Urolithiasis is a disease caused due to the precipitation and retention of crystal-forming salts within the kidneys. The disease is marked by a recurrence rate of approximately 40% in 5 years of initial treatment . Currently available medical expulsive therapy is a wait-and-watch kind of approach where success rate depends upon a number of factors such as location of the stone, size of the stone, associated obstructions, etc. . The high recurrence rate of urolithiasis  and risk factors associated with synthetic medicines  have necessitated the need for safer, reliable, cost-effective, and noninvasive alternatives to manage urolithiasis. During the last few decades, tremendous research has been focused on revealing the role of medicinal plants in the management of urolithiasis. Traditional medicines have been known to result in significant outcomes and play a critical role in treating and preventing human ailments along with paving the way for discovering new drugs. Antiurolithiatic herbs have been used to treat or prevent lithiasis worldwide throughout history.
The interaction of calcium oxalate monohydrate (COM) crystals with renal tubular cells elevate the production of reactive oxygen species (ROS) and stimulate epithelial cell injury, leading to inflammation and eventually cell death [5,6]. Renal epithelial cells injury by COM crystals provide adhesion sites for crystals, which serve as fixed particles for stone formation [7,8]. Epithelial cell injury also adds oxidative stress, further increasing the chances of stone formation . Considering the role of oxidative stress, treatment modality for urolithiasis should also include therapeutic agents possessing antioxidant abilities. It is a proven fact that the plants having diuretic and antioxidant activities possess inhibitory effects on crystallization, nucleation, and aggregation of crystals, thus having antiurolithiatic activity . Both the plants used in this study have been explored for their diuretic, antispasmodic, antioxidant, and other medicinal properties yet not well explored for their antiurolithiatic property [11–13]. These plants possess strong historical background for being used traditionally as antiurolithiatic agents [14–16]. Therefore, in the present investigation, we have compared the effect of hydroalcoholic extracts of two traditional medicinal plants Bryophyllum pinnatum (BPE) and Macrotyloma uniflorum (MUE) on ROS, COM crystal-induced cell injury, and wound healing to evaluate their antiurolithiatic potential.
2. MATERIALS AND METHODS
2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS), Dulbecco’s modified Eagles’s medium (DMEM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) were purchased from HiMedia Laboratories, India. Remaining chemicals were purchased from Central Drug House (P) Ltd (CDH) fine chemicals, India.
2.1.2. Plant materials
Plants and seeds of BPE and MUE, respectively, were collected locally and identified at the Department of Botany, University of Delhi. Voucher specimens of the authenticated BPE (accession number DUH14500) and MUE (accession number DUH14479) were submitted in the herbarium unit for future reference.
2.1.3. Cell culture
Vero cells were procured from the National Centre of Cell Sciences (NCCS, Pune, India). The cells were maintained as subconfluent monolayers in DMEM supplemented with 10% FBS and 1.2% antibiotics (penicillin-streptomycin) in a humidified incubator at 37ºC with 5% CO2.
2.2.1. Preparation of plant extracts
The leaves of BPE (50 g) were crushed to form a paste and mixed with 70% ethanol, while the powdered seeds of MUE (5 g) were mixed with 70% methanol to prepare hydroalcoholic extracts. After filtration and solvent evaporation, the obtained concentrate was lyophilized and stored in air-tight containers at 4ºC for further use. The percentage yield of each extract was calculated using the following formula:
where W1 is the weight of the extract after solvent evaporation and W2 is the weight of the leaves/seeds taken.
The powdered hydroalcoholic extracts of BPE and MUE were dissolved in distilled water to get desired concentrations before each experiment.
2.2.2. Phytochemical screening of the plant extracts
Qualitative phytochemical screening of BPE and MUE was carried out to detect the presence of phytoconstituents, such as tannins, flavonoids, saponins, alkaloids, glycosides, terpenes, and steroids, using standard procedures. Afterward, total phenolic content of the extracts was estimated spectrophotometrically at 723 nm using UV-Vis spectrophotometer and expressed as Gallic acid equivalent (GAE) milligrams per gram of the extract .
To confirm the presence of individual phytocompounds, gas chromatography–mass spectrometry (GC–MS) analysis of plant extracts was carried out. Model QP-2010 Plus (Shimadzu, Japan) of the gas chromatograph mass spectrometer was used for the GC–MS analysis. Samples of BPE and MUE were prepared by mixing them in ethanol and methanol, respectively. The samples were dissolved properly by vortexing. Sample solutions having a concentration of 1 mg/ml were used for analysis. Out of which 1 μl of sample was injected into an Rtx-5 MS capillary column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness) for analysis with injector–detector temperatures to be 250ºC and 280ºC respectively. The stepped temperature was programmed as held at 80ºC for 3 minutes and then from 80ºC to 280ºC at a ramp rate of 10ºC/minute. The retention time of components was compared with standard components provided by different databases such as WILEY8LIB and NIST11LIB for identification .
2.2.3. Determination of antioxidant activity
The free radical scavenging activity of extracts was evaluated using DPPH and ABTS assays as reported in the literature. DPPH and ABTS assays were carried out according to the method described by Jadid et al.  and Saeed et al. , respectively.
2.2.4. Effect of plant extracts on COM crystal-injured cells
COM crystals were prepared using the method described by Semangoen et al. . Before using, purity of harvested crystals was confirmed by Fourier transform infrared (FTIR) analysis (data not shown) and crystals were exposed to UV radiation to avoid any contamination.
Vero cells were divided into control, injured, and extract-treated groups. Injury was induced by adding fresh DMEM (without serum) containing COM crystals (200 μg/ml). For the extract-treated group, injured cells were exposed to different concentrations of BPE (10–400 μg/ml) and MUE (10–400 μg/ml) extracts. Cells exposed to serum-free DMEM without COM crystals were used as control. The effect of BPE and MUE on the repair of cell injury was assessed by measuring cell viability through MTT assay  against cells injured with COM crystals (injury group without treatment). Percentage cell viability was calculated using the following formula:
where At is the absorbance of test sample, Ac is the absorbance of control, and Ab is the absorbance of blank.
Finally, effective concentrations (EC50 and EC90) of both the extracts, i.e., BPE and MUE, were calculated using AAT bioquest EC50 calculator (https://www.aatbio.com/tools/ec50-calculator/).
2.2.5. Wound healing assay
Extracts were evaluated for their wound healing potential through scratch assay using the method reported by Danciu et al. . Cells were divided into test (with extract) and control (without extract) groups. For the test group, cells were exposed to serum-free DMEM containing BPE and MUE extracts at a concentration equivalent to calculated EC50 and EC90 values. Pictures of the scratched surface were taken at different time intervals (0, 3, 18, and 24 hours) under an inverted microscope (Olympus Life Science) at 10× magnification. Image Pro-Plus software (Media Cybernetics) was used to compare the images at different time intervals  and wound closure percentage was calculated  against control.
3. STATISTICAL ANALYSIS
Each experiment was carried out in triplicates (n = 3) and results were presented as mean + SD. GraphPad prism 6.0 software (GraphPad Software, San Diego, CA) was used for data presentation. One-way ANOVA was used to compare differences among different groups. Associated probability (p) value of < 0.05 was considered as statistically significant.
4.1. Preparation of plant extracts
The % yield of seeds extract of MUE was 11.4% as compared to 9.2% yield of leaves extract of BPE.
4.2. Phytochemical screening of the plant extracts
Preliminary phytochemical screening of BPE and MUE confirmed the presence of different classes of bioactive phytoconstituents in the extracts. Tannins, flavonoids, alkaloids, and steroids were found to be present in both the extracts. Unlike MUE, BPE was found to possess saponins. Cardiac glycosides were present in MUE and absent in BPE. Total phenolic contents of BPE and MUE were estimated to be 74.11 ± 0.003 and 34.11 ± 0.0015 μg/mg GAE, respectively.
Finally, the extracts were analyzed using GC–MS to screen out specific phytoconstituents. GC–MS analysis of BPE led to the identification of 62 compounds. Out of these, many compounds, such as eucalyptol, 1-tridecanol, terpinyl acetate, beta caryophyllene, squalene, vitamin E, gamma sitosterol, kolavenol acetate, alpha amyrin, etc., are of medical significance (Fig. 1, Table 1).
4.3. Determination of antioxidant activity
BPE exhibited significant radical scavenging activity against DPPH as well as ABTS free radicals. Percentage radical activity increased from 1.91% to 53.99% in DPPH assay and 39.26%–97.44% in ABTS assay with an increase in the concentration of BPE (12.5–400 μg/ml) (Fig. 3a).
Percentage radical scavenging activity of MUE was comparable with that of BPE. Different concentrations of the extract (12.5–400 μg/ml) resulted in dose-dependent antioxidant activity from 5.70% to 45.14% in DPPH assay and 17.96%–96.04% in ABTS assay (Fig. 3b).
4.4 Effect of plant extracts on COM crystal-injured cells
The effect of treatment with BPE and MUE was evaluated using cell viability assay against the injured group (without treatment). Exposure of the cells to COM crystals resulted in more than 40% decrease in cell viability against control (without COM crystal exposure). However, treatment of the injured cells with different concentrations of BPE and MUE extracts resulted in significantly improved cell viability which was at a comparable difference with the control group. BPE at a concentration of 200 μg/ml resulted in the retention of 98.5% viability after COM crystal exposure to Vero cells. The effect was found to be dose-dependent and more than 76.56% cell viability was retained at the lowest concentration of BPE (10 μg/ml).
|Figure 1: GC–MS chromatogram of hydroalcoholic extract of BPE leaves’ extract.|
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|Table 1: Chemical compositions of hydroalcoholic extract of BPE leaves (GC–MS).|
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|Figure 2: GC–MS chromatogram of hydroalcoholic extract of seeds of MUE.|
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Similarly, MUE also exhibited dose-dependent effect on cell viability against the injured group (Fig. 4b). Retained cell viability was found to be in the range of 77.4%–90.74% with different MUE concentrations. Both the extracts showed maximum activity at 200 μg/ ml.
Using the data of cell viability assay, EC50 and EC90 values were calculated for both the extracts. EC50 and EC90 for BPE were found to be 104.26 and 109.18 μg/ml, respectively. However, in case of MUE, EC50 and EC90 were found to be 74.64 and 246.09 μg/ml, respectively.
|Table 2: Chemical compositions of hydroalcoholic extract of seeds of MUE (GC–MS).|
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|Figure 3: Free radical scavenging potential of BPE (3a) and MUE (3b). Results are expressed as mean ± SD.|
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|Figure 4: Treatment of COM crystal injured Vero cells with BPE (4a) and MUE (4b). Activity of both plant extracts was dose-dependent. Values are expressed as mean ± SD. |
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4.5. Wound healing assay
Wound healing potential of both the extracts was evaluated at EC50 and EC90 concentrations. Treatment with EC50 of BPE (Fig. 5a) resulted in 84.48% wound closure after 24 hours which was significantly higher than the control group (73.79%). However, treatment with EC90 of BPE led to 74.08% wound closure which was not at a significant difference with the control (Fig. 5b). EC50 and EC90 of MUE resulted in 85.66% and 91.09% wound closure which were significantly higher than the control group (Fig. 5c), respectively.
Urolithiasis remains a medical challenge worldwide and the problem is increasing day by day due to the modern lifestyle. Moreover, the recurrence rate of urolithiasis is very high. In the present study, we evaluated the antioxidant, wound healing assays, and anti-urolithiatic potential of hydroalcoholic extracts of BPE and MUE. Both extracts exhibited dose-dependent radical scavenging activity against DPPH as well as ABTS free radicals. In the cell viability assay, it was observed that exposure of the Vero cells to COM crystals significantly reduced the cell viability to 57.4% with respect to 100% in case of control group (without COM crystal treatment). The data clearly indicated that exposure of the cells to COM crystals resulted in cytotoxicity and the results were found to be in agreement with the findings of Mittal et al. . Interestingly, the treatment of injured cells with the plant extracts resulted in a dose-dependent significant improvement (p < 0.0005) in the retained cell viability even at a lower concentration (10 μg/ml) for the BPE and MUE versus injured group. When we increased the extract concentration from 10 to 400 μg/ml, there was a significant increase in the retained cell viability. The results were found comparable to the control group at higher concentration of BPE (above 200 μg/ml). The exact mechanism responsible for this improved cytoprotective activity is not clear yet but it may be correlated with high antioxidant property and excellent wound healing potential of both the extracts.
MUE exhibited better wound healing efficacy than BPE when compared to control (untreated group). Treatment with EC50 of BPE resulted in significant improvement (p < 0.05) in wound closure. However, both EC50 (p < 0.0005) and EC90 (p < 0.0001) of MUE led to improved wound closure rates and were at comparable differences with the control. The results of the cell viability assay, antioxidant activity, and wound healing assay may be correlated with a number of pharmacologically active phytoconstituent identified during preliminary screening and GC–MS. Phytocompounds such as eucalyptol, 1-tridecanol, terpinyl acetate, beta caryophyllene, 3, 7, 11, 15-tetramethylhexadec-2-en-1-ol, squalene, vitamin E, gamma sitosterol, kolavenol acetate, and alpha amyrin are capable of influencing metabolic reactions in different ways. Eucalyptol is reported to possess ulcer healing abilities involving antioxidant and cytoprotective effect . A sesquiterpene, β-caryophyllene which was present in a significant concentration in BPE, has also been investigated for its cytoprotective effects and antioxidant activities [28,29]. Vitamin E along with another compound 3, 7, 11, 15-tetramethylhexadec-2-en-1-ol, which is also known as phytol, was found in significant concentrations in BPE. Phytol is a precursor of vitamin E and can be considered as a source of antioxidant [30,31]. Another polyunsaturated hydrocarbon squalene present in BPE is capable of preventing oxidative damage and act as a natural antioxidant [32,33]. Beta elemene and its derivatives, present in BPE, are already reported to possess potent antioxidant activity against ROS production . The presence of mome inositol as a major phytoconsituent along with terpinyl acetate, gamma sitosterol, and beta caryophyllene in smaller amounts in MUE also justifies its role in repairing the cell damage due to COM crystal exposure. The literature reveals the antioxidant activity of alpha amyrin . These phytocompounds could be responsible for providing the extract its antioxidant and wound healing abilities. Many medicinal properties have already been attributed to mome inositol, such as antialopecic, anticirrhotic, antineuropathic, cholesterolytic, lipotropic, and sweetener [36,37].
|Figure 5: Image showing wound closure at different time points (5a). Percentage of wound closure in BPE (5b) and MUE (5c) treated groups. Values are expressed as mean ± SD. |
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The presence of eucalyptol, β-caryophyllene, vitamin E, phytol, and squalene may be providing the antioxidative property to BPE. In MUE also, the presence of compounds like mome inositol, terpinyl acetate, β-caryophyllene, etc. could probably be the factors attributing its antioxidative potential.
In recent years, many surgical and nonsurgical treatment methods for urolithiasis have been emerged, but recurrence of the disease remains a challenge with modern treatment modalities. According to the literature, the generation of free radicals in kidneys plays the key role in the pathogenesis of renal calculi . In such conditions, using plant-derived antioxidants can be a wonderful approach to combat urolithiasis. Seeds of MUE and leaves of BPE are rich sources of antioxidants and have been used traditionally to treat kidney stones, thus serving the purpose.
In summary, hydroalcoholic extracts of MUE and BPE are rich in phytocompounds having antioxidative, cytoprotective, and wound healing properties. They are capable of protecting the renal cells which are injured with calcium oxalate crystals. However, specific phytochemicals of the extracts may be explored for therapeutic efficacy against urolithiasis.
The authors would like to acknowledge Jaypee Institute of Information Technology, Noida, India, for providing the facilities to conduct the research work.
8. AUTHORS’ CONTRIBUTIONS
We, the authors hereby declare that both authors have made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content and both agreed to submit to the current journal. Therefore, both the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
There is no funding to report.
10. CONFLICT OF INTEREST
The authors declare that they have no financial or any other conflicts of interest in this work.
11. ETHICAL APPROVAL
ABTS: 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
BPE: Bryophyllum pinnatum leaves’ extract
COM: Calcium oxalate monohydrate
DMSO: Dimethyl sulfoxide
GC–MS: Gas chromatography–mass spectrometry
MUE: Macrotyloma uniflorum seeds’ extract.
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