In vitro antiurolithiatic activity of c-phycocyanin isolated from Spirulina...

[Full title: In vitro antiurolithiatic activity of c-phycocyanin isolated from Spirulina platensis]

[Find the meaning and references behind the names: Change, Less, Class, India, Stones, Level, Better, Standard, Stone, Low, Active, Body, Shock, Mash, Present, Show, Wave, Mar, Main, Part, Original, Waves, Size, Data, High, Mass, Acid, Place, Sample, Reason, Due, Crystal, Bone, Rather, Light, Study, Strong, Serious, General, Factor, Jan, Shown]

Asian Journal of Pharmaceutic s • Jan-Mar 2021 • 15 (1) | 124 In vitro antiurolithiatic activity of c-phycocyanin isolated from Spirulina platensis N. J. P. Subhashini, Shilpika Nagula Department of Chemistry, University College of Science, Osmania University, Hyderabad, Telangana, India Abstract Background: Pashanabheda is used as antiurolithiatic in Ayurveda. In the present study, Spirulina platensis was selected for isolation of its active constituent, C-Phycocyanin (C-PC), and screening for in vitro antiurolithiatic potentials. Objective: Screening of compound isolated from S. platensis for antiurolithiatic potentials. Materials and Methods: The algae sample was subjected to preliminary analysis. Then, the sample was processed for the extraction of phycocyanin from S. platensis by various extraction methods, purified and then characterization was performed by reverse phase high-pressure liquid chromatograph y and by mass spectrometry. Finally, in vitro antiurolithiatic activity was screened by nucleation and aggregation assay Results: The isolated C-PC exhibited inhibitory action in both nucleation and aggregation assays to significant level. In the aggregation assay gradually decrease in the calcium oxalate (CaOx) crystal nucleation as well as growth was observed by light microscopy. The findings of the nucleation assay indicate that phytoconstituents inhibited the crystallization of CaOx in solution. There were less and smaller particles with increasing concentration of the phycocyanin. The increasing concentrations of C-PC (100, 200, 300, 400, and 500 µ g/ml) inhibited the CaOx crystal growth. C-PC demonstrated slightly better results compared to cystone standard solution to inhibit the formation of CaOx dihydrate crystals in the nucleation assay. Conclusion: The isolated C-PC has shown antiurolithiatic effect by significantly reducing the size and growth of calculi in the kidneys in the in vitro assays Keywords: aggregation assay, C-Phycocyanin, in vitro antiurolithiatic activity, nucleation assay Address for correspondence: Dr. N. J. P. Subhashini, Department of Chemistry, University College of Science, Osmania University, Hyderabad, Telangana - 500 007, India . E-mail: njsubhashini@yahoo.co.in Received: 09-12-2020 Revised: 11-02-2021 Accepted: 24-02-2021 INTRODUCTION U rinary calculi are the third most prevalent disorder of the urinary system. Approximately 80% of these calculi are composed of calcium oxalate (CaOx) [1-4] Urine is normally a supersaturated solution and only some individuals are prone to this disease. One reason for this is the presence of inhibitors of lithogenesis in urine, including macromolecules, citrate, and magnesium [4,5] Thus, an imbalance between the promoters such as low urine volume, calcium, oxalate, uric acid, phosphate, and inhibitors may represent a potential factor in lithogenesis Nowadays, stone formation is the oldest and serious painful urologic disease with significant prevalence in the population due to change in lifestyle and dietary factors. Stone formation or lithiasis is characterized by calculi formation. It has two main types such as nephrolithiasis and urolithiasis. Calculi formation in urinary bladder, ureter, or any part of urinary tract rather than kidney is known as urolithiasis while nephrolithiasis is characterized calculi formation in kidney [6-10] In general, calcification for the formation of bone and teeth takes place in controlled biological situations. Uncontrolled pathological crystallization occurs when solvent becomes supersaturated leading to the formation of precipitates in the body called as kidney stones Extracorporeal shock wave lithotripsy (ESWL) and drug treatment revolutionized urological practice almost became the standard procedure for eliminating kidney stones. However, shock waves had traumatic effects, residual stone fragments persisted and infection could occur. Moreover, ORIGINAL AR TICLE

[Find the meaning and references behind the names: Every, Foods, Debris, Plant, Bennett, Purity, Pellet, Cure, Kumar, Bind, Set, Tamil, Loss, Vivo, Six, Large, Bogard, Deep, Glass, Sampath, Tube, Chennai, Red, Cell, Room, Southern, Rate, Free, Green, Rich, Blue, Bath, Min, Nadu, Look]

Subhashini and Nagula: Antiurolithiatic activity of C-Phycocyanin Asian Journal of Pharmaceutic s • Jan-Mar 2021 • 15 (1) | 125 ESWL may cause acute renal injury, a decrease in renal function, hemorrhage, and hypertension. Therefore, it is worthwhile to look for alternative means such as medicinal plants or phytotherapy [11,12] Data from in vitro , in vivo studies and clinical trials reveal that phytotherapeutic agents could be useful as either an alternative or an adjunctive therapy in the management of urolithiasis [13-15] Oxalic acid is biosynthesized from ascorbic acid, glycolate, and glyoxylate in the metabolism of higher plants. A significant loss of minerals is more prevalent in the body when it is consumed in large content of oxalate rich foods. When calcium ions present in the body bind with free oxalic acid/oxalate, it precipitates as insoluble crystals [12] A large number of plants have been used in India since ancient times, which claim the efficient cure of urinary stone. C-Phycocyanin (C-PC), a phycobiliprotein, is a water-soluble and colored protein component of the photosynthetic light-harvesting antenna complexes present in cyanobacteria, red algae, and cryptomonads. Due to its anti-oxidant, anti-inflammatory, hepatoprotective, and radical-scavenging, lipid lowering, anti-arthritic properties C-PC are widely used in food, cosmetics, and pharmaceuticals. Spirulina, a type of cyanobacteria, has a high growth rate with as much as 25% (w/w) C-PC content in the biomass, and thus is considered as a promising and commercial C-PC source [14-16] Therefore, present study intends to establish the scientific rationality of the antiurolithiatic activity of C-PC isolated from Spirulina platensis by in vitro methods MATERIALS AND METHODS Collection and authentication of plant S. platensis is blue – green algae was purchased from Parrys Nutraceuticals, Chennai, Tamil Nadu, India, and was authenticated by Dr. V. Sampath Kumar (Scientist D in-charge), Botanical Survey of India, Southern regional center, Coimbatore, Tamil Nadu, India Extraction methods [14-16] Distilled water extraction 10 g of Spirulina biomass was suspended in the distilled water and kept in the deep freezer at −20°C for 24 h and then centrifuged at 6000 rpm for 10 min Homogenization 10 g of the wet biomass was homogenized with the help of mortar and pestle in the presence of glass beads in 50 mM of sodium phosphate buffer (pH 6.8) The extract was kept at −20°C for 24 h and centrifuged at 6000 rpm for 10 min. The pellet was re-extracted with buffer to ensure complete recovery of phycocyanin Freezing and thawing Phycocyanin was extracted by repeated freezing and thawing of cells by which the wet biomass (10 g) was mixed with 50 mM sodium phosphate buffer (pH 6.8) and kept at −20°C for 24 h. The above procedure was repeated 4 times. Then, the sample was centrifuged at 6000 rpm for 10 min. Then, the supernatant was taken for phycocyanin estimation Acid extraction 10 g of biomass was taken in two test tubes, in each test tube, 2 and 5 ml of the concentrated hydrochloric acid was added. The samples were kept in room temperature for 24 h. Samples were centrifuged at 6000 rpm for 10 min, and then supernatant was taken for phycocyanin estimation Heating 10 g of the wet biomass was taken in a test tube and kept inside the water bath, which was set at a temperature of about 60°C for 10 min. The sample was centrifuged at 1000 rpm for 10 min and supernatant was taken for phycocyanin estimation Every 1 h freezing and thawing 10 g of the wet biomass was taken in the beaker along with the 50 mM sodium phosphate buffer and kept at −20°C for 1 h and thawed at 4°C for 1 h. In the same way, 3–6 cycles were repeated .Then, the biomass was centrifuged at 6000 rpm for 10 min and supernatant was taken for phycocyanin estimation. Amount of C-PC was measured as described by Bennett and Bogard (1973) and purity was determined by using the formulae: Purity = A 620/A 280 Purification of C-PC The C-PC was extracted from blue-green algae by various methods Among the six methods, C-PC extracted by repeated freezing at −20°C and thawing at room temperature was found to be effective method. Cell debris was removed by centrifugation at 5000 rpm for 10 min and the extract thus obtained was termed as crude extract. Amount of C-PC was measured and purity was determined using the formulae: Purity = A 620 / A 280 Extraction yield = (C-PC) V/DB

[Find the meaning and references behind the names: Stage, New, Human, Normal, Final, Strain, Time, Triple, Tool, Api, Dry, Vis, Tris, Table, Ion]

Subhashini and Nagula: Antiurolithiatic activity of C-Phycocyanin Asian Journal of Pharmaceutic s • Jan-Mar 2021 • 15 (1) | 126 Where yield is the extraction yield of C-PC in mg C-PC/ dry biomass (g), V is the solvent volume (ml), and DB is the dry biomass (g). The optical density (OD) values were tabulated [17,18] Characterization of the C-PC Reverse phase high-pressure liquid chromatography (HPLC) To characterize the C-PC, reverse phase HPLC was performed using C 5 column. PDA detector set at 620 and 226 nm. The absorption spectra of these two chromatogram peaks were critically analyzed [19] Mass spectrometry (liquid chromatography [LC] MS /MS) The MS has been regarded as one of the most important analytical tool in studies of drug metabolism and biochemical toxicology. With the commercial introduction of new ionization methods such as API techniques in combination of LC-MS/MS, it has now become a truly indispensable technique in research. Triple stage quadrupole and ion trap MS are presently used for this purpose, because of their sensitivity and selectivity. It showed maximum identity with C-PC of S. platensis [20] In vitro antiurolithiatic activity Aggregation assay The rate of aggregation of the CaOx crystals was determined by a spectrophotometric assay with slight modifications. The CaOx monohydrate (COM) crystals were prepared by mixing both the solutions of calcium chloride and sodium oxalate of 50 mM each. Both solutions were then equilibrated. The solutions were then cooled to 37°C and then evaporated. The COM crystals were then dissolved with 0.5 ml of 0.05 mM Tris buffer and 0.5 ml of 0.15 mM NaCl solution at pH 6.5 to a final concentration of 1 mg/ml. Absorbance at 620 nm was recorded. The rate of aggregation was estimated by comparing the slope of turbidity in the presence of the extract against control [20,21] Nucleation assay (turbidity method) The inhibitory activity of the extracts on the nucleation of CaOx crystals was determined by a spectrophotometric assay. Crystallization was initiated by adding 100 µ l of 4 mM calcium chloride and 100 µ l of 50 mM sodium oxalate solutions to 0.5 ml of human normal urine, both prepared in a buffer containing 0.5 ml of 0.05 mM Tris buffer and 0.5 ml of 0.15 mM NaCl solution at pH 6.5 and 37°C and adjusted to volume by adding 1.5 ml of distilled water. The rate of nucleation was determined by comparing the induction time of crystals (time of appearance of crystals that reached a critical size and thus became optically detectable ) in the presence of the extract and that of the control with no extract. The OD was recorded at 620 nm, and the percentage inhibition calculated as (1-OD (experimental)/OD (control))/100 [22-24] RESULTS AND DISCUSSION Extraction of C-PC The C-PC was extracted from blue-green algae by various methods [Table 1 ]. Among the six methods, C-PC extracted by repeated freezing at −20°C and thawing at room temperature was found to be effective method. Cell debris was removed by centrifugation at 5000 rpm for 10 min and the extract thus obtained was termed as crude extract. Amount of C-PC was measured and purity was determined using the formulae: Purity = A 620/A 280. C-PC = (A 615)–(0.475 × A 652)/5.34. Extraction yield = (C-PC) V/DB Where yield is the extraction yield of C-PC in mg C-PC/dry biomass (g), Vis the solvent volume (ml), and DB is the dry biomass (g). The OD values were tabulated Figure 1: Reverse phase high-pressure liquid chromatography profile of C-Phycocyanin from Spirulina strain using PDA detector (620 nm) showing  and  subunit peaks a b

[Find the meaning and references behind the names: Fig, Ure, Cod, Under, Major, Grow, Peak]

Subhashini and Nagula: Antiurolithiatic activity of C-Phycocyanin Asian Journal of Pharmaceutic s • Jan-Mar 2021 • 15 (1) | 127 Purification of C-PC To characterize the C-PC, reverse phase HPLC was performed using C 5 column [Fig ure 1]. PDA detector set at 620 and 226 nm revealed two major peaks at 25.612 and 27.024 min. When absorption spectra of these two chromatogram peaks were critically analyzed, it was found that A 620:A 280 for the first peak RT = 25.612 min; which was approximately 1. It is due to the presence of one phycocyanobilin (PCB) chromophore, thus indicating that this peak corresponds to α subunit of PC, while the A 620:A 280 for the second peak RT = 27.024 min; it was approximately 2, which is due to the presence of two PCB chromophores, and therefore this peak corresponds to  subunit of PC Characterization of the C-PC by MS (LC MS/MS) The MS has been regarded as one of the most important analytical tool in studies of drug metabolism and biochemical toxicology. With the commercial introduction of new ionization methods such as API techniques in combination of LC-MS/MS, it has now become a truly indispensable technique in research. Triple stage quadrupole and ion trap MS are presently used for this purpose, because of their sensitivity and selectivity. It showed maximum identity with C-PC of S. platensis [ Fig ure 2] Aggregation assay CaOx crystals begin grow; aggregate with other crystals and retained in the kidney. This is aggregation process which causes renal injury. C-PC demonstrated slightly better compared to cystone standard solution to inhibit promoted the formation of COD crystals [25] [ Fig ure 3 ] Nucleation assay C-PC inhibited the crystallization by inhibiting nucleation of CaOx through disintegrating into smaller particles with increasing concentrations of the fraction. From the results Table 1: Extraction of C-Phycocyanin Method pH Temperature Optical density (OD) A 620 A 280 Concentrated HCl extract 1 Room temperature - - Normal freezing and thawing extract 6 −20°C 2.682 3.521 Homogenized freezing and thawing extract 7 −20°C 2.765 3.468 Water extract 6 −20°C 2.872 3.612 1 h freezing and thawing extract 6 −20°C 2.582 3.716 Figure 2: Mass spectrophotometer results Figure 3: Calcium oxalate (CaOx) crystals, observed under light microscope (×100), formed in the metastable solution of CaOx in the absence (a) which are larger and the presence of saponin rich fraction of C-Phycocyanin (b) 10 mg/ml, (c) 20 mg/ml, (d) 40 mg/ml, (e) 60 mg/ml, (f) 80 mg/ml, and (g) 100 mg/ml gradually decreases the CaOx crystals nucleation as well as growth d c g b f a e

[Find the meaning and references behind the names: Duron, Step, Natural, Man, Gilani, Khan, Kelly, Anand, Delhi, Ebu, Clin, Int, Enough, Single, Risk, Long, Bashir, Lab, Mumbai, Patnaik, Ser, Basavaraj, Blood, Bhat, Beutler, Baumann, Jammu, Genesis, Dhawan, Progress, Cartledge, Med, Role, Eau, Atmani, Browning, Herb, Property, Common]

Subhashini and Nagula: Antiurolithiatic activity of C-Phycocyanin Asian Journal of Pharmaceutic s • Jan-Mar 2021 • 15 (1) | 128 of the nucleation assay confirmed that the extract contained nucleation preventing agents [Fig ure 4] DISCUSSION Kidney stones are reportedly affecting mankind since long time and have been one of the causes for renal failure [24,26] As there is no single effective drug available for urolithiasis today, surgery is considered to be the best option especially when other alternatives fail. However, it is expensive and not affordable for common man. Hence, the natural drugs are considered to be next alternative. Pashanabheda plants are a group of medicinal plants which are used in Indian traditional medicinal system by Ayurveda practitioners as antiurolithiatic drugs and S. platensis is used conventionally as both antiurolithiatic and diuretic In the aggregation assay , CaOx crystals begin to grow; aggregate with other crystals, and retained in the kidney [27] This is aggregation process which causes renal injury. C-PC demonstrated slightly better compared to cystone standard solution to inhibit promoted the formation of COD crystals. COM has a stronger affinity with cell membranes; it may lead to become higher potential risk for renal calculi formation An in vitro crystallization study was performed, since nucleation is an important first step for the initiation of crystals, which then grow and form aggregates. The main findings of the present study were that cyanin rich fraction from plants inhibited the crystallization by inhibiting nucleation of CaOx in solution; less and smaller particles were formed with increasing concentrations of the fraction. The results of the nucleation assay confirmed that the extract contained nucleation-preventing agents. The limiting factors in stone formation could be those processes that affect crystal growth, because particles may become large enough to occlude the urinary tract, leading to stone formation [28-32] The herb extracts may contain substances that inhibit the growth of CaOx crystals. This property of plants may be important in preventing the growth of kidney stone. Aggregation may be an important factor in the genesis of stones [25,33] Recurrent calcium stone formers excrete clusters of crystals in their urine, caused by aggregation, also named agglomeration, whereas urine from normal people contains mainly single crystals [34-36] Again, percentage inhibition of crystals aggregation increased as the concentration of C-PC increased CONCLUSION Antiurolithiatic activity of S. platensis is mediated possibly through inhibition of CaOx crystal formation and its effect on the urinary concentration of stone forming constituents and nephrolithiasis inducing factors and this study rationalizes its medicinal use in urolithiasis REFERENCES 1. Anand R, Patnaik G, Kulshreshtha D, Dhawan B. Antiurolithiatic activity of lupeol, the active constituent isolated from Crateva nurvala. Phytother Res 1994;8:417-21 2. Anonymous. Indian Herbal Pharmacopoeia. Jammu Tawi: Indian Drug Manufacturers’ Association, Mumbai and Regional Research Laboratory; 1998 3. Anonymous. The Ayurvedic Pharmacopoeia of India. 1 st ed. New Delhi, India: Ministry of Health and Family Welfare; 2001 4. Bhat VB, Madyastha KM. C-phycocyanin: A potent peroxyl radical scavenger in vivo and in vitro . Biochem Biophys Res Commun 2000;275:20-5 5. Atmani F, Khan SR. Effects of an extract from Herniaria hirsuta on calcium oxalate crystallization in vitro . Br J Urol Int 2000;85:621-5 6. 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 Ser 2007;5:126-36 7. Bashir S, Gilani AH. Antiurolithic effect of Bergenia ligulata rhizome: An explanation of the underlying mechanisms. J Ethnopharmacol 2009;122:106-16 8. Baumann JM. Stone prevention: Why so little progress? Urol Res 1998;26:77-81 9. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med Figure 4: Effect of cystone and C-Phycocyanin on calcium oxalate (CaOx) crystal nucleation (a) effect of cystone on CaOx crystal nucleation (b) effect of C-Phycocyanin on CaOx crystal nucleation b a

[Find the meaning and references behind the names: Zhang, Liu, Lee, Fish, Malini, Pharm, Haroun, Four, Press, Vidya, Aulakh, Bouanani, Sheth, Touhami, Gupta, Wang, Parekh, Poindexter, Parmar, Wood, Hodgkinson, Arif, Joshi, Sweety, Henchiri, Sci, Chem, Hussain, Kuang, Mahmoud, Fruit, Jain, Aouf, Huang, Book, Chin, London, Root, Zarrouk, Baskar, Mohammady, Lam, Zhu, Hwang, Genes, Basu, Yagi, Simple, Nile, Vaidya, Hassaan, Rat, Pac, Prod, Swami, Turk, Zhou, Nil, Bai, Pan, Centre, Female, Chang, Karadi, Chen, Bhalla, Kok, Farouk, Pak, Ying, None]

Subhashini and Nagula: Antiurolithiatic activity of C-Phycocyanin Asian Journal of Pharmaceutic s • Jan-Mar 2021 • 15 (1) | 129 1963;61:882-8 10. Bouanani S, Henchiri C, Griffoni EM, Aouf N, Lecouvey M. Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats. J Ethnopharmacol 2010;129:38-45 11. Zhou ZP, Liu LN, Chen XL, Wang JX, Chen M, Zhang YZ, et al . Factors that effect antioxidant activity of C-Phycocyanin from Spirulina platensis . J Food Biochem 2005;29:313-22 12. Zhu Y, Chen B, Wang KB, Li YX, Bai KZ, Kuang TY, et al . A simple method for extracting C-phycocyanin from Spirulina platensis using Klebsiella pneumoniae . Appl Microbiol Biotechnol 2007;74:244-8 13. Fleisch H. Inhibitors and promoters of stone formation. Kidney Int 1978;13:361-71 14. Zhang YM, Chen F. A simple method for efficient separation and purification of C-phycocyanin and allophycocyanin from Spirulina platensis . Biotechnol Tech 1999;13:601-3 15. Gogte VM. Ayurvedic Pharmacology and Therapeutic Uses of Medicinal Plants (Dravyaguna Vignyan). Mumbai: Bharatiya Vidya Bhavan, Swami Prakashananda Ayurveda Research Centre; 2000 16. Ying J, Pan RW, Wang MF. Effects of phycocyanin on apoptosis of human laryngeal cancer HEP-2 cells. Chin J Pathophysiol 2015;7:1189-96 17. Minkova KM, Tchernov AA, Tchorbadjieva MI, Fournadjieva ST, Antova RE, Busheva MC. Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. J Biotechnol 2003;102:55-9 18. Hassaan MS, Mohammady EY, Soaudy MR, Sabae SA, Mahmoud AM, El-Haroun ER. Comparative study on the effect of dietary β-carotene and phycocyanin extracted from Spirulina platensis on immune-oxidative stress biomarkers, genes expression and intestinal enzymes, serum biochemical in Nile tilapia, Oreochromis niloticus. Fish Shellfish Immunol 2021;108:63-72 19. Stramarkou M, Papadaki S, Kyriakopoulou K, Tzovenis I, Chronis M, Krokida M. Comparative analysis of different drying techniques based on the qualitative characteristics of Spirulina platensis biomass. J Aquat Food Prod Technol 2021;24:1-9 20. Chofamba A. Cellular bioenergetics in Spirulina platensis towards growth and phycocyanin production under different photon flux densities using the modified Zarrouk’s medium. Turk J Agric Food Sci Technol 2021;9:28-34 21. Hodgkinson A. Determination of oxalic acid in biological material. Clin Chem 1970;16:547-57 22. Hodgkinson A. Oxalic Acid in Biology and Medicine. London: Academic Press; 1977. 23. Hussain T, Gupta RK, Sweety K, Khan MS, Hussain MS, Arif M, et al . Evaluation of antihepatotoxic potential of Solanum xanthocarpum fruit extract against antitubercular drugs induced hepatopathy in experimental rodents. Asian Pac J Trop Biomed 2012;2:454-60 24. Hwang T, Preminger G, Poindexter J, Pak C. Urinary glycosaminoglycans in normal subjects and patients with stones. J Urol 1988;139:995 25. Michelacci YM, Glashan RQ, Schor N. Urinary excretion of glycosaminoglycans in normal and stone forming subjects. Kidney Int 1989;36:1022-8 26. Iguchi M, Takamura C, Umekawa T, Kurita T, Kohri K. Inhibitory effects of female sex hormones on urinary stone formation in rats. Kidney Int 1999;56:479-85 27. Joshi V, Parekh B, Joshi M, Vaidya A. Herbal extracts of Tribulus terrestris and Bergenia ligulata inhibit growth of calcium oxalate monohydrate crystals in vitro . J Cryst Growth 2005;275:e 1403-8 28. Karadi RV, Gadge NB, Alagawadi KR, Savadi RV. Effect of Moringa oleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol 2006;105:306-11 29. Kiritikar KR, Basu BD. Indian Medicinal Plants. 2 nd ed. Dehradun, India: International Book Distributors; 1994 30. Kok DJ, Papapoulos SE, Bijvoet OL. Excessive crystal agglomeration with low citrate excretion in recurrent stone-formers. Lancet 1986;1:1056-8 31. Laroubi A, Touhami M, Farouk L, Zrara I, Aboufatima R, Benharref A, et al . Prophylaxis effect of Trigonella foenum graecum L. seeds on renal stone formation in rats. Phytother Res 2007;21:921-5 32. Lee YH, Huang WC, Huang JK, Chang LS. Testosterone enhances whereas estrogen inhibits calcium oxalate stone formation in ethylene glycol treated rats. J Urol 1996;156:502-5 33. Malini MM, Baskar R, Varalakshmi P. Effect of lupeol, a pentacyclic triterpene, on urinary enzymes in hyperoxaluric rats. Jpn J Med Sci Biol 1995;48:211-20 34. Mukharjee T, Bhalla N, Aulakh GS, Jain HC. Herbal drugs for urinary stones-literature appraisal. Indian Drugs 1984;21:224-8 35. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8 36. Parmar S, Gangwal A, Sheth N. Evaluation of antiasthmatic activity of a polyherbal formulation containing four plant extracts. J Curr Pharm Res 2010;2:40-4 Source of Support: Nil. Conflicts of Interest: None declared.

Other Health Sciences Concepts:

[back to top]

Discover the significance of concepts within the article: ‘In vitro antiurolithiatic activity of c-phycocyanin isolated from Spirulina...’. Further sources in the context of Health Sciences might help you critically compare this page with similair documents:

Ayurveda, Pashanabheda, Purity, Heating, Medicinal plant, Scientific rationality, Urinary calculi, Normal people, Urinary system, Urolithiasis, Urinary tract, Renal calculi, Lithogenesis, Antioxidant properties, Anti-inflammatory, Phytotherapy, In vitro, Optical density, Calcium oxalate, Mass spectrometry, Nephrolithiasis, Standard procedure, Stone formation, Antioxidant, Diuretic, Renal failure, Room temperature, Extraction method, Extracorporeal shock wave lithotripsy, Urinary stone, In vitro method, Kidney Stone, HPLC, Light microscopy, Spectrophotometric assay, Calcium chloride, PDA Detector, Reverse phase HPLC, Crude extract, Crystallization, Antiurolithiatic activity, Ethylene glycol, Reverse phase high pressure liquid chromatography, Phycocyanin, Renal injury, Antiurolithiatic drugs, Spirulina platensis, Light microscope, In vitro assay, Crystal growth, Absorption spectra, Freezing and thawing, Lithiasis, Crystal nucleation, Extraction yield, Cyanobacteria, Nucleation, Turbidity method, Single crystals, Sodium oxalate, Oxalic acid, Nucleation assay, Aggregation assay, Shock waves, Phytotherapeutic agent, Inhibitory action, Natural drug, C-phycocyanin, Kidney, Calcium oxalate monohydrate, Antiurolithiatic, Supersaturated solution, Homogenization, Renal calculi formation, Tris Buffer, Normal urine, Cell debris, S. platensis, Acid extraction, Smaller particles, Aggregation process, Crystal agglomeration, Dry biomass, Wet biomass.