Ethnopharmacological and Phytochemical Basis of Medicinal Plants in Urolithiasis: From Traditional Use to Molecular Mechanisms

Kidney stone disease is a uniquely challenging condition among the urological diseases; it is painful, expensive and often recurrent; and yet there is no single effective pharmaceutical treatment [1]. In humans, calcium oxalate is the most common lithogenic mineral (75%). The prevalence of nephrolithiasis in populations of European ancestry is estimated to be from 5 to 10% of adults and occurs approximately twice as often in men as in women under normal epidemiological conditions [2]. The high burden of the disorder on working population, which is compounded by climate-induced dehydration, obesity and metabolic syndrome, dietary transitions, has been proven in the GBD 2021 data; since 1990, the number of patients, cases and DALYs associated with urinary stones in the age group 20-54 has risen significantly across the globe [3]. It is not a single attack that makes this epidemiological course so difficult to treat, but the fact that the attacks tend to recur so much. The incidence of stone recurrence in established stone formers is 15%-20% per year with 27% to 50% cumulative at 5 years if no specific pharmacological treatment is applied [4].

There is a standard pharmacological armamentarium for preventing recurrent calcium oxalate stone formation: all of these drugs target one metabolic aspect of the risk of crystallization. Thiazide diuretics facilitate reabsorption of calcium in the tubules and have been found to decrease the recurrence of stones in some clinical trials, although a landmark NEJM trial showed that the dose required to achieve this effect should be carefully titrated, and a systematic review of the literature found evidence was directionally positive, albeit low-strength [5]. An acknowledged risk of allopurinol is an increased risk of acute kidney injury when compared with other drug classes (3.25-fold, according to analysis of the WHO adverse drug reaction database) and an increased risk of stone recurrence when compared with thiazides (32%, according to medical claims data). Thiazides and citrate must be co-administered with frequent potassium monitoring; liver enzyme monitoring is needed with allopurinol, which can present a monitoring burden that makes the drug difficult to adhere to in patients who are not particularly ill [6]. In addition to the safety issues, none of these agents acts at the basic level of the crystallization cascade the physical processes of nucleation, crystal growth and aggregation which determine whether a supersaturated urine will form a clinically relevant stone [7].

This therapeutic unmet need has maintained a continued scientific interest in the plant-derived bioactive compounds as multi-targeted alternative to the conventional prophylaxis. Medicinal plants have been used in traditional medicine system in South and Southeast Asian, Middle East and Latin American countries for centuries for the treatment of urinary stones and constitute an ethnopharmacological basis for the modern scientific study of antiurolithiatic activity; in many ways, such traditional use preceded and inspired the present-day research [8]. The pharmacological relevance of these plants is not only based on the empirical tradition, but also, on the structure and physicochemical properties of the phytochemicals, namely polyphenols, flavonoids, tannins, alkaloids and terpenoids, which have been reported in increasingly large experimental literature to directly interfere with calcium oxalate nucleation kinetics, modify crystal morphology, inhibit crystal aggregation, reduce the urinary supersaturation of calcium oxalate, and protect renal tubular epithelial cells from oxidative and inflammatory injury caused by crystal deposition. These compounds also seem to have convergent mechanisms of action affecting multiple steps of the stone formation cascade, as compared to conventional drugs that affect only a single metabolic parameter [9,10].

The present review summarizes the existing knowledge of the bioactive compounds of plants and their mode of action in the modulation of calcium oxalate crystallization. It consists of a sequence of lectures that gradually build the readers' knowledge from pathophysiology and crystallisation mechanics to the molecular biology of crystal-induced renal injury, the evidence base from in vivo and in vitro studies, and the importance of bioavailability issues for the credible translation of observed effects into real-world impact the "structural pharmacognosy" of antiurolithiatic compound activity. The aim of this book is to give pharmaceutical scientists and researchers in the area of natural product pharmacology a scientifically sound and mechanistic explanation of the present scenario and the most promising directions of research.

2. Pathophysiology of Calcium Oxalate Stone Formation: A Crystallization Perspective

The process of stone formation in the urinary tract is not a single event but is the result of a series of physicochemical and biological events, which take place in a given sequence in the nephron [11]. The main condition for urinary supersaturation is the condition of urinary calcium and oxalate ionic product exceeding the calcium oxalate solubility product under the urinary conditions of pH, ionic strength, and temperature [12]. Ironically, the condition of calcium oxalate being supersaturated in normal human urine is the rule, not the exception, but stones are the exception and don't form in the population in general. In physiological conditions, there is a complex endogenous system of crystallization inhibitors, which effectively inhibits the process of nucleation and subsequent crystal formation, thus resolving this apparent contradiction [13].

The initial event in stone formation is nucleation, which is the buildup of the smallest, thermodynamically stable cluster of mineral constituting the stone, either in the free solution or more likely, in the environment of the renal tubular lumen, on pre-existing surfaces like cellular debris, proteins, or already deposited mineral deposits [14]. When the initial nucleus is reached, crystal growth is achieved by the orderly deposition of calcium and oxalate ions onto the faces of the existing crystals, forming the characteristic shape of calcium oxalate monohydrate (COM) or the less clinically important calcium oxalate dihydrate (COD). Relative to the composition of the kidney stones, the ratio of COD to COM is found to be greater than 2:1 in the stone analysis and COM is the thermodynamically stable polymorph and accounts for the majority of mineral components of kidney stones in clinical practice [15]. This polymorphic distinction has also received considerable pathological significance: adhesive forces of COM crystals to the surface of renal epithelial cells have been shown to be significantly higher than those of COD crystals by atomic force microscopy, which can provide a molecular explanation for why COD-forming urine has a lower stone risk than COM-dominated crystalluria [16]. The process of crystal growth is followed by the physical aggregation (clustering) of individual microcrystals that leads to the formation of larger conglomerates, and it is the aggregation, not the crystal growth of the individual crystals that most directly determines whether a particle becomes large enough to obstruct the tubular lumen and begin the process of stone retention [17].

The interplay of crystallization promotion and inhibition determines whether the supersaturation is a clinical stone. Citrate is the most important inorganic inhibitor. Citrate interferes with calcium oxalate crystallization in two ways: citrate directly adsorbs to COM crystal step surfaces via its carboxylate functional groups, sterically preventing further ion deposition and citrate forms soluble complexes with calcium ions, which lowers the concentration of free calcium ions and thus the effective supersaturation. One of the most common and treatable metabolic abnormalities in nephrolithiasis is hypocitraturia (urinary citrate levels below 320 mg per day) [18]. Hypocitraturia has been seen in 20 to 60% of calcium stone formers. Magnesium also decreases urinary oxalate availability by forming complexes, and pyrophosphate decreases the growth of crystals by surface adsorption, but both have been found to be less important in the absence of which stone occurrence could be more significant than citrate in clinical stone disease [19]. Macromolecular inhibitors include the urinary protein osteopontin (OPN), which is an aspartic acid-rich inhibitor that strongly inhibits the nucleation and growth of COM crystals in the presence of normal urine at concentrations of about 150 µg per milliliter and by stereochemically specific interactions of the carboxylate groups with calcium ions, pinning step advancement on particular crystal faces [20]. The most abundant urinary protein Tamm-Horsfall protein, on the other hand, affects the ability of CaOx crystals to aggregate based on the state of its polymerization, and glycosaminoglycans also interfere with crystal–cell surface interactions by occupying the anionic binding sites of the tubular epithelial membranes [21].

Crystal retention in the renal tubule is the final and decisive step in stone formation, which is also regulated by the crystal–cell adhesion. COM crystals interact quickly with the epithelial cells lining the distal tubules at anionic sites, beginning within seconds after the crystals come in contact with the cell surface, as shown in culture models with radio-labeled COM. The luminal surface of the oxalate-injured tubular cell displays significantly higher levels of hyaluronan, osteopontin and CD44, which mediates crystal adhesion, resulting in a vicious cycle of crystal deposition and oxidative injury. A schematic of the crystallization cascade is provided in Figure 1, and the main urinary promoters and inhibitors of CaOx crystallization and the mechanisms and modulation of these by phytochemicals are presented in Table 1 [22].

Figure 1. Pathophysiology of Calcium Oxalate Stone Formation

Table 1. Urinary Promoters and Inhibitors of Calcium Oxalate Crystallization: Physicochemical Roles, Normal Urinary Levels, and Documented Modulation by Plant-Derived Phytochemicals [23–27]

Factor	Type	Role in CaOx Crystallization	Normal Urinary Level / Reference Range	Modulation by Phytochemicals
Calcium	Promoter	Provides ionic substrate for CaOx lattice formation; hypercalciuria drives supersaturation	100–300 mg/day	Flavonoids (e.g., quercetin) reduce urinary calcium in animal models via reduced intestinal absorption
Oxalate	Promoter	Primary anion in CaOx crystal lattice; hyperoxaluria is the most common lithogenic metabolic abnormality	20–45 mg/day	Polyphenols reduce hepatic oxalate synthesis; plant extracts reduce urinary oxalate in ethylene glycol models
Uric acid	Promoter	Promotes heterogeneous nucleation of CaOx by providing crystalline templates	< 800 mg/day (men); < 750 mg/day (women)	Alkaloids with xanthine oxidase inhibitory activity reduce urate production
Sodium	Promoter	Increases urinary calcium excretion; competes with citrate reabsorption	Variable; dietary-driven	Limited direct phytochemical evidence; diuretic terpenes indirectly lower concentration
Citrate	Inhibitor	Forms soluble calcium complexes; directly inhibits nucleation and aggregation via crystal face adsorption	Mean ~640 mg/day; hypocitraturia < 320 mg/day	Several flavonoids and organic acids in plants may raise urinary citrate levels in animal models
Magnesium	Inhibitor	Forms soluble MgOx complexes; reduces free oxalate ion activity	50–150 mg/day	Magnesium-rich plant extracts raise urinary magnesium in hyperoxaluric rats
Osteopontin (OPN)	Dual regulator	Inhibits COM nucleation and growth at normal urine levels; promotes crystal adhesion to CD44-expressing injured cells	> 100 nmol/L in normal urine	Kaempferol and related flavonoids downregulate OPN expression in crystal-injured HK-2 cells
Tamm-Horsfall protein (THP)	Inhibitor (physiological)	Inhibits crystal aggregation in monomeric form; promotes aggregation when polymerized under pathological conditions	Most abundant urinary protein; ~50 mg/day	Indirect modulation via reduction of renal tubular injury; no direct phytochemical evidence reported
Pyrophosphate	Inhibitor	Inhibits crystal growth through surface adsorption on COM crystal faces	~50–100 μmol/day	Not directly modulated by reported plant extracts
Glycosaminoglycans	Inhibitor	Occupy anionic sites on tubular epithelial surfaces, reducing COM crystal adhesion	Low nmol/L range	Saponins in plant extracts may mimic glycosaminoglycan surface competition

Figure 2. Mechanisms of Plant-Based Urolithiasis Inhibition.

8. Limitations of Current Evidence and Translational Gaps

The body of evidence reviewed in the preceding sections is broadly substantiated and mechanistically informative, but is structurally limited, in the manner in which antiurolithiatic phytochemical research has been designed and conducted in the majority of published studies. It is not a deprecation of the promise of the field to acknowledge these limitations but a necessary requirement for designing studies that will answer the question of whether the promise of the field can be realized clinically. The first and most obvious limitation is that there are differences between the in vitro inhibitory activity and attainable urinary levels in humans. Most spectrophotometric crystallization assay studies are performed at a concentration of 0.1–5.0 mg/mL or equivalent molar dosage, which are far higher than what can be reliably provided to the urinary compartment by the oral administration of standardized plant extracts or isolated compounds, especially in human subjects where bioavailability is poor for compounds like curcumin and berberine. Whereas the few studies that have tried to correlate assay concentrations with human blood or urine levels, like the quercetin study (2.5-160 μM), are remarkable for being rare exceptions. This disparity results in a significant percentage of positive in vitro data in the antiurolithiatic literature that correspond to a real effect in the physicochem domain but cannot be reached without formulation intervention in the pharmacokinet domain [59].

The second constraint is the lack of standardization of plant extracts in the experimental studies. The antiurolithiatic activity described for an extract is the sum total of the variations in qualitative and quantitative phytochemical profile that can occur between extracts of the same plant, but different extraction solvents, plant parts employed, geographic origin, harvest time, and processing method. Chemical fingerprinting, preferably by HR-LCMS, and reporting of the principal active constituent(s) and their concentration is required to compare activity data between studies and to confidently identify a compound or compounds that are responsible for the observed inhibition in order to make mechanistic claims. The inability to standardise across the heterogeneous studies is evident in the systematic review literature and is the reason why inferential power of meta-analytical approaches in this domain is limited.

The third constraint is the lack of a sex-specific analysis in the majority of pre-clinical studies. An epidemiological sex difference is apparent, with calcium oxalate stones being formed around twice as often in men than in women, and urinary citrate levels being lower in women than in men, thus setting up a different inhibitory environment. Sex hormones affect oxalate metabolism, calcium handling in the kidney and excretion of inhibitors of calcium handling in the urine in a biologically significant manner that is usually not accounted for in model studies using rats, which are typically male. This lack of generalizability of preclinical results to the broader patient population is a limitation.

A fourth issue is the fact that in the patients who will be actually taking conventional antiurolithiatic medicines, attention is limited to herb–drug interactions. High polyphenol loads can interact with cytochrome P450 enzymes (e.g. CYP3A4 and CYP2C9 are inhibited by quercetin and will change the plasma concentration of some drugs, including thiazide diuretics and allopurinol, which are used to treat nephrolithiasis; this interaction has not been studied in the context of nephrolithiasis pharmacotherapy). Lastly, there is not enough and not consistent enough clinical trial data to recommend at the guideline level. The limited randomized controlled trial evidence is directionally supportive, but consistently reports low quality of evidence, small sample sizes, and insufficient reporting of baseline urinary biochemistry to guide clinical practice [60].

9. Future Perspectives

The area of research of immediate relevance to the above mentioned translational bottlenecks is those that can provide the necessary methodological and clinical infrastructure to take phytochemical antiurolithiatic drugs into the evidence-based therapeutic recommendations in clinic. Network pharmacology and molecular docking have proven to be powerful front-end tools to define the multi-target pharmacological spectrum of complex plant extracts prior to investing in wet laboratory experiments. When applied to Choerospondias axillaris, quercetin, kaempferol, catechin, β-sitosterol and naringenin have been identified as major active compounds, AKT1, IL-6, TNF, TP53 and IL-1β have been emerged as main molecular targets and NF-κB and MAPK have been identified as the key signaling pathways involved by the application of PPI network analysis and KEGG pathway enrichment analysis. Importantly, these computational predictions were confirmed in experiment: experiments were performed in the 2-D CaOx agar gel system, where the extract at high concentrations exhibited a shift in crystal polymorph, from COH to COD, which is the polymorph that is associated with less epithelial adhesion and lower pathological risk. Incorporating this integrated computational-experimental approach systematically, across the most promising plant families that exhibit high densities of compounds that satisfy the criteria for good CaOx inhibition (pKa, LogD and hydrogen-bond) would significantly speed up the discovery of clinical lead compounds in antiurolithiatic medicinal chemistry. The case study of ellagic acid in Phyllanthus niruri is a typical success of such an approach, which was predicted using a network pharmacology approach to be targeting the proteins SQLE, SCD and HMGCS1 involved in CaOx-induced renal injury, and subsequently validated in vitro and in vivo [61].

The formulation challenge described in Section 7 requires a particular approach to research, which is the development of a delivery system to optimize the systemic bioavailability in a more conventional sense, but rather one that maximizes urinary excretion of an active or pro-active compound. There were no published studies that specifically characterized urinary pharmacokinetics of n LDs antiurolithiatic flavonoids (the urinary Cmax and the course of urinary inhibitory activity) as the main pharmacokinetic endpoint although the proof of concept of the oral absorption of these flavonoids by LDs with lipids and phytosomes was demonstrated. Such studies would bring about a pharmacokinetic–pharmacodynamic linkage for which the field is currently lacking, by combining the nano-enabled delivery with the validated in vitro crystallization assays performed at urine concentrations achieved in vivo. With proven regulatory expertise in nutraceutical and pharmaceutical applications, self-nanoemulsifying drug delivery systems have significantly enhanced the oral bioavailability of poorly soluble polyphenols such as curcumin and thus provide a scalable and practical system for this purpose.

Standardization of the conditions of the in vitro antiurolithiatic assay, such as crystallization buffer composition, pH, ionic strength, temperature, incubation time, and turbidity measurement parameters, within research groups would significantly enhance the comparability of the data reported from various laboratories, which would allow meaningful meta-analyses of concentration–response relationships. It is also critical that the use of extract characterization by HR-LCMS is established as a minimum reporting standard, which correlates observed activity to a defined phytochemical fingerprint, instead of a crude extract mass. From a clinical perspective, the design of Phase II randomized controlled trials of standardized, characterized phytochemical preparations in well-phenotyped cohorts of recurrent CaOx stone-formers with 24-hour urine biochemistry, stone recurrence imaging and urinary biomarkers of oxidative stress as co-primary outcomes represents the most straightforward pathway to guideline-relevant evidence. The promising preliminary results of clinical trials with clinical formulations that are rich in phytochemicals and have been shown to reduce the recurrence to a significant degree support the need to pursue such trials as a research priority [62].

CONCLUSION

The plant-based bioactive compounds are not only a mechanistically coherent approach to calcium oxalate stone disease, but they target multiple pathways simultaneously, such as the ability to inhibit the crystallization process, protect against oxidative stress, and suppress inflammation all of which are not provided by the pharmaceutical agents available today that target just one pathway. Based on the reviewed evidence, polyphenols, flavonoids, tannins, saponins, alkaloids and terpenoids have proven to possess structurally explicable antiurolithiatic effects backed by in vitro, preclinical and early clinical evidence. Gaps identified such as the concentration achievability problem, lack of extract standardization, lack of sex differentiated preclinical design, and lack of clinical trial evidence are research design issues, not biological in nature. Whether plant derived crystallization modulators can be incorporated in to evidence-based components of nephrolithiasis prevention will be determined by addressing them with HR-LCMS guided standardization, nano-enabled renal delivery strategies and adequately powered clinical trials.

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