiency clinically affects the epidermal, skeletal, central nervous, immune, gastrointestinal, and reproductive systems [2]. Zinc is an intrinsic part of more than 300 known enzymes [3].

Since the 15th century, Zinc, as Yashad Bhasma (YB) – a traditionally prepared zinc-based traditional medicine, is one of the prevailing essential trace metal elements used in Ayurveda to treat eye diseases, diabetes mellitus, anaemia, respiratory illnesses, skin problems, wound healing, and neurological disorders. Different methods are exemplified to prepare the YB. However, the product has diversified physicochemical characteristics depending on the specific method used for manufacturing or synthesis of the YB. Furthermore, the internal or external uses of YB have also been restricted to specified methods of preparations [1].

Published data indicates that Yashad Bhasma in Ayurveda consists of zinc oxide nanoparticles (ZnO NPs) along with trace amounts of other chemical impurities [4]. However, it is feasible to convert zinc (Zn) into zinc sulfide (ZnS) when preparing Bhasma, provided that there is strict temperature control throughout the incineration process. Furthermore, it is reported that the Bhasma of metals is safer in its sulfide forms than in oxides [5].

The preparation of YB involved several unit processes, including purification through Samanya Shodhana (general method of purification) and Vishesh Shodhana (specific method of purification), using various organic liquid media. Additionally, calcination processes, referred to as Puta, were applied until the desired final form was achieved. The fingerprinting of the final form of YB is possible with sophisticated characterization techniques such as XRD, XPS, SEM-EDX, TEM, DLS, TGA-DTA, and FT-IR. The fabrication or synthesis of the desired product is an art that involves a complete understanding of the traditional concept of Puta (calcination processes/incineration techniques).

The compaction of materials to the nano/micron scale can modify their structural, morphological, and chemical properties. Accordingly, these materials uniquely interact with cell biomolecules and facilitate their physical transport inside the cell membrane [6]. The chemical composition, surface chemistry, size, and shape of the end material also play a crucial role in cell toxicity [[7], [8], [9], [10]]. The characterization techniques can help to standardize the product by ensuring its quality control. Nanosized metal particles, due to their unique properties, exhibit various biological activities. This makes them promising for various biomedical applications.

In the present paper, a zinc-based traditional medicine was meticulously prepared using a specific traditional methodology, and its composition and physicochemical properties were comprehensively characterized through sophisticated analytical techniques. The study subsequently evaluated the antimicrobial, antioxidant, and anti-angiogenesis activity of YB, specifically assessing its effectiveness in inhibiting bacterial growth and preventing angiogenesis (as an indicator of invasion and metastasis).

2. Materials and Methods

2.1. Synthesis of YB

The YB was prepared in triplicate by following a specific traditional methodology available in the classical textbook of Rasa Shastra [11]. Amongst various available references, precise reference methods of Shodhana (purification) and Marana (calcination/incineration) are preferred by considering their feasibility and importance [11].

2.2. Reference methodology

The pharmaceutical processes were followed as per the standard references available in the classical literature of Ayurveda listed in the First Schedule of the Drugs and Cosmetics Act 1940 and Rules 1945 [11,12].

2.2.1. Shodhana

It is performed by following the Dhalana process (melting the metal and pouring it into the liquid media). Shodhana was completed in following two steps: Samanya Shodhana and Vishesh Shodhana.

Samanya Shodhana of Yashad: Ashodhita Yashad (raw zinc) was taken in an iron ladle and heated to get melted Yashad. Melted Yashad was then immediately quenched in Kanji (Sour gruel), Takra (buttermilk), Kulattha Kwatha (decoction of Dolichos biflorus), Gomutra (Cow urine), and Tila Taila (Sesame oil) (3 times in each liquid media), respectively.

Vishesh Shodhana of Yashad: Churnodaka (lime water) was prepared by mixing the limestone powder and water in a specified ratio. Raw Yashad was kept in an iron ladle over the gas flame for getting melted. After melting, Yashad was immediately poured into Churnodaka. The process was performed seven times using the same amount of Churnodaka each time.

2.2.2. Marana

The Marana process was completed in following two steps.

2.2.2.1. Pisti Nirman (Amalgamation of zinc and mercury)

Shodhit Yashad and Shodhit Parada are used to prepare the Pisti. 50 g of Shodhit Yashad was taken in a clean iron ladle and heated to liquefy it completely. Molten Yashad was soon poured into an iron-made mortar and pestle containing 50 g of Shodhit Parada. Soon the mixture was triturated rigorously to obtain the Pisti (amalgam). The Nimbu swarasa (lemon juice) was added in the required quantity to the Pisti, and the mixture was triturated thoroughly and washed carefully with water in the end of process.

2.2.2.2. Kajjali Nirman and Puta

50 g of Shodhit Gandhak was added to the Pisti and triturated thoroughly to obtain the Kajjali (blackish powder form). In this Kajjali, Kumari swarasa (aloe vera juice) was added and triturated to prepare the Chakrika (pellets) of Kajjali. These pellets were kept in the Sharava (earthen-made casserole), and the arrangement was sealed by keeping another Sharava over first. This arrangement was subjected to first Laghu Puta (traditional method of incineration). 12.5 g of Kajjali prepared by Parada and Gandhak only was added to the intermediate product after the first Puta, and pellets were prepared with Kumari swarasa for the second Puta. The same arrangement was made and subjected to the third and fourth Laghu Puta. A total of four Putas were given to Yashad to get the final form of YB. A light dull white-colored YB with smooth and soft consistency was collected after four cycles of the incineration process. The temperature pattern of Puta process was recorded in triplicate by using a thermocouple. The tip of the thermocouple was placed in contact with the Sharava and the temperature was noted at the intervals of 10 min. All the details of the Marana process and temperature pattern have been depicted in Supplementary Fig. 1. & Fig. 2.

Fig. 2.

XPS survey spectrum of YB (a) and core-level spectra of (b) Zinc, (c) Oxygen and (d) Sulfur.

2.3. Classical confirmatory tests

The classical confirmatory signs of any Bhasma ensure the quality and safety of the final product.

2.3.1. Varna (color)

The specific color of any Bhasma assures the chemical nature of the final product. In the case of YB, its color varies according to the material used during the incineration process.

2.3.2. Niswadu (tasteless)

Bhasma has no taste when kept over the tongue in fewer amounts.

2.3.3. Nischandratva (Lusterness)

It indicates the presence of free metal particles in Bhasma if luster is present under bright sunlight.

2.3.4. Varitara (Floating over water surface)

It indicates the lightness acquired in the Bhasma after proper incineration. The particles of Bhasma can float over the stagnant water without breaking its surface tension.

2.3.5. Unam (Floating of rice grains over Bhasma-covered water surface)

It also indicates the coating of Bhasma over the water surface due to its lightness.

2.3.6. Rekhapurnata (Fineness)

This simple test indicates the fineness of the Bhasma particles due to which Bhasma enter into the creases of the thumb and index fingers after rubbing of Bhasma particles in between them.

2.4. Characterizations

2.4.1. Physico-chemical analysis

To determine the total ash, acid-insoluble ash, water-soluble ash, 2 g accurately weighed YB was incinerated in a tared silica dish at a temperature not exceeding 450°C–600 °C. The loss on drying test was performed at 105 °C, and pH value was determined by using 5 % solution of YB. All the standard methods were adopted and followed as described in the Ayurvedic Pharmacopoeia of India [13].

2.4.2. Characterization methods

XRD analysis was performed (Bruker D8 ADVANCE) using Cu Kα irradiation (λ = 1.5406 Å) with 2θ values in the range of 20°–65° to determine the crystalline structure, crystallite size, and structural identification of YB. The elemental composition of YB was evaluated using XPS and measurements were carried out using a PHI 5000 Versa Probe III. Morphological analysis of the YB was done by employing SEM (CARL ZEISS Model EVO-18). Morphology at high magnification was obtained using TEM (FEI TECNAI 20 G2) operated at an accelerating voltage of 200 kV. Elemental analysis of the YB was carried out with the help of Energy Dispersive X-ray (EDAX) detector, (DS: 51N1000 – EDS System Company: Oxford Instruments Nanoanalysis) available with the SEM instrument. The DLS analysis was performed with Malvern Panalytical Zetasizer Ultra (ZSU5700) at 25°C to determine the particle size of the YB. The DLS technique is used to get a quick indication of particle size distribution. PerkinElmer Model- STA 6000 was employed to analyze the thermogravimetric property of the YB. FT-IR studies were conducted using FTIR-4700 JASCO, recorded from 4000 to 400 cm⁻¹ at a resolution of 2 cm⁻¹ [[14], [15], [16], [17], [18], [19], [20], [21], [22], [23]].

2.5. Antimicrobial activity

The bactericidal activity of synthesized YB was tested against Gram-positive Staphylococcus aureus (ATCC 24923), and gram-negative Escherichia coli (ATCC 25922). On the sterile nutrient agar plates, a homogeneous 35 μl of culture was spread using a pre-sterile L-shaped glass rod. The wells were prepared by sterile cork borer having a diameter of 2–3 mm. The YB having concentrations of 1 and 2 mg/mL were added into prepared wells. The incubation was done at 37 °C for various time intervals such as 0–72 h on a marked plate. The common standard antibiotic, streptomycin (1 μL) was used as a positive control. The experiment was repeated thrice, and the mean diameter of inhibitory zones was recorded [24,25].

2.6. Antioxidant activity

2.6.1. 1, 1-diphenyl-2-Picrylhydrazyl–Radical scavenging assay

YB was prepared at various concentrations (50–250 μg/mL) by reconstituting them in their respective solvents. One milliliter of each YB NPs concentration was then added to 5 mL of a 0.1 mM DPPH solution in methanol [26]. The mixture was vortexed and incubated for 20 min at 25 °C. After incubation, the absorbance was measured at 517 nm using a UV–Vis spectrophotometer against a blank containing only methanol. A separate control well containing only the 0.1 mM DPPH solution in methanol served as a reference for background absorbance. The DPPH scavenging activity of each YB concentration was determined by calculating its IC₅₀ value. The IC₅₀ represents the concentration of the YB required to scavenge 50 % of the DPPH radicals. Ascorbic acid was used as standard antioxidants for comparison. The experiment was performed three times (triplicate) for each concentration. The percentage of DPPH radical scavenging activity was calculated using the following formula:

$$\%\mathbf{DPPH}\mathbf{Scavenging}=\left[\left({\mathbf{A}}_{\mathbf{0}}-{\mathbf{A}}_{\mathbf{1}}\right)/{\mathbf{A}}_{\mathbf{0}}\right]\mathbf{x}\mathbf{100}$$ (1)

Here, A₀ represents the absorbance of the control well (containing only DPPH solution and solvent), and A₁ represents the absorbance of the well containing the YB standard mixture, DPPH solution, and solvent.

2.6.2. Nitric oxide radical scavenging assay

We prepared reaction mixtures in test tubes containing 10 mM sodium nitroprusside (SNP), phosphate-buffered saline (pH 7.4), and varying concentrations of the YB (50–250 μg/mL). The final volume of each mixture was adjusted to 3 mL, and they were incubated for 150 min at 25 °C [27,28].

Following incubation, 0.5 mL aliquots of the reaction mixtures were taken and mixed with 0.5 mL of Griess reagent (1 % sulfanilamide solution, 2 % H₃PO₄) for 5 min. Subsequently, 0.5 mL of NED (N-ethyl-N-(2-hydroxyethyl) diammonium chloride; 0.1 % w/v) was added, and the mixtures were incubated again for 30 min at 25°C. The formation of a pink chromophore during this reaction indicates NO scavenging activity. The absorbance of each mixture was measured spectrophotometrically at 546 nm against a blank YB. Curcumin was used as a standard for comparison. All experiments were performed in triplicate. The percentage inhibition of NO radical generation was calculated using the following formula:

$$\%\mathbf{Inhibition}=\left[\left({\mathbf{A}}_{\mathbf{0}}-{\mathbf{A}}_{\mathbf{1}}\right)/{\mathbf{A}}_{\mathbf{0}}\right]\mathbf{x}\mathbf{100}$$ (2)

Here, A₀ represents the absorbance of the control well (containing all components except the test) and A₁ represents the absorbance of the well containing the YB, SNP, and other reaction components.

2.7. Angiogenesis assay

The chick chorioallantoic membrane (CAM) assay was conducted on fertilized chick eggs, which were then incubated for 3 days at 37°C and 80% relative humidity. Throughout this incubation period, the eggs were positioned with the pointed end facing downward. After incubation, eggs were wiped with 70 % ethanol, and 1.5 ml albumin was aspirated through the eggshell and sealed with cello tape. Further, the egg was opened on the air sac side (blunt end), shell was removed carefully with forceps. The YB 0.5 mg/ml concentration was soaked in Whatman filter disc was applied on CAM. The photographs were taken and the cavity was covered with cello tape and parafilm. The eggs were incubated at 37 °C and relative humidity of 80 % for days 5 and 7. The phosphate buffer saline was used as a control. All experiment was carried out in laminar airflow to maintain sterile conditions [[29], [30], [31]].

2.8. Statistical analysis

One-way analysis of variance (ANOVA) followed by post hoc Tukey test was employed to compare the results of antimicrobial activity of YB. A p-value of ≤0.05 was considered statistically significant. The results are presented as mean ± SD.

3. Results

3.1. Classical confirmatory tests

The YB complied with all the classical parameters. The color of YB after the fourth Puta was greyish-white. The sample was tasteless. No shine or lustre was noted in the YB sample under bright sunlight. YB particles floated on the surface of the stagnant water. The paddy grains floated over the surface of YB floating on the stagnant water. The fine particles of YB entered the furrows of the fingers after rubbing it between the thumb and index fingers.

3.2. Physico-chemical analysis

YB exhibited a total ash content of 97.6 %, with an acid-insoluble ash content of 47.10 %, water-soluble ash content of 1.62 %, loss on drying of 0.5 %, and a pH value of 8.24. The final yield of YB was 44.66 % compared to the total weight of initial material.

3.3. Structural, elemental, and morphological characterization

The X-ray diffraction pattern of the sample is shown in Fig. 1. As observed, the prominent peaks at 2θ values of 28.50°, 47.46°, and 56.31° (marked as C) correspond to the (111), (220), and, (311) planes of the cubic phase of ZnS, respectively, in line with JCPDF 80–0020. Also, the XRD peaks found at 2θ values of 26.87°, 33.04°, and 39.54° marked as H, respectively are due to reflections from (100), (200), and (102) planes of hexagonal ZnS [14]. No characteristic peaks of ZnO were observed indicating high purity of ZnS. Moreover, the average crystallite size (D) was calculated using the Debye-Scherrer equation [15]:

$$D=K\lambda /\beta \mathit{Cos}\theta$$ (3)

where β represents full-width at half maximum (FWHM) and (λ = 1.5406 Å) is the wavelength of Cu Kα irradiation.

Fig. 1.

X-ray diffraction pattern of YB.

The average crystallite size of the YB particles was 32.66 nm. Further, it is observed that raw zinc metal has been converted into ZnS NPs with the proper incineration process.

The elemental composition and chemical state of the sample were investigated using X-ray photoelectron spectroscopy (XPS) measurements. Fig. 2a shows the XPS survey spectra of ZnS and the peaks corresponding to Zn 2p, O 1s, C 1s, and S 2p can be easily observed. The core-level XPS spectrum analysis was also performed. The peak at 1020 eV corresponds to the Zn 2p_3/2 presented in Fig. 2b [16]. The deconvoluted XPS spectra of O 1s into three peaks are shown in Fig. 2c. Consequently, the peaks at 529.5 eV and 530.5 eV correspond to the O₂⁻ ion in the ZnS structure and the oxygen-deficient regions within the ZnS respectively [17], and the peak at 531.6 eV corresponds to chemically adsorbed H₂O, O₂, or –CO₃, on the surface of ZnS [18]. The fitted peaks at 160.3 and 161.8 eV represent S 2p_3/2 and S 2p_1/2 (Fig. 2d) [19].

It is noted that the SEM image of YB NPs revealed the size in the range from 250 to 450 nm (Fig. 3a). Further, the TEM images (Fig. 3b and c) predominantly show cubic and spherical shaped morphology with sizes consistent with those observed through SEM analysis. The SAED pattern shown in Fig. 3d confirms the crystalline nature of the sample.

Fig. 3.

(a) SEM image, (b) and (c) shows the TEM images at different magnification scales, and (d) SAED pattern of the YB NPs.

The EDAX analysis attested to the considerable presence of Zn (37.2 %) and S (21.18 %). Also trace amounts of Na (2.94 %), Ca (1.17 %), and C (8.30 %) are also found in the sample as impurities.

Additionally, the particle size of the YB was analyzed by DLS as depicted in Supplementary Fig. 3. The mean particle size of the sample was found to be 361 nm by DLS analysis, which aligns with the findings of SEM and TEM.

The thermogravimetric analysis of YB is shown in Fig. 4. The total weight loss ( %) is 36.38 % in the range of 20 °C–1000 °C and the TGA curve can be divided into three steps. In the first step (20 °C–435.80 °C), a weight loss of 2.45 % i.e., 0.3232 mg was observed and is attributed to the removal of moisture and decomposition of carbon compounds. In the second stage (435.80 °C–667.17 °C), a weight loss of 19.06 % of the total mass occurs which is due to the decomposition of residual carbon compounds, accompanied by an exothermic reaction with a peak at 510.8 °C. The drastic decrease in mass beyond 667.17 °C indicates the decomposition of ZnS, which is an exothermic reaction as seen in the DTA curve at 732.62 °C [20]. The presence of 63.68 % of residual content successfully confirms the presence of ZnS NPs in the YB and the synthesized material is stable up to 435.80 °C.

Fig. 4.

The TGA/DTA analysis of YB heated at a temperature range of 20–1000 °C in an airflow.

The FTIR spectrum (Supplementary Fig. 4.) of the sample illustrates the surface functional properties of YB. The bands observed around 3445 cm⁻¹ and 1630 cm⁻¹ correspond to the O–H stretching and bending vibration of H₂O molecules adsorbed to the surface of ZnS [21]. Peak at 2330 cm⁻¹, corresponds to stretching vibration of S–H bonds. The intense absorption bands ranging between 450 and 650 cm⁻¹ represent the vibrational characteristics of Zn–S [22]. The peak at 1154 cm⁻¹ is assigned to the asymmetric SO₂ vibrations [23].

3.4. Antimicrobial activity

The antimicrobial activity of YB was investigated against Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial strains using a well diffusion assay. Fig. 5 & Supplementary Table 1 shows the antimicrobial activity of 1 and 2 mg concentrations of YB against E. coli (Fig. 5 A, B) and S. aureus (Fig. 5 C, D) at 0–72 h time incubation. At 0 h of incubation, no zone of inhibition was identified in the positive control, negative control, and YB due to the antibiotic substance not having time to react against the microorganisms, as incubation time proceeded from 0 to 72 h YB showed bactericidal activity.

Fig. 5.

Antimicrobial activity of YB against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria: A and B show the antimicrobial effect of YB against E. coli at 1 mg and 2 mg concentrations respectively at various time interval; C and D show antimicrobial effect of YB against S. aureus at 1 mg and 2 mg concentration respectively at various time interval.

3.5. Antioxidant activity

3.5.1. DPPH radical scavenging activity

The scavenging effect of the YB on DPPH (2,2-diphenyl-1-picrylhydrazyl) radical was evaluated. The % inhibition values of scavenging DPPH radicals for the YB from the concentration range of 50–200 μg/mL was found to be higher than ascorbic acid (a standard substance) (Table 1). The results of the antioxidant activity of the compound showed that the compound with low IC₅₀ values is a more potent scavenger of free radicals than the compound with high IC₅₀ values. The YB showed good free radical scavenging activity in the DPPH method. However, the antioxidant potential of YB was lower than that of standard antioxidant substance ascorbic acid in the present experiment (Table 1). As the concentration of YB NPs increases, the antioxidant activities increase simultaneously. The tested YB NPs exhibited significant antioxidant activity but less than the control standard compound ascorbic acid.

Table 1.

The mean % inhibition and IC₅₀ values of YB and standard (ascorbic acid).

Conc. (μg/ml)	Mean % inhibition YB	IC50 (μg/ml)	Mean % inhibition STD	IC50 (μg/ml)
50	11.21	3.06	5.17	1.54
100	19.093		9.047
150	26.263		22.317
200	47.367		40.903
250	66.3367		78.54

3.5.2. Nitric oxide radical scavenging activity

The YB NPs exhibited a scavenging effect on NO radicals and the result was concentration-dependent (50–250 μg/mL). However, the YB NPs displayed less NO scavenging activity compared with curcumin.

The study examined the nitric oxide (NO) scavenging activity of YB NPs using a spectrophotometric assay. The results demonstrated a concentration-dependent inhibition of NO radicals by YB NPs. The IC₅₀ values obtained for YB NPs were higher than those reported for curcumin, a known antioxidant (Table 2). This suggests that YB nanoparticles possess convincing NO scavenging activity. The observed increase in NO scavenging activity with increasing YB nanoparticle concentration further supports the assumption.

Table 2.

The mean % inhibition and IC₅₀ values of YB and standard (curcumin).

Conc. (μg/ml)	Mean % inhibition YB	IC50 (μg/ml)	Mean % inhibition STD	IC50 (μg/ml)
50	6.047	4.93	12.147	2.25
100	13.23		27.18
150	25.097		53.503
200	36.137		73.507
250	41.48		84.223

3.6. Angiogenesis assay

In the current study, eggs were treated with a concentration of 0.5 mg/mL of YB NPs and phosphate-buffered saline (PBS) as the test and control, respectively. The untreated CAM exhibited normal vasculature with well-developed branching of new blood vessels. However, the CAM treated with YB NPs at the concentration of 0.5 mg/mL showed disrupted branching or deformation of angiogenesis, as depicted in Fig. 6.

Fig. 6.

Anti-angiogenic activity of YB1 nanoparticles on CAM at days 3, 5, and 7.

4. Discussion

The melting point of zinc is 419.5 °C. During the Shodhana process, zinc melts and reacts with oxygen in the environment, partially converting into zinc oxide [32]. Repeated melting and immediate quenching in various liquid media with different acidic and alkaline pH levels lead to the separation of zinc metal particles due to increased brittleness and reduced ductility of zinc. Samanya and Vishesh methods of Shodhana can help increase the melting point of Yashad (zinc), making the material suitable for subsequent pharmaceutical processes [32,33].

The preparation of Pisti (amalgam) is a necessary step after Shodhana of Yashad. The Parada (mercury) is used to prepare the Pisti. Lemon juice was added to Pisti to make the process easier. The formation of amalgamated zinc (an alloy of mercury and zinc) is a prerequisite for proper incineration. In its metallic form, zinc has a limited surface area for reactions. The formation of Pisti allows the zinc molecules to be spread out evenly, which accelerates the reaction when Gandhak (sulfur) is added later on. Before adding the Gandhak, the amalgam is washed with water to remove any residual lemon juice from the surface of the Pisti. Subsequently, the Gandhak is added to Pisti to prepare Kajjali (a black fine powdered mixture of Zinc, mercury, and sulfur). It required the trituration of the mixture for 72 h [32].

Round-shaped flat pellets were prepared by combining Kajjali with Kumari swarasa (Aloe vera juice). Although the reference method does not mention this step, aloe vera juice is traditionally used to help grind the mixture and form the pellets and it is commonly used when more than one Puta is required to prepare Bhasma. This process ultimately leads to uniform heating and favorable reactions, resulting in the final form of Bhasma. The flat and circular structure makes them suitable for the Marana process (calcination) by exposing the maximum surface of the pellets to get uniform and constant heat. The presence of organic matter in the form of aloe vera juice may play a catalytic role and provide the organic coating over the YB particles. Furthermore, the physical and chemical transformation of the organometallic complex takes place during the entire process [33,34].

The repeated cycles of the Marana process make the material finer and obtain the definite shape and morphological characters, which could be analyzed by traditional confirmatory tests and sophisticated analytical tools and techniques. In the present sample of YB, it was observed that it passed all the classical confirmatory tests and confirmed its size reduction, fineness, and lightness by Rekhapurnatva, Varitaratva, and Unam tests, respectively. Further, the Nischandratva test assured about the absence of free metal particles of Zn in YB. Varna test ratified the color of YB and its possible chemical phase as zinc sulfide. The physicochemical analysis included the total ash of the YB to confirm the appropriate calcination of the zinc as improperly incinerated zinc leads to detrimental effects. Consequently, the sample showed proper incineration of zinc, as evidenced by the % values of total ash, acid-insoluble ash, water-soluble ash, and moisture content. Final weight of YB was 67 g after 4th Puta which is 44.66 % of total weight of initial raw materials.

The XRD analysis of YB revealed the presence of cubic and hexagonal crystallite peaks of ZnS. No characteristic peaks for crystalline zinc metal or ZnO were observed. The raw zinc metal was successfully converted into ZnS NPs, which had an average crystallite size of 32.66 nm due to the proper incineration process.

Earlier, it was reported that Yashad Bhasma is Zinc oxide in ZnO wurtzite crystals having different morphological forms and particle sizes ranging between 53.14 and 42.40 nm [4]. It was found that zinc oxide nanoparticles having the size of 100 nm and in a dose greater than 125 mg/kg body weight showed toxic effects when administered orally in Sprague Dawley rats for 90 days [35]. Furthermore, zinc oxide nanoparticles having a size of 20 nm exhibited organ toxicity at 2000 mg/kg body weight in the liver, pancreas, heart, and stomach [36]. However, sulfides are least toxic than oxides form of metal nanoparticles [5]. It is also applicable to zinc sulfide (ZnS) nanoparticles and zinc oxide (ZnO) nanoparticles. It is reported that the zinc sulfide nanoparticles exhibited selective cytotoxic effects on cancer cells. The ZnS NPs showed increased ROS and RNS-mediated oxidative stress, excess production of TNF-α, and altered cellular redox status due to the maximal internalization of Zn²⁺ ions in leukemic cells. However, minimal internalization of Zn²⁺ ions was observed in normal cells, which proved the selective toxic behavior of Zn NPs in the form of ZnS [6]. Additionally, the particle size of Yashad Bhasma has been reported to range from 250 to 450 nm, allowing for easy absorption by the intestine [37].

XPS data supports the formation of ZnS phase of YB along with XRD findings and reveals the role of Gandhak (processed sulfur) and aloe vera juice in the synthesis of YB.

In the process of Bhasmikarana, ultra-structural changes take place along with size reduction due to the redundant burning process in the presence of organic material. It was confirmed by SEM and TEM analysis. The particle size of the properly incinerated YB was noted in the range of 250–350 nm having cubic and spherical morphology, agranular and porous nature of YB [33].

It is reported that zinc oxide nanoparticles having sizes ranging from 13 to 68 nm and featuring hexagonal shapes, produced more toxicological effects than bulk zinc oxide particles with 94–199 nm sizes and characteristic spherical and elliptical shapes [38]. Furthermore, zinc oxide nanoparticles showed higher toxicity than the other metal oxide nanoparticles [5]. In the present study, the SEM and TEM data confirmed the size of YB NPs in a range of 250–350 nm, which exhibits safe pharmacological effects due to its ZnS phase and cubic and spherical morphology as shown in Fig. 3. The higher percentage of sulfur in EDAX analysis reveals that most of the zinc is converted into zinc sulfide. However, the presence of other elements in trace amounts may be attributed to aloe vera juice used during the process.

Particle size analysis by DLS of YB NPs also confirmed the size reduction, as the mean particle diameter of the YB NPs was 361 nm. The larger particle size of the YB observed through DLS analysis suggests that it may be due to the incineration cycles during the Bhasmikarana process [39]. Furthermore, when the sample disperses in aqueous media, colloid particles of YB come together and make a suspension of negatively charged hydrophobic particles. These particles come closer and cluster to a larger particle size due to repeated incineration and hydrophobicity [40].

The thermogravimetric analysis revealed the importance of exact quantum of heat required to get the desired final product. The maximum temperature recorded in the calcination process of YB was 600 °C (Supplementary Fig. 1). The specific arrangement in Laghu Puta (traditional method of incineration with cow dung cakes as specified fuel and quantum of heat) used for the synthesis of YB provides the exact quantum of heat that results into fabrication of desired chemical compound and prevents the further decomposition of material to another chemical entity. For each Laghu Puta, 2 kg of cow dung cakes were used, which restricted the temperature from exceeding 600 °C during each Puta. The TGA-DTA analysis provided a solid foundation for the Puta concept in Ayurveda, justifying the formation of a unique end product at a specific temperature range (Fig. 4.).

The FTIR spectrum confirms the presence of organic moieties along with ZnS phase of YB. The organo-metallic configuration of YB is due to different liquid medias used in the purification process of Zn and aloe vera juice used in each cycle of calcination during the synthesis of YB. The different functional groups may contribute to the therapeutic excellence of YB in various ailing conditions.

The smaller-size nanoparticles possess a high surface-to-volume ratio, which enables compelling antimicrobial activity against micro-organisms due to effective penetration into microbial cells. At 6–72 h incubation time zone of inhibition gets increased from 0.76 mm to 2.03 mm for S. aureus and at 6–24 h incubation time zone of inhibition gets increased from 1.66 mm to 1.96 mm for E. coli, as represented in Fig. 5 and Supplementary Table 1. After 24 h of incubation there was no significant increase in inhibitory zone for E. coli [41].

The antimicrobial potential of YB results from their small size, unique shapes, high surface-to-volume ratio, and the presence of sulfur and organic components. They likely work by generating reactive oxygen species, damaging cell walls, and disrupting bacterial metabolism [42,43]. In this study, YB demonstrated substantial antimicrobial efficacy against both gram-positive (S. aureus) and gram-negative (E. coli) bacteria, highlighting their potential as a promising antimicrobial agent.

Free radicals contribute to diseases like atherosclerosis and cancer. MNPs, due to their electron configuration, can neutralize free radicals [44,45]. It is reported that green-synthesized ZnO NPs possess antioxidant activity due to their free radical scavenging effects compared to l-ascorbic acid in the DPPH assay [46]. The antioxidant potential of wet-chemically synthesized zinc oxide (ZnO) and zinc sulfide (ZnS) nanoparticles (NPs) was compared, revealing that ZnS NPs exhibited greater antioxidant activity than ZnO NPs in scavenging DPPH free radicals. At a concentration of 20 μg/ml, the antioxidant activity for ZnO NPs was measured at 29 %, while ZnS NPs showed an activity of 34 %. At a higher concentration of 100 μg/ml, ZnO NPs demonstrated 65 % activity, and ZnS NPs reached 71 % [47].

The ability of YB NPs to scavenge DPPH and NO radicals is likely attributed to the presence of zinc sulfide in their composition. Zinc sulfide has been reported to exhibit antioxidant properties [48], including the ability to scavenge reactive oxygen species (ROS) such as DPPH and NO. These findings suggest that YB NPs could potentially be used as therapeutic agents for conditions associated with oxidative stress and excessive free radical production. However, further studies are required to elucidate the exact mechanisms underlying their DPPH and NO scavenging activity and to evaluate their efficacy in vivo.

YB demonstrated notable antioxidant activity, particularly in DPPH and NO scavenging. The synthesis method, biological reducing material, and capping agent influence this activity of nanoparticles [49].

Angiogenesis is the formation of new blood vessels from pre-existing ones. It is crucial for organ development, wound healing, and embryonic growth. However, it is also exploited by diseases like cancer to fuel tumor growth [29]. The anti-angiogenic potential of a test sample is often assessed by its ability to inhibit blood vessel growth, commonly using the chick embryo CAM assay, a reliable in vivo method. This assay also aids in studying tumor cell invasion and metastasis. Such in vivo analyses are crucial for developing novel therapies targeting angiogenesis [30]. The chorioallantoic membrane (CAM) is a complex structure composed of ectoderm, mesoderm, and endoderm layers, along with the allantoic sac. This membrane also contains various extracellular matrix (ECM) proteins such as collagen type I, fibronectin, integrin, and laminin. These ECM proteins play a crucial role in mimicking the physiological environment for tumor cells [31]. Due to its accessibility, cost-effectiveness, and ability to mimic the physiological tumor microenvironment, the CAM assay is widely utilized for studying angiogenesis and metastasis in various cancers. The disappearance of blood vessels beneath the sample-loaded disk after day 7, as depicted in Fig. 6, highlights the effective anti-angiogenic properties of YB.

The conversion of raw zinc metal into YB (NPs) led to the formation of cubic and hexagonal particles primarily composed of zinc sulfide, measuring between 250 and 350 nm. This process was achieved through a specific arrangement of Puta and strict temperature control during each step of the Bhasmikarana process. The limited oxygen access in the closed system of Puta, combined with the specific temperature pattern of the classical Puta method that has a finite maximum temperature, justified the final form of YB as zinc sulfide NPs. All advanced analytical tests highlight the significance of each step in the Bhasmikarana process for producing high-quality, safe, and therapeutic medicinal products.

5. Conclusion

The present study is the first report on Yashad Bhasma wherein the ZnS has been prepared as a final product by keeping all the parameters in reasonable control to get the best variety of Yashad Bhasma. The in-depth knowledge of each step of the Bhasmikarana process, especially the right quantum of heat and the material used during the entire process, are the most crucial factors for getting the final product of therapeutic excellence with utmost safety. The classical confirmatory tests and organoleptic examinations of ancient times give an idea about the quality of the final product. However, modern characterization techniques confirm the physicochemical and morphological characteristics of the final product and assure quality. In the present study, the various physicochemical characterization techniques reveal the significant transformation from the original zinc metal to YB as Zinc sulfide. XRD explains the step-wise conversion of zinc metal into crystallite having cubic and hexagonal ZnS phases. The characteristic peaks of Zn metal and ZnO were absent, which indicates the high purity of ZnS. The findings were also supported by XPS and EDAX results. DLS, SEM, and TEM analysis provide the basis for ancient nanotechnology by explaining the size reduction and serial morphological changes during the process. Furthermore, TGA and FT-IR support the organometallic nature of YB and prove the integral role of organic juices used during each step of Bhasmikarana. The YB displayed significant antimicrobial and prominent antioxidant properties, indicating their promising potential as therapeutic agents. Furthermore, the anti-angiogenesis activity of YB warrants attention for its possible anti-metastasis potential in treating cancer. Future experimental studies are warranted to evaluate the clinical importance and therapeutic targets of ZnS NPs over the ZnO NPs of YB by comparing their safety profile.

Authors contribution

GCN: Conceptualization, Writing-Original Draft, Formal analysis, Investigation, and Supervision MB: Investigation, Methodology, Data Curation, and Formal analysis. LNG: Visualization, Investigation, Supervision MA: Investigation, Writing-Review and Editing. CSPT: Investigation, Writing-Review and Editing. OSN: Investigation, Writing-Review and Editing. SKM: Investigation, Writing-Review and Editing. AV: Investigations. PK: Investigation, Data Curation. HR: Investigation. AS: Writing-Review and Editing. APT: Investigation, Writing-Review and Editing. AKC: Conceptualization, Visualization. All authors listed approved publication of the content and agreed to be accountable for all aspects of the work.

Declaration of generative AI in scientific writing

None.

Funding sources

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

Supplementary Fig. 1.

Pharmaceutical process of Bhasmikarana for the preparation of Yashad Bhasma (YB).

Supplementary Fig. 2.

Temperature pattern of Laghu Puta (calcination).

Supplementary Fig. 3.

Dynamic light scattering measurements.

Supplementary Fig. 4.

FTIR spectra of YB.