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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 7  |  Issue : 1  |  Page : 62-74

Comparative physicochemical analysis of Jalakumbhikshara prepared with water and cow urine


1 Ayurzon Most Morden Wellness Centre, Vadodara, India
2 Department of Rasa Shastra and Bhaishajya Kalpana Including Drug Research, Jamnagar, Gujarat, India
3 Department of Panchakarma, ITRA, Jamnagar, Gujarat, India

Date of Submission31-Jul-2021
Date of Decision07-Feb-2022
Date of Acceptance09-Feb-2022
Date of Web Publication07-Jul-2022

Correspondence Address:
Dr. Amisha P Patel
Panchakarma Consultant, Ayurzon Most Morden Wellness Centre, Vadodara 390012, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdras.JDRAS_37_21

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  Abstract 

INTRODUCTION: Kshara preparation is one of the prominent and widely practiced dosage forms of Ayurveda specifically indicated for the management of Kaphaja Galaganda (goiter). Varied descriptions regarding method of preparation and type of Kshara are mentioned in classics. Sushruta Samhita mentions the use of either water or cow urine as the solvent. No pharmaceutico-analytical study has been documented on Jalakumbhikshara till date. The aim of this study was to prepare Jalakumbhikshara with water (JKW) and cow’s urine (JKC) and to evaluate their analytical profile by physicochemical analysis, chromatographic pattern, and spectral method (Fourier transform infrared spectroscopy). MATERIALS AND METHODS: JKW and JKC were prepared by addition of six times of media (water or cow urine) to the ash of dried whole plant of Jalakumbhi followed by decantation and heating to dryness, which in turn is followed by analysis through physicochemical, sensory, spectral, and chromatographic methods. RESULTS: JKW and JKC were differentiable on the basis of color, taste, and smell. Physicochemical analysis of JKW and JKC reveals significant difference in ash value and water-soluble ash, whereas it shows insignificant difference in water-soluble extractives and acid-insoluble ash, respectively. Jalakumbhi Kshara prepared by water and cow urine predominantly contains 44.31%, 21.3% and 43.3%, 19.41% of potassium and chloride, respectively, with trace elements such as Na, Br, I, Cu, Mg, Ph, and Fe. Both the types of Kshara contain organic moieties, with comparatively 15.21% more organic content in Jalakumbhi Kshara prepared with cow’s urine. CONCLUSION: Change of extraction media for preparation of Jalakumbhi Kshara from water to cow urine alters organoleptic and physicochemical attributes of Kshara. Jalakumbhi Kshara prepared with water and cow’s urine has predominance of K and Cl ions. Cow’s urine is a better medium for extraction of inorganic alkaline earth elements in the preparation of Jalakumbhi Kshara than that of water.

Keywords: Cow’s urine, Jalakumbhikshara, physicochemical analysis, water


How to cite this article:
Patel AP, Bedarkar P, Thakar AB. Comparative physicochemical analysis of Jalakumbhikshara prepared with water and cow urine. J Drug Res Ayurvedic Sci 2022;7:62-74

How to cite this URL:
Patel AP, Bedarkar P, Thakar AB. Comparative physicochemical analysis of Jalakumbhikshara prepared with water and cow urine. J Drug Res Ayurvedic Sci [serial online] 2022 [cited 2022 Sep 27];7:62-74. Available from: http://www.jdrasccras.com/text.asp?2022/7/1/62/350052




  Introduction Top


Kshara Kalpana is one such formulation, in which the alkali present in the ash of the selected plants is extracted[1] and modified into formulation with specified desirable pharmacological attributes and physicochemical composition. As per Acharya Vagbhata, Kshara acts by extracting all the toxins from the site and cures the disease with negligible chance of recurrence.[2]Kshara is obtained by soaking the ash of the plant in suitable liquid medium; as a rule water is taken as liquid media for extraction but not exclusively. But for preparation of Jalakumbhi Kshara, cow’s urine is taken as liquid media for the process of Nimajjana, Ksharapaka (Kwathana), etc. As per Sushruta Samhita, water as well as cow’s urine may be used as the liquid media to extract Kshara.[3] Chemical constituents of cow’s urine are significantly different from plain water; as it is used for extraction in place of water, it may not just lead to addition of its constituents but may also influence prepared Kshara by changing the extent of extraction of solubles (qualitatively as well as quantitatively) and may form newer chemical constituents as a result of interaction of constituents of plant ash and cow’s urine. Ayurveda has described various evaluative procedures to judge the quality of the pharmaceutical products, which are based on physical qualities of the final product. With advent of the recent advancements in science, there evolved many procedures and tests which could help in proper judgment of the chemical composition (qualitative, quantitative), properties, and quality of the Ayurvedic drugs. These tests express the quality of the drug quantitatively and hence it makes feasible even for a layman to get an idea of the quality, purity, and strength of the formulation. As pharmaceutical yield (derived quantity) of Kshara in solid state as that of plant is very less when compared with other Panchavidha Kashaya Kalpana, and their Upakalpana from the same plant, it needs more time, economy, and labor for its production; there may be chances of its adulteration in view of saving time, economy, and labor. As Kshara owing to its peculiar pharmacological properties such as Tikshnatva (acrid), Ksharana (chemical cauterization), Dahana, Darana, Bhedana (piercing), Vilayana etc. is used as alternative of Shastra karma in susceptible cases in which surgery has relative risks in pediatric and elderly patients and pregnant ladies, diseases with surgical indication involving Marma (vital body structures) have major clinical adverse outcomes if they suffer damage during surgical management. Hence, their potency is of critical clinical significance. All of these factors necessitate to have developed parameters of standards of Kshara, as no comparative pharmaceutical-analytical study has been documented on Jalakumbhikshara till date. Two samples, i.e., Jalakumbhikshara prepared with water (JKW) and Jalakumbhikshara prepared with cow’s urine (JKC), were evaluated in terms of organoleptic characters including sensory evaluation by volunteers, preliminary pharmacopeial physicochemical parameters, quantitative inorganic elemental composition, chromatographic study, and qualitative functional group analysis, and the results were recorded and discussed.

Aim

The present study was aimed to prepare JKW and JKC and to evaluate their analytical profile by physicochemical analysis, sensory evaluation, chromatographic pattern, and Fourier transform infrared spectroscopy (FTIR).


  Materials and Methods Top


Collection of raw drugs

Jalakumbhi [Eichhornia crassipes (MART) Solms] required for the study was collected from various places such as Vadodara, Anand, Junagadh, and Mahisagar in fresh water ponds, pools, tanks, lakes, reservoirs, swamps, streams, rivers, irrigation channels, and paddy fields. The drug was collected during the period from November and January, when the plants attain maturity [Figure 1].
Figure 1: Wet drug (Jalakumbhi Panchanga)

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(1) Pharmaceutical preparation of two samples

The pharmaceutical process was carried out at the pharmaceutical unit of PG Institute of Ayurveda, as per the method mentioned in Sushruta Samhita.[3] Drug was checked for impurity, insects, eaten parts, and other extraneous materials. Two kilograms of selected Jalakumbhi from each batch was washed with fresh potable water. It was cut into small pieces and then kept in shade till it became completely dried [Figure 2]. It took an average of 15 days for completion of drying. The weight of the Jalakumbhi was found to be 200 g after drying. It was then burnt completely, and 40 g of grayish white-colored ash was obtained [Figure 3] and [Figure 4]. It was divided into two equal parts to prepare JKW and JKC samples. The ash was then soaked in six times of fresh potable water, macerated, and stirred well. Then it is allowed to stand still (undisturbed) overnight till settlement of suspending particles, and clear liquid was obtained, i.e., for an average of 1 h and filtration was carried out for 21 times through a three-folded cotton cloth (muslin cloth). The filtrate thus obtained was subjected to heating till the Kshara was obtained in the form of flakes adhering to the base of the vessel [Figure 5][Figure 6][Figure 7]. At the end, mild heating was carried out with continuous stirring so as to avoid overheating, sticking to the base of the vessel. Similarly, the JKC sample was prepared by soaking the ash in fresh cows urine. The process was repeated in four batches. The obtained moistened solid powdered Kshara averaging 14.8 and 11.2 g from JKW and JKC, respectively, was heated in an oven at 60°C till dryness (constant weight) to get JKW [Figure 8] and JKC [Figure 9], which were stored in airtight containers.
Figure 2: Dry drug (Jalakumbhi)

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Figure 3: Drug burnt completely

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Figure 4: Grayish white-colored ash soaked in water

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Figure 5: Appearance of clear liquid (overnight settlement)

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Figure 6: Heating Kshara Jala to concentrate

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Figure 7: Heating till the Kshara was obtained in the form of flakes

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Figure 8: Whitish Kshara obtained from water (JKW)

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Figure 9: Grayish Kshara obtained from cow’s urine (JKC)

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(2) Physicochemical study

The physicochemical analysis of two samples was carried out, and organoleptic characteristics such as color, odor, taste, and consistency were recorded along with the evaluation of the parameters such as loss on drying, total ash, water-soluble ash, acid-insoluble ash, pH, water-soluble extractive value, and alcohol-soluble extractive value by following the standard procedures.[4]

Also, the ICP AES analysis was carried out at Sophisticated Analytical Instrument Facility, Indian Institute of Technology, Bombay [Table 6] and FTIR spectroscopy at the Department of Pharmaceutical Sciences, Saurashtra University, Rajkot [Figure 7]. Quantitative inorganic elemental analysis of samples was done in aqueous solution, and powdered samples were analyzed for infrared spectroscopic evaluation for identification of constituent functional groups. The chromatographic pattern of JKC and JKW through high-performance thin-layer chromatography (HPTLC) was evaluated at the Pharmaceutical Chemistry Laboratory, ITRA, Gujarat Ayurveda University, Jamnagar, Gujarat, India, with application mode—Camag Linomat V, development chamber—Camag twin trough chamber, plates—precoated silica gel GF 254 plates, chamber saturation—30 min, development time—30 min, development distance—7 cm, scanner—Camag Scanner 3, detection—deuterium lamp, tungsten lamp, data system—winCATS software.

Differentiation of samples by sensory evaluation was carried out by a triangle test method[5],[6],[7] to evaluate detection of difference among the samples. A total of 30 healthy assessors (discriminators) without color blindness and anosmia were served four sets of samples, taking care of non-communication among them. Each set contained a total of three coded samples, two samples of one formulation, and one sample of another formulation. One sample among two samples of one formulation was mentioned as reference sample, and assessors were asked to identify odd (different, non-matching) sample on the basis of color, taste, and smell.


  Observations and Results Top


Significant precipitation, excessive smell of Gomutra, more and early darkening of color of Kshara Jala were observed during heating (Pachana) of Ksharajala in JKC batches. The weight of Jalakumbhi was found to be 200 g after drying (10% as that of fresh drug). It was then burnt completely and 40 g (20%) of grayish white-colored ash was obtained (as that of dried drug). Results of organoleptic characters of JKW and JKC expressed differentiability in odour [Table 1] which further upon subjecting to sensory evaluation through volunteers by triangle test exhibited statistically significant differentiation among organoleptic parameters [Table 2]. Both the samples of Kshara; JKC and JKW exhibited differences in physicochemical attributes [Table 3]. Physicochemical analysis of media cow urine and intermediate product, ash of Jalakumbhi are mentioned in [Table 4] and [Table 5], respectively . Physicochemical analysis of JKW and JKC reveals significant difference in ash value and water-soluble ash, i.e., 95.21% and 80%, pH 8 and 11, whereas insignificant difference was found in water-soluble extractives (99.98% and 99.26%), loss on drying 2.06% and 2.68%, and acid-insoluble ash, i.e., 0.40% and 0.20%, respectively. Analytical elemental analysis of all evaluated inorganic elements and quantity-wise major alkaline earth metals by ICP AES reveals insignificant difference, i.e., 44.31% and 43.37% of K, 21.3% and 19.41% of Cl in JKW and JKC, respectively [Table 6]. The HPTLC analysis of the formulation reveals comparatively more number of peaks in JKC than in JKW [Graph 1] three and five peaks in JKC at 254 and 366 nm, respectively. Peaks in JKW obtained at either wavelength where the total area under the curve (AUC) of separated contents was comparatively significantly more in JKC (15150.8, 5261.6) than that in JKW (7091.9, 4045.3) [Table 7]. Comparative FTIR study of JKC and JKW revealed different infrared spectra of JKC and JKW [Graph 2][Graph 3][Graph 4] different infrared spectra [Table 7] with comparatively more number of peaks, i.e., 26 in JKC than merely 21in JKW [Table 8]. Spectral region wise comparative distribution of observed peaks in FTIR study is mentioned in [Table 9] whereas assignment of peaks of JKW and JKC in [Table 10] and [Table 11], respectively. Similarity and dissimilarity of observed peaks of FTIR analysis of JKW and JKC assigned to different functional groups are placed in [Table 12].
Table 1: Organoleptic characters of the two samples

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Table 2: Sensory evaluation for differentiation of JKW and JKC

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Table 3: Results of physicochemical analysis of the two samples

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Table 4: Results of physicochemical analysis of cow urine

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Table 5: Results of physicochemical analysis of ash of JALAKUMBHI (JALAKUMBHI BHASMA)

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Graph 1: Densitogram and 3D graph of Jalakumbhi Kshara JKC and JKW at 254 and 366 nm from top to down respectively

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Table 6: Quantitative elemental analysis of various ions presents in the KSHARA by ICP AES

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Table 7: High-performance thin-layer chromatography (HPTLC) of JKW and JKC-(sample prepared method of methanolic extract and mobile phase-toluene and ethyl acetate 9:1)

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Graph 2: FTIR report of Jalakumbhi Kshara prepared by cow’s urine—JKC
Range: 4000-650 System; Apodization: Happ-Genze


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Graph 3: Comparative FTIR pattern of Jalakumbhi Kshara prepared by water—JKW (above) and JKC (below)
Range: 4000-650 System; Apodization: Happ-Genze


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Graph 4: FTIR report of Jalakumbhi Kshara prepared by water—JKW
Range: 4000-650 System; Apodization: Happ-Genze


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Table 8: FTIR peaks obtained in JKC & JKW

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Table 9: FTIR peaks of sample of JKC & JKW obtained in different regions

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Table 10: FTIR analysis of JKW

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Table 11: FTIR analysis of JKC

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Table 12: FTIR analysis of JKW and JKC

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  Discussion Top


Cow’s urine is judiciously and widely used in Ayurvedic pharmaceutics and therapeutics. It is used as solvent for extraction, liquid media for several pharmaceutical operations required in Ayurvedic formulations such as Bhavana, Shodhana, Marana, preparation of Kwatha, etc., and is used in therapeutics in the form of main treatment, as an adjuvant (Anupana). The average quantity of JKW and JKC samples obtained was 10.1 g (50.5%) and 13.1 g (65.5%) for ash and average quantity was 10.1% and 13.1% for dried drug, respectively. The higher quantity of JKC sample suggests that the solids present in Gomutra have contributed to increased yield of Kshara in JKC along with the Kshara present in the ash of the plant and 15% more yield of JKC when compared with JKW, which is in good agreement with total solid content (TSC) of cow’s urine, i.e., 5% in the present study. The color of JKW sample was whitish, whereas JKC was brownish in color which is probably due to the presence of the pigments present in the cow’s urine.[3] The taste of both the samples was acrid, but JKC was relatively acrider. The relatively acrid taste of the JKC was probably due to the presence of additional alkali compounds and salts presents in cow’s urine (nitrogenous compounds, uric acid, etc.), which were also present in the sample. The JKW sample had a peculiar smell of an alkali, whereas the JKC sample still retained the smell of cows urine, indicating the presence of the components of cows urine present in it. Both the samples were obtained in the form of thin flakes and coarse powder. The organoleptic characters of the two samples are shown in [Table 1].

Loss on drying is an important parameter to be assessed for the Kshara. It is hygroscopic in nature due to the presence of alkaline compounds and hence the loss on drying values denote the limit to which the sample has absorbed moisture.

Ayurvedic Pharmacopeia of India has mentioned a wide acceptable range of ash values of dried Panchanga of Jalakumbhi, i.e., <52%,[8] which supports the presence of significant quantity of Kshara in plants and thus justifies its dosage form Kshara. The percent yield of Jalakumbhikshara from plant ash when compared with that of ash of dried Panchanga in the present study (i.e., avg. 50.5% and 65.5% in JKW and JKC, respectively) is also comparatively more. The percent yield of Kshara from ash of dried Panchanga of other plants of Kshara Varga is also shown.[9] More ash value of dried plant of Jalakumbhi when compared with that of other plants of Kshara Varga Dravya supports this observation. The ash values denote the amount of inorganic and silacious materials present in the given sample.[10] Total ash values for JKW and JKC were found to be 95.21% and 80%, respectively. There is 15.21% less ash value in JKC sample, compared with that of JKW (in spite of merely 0.62% more loss on drying in JKC). Thus around 15% more ash value in JKW as that of JKC may suggest more organic content in it, which may be a result of the TSC of cow’s urine and more extraction (specifically organic matter) from ash. Addition of contents of cow’s urine is supported by the presence of comparatively more numbers of peaks in organic functional groups in JKC, i.e., 26 peaks compared with 21 in JKW.

As discussed, comparatively less ash value (by 15.21%) in JKC than in JKW is suggestive of more organic content in JKC than in JKW. If both media have similar extraction pattern, inorganic elemental composition in JKC should have been significantly less than that in JKW, but there is insignificant difference in all inorganic elemental compositions and principle alkaline earth elements such as potassium and chlorine between both the samples of Kshara. This is suggestive of better extraction of these inorganic elements by using cow’s urine as media for extraction in JKC than that of water media in JKW. The possibility for the presence of almost similar potassium content in JKC and JKW, i.e., 43.37% and 44.31%, respectively (in spite of addition of TSC of cow’s urine to JKC), may be due to high K content of cow’s urine.[11]

In spite of addition of TSC of cow’s urine to JKC, almost similar chlorine content in JKC and JKW (19.41% and 21.3%, respectively) cannot be justified by the mere addition in view of insignificant chlorine content of cow’s urine.[12],[13],[14] Thus, it suggests comparatively better extraction of these elements (K and Cl) by cow’s urine in JKC than that of water.

The values obtained for acid-insoluble ash for JKW and JKC were 0.40% and 0.20%, respectively, which are suggestive of negligible insoluble substances such as silica, indicating sample free of dust, clay, silacious matter, etc. Fairly water-soluble substances and drugs of alkaline nature should be soluble in acidic media, but findings of the present study may suggest formation of insoluble complexes in traces upon heat treatment with dilute acid. The water-soluble ash values for JKW and JKC were 95.21% and 80%, respectively, which suggest high water solubility of either Kshara at room temperature.

pH depicts the relative acidity or alkalinity of any sample. The pH of both the samples JKC and JKW was found to be 8 and 11, respectively, which clearly intimates about the alkaline nature of the Kshara. Comparatively less alkalinity of JKC may be due to contents in cow’s urine such as carbonic acids, sulfur, uric acid, amino acids, generation of formaldehyde, sulfinol, ketones, etc. or due to the formation of complexes of contents of ash with them. Comparatively more alkaline nature of JKW may be claimed to have more Ksharana Karma, but other physicochemical parameters such as total acid neutralizing capacity, conductivity (potential difference), etc. and/or biological parameters such as Dagdha lakshana[15] (after application of Kshara), irritation, and corrosiveness may determine it in a better manner.

Water-soluble extractive values for JKW and JKC were 99.98% and 99.26%, respectively. The results obtained from the test show that both the samples consisted of the constituents which are soluble in water. This is in perfect agreement with the classics where it is been told that Kshara is extracted from the ash of the plant after soaking it in water.

The results of physicochemical analysis are tabulated in [Table 3].

Studies have advocated for repeated washing of the ash as Kshara can be obtained after subsequent washing also. The quantity of Kshara obtained will be less when compared with the first wash; to extract the whole of Kshara, repeated washing may prove to be beneficial.[16]Jalakumbhikshara has also proven utility for the treatment of Amlapitta[17] and goiter.[18] The authors discovered that the molecular basis for this is that potassium channel, KCNQ1-KCNE2, is expressed in the basolateral membrane of thyrocytes and is required for the normal production of thyroid hormone by the thyroid gland. KCNQ1-KCNE2 is required for efficient function of basolateral sodium iodide symporter (NIS) in this fashion and thus for efficient I uptake. It seems that NIS can function adequately without KCNQ1-KCNE2 in, pups of Kcne2−/− dams that are severely affected, exhibiting early cardiac hypertrophy, growth retardation, and alopecia, largely because the dams hypothyroidism impairs milk ejection. The presence of significant quantity of K in Kshara prepared by either methods and iodine in JKW may suggest action of organo-inorganic complexes of potassium, iodides at these channels, and at basolateral membrane of thyrocytes (target organ, site, receptor delivery of active pharmaceuticals), assisting sequestration of iodide in the thyroid gland, using the sodium gradient to facilitate influx of iodide ions (I). The FTIR analysis reveals the presence of organic compounds even in JKW, suggesting persistence of organic matter which, although very negligible, is evident from very high ash value (95.21%) as they are probably in the form of their salts with observed ions of alkaline earth metals and elements such as potassium, chloride, sodium, bromide, etc. It suggests the presence of organic moieties with common functional groups such as carboxylic acids and derivatives, alcohols, phenols, amines, amides, phosphines, alkanes, and alkynes. More number of peaks in FTIR analysis of JKC may be due to diverse organic constituents of cow urine, i.e., carbolic acid, phenolic acids, amino acids, allantoin, hormones, enzymes, salts, etc.[19],[20]

The salts of alkaline earth metals (NaCl, KCl, CsCl, MgCl2, NaBr, KBr, NaI, KI), which are completely ionized in water, do not absorb in the IR and only their interactions with water are observed and analyzed; still, the FTIR peak in JKW revealed phosphine bonds. Their analysis poses challenge due to interference by water (shift of spectra, masking, etc.), owing to their hygroscopicity as well as their hydroxide forms. Thus, the broader peaks in the range of 3200–3550 in either samples, which are designated to the OH group, are probably due to the surface water present predominantly on KCl or hydrated KOH, which is supported by the hygroscopic nature of either sample of Kshara reflected by 2.06% and 2.68% of loss on drying of samples of JKW and JKC, respectively.

Comparison of sensory analysis of samples by healthy volunteers suggests that both the samples of Kshara JKW and JKC are differentiable on the basis of color, taste, and smell with 0.1% significance (one-tailed test)[21],[22],[23],[24],[25] [Table 2].

Stoichiometric analysis of results of elemental analysis by ICP AES of both the Kshara suggests the presence of potassium chloride[26] or potassium chlorate as predominant salts with utilization of 34.04% or 21.4% of K, and the remaining 10.27% or 21.97% of K ions might be in the form of other salts and complex compounds with probably hydroxides of K. This is supported by significantly high ash values, free solubility in water and alcohol, and non-melting upon heating in the process of ash value along with organoleptic attributes (saline, alkaline taste, odorless, soapy nature, hygroscopicity, etc.). However, the possibility of formation of traces of potassium bromide, potassium iodide in JKW, and chlorides like NaCl, CaCl2, FeCl3, oxides like CaO, CuO, Cu2O, MgO, MnO2, Mn2O3, traces of hydroxides NaOH, KOH, CaOH in either Kshara and formation of ferric ammonium chloride and boron trinitride in JKC Kshara cannot be denied. Owing to their possible negligible concentrations, their peaks might not be differentiable in the FTIR analysis. General properties of Kshara such as Ksharana (cauterization), Tikshnatva, Krimighna (antimicrobial property), Shodhana, Darana, and Bhedana might be justified by the above-mentioned proposed chemical constitution of either Kshara.[27] Observation of peaks in either samples of Jalakumbhikshara probably suggests the presence of organic content in Kshara and/or formation of salts of organic moieties. Comparatively, a greater number of peaks in FTIR as well as chromatographic study (eight peaks against merely four in JKW), along with comparatively more total AUCs of separated contents in JKC (20,412) than that of JKW (11,137.2), suggest more organic content, as cited earlier, or salts or compounds of organic moieties, predominantly potassium and chlorine in JKC than that in JKW.


  Conclusion Top


Jalakumbhikshara prepared by extraction with water and cow’s urine has 10.1% and 13.1% yield as that of dry drug and possesses differential color, taste, and smell and chromatographic pattern. They contain predominantly potassium and chloride ions in the concentration of 44.31%, 21.3% and 43.37%, 19.41%, respectively, with probable predominance of KCl salt along with trace elements such as Na, Br, I, Cu, Mg, Ph, and Fe. Both the Jalakumbhikshara contain more numbers of organic moieties with comparatively more organic mass in Jalakumbhikshara prepared with cow’s urine (15.21%) than that with water.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.





 
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    Figures

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    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]



 

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