Pharmaceutico-Analytical Standardization of Amavatari Rasa – A Herbo-Mineral Formulation : Journal of Ayurveda

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ANALYTICAL STUDY: Original Article

Pharmaceutico-Analytical Standardization of Amavatari Rasa – A Herbo-Mineral Formulation

Bhinde, Sonam Sagar; Patgiri, Biswajyoti J.

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Journal of Ayurveda 17(1):p 22-30, Jan–Mar 2023. | DOI: 10.4103/joa.joa_108_21
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The increased popularity of Ayurvedic medicines has also brought concerns regarding the quality, and safety of the raw materials and final products. Despite the existence and use of traditional medicines over many centuries, data on the safety, efficacy, and quality parameters of Ayurvedic formulations are not available up to the mark to support their use worldwide.[1] The metal content of Ayurvedic preparations is currently a hot topic of discussion, especially regarding its safety. Therefore, Ayurvedic preparations need stringent quality control of the finished products, as many of them contain inorganic elements such as arsenic, mercury, and lead, which are known to be highly toxic if not processed properly.[2,3] As many Ayurvedic pharmacies are preparing such formulations on a large scale, it is a need to standardize their manufacturing process on a laboratory scale so that equal standards can be maintained on large-scale production, too.

Amavatari Rasa (AR) is mercury-containing herbo-mineral drug, widely used in the management of Amavata (rheumatoid arthritis). AR helps to relieve pain, and inflammation, improve joint movement and flexibility, work on vitiated Vata and Kapha Dosha, and reduce stiffness.[4] It is being manufactured by many GMP-approved pharmaceutical companies such as Arya Vaidya Nilayam, Baidyanath, Dhootapapeshvara, Patanjali, and Dabur, in the commercial market, but standard manufacturing process (SMP) and quality parameters of this drug are still not available. Hence, the present study was carried out to develop the SMP of AR along with its quality parameters through physico-chemical, high-performance thin-layer chromatography (HPTLC) and inductively coupled plasma atomic emission spectroscopy (ICPAES) findings.

Materials and Methods

Collection and authentication of raw drugs

Ashuddha Parada (unprocessed mercury); Ashuddha Gandhaka (unprocessed Sulphur); fruits of Haritaki (Terminalia chebula Linn.), Bibhitaki (Terminalia bellirica Linn.) and Amalaki (Embelica officinalis Gaertn.); Chitraka (Plambago zeylanica Linn) roots were procured from the pharmacy, ITRA, Jamnagar. Guggulu (Commiphora wightiiArn.) was purchased from the forest department, Jamnagar. Eranda (Ricinus communis Linn.) Taila, garlic, lime powder and rock salt were purchased from the local market of Jamnagar [Table 1].

Table 1:
Contents of Amavatari Rasa

Parada[5] and Gandhaka[6] were authenticated as per Grahya Lakshana (acceptable qualities) mentioned in Rasa classics at the Department of RSBK, ITRA. All the herbal raw drugs were authenticated at the pharmacognosy laboratory, ITRA, Jamnagar.

Pharmaceutical procedures

All required pharmaceutical procedures were carried out in three batches at the department of Rasa Shastra & Bhaishajya Kalpana, ITRA, Jamnagar.

Parada Shodhana

One thousand grams unprocessed mercury was taken in a clean black stone mortar. An equal quantity of lime powder was added and triturated for 24 h. The changes in the color and texture of the mixture were noted. The mixture was filtered through a two-layered cotton cloths. The solid lime was separated from the top of the cloth and mixed with water. This mixture was allowed to settle so that Parada settles at the bottom. This portion of the Parada was mixed with the Parada obtained earlier through filtration with a cotton cloth. Garlic cloves were separated from garlic bulbs (1000 g). Cleaned and peeled cloves weighing 750 g were crushed in an electric mixer (SS pot) to obtain Rasona Kalka (750 g). Parada (750 g), Rasona Kalka (750 g), and Saindhav (375 g) were taken in the Khalva Yantra and triturated for 8 h. The Parada obtained was washed with lukewarm water (40°C). This Shodhita Parada was stored in a transparent glass bottle.[7]


Three hundred and fifty grams of Ashuddha Amalasara Gandhaka was powdered with the help of mortar and pestle. Seven hundred and milliliters of cow milk was taken in a cylindrical shape steel vessel and heated up to 70°C. The mouth of this vessel was covered with cotton cloth, and cotton cloth was smeared by cow ghee. 43.75 g of cow ghee was taken in steel vessel and heated on mild heat. Powdered Gandhaka was poured into this vessel, and continuous stirring was done with heating. When Gandhaka was totally melted, it was poured (Dhalana) into the vessel containing preheated cow milk through the cotton cloth. Vessel containing milk was shaken continuously during the pouring of melted Gandhaka to avoid lump formation. Globular spherical granules of Gandhaka were collected from the bottom of vessel. The Gandhaka thus procured was washed with hot water and kept for drying. The procured Gandhaka was again processed through a similar process for six more times. The product obtained after these processes was called Shuddha Gandhaka.[8]

Preparation of lime water

Lime water was prepared with a classical ratio 1:240 of lime powder and water.[9] For 10 l lime water, 41.66 g lime powder was added to 10 l of water. It was kept for 12 h. After 12 h it was filtered through cotton cloth.

Chitraka Shodhana

Chitraka roots were cleaned manually. Physical impurities such as stone, mud, and wood were removed and washed with tap water. After cleaning, they were immersed in lime water. It was kept for 9 h. After that Chitraka root were washed three times with lukewarm water. Then, it was sun-dried completely to prevent any fungal overgrowth. Shodhita Chitraka roots were pulverized and sieved using a 72# mesh sieve once they were completely dry.[10]

Triphala Kwatha preparation

Two kilograms of Triphala coarse powder was kept soaked in 32 l of water for overnight in stainless steel vessels. Next day it was boiled on mild heat without covering its mouth. Water was evaporated slowly and reduced till the quantity became one-fourth. It was filtered with a clean cotton cloth, and the filtered liquid was collected as Triphala Kwatha. It took approximately 6 h heating to reduce the water to one-fourth quantity from 32 to 8 l.

Guggulu Shodhana

Physical impurities such as stone, bark, and wood were removed manually from Ashuddha Guggulu. Ashuddha Guggulu was mixed with Triphala Kwatha in stainless steel vessel to immerse completely. The vessel was placed over the gas stove. Heating was given to it by maintaining the temperature at 70°C. The heating process was carried out till the maximum dissolution of Guggulu in Triphala Kwatha was done. After that, the residue was removed by filtering it and the filtered part was heated again to evaporate the liquid portion. After obtaining semisolid consistency, the mass was shifted into a steel tray smeared with ghee and made into a uniform layer. This was allowed to dry in sunlight. After completely drying of Guggulu, it was ground in a mixture for fine powder and sieved through 72#.[11]

Preparation of Dwiguna Kajjali

The mentioned amount of Shuddha Parada and Shuddha Gandhaka was taken in a Khalva Yantra and triturated carefully (average 40 times per min.) until the formation of Nischandra (luster less; without shining particles) Kajjali.

Preparation of Amavatari Rasa

Dwiguna Kajjali was taken in Khalva Yantra and Mardana was done for 10 min. Triphala Churna and Chitraka Churna were added in Kajjali and triturated till the homogeneous mixture was achieved. Then, Shuddha Guggulu powder was added, and Mardana was done till complete mixing was achieved and a uniform blend was obtained. Then, step by step, castor oil was mixed in the above mixture and triturated till the soft, smooth homogenous mixture was obtained. Thus prepared AR was kept in airtight glass jar[12] [Figure 1].

Figure 1:
Amavatari Rasa preparation. (a) Preparation Dviguna Kajjali, (b) Kallali, (c) Triphala power, (d) Chitraka powder, (e) Guggulu power, (f) Eranda Taila, (g) addition of Kajjali into Kharala, (h) addition of Triphala power, (i) addition of Chitraka powder, (j) addition of Guggulu power, (k) addition of Eranda Taila, (l) Final product Amavatari Rasa

Analysis of final product

Physicochemical parameters like loss on drying at 110°C,[13] ash value,[14] acid insoluble ash, water soluble extractive, and alcohol soluble extractive were carried for all three batches as methods mentioned in Ayurvedic pharmacopeia of India.

High-performance thin-layer chromatography

HPTLC was done to develop chromatographic pattern of AR by following the standard procedure at 254 nm and 356 nm. Solution was prepared by taking a 2.5 g sample in the conical flask. 50 ml methanol was added to it and shaken well. Then, it was kept for 24 h. Then, it was filtered and evaporated in water bath and then used for HPTLC profiling. HPTLC was carried out at analytical laboratory, ITRA on the AR sample of first batch only. The chromatographic pattern of HPTLC is given in Table 2.

Table 2:
Chromatographic conditions during high-performance thin-layer chromatography

Inductively coupled plasma atomic emission spectroscopy

ICPAES[15] was done at Sophisticated Analytical Instrument Facility, IIT, Bombay through an instrument with specifications mentioned in Table 3.

Table 3:
Inductively coupled plasma atomic emission spectroscopy instrument specification


An average 28.2% Parada loss was observed after the completion of Shodhana process [Table 4]. An average of 8.35% Gandhaka loss was observed after the completion of Shodhana process [Table 5]. The average 98.06% yield of Shuddha Chitraka was found after Shodhana in three batches [Table 6]. An average of 103.33 g residue was found in Guggulu Shodhana. An average 15.2% increase in weight was found in three batches of Shodhita Guggulu [Table 7]. Average of 54.33 g Eranda Taila was required in 354.33 g powder mixture (20, 40, 60, 80, 100, and 54.33 g of Parada, Gandhaka, Triphala, Chitraka, Guggulu, and Eranda Taila, respectively) of AR [Table 8]. Final product was dark black in color, odorless, Bitter and astringent in taste, and granular and sticky in touch [Table 9]. Results of the physicochemical parameters of three batches of AR are given in Table 10. HPTLC study reveals that AR has 4 peaks in 254 nm and 5 peaks in 366 nm ultraviolet (UV) lights [Table 11 and Figure 2].

Table 4:
Results of Parada Shodhana
Table 5:
Results of Gandhaka Shodhana
Table 6:
Results of Chitraka Shodhana
Table 7:
Result of Guggulu Shodhana
Table 8:
Observations and results of Amavatari Rasa
Table 9:
Organoleptic characteristics of all the ingredients and final Amavatari Rasa
Table 10:
Physicochemical parameters of three batches of Amavatari Rasa
Table 11:
Results of high-performance thin-layer chromatography of Amavatari Rasa
Figure 2:
Densitogram of HPTLC. (a) Densitogram at 254 nm. (b) Densitogram 366 nm. HPLC: High-performance thin-layer chromatography

ICP AES study reveals that the sample of AR contains 7.38% of mercury, 0.0031% of As (arsenic), and 0.0032% of Pb (lead) as heavy metals [Table 12].

Table 12:
Results of inductively coupled plasma atomic emission spectroscopy heavy metal analysis of Amavatari Rasa


AR is Sagandha (with sulphur) Niragni (without heat) Murchhana (swooning or making mercury lose its form) of Parada. It is a Khalviya Rasayana prepared by trituration of multiple herbo-mineral compounds with Eranda Taila.[16] However, this formulation requires multiple pharmaceutical procedures such as Shodhana of Parada, Gandhaka, Chitraka, and Guggulu before preparing AR as Khalviya Rasayana.

Initially, Ashuddha Parada was silverish white, which turned to silver color after being processed with lime powder. After Shodhana with garlic paste and rock salt, complete Nashtapishtatva (loses cohesiveness and breaks into globules) was observed in Parada, and garlic paste became dark black in color after 8 h. Finally, Shodhita Parada was turned to bright silver. Luster of Parada was also increased after Shodhana process [Table 9]. Parada Gaties might be responsible for the 28.2% loss in the present study.[17]

Gandhaka was powdered, to increase the surface area for easy melting without burning, during the process of heating. Mandagni was given to avoid the burning of Sulfur. The filtering cloth was smeared with cow ghee to avoid sticking of Gandhaka to the cloth. In the classical reference of Rasatarangini, equal amount of ghee is mentioned,[18] while in AFI part-I (quoting the reference of Rasamritam), ghee is not mentioned for the purification of Gandhaka.[19] Taking this in to consideration, in this study, 1/8th amount of ghee was taken for the first melting, followed by 1/16th of amount of ghee for another six Dhalana as Gandhaka became sticky after the first Dhalana. A total of 175 g of ghee was taken for 350g of Gandhaka for 7 melting. Cow milk was taken in double quantity of Gandhaka because Gandhaka should be immersed completely during Dhalana. After Dhalana and Swedana, Gandhaka was thoroughly washed with hot water to remove the fat contents of milk and Ghee. Average 8.35% loss of Gandhaka was observed after Shodhana process [Table 5]. Reason for loss may be procedural, like repeatedly washing with hot water, and adherence of some Gandhaka particles to cotton cloth.

During Chitraka Shodhana of each batch, fresh Churnodaka was prepared. Chitraka roots were immersed completely in Churnodaka. Chitraka roots became dark red after Shodhana. Colour of Churnodaka became dark red from white. Characteristic (Tikshna) smell of Chitraka was smelt during grinding.

During the preparation of Triphala Kwatha little frothing was observed. It is because of the boiling process, in which water molecules try to escape the liquid in the form of vapor, resulting in the formation of bubbles. The method of Kwatha preparation to boil the water with Kwathadravya still it remains ¼th, might be to provide adequate time to yield maximum water soluble content from Kwathya Dravya.

For the present study, Guggulu Shodhana was carried out as per the opinion of the subject experts, in which Ashuddha Guggulu was directly immersed into Triphala Kwatha instead of making Pottali. The color of Triphala Kwatha was brown initially; after dissolving Guggulu, the solution became dark brown. It took ½ to 1 h for the complete dissolution of Guggulu. The orange-yellow colored layer was observed on top of Triphala Kwatha. 2 l of Triphala Kwatha was used for Shodhana process of 500 g of Guggulu[20] [Table 7]. Aim for Shodhana process of Guggulu is to separate the liquid soluble and insoluble parts from the Ashuddha Guggulu and transfer of virtue of Shodhana media into the Guggulu. Guggulu was found very sticky during handling, so utensils and hands were smeared with ghee to minimize the loss. Boiling of filtrate above 70°C caused intense frothing, which needs proper care. To avoid this, the heat was reduced and maintains around 70°C. Constant stirring of the contents throughout the process is needed. The color of Shuddha Guggulu was found dark black in each batch. For 1 part (500 g) of Ashuddha Guggulu 4 part (2 l) of Triphala Kwatha is required. Average of 15.2% of yield from Triphala Kwatha was observed at the end of Guggulu Shodhana. This yield is due to the solid content of Triphala Kwatha.

Dwiguna Kajjali was prepared by trituration of Shuddha Parada and Shuddha Gandhaka, till the Nischandratva state was obtained, which indicate the complete reaction in between Parada and Gandhaka. This process of mechanical dispersion cause the reduction in the size of the mercury drops; and as reaction time increases the sulfur molecules form an interface with the microspheres of Hg and yield the formation of Kajjali.[21] It took 48 h to prepare Nishchandratva Kajjali in the present study. A small quantity of water was sprinkled[22] to the Kajjali to the prevention of loss during trituration. Preparation of Kajjali form serves many purposes, namely, formation of mercury sulfide, reduction of its particle size as well as reducing the presence of free mercury.[23]Rekhapurnatva (filling the furrows when rubbed between two fingers) and Nischandratva[24] and Varitaratva (floating on stagnant water) features appeared in the compound after 45 h, 48 h and 48 h of trituration, respectively, it indicates the fineness and absence of free mercury in the Kajjali.

During AR preparation, step-by-step Eranda Taila was mixed in a powder mixture and levigated till the soft, smooth homogenous mixture was obtained. Here, Bhavana has played an important role in initiating a combination reaction where multiple elements combine to form a single compound. It may also add some organic and inorganic trace elements into the final compound along with enhancement of therapeutic qualities of the compound. This mixing mechanism can be compared with Geometric Dilution.[25]

If there is any decoction or juice to be used in Bhavana process, then one cannot consider the total weight of Bhavana Dravya in the final product. Reduction in the yields in the final product is common because of the evaporation of water content during levigation. However in AR, due to oil as Bhavana Dravya, evaporation did not take place and thus very less loss was observed during drug preparation due to Snigdha (oily) property of castor oil, the final product becomes sticky, and hence, tablet punching was not possible. Hence, it was kept in powder form.

In physico-chemical analysis of AR, it was found that the mean ± SD of pH is 3.77 ± 0.20, it might be because of the presence of the acidic nature of the component of AR. As it was earlier established the pH of Triphala powder[26] and Kajjali[27] is 3.2 and 4.6, respectively.

6.29 ± 0.41%w/w loss on drying suggests the presence of moisture in AR. However previous researchers[28] had found a 0.5% loss on drying (LOD) in their research. This vast difference could be because of a difference in pharmaceutical procedure or might be due to changes in the method of evaluation and also seasonal variation. Although this particular parameter requires further validation, the present study findings appear more relevant. Because Guggulu, the main ingredient of AR, should have LOD less than 14 as per API, and 12.5 was observed in the present study too. It is also important to note that Chitraka and Triphala Churna also had LOD 10.84 and 5.25, respectively. Hence, LOD near to 6.29% should be considered as the standard one for this given formulation.

5.46 ± 0.72% w/w ash value indicates that 5% is contributed by mineral content [20 g of Parada and 40 g of Gandhaka total of 60 g in 354.33 g of AR-Table 7] and the rest of solid residue were from herbal part.

The Acid Insoluble Ash content is the proportion of a sample that is not hydrolyzed by 72% sulphuric acid and is not subsequently volatilized upon the incineration of this Acid Insoluble Residue. 0.40 ± 0.15 acid insoluble ash indicates that most of the components (approximately 99%) of AR are acid soluble. This particular parameter indicates that AR when taken orally, majority component of AR might start dissolving by mixing with stomach acid. 23.20 ± 5.15 Water soluble extractive and 30.13 ± 7.19 alcohol soluble extractive might be because of more proportion of herbal drugs in the sample compared to inorganic drugs. Kajjali is neither soluble in water nor in alcohol.

Four peaks in 254 nm and 5 peaks in 366 nm UV lights in HPTLC are helpful for the fingerprinting of this SOP in the present chromatographic condition.

ICP-AES qualitatively and quantitatively finds the trace elements from the given sample. The presence of 7.38% mercury is acceptable because it is an integral part of the formulation. According to classical reference, the weightage of Parada is 6.66% (1 part out of a total of 15 parts of ingredients) in AR. The present finding is in line with the proportion of raw ingredients. A slight increase in this might be because of the decreased share of other ingredients during the pharmaceutical procedure. 0.0031% of As and 0.0032% of Pb as heavy metals are also within permissible limits mentioned by API[29] and might be introduced because of cross-contamination during formulation preparation.


The adopted method for preparation of AR can be considered an easy, convenient, and SMP. To avoid the batch-to-batch variation, this SMP may be applied to large-scale production of AR. The values of physicochemical parameters, HPTLC and ICP-AES results can be taken for quality assurance, as it is not mentioned in API, could not compared with standard values.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.



The author would like to acknowledge Dr. Sagar Bhinde, Assistant Professor, Department of Kaumarbhritya, IPGT&RA, GAU, Jamnagar, for his help with suggestions and corrections.


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High-performance thin-layer chromatography; inductively coupled plasma atomic emission spectroscopy; quality control; standard manufacture process

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