1. INTRODUCTION:

In present era, incorporation of modernization in daily life suppressing of the natural urges by human being is observed on large scales which subsequently act as a leading precursor to illness. According to Ayurveda the three sutras are there for compounding any pharmaceutical dosage form i.e. ‘Hetu Sutra’ (precursors and factors for the formation of the disease), ‘Linga Sutra’ (symptoms and risk factors of illness) and ‘Aushadha Gyana Sutra’, (know how, when, where and what kind of drug and its combination treat disease).

Aushadha Gyana Sutra’ Third one is more important due to the fact the success of remedy (Chikitsa) depends on medicine (Aushadha) without which the physician is helpless to deal with the patients and that’s why medication (Aushadha) occupies a crucial role in Chikitsa Chatushpada^1-3.

Urolithiasis (Mutrakrishra) is one of the oldest causes of distress in world. A large number of humans are suffering from urolithiasis (Mutrakrishra)⁴. Kidney stone is a deposition of hard mass that forms in the kidney, consisting mainly of phosphates, oxalates, and urates salts. The process of crystallization expanded slowly over a number of months to years^5,6. The size might be differing in case of men or women. Kidney stone removal through a ureter or the urethra might be trouble-free or might be associated with severe pain depending upon size of the stone⁶. A medical management of kidney stone recommended if the stones cannot pass on their own. If untreated, it might cause substantial kidney damage and moreover in extreme cases severe renal impairment or failure occurs. Early incidence recognition is always advantageous in the management of kidney stone^7,8.

Urolithiasis (Mutrakrishra) prevalence relies upon geographical, climatic, ethnic, nutritional dietary and genetic elements^1-3. The chances of recurrence threats are also there. It is a global problem afflicting human for diverse spanning all geographic areas with an envisioned annual incidence of 1% and lifetime threat of 15-25%. Once afflicted, urolithiasis (Mutrakrishra) tends to be recurrent in the majority of cases. Recurrence chances after the first stone episode are 14% (after 1 year), 35% (after 5 years) and 52% (after 10 years). Vedas, Puraņas and Samhita discussed about anatomy, pathophysiology of urolithiasis (Mutrakrishra) with precautionary measures and remedies for the patients^9,10. Charak Samhita described the anatomy, physiology, pathology, prognosis and remedies of urolithiasis (Mutrakrishra) in scattered manner however in Aşhmari Nidana (third chapter) of Sushruta Samhita presents a scientific explanation about types, characteristics, etiology and symptoms of urolithiasis (Mutrakrishra), whereas the treatment of urolithiasis (Mutrakrishra) is described at seventh chapter of Aşhmari Chikitsita Sthana^2,11,12.

Apamarg kshar is an off-white alkaline preparation derived from the water-soluble ash of Apamarg Panchanga (Achyranthes aspera Linn.) and claimed for the treatment of many pathophysiological conditions i.e. Gulma (Abdominal lump); Udarasula (Abdomen pain); Grahani (Malfunctioning of duodenum or small intestine); Visuchka (Gastro-enteritis with piercing pain); Alasaka (Intestinal atony); Ajirna (Dyspepsia); Aruchi (Tastlessness); Anaha (Distention of abdomen due to obstruction to passage of urine and stool); Arsha and Bhagandara (Piles and fistula); Sarkara (Gravel in urine); Asmari (Calculus); Krmi (Helminthiasis); Antarvidradhi (Hernia); Swasa (Asthma); Karnaroga (Ear disorder) etc. Different techniques for the preparation of Kshar differs by the ratio of water added to prepared ash, soaking time, folds of cloth and number of times of filtration described by various authors from time to time^13-16. In the present study Apamarg kshar was prepared as per method prescribed in Rasa Tarangini¹⁶ and compressed into tablet dosage from. The standardisation of the prepared tablets was done by employing tests like organoleptic, physicochemical, powder flow properties. The evaluation parameters for prepared tablet i.e. Shape, Appearance, Thickness, diameter, Weight variation, Hardness, Friability, Disintegration, Dissolution, Infrared spectroscopy, Microbial load was performed and antiurilthatic activity of tablet was evaluated by in-vitro method.

Description of Apamarg Panchang in Ayurvdic text:

Various opinion regarding gana, therapeutic uses, synonyms and variety mentioned by ancient scholar details of the same is tabulated in the Table 1.

Table 1: Description of Apamarg Panchang in Ayurvdic text Ayurvedic literature

Sr. No.	Text Name	*Gana* (group)	Synonyms	Variety	Therapeutic Uses
1	Charka Samhita ^[2]	Krimighna, Vamanopaga, Sirovirecanopaga	Apamarga, Pratyakpuspi, Mayuraka, Sikhari	Shweta and Rakta	Nasya,Shirovirechana, Bhasmakaroga
2	Sushrut Samhita ^[3]	Varunadigana, Viratarvadigana, Arkadigana	Kharamanjari, Pratyakpuspi, Mayuraka, Vasir	Shweta and Rakta	Bhasmakaroga,
3	Bhavprakash Nighantu ^[5]	Guduchyadi varga	Shikhari, Adhahshalya, Mayurak, Markati, Durgraha, Kinihi, Kharmanjari	Shweta and Rakta	Bhasmakaroga, Dipan, Pachan, Pittavirechak, Vamak, Mutrajanan, Kaphaghna,Vishaghna, Kramighna, Amltanashak
4	Astanga Sangraha ^[17]	Sodhanadigana as Sirovirecana dravya, Tikta Skanda drugs	Kinihi, Kharamanjari, Nandi, Mayuraka, Sikhari, Vasir, Pratyakpuspi	Shweta and Rakta	Ashmari
5	Shodhala Nighantu ^[18]	Guduchyadi varga	Shikhari, Pratyakpuspi, Mayurak, Adhahshalya, Kinihi, Kanti, Markatpippali, Kshav, Adhomarkava, Ghanta, Markati, Durabhigraha, Pratyakshreni, Vashir, Katu, Kharmanjari	Shweta and Rakta	-
6	Shaligrama Nighantu ^[19]	Guduchyadi varga	Shaikharik, Dhamrgava, Mayurak, Pratyakaparni, Kishparni, Kharmanjari	Shweta and Rakta	Vishamajwara, Shiroruj
7	Dhanvantari Nighantu ^[20]	Guduchyadi varga	Apamarga, Shikhari, Pratyakpuspi, Mayurak, Adhahshalya, Kinihi, Durgraha, Kharmanjari, Shaikharik, Markati, Durabhigraha, Parakpuspi, Vashir, Kanti, Markatpippali.	Shweta and Rakta	Bhasmakaroga, Kandu, Udararoga, Raktatisara
8	Madanpal Nighantu ^[21]	Abhayadi varga	Shikhari, Kinihi, Kharmanjari, Adhahshalya, Shaikharik, Pratyakpuspi, Mayurak	Shweta and Rakta	Bhasmakaroga, Kandu, Udararoga, Kushta
9	Aadarsh Nighantu ^[22]	Apamargadi varga	-	Sveta, Rakta, Krishna	-
10	Kaiyadeva Nighantu ^[23]	Oshadhi varga	Shaikharik, Shikhari, Kharmanjari, Adhahshalya, Ksharmadhya, Durgraha, Durabhigraha, Aaghat, Kinihi, Marga, Mayurak, Pratyakapuspi	Apamarga. Vashira. Ramatha (Jalapamarga)	Udararoga, Kandu, Dadru, Apach, Arshas, Shoola
11	Raja Nighant/ Nighsntu Raja or Abhidhana Cudamani ^[24]	Shatahwaadi varga	Shikhari,Kinihi,Kharmanjari, Durgraha, Adhahshalya, Pratyakpuspi, Mayurak, Kaandkant,Shaikharik, Kubja, Markati, Durabhigraha, Vashir, Parakpuspi, Kanti, Markatpippali, Katu, Manjari, Nandi, Kshavak, Panktikantaka, Malakant	Apamarga, Raktapamarga, Kshudrapamarga	Bhasmakaroga, Ashmari, Kandu,Udararoga, Adhmana, Raktatisara, Kasa, Shwasa, Pandu, Pradara, Jwara, Krimi, Karnanaada
12	Hridayadipaka Nighanṭu ^[25]	Tripaad varga	Pratyakapuspi, Mayurak, Marga, Aaghat, Shikhari, Kharmanjari	Shweta and Rakta	-
13	Priya Nighantu ^[26]	Shatpuspadi Varga	Pratyakapuspi,Adhahshalya	Shweta and Rakta	-
14	Sausruta Nighantu ^[27]	Arkadi gana		Shweta and Rakta	-
15	Ashtanga Nighantu^[28]	Arkadi Gana	Apamarga, Shaikharik, Pratyakpuspi, Mayurak	Shweta and Rakta	-
16	Madanadi Nighantu^[29]	Chaturtha gana	Apamarga, Shaikharik, Pratyakpuspi, Mayurak, Kharmanjari, Adhahshalya, Kshudhaapamarga	Shweta and Rakta	-

2. MATERIALS AND METHOD:

The raw herb Apamarg pachang (Achyranthes aspera) was collected from the local market of Jalandhar and authentication is carried out by Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar under reference no. 1453.

2.1 Preliminary Qualitative Phytochemical Analysis:

This study was carried out to identify the presence of secondary metabolites in plant part. The aqueous extracts of fruits of Apamarg Panchang was prepared and preliminary phytochemical analysis were performed by using the following standard methods^30,31.

2.1.1 Tests for Tannins:

Ferric Chloride Test:

Drug extract when treated with ‎FeCl₃ [ferric chloride] solution may develop with the intense green, purple, blue or black colour that confirms the presence of tannins.

Lead Acetate Test:

Drug extract when treated with few drops of 10% Pb(C₂H₃O₂)₂ [lead acetate]. Precipitate was formed, confirms the presence of tannins.

Bromine water:

Drug extract when treated with 10 ml of Br₂ [bromine] water. Decolouration of bromine water confirms the presence of tannins^30,31.

2.1.2 Tests for Glycosides:

Borntrager’s test:

Boiled 200mg of drug with 2ml of H₂SO₄ [sulphuric acid] in a test tube for 5 minutes. Filtered it while hot. The filtrate was cooled and shaken with equal volume of CHCl₃ [chloroform]. Separate the lower layer of chloroform and shake it with half of its volume of dilute NH₃ [ammonia]. A rose pink to red colour was developed in the ammoniacal layer confirms the presence of glycoside.

Liebermann’s Test:

Two ml of CH₃COOH [acetic acid] was added in CHCl₃ [chloroform] and mixed with 2 ml of drug extract. The lower layer of CHCl₃ [chloroform] was separated and shaken. The mixture was then cooled and added a few drops of concentrated H₂SO₄ [sulphuric acid]. Green colour showed the presence of glycoside.

Keller-Kiliani Test:

Four ml of CH₃COOH [glacial acetic acid] and 1 drop of 2% FeCl₃ [Ferric chloride‎] mixture was mixed with 10 ml of aqueous plant extract and 1ml concentrated H₂SO₄[sulphuric acid]. A brown ring formed between the layers which confirms the presence of glycoside.

Salkowski’s Test:

Two ml concentrated H₂SO₄[sulphuric acid] was added to drug extract. A reddish-brown colour formed which confirms the presence of glycoside^30,31.

2.1.3 Test for Saponin:

Froth/Foam test:

A pinch of the dried powder plant was added to 2-3ml of distilled water. The mixture was shaken vigorously. The froth was mixed with few drops of olive oil and mixed vigorously. Appearance of foam confirms the presence of saponin^30,31.

2.1.4 Tests for Protein:

Millon's test:

Millon’s reagent (2ml) was added to drug extract. A white precipitate appeared, which turned red upon gentle heating indicating the presence of amino acids.

Biuret test:

Biuret reagent (2ml) was added to drug extract (2ml). Appearance of violet colour indicated the presence of amino acids.

Ninhydrin test:

Amino acid, when boiled with few drops of Ninhydrin solution (5%). Appearance of violet colour confirms the presence of amino acids^30,31.

2.1.5 Tests for Carbohydrates:

Benedict's solution test:

Benedict's reagent (2ml) and drug extract (1ml) mix and heated in a boiling water (3 minutes). A colour change from yellowish to bright yellow or bright orange indicated the presence of carbohydrates.

Fehling’s test:

Fehling’s A (10ml) and Fehling’s B (10ml) reagents were mixed and boiled with drug extract (2ml). A brick red precipitate of cuprous oxide indicated the presence of carbohydrates.

Molisch’s test:

One ml of drug extract was treated with few drops of alcoholic α-naphthol. Concentrated H₂SO₄[sulphuric acid] were added slowly through sides of test tube, purple to violet colour ring appeared at the junction indicated the presence of carbohydrates^30,31.

2.1.6 Tests for Alkaloids:

Mayer’s test:

Mayer’s reagent was added to drug extract. Formation of cream coloured precipitates indicated the presence of alkaloids

Dragendorff’s test:

Dragendorff’s reagent was added to drug extract. Formation of reddish-brown precipitate indicated the presence of alkaloids.

Wagner’s test:

Wagner’s reagent was added to drug extract. Formation of reddish-brown precipitate indicated the presence of alkaloids.

Hager’s test:

Hager’s reagent was added to drug extract. Formation of yellow precipitate indicated the presence of alkaloids^30,31.

2.1.7 Tests for Steroids:

Libermann-Burchard test:

Two ml of drug extract was treated with few drops of acetic anhydride [C₄H₆O₃], boiled and cool. Then added concentrated sulphuric acid [H₂SO₄] from the sides of the test tube. A brown ring was formed at the junction two layers and upper layer turned green which showed presence of steroids.

Salkowski test:

Two ml drug extract was treated with few drops of concentrated H₂SO₄ [sulphuric acid]. Red colour at lower layer indicates presence of steroids^30,31.

2.1.8 Tests for Phenols:

Ferric Chloride test:

To the drug extract added a few drops of neutral 5% FeCl₃ [ferric chloride] solution. A dark green colour indicated the presence of phenolic compounds.

Liebermann's nitroso reaction:

The sample was treated with sodium nitrite [NaNO₂] and concentrated H₂SO₄ [sulphuric acid]. Deep green or blue colour which changed to red on dilution with water indicated the presence of phenolic compounds.

Lead Acetate test:

To the test solution, added a few drops of 10% Pb(C₂H₃O₂)₂ [lead acetate]. Formation of white precipitate indicated the presence of phenolic compounds.

Gelatin test:

To the test solution, added a few drops of 10% gelatin solution. White precipitates indicated the presence of phenolic compounds^30,31.

2.1.9 Tests for Flavonoids:

Alkaline reagent test:

To the drug extract, added a few drops of NaOH [sodium hydroxide] solution. Intense yellow colour was formed which turned to colourless on addition of a few drops of dilute acid indicated the presence of flavonoids.

Zinc hydrochloride test:

To the drug extract, added a mixture of zinc dust and concentrated HCl [hydrochloric acid]. It gave red colour after few minutes indicating the presence of flavonoids^30,31.

2.2 Preparation Apamarg Kshar:

Apamarga Panchanga was collected and after shad drying the foreign matter was removed. The dried Panchanga was incinerated to obtain white colour ash. After swangsheetikaran (self-cooling) prepared ash were collected. Add water to the ash in the ratio (1:6). The contents were properly mashed and kept undisturbed for six hours. Afterwards the clear and supernatant liquid layers were collected and filtered. The residual ash was again mashed by adding fresh water into it and kept undisturbed for the next six hours, followed by a collection of the 2^nd and 3^rd illustration as Ksharajala was collected. All the illustrations mixed properly and subjected into Mandagni to evaporate the water portion to obtain Apamarg Kshara. By following this method three batches of Apamarg Kshara was prepared¹⁶.

2.3 Physicochemical Parameters:

2.3.1 Determination of Foreign Matter:

Drug sample (500 g) was taken and spread into tray. Separate out the unwanted material by visual inspection, using a magnifying lens. Weight it and calculate the percentage of foreign matter^32,33.

2.3.2 Determination of Moisture Content (Loss on Drying at 105⁰C):

10gm of the drug sample was taken and dried it at 105⁰C for 5 hours in hot air oven and weighed after cooling in desiccator. It was then dried until the difference between two progressive readings was not more than 0.25 percent and computed the percentage of LOD^32,33.

2.3.3 Determination of Total Ash:

Powered two g sample drug was incinerated in tarred silica crucible at 450⁰C for 5 hrs. in a muffle furnace until it turned white, indicating the absence of carbon. This was cooled in a desiccator and weighed. The percentage of total ash was calculated with the reference to the air-dried sample^32,33.

2.3.4 Determination of Acid Insoluble Ash:

The acquired ash was boiled for 5 minutes with 25ml of 6N HCl [hydrochloric acid], filtered through ash less filter paper. The insoluble matter was washed with hot water until the filtrate becomes chlorine free afterword gathered the insoluble matter in a crucible. It was ignited to constant weight and then calculated the percentage^32,33.

2.3.5 Determination of Alcohol Soluble Extractive:

Five g of coarsely powdered sample drug was macerated with 100ml of alcohol in a closed conical flask for twenty-four hours. Shaking was done frequently for 6 hours and then allowed to stand for 18 hours. It was filtered with taking precautions against loss of liquid. Twenty-five ml of filtrate was evaporated to dryness in a tarred flat evaporating dish, and dried at 105⁰C to consistent weight and then weighed it. Calculated the percentages of alcohol soluble extractive with reference to air dried sample^32,33.

2.3.6 Determination of Water-Soluble Extractive:

The same procedure was followed as that of alcohol soluble extractive replacing alcohol with water^32,33.

2.4 Pre-Compression Characterization:

It included recording of organoleptic characteristics of the drug using descriptive terminologies since record of colour and odour of early batches is very useful in establishing appropriate specifications for production later on^32,34.

2.4.1 Density:

Powder density may influence compressibility, sphericity, pellet porosity, dissolution^30-32,34,36.

2.4.2 Bulk density (BD):

Bulk density is ratio of mass of powder to bulk volume of powder. The parameter was measured following standard procedure. The equation for determining bulk density is

BD (ρ_b)= m/ V_b, where, ρ_b = Bulk density, m = Mass of powder, vb = Bulk Volume ^{[30-32, 34-36]}

2.4.3 Tapped density (TD):

It is a measure used to describe void space of powder. The pre-weighed powder was filled in measuring cylinder. Then it was tapped in bulk density test apparatus. After 100 taps the volume was measured. The equation for determining tapped density is

TP (ρ_t)= m/ V_t, where, ρ_t = Tapped density, m = Mass of powder, vt = Tapped volume ^30-32,34-36

2.4.4 Carr’s (Compressibility) Index (CI):

Compressibility is indirectly related to the relative flow rate, cohesiveness and particle size distribution of the powder. Tapped density (ρ_t) and bulk density (ρ_b) of powder material was used to measure compressibility of a powder material. The equation for determining Carr’s index is

CI (%) = (ρ_t-ρ_b)/ρt*100, Where, ρ_b = Bulk density, ρ_t = Tapped density ^{30-32, 34-36}

2.4.5 Hausner’s Ratio (HR):

It is the ratio of bulk volume to tapped volume or tapped density to bulk density. It is a measure of compressibility of powder. Tapped density (ρt) and bulk density (ρb) of powder material were used to measure Hausner’s Ratio ^30-32,
34-36.

2.4.6 Angle of Repose:

Angle of repose is the maximum angle possible between the surface of a pile of powder and the horizontal plane. The angle of repose of powder blend was determined by “fixed funnel and free-standing cone method”. The accurately weighed powder blend was taken in the funnel and tip of funnel was blocked by thumb initially. The height of the funnel was adjusted in such a way the tip of the funnel just touched the apex of the powder blend (fixed at approximately 2 cm from plane to tip of funnel). The powder blend was allowed to flow through the funnel freely on to the surface. It is used to describe flow ability of the powder material. Angle of Repose is determined by

θ = tan^-1 (h/r), Where, θ = Max. angle between pile of powder and horizontal plane, h = Height of pile of powder, r = Radius of the base of conical pile^{30-32, 34-36}

2.5 Post-Compression Parameters:

2.5.1 Shape and Appearance:

Shape and appearance were observed by visual inspection^30-32,34-36.

2.5.2 Diameter and Thickness:

Dimension of the tablets was measured by using a calibrated dial caliper. Five tablets were picked out randomly and their diameter and thickness were measured individually^{30-32, 34-36}.

2.5.3 Hardness:

The prepared tablets were subjected to hardness test. It was carried out by using Monsanto hardness tester and the observation were expressed in kg/cm^{2
30-32, 34-36}.

2.5.4 Friability (F):

The friability was determined using Roche friabilator and expressed in percentage (%). Twenty tablets from batch were weighed separately (W_initial) and placed in the friabilator, which was then operated for 100 revolutions at 25 rpm. The tablets were reweighed (W_final)and the percentage friability was calculated for each batch by using the following formula – F = (W_initial - W_final) / W_initial X 100 ^30-32,
34-36.

2.5.5 Weight Variation Test:

The weight variation test was done by taking 20 tablets randomly and weighing them accurately. The composite weight divided by 20 provided an average weight of a tablet. The average weight and standard deviation of the tablets were calculated^{30-32, 34-36}.

2.5.6 Disintegration Time:

Six tablets were placed individually in each tube of disintegration test apparatus and discs were placed. Disintegration time was measured in distilled water at 37 ±2⁰C. The tablets were considered as completely disintegrated when all particles passed through the wire mesh^{30-32, 34-36}.

2.5.7 Percentage Drug Release:

Percentage drug release was measured by using UV-VIS spectrophotometry method. The dissolution study was carried out in USP type II (paddle) apparatus. For this study, 900m of 6.8 phosphate buffer was maintained at 37±0.5^oC temp. the study was carried out for 60 minutes with a paddle speed 50 RPM. Five ml of samples were withdrawn at different time intervals and replaced with same volume of fresh buffer to maintain in-vitro sink conditions^{30-32, 34-36}.

2.6 Stability Studies of Optimized Formulation:

Stability of pharmaceutical product may be defined as the capability of a particular formulation, in a specific container/package, to remain within its physical, chemical, therapeutic and toxicological specifications throughout its shelf life. Stability study was carried out for 6 months at accelerated storage conditions (40±2⁰C/ 75% RH ± 5%) following ICH guidelines^30,31,35.

2.7 FTIR Analysis:

FTIR of Apamarg panchang and Apamarg kshar was recorded on Shimazu made instrument using KBr disc method. IR radiations was passed through compressed disc to record down the molecular fingerprint (spectrum) of sample ^30,31,35,37.

2.8 Antiurolithic Activity:

The antiurolithic effect of Apamarg Kshar Tablet on calcium oxalate crystallization was determined by the time course measurement of turbidity changes owing to the crystallization in artificial urine on adding 0.01M sodium oxalate solution. The precipitation of calcium oxalate was measured in terms of turbidity using UV spectrophotometer (620 nm)^{30,31,35,38,40}.

2.8.1 Synthesis of Calcium Oxalate crystals:

The inhibitory effect of aqueous extracts on calcium oxalate crystallization was observed in the form of turbidity due to the crystal nucleation and aggregation while adding 0.01M sodium oxalate to artificial urine, it was observed that calcium oxalate was precipitated at pH 6.8, temperature 37⁰C and wavelength 620 nm with the help of UV spectrophotometer in the form of turbidity^{30,31,35,38-40}.

2.8.2 Preparation of artificial urine:

The artificial urine was prepared by following the reported method of Finlayson et al., 1978⁴⁵, at a constant temperature of 37^oC in capped bottle. Following formula was followed for making artificial urine. All the chemical reagents (sodium chloride 105.5 mmol/litre, sodium phosphate 32.3 mmol/litre, sodium citrate 3.21 mmol/litre, magnesium sulfate 3.85 mmol/litre, sodium sulfate 16.95 mmol/litre, potassium chloride 63.7, calcium chloride 4.5 mmol/litre, sodium oxalate 0.32 mmol/litre, ammonium hydroxide 17.9 mmol/litre and ammonium chloride 0.0028 mmol/litre) were dissolved in deionized water and the pH was adjusted to 6.0^{30,31,35,38-40}.

2.8.3 Observation without the addition of plant extract:

One ml of artificial urine and 0.5 ml distilled water were transferred into the cell and blank reading was taken on a spectrophotometer. Then 0.5 ml of 0.01M sodium oxalate was added and readings were taken after a time period of 10 minutes ^{30,31,35,38-40}.

2.8.4 Observation in the presence of Apamarg Kshar tablet:

Different concentrations of Apamarg kshar tablet i.e. 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 and 550 µg/ml were tested for calcium oxalate crystallization inhibition. Half ml of each concentration was added to 1 ml of artificial urine and blank reading was taken through UV spectrophotometer at 620 nm. Then half ml of 0.01M sodium oxalate was further added and the measurement was done after a period of 10 minutes. Three replicates were run for each experiment ^{30,31,35,38-40}.

2.8.5 Microscopic study:

All the above-mentioned samples concentrations were studied under a trinocular microscope (45X) for the appearance of calcium oxalate crystals and the pictures were taken using digital camera^{30,31,35,38-40}.

2.9 Heavy Metals Determination:

Atomic absorption spectrophotometer was used in the determination of heavy metal elements i.e. Lead, Mercury, Arsenic and Cadmium^30,31,35.

2.10 Microbial Load:

The presence of microbial load was carried out as per the method described in The Ayurvedic Pharmacopoeia of India ^30,31,35.

3. RESULT AND DISCUSSION:

3.1 Qualitative phytochemical of Apamarg panchang:

The preliminary phytochemical characterization of Apamarg panchang revealed the presence of phytochemicals [Table 2].

Table 2 Qualitative test for phytochemicals of Apamarg panchang

Sr. No.	Qualitative Test	Phytochemical constituents	Observation
1	Ferric chloride test	Tannins	Present
	Lead acetate test		Present
	Bromine water		Present
2	Borntrager’s test	Glycoside	Present
	Liebermann’s Test		Present
	Keller-Kiliani Test		Present
	Salkowski’s Test		Present
3	Foam test	Saponin	Present
4	Millon's test	Protein	Present
	Biuret Reagent test		Present
	Ninhydrin Test		Present
5	Benedict's solution test	Carbohydrates	Absent
	Fehling’s test		Absent
	Molisch’s test		Absent
6	Mayer’s reagent	Alkaloids	Present
	Dragendroff reagent		Present
	Wagner’s reagent		Present
	Hager’s reagent		Present
7	Liebermann Burchard’s reaction	Steroids	Present
7	Salkowski test	Steroids	Present
8	Ferric Chloride Test	Phenols	Present
	Liebermann's nitroso reaction		Present
	Lead Acetate test		Present
	Gelatin test		Present
9	Alkaline reagent test	Flavonoids	Present
9	Zinc hydrochloride test	Flavonoids	Present

3.2 Preparation Apamarg kshar:

Apamarg kshar was prepared according to the procedure mentioned in the Rasatarangini and finding are tabulated in Table 3.

Table 3 Observation and results obtained during preparation of Apamarg kshar

Sr. No.	Observation	Results
Sr. No.	Observation	Batch I	Batch II	Batch III
1	Weight of dried Apamarg panchag	15 kg	15 kg	15 kg
2	Weight of ash obtain	2.5 kg	2.3 kg	2.2 kg
3	Ksharjal collected (1^stillustration)	8 lit.	8 lit.	8 lit.
4	Ksharjal collected (2^nd illustration)	8 lit.	8 lit.	8 lit.
5	Ksharjal collected (3^rd illustration)	8 lit.	8 lit.	8 lit.
6	Total Ksharjal collected	24 lit.	24 lit.	24 lit.
7	Total Apamarg kshar obtained (after ghankriya)	435 g	418 g	405 g

3.3 Physicochemical properties:

The various physicochemical parameters i.e. foreign matter, total ash, acid insoluble ash, alcohol soluble extractive, water-soluble extractive of raw herb and loss on drying, total ash, acid insoluble ash, water-soluble extractive, percentage (w/w) of sodium ions and potassium ions were calculated based on standard procedures mentioned in API and findings were tabulated in Table 4.

Table 4: Physico-chemical parameters

Sr. No.	Parameters	*Apamarg panchang*		*Apamarg Kshar*
Sr. No.	Parameters	Mean ± SD	Standard value as per API	Mean ± SD	Standard value as per API
1	Foreign matter (% W/W)	1 ±0.15	NMT 2 %	-	-
2	Total ash (% W/W)	15.2 ±0.20	NMT 17 %	94.80 ±0.36	-
3	Acid-insoluble ash (% W/W)	3.6 ±0.25	NMT 5 %	0.52 ±0.04	NMT 1 %
4	Alcohol-soluble extractive (% V/W)	8.3 ±0.26	NLT 2 %	-	-
5	Water-soluble extractive (% V/W)	25.8 ±0.15	NLT 12 %	95.45 ±0.07	-
6	LOD at 105⁰C (% W/W)	-	-	2.37 ±0.12	NMT 4 %
7	pH (10% aqueous solution)	-	-	10.43 ±0.09	10 to 11
8	Sodium (% W/W)	-	-	6.29 ±0.03	NLT 4 %
9	Potassium (% W/W)	-	-	34.58 ±0.14	NLT 29 %

All values are expressed as mean (±) n=3

3.4 Evaluation parameters of powder blend for tablet:

Powder blend was evaluated for the following pre-compression parameters i.e. bulk density, tapped density, Carr’s compressibility index, Hausner ratio and angle of repose. Ten grams of sample was taken for the studies. The results are given in Table 5.

Table 5: Powder flow properties Weight of sample = 10 gm

Sr. No	Parameters	*Apamarg* kshar (Zero day)	*Apamarg* kshar (6 Months)	Inter pretation
1	Bulk density (g/cm³)	0.676	0.676	Fair
2	Tapped density (g/cm³)	1.250	1.250	Fair
3	Compressibility index	45.9	45.9	Poor Flow
4	Hausner ratio	1.85	1.85	Poor Flow
5	Angle of repose	38.65	37.65	Fair

All values are expressed as mean (±) n=3

3.5 Preparation of Apamarg kshar Tablet:

Tablets were prepared by using direct compression technique. Prepared tablets (weighing 300 mg) were evaluated for the post-compression parameters i.e. shape, diameter, thickness, hardness, friability, weight variation test, disintegration time and percentage drug release. The observation tabulated in Table 6.

Fig. 1 Calibration curve of Apamarg Kshar Tablet

Fig. 2 (A) Percentage drug release of Apamarg Kshar Tablet (zero day)

Fig. 2 (B) Percentage drug release of Apamarg Kshar Tablet (after 6 months)

Table 6 Evaluation parameters for prepared Tablets

Sr.No	Parameters	*Apamarg* *kshar tablet* (zero day)	*Apamarg* *kshar tablet* (6 month) (40^oC ± 2^oC/ 75% RH ± 5%)
1	Shape and appearance	Round	Round
2	Diameter	6.07 ± 0.15	6.07 ± 0.15
3	Thickness	1.13 ± 0.20	1.13 ± 0.20
4	Hardness (kg/cm²)	3 ± 0.43	2 ± 0.43
5	Friability (%w/w)	0.85 ± 0.11	0.85 ± 0.11
6	Weight variation test (%w/w)	1.8 ± 0.26	1.8 ± 0.26
7	Disintegration time (minutes)	3 ±1	2 ±1
8	Percentage drugs release after 60 Min.	80.90 ± 1	78.22 ± 1

All values are expressed as mean (±) n=3

3.6 FTIR spectroscopy:

FTIR study was done by using shimadzu IR spectrophotometer. The spectra of Apamarg Panchang powder and Apamarg Kshar were recorded. The observed functional groups were tabulated in Table 7.

Table 7 Interpretation of IR spectra

Sr. No.	Wave numbers (Cm^-1)	Intensity	Observations
1	3176.87	Variable	O-H stretching vibration (associated)
2	1562.39	Variable	C=C stretching vibration
3	1633.76	Weak-medium	C=C stretching vibration
4	1361.79	Strong	C-H sym. deformation vibration
5	995.30	Weak	C-C skeleton vibration
6	844.85	Weak	COC stretching vibration

Fig. 3 Apamarg Panchang

Fig. 4 Apamarg Kshar

3.7 In vitro study:

The prepared sample of Apamarg kshar tablet started showing inhibition of crystal growth from zero minute. With the passage of time the percentage (%) inhibition also changed. Percentage of inhibitions was calculated as

Percentage of inhibition = (1-OD (experimental)/OD(control)X100. The 550µg/ml concentration of drug tablet from showed 82 % inhibition. The observations are tabulated in Table 8 and [Figure 5-6].

Table 8 In vitro inhibitory activity of CaOx crystals growth by UV spectrophotometer 620 nm

Sr. No.	Drug Conc. (µg/ml)	Absorption	Percentage inhibition
1	50	0.342	2.29
2	100	0.316	9.72
3	150	0.285	18.58
4	200	0.272	22.29
5	250	0.253	27.72
6	300	0.238	32
7	350	0.223	36.29
8	400	0.190	45.72
9	450	0.140	60
10	500	0.075	78.58
11	550	0.063	82

* Control sample Absorption without drug 0.350

Fig. 5: Crystal growth (Control)

Fig. 6: Crystal inhibition at 550 µg/ml

Figure 5-6 In vitro microscopic studies of formulations

3.8 Heavy Metals Determination:

The determination of heavy metals in the prepared Apamarg kshar tablets was carried out using atomic absorption spectroscopy and results are tabulated in table 9.

3.9 Microbial Load:

The presence of microbial load was carried out as per the method described in The Ayurvedic Pharmacopoeia of India in table 10.

4. CONCLUSION:

This work involved evaluation and assessment of in vitro potential of prepared Apamarg Kshar tablets against urolithiasis. The preliminary phytochemical study of Apamarg Panchanga (A. aspera) revealed the presence of tannins, glycoside, saponin, protein, alkaloids, steroids, phenols and flavonoid. As a precautionary measure the dried Apamarg pachang are burnt in a vessel which help us to prevent the material form any contamination during burning. The RO-water was used to counter the interference of inorganic salts which may present in tap water. Mixture of water and prepared ash rubbed homogenously and wort was allowed to settle down for 6 h. After that supernatant water layer is collected as Ksharajal and performed the ghankriya for obtaining the Apamarg Kshar. The drug and prepared sample were subjected to physicochemical evaluation parameters like foreign matter, total ash, moisture content, alcohol and water-soluble extractives, pH, sodium and potassium percentage and the results were found to be within the limits mentioned in API. Drug and excipients were thoroughly mixed and subjected to preformulation studies. Carr’s index, Hausner’s ratio and angle of repose were found to be satisfactory. The compressed tablets were evaluated for post-compression parameters like shape, thickness, hardness, friability, weight variation, and disintegration time. Prepared tablets were able to comply with the pharmacopoeial standards. The tablets were able to release more than 80 % drug release over a period of 60 minutes. FTIR study of formulation revealed presence of all the functional groups without any noticeable interaction with the ingredients. Crystal growth inhibition started at a concentration of 50 µg/ml but 550 µg/ml of drug showed maximum inhibition of 82 %. The microbial load of the prepared Apamarg Kshar tablet was under the limits prescribed by The Ayurvedic Pharmacopoeia of India. Tablets were stable over a period of 6 months when exposed to accelerated stability studies. It can be concluded that prepared tablet dosage form of Apamarg Kshar was effective in the management of urolithiasis (Mutrakricchra) by in-vitro technique.

5. ACKNOWLEDGEMENT:

Authors are thankful to Second International Conference of Pharmacy, held at School of Pharmaceutical Sciences, Lovely Professional University on September 13-14, 2019 to fund the publication of this manuscript. Authors are also thankful to M/S Ashirvad Pharmaceuticals Varanasi, Uttar Pradesh for carryout the Microbial analysis, and AAS studies.

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Received on 28.11.2019 Modified on 10.12.2020

Research J. Pharm. and Tech. 2022; 15(5):2017-2027.

DOI: 10.52711/0974-360X.2022.00334

Sr. No.	Metals	Lead	Mercury	Arsenic	Cadmium
1	Observed values	1.1 ppm	Not detected	2.1 ppm	Not detected
2	Limit as per API	10 ppm	1 ppm	3 ppm	0.3 ppm