Evaluation of Coscinium fenestratum (Goetgh.) Colebr. stem extracts for urolithiasis and quantification of bioactive alkaloids to validate the traditional claims

Abstract Coscinium fenestratum (Goetgh.) Colebr. is widely used for urinary disorders and kidney stones by ethnic communities in southern India. The species is documented in various ancient Indian Ayurvedic literatures having therapeutic use in ‘Ashmari’ i.e., urolithiasis. The present study aims at validation of in-vitro anti-urolithiatic potential of various extracts of C. fenestratum stem along with identification and quantification of major bioactive alkaloids, i.e., berberine and palmatine through HPTLC and LC-MS/MS. Water extract showed maximum anti-urolithiatic activity which on further kinetic analysis, showed concentration dependent inhibitory delay in nucleation and aggregation of calcium oxalate crystals. Berberine and palmatine were quantified with maximum content in methanolic extract (0.478 ± 0.003 and 0.0358 ± 0.001) followed by chloroform and petroleum ether extracts. The study validates ethnobotanical use of C. fenestratum as anti-urolithiatic agent. Further, species can also be explored as a substitute for Berberis spp. for the alkaloid metabolites i.e., berberine and palmatine. Graphical Abstract


Introduction
Coscinium fenestratum (Goetgh.) Colebr. (Menispermaceae) is a woody climber, traditionally used in various systems of medicine and is synonymously known as 'Daruharidra'. The species is found in several parts of the world, mainly in Vietnam, Thailand, Sri Lanka, Malaysia and Southern India. The plant is mentioned in the Ayurveda and Siddha for the treatment of diabetes, ulcers, jaundice, piles, fever and snakebite (Taher et al. 2019). The ancient traditional literature of India advocates the use of species in stones related disorders, in Ayurvedic Pharmacopoeia of India 3-6 g of Coscinium (root and stem) powder has therapeutic use in 'Ashmari' i.e., urolithiasis or urinary stones (Anonymous 2006). Besides, another classical text Bhava Prakasha (Prakasha and Mishra 2009) suggest the use of Coscinium to destroy the calculus. The species is a rich source of isoquinoline alkaloids i.e., berberine, tetra-hydroberberine, palmatine, tetra-hydropalmatine, jatrorrhizine (Pinho et al. 1992). Due to the presence of pharmacologically active metabolite berberine, the species is also used as substitute of Berberis spp. in India and Sri Lanka. Ethnic communities in southern part of India use decoction of this plant to treat patients suffering from kidney stone and related urinary disorders (Perera and Kulatunga 2018).
Urolithiasis is the most prevalent urologic disease, frequency ranging from 1-5% in Asia, 5-9% in Europe and 7 À 13% in North America (Bashir and Gilani 2011). There are various types of kidney stones, in which calcium oxalate are in majority (80%). Berberine exhibits potential anti-urolithiatic effect and has been thoroughly studied in drug development (Sorokin et al. 2017). Therefore, berberine containing species are under constant threat of biodiversity loss due to its multifarious medicinal properties and it is relevant to explore the substitute. C. fenestratum contains berberine as a major metabolite and was exploited for the same, leading to its bio-diversity threat. Further, less availability of wild population decelerate the scientific studies on species and consequently, the traditional claims were not explored up to its potential. Hence, in this study various extracts of C. fenestratum were evaluated for in-vitro antiurolithiatic activity and were characterised through validated HPTLC method for simultaneous quantification of bioactive alkaloids, berberine and palmatine.

Results and discussion
2.1. Anti-urolithiatic assay and reaction kinetics The first step in renal (CaOx) stone formation is nucleation, where CaCl 2 and Na 2 C 2 O 4 react to forms CaOx crystals. These when adhere together lead to formation of loose aggregate of crystals and develop into renal calculi. In nucleation assay, water soluble extract exhibited maximum anti-urolithiatic activity with least IC 50 i.e., 79.04 mg/mL followed by methanol, chloroform, toluene, butanol and petroleum ether soluble extract. In the aggregation assay also, water extract showed maximum inhibition (IC 50 at 36.35 mg/mL) followed by butanol, chloroform, toluene, petroleum ether, and methanolic extract (Table S2).
The whole urine assay was performed with berberine, palmatine ( Fig. S3) and different extracts of C. fenestratum stem (Fig. S3a). In the control, large aggregated stonelike structures were observed along with bi-concave, oval and dumbbell shaped calcium oxalate monohydrate (COM) crystals and few octahedral calcium oxalate dihydrate (COD) crystals with significant aggregation (Fig. S3A & B). In standard i.e., berberine and palmatine significant inhibition was observed in the formation of COM crystals, whereas COD crystals were found more prominent. As shown in fig. S3C, the lowest concentration of berberine showed small COM crystals most of which were aggregated and few COD crystals; while as the concentration increased, COM crystals become rare with an increase in number of COD crystals ranging small to medium in size ( Fig. S3D & E). Similar observations were found in palmatine (Fig. S3F,G & H). In all the samples, aggregation of crystals was observed in the reactant mixture having least extract concentration and it gets reduced along with increase in concentration. As the concentrations of the plant extracts were increased in the reactant mixture, the large COM crystals become small, more numerous, spherical in shape and less aggregated. Water extracts of C. fenestratum show potential activity, in which all the type of crystals were small in size. At higher concentration i.e., 50 mg/mL, COD crystals were observed to be of medium size while COM crystals were numerous, smaller, rounded in shape and less aggregated (Fig. S3a E). The decrease in aggregation was accompanied with or may be responsible for appearance of numerous small crystals. Methanol extract at lower concentrations showed both COD and COM crystals, larger in size and of characterstics octahedral and orthorhombic shapes, respectively (Fig. S3a L & M). But, at higher concentration, it showed good potential as well, since; the crystals observed were very less in number and minimal aggregation as compared to water (Fig. S3a N). Butanol, chloroform, petroleum ether and toluene extracts showed larger octahedral COD crystals in comparison to water extract and COM crystals were orthorhombic, bi-concave, dumbbell shaped and more aggregated (Fig. S3a). Based on their pathogenicity, COM crystals are reported to be more dangerous than COD, due to the former's higher affinity with renal tubular cells. In the microscopic observation, significant changes were observed in the morphology and size of COM crystals in presence of plant extracts. With increasing plant extract concentration, the occurrence of numerous small rounded COM crystals suggest that the plant extracts might be promoting the transition of COM crystals from hexagonal to spherical shape, as suggested in previous reports (Mittal et al. 2015;De Bellis et al. 2019). These rounded, smaller COM crystals have been reported previously to be less stable thermodynamically and demonstrate reduced affinity and adhesion for the renal cell membrane as compared to hexagonal COM crystal. The resulting modification in the shape of COM crystals assists in their easy discharge via urinary tract, thus preventing the formation of kidney stones (Mittal et al. 2015).
Based on above results, water extract of C. fenestratum (having pronounced effect) was selected for time-course study (reaction kinetics) for nucleation and aggregation of calcium oxalate crystals. Four variable concentrations of standards (berberine and palmatine) i.e., 2.5, 5, 7.5, 10 mg/mL and sample i.e., 25, 50, 75,100 mg/mL were used. A typical time-course kinetic graph of CaOx crystallisation (Hess et al., 1995) was observed in each dilution of extract, including the control (Fig. S4). Maximum nucleation in least time interval (slope of nucleation) was observed in control, followed by the extracts in increasing order of concentration. In the control, the increase in absorbance i.e., nucleation was stopped at 7 min, but in the presence of plant extracts, increase in absorbance ceased at about 5-6 min only, indicating that nucleation was inhibited early. The slope of aggregation also follows the similar pattern, maximum slope was in control, followed by the extracts in increasing concentration. In control, the aggregation was complete at around 27 min only whereas in the presence of water extract, the decrease in absorbance kept continuing even after 30 min and thus indicating aggregation inhibition. The time duration for complete aggregation (i.e., decrease in absorbance post nucleation) kept increasing with increase in plant extract/ berberine concentration.

Quantitative estimation of berberine and palmatine in C. fenestratum
HPTLC is a widely accepted, reliable and cost-effective method for identification and quantification of biomarker compounds in standardisation and quality control of plant material. It is a crucial analytical technique useful in the identification of a plant species through its fingerprinting profiling, identification of intraspecific population vis-avis variation in their metabolites. Besides, the species from different genera can also be identified through HPTLC (Toniolo et al. 2014). Therefore, HPTLC has been adopted in this study for the quantification of bioactive alkaloids. The chromatographic separation of berberine and palmatine was done in the tertiary solvent system of n-propanol: water: formic acid (9:1:0.1 v/v). After the development of chromatogram (Fig.  S6), identification of berberine and palmatine was done by comparing the R f and UV absorption spectra of standards and sample(s) (Fig. S5). Berberine and palmatine were identified at R f 0.334 ± 0.07 and 0.216 ± 0.05. Densitometric scanning and quantification at 350 nm (Fig. S6) reveal that the content of berberine and palmatine was found to be maximum in methanol (0.478 ± 0.003 and 0.0358 ± 0.001) and minimum in toluene and petroleum ether extracts (Table S3).
In the present study, berberine and palmatine as well as the water extract have shown excellent anti-urolithiatic activity. Also, the HPTLC simultaneous quantification reveals the presence of both berberine and palmatine in the water extract. Water extract was further studied through LC-MS/MS for the elucidation of chemical constituents. The spectra revealed a number of chemical compounds (Fig. S8 & S9); however, berberine and palmatine were present predominantly in the extract along with other unknown compounds (Fig. S10). Thus, in the present study, it can be deduced that the anti-urolithiatic potential of berberine, palmatine, as well as other water soluble metabolites might be synergistically responsible for the excellent activity of water extract.

Conclusions
C. fenestratum exhibits potential anti-urolithiatic effect in multifarious ways i.e., via inhibition of CaOx crystal growth, inhibition of crystal aggregation and/or due to alteration in the morphology of COM crystals and enhanced production of COD crystals. Water extract exhibited maximum potential in all the in-vitro assays (whole urine, nucleation and aggregation) and kinetic analysis revealed a concentration dependent inhibitory delay of both nucleation and aggregation of CaOx crystals. HPTLC quantification data revealed the presence of both berberine and palmatine in all the prepared extracts. However, the content of palmatine in water extract was found to be below detection limit indicating towards the synergistic anti-urolithiatic potential of other water soluble metabolites in the extract. Thus, the study validates the ethnobotanical claim of C. fenestratum as anti-urolithiatic agent and the identification and quantification of berberine and palmatine also establish the species as a suitable substitute for Berberis spp.