Anti-urolithiatic activity of Salvia hispanica L. seeds in ethylene glycol induced urolithiasis rat’s model

: Urolithiasis is a disorder of kidneys in which stones formation occur due to the excessive deposition of minerals in the urinary tract. It affects 12% of the population worldwide. Salvia hispanica seeds are rich source of quercetin which has preventive role in renal stone formation. The study objective was to explore scientifi cally the antiurolithiatic effect of Salvia hispanica seed’s methanol extract using in vitro and in vivo urolithiasis models. For in-vitro study nucleation, growth and aggregation assays were performed. In vivo study was performed on rats and they were divided into six groups (n=6). Group-I was given vehicle only. Group-II was disease control, treated with 0.75% EG in drinking water which triggered urolithiasis. Groups-III received cystone (750 mg/ kg, orally). Groups IV–VI were treated with extract at 100, 300 and 700 mg/kg doses orally once daily. Groups III-VI additionally received 0.75% EG in drinking water. In vitro study revealed concentration dependent increase in percentage inhibition of crystal’s nucleation, growth and aggregation. In vivo study revealed anti-urolithiatic activity by lowering oxalate, calcium, phosphate, sodium and potassium levels in the urine and the serum uric acid, blood urea nitrogen, total proteins and total albumin. Salvia hispanica seeds are good alterative of allopathic anti-urolithiatic drugs to treat urolithiasis.


INTRODUCTION
Urolithiasis is a type of urinary system disorder in which crystals development occur when minerals get deposited in the renal, urinary bladder or ureter (Mamillapalli et al. 2016a). Urolithiasis develops when inhibitors (e.g. magnesium) and promotors (e.g. uric acid) of kidney stones lose their balance in the kidneys (Gupta et al. 2011, Alelign & Petros 2018. It has been reported that 80% renal stones are composed of calcium oxalate and calcium phosphate, 10% are struvite containing magnesium ammonium phosphate. Whereas, the drug related renal stones are caused by uric acid (9%) and cystine or ammonium acid urate (1%) (Coe et al. 2005, Divakar et al. 2010. The worldwide prevalence of urolithiasis is recorded as 12% and it affects both male and female of age 20 to 49 years (Alelign & Petros 2018). But it is 2 to 4 times less common in women than in men (Sofi a & Walter 2016, Alelign & Petros 2018. Data showed that the prevalence of urolithiasis in Pakistan is 40-50% (Desai et al. 2011, Ahmad et al. 2016). The non-pharmacological and pharmacological therapies are available but infertility as major side effect and 50% to 80% relapse rats diverted attention of researchers to fi nd out new drugs having minor side effects and minimum relapse cases (Mikawlrawng et al. 2014, Raheem et al. 2017, Türk et al. 2013, Erickson et al. 2011. Many crude drugs have diuretic effect which are considered to be effective in abnormal renal functions, providing a vast scope in the prevention and cure of urolithiasis (Brancalion et al. 2012). The consumption of phytomedicines is increasing day by day because of people believe that natural products have low toxicity or rather no side effect (Mikawlrawng et al. 2014).
Salvia hispanica (S. hispanica) belongs to lamiaceae family which is comprised of 900 species and grow in America (natively), northern Guatamala and Maxico and also in steppe regions (Uribe et al. 2011). S. hispanica seeds have multiple folklore use including antiarthritis, antidiabetic, antihypertensive, anticancer, antihyperlipidemic, antiaging, antianxiety, laxative, immune-booster and also being used in cosmetics and paints (Muñoz et al. 2013, Sandoval-Oliveros & Paredes-López 2012, Ulbricht et al. 2009, Ullah et al. 2016. Literature showed S. hispanica seeds are the richest source of quercetin which belongs to flavonoid class of compounds which are involved in the prevention of renal stones formation due to its strong anti-oxidant activity (Zeng et al. 2018). However, there is no study available in the literature about anti-urolithiatic activity of S. hispanica seeds. Based on the antioxidant potential and the presence of flavonoids in the seeds, the current study was aimed to determine physicochemical and phytochemical properties, in vitro anti-urolithiatic activity by spectrophotometric method and explore in vivo anti-urolithiatic activity of the extract in ethylene glycol induced urolithiasis rats' model.

S. hispanica seeds collection and preparation of extract
Seeds were obtained from the Botanist of Govt. College University, Faisalabad. After cleaning, washing and drying, seeds were ground to powder. One kg seed's powder was soaked in 5 liters of methanol for five days with occasion shaking. Filtrate was obtained by draining extraction solvent via filter paper. Filtrate was passed through rotary evaporator at 40 o C to get semisolid S. hispanica methanol extract which was further used in analysis. Percentage yield was calculated with formula:

=
Actual yield Percentage yield * Theoretical yield

Physiochemical analysis
Total moisture content, total ash content, acid insoluble ash, water insoluble ash, sulphated ash, water and alcohol soluble extractives were quantified (Figure 1) by following methods given in United States Pharmacopoeia-National Formulary (2003).

Phytochemical analysis
Total alkaloid content, phenolic content and flavonoid content in the extract were quantified for phytochemical analysis of S. hispanica seeds methanol extract.

Determination of total alkaloid content
The extract; 1 mg/mL; was mixed with (2 N) HCl and filtered to get clear filtrate. Bromocresol green and phosphate buffer (pH 4.7); each 5 mL; were added to filtrate and mixture was shaken well. Trichloromethane was added to this mixture. Different concentrations of standard atropine (0.5, 1, 1.5, 2 and 2.5 µg/mL) solution was prepared in an identical manner as that of sample in order to construct standard curve.
The absorbance of sample and standard were recorded at 470 nm by UV spectrophotometer. Blank was prepared similarly but without sample and standard. Results were displayed as total alkaloid content in the extract as mg of atropine equivalent (AEq/g) (Das et al. 2018, Tabasum et al. 2016.

Determination of total flavonoid content
Saleem et al. study was followed to quantify flavonoid content. Briefly, sample/standard (1 mg/kg, volume: 200 µL) was mixed with 0.1 mL potassium acetate (1 M) and 100 µL aluminum nitrate solution (10%) and distilled water (100 µL). The sample/standard (querecetin; QTN) was incubated at room temperature for 45 minutes. Blank was prepared similarly except analyte. Absorbance was recorded at 415 nm. Linear regression equation was constructed to calculate flavonoid content in the sample. Total

Determination of total phenolic content
Gallic acid was used as standard to draw the standard curve. 200 µL of Folin-Ciocalteu's phenol reagent was added to 200 µL of sample (1 mg/kg) and standard (1 mg/kg) and mixed well. After 4 minutes, 1 mL of Na 2 CO 3 (15%) was added and mixture was incubated at room temperature for 2 hours. Absorbance was measure at 760 nm. Blank was prepared similarly but it did not contain analyte. Gallic acid equivalents in the sample were quantified using linear regression line of gallic acid. Total phnolic content in the sample were calculated by putting values in the following equation (Saleem et al. 2014): Gallic acid equivalents µg per mL X extract volume Total Phenolic Content Sample g

Figure 1.
Scheme for physicochemical analysis.

Determination of antioxidant activity of extract by in vitro DPPH assay
Sample, control and DPPH were prepared in methanol. DPPH (0.1 mM) 3.5 mL was added to 500 µL of sample and control (ascorbic acid). Sample was prepared in concentration ranging from 0.01 to 1 mM. Blank was prepared identically but 500 µL of methanol was added in place of sample/control. Samples, control and blank were incubated at room temperature for 30 minutes and absorbance was measured in triplicate at 517 nm (Das et al. 2018). Radical scavenging activity was calculated with equation:

100
Absorbance of Sample Absorbance of Blank % scavenging activity * Absorbance of Blank − = IC 50 of sample and control were calculated by drawing graph between percent scavenging activity vs sample concentrations/control.

Nucleation assay
CaCl 2 (0.5 g/L) and sodium oxalate (0.75 g/L) solutions were prepared in Tris HCl buffer (5 g/L; pH 6.5). Cystone was used as standard drug. Two fold serial dilutions were prepared for standard and sample. 100 µg of extract at different concentrations was added to 950 µL CaCl 2 solutions. Next, solution of sodium oxalate (950 µL) was added to it and crystal aggregation started immediately. Then this solution was incubated at 37 o C for 30 minutes. The mixture's optical density (OD) was measured at 620 nm. The percentage inhibition of nucleation by the extract was calculated against standard using following formula (Mamillapalli et al. 2016b, Nirmaladevi et al. 2012, Bawari et al. 2018:

Growth assay
NaC 2 O 4 (4 mM) and CaCl 2 (4 mM) solutions (1 mL each) were added to 1.5 mL of Tris HCL (10 mM) and NaCl (10 mM) buffer having pH 7.2. CaOx crystals (30 μL) were added to this solution. Two fold serial dilutions (200-100 µg/mL) of sample and standard were mixed with mixture having CaOx crystals and incubated for 30 minutes at 37 °C. Absorbance was recorded at 240 nm. The absorption of sample was compared with absorption of standard. The percentage inhibition of growth assay was calculated using formula (Chaudhary et al. 2008):

Aggregation assay
Aggregation assay was performed according to method described by Bawari et al. NaC 2 O 4 and CaCl 2 (each 50 mmol/L) were mixed and placed in water bath at 60 o C for one hour then incubated at 37 °C for 12 hours. Next mixture was evaporated to yield CaOx crystals. One mL Tris HCL (0.05 mol/L) and NaCl (0.15 mol/L) buffer having pH 6.5 was added to 1 mg of CaOX crystals. CaOx solution (3 mL) was added to each concentration (20-80 mg/mL) of extract/standard (cystone) and incubated at 37 °C for 30 minutes. Absorbance of sample and standard was measured at 620 nm. The percentage inhibition of CaOx crystals aggregation was calculated by formula given in the above mentioned nucleation assay (Bawari et al. 2018).

Animals
Healthy albino rats both sexes, weighing 150-200 g, were used for in-vivo anti-urolithiatic activity. Rats were housed in polypropylene cages as six rats/cage (n=6) in the animal house of Govt. College University, Faisalabad-Pakistan. The temperature was maintained at 23 ± 2 ⁰C following a 12 h light & dark cycle. All animals were provided chao and water ad libitum. Rats were placed in the animal house seven days before the start of experimental work in order to acclimatize them with the environment.

Ethical Approval
Ethical approval for in vivo study was obtained from Institutional Review Board of Govt. College University Faisalabad. Study reference was GCUF/ERC/2034.

Induction of urolithiasis in rats
Ethylene glycol (0.75% v/v) and ammonium chloride (1% w/v) were given to animals in drinking water for the initial three days followed by ethylene glycol (0.75% v/v) alone for the next eighteen days for induction of urolithiasis in rats (Iman et al. 2020a, b).

Study design
Thirty six rats were classified into six groups (n=6). Group-I was normal control, receiving vehicle only. Group-II was served as disease control group, receiving ethylene glycol (0.75% v/v). Group-III served as standard and was given Cystone ® (750 mg/kg) orally for three weeks. Groups IV-VI were treatment groups, administered with extract at doses (100, 300 and 700 mg /kg, orally), respectively for 21 days.

Urine collection and measurement of urine parameters
The urine samples were collected at 22 nd day of the study for biochemical analysis, such, as oxalate, phosphate, magnesium, sodium and calcium which were analyzed using standard diagnostic kits.

Determination of the serum parameters
The blood specimens were collected by cardiac puncture at 22 nd day of study under anesthesia and serum was separated for biochemical analysis (serum creatinine, serum uric acid, blood urea nitrogen, serum protein and serum albumin) using standard diagnostic kits.

Histopathological analysis of kidneys
The rats were killed humanely by cervical dislocation after blood samples collection. Both kidneys were removed by incising the abdominal part of the each rat and each kidney was weighed on the weighing balance. Isolated kidneys were preserved in formalin 10% solution. Slides were prepared with hematoxylin and eosin staining solutions. Slides were observed under light microscope and images were captured at 10X.

Statistical analysis
Results were expressed as mean ± S.E.M. Data were analyzed by applying two way ANOVA followed by Bonferooni posthoc test by using Graph pad prism version 5.0. p < 0.05 was set as statistically significant value.

RESULTS
Physical properties and percent yield of the extract The extract was highly viscous with deep green color and specific odor. The percent yield of the extract was found 3.68% (Table I).

Solubility of the extract
The solubility of the extract was assessed in different types of vehicles. The extract was soluble when it was dissolved in vegetable oil, normal saline, distilled water, tween 20-, 60-and 80, volatile solvents like ethanol and methanol (Table II).

Phytochemical analysis
The extract contained 119.08 mg of quercetin equivalents/g of total flavonoid content which were quantified using linear regression equation y = 0.0009x + 0.5989 (R 2 = 0.9711) of the quercetin standard curve. Total alkaloid content in extract were 48.55 mg of atropine equivalents/g of total alkaloid content which were calculated using linear regression equation y = 0.0009x + 0.3215 (R 2 = 0.9748) of the atropine standard curve. Moreover, extract had 7.45 mg of gallic acid equivalents/g of total phenolic content which were determined applying linear regression equation y = 0.0014x + 0.4462 (R 2 = 0.9764) of gallic acid standard curve ( Figure 2; Table IV).

Determination of antioxidant activity of extract by in vitro DPPH assay
The extact and standard (ascorbic acid) showed dose dependent increase in percent scavenging effect but the results of ascorbic acid were more pronounced as compared to extract. IC 50 value of extract was 214.01 µg/mL which was higher than ascorbic acid value i.e. 163.73 µg/mL indicating antioxidant potential of extract is less as compared to ascorbic acid ( Table V).

Determination of anti-urolithiatic activity: in vitro study
Percentage inhibition of nucleation assay, growth assay and aggregation assay was increased dose dependently with extract and standard. Maximally significant percentage inhibition was noted at 1000 mg/kg of extract and standard. The results of extract in nucleation and growth assays at 1000 mg/kg were comparable with that of standard values but extract had more aggregation inhibition than standard at highest selected concentration i.e. 1000 mg/kg (Table VI).

Effect of extract on the body and kidney weights
Body weight of rats in extract and standard treated groups increased significantly (p < 0.001) during study as compared to disease control whereas body weight of disease control was decreased significantly (p < 0.001) during experiment with reference to normal control (Table VII).

Effect of extract on the serum parameters
In disease control group, the levels of serum uric acid, creatinine, blood urea nitrogen, total protein and albumin (4.56 ± 0.02 mg/dL, 0.84 ± 0.02 mg/dL, 14.17 ± 0.02 mg/dL, 6.14 ± 0.02 g/dL summarized in figure 3. No calcium oxalate aggregates and interstitial inflammation were seen in normal control (Figure 3a). Prominent calcium oxalate stone deposition and interstitial inflammation were observed in the disease control (Figure 3b). Standard treated group had histology picture identical to that of normal control (Figure 3c). Extract at 100 mg/kg showed moderate tubular injury with inflammation ( Figure 3d). Extract at 300 mg/kg showed slight interstitial inflammation and tissue damage (Figure 3e). Extract at 700 mg/kg revealed no cellular injury and no interstitial inflammation ( Figure 3f). Dissolution of calcium oxalate crystals was clear at all concentrations of extracts.

DISCUSSION
In current study, physicochemical and phytochemical analyses of S. hispanica seeds powder and extract were performed in order to characterize the seeds. Extract was enriched with flavonoid content followed by alkaloid content and phenolic content which prevent occurrence of urolithiasis by protecting the CaOx crystals formation (Nirumand et al. 2018). Mostly CaOx crystals aggregate and grow in the kidney and resulted in stone formation (Aggarwal et al. 2013). Nucleation, aggregation and growth are three main steps in the formation of CaOx crystals. In vitro study showing dose related increase in percentage inhibition of these three main steps of CaOx crystal formation which is indicative of antiurolithiatic activity of the extract. Our result is in agreement with Bawari et al. study on Daucus carota (Bawari et al. 2018).
For in vivo study, ethylene glycol induced urolithiasis rats model was used. The addition of ammonium chloride with ethylene glycol accelerates the stone formation. Extract treated  and 3.74 ± 0.03 g/dL respectively) were increased significantly (p < 0.001) than normal control values (2.75 ± 0.01 mg/dL, 0.56 ± 0.02 mg/dL, 9.14 ± 0.02 mg/dL, 5.44 ± 0.02 g/dL and 3.06 ± 0.02 g/dL respectively). But in standard and extract treated groups, serum uric acid, creatinine, blood urea nitrogen, total protein and albumin were parallel to normal control values as shown in Table VIII.

Effect of extract on histopathology of kidneys
The results of histopathological examination of the kidneys of all the six groups have been   3.85 ± 0.02*** 0.74 ± 0.02** 12.15 ± 0.02*** 5.74 ± 0.02*** 3.44 ± 0.02*** 700 3.44 ± 0.02*** 0.65 ± 0.02*** 11.05 ± 0.02*** 5.55 ± 0.02*** 3.24 ± 0.02*** increased urinary magnesium level indicating dissolution of CaOx crystals (Dinnimath et al. 2017, Ramaswamy et al. 2015. In this study, urine samples were collected and body weight along with kidney weight (separately) were measured and both of these parameters in extract treated groups were found as that of normal control values. Urine volume was increased in extract treated groups that revealed diuretic activity of the understudied seeds. The weight of kidneys was higher in disease control group due to CaOx crystal precipitation as compared to extract treated groups (Dinnimath et al. 2017). Histopathological analysis also supported whole this data.

CONCLUSION
The present study unveiled anti-urolithiatic activity of S. hispanica seeds methanol extract in EG induced urolithiasis rat model. The extract prevented the formation of calcium oxalate stones by inhibiting early steps of stone formation i.e. nucleation, aggregation and growth. Moreover, the extract displayed anti-urolithiatic activity by decreasing oxalate, calcium, phosphate, total proteins, albumin, blood urea nitrogen and uric acid levels.