In vitro Inhibition of Calcium Oxalate Nucleation by Extract-based Fractions of Aerial Parts and Roots of Aerva lanata (Linn.) Juss. ex Schult

*Corresponding Author:

Swati Madan
Department of Pharmacognosy, Amity Institute of Pharmacy, Amity University, Noida-201 313, India
E-mail: smadan3@amity.edu

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Abstract

The current investigation is an attempt to explore the comparative in vitro antiurolithic potential of various phytoconstituent-rich fractions derived from the hydroalcoholic extract of aerial parts and roots of Aerva lanata (Linn.) Juss. ex Schult (Amaranthaceae). The antiurolithic assay was performed by nucleation method. The analysis revealed the phenolic and flavonoid containing fraction from aerial parts to be the most active (71.01±1.13%) amongst the different extract-derived fractions compared to the one obtained from roots (54.61±2.30%). Besides, fluorescence analysis and physicochemical evaluation were carried out according to the official guidelines and supported the quality control of the plant material. Preliminary phytochemical screening of the fractions was also performed and confirmed the presence of various metabolites. Additionally, the content of tannins, total phenolics and flavonoids were also determined spectrophotometrically and found to be comparatively higher in aerial parts (tannin content of 4.34±0.63 mg tannic acid equivalent/g extract, total phenolic content of 127.84±1.50 gallic acid equivalent/g extract and total flavonoid content of 77.61±3.78 rutin equivalent/g extract) than roots (tannin content of 3.03±0.63 mg tannic acid equivalent/g extract, total phenolic content of 98.09±1.10 gallic acid equivalent/ g extract and total flavonoid content of 62.81±5.69 rutin equivalent/g extract). Hence, the present study aids in screening out the best active extract derived fraction exerting litholytic efficacy, which provides a route to further isolation of lead bioactive compounds contributing to significant antiurolithic potency of this traditionally employed herb of immense pharmacological spectrum.

Keywords

Aerva lanata (Linn.) Juss. ex Schult, antiurolithiatic, fraction, phenolics, flavonoids

Urolithiasis refers to the formation of calculi anywhere in the urinary tract afflicting the kidneys, ureters, bladder or urethra [1]. It is the third most prevalent urological disorder after urinary tract infection and prostate conditions [2]. Approximately 12 % of the world population is affected by the incidence of urinary stones with a comparatively higher frequency of recurrence in males (70-81 %) than in females (47- 60 %) [3]. Depending upon the chemical nature, there are various types of stones such as calcium oxalate monohydrate, calcium oxalate dihydrate, basic calcium phosphate (brushite), magnesium ammonium phosphate (struvite), uric acid and cystone stones [4]. Manifestations include colicky pain, vomiting, dysuria, haematuria, pyuria and oliguria [5]. These calculi arise as an outcome of supersaturation of urine with stone forming constituents resulting in crystallization followed by crystal nucleation, aggregation and growth leading to their adherence to the renal tubules [6].

Numerous surgical strategies exist in scenario for management of urinary calculi such as shock wave lithotripsy (SWL), ureteroscopic lithotripsy (URS), digital endoscopy, percutaneous nephrolithotomy (PNL) and robotic surgery [7]. However, many of these techniques bring along with them several drawbacks such as renal casualties in the long run, hypertension and repeated episodes of stones [8]. Several allopathic interventions are available but are increasingly being overtaken by herbal therapeutics on account of their safety, minimal side effects, comparatively greater efficacy in dissolving stones and preventing the chances of recurrence [9]. Moreover, the applications of medicinal plants in combating urinary stones have been extensively mentioned in the ancient medical literature as well as Ayurvedic system of medicine in addition to their relevance as folk remedies [10].

Aerva lanata (Linn.) Juss. ex Schult. (family: Amaranthaceae) is one such efficacious medicinal plant amongst the diverse pool of litholytic herbs bestowed by nature. It is commonly referred as Mountain knotgrass in English and Gorkhabundi or Kapurijadi in Hindi, which is an erect or prostrate herb with a long tap-root and many wolly-tomentose branches [11]. It grows up to an elevation of 900 m and is indigenous to India, Africa and Australia and considered as Pashanbheda in Ayurveda, which is a collective term assigned to a group of medicinal herbs with a potential for dissolving kidney stones [12]. It is a reservoir of diverse phytoconstituents such as canthin- 6-one (aervine, aervoside) and β-carboline alkaloids (aervolanine), flavonoids (kaempferol, quercetin), β-sitosterol, phenolic acids (syringic acid, vanillic acid), betulin [13]. Literature elaborates a tremendous in vitro and in vivo antiurolithic potency of the extract from whole plant [14-18]. However, evaluating the extracts for biological activity provides generalized information regarding the efficacy of a particular medicinal plant regardless of the specific phytoconstituents. The current era of phytopharmaceuticals has led to an emerging curiosity towards an in depth scrutiny of bioactive phytoconstituents contributing to the therapeutic efficacy of herbs and to achieve this bioassay-guided fractionation is a mandatory step, which implies stepby- step separation of extracted components based on the differences in their physicochemical properties using solvents of different polarities and assessing the biological activity. A recent study reported about the efficacy of various extracts derived from aerial parts using different solvents [19]. The research highlighted that the aqueous extract exhibited the best activity which however, does not establishes the basis regarding the contribution of phenolics and flavonoids towards antiurolithic ability of A. lanata as a large number of phytocomponents are soluble in water apart from phenolic and flavonoid compounds. Additionally, the activity was performed at only one dose level, which cannot ascertain the degree of efficacy as a dose range is more reliable in analysing the maximal efficacy of a particular pharmaceutical agent. Statistical analysis was missing in the study, which is a mandatory step while assessing the biological activity to assure any significant differences among the different extract groups. Another protocol was conducted with the objective of in silico antiurolithic screening of the isolated components [20]. But, the study did not reported a systematic in vitro screening of different fractions for bioactivity followed by an in-depth evaluation of various altered biochemical parameters in vivo.

From the literature, it is evident that a systematized bioactivity guided fractionation of A. lanata has not been reported to provide a scientific basis for identification of actual active principles responsible for its antiurolithic activity. Keeping this in view, we have reported the in vitro nucleation assay of various extract derived fractions of aerial parts and roots at various dose levels in our present study to screen out the best bioactive fraction contributing towards the antiurolithic potency of this miraculous herb of nature. This gives a better evidence for antiurolithic activity of various fractions of A. lanata at different dose levels. Hence, the present study constitutes a novel research, which has not been reported earlier. The current research also aims at carrying out the fluorescence analysis as well as physicochemical assessment, which is a mandatory step to ensure fulfilment of the plant material towards the fundamental criteria for identity, quality and purity. Additionally, it also objects towards the preliminary phytochemical screening of the fractions derived from them to ensure the presence of intended phytochemicals and spectrophotometric estimation of the content of tannins, total phenolics and flavonoids to correlate their influence on the antiurolithiatic efficacy of this medicinal plant.

Materials and Methods

Folin-Ciocalteu reagent, calcium chloride dihydrate and aluminium chloride were procured from Central Drug House Pvt. Ltd., New Delhi. Methanol was purchased from Rankem, Gujarat. Diethyl ether was procured from High Purity Laboratory Chemicals Pvt. Ltd., Mumbai. Ethyl acetate, chloroform, sodium chloride and sodium hydroxide were obtained from Thermo Fisher Scientific India Pvt. Ltd., Mumbai. Tris was purchased from Ubichem Plc., Hampshire. Sodium carbonate was obtained from Loba Chemie Pvt. Ltd., Mumbai. Sodium oxalate and sodium nitrite were procured from Qualigens Fine Chemicals, Mumbai. Tannic acid and gallic acid were purchased from Hi Media Laboratories Pvt. Ltd., Mumbai. Rutin was purchased from Search Chem, Mumbai. All other chemicals employed were of analytical grade.

Collection, identification and authentication of plant material

Dried whole plant of A. lanata was procured from Balaji Traders (Tamil Nadu, India) during the month of March, 2015. The identity of the plant was validated at the Raw Materials Herbarium and Museum, NISCAIR, New Delhi and a voucher specimen number NISCAIR/ RHMD/Consult/2014/2792/171 for the same was deposited at the Herbarium of National Institute of Science Communication and Information Resources, New Delhi. Subsequently, the roots and aerial parts were separated, dried and powdered.

Fluorescence analysis

A small portion of the powdered aerial parts and roots was placed on a microscopic slide and examined under short wave UV light (254 nm), long wave UV light (366 nm) and day light as such as well as following their treatment with various reagents [21].

Physicochemical evaluation

The physicochemical parameters were performed as per the official guidelines and included foreign matter, extractive values (water soluble, alcohol soluble, ether soluble), ash values (total, water soluble, acid insoluble), loss on drying, foaming index and swelling index [22,23].

Extraction of plant material

About 200 g of the powdered aerial parts and roots were weighed and macerated separately in a solvent mixture of water:methanol (2:1) overnight followed by refluxing the hydromethanol blend for 5 h. The extracts thus obtained from aerial parts and roots were then evaporated to dryness to one-fourth of their original volume and further divided into various portions for the generation of fractions.

Preparation of fractions

The hydromethanol extract, separately for both roots and aerial parts, was divided into three portions out of which one-fourth part was evaporated to dryness below 60° and considered as the mother extract. One-half of the volume was made acidic and partitioned with equal volume of ether in a separating funnel followed by separating the ether layer and evaporating it to dryness which led to fraction I, which was supposed to contain fatty material, steroidal aglycones, terpenoids and coloring matter. The remaining aqueous layer was rendered alkaline with ammonia solution and partitioned with equal volume of chloroform thrice after which it was separated and evaporated to dryness resulting in fraction II expected to be composed of alkaloids. The remaining alkaline aqueous layer was made acidic with dilute hydrochloric acid. Thereafter, it was refluxed for two hours followed by further fractionation with equal volume of ethyl acetate thrice, which was evaporated to dryness consequently leading to fraction III supposed to contain flavonoid and phenolic aglycones. Finally, the remaining one-fourth volume of the extract was passed with equal volume of ether and the aqueous layer left behind was passed with ethyl acetate thrice, which was evaporated to dryness giving rise to fraction IV expected to be composed of glycosides [24,25].

Preliminary phytochemical screening

Various qualitative tests were performed to confirm the presence of steroids, terpenoids, phenolics, flavonoids, glycosides and alkaloids in the distinctive fractions generated from the hydromethanolic extracts of roots and aerial parts [26,27].

Nucleation assay

In vitro antiurolithiatic assay was conducted by the procedure as mentioned in literature [28,29]. The assay was carried out in the absence (control) and presence of inhibitor (standard/extract/fraction). About 5 mM and 7.5 mM calcium chloride and sodium oxalate solutions were made in a buffer composed of 50 mM Tris and 150 mM sodium chloride at pH 6.5. Stock solutions of standard (cystone) and samples (extracts of aerial parts and roots along with their fractions) were prepared at a concentration of 10 mg/ml. About 1 ml of calcium chloride was added in both the control and sample sets. Additionally, 1 ml of distilled water was added in the control set. On the contrary, 1 ml of various dilutions of sample (200, 400, 600, 800, 100 μg/ml) were added in the sample set instead of distilled water. The onset of crystallization was achieved by the addition of 1 ml of sodium oxalate solution to all the sets. The tubes were incubated at 37° for 30 min after which the absorbance was read at 620 nm using Perkin Elmer Lambda 25 UV/Vis spectrophotometer. The percentage inhibition of nucleation was calculated as follows: percent inhibition=[(C-S)/C]×100, where, C=turbidity of control set, S=turbidity of sample set.

Determination of tannins

Tannin content was assessed by Folin-Ciocalteu reagent method [30]. About 0.5 g each of powdered aerial parts and roots were extracted by boiling in water, filtered and concentrated on a water bath and dried in an oven below 60°. Stock solutions of the extracts were prepared (1 mg/ml) in distilled water. Thereafter, 0.1 ml of the extracts were mixed with 0.5 ml of Folin-Ciocalteu reagent, 1 ml of 35 % sodium carbonate solution and the volume was made up to 10 ml with distilled water. The samples were left for colour development for 30 min and absorbance was read at 725 nm using Perkin Elmer Lambda 25 UV/ Vis spectrophotometer. Stock solution of tannic acid was prepared in water (100 mg/100 ml) from which appropriate dilutions were made (2, 4, 6, 8 and 10 μg/ ml) and standard curve was achieved. Tannin content was expressed as mg of tannic acid equivalents per g extract (mg TAE/g).The experiment was carried out in triplicate.

Determination of total phenolics

Total phenolic content was analysed by spectrophotometric method [31]. Stock solutions of the extracts of roots and aerial parts were made in distilled water (1 mg/ml). About 1 ml of the extracts from stock was admixed with 1 ml of Folin’s reagent. After 5 min, 4 ml of 7 % sodium carbonate solution was added along with 4 ml of distilled water. The samples were prepared in triplicate. The mixtures were incubated for 60 min in dark and the absorbance was taken at 750 nm against a reagent blank. Stock solution of gallic acid in water was prepared (100 mg/100 ml) from which appropriate dilutions were obtained (2, 4, 6, 8 and 10 μg/ml) and standard curve was established. Total phenolic content was expressed as mg of gallic acid equivalents per g extract (mg GAE/g).

Determination of total flavonoids

The content of total flavonoids was evaluated by aluminium chloride colorimetric assay [32]. Stock solutions of extracts were prepared (1 mg/ml) in distilled water. About 0.5 ml of the extracts from stock solutions was mixed with 2 ml distilled water to which 0.15 ml of 5 % sodium nitrite solution was added. After 6 min, 0.15 ml of 10 % aluminium chloride solution was added to the mixture and placed for another 6 min. About 2 ml of 4 % sodium hydroxide solution was added to this and final volume was made up to 5 ml by adding 0.2 ml of distilled water. The experiment was conducted in triplicate. All the reaction mixtures were mixed well and left to stand for 15 min and the absorbance was recorded against a reagent blank at 510 nm. Stock solution of rutin in water was prepared (100 mg/100 ml) from which appropriate dilutions were made (2, 4, 6, 8 and 10 μg/ml) and standard curve was obtained. Total flavonoid content was expressed as mg of rutin equivalents per g extract (mg RE/g).

Statistical analysis

The experiments were carried out in triplicate and data were expressed as mean±standard deviation. Detection of significant differences between the groups was done by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The results were recognized significant when P<0.05.

Results and Discussion

The results of fluorescence analysis of the powdered aerial parts and roots are mentioned in Table 1 and Table 2. It served as a relevant criterion for their identification on account of the characteristic fluorescence displayed by the phytoconstituents in them. The values for physicochemical evaluation are given in Table 3. The foreign matter was slightly higher in roots (0.54±0.04 %) than aerial parts (0.48±0.03 %). The water soluble, alcohol soluble and ether soluble extractives were found to be 13.41±0.46, 4.02±0.42 and 1.62±0.06 %, respectively in aerial parts while in roots they were found to be 13.20±0.82, 5.01±0.51 and 5.61±0.17 %, respectively. The moisture content (loss on drying) was within the limit in both the aerial parts (4.80±0.30 %) and roots (4.32±0.26 %), which assures the acceptable quality of the plant material. The levels of total ash, acid insoluble ash and water-soluble ash values were also agreeable and reported to be 9.50±0.47, 2.23±0.21 and 8.51±0.12 %, respectively in aerial parts whereas, 7.50±0.46, 3.15±0.08 and 7.48±0.18 %, respectively in roots. The foaming index was found to be less than 100 in both the aerial parts and roots but, the swelling index was comparatively higher in roots (7.65±0.72 %) as compared to aerial parts (4.11±0.66 %). Preliminary qualitative tests of the fractions obtained from aerial parts and roots revealed the presence of various phytoconstituents such as steroidal compounds, terpenoids, flavonoids, phenolic substances, alkaloids and glycosides thereby, supporting the phytochemical diversity of the plant material. The results are shown in Table 4.

Table 1: Fluorescence analysis of Aerial Parts of Aerva Lanata

Table 2: Fluorescence analysis of Roots of Aerva Lanata

Table 3: Physicochemical Parameters of Aerva Lanata

Table 4: Preliminary Phytochemical Screening of Various Fractions of Aerial Parts and Roots of Aerva Lanata

In vitro nucleation assay demonstrated a significant dose-dependent enhancement of the percentage inhibitory activity as exhibited by the extracts from aerial parts, roots and diverse fractions derived from them compared to standard (cystone). The percentage inhibition shown by aerial parts and roots is mentioned in Table 5 and Table 6. Fraction III from both aerial parts and roots exerted the maximum inhibition on calcium oxalate nucleation at the concentration of 1000 μg/ml amongst the various generated fractions (71.01±1.13 % for aerial parts and 54.61±2.30 % for roots). Moreover, the extract from aerial parts and roots gave a reasonable activity compared to standard at 1000 μg/ml (74.18±2.57 %) with the aerial parts exhibiting a higher inhibition (72.96±1.48 %) than roots (67.82±0.43 %). The least hindrance to nucleation of calcium oxalate was demonstrated by fraction I in case of aerial parts (36.37±2.11 %) and fraction IV in case of roots (27.42±2.12 %).

Table 5: Nucleation Assay of Aerial Parts of Aerva Lanata

Table 6: Nucleation Assay of Roots of Aerva Lanata

The quantity of tannins, total phenolics and total flavonoids were found to be 4.34±0.63 mg TAE/g extract, 127.84±1.50 mg GAE/g extract and 77.61±3.78 mg RE/g extract, respectively in aerial parts, which were comparatively higher than roots being 3.03±0.63 mg TAE/g extract, 98.09±1.10 mg GAE/g extract and 62.81±5.69 mg RE/g extract, respectively. The results are reported in Table 7.

Table 7: Tannin, Total Phenolic and Total Flavonoid Content of Aerva Lanata

The fluorescence characteristic displayed by the aerial parts and roots either as such or after treatment with various reagents are emanated by the specific phytochemicals contained in them and is an exclusive feature of the crude drug thereby, serving as a crucial factor for pharmacognostic identification [33]. The results of fluorescence behaviour from the current study were unique to the plant material.

Physicochemical inspection is an imperative tool to ascertain the identity, quality and purity of a crude drug. It plays a vital role in the detection of adulteration, substandard variety and discrepant handling of crude drugs [34]. Failure to perform physicochemical studies is associated with deterioration of the potency and stability of medicinal herbs. The values of foreign matter, moisture content, ash values, extractive values, foaming index and swelling index in the present work were in accordance with the official standards thus supporting the purity and quality of the plant material. Hence, the physicochemical parameters of roots and aerial parts of A. lanata complied with the official limits, which can serve as a reference for carrying out standardization of the plant material for future studies.

In vitro antiurolithiatic activity was conducted through solvent-solvent partitioning based fractionation with the objective of achieving the concept regarding the category of phytoconstituents responsible for the litholytic efficacy of this herb. Nucleation refers to the formation of a solid crystal state in a solution and is the initial stage in crystal formation [35]. The process of nucleation involves either homogenous nucleation or heterogenous nucleation. The former occurs in pure solution whereas, the latter takes place in divergent chemical environment such as the human urinary system and refers to epitaxial accumulation of crystals on pre-formed urinary crystals, cellular material or urinary casts [36]. Stone formation commences with the urinary supersaturation with stone forming components such as calcium, oxalate and phosphate leading to the formation of crystals, which serve as a nidus for crystal nucleation, growth, epitaxial growth and aggregation progressively leading to development of calculi [37]. Therefore, a phytopharmaceutical agent capable of impeding the nucleation phase will also intervene with further stages of stone formation thereby preventing lithiasis. Restoration of the balance between urinary stone promoters (calcium, oxalate, uric acid, phosphate) and inhibitors (citrate and magnesium) needs to be attained by the intended antiurolithiatic agent as the imbalance contributes to lithiasis [38]. Besides this, raising the urine volume, pH and anticalcifying activity, enhancing renal function and oxalate metabolism, lessening the oxidative stress on renal tissues are other mechanisms exhibited by medicinal herbs for alleviating the incidence of calculi [39].

In the current investigation, fraction III demonstrated the maximum inhibition on nucleation of calcium oxalate as compared to other extract-derived fractions of aerial parts and roots. This fraction was chiefly composed of flavonoids and phenolic compounds as evidenced from phytochemical screening. Flavonoids and phenolics chiefly exhibit antioxidant activity and hence, can serve as antiurolithiatic agents by averting the crystal adhesion and consecutive formation of urinary stones by alleviating the injury caused by crystal accumulation in the urinary system that otherwise enhances the formation of free radicals resulting in destruction of membrane surface of kidney tubules via lipid peroxidation [40,41]. In the present research, the extract and the flavonoid enriched fraction of aerial parts exerted a better efficacy in comparison to those from roots, which is likely attributed to the existence of highly efficacious antiurolithiatic moieties in aerial parts compared to roots. This can be further ascertained by the isolation and quantification of active phytocomponents from aerial parts. Moreover, these phytometabolites exist in varying concentrations in different parts of a medicinal plant and thus, exert variable degrees of activity because of which it becomes indispensable in exploring the botanical part majorly contributing towards the remedial effect. Both the extracts from aerial parts and roots exhibited a higher impediment to nucleation, which was comparable to that by the standard cystone, which is a polyherbal blend and an established potent marketed formulation. Fraction III from aerial parts demonstrated an immense hindrance to nucleation which was as high as the standard formulation and the extract while fraction III from roots exhibited a lower activity than the standard and the extract. This highlights the potency and tremendous efficacy of various antiurolithic flavonoid and phenolic aglycones in the aerial parts compared to roots. However, the greater antiurolithic ability of the extract as a whole is likely associated with the synergistic effect of various phytoconstituents present. On the contrary, fraction I composed of steroidal moieties as well as terpenoidal compounds derived from aerial parts and fraction IV from roots possessing glycosidal compounds displayed the least inhibition amongst the various fractions. This is reasonably attributed to the lower concentration and limited antiurolithic efficiency of those specific metabolites in aerial parts and roots, respectively. Fraction II from aerial parts and roots composed of alkaloidal compounds also demonstrated lower activity compared to flavonoid and phenolic containing fraction. This implies that flavonoids and phenolics have a dominating contribution in lessening the incidence of stone formation as compared to other metabolites. An earlier study reported that both flavonoids (quercetin) and terpenoids (betulin) have a significant role in antiurolithic activity of A. lanata [20] unlike the present study, which shows the fraction rich in steroids and terpenoids exhibiting much inferior activity compared to the flavonoid rich fraction displaying the best activity.

As evident from the current study, the amount of tannins, total phenolics and total flavonoids in aerial parts were also relatively greater than roots. This accounts for the higher antiurolithic efficacy of aerial parts over roots. Hence, the present work provides the concept regarding the role of flavonoids and phenolics towards the litholytic efficacy of this medicinal plant of immense pharmacological scope. Further investigation is mandatory with respect to in vivo studies of the fraction followed by thorough phytochemical evaluation and isolation of the phytoconstituents from the same by column chromatography, flash chromatography, HPLC and other techniques to establish the validity of the current finding. This miraculous herb can be a source of novel leads, which can exhibit a crucial role in alleviating the prevalence of this affliction amongst the general population.

Acknowledgements

The authors thank Dr. Sunita Garg, Head, Raw Materials Herbarium and Museum, NISCAIR, New Delhi for authenticating the plant material and the Amity Institute of Pharmacy, Amity University, Noida for providing the required facilities needed for the present research work.

Conflicts of interest

There are no conflicts of interest.

Financial support and sponsorship

Nil.

References

1. Vijaya T, Kumar MS, Ramarao NV, Babu AN, Ramarao N. Urolithiasis and its causes- short review. J Phytopharmacol 2013;2:1-6.
2. Al-Jebouri MM, Atalah N. A study on the interrelationship between renal calculi, hormonal abnormalities and urinary tract infections in Iraqi patients. Open J Urol 2012;2:6-10.
3. Joy JM, Prathyusha S, Mohanalakshmi S, Praveen Kumar AVS, Ashok Kumar CK. Potent herbal wealth with litholytic activity: a review.IJIDD 2012;2:66-75.
4. Nagal A, Singla RK. Herbal resources with antiurolithiatic effects: a review. Indo Global J Pharm Sci2013;3:6-14.
5. Tiwari A, Soni V, Londhe V, Bhandarkar A, Bandawane D, Nipate S. An overview on potent indigenous herbs for urinary tract infirmity: urolithiasis. Asian J Pharm Clin Res 2012;5:7-12.
6. Aggarwal KP, Narula S, Kakkar M, Tandon C. Nephrolithiasis: molecular mechanism of renal stone formation and the critical role played by modulators. Biomed Res Int2013;2013:292-953.
7. Rosa M, Usai P, Miano R, Kim FJ, Agrò EF, Bove P, et al. Recent findings and new technologies in nephrolithiasis: a review of the recent literature. BMC Urol 2013;13:1-11.
8. Begun FP, Knoll CE, Gottlieb M, Lawson RK. Chronic effects of focused electrohydraulic shock-waves on renal function and hypertension. J Urol 1991;145:635-9.
9. Alok S, Jain SK, Verma A, Kumar M, Sabharwal M. Pathophysiology of kidney, gall bladder and urinary stones treatment with herbal and allopathic medicine: a review. Asian Pac J Trop Dis 2013;3:496-504.
10. Chitme HR, Alok S, Jain SK, Sabharwal M. Herbal treatment for urinary stones. Int J Pharm Sci Res 2010;1:24-31.
11. Goyal M, Pareek A, Nagori BP, Sasmal D. Aervalanata: a review on phytochemistry and pharmacological aspects. Pharmacogn Rev 2011;5:195-8.
12. Nagaratna A, Prakash LH, Harini A. A pharmacological review on Gorakhaganja(Aervalanata(Linn) Juss. Ex. Schult). J PharmacognPhytochem2014;3:253-7.
13. Rajesh R, Chitra K, Paarakh PM. Aervalanata(Linn.) Juss. exSchult.- An overview. Indian J Nat Prod Resour 2011;2:5-9.
14. Soundararajan P, Mahesh R, Ramesh T, Hazeena Begum V. Effect of Aervalanataon calcium oxalate urolithiasis in rats. Indian J ExpBiol 2006;44:981-6.
15. Selvam R, Kalaiselvi P, Govindaraj A, BalaMurugan V, Sathish Kumar AS. Effect of Aervalanataleaf extract and VediuppuChunnamon the urinary risk factors of calcium oxalate urolithiasis during experimental hyperoxaluria. Pharmacol Res 2001;43:89-93.
16. Sunil Kumar KN, Prabhu SN, Ravishankar B, Sahana, Yashovarma B. Chemical analysis and in vitroevaluation of antiurolithiatic activity of Aervalanata(Linn.) Juss. Ex Schult. roots. Research and Reviews: J PharmacognPhytochem 2015;3(3):1-7.
17. Nirmaladevi R, Chandirika JU, Annadurai G, Kalpana S, ShrinidhiRai T. Evaluation of Aervalanataflower extract for its antilithiatic potential in vitro and in vivo. Int J Pharm PharmSci Res 2013;3:67-71.
18. Arthi I, Ravichandiran V, Sampath Kumar KP, Subburaju T. Antiurolithiatic effect of AervalanataLinn extract on ethylene glycol induced urinary calculi model in rats. Int J Pharm Sci Rev Res 2012;17:46-50.
19. Saravanasingh K, Ramamurthy M, Parthiban P. Invitroanti-urolithiatic activity of aerial parts of Aervalanata (L.) Juss. Int J Curr Res Med Sci 2016;2(3):24-7.
20. Dinnimath BM, Jalalpure SS. In silicoantiurolithiatic screening of Aervalanata(L) isolated constituents. Indian J Pharm Educ Res 2015;49(1):1-8.
21. Kokoski CJ, Kokoski RJ, Slama FJ. Fluorescence of powdered vegetable drugs under ultraviolet radiation. J Am PharmAssoc1958;47:715-7.
22. http://www.ayurveda.hu/api/API-Vol-1.pdf.
23. http://apps.who.int/iris/bitstream/10665/41986/1/9241545100.pdf.
24. Soni S, Anandjiwala S, Patel G, Rajani M. Validation of different methods of preparation of Adhatodavasicaleaf juice by quantification of total alkaloids and vasicine. Indian J Pharm Sci 2008;70:36-42.
25. Yadav S, Kumar P. Production, isolation and identification of flavonoids from aerial parts of Hiptagebenghalensis. Int J Life Sci Pharma Res 2012;2:1-5.
26. Harborne JB. Phytochemical methods: A guide to modern techniques of plant analysis. 3rd ed. London: Chapman and Hall; 1998.
27. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 13th ed. Pune: NiraliPrakashan; 2005.
28. Saranya R, Geetha N. Inhibition of calcium oxalate (CaOx) crystallization in vitroby the extract of beet root (Beta vulgaris L.). Int J Pharm PharmSci 2014;6:361-5.
29. Atmani F, Khan SR. Effects of an extract from Herniariahirsutaon calcium oxalate crystallization in vitro. BJU Int 2000;85:621-5.
30. Tambe VD, Bhambar RS. Estimation of total phenol, tannin, alkaloid and flavonoid in Hibiscus tiliaceusLinn. wood extracts. Res Rev: J PharmacognPhytochem 2014;2:41-7.
31. Shafei AA. Qualitative and quantitative estimation of flavonoids and phenolic compounds and the biological activities of Colvillearacemosacultivated in Egypt. Int J PharmacognPhytochem Res 2016;8:836-40.
32. Sharma V, Agarwal A. Physicochemical and antioxidant assays of methanol and hydromethanol extract of aerial parts of IndigoferatinctoriaLinn. Indian J Pharm Sci 2015;77:729-34.
33. Kumar M, Mondal P, Borah S, Mahato K. Physico-chemical evaluation, preliminary phytochemical investigation, fluorescence and TLC analysis of leaves of the plant Lasiaspinosa(Lour) Thwaites. Int J Pharm PharmSci 2013;5:306-10.
34. Regupathi T, Chitra K. Physicochemical analysis of medicinal herbs, Ecliptaalba(L.) Hassk and Lippianodiflora(Linn.). Int J Pharm Phytopharm Res 2015;4:249-51.
35. Basavaraj DR, Biyani CS, Browning AJ, Cartledge JJ. The role of urinary kidney stone inhibitors and promoters in the pathogenesis of calcium containing renal stones. EAU-EBU Update Series 2007;5:126-36.
36. Matlaga, BR. Pathogenesis of renal calculi. In: Pearle MS, Nakada SY, editors. Urolithiasis: Medical and Surgical Management. 1st ed. London: Informa; 2009. p. 10.
37. Charles YC, Pak MD. Calcium urolithiasis: Pathogenesis, Diagnosis and Management. 1st ed. New York: Plenum Publishing Corporation; 1978.
38. Jawalekar S, Survey VT, Bhutey AK. Inhibition efficiency of urine towards stone forming minerals. Int J PharmaSci Res 2010;1:23-7.
39. Gupta SK, Baghel MS, Bhuyan C, Ravishankar B, Ashok BK, Patil PD. Evaluation of anti-urolithiatic activity of PashanabhedadiGhritaagainst experimentally induced renal calculi in rats. Ayu 2012;33:429-34.
40. Shirfule AL, Racharla V, Qadri SSYH, Khandare AL. Exploring antiurolithic effects of GokshuradipolyherbalAyurvedic formulation in ethylene-glycol-induced urolithic rats. Evid Based Complement Alternat Med 2013;2013:1-9.
41. Padala K, Ragini V. Anti-urolithiatic activity of extracts of Aervajavanicain rats. Int J Drug Dev Res 2014;6:35-45.