Formulation, Evaluation and Assessment of In Vitro Potential of
Gokshur Ghan Tablet against Urolithiasis (Mutrakrichra)

 

Dileep Singh Baghel¹, Amit Mittal^1*, Saurabh Singh¹, Anand Kumar Chaudhary², Amit Bhatia³, Shruti Chopra⁴

¹School of Pharmaceutical Sciences, Lovely Professional University, Jalandhar - Delhi G.T. Road,

Phagwara, Punjab (India) -144411.

²Department of Rasa Shastra and Bhaishjya Kalpana (Ayurvedic Pharmaceutics),

Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Uttar Pradesh, India.

³Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical University, Punjab, India.

⁴Amity Institute of Pharmacy, Amity University, Uttar Pradesh, India.

 

ABSTRACT:

Background: Tribulus species spread in the warm, temperate regions and prevalent in zone where hot summers and dry soil present. Tribulus species comprises of more than 25 species. Tribulus terrestris L. (Zygophyllaceae) consists of ripe, dried, whole fruit and an annual herb, rarely perennial that grows worldwide, particularly in the temperate regions. It is used as traditional medicines in India, China, South Africa, Bulgaria and many other countries. It is used for strengthening (balya), nutritive (brimhan), rejuvenator (rasayan), diuretic (mutral), anti-inflammatory (shothahara), renal calculi (ashmari) and urolithiasis (Mutrakrichra). Objective: To prepare Gokshur tablet and evaluate its potential against Urolithiasis (Mutrakrichra) by in vitro technique. Materials and Methods: Gokshur kvatha churna was prepared as per the methodology mentioned in Sharandhar samhita and ghan was prepared by evaporating the watery portion from prepared kvatha. Prepared ghan was compressed into tablet, and quality of tablets were evaluated. Results: Physicochemical and stability studies have not shown any remarkable variations with prepared tablet dosage form. In vitro studies showed 58.24% of crystal inhibition at 650µg/ml. Conclusion: The prepared tablets of Gokshur ghan did not have remarkable variation during physicochemical and stability studies. The prepared tablets were able to show remarkable in vitro activity against Urolithiasis (Mutrakrichra).

 

 

 

INTRODUCTION:

Nearly all the ethnicity of the world is mentioned the therapeutic implementation plants or their derivatives as a primary source of medicine. WHO (World Health Organization) reported that the 80% of the total population of the world have consuming traditional medicine based on plants or their derivatives^1,2.

 

Urolithiasis (Mutrakrichra) ranked third among the most prevalent life-threatening disorders after cancer and cardiac ailments. It is a collective process of nucleation, growth, aggregation of crystals and attachment of the crystals to renal cells^3,4,5. The etiology of Urolithiasis (Mutrakrichra) is multifactorial and might be based on diet, nutrient, low physical activity and genetics. Calcium-oxalate and phosphate are the calcium containing salts which are commonest type of kidney stones (75-90%). The prevalence of Calcium oxalate stones is more than calcium phosphate stones followed by struvite (magnesium ammonium phosphate stone) (10–15%), uric acid (3–10%), and cystine (0.5–1%)^6-10.

 

In Ayurveda, the causes of renal disorders are vitiation of Mutravahasrotas (channels carrying urine) the common etiological factors which are accountable for mutravahasrotodushti. The probable causes of Urolithiasis (Mutrakrichra) are Ati-vyayam (excessive exercise), Tikshna aushadha and aahara seevan (intake of sharp medicine and dry food), Rukshamadya prasanga (excessive consumption of dry variety of alcohol), Nitya druta prishthayanat (riding on the back of fast-moving animals regularly), Anupa matsya (intake of flesh of wet land fish), Adhyashana and Ajirnat (eating before the digestion of previous meal and indigestion), Katiskandha tidharanat (weight lifting), Mutravaha srotodushti (urinary tract infection), Mutravega nigraha / Mutra vegaavarodha (suppression of urge) and Abhikshata (person suffering with injury to the organs of mootravaha srotasa)^11,12.

 

Gokshur (Tribulus terrestris) is a one of the potential medicinal plant described in Ayurvedic literature with the properties to destroy diseases of mutravaha srotas (urinary system) due to presence of nitrates, essential oil and potassium in its fruits^11,12.

 

Table 1: Gokshur gana (group) classification by Ayurvedic literature

 

Table 2: Rasapanchaka (Properties and actions mentioned in Nighantus)

 

It shows the pharmacological activities such as amavata (rheumatism), amlapitta (hyperacidity), Antravrddhi (hernia), Mutrakrichra (urolithiasis), ashmari (calculus), arhsa (piles), hridyaroga (heart disease), jvara (fever), mutraghata (urinary obstruction), pradar (excessive vaginal discharge), prameha (metabolic disorder), raktapitta (bleeding disorder), sula (pain) and svasa (asthma)^13-25. In this present study, Gokshur ghan tablets were prepared and evaluated for their in vitro potential against urolithiasis (Mutrakrichra).

 

Description of Gokshur in Ayurvdic text:

Various opinion regarding gana, dosha-prabhava, rasapanchaka and synonyms mentioned by ancient scholar details of the same is tabulated in the Table 1 and Table 2.

 

MATERIAL AND METHOD:

The fruits of Gokshur (T. terrestris) 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^26-29:

This study was carried out to identify the presence of secondary metabolites in plant part. The aqueous extracts of fruits of Gokshur (T. terrestris) was prepared and preliminary phytochemical analysis were performed by using the following standard methods.^26-29

 

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 10ml 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 2ml 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^30-32:

Determination of Foreign Matter:

Drug sample (500g) 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 105°C 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 450°C 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 105⁰C 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^33-37:

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 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

 

POST-COMPRESSION PARAMETERS^33-37:

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 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.

 

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 ±2⁰C. The tablets were considered as completely disintegrated when all particles passed through the wire mesh.

 

STABILITY STUDIES OF OPTIMIZED FORMULATION^33-37:

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.

 

ANTIUROLITHIC ACTIVITY^38-39:

The antiurolithic effect of Gokshur 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., 1978⁴⁰, at a constant temperature of 37^oC in capped bottle. Following formula was followed for making artificial urine. All the chemical reagents (sodium chloride 105.5mmol/litre, sodium phosphate 32.3mmol/litre, sodium citrate 3.21 mmol/litre, magnesium sulfate 3.85 mmol/litre, sodium sulfate 16.95mmol/litre, potassium chloride 63.7 mmol/litre, calcium chloride 4.5mmol/litre, sodium oxalate 0.32mmol/litre, ammonium hydroxide 17.9 mmol/litre and ammonium chloride 0.0028mmol/litre) 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 Gokshur ghan tablet:

Different concentrations of Gokshur ghan tablet i.e. 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 and 650 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 0.01 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 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.

 

HEAVY METALS DETERMINATION^30,31:

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

 

MICROBIAL LOAD^30,31

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

 

 

 

RESULT AND DISCUSSION:

The preliminary phytochemical characterization of Gokshur (T. terrestris) fruits revealed the presence of phytochemicals. [Table 3]^26-29

 

Table 3: Qualitative test for phytochemicals of Gokshur fruit 

 

PHYSICOCHEMICAL PROPERTIES:

The various physicochemical parameters i.e. foreign matter, 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 4^30-33.

 

Table 4 Physiochemical parameters for Gokshur fruit

 

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 5^34-37

Table 5 Powder flow properties

*±(n=3)

 

PREPARATION OF GOKSHUR GHAN TABLET:

Tablets were prepared by using direct compression technique. Prepared tablets (weighing 300mg) 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 6^34-37.

 

Table 6 Evaluation parameters for prepared Tablets

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

 

Table 7 In vitro inhibitory activity of CaOx crystals growth by UV spectrophotometer 620nm

* Control sample Absorption without drug 0.522

IN VITRO STUDY:

The prepared sample of Gokshur 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 58.24 % inhibition^38,39. The observations are tabulated in Table 7 and Figure 1-3.

 

Microscopic observation:

 

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 Gokshur ghan tablets was carried out using atomic absorption spectroscopy and results are tabulated in table 8^30,31.

 

Table 8 Heavy metal concentrations in Gokshur ghan

 

MICROBIAL LOAD:

The presence of microbial load was carried out as per the method described in The Ayurvedic Pharmacopoeia of India in table 9 ^30,31.

 

 

 

Table 9 Observation of Microbial load

 

 

CONCLUSION:

This work involved evaluation and assessment of in vitro potential of prepared Gokshur ghan tablets against urolithiasis. The preliminary phytochemical study of Gokshur (T. terrestris) fruit revealed the presence of Tannins, Glycoside, Saponin, Alkaloids, 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. Gokshur powder 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. Crystal growth inhibition started at a concentration of 50µg/ml but 650 µg/ml of drug showed maximum inhibition of 58.24%. The microbial load and heavy metal analysis of the prepared Gokshur ghan tablet was found 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 Gokshur 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.

 

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Received on 26.11.2019            Modified on 27.03.2020

Accepted on 02.06.2020         © RJPT All right reserved