Chemical Characterization of an Ayurvedic Herbo-Mineral Formulation - Vasantakusumākara Rasa: A Potential Tool for Quality Assurance.

BACKGROUND: Herbo-mineral formulations of Ayurveda contain specified metals or minerals as composition, which have their beneficial effects on biological systems. These metals or minerals are transformed into non-toxic forms through meticulous procedures explained in Ayurveda. Though literature is available on quality aspects of such herbo-mineral formulations; contemporary science is raising concerns at regular intervals on such formulations. Thus, it becomes mandate to develop quality profiles of all formulations that contain metals or minerals in their composition. Considering this, it is planned to evaluate analytical profile of Vasantakusumākara Rasa.
OBJECTIVE: To prepare Vasantakusumākara Rasa as per Standard operating Procedures (SoP) mentioned in classical text and to characterize it chemically using modern analytical techniques.
MATERIALS AND METHODS: The drug (Vasantakusumākara Rasa) in three batches was prepared in GMP certified pharmacy. Physico-chemical analysis, Assay of elements and HPTLC were carried out as per API. XRD was conducted using Rigaku Ultima-IV X-ray diffractometer.
RESULTS: The analysis shown the presence of Mercury, Tin, Gold, Silver, Iron, Zinc and Calcium etc., and HPTLC revealed presence of organic constituents from plant material. The XRD indicated the presence of cinnabar (mercury sulphide from Rasa Sindhura), cassiterite (tin oxide from Vaṅga Bhasma), massicot (lead oxide from Nāga bhasma) and Magnetite (di-iron oxide from Loha bhasma).
CONCLUSION: The physico chemical analysis reveals that VKR prepared by following classical guidelines is very effective in converting the macro elements into therapeutically effective medicines in micro form. Well prepared herbo-mineral drugs offer many advantages over plant medicines due to their longer shelf life, lesser doses, easy storing facilities, better palatability etc. The inferences and the standards laid down in this study certainly can be utilized as baseline data of standardization and QC.

Introduction

Vasantakusumākara Rasa, a classical herbo-mineral formulation is in practice since long for various therapeutic purposes.[12] The drug said to bring new positive energy in body and life, glow to skin etc., and is known for its Rasāyana (rejuvenative), vājīkaraṇa (aphrodisiac), Pramehanāśaka (anti-diabetic) and Jarā vyādhi nāśaka (anti aging) properties. It is therapeutically known for Prameha (diabetes), Vṛkkāmaya (diseases of urinary tract), Smṛti bhraṃśa (memory loss), Kārṣya (general debility), Unmāda (insanity), Śvāsa (bronchial asthma) etc.[34] Though it is an important formulation; standard manufacturing procedure (SMP) and preliminary analytical profiles are unavailable. Considering this, we planned to develop the physico-chemical profile along with possible characterization of Vasantakusumākara Rasa

Materials and Methods

Composition and preparation of Vasantakusumākara Rasa

Raw materials were procured from M/s. Maharishi Ayurveda Pharmacy, Noida and authenticated by Dravyaguna expert/Botanist, while the metal/minerals were certified by Rasa Śāstra experts. The composition of the formulation is mentioned in Table 1.

Table 1

Formulation composition of Vasantakusumakara Rasa

Preparation of Vasantakusumākara Rasa

The drug was prepared in the following steps.

Preparation of Rasa Sindhura

Preparation of Pravāla, Abhraka, Rajata, Svarṇa, Lauha, Nāga and Vaṅga Bhasmas

Preparation of Muktā piṣṭi

Levigation of the blend with specified liquids and converting into tablet.

Preparation of Rasa Sindhura

Preparation of Rasa Sindhura involves preparation Kajjalī, Bhāvanā (levigation) with Vaṭāṅkura jala (decoction of leaf buds of Ficus benghalensis Linn.) and processing in vāluka yantra Annexure 1.[5]

Kajjalī was prepared by triturating equal quantities i.e. 180 gm each of Hiṅgulottha Pārada Annexure 1 (Mercury obtained from cinnabar) and Śuddha Gandhaka (processed sulphur) in a khalva yantra (mortar pestle), till the formation of a black coloured, soft, lustreless fine collyrium like powder.[6789] This was further levigated with Vaṭāṅkura jala and then dried. This was filled in a strong amber coloured kāca kūpī (glass bottle) in Vāluka yantra (heating device) and subjected to increasing heat gradually. Mild heat was applied for first 6 hours, followed by moderate heat. When the bottom of the bottle became red, the mouth of the bottle was blocked with cork and sealed with mud mixed with lime and jaggery smeared cloth. This was followed by application of strong heat for the next six hours. Thereafter, the Vāluka yantra was allowed to cool down on its own [Figure 1]. The bottle was then removed from the Vāluka yantra and the mud smeared cloth was scraped out using a knife. The bottle was broken down carefully to collect crystallized Rasa Sindhura of 180 gm from the neck of the bottle.

Figure 1

Showing the temperature pattern for the preparation of Rasa Sindura

Preparation of Bhasmas

Preparation of Pravāla Bhasma

260 gm of śodhita Pravāla (coral processed in Sarjikṣāra water for 3 hours) was levigated in the juice of Aloe vera, flat thin pellets were prepared, dried, placed in śarāva sampuṭa Annexure 1 and as heated in Gajapuṭa Annexure 1. At the end, 180 gm of Pravāla bhasma of desired quality was obtained.[1011]

Preparation of Abhraka Bhasma

Vajrābhraka was processed in presence of Triphalā (three myrobalans) decoction by following classical guidelines. This was bundled in a jute bag with 1/4th quantity of paddy and immersed in kāñji Annexure 1 for 3 days, followed by thorough rubbing in Kāñji, so that fine Abhraka particles can enter into the liquid. The settled down contents known as Dhānyābhraka were collected carefully by decanting the liquid.

300 gm of Dhānyābhraka was levigated with Arka kṣīra (latex of Calotropis procera), flat thin pellets were prepared, dried, placed in śarāva sampuṭa and heated in Gajapuṭa. At the end of seventh puṭa, the contents were levigated with Nyagrodha mūla kvātha (decoction of Ficus benghalensis roots), dried and was subjected to gaja puṭa. The process was repeated three times followed by levigation with Rambhā rasa (juice of rhizome of Musa paradisiaca) and seven gaja puṭas. Finally, 180 gm of Abhraka Bhasma was obtained.[121314]

Preparation of Rajata Bhasma

Rajata Patras were heated till red hot and immersed consecutively in Tila taila, takra, kāñji, gomūtra and decoction of the seeds of kulattha. The whole process was repeated three times. Śuddha Rajata patra (70 g) were further processed in Agastya svarasa, Śuddha Hiṅgula (processed cinnabar) was added to it and ground well to prepare a homogenous mass, placed in Ūrdhva patana yantra and heated for six hours. On cooling, the pot was opened to collect 90 g of Rajata bhasma.[151617]

Preparation of Svarṇa Bhasma

Svarṇa patras were heated till red hot and immersed consecutively in Tila taila, takra, kāñji, gomūtra and decoction of the seeds of kulattha. This was amalgamated by triturating with Śuddha pārada. 95 g of śuddha svarṇa patra was placed in a śarāva along with half quantity of Gandhaka and covered by another śarāva. This sampuṭa was dried under sun and subjected to a puṭa with 30 cow dung cakes adding one part of Gandhaka each time. The process was repeated 14 times and 90 g. of Svarṇa bhasma was obtained.[18]

Preparation of Lauha Bhasma

Lauha was heated to red hot and immersed consecutively in Tila taila, takra, kāñji, gomūtra and decoction of the seeds of kulattha. This was further processed in equal quantities of Triphalā kaṣāya and Gomūtra. Lauha cūrṇa thus obtained was further processed through Bhānupāka (processed in sun rays), followed by Sthālīpāka (heated with decoction of Triphalā in stainless steel vessel). Śuddha lauha patra (125 g) was levigated with Triphalā kvātha; Cakrikās were prepared, dried and placed in śarāva sampuṭa subjected to Gaja puṭa. The same procedure was repeated for 60 times and 135 g of Lauha bhasma was obtained.[19202122]

Preparation of Nāga Bhasma

Molten Nāga was poured consecutively in Tila taila, takra, kāñji, gomūtra and decoction of the seeds of Kulattha for three times each in all the liquids. This was further collected in an iron pan and heated. On melting; powders of Cincā tvak (Tamarindus indica Linn.) and Aśvattha tvak (Ficus religiosa Linn.) were sprinkled in small quantities and stirred with loha darvi (Iron spatula). This process was continued till the molten Nāga is reduced to powder form.

165 g of jarita nāga was added to equal quantity of Manaḥśilā and levigated with kāñji; small cakrikās were prepared, dried and placed in śarāva sampuṭa and subjected to ardha gaja puṭa Annexure 1.[23] This process was repeated 60 times and 135 g. of Nāga bhasma was obtained.[24]

Preparation of Vaṅga Bhasma

Molten Vaṅga was poured consecutively three times each in Tila taila, takra, kāñji, gomūtra and decoction of the seeds of kulattha. In further processing, this was again melted and poured in Sinduvāra svarasa (juice of Vitex negundo Linn.) added with haridrā cūrṇa (powder of Curcuma longa Linn.) three times. The material obtained at the end of this process was converted into a powder (Jarita Vaṅga) by treating with powders of Cincā and Aśvattha tvak. 115 gm of jarita Vaṅga was added with equal quantity of orpiment powder and levigated with lime juice. Small cakrikās were prepared, dried, placed in śarāva sampuṭa and subjected to ardha gaja puṭa. This process was repeated 10 times and 13g of Vaṅga bhasma was obtained.[2526]

Preparation of Muktā Piṣṭi

190 g of Muktā (pearl) was made into a bundle in a piece of cloth, suspended and heated in a vessel containing Jayantī svarasa. This was triturated with rose water for 21 days and 180 g. of Muktā Piṣṭi was obtained.[2728]

Levigation of the blend with specified liquids and converting into tablet

All above ingredients were mixed together (1.305 kg) thoroughly to make a uniform blend and levigated with nine Bhāvanā dravyas in an end runner [Table 2]. At the end of this, 1.895 kg of contents were carefully collected, shifted to a tray drier and dried at 50°C. Dried material was shifted to tablet section and granules were prepared. 2% talcum powder was added to the granules and compressed into tablets of 125 mg size by passing through a Rotary Tablet Punching Machine.

Table 2

Brief details of levigation process

Physico-Chemical Analysis

Physicochemical analyses, viz. estimation of loss on drying, ash content, acid insoluble ash, water/alcohol soluble extractive, pH, etc., qualitative/quantitative elemental testing, residual pesticide, microbiological examination and tablet parameters viz. hardness, friability, average weight, dissolution time etc., were carried out by following standard methods as per Ayurvedic Pharmacopoeia of India (API) guidelines.[293031323334] The quantitative estimation of heavy metals viz. Pb, Cd, As, Hg and Cr was carried out by Atomic Absorption Spectrometer (Perkin Elmer (USA) Analyst 400) and the other elements viz., Mg, Cu, B, Mn, Al, were analyzed on ICP-AES (Thermo Electron Corporation’s model Iris Intreprid II XDL). However, Sulphur, Silica, Sn, Au, Ag, Fe, Zn, and Ca, were quantified by using conventional methods.[34]

High performance thin layer chromatography method

Sample preparation: 2 g powder each of three batches of VKR were soaked overnight separately in 20 ml of methanol. The solutions were continuously stirred for 6 hr and kept for next 18 hr and then the filtered samples were dried and made into 10% solution.

High Performance Thin Layer Chromatography was performed on TLC plates pre-coated with 0.25 μm thin layers of silica gel 60 F254 (E. Merck). 10 μL methanolic solution of formulation (three batches) were applied on the plates as bands 8.0 mm wide by use of a Linomat-V applicator (CAMAG, Switzerland) fitted with a 100 μL syringe (Hamilton, Switzerland). The application positions X and Y were both 10 mm, to avoid edge effects. Linear ascending development to a distance of 80 mm with mobile phase-Toluene: Ethyl acetate: formic acid 10:3:1 (v/v) was performed in a twin-trough glass chamber previously saturated with vapours of mobile phase for 20 min. The plates were dried in air and visualized under 254 nm and 366 nm for ultra violet detection and the fingerprints were taken. The same TLC plate was also derivatized with anisaldehyde-sulphuric acid reagent and visualized in white light.

X-ray diffraction study

Powder X-ray diffraction (XRD) analysis was carried out using Rigaku Ultima-IV X-ray diffractometer with CuK α radiation (λ=1.54 A°) operating at 40 kV and 30 mA. Pattern was recorded for angle (2θ) ranging from 10-100 degree at a scanning rate of 1 degree/second and scan step of 0.1 degree. XRD pattern of VKR (3 batches) is shown in spectra. Sample identification was done by matching d-spacing with the standard database.

Results and Discussion

The organo-leptic observation shows that the prepared VKR is a Brown colored tablet with aromatic odour and bitter, astringent taste. The qualitative analysis shows positive tests for the presence of Mercury, Tin, Gold, Silver, Iron, Zinc and Calcium. Chemical analysis revealed presence of 6.27% of Tin, 10.79% of Mercury, 2.89% of Sulphur, 5.66% of Iron, 3.67% of Gold, 4.47% of Silver, 9.5% of Calcium, 4.95% of Zinc, 1.196% of Magnesium, 1.05% of Lead and 2.08% of Silica with other trace elements such as Aluminum, Manganese, Copper, Boron, Chromium, Cadmium and Arsenic etc., Moisture content (4.18%), total ash (57.61%) is left after burning of organic/volatile matter (39.29%), water soluble (13.54%) and alcohol soluble (11.48%) extracts were evaluated (all these values are average analytical values of three batches). The drug was tested for residual pesticides and microbiological examination and they were found to be in permissible limits [Table 3]. HPTLC revealed presence of organic constituents from plant material [Figure 2 and Table 4].

Table 3

Analytical profile of Vasantakusumākara Rasa

Figure 2

High performance thin layer chromatography Profiles of Vasantakusumakara Rasa

Table 4

Observations/visualization/detection (Rf values)

The XRD patterns of the three batches of VKR as shown in spectra are nearly identical. Batch II showed slightly different phases. XRD results as shown in Table 5 indicate that all the samples contained cinnabar (mercury sulphide added as Rasa Sindhura); cassiterite (tin oxide, Vaṅga Bhasma); and, Massicot (lead oxide; Nāga bhasma) and Magnetite (di-iron oxide; Loha bhasma). No signature of Raupya or Svarṇa Bhasma could be identified in these spectra, indicating that the methods used for their preparation, did not yield any crystalline products or their signature is buried under the strong lines of other constituents. The XRD lines at 38.3, 44.4 and 64.7 degrees could show the presence of elemental gold. This has to be examined under light microcopy, for the presence of any shiny particles, which, if present, can indicate the reduction of Svarṇa bhasma added into metallic gold in the presence of organics. Elemental chemical analysis has shown the presence of gold and silver. The crystalline form of Abhraka bhasma appears to be not present [Figures 3 and 4].

Table 5

d-spacing and 2θ (°) values of X-ray diffraction analysis

Figure 3

X-ray diffraction pattern of Vasantakusumakara Rasa (Batch –I, II, III)

Figure 4

Overlay of X-ray diffraction pattern of Vasantakusumakara Rasa (Batch –I, II, III)

Despite the addition of Pravāla bhasma and Mauktika piṣṭi (essentially Calcium carbonate) has not shown any crystalline form such as aragonite or calcite in the XRD pattern despite high presence of calcium in the chemical analysis. The presence of crystalline magnesium oxide is not explained by the added inorganic chemicals in the preparation of VKR.

It can be seen that the three XRD patterns on the samples are qualitatively same in the relative intensities of the peaks. The assay of total ash content in these samples (app. 56% - 59% almost matching the expected fraction of inorganic constituents by weight) and the XRD results indicate that the inorganic contents have remained intact over time.

Conclusion

The physico chemical analysis reveal that VKR prepared by following classical guidelines is very effective in converting the macro elements into therapeutically effective medicines in micro form. Well prepared herbo-mineral drugs offer many advantages over plant medicines due to their longer shelf life, lesser doses, easy storing facilities, better palatability etc., The inferences and the standards laid down in this study certainly can be utilized as baseline data of standardization and quality assurance of this herbo-mineral formulation. It will be helpful laying down the further pharmacopoeial standards of Vasantakusumaākara Rasa.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.