Experimental Urolithiasis Model to assess Phyto-fractions as Anti-lithiatic Contributors: A Herbaceutical Approach

Life-style disorders have bought a serious burden on the maintenance of health in animals and humans. Lithiasis specifically nephro- and urolithiasis is no exception and needs urgent attention. Currently, only semi-invasive and surgical methods are widely employed which leads to trauma and reoccurrence of kidney stones. Hence complementary and alternative herbal medicine could pave newer ways in exploring anti-lithiatic contributors. The current study attempts to screen twenty herbal hot aqueous leaf extracts for assessing their antioxidant potency (anti-stress) and efficiency against urolithiasis in an experimental calcium oxalate-induced in vitro (chicken egg membrane) model. The study was further validated by In silico molecular docking studies using the Molegro software package on enzymatic biomarkers involved in scavenging oxidants in the host and regulating oxalate metabolism at a cellular level. Among the screened botanicals Kalanchoe pinnata exhibited promising results compared to the standard chemical (potassium-magnesium citrate) and phyto-formulation drug (cystone) currently used by clinicians for treating urolithiasis. The phytochemical profiling (qualitative and quantitative) and virtual studies indicated rutin from Kalanchoe pinnata as a potential candidate for preventing kidney stones. The results of the current study provide better insights into the design and development of newer, smart, and cost-effective herbal therapeutics making food as medicine. Graphical Abstract


Introduction
Food and health are the two integral components of human wellbeing. However, in the urge of adapting to newer technologies and advanced niches both these components are highly neglected. In consequence, many lifestyle, non-communicative disorders, or diseases has become a part of human health. Indian folklore medicine and Ayurveda proposes the implications of "food as medicine" from time immemorial. Further, complementary and alternative medicine (CAM) has been successful in exploring the scientific proof and mechanism of actions of these phyto-formulations leading to the development of newer drug targets and therapeutics. Phyto-cocktails are effective in many complications from venom antidots (Janardhan et al., 2019;Vineetha et al., 2020, Bhavya et al., 2021, neurodegenerative disorders (Kunnel et al., 2019;Satapathy et al., 2020), diabetes (Putta et al., 2016), ulcers (Prasad et al., 2019), cancer (Rakesh et al., 2015), infection  to inflammation (Rakesh et al., 2016). With all the set examples from our previous studies, one major disorder caught our attention which was a common lifestyle disorder yet very painful, which had no structured therapy and was found in folks of all ages but most prevalent in men known as lithiasis (stone formation).
Lithiasis is a condition in which the formation of stones or calculi is observed due to the concentration of mineral salts (Grases et al., 2007). The kidney, urinary tract, pancreas, and gallbladder are the most commonly affected organs by lithiasis, and they are categorized based on their site of formation leading to nephrolithiasis, urolithiasis, pancreatolithiasis, and cholecystolithiasis respectively (Tatapudi et al., 2020). Stones containing calcium are a common type of calculi with a prevalence of 70-80%, of which calcium phosphate and calcium oxalate dominate (Han et al., 2015). Supersaturation of urine in presence of calcium and oxalate facilitates calcium oxalate stone development (Paliouras et al., 2012). Following are the factors which affect the formation of calcium oxalate stones; acidic urine, low volume of urine, hypercalciuria (increased concentration of calcium in urine leads to precipitation of calcium salts), hyperoxaluria (high concentration of oxalate excreted in urine), hypocitraturia (excretion of a lower amount of citrate in urine which leads to high pH) and hyperuricosuria (acidic urine dissolves uric acid, leading to stone formation). Apart from calcium, magnesium phosphate, uric acid, cysteine, silica, xanthine, and 2, 8-dihydroxyadenine also account for the stone formation (Moe et al., 2006;Evan et al., 2015). Further, with all the above understanding of biochemistry and pathophysiology of urolithiasis, an optimized and standardized model system is a prime necessity that should be rapid, reliable, and reproducible. To serve this purpose, the chicken egg membrane model was employed to study the dissolution of calcium oxalate with nucleation and aggregation assessments (Phatak and Hendre, 2015). Indian traditional system of medicine (Ayurveda) strongly emphasizes the use of phyto-formulations for almost any ailments and urolithiasis, is no exception. Hence an attempt was made to explore the less exploited plants as anti-urolithiatic agents (table 1).
Further, phytobioactives in different forms crude, partially purified, purified, or in cocktails play a vital role either as a single drug or complex provide synergy in ameliorating health complications from a simple infection, inflammation to a cascade of disorders/diseases (Aishwarya et al., 2020). These various activities are mainly due to the different classes of phytochemicals predominately secondary metabolites of polyphenolic umbrella namely phenolics, flavonoids, alkaloids, terpenoids, tannins, saponins, and sterols (Pankaj et al., 2020;Khan et al., 2020). According to Gulcin (2020), a detailed correlation of phytochemicals with antioxidant efficacy is well illustrated and this property could be explored for stress and inflammation which is usually encountered during urolithiasis. Apart from the antioxidant phytobioactives, enzymatic biomarkers such as catalase (CAT), superoxide dismutase (SOD), peroxidase (PER), glutathione S-transferase (GST), contribute against redox reaction, stress, and inflammation respectively (Gopal et al., 2009). Besides, metabolic enzymes such as alanine-glyoxlate aminotransferase, oxalyl-coA decarboxylase, D-glycerate dehydrogenase, and lactate dehydrogenase (LDH) regulate a crucial role in stone formation (Ramu et al., 2017). Elucidation of the mechanism of action of these biomarkers in urolithiasis is very much essential. Structure-activity relationship (SAR) studies were employed to decipher the above set objective using molecular docking studies (Madhusudhan et al., 2016).
Henceforth, in the current study, an attempt has been employed to profile phytochemicals (qualitative), to evaluate the antioxidant potency, and to adapt a simple experimental model for kidney stones (chicken egg membrane) to screen anti-urolithiatic effect (in vitro) of selected 20 plants (aqueous extracts) supported by the in silico evaluation for proof-of-concept in search of highly-efficient and cost-effective herbaceuticals to ameliorate urolithiasis.

Materials & Methods
All chemicals, solvents, and reagents used for the study were of analytical grade and were purchased from Himedia Pvt. Ltd, Mumbai, India. The standard drug Cystone ® was purchased from Himalaya Drug Company, Bangalore, India. Unfertilized chicken eggs were procured from local poultry in Mysuru and membranes were isolated. harvested, leaves were surface sterilized and dried in a hot air oven at 45°C for 48 hours and powdered to 60 mesh packed in an air-tight container until further experimentation.
For extraction: The hot aqueous extract was prepared for all the twenty plants, 20g of finely powdered leaves of the individual plant were mixed with 100 mL of distilled water (pH 5.8) and kept over a magnetic stirrer for 3 hours with a constant heat around 50 ± 2°C (Meghashri et al., 2011). Only aqueous extracts were targeted in the study, intending to isolate polar/hydrophilic phyto-molecules which will be much more practical to suggest patients with urological complications as a homemade decoction. Muslin cloth was used to filter; later, the filtrate was subjected to centrifugation for 10 min at 8000 rpm, after which the supernatant was collected and stored in a freezer for further use.

Phytochemical analysis
All the qualitative phytochemical profiling was performed according to the AOAC method (Shameh et al., 2018) and is represented in supplementary table 1.

Estimation of total phenol content (TPC)
All aqueous plant extracts were assessed by Folin-Ciocalteau (FC) method with a slight modification (Meghashri et al., 2010). Briefly, various volumes of the extracts were made up to 3 mL with distilled water. Folin-Ciocalteau reagent of (1:1 diluted) 0.5 mL was added and incubated for 5 min at room temperature, 1.5 mL of sodium carbonate (20%) was added followed by incubation in a boiling water bath for 5 min. The absorbance was measured at 640 nm. Gallic acid (1mg/mL) equivalents were used as the standard to estimate the phenolic content.

Assessment of In vitro antioxidant activities for aqueous phyto-fractions:
DPPH free radical scavenging assay The free radical scavenging activity of all the plant extracts was measured by the DPPH (1,1-diphenyl-2picrylhydrazyl) method (Meghashri et al., 2010). Varied concentrations of the extracts [5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL, and 25 µg/mL of gallic acid equivalent] followed by 1 mL of DPPH solution, shaken and was incubated at room temperature for 20 min in the dark after which absorbance was measured at 517nm.
Gallic acid was used as standard. The activity of the radical scavenging was calculated by the following equation:

Metal ion chelating assay
The complex ferrous iron -ferrozine can chelate Fe 2+, which can be observed at 562 nm (Meghashri et al., 2010). Different concentrations of all extracts were taken, 50 µL of FeCl 2 and 200 µL of ferrozine were mixed and incubated at room temperature in the dark for 10 min; finally, absorbance at 562 nm against blank was measured. The same equation used in DPPH scavenging activity is used here to know the capacity of the extract in cheating ferrous ions with EDTA considered as standard.

Reducing power assay
Reduction of iron (III) by the extracts was measured with slight modification (Nedamani et al., 2015) by adding the various concentration of all extracts and made up the volume to 500 µL with phosphate buffer (20 mM) and then 500 µL of sodium ferricyanide (1%) was added and incubated at 50°C for 20 min. 500 µL of trichloroacetic acid (10%) was added to the solution to terminate the reaction followed by 10 min centrifugation. Finally, 1.5 mL of distilled water along with 300 µL of ferric chloride (0.1%) was added to the supernatant, and absorbance at 700nm was measured using gallic acid as standard.

Anti-lithiatic activity
The anti-lithiatic activity was assessed for all the extracts according to Phatak and Hendre, (2015). Calcium chloride (25 mM) 1mL of the solution was mixed with 2 mL of tris buffer (pH 7.4). Water was considered as control, and extracts were added to the above mixture; finally, in the end, 1 mL of sodium oxalate (25 mM) was added, and the clock was started. Absorbance at 620 nm for 10 min was measured and a standard graph.

Preparation of semi-permeable membrane from chicken eggs
Among the 20 extracts screened, only the top five extracts (Kalanchoe pinnata, Musa paradisiaca, Punica granatum, Coriandrum sativum, and Moringa oleifera) were used for further in vitro anti-lithiatic egg membrane assay. The complete content of the eggs was removed by piercing with a glass rod on top of the eggs. Then these eggs were washed carefully with distilled water and were immersed in a beaker containing 2N HCL overnight for decalcification. The following day the semi-permeable membrane was cautiously removed from the shells rinsed with distilled water and neutralized with ammonia solution to remove any traces of acid from the membranes and finally rinsed with distilled water.

Synthesis of calcium oxalate through homogenous precipitation
Calcium chloride dihydrate weighing 1.47g was dissolved using 100mL of distilled water, and 100mL of 2N H 2 SO 4 was used to dissolve 1.34g of sodium oxalate. Calcium oxalate starts precipitating with constant stirring when both the above solutions are mixed.
Traces of sulphuric acid is removed by rinsing with ammonia solution and then finally rinsed with distilled water and kept for drying at 60°C for 4 hours (Monika et al., 2012). Calcium oxalate 1 mg/mL served as a negative control and a Cystone concentration of 20 mg/mL was set as a positive control (Phatak and Hendre, 2015). Further, the experimental groups were as follows, Negative control: 1 mL calcium oxalate + 1mL of distilled water, Positive control: 1 mL of calcium oxalate + 1 mL of Cystone solution, Kalanchoe pinnata: 1 mL of calcium oxalate + 500 µL of extract, Punica granatum: 1 mL of calcium oxalate + 500 µL of extract, Musa paradisiaca: 1 mL of calcium oxalate + 500 µL of extract, Coriandrum sativum: 1 mL of calcium oxalate + 500 µL of extract and Moringa oleifera: 1 mL of calcium oxalate + 500 µL of extract respectively.
The above-mentioned groups consisting of both positive and negative controls along with extracts were packed into respective semi-permeable membranes further the mouth of the membrane was tied with the help of thread and was immersed in a conical flask containing 100 mL of 0.1 M tris buffer. Incubator was preheated for 2 hours to obtain a constant temperature of 37°C; all the groups were placed inside the incubator for 7-8 hours (Phatak and Hendre, 2015). The contents of each group were transferred into a fresh flask, and 2 mL of 1N sulphuric acid was added and was titrated against 0.9494 N KMnO 4 till the endpoint that is light pink color is observed. Percentage dissolution was calculated based on titration (Monika et al., 2012). Based on these results the best aqueous extract exhibiting antilithiatic activity was found to be Kalanchoe pinnata and further studies focused on this single phyto-fraction only.

Standard curve of calcium oxalate
Potassium permanganate 3.2 g was dissolved in 1000 mL of distilled water and then boiled for 30 min. Later, filtered using Whatman filter paper to obtain 0.02M of KMnO 4 . Different concentrations of calcium oxalate (0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1 mg/mL) was used and the volume to 1 mL using distilled water. A 4 mL of sulphuric acid was added with 80 µL of 0.02M KMnO 4 . The above mixture was mixed well and incubated for 2 hours after which absorbance was read using a spectrophotometer at 620 nm.
For all further analyses, only the best extract (Kalanchoe pinnata) was intervened to deduce the probable mechanism of calcium oxalate dissolution.

Nucleation assay
The nucleation of calcium oxalate crystals was estimated using a spectrophotometer, and Kalanchoe pinnata inhibiting potency was determined by the method of Saha and Verma (2013), with minor alteration. Calcium chloride 4 mmol/L and sodium oxalate 50 mmol/L were mixed to initiate crystallization, and this was added to artificial urine. The solutions were prepared in Tris 0.05mol/L for calcium chloride and NaCl 0.15mol/L at pH 6.5 and 37°C for sodium oxalate. Nucleation rate was obtained by equating the time of crystal formation about the presence of varying concentrations of Kalanchoe pinnata and with no extract in another and also Cystone was used as the positive control, absorbance was recorded at 1 hour, 3 hours, and 24 hours at 620 nm, percentage inhibition was calculated accordingly.

Aggregation assay
Saha and Verma (2013) method was tailored slightly to obtain the rate of aggregation of crystals of calcium oxalate. Calcium chloride and sodium oxalate 50 mmol/L solutions were mixed to obtain COM crystals. The two solutions were equalized by incubating in a water bath at 60°C for 1 hour and then brought to 37°C followed by evaporation. These crystals were brought to a concentration of 1 mg/mL by dissolving in 0.05 mol/L Tris and 0.15 mol/L NaCl at 6.5 pH. Different concentrations of Kalanchoe pinnata were used along with negative control and positive control being Cystone were observed under a light microscope.

RBC protection assay by Kalanchoe pinnata extract
Consent from the healthy individual to procure erythrocytes was taken. The blood which was heparinized was centrifuged for 15 min at 1000g through which the buffy coat and plasma were separated, and the erythrocytes with the help of PBS were rinsed thrice at room temperature reintroduced into PBS for further analysis, and the volume was made up four times. For 5 min, Kalanchoe pinnata was incubated with erythrocytes and then further incubated for 1 hour at 37°C with hydrogen peroxide, ferric chloride, and ascorbic acid and kept in a shaker incubator for incubation and was observed under an optical microscope for any changes in the morphology (Beulah et al., 2015).

DNA protection assay by Kalanchoe pinnata extract
Protocol from Meghashri et al., (2010) with slight variations was adapted to check the efficacy of Kalanchoe pinnata in protecting the DNA was checked using lambda phage DNA (Meghashri et al., 2010). In the presence or lack of Kalanchoe pinnata and gallic acid, the oxidation with the help of Fenton's reagent along with lambda DNA was estimated for 2 hours at 37°C. Electrophoresis was performed for the samples using 1% agarose gel at 50V DC for 2 hours and stained the gels using ethidium bromide and visualized using gel documentation.

Molecular Docking
In silico analyses provides molecular insights (Ranganatha et al., 2014;Satapathy et al., 2020) on the interaction of ligands (phyto-molecules) with that of the receptors (target enzymes). In the current study, two major classes of targets were analyzed (1). The enzymes regulating redox machinery and (2). Enzymes involved in calcium oxalate regulation. gliding values (Rakesh et al., 2015(Rakesh et al., , 2016Satapathy et al., 2020).

Statistical analysis
All data were expressed as mean ± standard deviation (n = 3). Results were determined using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test using GraphPad Software, Inc (version 6.0, California, USA). The results were considered statistically significant if the P < 0.05. The minimum dosage of extract that is necessary to produce 50% inhibition was known as the effective dose (ED 50 ), which is calculated using regression analysis.

Results & Discussion
Phytofractions are extensively used as nutraceuticals in complementary and alternative medicine to boost health and to prevent stress, inflammation, and secondary lifestyle diseases. The current study investigates the interconnection of antioxidants and regulation of calcium oxalate which is a burgeoning area of research that is gaining increased attention due to the increased number of urolithiasis cases globally. We examined the ability of naturally occurring plant-derived aqueous fractions for their anti-stress and anti-urolithiatic efficacy in the prevention and treatment of the disease using in vitro and in silico platforms.
With the background of Indian traditional medicine (Ayurveda) and folklore, twenty priority botanicals with various other bioactivities were selected namely Aegle marmelos,  (table 1) and their leaves component were screened for their antioxidant and anti-urolithiatic potency using aqueous hot extraction method. This extraction method was intentionally used with the purpose to formulate homemade decoctions with the motto of making food as medicine. The phyto-cocktails from all 20 extracts were subjected to qualitative phytochemical profiling for the presence of carbohydrates, glycosides, alkaloids, phenols, flavonoids, tannins, triterpenoids, steroids, saponin, and gums (supplementary table   1). Among them, most of the aqueous extract was found to be composed of the abovementioned phytochemical classes. However, glycosides and steroids were absent in most of the extracts indicating the non-dissolution of hydrophobic phyto-molecules. Most of the hydrophilic phyto-molecules (tight and loosely bound secondary metabolites) were extracted during the procedure. This observation implies an increased bioavailability of the phytococktail with enhanced bioabsorption within the system. All aqueous extracts were further subjected to the estimation of total phenolic content (TPC) to ensure the quantitative measurements (figure 1). Among the 20 leaves extracts P.granatum, M.oleifera, P.guajava, and C.sativum contented higher levels (nearing 2 mg/mL) whereas, M.charantia and C.sativus exhibited the least (nearing 0.5 mg/mL) phenolic content. The rest of the 14 extracts possessed (a range between1 to 1.5 mg/mL) of phenolics. For all dose-dependent studies, TPC was considered for quantitation.
Oxyradicals majorly reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated during metabolic insult and are the prime cause for biomolecular dysregulation, cellular damage, accounting for cell membrane disruption, mitochondrial dysbiosis, protein folding, inflammation leading to a high frequency of time-dependent diseases like early aging, cancer and neurodegenerative disorders which hampers general health maintenance. According to Vina and co-workers (2005), oxidants are the primordial cause for any disorders. Striking a proper equilibrium with antioxidant molecules will largely facilitate the chances of better survival. Hence a quest for exploring potent phyto-antioxidants is an urgent necessity to overcome lifestyle-related diseases (Garg et al., 2020). In the current study, all the 20 aqueous hot leaf extracts were subjected for their efficacy to quench free radical scavenging (figure 2) in a dose-dependent manner at 5µg/mL, 15µg/mL, and 25µg/mL of TPC respectively. DPPH (figure 2A), metal chelating (figure 2B), and reducing power (figure 2C) assays were employed. Ethylenediaminetetraacetic acid (EDTA) was used as a standard for metal chelating assay and gallic acid was used as the standard for the other two assays. In DPPH assay, A.indica, K.pinnata, P.granatum, and P.emblica were found to have better free radical scavenging activity. Whereas, A.graveolens, C.sativum, C.sativus, and P.amboinicus (Bidchol et al., 2011;Wong et al., 2014;Nedamani et al., 2015) have been well document by the previous researchers.
Besides, the antioxidant potency, the 20 selected botanicals with a concentration of 25µg/mL were subjected to spectral studies, to assess their ability in inhibiting calcium oxalate crystal formation with cystone as the standard herbal drug (figure 3). The results exhibited that among the screened extracts C.sativum, K.pinnata, M.oleifera, and M.paradisiaca, were found to have evidence in inhibiting calcium oxalate crystal formation.
Henceforth for all further studies, these five aqueous phytoextracts were considered. Further, the top five extracts were subjected to an in vitro experimental chicken egg membrane model to mimic the dissolution across the biological membrane (figure 4) of which K.pinnata exhibited 71% dissolution than other extracts in comparison to cystone standard (86%).
Hence for all the further experimentation leaves aqueous hot extract of K.pinnata was used and subjected to critical analyses.
Nucleation and aggregation are the two major phases during lithiasis. To assess the sequential effectiveness of K.pinnata, the extract was subjected in a dose-dependent manner at a range of concentrations (5µg/mL, 10µg/mL, 15µg/mL, 20µg/mL, and 25µg/mL) respectively with 25µg/mL of cystone as standard drug formulation. The calcium oxalate curve was considered as untreated standard (supplementary figure 1) The nucleation assay was assessed at 1h, 3h, and 24 h after incubation (figure 5A). The results provided sufficient evidence that after 24 h of incubation a promising inhibition (70%) of calcium oxalate was observed with K.pinnata extract compared to cystone (76%). Further, aggregation assessment was also performed to have a microscopic birds' eye view on the crystal dissolution ( figure   5B). Thus, K.pinnata extract was found to be a potent anti-crystallization contributor. This could be probably due to higher concentrations of flavonoids, saponins, and gums ( Van-Dooren et al., 2016) which has a shred of qualitative evidence with phytochemical profiling (supplementary table 1).
Further, to evaluate the toxicity of the K.pinnata extract RBC (figure 6: Panel A) and DNA (figure 6: Panel B) protection assay were performed. The K.pinnata phyto-cocktail exhibited potential hampering of the induced oxidant partially at 5µg/mL and completely at 25µg/mL in comparison to gallic acid (pure drug) and cystone (formulation) as standard drug.
Similar kind of studies has been evident with Beulah et al., (2015) and Meghashri et al., (2010) respectively. HPLC profiling was performed (figure 7) on aqueous K.pinnata leaves extract. The sample was found to contain gallic acid at 272nm, rutin at 257nm, and vanillic acid at 261nm whereas, ferulic acid and quercetin were present in minor quantities. The chromatogram for standards has been provided in supplementary figure 2.
Inside a cell, biomarkers such as antioxidant, inflammatory, and calcium oxalate regulating metabolic enzymes play a pivitol role in maintaining cellular homeostasis during lithiasis. Hence an attempt was made to investigate these two classes of enzymes namely catalase (CAT), superoxide dismutase (SOD), peroxidase (PER), glutathione S-transferase (GST), contributing towards redox machinery and stress respectively. Besides, metabolic enzymes such as alanine-glyoxlate aminotransferase, oxalyl-coA decarboxylase, D-glycerate dehydrogenase, and lactate dehydrogenase (LDH) regulate a crucial role in stone formation, and inflammation was considered for the study. Three major ligands namely, gallic acid, rutin, and vanillic acid were considered with potassium-magnesium citrate as the standardcurrently used drug for urolithiasis. Structure-activity relationship (SAR) studies were performed for antioxidant (figure 8) and calcium oxalate regulating enzymes (figure 9) with the best-docked pose. Further, the data have been tabulated in table 2 and table 3 respectively indicating the specific amino acid which comprises the active site for the target and ligand.
Among the docked ligands rutin (molar mass: 610.517 g/mol) was found to be active and best docked on both the classes of the target enzymes than compared to the available drug currently used by clinicians to treat uro/nephrolithiasis. Previous studies on rutin as a potent antioxidant and anti-urolithiatic agent have been well document by Enogieru, et al., (2018) and Ghodasara et al., (2010) respectively. Further, Radwan et al (2021) and Santos et al., (2021) have explored the possible role of the K. pinnata attributes in hemostasis and oxytocin signaling pathways respectively.
Finally, to summarize, the current study has made an exciting observation that K.pinnata aqueous leaves extract can completely suppress the clinical disease in an experimental in vitro model of urolithiasis. However, further studies at a molecular level are needed to test the central hypothesis that K. pinnata has a profound effect on pro-and antiinflammatory responses regulating transcriptional activity which may lead to the amelioration of the disease. Newer in silico algorithms have to be generated which can target gene prediction and pathways that are dysregulated during urolithiasis. In conclusion, these studies should provide novel insights into the basic mechanisms through which phyto-cocktails exhibits anti-lithiatic properties so that it can be used against a wide range of lifestyle diseases leading to better health and wellbeing.

Conflict of Interest:
All authors declare no conflict of interest.