MATERIALS AND METHOD:

The roots of Punarnava (B. diffusa) were collected from the local market of Jalandhar and authentication was carried out by Herbal Health Research Consortium Pvt. Ltd., Amritsar.

Preliminary qualitative phytochemical analysis^27-30:

This study was carried out to identify the presence of secondary metabolites in plant part. The aqueous extracts of root parts of Punarnava (B. diffusa) was prepared and preliminary phytochemical analysis were performed by using the following standard methods.

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.

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 10ml 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.

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 saponins.

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.

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.

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.

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.

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

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.

PHYSICOCHEMICAL PARAMETERS^31-32:

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.

DETERMINATION OF MOISTURE CONTENT (LOSS ON DRYING AT 105 C):

Ten g 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.

DETERMINATION OF TOTAL ASH:

Powered two g sample drug was incinerated in tarred silica crucible at 450C 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.

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.

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 105C to consistent weight and then weighed it. Calculated the percentages of alcohol soluble extractive with reference to air dried sample.

DETERMINATION OF WATER-SOLUBLE EXTRACTIVE:

The same procedure was followed as that of alcohol soluble extractive replacing alcohol with water.

PRE-COMPRESSION CHARACTERIZATION^33-36:

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.

DENSITY:

Powder density may influence compressibility, sphericity, pellet porosity, dissolution.

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

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

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

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.

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 2cm 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

POST-COMPRESSION PARAMETERS^33-36:

SHAPE AND APPEARANCE:

Shape and appearance were observed by visual inspection.

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.

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/cm².

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 25rpm. 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.

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.

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±2°C. The tablets were considered as completely disintegrated when all particles passed through the wire mesh.

STABILITY STUDIES OF OPTIMIZED FORMULATION^33-35:

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±2 C/ 75% RH ± 5%) following ICH guidelines.

ANTIUROLITHIC ACTIVITY^37-38:

The antiurolithic effect of Punarnava ghan 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 (620nm).

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 620nm with the help of UV spectrophotometer in the form of turbidity.

Preparation of artificial urine:

The artificial urine was prepared by following the reported method of Finlayson et al., 1978³⁹, at a constant temperature of 37⁰C in capped bottle. Following formula was followed for making artificial urine. All the chemical reagents (sodium chloride 105.5mmol/liter, sodium phosphate 32.3mmol/liter, sodium citrate 3.21 mmol/liter, magnesium sulfate 3.85 mmol/liter, sodium sulfate 16.95mmol/liter, potassium chloride 63.7, calcium chloride 4.5mmol/liter, sodium oxalate 0.32 mmol/liter, ammonium hydroxide 17.9mmol/liter and ammonium chloride 0.0028mmol/liter) were dissolved in deionized water and the pH was adjusted to 6.0.

Observation without the addition of plant extract:

One ml of artificial urine and 0.5ml distilled water were transferred into the cell and blank reading was taken on a spectrophotometer. Then 0.5ml of 0.01M sodium oxalate was added and readings were taken after a time period of 10 minutes.

Observation in the presence of Punarnava ghan tablet:

Different concentrations of Punarnava ghan tablet i.e. 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 and 650µg/ml were tested for calcium oxalate crystallization inhibition. Half ml of each concentration was added to 1ml of artificial urine and blank reading was taken through UV spectrophotometer at 620nm. Then half ml of 0.01 M sodium oxalate was further added and the measurement was done after a period of 10 minutes. Three replicates were run for each experiment.

Microscopic examination:

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.

HEAVY METALS DETERMINATION³⁰:

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

MICROBIAL LOAD³⁰:

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

RESULT AND DISCUSSION:

Qualitative phytochemical analysis of Punarnava:

The preliminary phytochemical characterization of Punarnava (B. diffusa) roots revealed the presence of phytochemical were tabulated in Table 2.^27-30

Table 2: Qualitative test for phytochemicals of Punarnava root

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

Physicochemical properties:

The various physicochemical parameters i.e. foreign matter, loss on drying, total ash, acid insoluble ash, alcohol soluble extractive, and water-soluble extractive were determined as per the standard procedures mentioned in API and findings were tabulated in Table 3 ^31-32.

Evaluation parameters of powder blend of 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 4 ^33-36.

Table 3: Physiochemical parameters for Punarnava root

Sr. No.	Parameters	Observation (Mean ± SD)	Standard value as per API
1	Foreign matter % W/W	2.1 ±0.31	NMT 2 %
2	Loss on drying	9.3 ±0.53	NMT 15 %
3	Total Ash % W/W	10.3 ±1.20	NMT 15 %
4	Acid-insoluble ash % W/W	2.2 ±0.26	NMT 3 %
5	Alcohol-soluble extractive % V/W	7.2 ±0.70	NLT 5 %
6	Water-soluble extractive % V/W	15.2 ±0.045	NLT 8 %

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

Table 4: Powder flow properties

Sr. No.	Parameters	*Punarnava* ghan (Zero day)*	*Punarnava* ghan (6 Months)*	Interpretation
1	Bulk density (g/cm³)	0.763 ±0.15	0.770 ±0.21	Fair
2	Tapped density (g/cm³)	1.087 ±0.13	1.052 ±0.32	Fair
3	Compressibility index	29.8 ±0.17	28.9 ±0.12	Poor
4	Hausner ratio	1.42 ±0.13	1.41 ±0.21	Poor
5	Angle of repose	39.79 ±0.19	40.05 ±0.22	Passable

*±(n=3)

Preparation of Punarnava ghan 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 and disintegration time. The observation tabulated in Table 5 ^33-36.

Table 5: Post-compression evaluation parameters for tablets

Sr. No	Parameters	*Punarnava ghan* *tablet* (zero day)	*Punarnava ghan* *tablet* (After 6 month) (40^oC ± 2^oC/ 75% RH ± 5%)
1	Shape	Round	Round
2	Diameter	6.06 ± 0.07	6.07 ± 0.04
3	Thickness	1.13 ± 0.02	1.10 ± 0.12
4	Hardness (kg/cm²)	4 ± 1	3 ± 1
5	Friability (%)	0.83 ± 0.02	0.89 ± 0.32
6	Weight variation test	1.8 ± 0.33	1.8 ± 0.23
7	Disintegration time (minutes)	4 ± 1	3 ± 1

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

In vitro study:

The prepared sample of Punarnava ghan 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 650µg/ml concentration of drug tablet from showed 50% inhibition. The observations are tabulated in Table 6 and Microscopic examination were shown in Figure 1-3^38-39.

Table 6: In-vitro inhibitory activity of CaOx crystals growth by UV spectrophotometer 620 nm

Sr. No.	Drug Conc. (µg/ml)	Absorption (UV spectrophotometer)	Percentage inhibition
1	50	0.454	4.62
2	100	0.435	8.61
3	150	0.415	12.881
4	200	0.397	16.59
5	250	0.385	19.11
6	300	0.357	25
7	350	0.332	30
8	400	0.323	32.14
9	450	0.308	35.29
10	500	0.293	38.44
11	550	0.287	39.70
12	600	0.258	45.79
13	650	0.238	50

* Control sample Absorption without drug 0.476

Microscopic examination:

In-vitro inhibitory activity of CaOx crystals growth.

Figure 1: Crystal Growth (Control)

Figure 2: Crystal inhibition at 50 µg/ml

Figure 3: Crystal inhibition at 650 µg/ml

HEAVY METALS DETERMINATION:

The determination of heavy metals in the prepared Punarnava ghan tablets was carried out using atomic absorption spectroscopy and results are tabulated in table 7³⁰.

MICROBIAL LOAD:

The presence of microbial load was carried out as per the method described in The Ayurvedic Pharmacopoeia of India in table 8⁽³⁰⁾.

CONCLUSION:

This work involved evaluation and assessment of in-vitro potential of prepared Punarnava ghan tablets against urolithiasis. The preliminary phytochemical study of Punarnava (B. diffusa) root revealed the presence of Tannins, Saponin, Protein, Carbohydrates, Alkaloids, Steroids, Phenols and Flavonoids. The drug sample was subjected to physicochemical evaluation parameters like foreign matter, total ash, moisture content, alcohol and water-soluble extractives and the results were found to be within the limits. The aqueous extract was thoroughly mixed and subjected to preformulation studies. Bulk density, tapped density, Compressibility index, Hausner 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. Crystal growth inhibition started at a concentration of 50 µg/ml but at 650 µg/ml of drug concertation showed maximum crystal inhibition of 50%. The microbial load and heavy metal analysis of the prepared Punarnava ghan 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. So, It can be concluded that the prepared tablet dosage form of Punarnava ghan was effective in the management of urolithiasis (Mutrakricchra) by in-vitro technique.

ACKNOWLEDGEMENT:

Authors are thankful to Second International Conference of Pharmacy, held by 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.

REFERENCES:

1. Khan, Saeed R., Margaret S. Pearle, William G. Robertson, Giovanni Gambaro, Benjamin K. Canales, Steeve Doizi, Olivier Traxer, and Hans-Göran Tiselius. Kidney stones. Nature Reviews Disease Primers 2 (2016): 16008

2. Selvam, R., P. Kalaiselvi, A. Govindaraj, V. Bala Murugan, and AS Sathish Kumar. Effect of A. lanata leaf extract and Vediuppu chunnam on the urinary risk factors of calcium oxalate urolithiasis during experimental hyperoxaluria. Pharmacological Research 43, no. 1 (2001): 89-93

3. Moe, Orson W. Uric acid nephrolithiasis: proton titration of an essential molecule? Current opinion in nephrology and hypertension 15, no. 4 (2006): 366-373

4. Bhasin, Bhavna, Hatice Melda Ürekli, and Mohamed G. Atta. Primary and secondary hyperoxaluria: understanding the enigma. World journal of nephrology 4, no. 2 (2015): 235

5. Hoppe, Bernd, Ernst Leumann, Gerd von Unruh, Norbert Laube, and Albrecht Hesse. Diagnostic and therapeutic approaches in patients with secondary hyperoxaluria. Front Biosci 8, no. 1-3 (2003): e437-e443

6. Moe, Orson W. Kidney stones: pathophysiology and medical management. The lancet 367, no. 9507 (2006): 333-344

7. Awasthi, Madhvi, S. R. Malhotra, and Rajni Modgil. Dietary Habits of Kidney Stone Patients of Kangra District, Himachal Pradesh, North India. Journal of Human Ecology 34, no. 3 (2011): 163-169

8. Evan, Andrew P. Physiopathology and etiology of stone formation in the kidney and the urinary tract. Pediatric Nephrology 25, no. 5 (2010): 831-841

9. Singh, Kshetrimayum Birla, and Saitluangpuii Sailo. Understanding epidemiology and etiologic factors of urolithiasis: an overview. Sci Vis 13, no. 4 (2013): 169-174

10. Gault, M. H., and L. Chafe. Relationship of frequency, age, sex, stone weight and composition in 15,624 stones: comparison of results for 1980 to 1983 and 1995 to 1998. The Journal of urology 164, no. 2 (2000): 302-307

11. Coe FL, Evan A, Worcester E. Kidney stone disease. J Clin Invest 2005; 115:2598–2608

12. Khan F, Haider MF, Singh MK, et al. A comprehensive review on kidney stones, its diagnosis and treatment with allopathic and ayurvedic medicines. Urol Nephrol Open Access J. 2019;7(4):69‒74

13. Romero, Victoriano, Haluk Akpinar, and Dean G. Assimos. Kidney stones: a global picture of prevalence, incidence, and associated risk factors. Reviews in urology 12, no. 2-3 (2010): e86.

14. Heywood, Vernon Hilton, D. M. Moore, Judith Dunkley, and Christabel King, eds. Flowering plants of the world. Vol. 336. Oxford: Oxford University Press, 1978.

15. Samhita, Charak, and Kashinath Sastri. Chaukhambha Bharti Academy. Varansi, Reprint 79 (2013): 80-84

16. Sushruta. Sutrasthana, chapter 38, verse 4, 13, 67 and chapter 42, verse 11. In: Sushruta Samhita. Shashtri AD. editor. Varanasi: Chaukhambha Sanskrita Sansthan, 2001

17. Vagbhata, Astanga Sangraha. Sutrasthana 178/103. Saroj Hindi commentary by Indradev Tripathi, Chaukhamba Sanskrit Pratisthan, Varanasi 5 (2005): 27.

18. Chunekar, Krsihna Chand, and G. S. Pandey. Bhavprakash nighantu. Hindi Commentary, Chukhamba Bharti Academy Varanasi, Reprint 314 (2004): 212.

19. Shodhala, Nighantu of Shodhala by Prof. P.V. Sharma publisher Chaukhambha Vidyabhawan,Varanasi,Reprint-1972

20. Kamat, S. D., and S. D. Kamat. Dhanvantari nighantu. Delhi: Chaukhambha Sanskrit Pratisthan (2002): 1.

21. Pandey, G. Madanpal Nighantu. Varanasi: Chowkhambha orientalia (2012)

22. Vaidya, Bapalal G. Nighantu adarsha. Chaukhambha Bharati Academy, Vanarasi, 2nd Ed. I (1998): 211-214

23. Sharma, Acharya Priyavrata. Commentary on Kaidev Nighantu (Gavya Ghrita Guna-271)-Chaukhambha Orientalia. (2009).

24. Narahari, Pandit, and Raj Nighantu. Indradev Tripathi. Krishna Das Academy, Varanasi, p.-29 416 (2003): 443.

25. Vaidyacarya Bopadeva, Hridayadipaka Nighantu with PRAKASA commentary of Vopadeva By Prof. P.V. Sharma, Publisher chaukhambha Amarabharati prakashan,Varanasi,Print-1977

26. Sharma, Priya Vrat. Priya Nighantu. Chaukamba Bharti Academy, Varanasi (1983).

27. Evans, William Charles. Trease and Evans Pharmacognosy E-Book. Elsevier Health Sciences, 2009.

28. Sharma, P. C., M. B. Yelne, and T. J. Dennis. Database on medicinal plants used in Ayurveda. Vol. 1-3. New Delhi: CCRAS, 2005

29. Kirtikar, K. R., and B. D. Basu. Indian Medicinal Plants: periodical book agency. New Delhi 1 (1998): 686-689.

30. Anonymous. The Ayurvedic Pharmacopoeia of India, Part I, Vol 1-Vol 9 and Part II, Vol 1-Vol 3, Government of India, Ministry of Health and Family Welfare. Department of AYUSH, Delhi

31. Lohar, D. R. Protocol for testing Ayurvedic, Siddha and Unani medicines, Government of India Department of AYUSH. Ghaziabad: Ministry of Health & Family Welfare, Pharmacopoeial Laboratory for Indian Medicines (2007): 21.

32. Gupta, A.K., Neraj Tandon., and Madhu Sharma. Quality Standard of Indian Medicinal Plants (Vol. I, II, III, IV, VII, VIII. Indian Council of Medicinal Research, 2003, 2005, 2006, 2008, 2010

33. Mannhold, Raimund, Helmut Buschmann, and Jorg Holenz. Innovative Dosage Forms: Design and Development at Early Stage. Vol. 76. John Wiley & Sons, 2019

34. Lachman, Leon, Herbert A. Lieberman, and Joseph L. Kanig. The theory and practice of industrial pharmacy. Lea & Febiger, 1986

35. Rowe, Raymond C., Paul Sheskey, and Marian Quinn. Handbook of pharmaceutical excipients. Libros Digitales-Pharmaceutical Press, 2009.

36. Haritha, B. A Review on Evaluation of Tablets. Journal of Formulation Science & Bioavailability 1, no. 1 (2017): 1-5

37. Ahmed, Salman, Muhammad Mohtasheemul Hasan, and Zafar Alam Mahmood. In vitro urolithiasis models: An evaluation of prophylactic management against kidney stones. Journal of Pharmacognosy and Phytochemistry 5, no. 3 (2016): 28

38. Shah, Muhammad Ajmal, Sikandar Khan Sherwani, Mohammad Sualeh, Sandaleen Kanwal, Haq Nawaz Khan, and Shahana Urooj Kazmi. In vitro anthelmintic and antiurolithic assessment of Berberis lycium root bark. Journal of Pharmacognosy and Phytochemistry 3, no. 2 (2014)

Received on 22.11.2019 Modified on 30.03.2020

Research J. Pharm. and Tech 2021; 14(3):1469-1476.

DOI: 10.5958/0974-360X.2021.00261.4

Sr. No.	Text Name	*Gana* (group)	Synonyms
1	Charka Samhita¹⁵	Swedopaga, Anuvasanopaga, Kasahara, Vayasthapana,	Punarnava, Mahavarshabhu, Vrishchiva, Dirghavarshabhu, Shvetamula, Raktavrinta, Vaishakha, Shinati, Varshaketu
2	Sushrut Samhita¹⁶	Vidaarigandhadi,Vatashanshamana varga, Tikta varga, Shaka varga	Punarnava, Rishabhketu, Mahavarshabhu, Vrishchiva, Dirghavarshabhu, Shvetamula, Raktavrinta, athillaka, Vaishakha, Shinati, Kshudravarshabhu
3	Astanga Sangraha ¹⁷	Viprakirna varga, Vidarigandhadi Gana,	Punarnava, Varshaketu, Vrishchiva, Shvetamulaka, Varshabhu, Dirghapatra,Vikasa, Kathillaka, Sunadik, Raktapushpa, Vishakha, Mandalacchada
4	Bhavprakash nighantu¹⁸	Guduchyadi varga	Arunaa, Kshudravarshabhu, Raktapushpaka, Shilatika, Shothaghni, Varshaketu,
5	Shodhala Nighantu¹⁹	Guduchyaadi varga	Mahavarshabhu, Pravrushaayani, Raktapushpaka,Shivatika, Shophaghni, Varshabhu, Varshaketu
6	Dhanvantari Nighantu²⁰	Guduchyaadi varga	Deerghapatraka, Kathillaka, Kshudravarshabhu, Shivatika, Vrushchira
7	Madanpal Nighantu²¹	Abhayadi varga	Arunaa, Kathillaka Kruraka Kshudravarshabhu, Raktapushpaka, Tikta, Shivatika, Varshaketu
8	Aadarsh Nighantu²²	Punarnavadi Varga	Raktapunarnava, Vishakha,Katilla, Kathila, Shothaghni, Mahavarsabhu
9	Kaiyadeva Nighantu²³	Aushadhi varga	Deerghapatraka, Shophaghni, Varshabhu, Vrushchiva
10	Raja Nighant/ Nighsntu Raja or Abhidhana Cudamani²⁴	Parpatadi varga	Raktapunarnava, Krura, Mandalpatrika,Raktakanda, Varshketu, Lohita, Raktapatrika, Vaishakhi, Raktavarshabhu, Shophaghni, Raktapushpika, Viksvara, Vishaghni, Pravrishenya, Sarini, Varshabhav, Shorapatra, Sammilitadruma, Punarnav, Nav,Nachya
11	Hridayadipaka Nighanṭu²⁵	Dvipadi Varga	Punarnava,Vrishchiva, Varshabhu, Shivatika
12	Priya Nighantu²⁶	Shatpushpadi Varga	Punarnava, Varshabhu

Sr. No.	Metals	Lead	Mercury	Arsenic	Cadmium
1	Observed values	1 ppm	Not detected	1.8 ppm	Not detected
2	Limit as per API	10 ppm	1 ppm	3 ppm	0.3 ppm

Microbial analysis	Total bacterial count	Total yeast and mould	*E. coli*	*S. spp.*	*S. aureus*	*P. aeruginosa*
Limit as per API	NMT 10⁵ CFU/ml	NMT 10³ CFU/ml	Absent	Absent	Absent	Absent
Observed values (Punarnava ghan)	69000 CFU/ml	500 CFU/ml	Absent	Absent	Absent	Absent