In vitro Antiurolithiatic activity of the leaves and flowers extracts of Paronychia argentea, a plant used in traditional medicine in Algeria

 

Omar Mechraoui¹, Ali Imessaoudene², Mohamed Y. Maiz², Hicham Banouh², Lotfi Mouni², Abdelkrim Rebiai³,

Mohamed L. Belfar⁴, Noureddine Elboughdiri^5,6, Djamel Ghernaout^5,7, Bachir Ben Seghir^8,9,10*

¹Faculty of Technology, University Amar Telidji-Laghouat, P.O. Box 37G, Laghouat 03000, Algeria

²Laboratory of Management and Valorization of Natural Resources and Quality Assurance,

SNVST Faculty, University of Bouira, Bouira 10000, Algeria

³Chemistry Department, Faculty of Exact Sciences, University of El Oued,

P.O. Box 789, El Oued 39000, Algeria

⁴Department of Chemistry, Faculty of Mathematics and Material Science,

University Kasdi Merbah Ouargla, P.O. Box 511, Ouargla 30000, Algeria

⁵Chemical Engineering Department, College of Engineering, University of Ha’il,

P.O. Box 2440, Ha'il 81441, Saudi Arabia

⁶Chemical Engineering Process Department, National School of Engineering Gabes,

University of Gabes, Gabes 6011, Tunisia

⁷Chemical Engineering Department, Faculty of Engineering, University of Blida,

P.O. Box 270, Blida 09000, Algeria

⁸Laboratory of Industrial Analysis and Materials Engineering (LAIGM),

University 8 May 1945 Guelma, BP 401, Guelma 24000, Algeria

⁹Department of Process Engineering and Petrochemical, Faculty of Technology,

University of Echahid Hamma Lakhdar, El Oued 39000, Algeria

¹⁰Renewable Energy Development unit in Arid Zones (UDERZA),

University of Echahid Hamma Lakhdar, El Oued 39000, Algeria

 

ABSTRACT:

Plants are a large source of new bioactive molecules with therapeutic potentials. However, only a small amount of worldwide plants has been phytochemically investigated. The ethanolic extracts from leaves and flowers of Paronychia argentea were evaluated for their antilithiasic activity in vitro. The effect of extract (0.1, 0.2, 0.3, 1, 2, and 5mg/mL) was studied by the measurement of turbidity in presence or absence of extract at 620nm using UV/Vis spectrophotometer. Total phenol and flavonoid contents were also evaluated. Polyphenol content was found to be more present in the leaves extract (9.29±0.009mg of Gallic acid equivalent (GAE)/g) compared to the flowers extract (5.92±0.14mg GAE/g). Flavonoids content was also found to be more present in the floral extract that is estimated at 0.18±0.01 mg QE/g compared to the flowers extract (0.47±0.0035mg QE/g). For the antilithiasis activity, the results clearly shown that P. argentia extracts inhibited calcium oxalate crystallization by concentration-dependent manner. The maximum percent inhibition of calcium oxalate by flowers extract was found to be 70.97%  at 5mg/mL. Further, P. argentea leaf extract has shown antilithiasic properties and may be used for the prevention of kidneys stones. Plants are a large source of new bioactive molecules with therapeutic potentials. However, only a small amount of worldwide plants has been phytochemically investigated. The ethanolic extracts from leaves and flowers of Paronychia argentea were evaluated for their antilithiasic activity in vitro. The effect of extract (0.1, 0.2, 0.3, 1, 2, and 5mg/mL) was studied by the measurement of turbidity in presence or absence of extract at 620 nm using UV/Vis spectrophotometer. Total phenol and flavonoid contents were also evaluated. Polyphenol content was found to be more present in the leaves extract (9.29±0.009mg of Gallic acid equivalent (GAE)/g) compared to the flowers extract (5.92±0.14mg GAE/g). Flavonoids content was also found to be more present in the floral extract that is estimated at 0.18±0.01mg QE/g compared to the flowers extract (0.47±0.0035mg QE/g). For the antilithiasis activity, the results clearly shown that P. argentia extracts inhibited calcium oxalate crystallization by concentration-dependent manner. The maximum percent inhibition of calcium oxalate by flowers extract was found to be 70.97% at 5mg/mL. Further, P. argentea leaf extract has shown antilithiasic properties and may be used for the prevention of kidneys stones.

 

 

 

INTRODUCTION:

Oxalocalcic lithiasis is the most common form of urinary stones  present in more than 80% of stones as oxalate calcium monohydrate (whewellite) and/or dihydrate (weddellite)^1,2. Calcium oxalate is the main constituent of around 50% of stones in women and 75% in men³. The formation of urinary stones is due to the imbalance between crystallization promoters such as calcium oxalate and inhibitors such as citrate, magnesium or urinary macromolecules.

 

Although the difficult procedures used to treat urolithiasis is time requiring and cost-effective, the need for new bioactive molecules to treat such diseases is crucial. Therefore, scientists are seeking for new drugs from nature to enhance the treatment’s cost/efficiency. Recently, herbal medicine has become the main source of discovery, selection and purification of new bioactive compounds to be used in modern medicine³.

 

Algeria, by its geographical position, offers a great variety of flora, but a strong ethno-medicinal tradition is still in force.

 

Paronychia argentea is a plant traditionally prescribed by herbalists to treat urinary stones. It is a plant native to lower Morocco, Egypt and the basin Mediterranean. It was used in ancient Egypt to treat asthma, kidney stones and renal colic. It belongs to the family of Caryophyllaceae and is commonly known as Arabic tea. Medicinal uses of its aerial parts are used in Algerian folk medicine to treat kidney disease^4-6 and diabetes⁷. This plant is also used to treat stomach ulcers, anorexia, bladder disease, and prostate ^{8, 9}, as well as kidney stones ⁹ and heart pain^{10, 11}.

 

It is thus our duty to establish the pharmacological effect of this eco-friendly medicinal plant for the removal of human suffering and miseries from kidney diseases. Hence, this study has evaluated the in vitro anti-lithiatic effect of P. argentia leaves and flowers extracts.

 

MATERIALS AND METHODS:

Chemical and Reagents:

The chemicals, biochemicals and solvents used in this study were of analytical grade. These include ethanol, sodium carbonate solution (NaCO₃), aluminum trichloride (AlCl₃), sodium chloride (NaCl), trisodium citrate dehydrate and disodium oxalate, all obtained from Scharlau. However, tris-hydroxymethylmethalamine (Tris) was obtained from Sigma. Calcium chloride dihydrate was obtained from Biochem and Folin-Ciocalteu (FCR). Gallic acid and quercetin standards were obtained from Sigma-Aldrich.

 

Plant materials:

P. argentea samples consisting of leaf and flower were collected between March and April 2019 from Tikdjda, Bouira (North of Algeria). The samples were identified in the herbarium room at the Botanical Department, Higher National Agronomic School of Algiers.

 

Preparation of Extracts:

The plant samples consisting of different parts (flower and leaf) were washed with tap water, rinsed with distilled water, and then air dried for 7 days. The dried samples were ground into powder form, and each powdered sample was weighed 6g and soaked in ethanol at the ratio of 1:10 (w/v) for 24 h during which time the mixture was sonicated for 30 min in an ultrasonic bath. The extracts were decanted and filtered through Whatman filter paper. The filtrate was then concentrated in a rotary evaporator at temperature 60°C and reduced pressure. The filtrates were frozen and lyophilized in a lyophiliser. The lyophilized powder was stored at −30°C until using for analysis. The powder obtained was used to prepare solutions at different concentrations (0.1, 0.2, 0.5, 1, 2 and 5mg/mL) in distilled water. The extraction yield Y(%) was calculated according to Equation (1):

where:   

Y(%): Extraction yield in %; M₀: Mass of the empty beaker (g); M₁: Mass of the beaker after evaporation (g); M: Mass of dry matter (g).

                                                  

Determination of total phenolic content (TPC):

The concentration of total phenolic content (TPC) in the different parts of plant extracts was determined according to the method explained in¹² with slight modifications. A volume of 500 μL of diluted extracts was mixed with 2.5 mL of Folin-Ciocalteu reagent (diluted 10 times with distilled water) and 2mL of Na₂CO₃ (7.5%). The samples were incubated for 5 min at 50ºC and then cooled. Distilled water (500μL) was used as a negative control for the experiment. The absorbance was recorded spectrophotometrically at 765nm using UV/Vis spectrophotometer. The TPC was determined using Gallic acid as a standard and expressed as mg/g from the calibration curve (y = 2.3214x + 0.0349, R² = 0.9987).

 

Total Flavonoid Content (TFC):

The determination of total flavonoid content (TFC) was conducted according to the methods described in¹³. A volume of 1mL of plant extract is mixed with 1 mL of the 2% aluminum trichloride (AlCl₃) solution. The mixtures were incubated at room temperature for 10 min and then the absorbance was recorded spectrophotometric ally at 415nm. The TFC was calculated using quercetin as a standard and expressed as mg/g from the calibration curve (y = 35.927x + 0.0015, R² = 0.9998).

 

Study of Inhibition of Calcium Oxalate Crystal Growth:

In vitro anti-urolithiatic activity test was carried out by turbidity method using the method of Sasikala et al. ¹⁴ with slight modification.

 

The inhibition of calcium oxalate formation in the presence of the extract was compared with the inhibition of calcium oxalate formation in the presence of the standard (citric acid). The precipitation of calcium oxalate at 37°C and pH 6.5 was studied by the measurement of turbidity at 620nm using UV/Vis spectrophotometer. The turbidity is caused by the formation of calcium oxalate that is due to the reaction of calcium chloride (CaCl₂) with sodium oxalate.

 

In absence of inhibitor:

We chose the turbidity model for the study of calcium oxalate crystallization because of its simplicity, satisfactory and reproducibility. This model includes the study of crystallization without inhibitor and with it, in order to assess the inhibiting capacity of any chemical species used. Solution of calcium chloride and sodium oxalate were prepared at the final concentrations of 5 mmol/L and 7.5mmol/L, respectively in a buffer containing Tris 0.05mol/L and NaCl 0.15mol/L at pH 6.5. For this, a volume of 1mL of calcium chloride solution is mixed with 1mL of distilled water. Crystallization was started by adding 1mL of sodium oxalate solution. The temperature was maintained at 37°C. The measurement of turbidity was done by measuring the absorption by UV/Vis spectrophotometer (Agilent technologies Cary 60 UV-vis) at 620nm. Then, the measurement of the absorbance was carried out to a period of 10 min¹⁴.

 

In presence of plant extract:

The study was continued to know the effect of the different parts of the plant extracts against in vitro stone nucleus formation (formation of calcium oxalate stone). In this experiment, the effect of the extract on inhibition was carried out in five concentrations of the extract (0.1, 0.2, 0.5, 1, 2, and 5mg/mL). For this, a volume of 1ml of 5mM CaCl₂ (in Tris/NaCl buffer, pH 6.5) and 1mL of extract were added to read the blank, and then 1mL of sodium oxalate 7.5mM (in Tris/NaCl buffer, pH 6.5) was added. The measurement of turbidity was done by measuring the absorption by UV/Vis spectrophotometer (Agilent Technologies Cary 60 UV-vis) at 620. Then the measurement of the absorbance was carried out to a period of 10 min ¹⁴.

 

The percentage of inhibition I (%) is calculated by Equation (2) :

 

Where S_i: slope of graph in the presence of inhibitor (drugs/extracts);S_c: slope of graphe without inhibitor (control test).

 

In presence of citric acid (positive control)

1mL of calcium chloride solution is mixed with 1mL of 1.15mg/mL citric acid. Crystallization was started by adding 1mL of sodium oxalate solution. The temperature was maintained at 37°C. The absorbance of the solution was measured at 620nm¹⁵.

 

Microscopic study:

The calcium oxalate crystals growth inhibition study of M. malabathricum was also carried out with the aid of microscopic examination (OPTIKA B-192 binocular microscope 4x, 10x, 40x, 100x). The photographs of calcium oxalate crystals formed in the presence and absence of inhibitors were observed using a light microscope, equipped with digital camera and connected to a computer. The photographs were taken at (G×40) magnification objective lens.

 

Statistical analysis:

The statistical study was carried out using statistical software Minitab® 17. All experiments were performed in triplicate. The results are expressed as the mean± standard deviation, and compared by the Anova test, followed by the Tukey test. P values less than or equal to 0.05 are considered statistically significant.

 

RESULT:

Extraction yield:

In this study, the extraction yield Y(%) (the dry extract, obtained after evaporation) was determined relatively to 6 g of the plant material (leaves, flowers). The weight of the dried extract was determined by the difference between the weight of the full beaker (after evaporation) and the weight of the empty beaker (before evaporation). The results are shown in Table 1.

 

Table 1. Extraction yield Y(%) of the different organs extract of P. argentea.

 

It appears from the observation of the extraction yields Y(%) listed in Table I that the flowers extract give the higher extraction yield with a percentage of 13.83%, while the leaves extract give the yield of 13.41%. The results reported by Adjadj et al.¹⁶ on P. argentea concerning the extraction yield by methanolic maceration is 10.97%. These results are close to those obtained in the previous study on Chinese herbs that the extraction yield depends on the methods used during extraction¹⁷, because the constituents of the cells are released by rupture of the cell walls towards to the solvent¹⁸. It is generally in a crushed form, the plant material will present a greater surface of contact with the solvent, thus making it possible to accelerate its solubility¹⁹. According to Bruneton²⁰, the variation in results from one extract to another is mainly due to the extraction solvents used. Indeed, polar solvents show a better extraction yield compared to less polar solvents. The difference in polarity of the solvents used allows the extraction of a wide range of secondary metabolites²¹. The pH, temperature, extraction time, and phytochemical composition of the sample also affect the variation in extraction results²².

 

Polyphenol and flavonoid contents

Among all the chemical constituents of plants, phenolic compounds occupy only a modest quantitative place. They have multiple properties that have always been sought after by human beings²³. Plant polyphenols and flavonoids could effectively inhibit the formation of CaOx stones in vitro and in vivo, correlating with their diuretic, antioxidant, anti-inflammatory, antibacterial and other protective effects²⁴. For this reason, they are used in many fields including, for example, medicine, nutrition, dyes and cosmetics. In this regard, the determination of the phenolic compounds in the leaves and flowers of P. argentea and the study of their antilithiasic capacity were approached in order to assess the capacity of this plant to synthesize these compounds and assess their antilithiasic activity. All the values ​​illustrated in the levels of Figures 1 and 2 are the average of three repetitions for each part of the plant studied.

 

Polyphenol content:

Figure 1 illustrates the contents of the total polyphenols in the leaf and flowers extracts of P. argentia. These results are expressed in mg of Gallic acid equivalent (GAE) per gram of extract (mg GAE/g extract) using the standard curve equation of Gallic acid.

 

The TPC for the leaves extracts was found to be at 9.29 ± 0.45 mg GAE/g. While the flowers extract was found to be 5.92 ± 0.14 mg GAE/g. The highest phenolic content appeared in the leaves extracts.

 

Figure 1. Variations in the content of total polyphenols in the leaves and flowers ethanolic extracts of P. argentea.

 

The yield of this extraction strongly depends on the nature of the solvent and the extraction method. However, it also depends on the plant material and the richness of bioactive compounds ²⁵. The comparison of our results with those of the literature shows that the polyphenol contents of our extracts are lower than that obtained by Ammor ²⁶ (71.39±1.13mg/g with ethanolic extracts of Herniaria hirsuta). Nevertheless, our results are better than those obtained by Adjadj et al.¹⁶. (0.56± 0.25mg/g with methanolic extracts of Paronychia argontea). Several factors could influence the content of phenolic compounds. Recent studies have shown that extrinsic factors (such as geographic and climatic factors), genetic factors, but also the degrees of maturation of the plant and the duration of storage have a strong influence on the polyphenol content ^27-29.

 

Flavonoids content:

Figure 2 illustrates the contents of the total flavonoids in the leaves and flowers extracts of P. argentia. These results are expressed in mg of quercetin equivalent per gram of dried matter (mg QE/g) using the standard curve equation of quercetin.

 

As shown in figure 2, the leaves extract contain a higher flavonoid content than that of the floral extract estimated at 0.18±0.01mg QE/g while the leaves extract contains 0.47±0.0035mg QE/g.

 

Figure 2. Variations in flavonoid contents in the ethanolic leaves and flowers extracts of P. argentia.

 

Ammor²⁶ found a higher level of flavonoids content in the ethanolic extracts of Herniaria hirsuta (13.93±0.12 mg/g). These results are higher than ours. Adjaje et al.¹⁶ also recorded a higher content of flavonoids than our study with the methanolic extracts of Paronychiya argantia (3.24±0.03mg/g).

 

The differences in flavonoid content may be due to the growing conditions of the plant such as soil, geographic location, environmental conditions during organ development, degree of maturity, harvest and genetic differences³⁰.

 

Antilithiasic activity:

The in vitro antilithiasic activity of the extracts was evaluated by the turbidity method (inhibition of the formation of calcium oxalate). The inhibition of calcium oxalate formation was measured in terms of turbidity using a spectrophotometer. The lower the turbidity, the stronger the inhibition and the less the absorbance will be observed in the spectrophotometer. The inhibition of calcium oxalate formation in the presence of the extract was compared to a negative and a positive control (citric acid). The study was carried out at 37°C with constant agitation at pH 6.5. First, crystal growth in the absence of any inhibitors was performed (negative control) and turbidity was formed immediately after mixing the chemicals. The data obtained were used as a control for the comparison of the growth in the presence of the different parts of plant extracts.

 

The inhibition of crystals formation was calculated by the graphical method using the mathematical Equation (2). The increasing concentration of extracts (0.1, 0.2, 0.5, 1, 2 and 5mg/mL) had inhibited crystal growth by inhibiting the nucleation of calcium oxalate. The results of the nucleation analysis confirmed that the extract contained nucleation-preventing agents.

 

Figure 3 represents the variation of absorbance according to time for the tries with inhibitors 0.1 to 5mg/mL, and Table 2 show the maximum values of the variation of absorbance, and the turbidimetric slopes relating to the concentration of extracts.

 

It was observed that the lower concentration of leaf extract (0.1mg/mL) showed inhibition of 25.87%, while the highest concentration (5mg/mL) showed 50.56% inhibition (Table 2 and Figure 3). On the other hand, the extract of the flowers gave an inhibition of 46.58%, for the lowest concentration (0.1mg/mL) and the highest dose (5mg/mL) gave 70.97% inhibition (Table 3 and Figure 4). After exploring these results, it is found that the floral part has the highest percentage of inhibition compared to the leaf extract.

 

Table 3. Turbidimetric parameters of the oxalocalcic crystallization in the presence and absence of leaves extract.

  

 

 

Figure 3. Crystallization curves in the absence and presence of the leaf extract at different concentrations.

 

Figure 3. Crystallization curves in the absence and presence of the leaf extract at different concentrations.

 

   

Figure 4. Crystallization curves in the presence  of the flowers extract at different concentrations

 

 

Figure 5. Variation of absorbance according to time for tries with the presence of citric acid 1.15 mg/mL

 

Table 4. Turbidimetric parameters of the oxalocalcic crystallization in the presence of flowers extract.

Extract flowers (mg/mL)	Ti (min)	slope (Do/min)	I (%)
0.00	0.012 ±0.002	1.1069±0.321	---------
0.10	0.101±0.028	0.5912±0.0023	46.58
0.20	0.113±0.004	0.5435±0.0114	50.89
0.50	0.121±0.007	0.5226±0.096	52.78
1.00	0.213±0.069	0.4757±0.043	57.02
2.00	0.345±0.063	0.4047±0.029	63.43
5.00	0.486±0.24	0.3231±0.055	70.97
Citric acid 1.15 mg/mL	1.14±0.02	0.0337±0.0037	96.95

 

These results are superior to those obtained by Rajeshwari et al. ³¹ Who worked on the aqueous extract of the leaves and the flowers of Convolvulus arvensis (the higher concentration 100 mg/mL gave 92.27% of inhibition for the leaves extract, and 90.41% for the flowers extract). On the other hand, our findings are lower than  those obtained by Sasikala et al.¹⁴ with the methanolic extract of the roots and stems of Rotula aquatica (the higher concentration 0.5mg/mL gave 70% of inhibition for the roots extract, 50% of inhibition for the stems extract). These differences may be due to the phytochemical composition of the plant, conditions of growth of the plant like the soil, the geolocation of the plant, ambient conditions during the development of the organ, degree of maturity, harvest and genetic differences and the extraction solvent.

 

These results are considered positive because the extract of the leaves and flowers inhibits crystallization and prevents the formation of stones. This property of the extracts is therefore advantageous because it prevents the formation of urinary stones by inducing the excretion of small particles from the kidney and reduces the risk of retention in the urinary tract³². The herb extract contains substances that inhibit the growth of COM crystals. This property of the plant extract can play an important role in preventing the formation of kidney stones¹⁴. If the extract keeps the CaOx particles dispersed in solution, they will be more easily eliminated by the kidneys. Plant extract decreases crystals in solution and reduces supersaturation and particle size.

 

These results are superior to those obtained by Rajeshwari et al.³¹ Who worked on the aqueous extract of the leaves and the flowers of Convolvulus arvensis (the higher concentration 100mg/mL gave 92.27% of inhibition for the leaves extract, and 90.41% for the flowers extract). On the other hand, our findings are lower than  those obtained by Sasikala et al. ¹⁴ with the methanolic extract of the roots and stems of Rotula aquatica (the higher concentration 0.5mg/mL gave 70% of inhibition for the roots extract, 50% of inhibition for the stems extract). These differences may be due to the phytochemical composition of the plant, conditions of growth of the plant like the soil, the geolocation of the plant, ambient conditions during the development of the organ, degree of maturity, harvest and genetic differences and the extraction solvent.

 

These results are considered positive because the extract of the leaves and flowers inhibits crystallization and prevents the formation of stones. This property of the extracts is therefore advantageous because it prevents the formation of urinary stones by inducing the excretion of small particles from the kidney and reduces the risk of retention in the urinary tract³². The herb extract contains substances that inhibit the growth of COM crystals. This property of the plant extract can play an important role in preventing the formation of kidney stones¹⁴. If the extract keeps the CaOx particles dispersed in solution, they will be more easily eliminated by the kidneys. Plant extract decreases crystals in solution and reduces supersaturation and particle size.

 

Study of crystallization by optical microscopy:

The times of the photographs (t = 2 min) correspond respectively to the stage of growth for the tests with and without inhibitor are represented in Figures 6 and 7. Several studies are carried out using an optical microscope to validate the results obtained by the turbidimetric method. In this work we followed the number and the size of oxalocalcic crystals in absence and in the presence of inhibitor. The results obtained are compatible with those achieved by Bensatal and Ouahrani³³. Photographs G, A, B, H and K presented in Figures 6 and7 correspond to the growth stage for the crystallization of calcium oxalate in the absence and in the presence of extracts from the leaves and flowers at low concentrations (0-0.5mg/mL). The comparison between these photographs shows that the decrease in the number and size of the crystals with low concentrations of extracts is not significant, which is why the inhibition increases according to the concentration in the presence of extract compared to the photographs of the concentrations (1, 2, and 5mg/mL).

 

 

Figure 6. Photomicrograph of CaOx crystals in presence and in absence of leaves extract of P. argentia on different concentrations (A: 0.1mg/mL, B: 0.2mg/mL, C: 0.5mg/mL, D: 1mg/mL, E: 2mg/mL, F: 5mg/mL, G:0mg/mL) (G × 40).

 

    

Figure 7. Photomicrograph of CaOx crystals in presence of flowers extract of P. argentia on different concentrations (H: 0.1 mg/mL, K: 0.2mg/mL, L: 0.5mg/mL, M: 1mg/mL, N: 2mg/mL, O: 5mg/mL) (G × 40).

 

The number and the size of the crystals existing in Figures 6 and 7 correspond to the growth stage for the tests in the presence of extract are reduced compared to the growth stage for the tests in the absence of inhibitor. Which explains why the ethanolic extract of the leaves and flowers of P. argentea induces a significant inhibition on the growth of crystals. Numerous studies have shown that extracts from medicinal plants are reacted at the growth stage^34,35. The photographs obtained by the light microscope clearly show that the inhibition manifests at the stage of growth.

 

The plant extract contains substances that inhibit the growth of COM crystals. This property of the plant extract can play an important role in preventing the formation of kidney stones¹⁴.

 

CONCLUSION:

We conclude that P. argentea extract has shown an inhibition activity on the crystal formation when compared to the standard crystal formation using supersaturated calcium oxalate. The flowers extract has shown better urolithiolytic activity than that of the leaves extract. Thus, the extract of P. argentea could be further analyzed in vivo and further characterization of its active compound could lead to the discovery of a new candidate drug for the patients suffering with urolithiasis. These extracts reduce the crystallization of calcium oxalate and can therefore constitute an interesting prophylactic treatment for oxalocalcic lithiasis. It is necessary to carry out more studies in vivo in order to evaluate the cytotoxicity of these compounds and to make pharmacokinetic and pharmacodynamic tests to exploit the extract as a possible anti-lithiasic treatment in the future. It is also important to fractionate the extracts used by chromatography on silica gel in order to know exactly which chemical compounds are responsible for this inhibition.                

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors would like to thank all those who contributed to this work.

 

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Received on 07.09.2021                    Modified on 20.09.2021

Accepted on 28.09.2021                   ©AJRC All right reserved