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PHARMACEUTICAL STUDY
Year : 2017  |  Volume : 38  |  Issue : 2  |  Page : 158-164  

Analytical profile of Kukkutanda Tvak Bhasma (incinerated hen egg shells) prepared by two different methods


1 Department of Rasashastra and Bhaishajya Kalpana, Government Ayurved College, Raipur, Chhattisgarh, India
2 Ayurved Consultant Ananatlala Tribhuvandas Hospital, Bharuch, Gujarat, India
3 Department of Rasashastra and Bhaishajya Kalpana including Drug Research, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
4 Department of Rasashastra and Bhaishajya Kalpana, All India Institute of Ayurveda, New Delhi, India

Date of Web Publication28-Aug-2018

Correspondence Address:
Dr. B J Patgiri
Department of Rasashastra and Bhaishajya Kalpana including Drug Research, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ayu.AYU_209_17

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   Abstract 


Background: Kukkutanda Tvak Bhasma (KTB) (incinerated hen egg shells) is one of the important calcium-rich medicines used to treat leukorrhea, urinary tract infections, etc. Ancient scholars suggested that KTB prepared with processed Hingula (cinnabar) is more potent than Bhasma prepared without Hingula. Hence, in the present study, an attempt has been made to prepare incinerated hen egg shells using two different methods with and without cinnabar and their analytical profiles have been developed. Aims and Objectives: To develop analytical profile of KTB prepared by two different methods. Materials and Methods: Two samples of KTB were prepared. Sample KTB-A was prepared by Kumari Swarasa (juice of Aloe vera Tourn. Linn.) and sample KTB-B was prepared in the presence of Hingula as a medium for Marana using electric muffle furnace. The final product of both the samples of Bhasma were analyzed by organoleptic characteristics, physicochemical parameters and advanced sophisticated instrumental technologies such as particle size detection, inductively coupled plasma-atomic emission spectroscopy, Fourier transfer infrared spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy. Observations and Results: 22.75% and 41.16% of Calcium was detected in samples KTB-A and KTB-B, respectively. 0.29% and 0.15% of magnesium was found in samples KTB-A and KTB-B respectively. Both the samples of Bhasma were found to contain calcium hydroxide Ca(OH)2. Conclusions: Minimum four Puta (incineration cycles) with average 800°C temperature is required to prepare KTB through electric muffle furnace using Kumari Swarasa and processed Hingula as a medium. An average particle size was found as 9.35 μm and 9.97 μm in samples KTB-A and KTB-B, respectively. XRD study reveals that raw Kukkutanda Tvak is CaCO3 (calcite) and CaCO3 (calcium carbonate) whereas both the Bhasma contain CaH2O2 (portlandite syn) and Ca(OH)2.

Keywords: Hingula, Kukkutanda Tvak Bhasma, Kumari Swarasa


How to cite this article:
Singh TR, Fanasiya KM, Bedarkar P, Patgiri B J, Prajapati P K. Analytical profile of Kukkutanda Tvak Bhasma (incinerated hen egg shells) prepared by two different methods. AYU 2017;38:158-64

How to cite this URL:
Singh TR, Fanasiya KM, Bedarkar P, Patgiri B J, Prajapati P K. Analytical profile of Kukkutanda Tvak Bhasma (incinerated hen egg shells) prepared by two different methods. AYU [serial online] 2017 [cited 2019 Sep 18];38:158-64. Available from: http://www.ayujournal.org/text.asp?2017/38/2/158/239952


   Introduction Top


Ayurveda is one of the ancient Indian traditional systems of medicine practiced over thousands of years. Rasashastra (an ancient Indian pharmaceutical science dealing with metals, minerals and ores) is one of the parts of Ayurveda that deals with metals/nonmetals/herbomineral preparations called as Bhasma.[1]Bhasma, literally means 'ash' are inorganic preparations produced by alchemic process, which converts a metal or mineral into its compounds such as carbonates and oxides.[2] The advantages of these preparations over plant preparations are their stability, lower therapeutic dose and potency.[3] The lack of validation of traditional medicines raises concerns on their authenticity. The Kukkutanda Tvak Bhasma (KTB) (incinerated hen egg shells) is one of the calcium-rich mineral medicinal formulations mentioned in Ayurveda.[4] It is used to treat ailments such as Shweta Pradara (leukorrhea), Rakta Pradara (menorrhagia), Prameha (diabetes mellitus), Mutraroga (urinary tract infection) and Manasika Daurbalya (mental disorders). It also has the properties such as Rasayana (immunomodulation) and Balya (strength). The Bhasma were administered with milk as an Anupana.[5] It is also used as a key ingredient in many patent drugs and proprietory.[6],[7] There are different methods of preparation for KTB mentioned in classical texts based on the media used during Marana (incineration) process.[8],[9],[10],[11]Kumari Swarasa (juice of Aloe vera Tourn. Linn.) and processed Hingula (cinnabar) are frequently used during preparation of KTB as a medium.[11] To find out the analytical profile of Ayurvedic medicine is the need of hour to identify nature of final product. By considering this fact, the present work was carried out to establish analytical profile of KTB prepared by Kumari Swarasa and processed Hingula as media by evaluating physicochemical parameters and using sophisticated analytical techniques such as particle size detection, inductively coupled plasma-atomic emission spectroscopy (ICP, AES), Fourier transfer infra-red spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM).


   Materials and Methods Top


Collection of raw materials

Hen eggshells (Kukkutanda Tvak [KT]) were collected from local market of Jamnagar. Hingula (cinnabar) and Saindhava Lavana (rock salt) were procured from the Pharmacy, Gujarat Ayurved University, Jamnagar and authentication was done as per Grahyalakshana (acceptable quality of the drug) mentioned in the Ayurvedic classical texts.[12]Kumari (A. vera Tourn. Linn.) was collected from botanical garden, Gujarat Ayurved University, Jamnagar and authentication was done at Pharmacognosy Laboratory, IPGT and RA, Jamnagar.

Shodhana of raw materials

Purification of KT was done by following classical guidelines.[13] Raw KT was taken and washed with potable water. Purification was carried out by Swedana (boiling) in saltwater [14] for 3 hr. and then, it was left for self-cooling. After self-cooling, it was rubbed with hands and washed with hot water until separation of the inner layer from KT. Purification of cinnabar was carried out as per the reference of Rasatarangini.[15] Three kilograms cinnabar was made into powder and levigated with sufficient quantity of Nimbu Swarasa (lemon juice) for 6 h. The same procedure was repeated for six times more.

Preparation of Kukkutanda Tvak Bhasma

Marana (incineration) of Shodhita Kukkutanda Tvak without cinnabar (Kukkutanda Tvak Bhasma-A)

For first incineration, purified Shodhita KT powder was taken in Sharava (earthen saucer) and covered with another earthen saucer. The junctions of the two earthen saucers were sealed with cotton cloth, which was smeared with clay. Sealed saucer was allowed to dry under sunlight. These earthen saucers were subjected for incineration in electric muffle furnaces (EMFs) at 800°C temperature. Temperature at 800°C was maintained for 15 min. Next day, after self-cooling, earthen saucers were withdrawn and KT powder was collected. For second incineration cycle, collected KT powder was levigated with juice of Kumari for 6 h, followed by preparation of pellets and subjected for sun drying. The dried pellets of KT powder were taken in Sharava (earthen saucer) and covered with another earthen saucer sealed with clay-smeared cloth. After sun drying, it was subjected to incineration at 800°C for 15 min. The same procedure was repeated to obtain desired quality of KTB.[11]

Marana of Shodhita Kukkutanda Tvak with Cinnabar (Kukkutanda Tvak Bhasma-B)

First incineration cycle was carried out same as that of KTB-A. For second incineration, one part of first incinerated powder of KT was triturated with purified cinnabar (¼th part) in Khalva Yantra (mortar and pestle) till homogenous mixing. Then, mixture was levigated with juice of Kumari and it was followed by preparation of pellets and subjected for sun drying. The dried pellets were placed in Sharava (earthen saucer) and covered with another earthen saucer sealed with clay-smeared cloth. After sun drying it was subjected for incineration at 800°C for 15 min. The same procedure was repeated to obtain desired quality of KTB.[11]

Analytical study

Analytical studies of both the samples of KT Bhasma were carried out by classical parameters such as Varna (color), Sparsha (touch), Rasa (taste), Gandha (odor), Shabda (sound), Rekhapurnatwa and Nishchandratva (incinerated powder of KT should enter between the lines of finger and should not glitter), Sukshmatwa (sufficient fineness), Mridutwa (softness) and Nirgandhatwa (having no specific smell).[16] Modern physicochemical parameters such as determination of pH,[17] specific gravity,[18] loss on drying,[19] total solid contents,[20] ash value,[21] acid-insoluble ash,[21] water-soluble extractives,[19] alcohol-soluble extractives [19] and acid-neutralizing capacity were also done.[22] In addition, particle size distribution, atomic emission spectroscopy with inductively coupled plasma, FTIR, XRD, and SEM were carried out.


   Results Top


During purification of KT, average 10 l of saltwater was required to complete purification of 2 kg of KT. The pH of saltwater increased after purification and the solid contents of salt water also increased from 6.3 to 6.9% [Table 1]. A total of 629 g, i.e., 31.45% weight loss, was observed during purification process of KT [Table 2] and [Figure 1]a, [Figure 1]b. After purification of Hingula, no significant changes in weight were observed, but color changed from reddish brown to brick red [Figure 1]c and [Figure 1]d. For complete conversion into Bhasma of desired quality, minimum four incineration cycles were required to prepare KTB-A and KTB-B [Figure 2]. Average weight loss of 183 g, (36.6%) and 118.4 g, (23.68%) was found in incineration process during preparation of KTB-A and KTB-B respectively [Table 3]. The pH of KTB-A and KTB-B was 10.5 and 10 respectively [Table 4].
Table 1: Physicochemical analysis of saltwater before and after purification of Kukkutanda Tvak

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Table 2: Observations made during Shodhana of Kukkutanda Tvak

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Figure 1: Shodhana of raw materials. (a) Raw Kukkutanda Tvak (b) Shodhita Kukkutanda Tvak (c) Raw Hingula (d) Shodhita Hingula

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Figure 2: Marana of Shodhita Kukkutanda Tvak. (a) Kukkutanda Tvak Churna after first Puta, (b) Kumari Swarasa, (c) Levigation, (d) Kukkutanda Tvak Chakrika before second Puta, (e) Kukkutanda Tvak Bhasma A after second Puta, (f) Kukkutanda Tvak Bhasma A Chakrika after fourth Puta, (g) Kukkutanda Tvak Bhasma B after fourth Puta, (h) Kukkutanda Tvak Bhasma B Chakrika after fourth Puta

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Table 3: Observations made during Marana of Shodhita Kukkutanda Tvak powder

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Table 4: Results of physicochemical parameters of Kukkutanda Tvak Bhasma

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Acid-neutralizing capacity

There was no significant change in pH of KTB-A till 1 h, while testing acid-neutralizing capacity, but in pH of KTB-B, there was comparatively significant increase. 0.1 N NaOH was required slightly more in quantity for neutralization of KTB-A in comparison to KTB-B. The results of acid-neutralizing capacity are summarized in [Table 5] and [Table 6].
Table 5: Average alkali requirement for Kukkutanda Tvak Bhasma to adjusting pH to 3.5 and for neutralization

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Table 6: Average comparative acid-neutralizing capacity of two samples of Kukkutanda Tvak Bhasma

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Comparatively better acid neutralizing capacity of KTB-B is supported with less alkalinity and presence of polysulphides (FTIR) due to cinnabar.

Particle size distribution

It was observed that the particle size of KTB-B is less than KTB-A. The detailed results of particle size distribution are depicted in [Table 7].
Table 7: Results of particle size distribution analysis of Kukkutanda Tvak Bhasma

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Inductively coupled plasma-atomic emission spectroscopic analysis

22.706 ppm and 4383.5 ppm of arsenic were detected in sample KTB-A and KTB-B, respectively, whereas 0.0591 ppm and 82.572 ppm of cadmium were detected in Sample KTB-A and KTB-B, respectively. 88547.0 ppm of mercury was detected in only sample KTB-B. Lead was not detected in sample of KTB-A. The results of ICP-AES analysis are tabulated in [Table 8].
Table 8: Results of inductively coupled plasma-atomic emission spectroscopic analysis of Kukkutanda Tvak Bhasma

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Scanning electron microscopic analysis

As per the stoichiometry of the compounds identified in sample KTB-A and KTB-B, the percentages of calcium were 22.75 and 41.16%, respectively. Magnesium and sulfur were 0.29% and 12.45% and 0.15% and 1.93% in KTB-A and KTB-B, respectively. When XRD reports matched with SEM, it revealed that there is a formation of multiple compounds. The detailed results of SEM analysis are given in [Table 9] and [Table 10].
Table 9: Result of scanning electron microscopy of Kukkutanda Tvak Bhasma-A (full scale 3484 cts)

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Table 10: Result of scanning electron microscopy of Kukkutanda Tvak Bhasma-B (full scale 7145 cts)

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Fourier transform infrared spectroscopy

FTIR spectra of both samples were taken in the region of 400–4000 cm −1. General overview indicates the presence of large number of functional groups. In KTB-A, fairly sharp peaks were obtained at and around 3440.41, 1500–1400, 1446.56, 1056.50 and 875.56. These peaks indicate the presence of organic compounds such as OH, Ca, sulfate, CO2 and carbonation. In sample KTB-B, fairly sharp peaks were obtained at and around 3643–3441.95, 2924–2854.04, 2135.24, 1632.24–1558.62, 1453.84, 1122.32, 875.81, 674.88–612.88 and 416.36. These peaks indicate the presence of organic compounds such as OH, methyl, cyanide, Ca, sulfate, carbonate and polysulfide ion [Table 11] and [Table 12].
Table 11: Results of Fourier transfer infra-red spectroscopy analysis of Kukkutanda Tvak Bhasma-A

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Table 12: Results of Fourier transfer infra-red spectroscopy analysis of Kukkutanda Tvak Bhasma-B


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X-ray diffraction analysis

Raw KT was found to be in calcite (calcium carbonate [CaCO3]), and the crystal structure was rhombohedral in nature. Both the samples of KTB were found to contain portlandite and calcium hydroxide Ca(OH)2) and hexagonal in nature [Table 13], [Table 14], [Table 15], [Table 16], [Table 17] and [Figure 3].
Table 13: X-ray diffraction peak report of raw Kukkutanda Tvak

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Table 14: X-ray diffraction report of Kukkutanda Tvak Bhasma-A

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Table 15: X-ray diffraction peak report of Kukkutanda Tvak Bhasma-A

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Table 16: X-ray diffraction report of Kukkutanda Tvak Bhasma-B

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Table 17: X-ray diffraction peak report of Kukkutanda Tvak Bhasma-B

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Figure 3: X-ray diffraction image of Kukkutanda Tvak Bhasma. (a) Raw Kukkutanda Tvak, (b) Kukkutanda Tvak Bhasma A, (c) Kukkutanda Tvak Bhasma B

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   Discussion Top


KTB is one of the important calcium-rich medicines mentioned under Sudha Varga/Shukla Varga in different classics of Ayurveda.[4] It is used to treat ailments such as Rakta Pradara (menorrhagia), Shweta Pradara (leuchorrhea), Shwasa (bronchial asthma), Kasa (cough), Napunsakata (impontancy) and Prameha (diabetes).[9],[10],[11] For preparation of KTB, it is necessary to purify KT as it contain impurities such as excreta and tissues of creatures, which are of heterogeneous nature. Purification of KT was carried out by applying bioling process in salt water. It was prepared in the ratio of 1:4 of rock salt and portable water.[13],[14] Average 10 l salt water was required to complete purification process of 2 kg KT. Average 629 g, i.e., 31.45%, weight loss was observed during the purification process of KT [Table 2]; it may be due to removal of impurities and inner side albumin layer of eggshell. The average pH of saltwater before and after purification was 7.36 and 8.25 respectively. The calcium component present in KT causes its basic nature; hence, during steam heating process due to dissociation of solid contents of the KT in the media, the alkalinity may be increased. It is also evident by observing the increase in total solid content of the media after purification. Total solid contents of purification media increased from 6.30 to 6.90; after purification, it may be due to addition of protein matter from KT besides simple concentration due to heating [Table 1].

Incineration of KT was carried out as per the reference of Bhasma Vigyaneeyam.[11] For incineration process, conventional heating method of EMF was used due to convenience in temperature regulation and recording. For both incineration methods, the first incineration process was given Puta without levigation at 800°C, which was maintained for 15 min. From the second incineration, Kumari Swarasa (KTB-A) and processed Hingula (KTB-B) were used as media for levigation and the same temperature was given as that of first Puta. For KTB-A and KTB-B, minimum 4 and 8 incineration cycles (Puta) were required to obtain desired quality of Bhasma, respectively [Figure 4]. An average weight loss was found 183 g, i.e., 36.6%, and 118.4 g, i.e., 23.68%, during preparation of KTB-A and KTB-B, respectively [Table 3]. This loss may be due to evaporation of water and burning of some organic or organo-inorganic materials. Both the samples were smooth, tasteless and lusterless without any specific odor and produced no perceptible sound during chewing, indicating that they passed all the classical tests of Bhasma Parisksha.[16]Varitara Pariksha (floating over water surface) was negative in KTB due to hygroscopic nature of the KTB.
Figure 4: Kukkutanda Tvak Bhasma. (a) Kukkutanda Tvak Bhasma A, (b) Kukkutanda Tvak Bhasma B

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The physicochemical parameters of all the batches of KTB revealed that pH of KTB-A and KTB-B were 10.5 and 10 respectively [Table 4]. It is evident from previous research work that KTB is in CaCO3, CaO, or CaPO4 form.[23] Hence, calcium compounds are inherently basic in nature. The ash values were found 74.34% and 96.41% in samples KTB-A and KTB-B respectively [Table 4]. It is suggestive of remnant unburnt and evaporative substances in the prepared samples. These tests revealed that among two samples, KTB-B has least unburnt and evaporative constituents.[Table 4] Juice of Kumari was used as a medium for preparing KTB-A, whereas in other sample KTB-B cinnabar was used as a medium during incineration; hence, inorganic compound of cinnabar might have increased the ash value of sample KTB-B as compared to KTB-A. An average loss on drying observed in KTB-A and KTB-B was 1.54 and 1.29 respectively, which indicates the presence of least amount of moisture content. The acid-insoluble ash value was carried out to evaluate the percentage of insoluble inorganic content of KTB in dilute acid, which provides a step toward the bioavailability of the Bhasma. The acid-insoluble ash of KTB-A (0.67%) was higher in comparison to KTB-B (0.37%), but both Bhasma are highly soluble in acidic media suggestive of good bio-availability. It can be inferred that bioavailability of some constituents of KTB-A might be comparatively less than KTB-B. Water-soluble extractives were 7.51% in KTB-A and 7.38% in KTB-B and alcohol-soluble extractives were 0.004% in KTB-A and 1.44% in KTB-B [Table 4]. Less alcohol-soluble extractive indicates less extraction of polar organic matter. In acid-neutralizing capacity, there was no significant change in pH of KTB-A, whereas pH of KTB-B was comparatively less alkeline [Table 4]. It may indicate the KTB prepared without cinnabar (KTB-A) has faster and better acid-neutralizing capacity [Table 5] and [Table 6]. It was observed that the particle size of KTB-B is less than KTB-A [Table 7]. Small particle size enhances the absorption;[17] hence, the bioavailability and potency of the drug increases resulting in to decrease in its therapeutic dose. This eventually leads to decrease in dose related side effects of the drug. Normal particle size distribution is found in KTB-A, while it shows minor skewness at the particle size of 3–4 μm in KTB-B [Table 7]. SEM analysis showed the presence of calcium, magnesium and sulfur in both the samples of KTB [Table 9] and [Table 10]. Various other elements such as Cl and Fe might be due to juice of Kumari used for levigation. It is evident from previous research work that raw KT contains Mg and S in trace amounts, which were found in the analysis of end product. FTIR study revealed the presence of large number of functional groups such as OH, Ca, sulfate and CO2 in both the samples of KTB, which might be because of herbs used during preparation. In KTB-B, methyl and cyanide groups were observed, which may be due to the use of cinnabar [Table 11] and [Table 12]. In XRD study, KT was found contain calcite (CCaCO3) form whereas both the samples of KTB were found to have Ca(OH)2 form [Table 13] to [Table 14], [Table 15], [Table 16], [Table 17] and [Figure 3].


   Conclusions Top


For KTB preparation, minimum four incineration cycles are required using EMF with an average 800°C temperature without and with cinnabar as a medium. The average particle size of KTB are 9.35 μm (KTB-A) and 9.97 μm (KTB-B). XRD reveals that raw KT is rhombohedral in nature with CCaCO3 (calcite) and CaCO3 form, whereas both the Bhasma contain hexagonal CaH2O2 (portlandite syn) and Ca(OH)2 form. SEM, FTIR, and XRD reports suggest that both samples are crystalline in nature with mixture of different organic and inorganic compounds.

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Conflicts of interest

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   References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12], [Table 13], [Table 14], [Table 15], [Table 16], [Table 17]



 

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