Introduction

Kidney stones are the third most common urinary tract problem with around ten to twelve percent of the population in industrialized countries reporting kidney stones with a high rate of recurrence [1, 2]. Kidney stones patients suffer from severe colic, which cannot be treated with traditional pain killers requiring the use of narcotic analgesics [3]. In addition to pain, obstruction of the urinary tract, urinary tract infections, hydronephrosis, and severe bleeding may occur, and in some cases, surgery may be necessary to remove or break the stones [4]. Kidney stone normally begins as a small crystal-like material, and it gradually builds up into a larger, solid mass. Based on the chemical nature and its composition, commonly occurred urinary stones are referred as calcium oxalate/ phosphate, struvite, uric acid, and ammonium acid [5]. Among these, about 80% of stones are calcium based, and those are calcium oxalate stones [6]. They are usually treated with medications that may cause a number of side-effects. The use of herbs in the prevention and treatment of kidney stone is a useful strategy and safer than synthetic drugs. It has been estimated that 80% of the world’s population relies on traditional medicine for treatment [7].

The stone breaking plants are a group of medicinal plants which are used in Indian traditional medicinal system, claimed to be useful in the treatment of kidney stone. Therapeutic effects of medicinal plants on kidney and urinary tract disorders have been studied in various ways, and their effectiveness have been proven [8]. In recent years, the antiurolithiatic potential of several extracts from different parts of various medicinal plants such as Prunus cerasoides [9], Boldoa purpurascens [10], Micromeria fruticosa [11], and Pleroma pereirae [12] has been reported. The use of such extracts may lead to the complementary and alternative medicine against synthetic drugs. In this scenario, this study aims to elucidate the antiurolithiatic ability of Pleurolobus gangeticus (L.) J. St.- Hil. ex H. Ohashi & K. Ohashi root along with its antioxidant and antibacterial properties.

This plant species, formerly Desmodium gangeticum (L.) DC, has emerged as a vital ingredient of traditional medicine, and it belongs to the Fabaceae family [13]. In Ayurvedic System of Medicine (ASM), the various parts of this species are used as febrifuge, bitter tonic, digestive medicine, anticatarrhal, and antiemetic, in inflammatory conditions of the chest and in various other inflammatory conditions [14, 15]. Many of the Ayurvedic formulations such as “Dashmoola Kwatha” and “Dashamoolarishta” contain this medicinal plant, and it is often known by the moniker “Master of medicinal plants” [14, 16]. The plant is known to be a rich source of important phyto-constituents such as flavonoids, alkaloids, sterols, glycolipids, terpenoids, pterocarpans and coumarins [17]. In addition, elements such as calcium (Ca), phosphorus (P), and magnesium (Mg) were reported from the plant. This chemical variety has made this species highly medicinal, and the plant has been reported for its antidiabetic [18] antiamnesic [19], antibacterial [20], antiinflammatory [21], antinociceptive [22], antioxidant [23], cardioprotective [24], hepatoprotective [25] and antileishmanial [26] properties. Till date, there has been no report on the antiurolithiatic properties of this plant species to our knowledge. This preliminary investigation may help the identification of a new antiurolithiatic agent from this plant species.

Materials and Methods

Collection and Identification of Plant Sample

Pleurolobus gangeticus (L.) J. St.- Hil. ex H. Ohashi & K. Ohashi (Fabaceae) was collected from the Idukki (Latitude, 10.0891° N/Longitude, 77.0597° E) District of Kerala, India. The plant was identified from Botanical Survey of India (Southern Region Center) Voucher Number: BSI/SRC/5/23/2021/Tech/156.

Preparation of Chloroform Fraction (CF)

Fresh roots of P. gangeticus were collected and washed thoroughly with water and dried in shade. One hundred grams of air-dried roots was ground to fine powder and soaked in 80% aqueous ethanol for 48 h with continuous stirring. After soaking, the mixture was then filtered with Whatman No. 1 filter paper. The filtrate obtained was centrifuged at 10,000 rpm at room temperature (25 °C), and the pellet was discarded. The supernatant was collected and concentrated in vacuo by means of rotavapor.The concentrated extract was then washed with n-hexane; further, it was suspended in distilled water and fractionated with chloroform in a stepwise manner [27].

High-Performance Thin Layer Chromatography (HPTLC) Profiling

High-performance thin layer chromatography (HPTLC) analysis was carried out on a HPTLC (Camag, Switzerland) system with chloroform fraction of P. gangeticus. TLC plates precoated with silica gel 60 F254 (20 × 10 cm with 200-μm layer thicknesses) from Merck, Germany, were used for the chromatographic separation of the sample. Five microliter of CF (2 mg/mL) was spotted onto the plates with 8 mm bandwidth using Camag 100 μL sample syringe (Hamilton, Switzerland) and Camag Linomat 5 applicator (Camag, Switzerland). Linear ascending development was carried out in a twin trough glass chamber with Hexane:Ethyl acetate (8:2). Scanning was performed using Camag TLC scanner 3 at 254 nm and 366 nm through fluorescence mode and operated by win CATS software (version 1.4.1, Camag). Plates were visualized under UV 254 nm, UV 366 nm and in visible light.

Fourier Transform Infrared (FTIR) Profiling

The spectra used in obtaining the structural properties of the chloroform fraction of P. gangeticus were obtained from the FTIR spectrometer equipped with an attenuated total reflectance (ATR-FTIR), model Perkin-Elmer Spectrum 400. In the ATR-FTIR method, the sample to be analyzed is placed directly into the sample cell, where a good and reproducible contact between the sample and the crystal of reflection is obtained non-destructively, producing good quality infrared spectra. The FTIR spectra were recorded in the range of 4,000,700 cm−1.

Gas Chromatography-Mass Spectrometry (GC–MS) Profiling

The identification of chemical constituents present in the CF of P. gangeticus was determined by GC–MS (Agilent 7250 GC/Q-TOF). The high-resolution GC/Q-TOF enables accurate mass screening by GC/MS and enhanced compound identification through MS/MS (Detector: Microchannel plate/scintillator/PMT; ADC electronics), equipped with low-energy electron ionization and complimentary chemical ionization techniques at an initial column temperature of 30 °C heated up to 300 °C at 10 °C per 4 min. Helium was used as the carrier gas. Exactly 1 µL of the sample was injected with splitless mode. The ionization voltage was 70 eV. MS scan range was set at 45–450 (MHz). The total running time for a sample was 55 min. The chemical constituents were identified by GC–MS. The compounds fragmentation patterns of mass spectra were compared with those stored in the spectrometer database from the National Institute of Standards and Technology (NIST) Mass Spectral Library.

Pharmacokinetics and Drug-Likeness Profiling

Pharmacokinetics and drug-likeness prediction for the compound stigmasterol (identified from CF through GC/MS profiling) was performed by online tool Swiss-ADME [28] of the Swiss Institute of Bioinformatics (http://www.sib.swiss) [29]. 2D structural models were drawn in ChemBioDraw Ultra version 15.0 (Cambridge Software), and SMILES of stigmasterol was translated into molfile by online SMILES translator and structure file generator found in online tool Swiss-ADME. The analysis task was done to check whether the compound was an inhibitor of isoforms of the Cytochrome P450 (CYP) family, such as CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4. Also, pharmacokinetics (such as gastrointestinal absorption, P-glycoprotein, and blood–brain barrier) and drug-likeness prediction such as Lipinski and Ghose rules and bioavailability score were done [30,31,32]. The Lipinski and Ghose rules were applied to assess drug-likeness to predict whether a compound was likely to be bioactive as per important parameters such as molecular weight, Log P, number of HPA, and HBD. The Swiss-ADME tool used a vector machine algorithm (SVM) [33] with fastidiously cleaned large datasets of known inhibitors/non-inhibitors as well as substrates/non-substrates.

Antiurolithiatic Assay

(a) Nucleation Assay

Calcium chloride (CaCl2) and sodium oxalate (Na2C2O4) solutions were prepared separately, at final concentrations of 5 and 7.5 mmol/L, respectively, in a buffer (0.05 mol/L Tris-HCl and 0.15 mol/L NaCl) at pH 6.5; both solutions were filtered with a Millipore filter paper. For the assay, 950 µL of calcium chloride and 100 µL of CF (2, 4, 6, 8, and 10 mg/mL) were mixed. To this mixture, 950 µL of sodium oxalate was added and stirred well. Afterwards, it was incubated at 37 °C for 30 min. The absorbance was measured at 620 nm with an ultraviolet (UV) visible spectrophotometer (Jasco, UV/VIS Spectrophotometer). Reaction mixture was prepared without the extract was considered as the blank. Experiments with each test sample were carried out in triplicates. Morphology and number of the CaOx crystals formed were also observed using a Leica DM 2500 LED microscope [34, 35].

(b) Aggregation Assay

Solutions of calcium chloride and sodium oxalate at 50 mmol/L were prepared with distilled water and mixed well to form the calcium oxalate (CaOx) crystals. The mixture was then heated to 60 °C on a water bath for 1 h and then allowed to cool at 37 °C overnight. The mixture was then centrifuged at 2500 rpm for 10 min, and the supernatant decanted and the pellets were collected to get the CaOx crystals. A 1 mg/mL solution of CaOx crystals was prepared in Tris–HCl (0.05 mol/L) and NaCl (0.15 mol/L) buffer at pH 6.5. About 100 µL of CF (2, 4, 6, 8, and 10 mg/mL) was added to the buffered solution of CaOx crystals and was mixed properly and the absorbance read at 620 nm wavelength. Experiment with each test sample was carried out in triplicates [36, 37].

Antioxidant Assay

(a) DPPH Radical Scavenging Assay

The free radical scavenging activity of CF of P. gangeticus roots was determined by using DPPH method [38]. Briefly, 0.1 mM solution of DPPH was prepared in 95% methanol and 1 mL of this solution was added to 3.0 mL of CF of P. gangeticus roots at different concentrations (100, 200, 300, 400, and 500 µg/mL). Ascorbic acid was used as the standard for the assay. The reaction mixtures were incubated for 30 min under dark condition at room temperature. Then the absorbance was measured at 517 nm, and the IC50 values were calculated. The experiment was carried out in triplicate, and the DPPH scavenging potential was evaluated.

(b) NO Radical Scavenging Assay

One hundred microliter of CF and standard ascorbic acid at 100–500 µg/mL concentration were added separately to 1 mL of 10 mM phosphate buffered saline at pH 7.4 containing 10 mM sodium nitroprusside and mixed well. This reaction mixture was kept under incubation for 2 h at 30 °C. After incubation, 500 µL of Griess reagent (1% sulfanilamide, 2% H3P04, and 0.1% N-(1- naphthyl) ethylenediamine dihydrochloride) was added. Then the absorbance was read at 550 nm. [39].

(c) Superoxide Radical Scavenging Assay

Nitro Blue Tetrazolium (NBT) solutions (156 mM NBT in 100 mM phosphate buffer, pH 8.0) were prepared, and 1 mL of it is mixed with 1 mL of nicotinamide adenine dinucleotide (NADH) solution (468 mM in 100 mM phosphate buffer, pH 8.0). To this mixture, 100 µL of 100 to 500 µg/mL of CF and standard ascorbic acid was added separately. To the above reaction mixture, 100 µL phenanzine methosulfate (PMS) solution (60 mM PMS in 100 mM phosphate buffer, pH 8.0) was added and incubated at 25 °C for 5 min. After incubation, the absorbance was measured at 560 nm [40].

(d) Total Reduction Capability

The total reducing capacity of the CF was determined according to the method adopted by [41]. Briefly, 2.5 mL of 0.2 M phosphate buffer at pH 6.6 containing (1% w/v) potassium ferricyanide (K3Fe(CN)6) was added separately to 100 µL of different concentrations (100–500 µg/mL) of CF and standard ascorbic acid. This reaction mixture was incubated at 50 °C for 20 min. After incubation, 2.5 mL of (10% w/v) trichloroacetic acid (TCA) was added to each tube, and the reaction mixture was centrifuged at 3000 rpm for 10 min, and the supernatant was collected. 2.5 mL of the supernatant was mixed with 2.5 mL of distilled water, and 0.5 mL of FeCl3 (0.1%, w/v) solution was added, and the absorbance was read at 700 nm.

Antibacterial Assay

(a) Test Pathogens

Pure bacterial cultures of Escherichia coli (MTCC-1687), Klebsiella pneumonia (MTCC-3384), and Staphylococcus aureus (MTCC-3163) were obtained from Microbial Type Culture Collection (MTCC).

(b) Agar Well Diffusion Method

The antibacterial activity of CF of P. gangeticus against selected UTI pathogens was evaluated by agar well diffusion method [42]. The overnight inoculated bacterial cultures were spread over freshly prepared Muller-Hinton agar plates using sterile cotton swabs. Wells of 6 mm in size were prepared using a sterile cork borer (HiMedia), and the wells were loaded with diluted sample (100 μL of 10 mg/mL concentration) of CF. Ampicillin (15 μg) were used as the positive control for the study. All the plates were incubated at 37 °C for 24 h. After the completion of incubation period, the zones of inhibition (ZOI) (mm) were measured and noted.

Statistical Analysis

The results were expressed as Mean ± SD. All the values were statistically evaluated using SPSS version 16.0 by means of one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). Mean values were considered statistically significant when p < 0.05.

Results and Discussion

Plants contain a diverse range of bioactive compounds having definite applications, especially in the fields of medicine [43]. Pharmacologically active compounds are identified from plants mostly through biological activity guided extraction and isolation from crude drugs leading to the discovery of many new drug candidates. Moreover, the use of herbal medicine has many advantages over synthetic drugs including being inexpensive and eco-friendly.

The chemical profile of a crude drug depends on its genetic constitution, geographical location, growth conditions, soil chemistry, and season of harvest of the plant [44]. Extraction is the main process by which bioactive molecules can be obtained from biomass, and the main objective of extraction technique is to maximize the amount of target molecules and to obtain the highest chemical biology of these extracts [45]. The extraction yield and chemical biology of the resultant extract is not only affected by extraction techniques like microwaves, ultrasonic, pressurized liquids, supercritical fluids, and electric fields [46] but also by the extraction solvent [47]. Scientists have reported the impact of different type of solvents on bio-molecule extraction [48, 49]. Generally, polar (ethyl acetate, chloroform, acetone, ethanol, methanol, and water) and non-polar (hexane, benzene, and petroleum ether) solvents are used for extracting bioactive compounds from plant materials [50]. Chloroform extract or fraction derived from P. gangeticus has various pharmacological activities, including antiamnesic [51], inotropic [52], antileishmanial and immunomodulatory [53], antiinflammatory [54], and psychopharmacological [55] activities.

This study was conducted to elucidate the antiurolithiatic ability of the chloroform fraction (CF) of P. gangeticus root against calcium oxalate (CaOx) crystals as part of our pursuit of new and safe antiurolithiatic agents. Moreover, we have also attempted to evaluate the antioxidant and antibacterial potential of CF to establish a possible mechanism for the same. For CF, air-dried roots were pulverized using a milling machine and extracted with 80% aqueous ethanol by using a shaker for 48 h at room temperature (RT). The extract was filtered and concentrated, washed with n-hexane, and was subsequently suspended in distilled water and fractionated with chloroform in a stepwise manner, resulting in a brownish gummy CF with 6.2% yield. Its preliminary phytochemical profiling indicated the presence of amino acids, phenols, alkaloids, flavonoids, coumarins and triterpenoids (Table 1). Many previous phyto-chemical studies on CF of various plant materials were in agreement with the present findings and supported it [56,57,58].

Table 1 Preliminary phytochemical profiling of CF of P. gangeticus
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High-performance thin layer chromatography (HPTLC) is appropriate for the expansion of chromatographic fingerprints to determine major active phyto-constituents through phytochemical marker profiling [59]. The separation and resolution are much better, and the results are much more reliable and reproducible than TLC [60]. It is integrated with digital scanning profiling, densitometry scanning being the main advantage of in situ qualitative and quantitative measurements. Also, a colorful pictorial HPTLC image provides additional and intuitive visual color and/or fluorescence parameters for parallel evaluation of the same TLC plate. It also provided better differentiation of individual/single secondary metabolites [59]. Extracts from various medicinal/aromatic plants have been reported through HPTLC profiling in the past [61, 62]. The present study on HPTLC profiling of CF indicates the occurrence of at least 14 different polyvalent components (Fig. 1). The developed chromatogram will be specific with selected solvent system as hexane: ethyl acetate (8:2) and scanned at 254 nm. The peaks with Rf values of 0.02 (11.10%), 0.15 (10.75%), 0.21 (11.88%), and 0.54 (16.61%) were found to be more prominent. The remaining components are less in quantity; the percentage area of all peaks ranges between 0.96 and 8.99%. The details of major peak are described in Table 2. These sets of information are helpful for the further isolation of individual components from this fraction.

Fig. 1
figure 1

HPTLC profiling of CF of P. gangeticus. a TLC plate visualized at visible light, b TLC plate visualized at 366 nm, c TLC plate visualized at 254 nm

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Table 2 HPTLC major peak details of CF of P. gangeticus
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FTIR offers a rapid and non-destructive investigation into fingerprint of plant extracts. In general, the phyto-extracts contains various classes of compounds which have different bonds (C–C, C = C, C \(\equiv\) C, C–O, C = O, O–H and N–H). These kinds of bonds can be identified by detecting the characteristic frequency absorption band in the infrared spectrum [63, 64]. In the present study, the functional group (FG) identification was made by FTIR profiling, and seven characteristic peaks were found (Fig. 2). The strong intense peaks are observed at 2924.18 cm−1 (range 2935–2915 cm−1) assigned to the H-bonded/O–H stretching vibration, and it indicated the presence of hydroxyl/alcohol/phenolic compounds in the fraction. The peak at 2853.96 cm−1 implied a symmetric stretching of –CH-(CH2)- vibrations corresponding to fatty acids/lipids/protein. Moreover, characteristic peak at 1711.50 cm−1 (range 2100–1800 cm−1) and 1464.92 cm−1 (range 1510–1450 cm−1) was assigned to the carbonyl compound and aromatic ring vibration, demonstrating the existence of some carbonyl and aromatic compounds in the chloroform fraction.

Fig. 2
figure 2

FTIR spectra of CF of P. gangeticus

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The results of the GC–MS analysis of the CF revealed the presence of 25 compounds based on the separation of the individual peaks by the GC (Fig. 3). The mass spectra of these compounds were matched with the spectra of known compounds listed in NIST spectral databases and the main components of which are illustrated in Table 3. The major component in the CF of P. gangeticus was identified as stigmasterol with 45.06% followed by 14-methyl palmitic acid (7.28%), octyl phthalate (5.17%), palmitic acid (5.14%), and 2,4-di-tert-butylphenol (5.12%).

Fig. 3
figure 3

Chromatogram for the GC–MS profile of P. gangeticus

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Table 3 GC–MS profiling of CF of P. gangeticus
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Stigmasterol, also known as stigmasterin, is an unsaturated phytosterol present in various medicinal plants. In the present chemical investigation, stigmasterol was found to be a major component of the CF of P. gangeticus. Stigmasterol is involved in the synthesis of many hormones like androgens, estrogens, corticoids, and progesterone. Moreover, this molecule is already reported for several biological activities such as larvicidal activity [65], antimicrobial [66], antiinflammatory [67], and antioxidant [68]. In addition to stigmasterol, many of its derivatives like stigmasterol glucoside, fucosterol, fluorostigmasterol, cyasterone, spinasterol, and fucosterol epoxide have also demonstrated potent pharmacological properties [69].

In this context, we tried to understand the pharmacokinetics and drug-likeness properties of stigmasterol through an in silico study. The computer modeling of ADME-PK properties of compounds provides an idea about structure–property relationships and drug metabolism and pharmacokinetics properties based on its chemical structure [70]. We recently successfully demonstrated the pharmacokinetics and drug-likeness properties of pterocarpans [44], quinones [71], and phenothiazines [72]. In silico approaches are applied at an early phase of the drug development process, in order to remove molecules with poor ADME-PK properties and making significant savings in research and development costs.

The chemical structure and physicochemical properties (Lipinski’s rule) of stigmasterol were analyzed as per our previous study [73], and the same is illustrated in Table 4. These information help to determine whether a biologically active chemical is having the physico-chemical properties to be orally bioavailable. The ADME-PK studies of stigmasterol revealed low gastrointestinal (GI) absorption and lack blood–brain barrier (BBB) permeability and P-glycoprotein (P-gp) permeability (Table 5). Moreover, the lipophilicity (log Po/w) and skin permeation (log Kp) of stigmasterol was found to be 6.97 and − 2.74, respectively. Furthermore, the compound showed inhibition of CYP2C19 (cytochrome P450 isomer). The topological polar surface area (TPSA) was 20.23 Å (≤ 140 Å), indicating that compounds have appropriate oral bioavailability (0.55). The compound meets the criteria of drug-likeness assessment based on Lipinski and Ghose rule (Table 6).

Table 4 Chemical structure and physicochemical properties of stigmasterol identified from the CF of P. gangeticus
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Table 5 Pharmacokinetic studies of stigmasterol identified from the CF of P. gangeticus
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Table 6 Drug-likeness and medicinal chemistry of stigmasterol identified from the CF of P. gangeticus
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Urine is supersaturated in calcium oxalate (CaOx) implying that it will contain CaOx crystalline particles that are formed spontaneously. Nucleation is an initial step in this where the supersaturated solution is converted to solid phase [74].When crystalline particles are retained within the kidney, they can grow to become full-size stones through growth and aggregation processes. Our in vitro inhibitory effect of CF on CaOx crystallization begins with nucleation assay. For this, different concentrations (2, 4, 6, 8, and 10 mg/mL) of test solution of CF were prepared, and the inhibitory effect was tested and depicted in Table 7. The minimal turbidity at 2 mg/mL of the extract was observed to be 0.377 ± 0.006, and the maximum absorbance at 10 mg/mL was observed to be 0.652 ± 0.006. In control, the addition of sodium oxalate solution into CaCl2 solution results in the formation of numerous large CaOx crystalline particles (Fig. 4A). Similarly, the presence of CF leads to an increase in turbidity with the formation of small fine crystals (Fig. 4B–F). When compared to the control, the absorbance was observed to be more with increased concentration of CF (Table 7). Results of our microscopic evaluation corroborate the findings of the assay. It showed a gradual increase in the number of crystals with reduced size in the presence of the CF (Fig. 5). Stones with smaller size have the possibility of spontaneous passage and elimination through the urinary tract without any discomfort [75, 76]. These results showed a promising outcome for the study. Bawari and co-workers reported antiurolithiatic potential of wild Himalayan cherry (Prunus cerasoides) through CaOx crystal. In this assay, they found turbidity and formation of small fine crystalline particles depend up on the concentration of test solution [9]. Likewise, similar observations also found in Desmodium styracifolium [77] and Betula alba [78].

Table 7 Effects of CF of P. gangeticus on CaOx crystal nucleation
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Fig. 4
figure 4

Representative photographs of CaOx crystals from in vitro crystallization experiment as observed under light microscope (× 40). A Control, B 2 mg/mL, C 4 mg/mL, D 6 mg/mL, E 8 mg/mL, F 10 mg/mL

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Fig. 5
figure 5

Number of crystals observed in the CaOx crystallization assay

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The microscopic evaluation showed a morphological conversion of calcium oxalate monohydrate (COM) crystals to calcium oxalate dihydrate (COD) crystals in the test solution. The transformation of pointy edged dendritic COM crystals to octahedral shaped COD crystals with reduced size and number through the inhibition of COM crystals showed in Fig. 4. This clearly indicates the COM crystal inhibitory potential of the CF. The COM crystals are thermodynamically more stable and bound tightly to the renal epithelial cell surface than the COD crystals, and this will cause renal tubular damage and form difficulties [79]. Moreover, COM crystals show more affinity and strong attachment to the renal epithelial cells than the COD crystals, and this leads to the retention of stones in the kidney [80].So, the transformation from COM to COD is considered as an important step in the treatment of urolithiasis. Similar findings are reported in various plant extracts [80,81,82]. For example, hydro-alcoholic extract from the Bergenia ciliate significantly inhibited the formation and reduction of size of the COM crystals, and meanwhile, it promotes the COD crystals with reduced size [81].

Aggregation is considered as a crucial and final step in urolithiasis, and it is the key factor behind stone retention. During the process, stones get aggregated within a short time, and its elimination through urethra is obstructed [83]. Our results showed gradual increase in absorbance in a dose-dependent manner at all concentrations (Table 8). The turbidity was found to be 0.349 ± 0.007 at 2 mg/mL concentration of CF, whereas the absorbance was found to be increasing to 0.761 ± 0.005 at 10 mg /mL concentration. Absorbance increases due to the breakdown of preformed calcium oxalate crystals into fine particles in the solution. The CF also allows the CaOx crystals get dispersed in the solution and this will help its elimination during the urination and reducing the severity and retention of the stones. The results showed clear inhibitory action against the agglomeration of crystals. Similar findings were previously recorded by several authors and found the clear inhibition of the aggregation of crystals by various plant extracts. Recent reports on the antiurolithiatic potential of betulin obtained from Betula alba found to be efficient in preventing the crystal aggregation, and the results showed significant increase in turbidity along with the increase in concentration of the extract [78]. The aqueous extract obtained from Phyllanthus niruri found to be effective in preventing the CaOx aggregation and is capable of interfering with the early stages of stone formation [84]. Similar results also observed in the extract of Eysenhardtia polystachya [85]. All these findings supported the outcome of the present study.

Table 8 Effects of CF of P. gangeticus on CaOx crystal Aggregation
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The oxalate triggered damages occurring in the renal epithelial cells may also be a reason for urolithiasis [78]. The exposure of renal epithelial cells to different type of crystals like CaOx, calcium phosphate, and uric acid produces reactive oxygen species (ROS) [86]. The production of reactive oxygen species (ROS) is also found to be a reason for the development of kidney stones, and it causes damages to the renal epithelial cells through the collapse of oxidant–antioxidant balance in the kidney [87]. The crystal attachment to the tubular cells causes oxidative stress, and it leads to the renal cell injury [88]. The membrane damage also occurs as a result of the absence or reduction of antioxidant enzymes [89]. Thus, the antioxidants have the capacity to prevent the ROS production and to protect the renal epithelial cells from further damage. Plant secondary metabolites are well known for their potential to ameliorate ROS-mediated oxidative stress, which could be beneficial in the management of urolithiasis [88].

In the DPPH assay, the CF of P. gangeticus showed strong inhibition of DPPH radical when compared to the standard ascorbic acid. The fractions showed the capacity to reduce the stable DPPH radical (purple) to its non-radical form DPPH (yellow). Maximum percentage of inhibition of 71.33% was observed at 500 µg/mL, and minimum percentage of inhibition of 37.66% was observed at 100 µg/mL. The half maximal inhibitory concentration (IC50) is 415.9327 µg/mL. The DPPH scavenging potential of CF is depicted in Table 9.

Table 9 Effect on different radical-scavenging activities of CF of P. gangeticus
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The CF of P. gangeticus showed strong promising reduction of the nitrite levels formed due to the decomposition of nitroprusside. Maximum percentage of inhibition of 52.66% at 500 µg/mL concentration and minimum percentage of inhibition of 18.33% at 100 µg/mL concentration were estimated. The half maximal inhibitory concentration (IC50) was 391.729 µg/mL. The NO radical scavenging potential of CF is depicted in Table 9.

In the superoxide radical–scavenging assay, the generated super oxide anion radicals react with NBT and form coloration, measured at 560 nm. The results observed are depicted in Table 9. Maximum percentage of inhibition of 56.66% at 500 µg/mL concentration and minimum percentage of inhibition of 14.66% at 100 µg/mL concentration were observed. The half maximal inhibitory concentration (IC50) was 275.971 µg/mL.

The total reduction capability of the CF showed promising results for its antioxidant activities implying that it can protect the tissues from oxidative stress. Our result showed an increase in the absorbance at 700 nm and is depicted in Table 9. The minimum inhibitory percentage was observed at 100 µg/mL concentration is 0.30%, and the maximum inhibitory concentration at 500 µg/mL is 0.90%. The IC50 value observed was 419.14 µg/mL. This result demonstrated the capacity of CF to prevent the ROS formation and reduce the possibilities of stone formation.

Urolithiasis has a strong connection with the bacterial infections. Patients suffering from urolithiasis may have higher chances of urinary tract infections (UTI), and in some cases, the UTI may lead to the formation of kidney stones [90]. The term “infection stones” refer to the subset of urinary stones that consist of stone components associated with the UTIs [91]. Uropathogens such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa lead to the formation of UTIs and may cause the formation of kidney stones. The result of the antibacterial assay is depicted in Table 10. The maximum activity was observed in Klebsiella pneumoniae with a zone of inhibition (ZOI) of 12.33 ± 1.05 mm, and the minimum activity was observed in Staphylococcus aureus with a ZOI of 8.46 ± 0.27 mm. Our results suggest strong antibacterial potential of the CF against the selected uropathogens, also preventing UTI and stone formation. Several reports suggest the promising antibacterial potential of plants against uropathogens [92, 93].

Table 10 Antibacterial activity of CF of P. gangeticus
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Conclusion

P. gangeticus is an important medicinal plant having various pharmacological properties. In the present study, the CF of P. gangeticus showed good inhibition against CaOx crystal formation through nucleation and aggregation. The HPTLC profiling of CF showed the presence of many polyvalent compounds, and FTIR profiling gave the idea about the functional groups and nature of these polyvalent compounds. Moreover, the GC/MS analysis indicated that CF contains many biologically important phyto-constituents. The stigmasterol was found to be a major compound in CF. In this context, ADME-PK property of this molecule was established. To support antiurolithiatic activity of CF, its antioxidant and antibacterial properties were investigated. This is the first record of antiurolithiatic potential of P. gangeticus along with the antioxidant and antibacterial activity. In our laboratory, bioassay-guided studies are undergoing, which may lead to the identification of new antiurolithiatic agents from this plant species.