156 List of Publication

List of Publication

Jaymin Mendpara, Vivek Parekh, Sudhir Vaghela, Atul Makasana, Prashant D.

Kunjadia, Gaurav Sanghvi, Devendra Vaishnav, and Gaurav S. Dave, “Isolation

and characterization of high salt tolerant bacteria from agricultural soil”. European

Journal of Experimental Biology, 2013, 3(6):351-358.

Atul Makasana, Vishvas Ranpariya, Dishant Desai, Jaymin Mendpara, Vivek

Parekh, “Evaluation for the antiurolithiatic activity of Launaea procumbens against

ethylene glycol-induced renal calculi in rats”, Toxicology Report, 2014, 1: 46-52.

Available online at www.pelagiaresearchlibrary.com

Pelagia Research Library

European Journal of Experimental Biology, 2013, 3(6):351-358

ISSN: 2248 –9215

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351 Pelagia Research Library

Isolation and characterization of high salt tolerant bacteria from agricultural soil

Jaymin Mendpara1, Vivek Parekh1, Sudhir Vaghela1, Atul Makasana1, Prashant D.

Kunjadia 2, Gaurav Sanghvi3, Devendra Vaishnav3 and Gaurav S. Dave1*

1Department of Biochemistry, Saurashtra University, Rajkot, Gujarat, India 2B. N. Patel Institute of Paramedical and Science, Bhalej Road, Anand, India

3Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India _____________________________________________________________________________________________ ABSTRACT Adaptation characteristic of microorganisms in different extreme conditions seeks discoveries in field of pharma, food, & bioenergy sectors. In the present study, new experimental approach was proposed to prove adaptation ability in extreme conditions with growth and development of agricultural soil bacterial species in stepwise adaptation in high salt (NaCl) conditions. Six different bacterial species were isolated from agricultural soil surrounding the Rajkot region. Out of six isolates, two showed salt tolerance up to 10% NaCl concentration. Biochemical and molecular (16S rDNA sequencing) characterization revealed the strains to be Exiguobacterium sp. and Serratia sp. designated as GSD1 and GSD 2 strains. Species were able to adapt upto 10% of NaCl in nutrient broth growth medium. Primary screening for extracellular enzymes (protease, amylase, lipase) secretion unveiled activity of enzymes in nutrient agar plates containing skim milk or starch or mineral oil for protease, amylase and lipase production respectively. This experiment provides the base to link the adaptation capabilities of agricultural soil microorganisms in high salty environment and vice a versa. Keywords: Exiguobacterium, Serratia, Salt Adaptation, Extracellular enzymes _____________________________________________________________________________________________

INTRODUCTION Adaptation is an evolutionary process through which living organisms develop to live in its changed habitats. Lower to higher living organisms are influenced but developed to adjust with different abiotic stresses i.e. changes in salinity of soil and water, temperature, pH, atmospheric humidity, air circulation and radiation [1,2]. Amongst these all soil and water salinity is the major stress impacts on life, and it becomes more prominent with time. Global warming is the key issue of current world environmental problems, its main emphasis on level and surface temperature of sea. The scientific committee on Antarctic research proposed a rise in mean sea level of up to 1.4 m by 2100 century and other scientists anticipated sea level rise of one to several meters[3-5] in the same phase. Reports year[6].Gujarat has 1663 km, coastal region and rise in 0.5 meter sea level can cover roughly 8452.971 Km2 land nearby coastal area[6]. This can be even more anti-agricultural if sea level rises about 0.5 to 1.4 meters[7].

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Similarly, with global warming, natural disasters like earthquake and tsunami will cost more deadly effects on microorganisms of agricultural soil. Recently, stroked tsunamis in South Asia has affected 11,000 Ha agricultural land [8], with 4067 Ha cropped area and 2260 km of coastal area in India alone[9]. Tsunami contaminated the agricultural soil with salts of Kerala, Andhra Pradesh, Tamilnadu and Pondicherry states of India. In future, natural disasters like tsunami if strike in Gujarat it may contaminate the agricultural soil of Rajkot, Jamnagar, Junagadh, Porbandar, Surat and Kutch districts and it will affects the microorganisms population in agricultural soil. In future high salt contaminated soil, either it will lose some of the microorganisms’ species or microorganisms have to adapt to salty soil. In view of this, we have focused on microorganisms of non-saline soil to adapt it in high salt concentration in artificial synthetic media. We have isolated the total six bacterial species from agricultural garden soil of Saurashtra University campus and experimented to evaluate its adaptability in high salinity medium. Out of total six isolates we have found two isolates Exiguobacterium sp. GSD1 (JN020918) and Serratia sp. GSD2 (JN020917) with capability to adapt upto 10% of NaCl salt containing medium. Exiguobacterium sp. have been isolated from markedly diverse sources, including Greenland glacial ice, hot springs at Yellowstone park, rhizosphere of plant and environment of food processing unit[10].Gram negative bacteria of the genus Serratia are opportunistic human, plant and insect pathogens[11]and member of the family enterobacteriaceae [12]. Salinity has an effect on microbial cell membrane[13] and synthesis, structure and function of protein [14,15] as well as the growth of the microorganisms [16]. Bacteria develop different defense mechanisms to survive in high salinity environment [17].Present study evaluates the adaptation capabilities of agricultural soil microorganisms in high NaCl containing medium to propose its futuristic adaption in salt contaminated soil, if tsunami like natural disaster strikes or rise in sea level, change the fertility of soil.

MATERIALS AND METHODS Collection of soil sample and physico-chemical analysis: Soil sample was collected from provinces of “Van-Vibhag” (agricultural soil) Saurashtra University campus, Rajkot, India. Soil sample was collected from the depth of 10-12 inch in sterile polythene bag and samples were kept at room temperature until used. Soil analysis was conducted to measure electro conductivity, pH, total nitrogen, K2O, P2O5 content at the government soil analysis laboratory, Rajkot. Isolation and Screening of Bacteria: Soil suspension was prepared with 5g of soil in 20ml of sterile double distilled water and vortexed. Loop full of soil suspension was streaked on N-Agar plates (Himedia) and incubated for 24h at 37°C for isolation of different bacteria. Total six different isolates were found from primary screening and further grown on N-agar plate containing 2%, 4%, 6%, 8% & 10% NaCl separately for 72h at 37°C. Biochemical Characterization: Biochemical analysis of two isolates were carried out according to Bergey’s Manual of Determinative Bacteriology[18] and classified primarily through morphological, physiological and biochemical observation. Amplification and sequencing of 16S rDNA encoding genes: 16S rDNA genes, from the purified genomic DNA of the 2 isolates were amplified by PCR with the following set of primers: F (5´-AGAGTTTGATCCTGGCTCAG-3´) and R (5´-GGTTACCTTGTTACGACTT-3´). Each 50-µl reaction mixture contained 30 mM Tris (pH 8.4), 50mM KCl, 1.5 mM MgCl2, 50mM concentrations of each dNTPs, 10 pmol of each primer, and 1U of Taq polymerase. The PCR was performed with following specification: first step of denaturation for 5 min at 95°C, 30 cycles were performed, denaturation for 1 min at 95°C, annealing for 1 min at 55°C, and polymerization for 1 min at 72°C. The amplified genes were sequenced using sequence scanner v1.0 (Inst Model/Name: 3730XI/ABI3730XL-1414-008) (Applied Biosystems). Phylogenetic analysis: The 16S rDNA gene sequences of two isolates (Exiguobacterium sp. GSD1, Serratia sp.GSD2) were compared to references of 16S rDNA gene sequences retrieved from GenBank database. BLASTN 2.2.26+ used to retrieve similar reference sequences and aligned for phylogenetic relationship with our isolated strain by neighbor-joining method using treedyn online software.

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GeneBank Submission and nucleotide accession number: 16S rDNA gene sequences of Exiguobacterium and Serratia were deposited in the NCBI GenBank under accession numbers JN020918 and JN020917 respectively. Growth Curve: Vegetative cells of Exiguobacterium sp. GSD1 and Serratia sp. GSD2 were grown in a nutrient broth containing 2%, 4%, 6%, 8% & 10% of NaCl. Control tubes were served without addition of NaCl. 0.1 ml of fresh culture (O.D near about 1.000 at 660nm) was inoculated in medium. O.D was measured of the sample at 660nm with (UV-1800, SHIMADZU) at duration of 1h for 33h. Extracellular enzyme production at different salt concentration Amylase production: Amylase production was analyzed by starch hydrolyzing method. Starch agar plate was prepared containing 4% and 8% NaCl. Organisms were inoculated on starch agar plate and incubated at 37°C for 24h. Amylase production was detected as a colorless zone on surrounding of colony on addition of iodine. Proteolysis production: Skim milk agar plates were prepared containing 4% and 8% NaCl, organisms were inoculated. Dissolved casein surrounding the colony resulted on secretion of protease enzyme. Lipase production: Nutrient agar plates containing vegetable oil prepared to study extracellular lipase activity. Test organisms were inoculated and incubated for 24h at 37°C. Addition of CuSO4 in plates on next day develops bluish green appearance surrounding the colony, confirms the hydrolysis of fat in glycerol & fatty acid.

RESULTS Soil Analysis: Soil analysis data showed in (Table 1) confirms quality as on agricultural soil.

Table 1 Analysis for different physicochemical parameter of soil

Sample E.C. mS/M pH % Nitrogen P2O5 Kg/ha K2O Kg/ha

B 0.62 8.7 0.38 28 400

Bacterial Identification, Screening and Biochemical test: Gram staining revealed Exiguobacterium sp. as a grampositive aerobic microorganism with orange, nontransparent, circular colony characteristic grown on N-agar after48h of incubation. Exiguobacterium sp. exhibited negative test for indole production, citrate utilization, ammonia and urea reduction as well as acid production, whereas nitrate reduction and carbohydrate fermentation was found to be positive (Table 2). However, the other strain (Serratia sp.) is gram negative aerobic bacteria exhibiting off-white, non-transparent and irregular shape colony. Biochemical test for Serratia sp. showed negative indole production, ammonia and urea reduction as well as acid production test, whereas citrate utilization and carbohydrate fermentation result confirmed positive test (Table 2).

Table 2 Biochemical Characterization

Tests Exiguobacterium sp. GSD1 Serratia sp. GSD2 Indol production - - Urease test - - Methyl red test + + Voges proskauer - - Simmon Citrate test - - Ammonia reduction - - Nitrate reduction + +

Triple sugar iron agar test Slant yellow, Butt yellow,

No H2S production Fermentation observed

Slant yellow, Butt yellowish red, No H2S production

Fermentation observed * + indicates positive results, − indicates negative results.

Phylogenetic analysis on the basis of 16s rDNA analysis: Distance phylogenetic trees for two isolates were constructed by the neighbor-joining method using TreeDyn 198.3 and the topology of the trees was evaluated by bootstrapping score over 1,000. Alignment positions with gaps were excluded from the calculations (Figure 1 & 2). Phylogenetic tree showed the position of Exiguobacterium GSD1 and Serratia sp. GSD2 with respect to other GenBank 16S rDNA sequences. The tree was constructed by Neighbor joining method and minimum possibility with alignment of 812 and 1172 base pairs, with bootstrap support values greater than 90% and 95% (Figure 1 & 2).

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Figure

Figure

Figure 3 Growth curve♦0% NaCl,

Growth curve: As Observed in Figure 3, Exiguobacterium time periods grow in 6% NaCl concentration, where as culture grown in 0%, 2% and 4% NaCl showed log phase entry earlier then 6%, 8% and 10% NaCl, In case of organism grown in 8% and10% N

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Figure 1 Phylogenetic tree analysis of Exiguobacterium sp. GSD1

Figure 2 Phylogenetic tree analysis of Serratia sp. GSD2

Growth curve of Exiguobacterium sp. GSD1 incubated under various NaCl concentrations

0% NaCl, ■2% NaCl, ▲4% NaCl, ●6% NaCl, ▬8% NaCl, 10% NaCl

Exiguobacterium sp. GSD1 showed log phase after the three to four hours of incubation time periods grow in 6% NaCl concentration, where as culture grown in 0%, 2% and 4% NaCl showed log phase entry earlier then 6%, 8% and 10% NaCl, In case of organism grown in 8% and10% NaCl showed no growth even

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sp. GSD1 showed log phase after the three to four hours of incubation time periods grow in 6% NaCl concentration, where as culture grown in 0%, 2% and 4% NaCl showed log phase

aCl showed no growth even

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after the 32h incubation. Similar observation has been noticed with Serratia sp. GSD2 grown in 0% to 10% NaCl (Figure 4).

.

Figure 4 Growth curve of Serratia sp. GSD2 incubated under various NaCl concentrations ♦0% NaCl, ■2% NaCl, ▲4% NaCl, ●6% NaCl, ▬8% NaCl, 10% NaCl

Screening for Extracellular enzyme production on agar plates: Exiguobacterium sp. GSD1 and Serratia sp. GSD2 exhibited protease, amylase and lipase activity in 0% and 4% NaCl containing medium, whereas Serratia sp. GSD2 exhibited amylase and lipase activity in 8% of NaCl medium (Table 3).

Table 3 Qualitative enzyme production test for Caseinase, Amylase and Lipase

Enzyme production Exiguobacterium sp. GSD1 Serratia sp. GSD2

0% 4% 8% 0% 4% 8% Caseinase + + - + - - Amylase + + + + + + Lipase + + - + + +

* + indicates positive results, − indicates negative results

DISCUSSION

The present study was designed to evaluate the adaptation properties of agricultural soil microorganism under the high salt (NaCl) growth medium to establish the adaptability characteristics of non-saline agricultural soil microorganism in saline medium. Exiguobacterium sp. GSD1 strain and Serratia sp. GSD2 were the isolates utilized for experiments. Exiguobacterium sp. has been found from much wide range of habitats of all over the world. Exiguobacterium sp. exhibits in wide range of habitats including cold and hot environments with temperature range from -12°C to 55°C[19]. This diversification in habitats of Exiguobacterium sp. is responsible of its adaptation in diverse conditions and beneficial to survive against extreme environmental factors. Exiguobacterium genus comprises psychrotrophic, mesophilic, and moderate thermophilic species[20,21], with pronounced morphological diversity (ovoid, rods, double rods, and chains) depending on species, strain, and environmental conditions[22]. Our experimental results of growth curve of Exiguobacterium sp. GSD1, suggest the down-regulation of growth under successive increase of NaCl concentration. As shown in figure 3, without additional NaCl concentration in growth medium it did not affect the growth of organism but as the concentration of NaCl increases, growth of organisms decreased with maximum tolerance upto 10% NaCl concentration. Several Exiguobacterium strains possess unique properties of interest for application in biotechnology, bioremediation, industry and agriculture. Exiguobacterium sp. are capable of neutralizing highly alkaline textile industry wastewater[23], high potential for pesticide removal[24]

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and reducing arsenate to arsenite[25]. Furthermore, several enzymes (alkaline protease, EKTA catalase, guanosine kinase, ATPases, dehydrogenase, esterase) with stability at a broad range of temperatures were purified from different Exiguobacterium strains [26-31]. Our isolates Exiguobacterium sp.GSD1 showed extracellular enzyme production of protease, lipase and amylase on N-agar plate with additional food material respectively casein, mineral oil and starch. Plate results reveal that enzyme secretion was decreased with increase in salt concentration with 4% and 8% NaCl in comparison to 0% NaCl, this observation was made on the basis of zone of enzyme production. In view of this we can assume that enzymes would be secreting in extracellular environment but because of high salt concentration in medium, enzymes could not be active fully or decrease in growth of microorganism directly affects the production of extracellular enzymes. From the observation on production of enzyme, we can deduce that as salt concentration increases, it decreases the activity of enzyme, but high salt concentration could not inhibit the enzyme activity completely. These results are in hope and favor to develop and upgrade strategies for enzyme production sustainable in high saline medium. Report on Exiguobacterium 255-15 isolated from Siberian permafrost showed potential stress responses with changing growth rate, cellular morphology, cell membrane composition, polysaccharide composition, and carbon utilization under different temperature conditions and wide range of salt concentration makes Exiguobacterium dynamic organism to survive and study for evolutionary linkages[32]. Our isolated Serratia sp. GSD2showed growth upto 10% NaCl in growth medium, with supporting results same on agar plate at 10% NaCl. Previously, Serratia species has been isolated from water, soil, skin of animals (including man) and from the surfaces of plants. Serratia sp. grows at temperature range from 5–40° and pH range from 5 to 9[33,34]. Strains of this species produce plant-growth-promoting chemicals, have anti-fungal and anti parasitic properties, encourage the establishment of nitrogen-fixing symbionts and act as insect pathogens [35-38]. These observations revealed that in future if any agricultural soil area covered with saline environment by any natural disaster, our isolated Serratia sp. will be a natural remedy for removal of NaCl to keep agricultural soil fertile. A subspecies of Serratia marcescens (S. marcescens subsp. sakuensis) and a urea-dissolving species (Serratia ureilytica) have been described previously [39-40]. In our isolated Serratia sp.GSD2 species, we have found biofilm production[41], which increase with increase in salt concentration from 0 % to 10 % NaCl salt concentration. In this present experiment Serratia sp. GSD2 also produces extracellular enzymes amylase, protease and lipase. We have found decrease in extracellular enzyme activity as increase in salt concentration ranging from 0%, 4%, 8% NaCl. Protein content in terms of total gene expression was increased with increase in salt concentration as shown in Exiguobacterium sp. GSD1 & Serratia sp. GSD2, as well as gradual decline in extracellular enzyme activity was also observed in proteinase, lipase and amylase upto 8% NaCl in solid medium. Biochemical and biophysical data have been postulated for several salt-tolerant enzymes, including glutaminase from Lactobacillus rhamnosus[42] α-amylase from Bacillus dipsosauri[43], protease from Aspergillus sp. FC-10[44], α-type carbonic anhydrase from Dunaliella Salina[45]and thermolysin from B. thermoproteolyticus[46], Lipase from Pichia anamola[47]. Halophilic and halotolerant microorganisms have various mechanisms to adapt against high salt concentration i.e. synthesis of betaine, Ectoine and hydroxyectoine, β-Glutamate, trehalose etc.[48,49], any of or all of these mechanism would be followed in our isolated microoorganisms under high salt media. Diminished extracellular enzyme activity on increase in salt concentration in our experiments are in support that halophilic enzymes are found to have multilayered hydration shells that are of considerably of greater size and order compared to non-halophilic enzymes[50]. In conclusion, this study provides the futuristic aspect of agricultural soil microorganisms on adaptation and use as a natural fertilizer, whenever any nature disaster like tsunami strikes the coastal region of Saurashtra (Gujarat). The above studied microorganism Exiguobacterium and Serratia will able to survive in high salt condition and could be the possible factor to revive fertility of agricultural soil and removal of salt contamination. Further, extracellular enzyme production by Exiguobacterium and Serratia opens the doors for its application in food, pharma and bioenergy industries. In view of the above, present study provides the possible natural bioremediation approach for famers of Saurashtra region of Gujarat state as well as possible microbiological survivor in future under any natural disaster like tsunami or earthquake. Acknowledgement Authors are thankful to Dr. Navin R, Sheth, Head, Department of Biochemistry for his valuable guidance and support throughout the experimental work.

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REFERENCES [1] Lynn JR, Rocco LM, Nature, 2001, 409, 1101. [2] Duran RE, Budak B, Yolcu O, European Journal of Experimental Biology,2013, 3, 110. [3]Rignot E, Kanagaratnam P, Science, 2006, 311, 990. [4]Ivins RE, Science, 2009, 324, 889. [5]SCAR, Antarctic Climate Change and the Environment, in Scientific Committee Antarctic Research, Scott Polar Research Institute, Cambridge, UK, 2009. [6]Dwivedi DN, Sharma VK, In: Proceedings of the 14th Biennial Coastal Zone Conference, 2005, India. [7]Rahmstorf S, Science, 2007, 315, 370. [8]Niino Y, International Workshop on Post Tsunami Soil Management, 2008. [9]Rasheed A, Das VK, Revichandran C, et al, Science of Tsunami Hazards, 2006, 24, 33. [10]Vishnivetskaya TA, Kathariou S, Tiedje JM, Extremophiles, 2009, 13, 555. [11]Fineran PC, Williamson NR, Lilley KS et al, Journal of Bacteriology, 2007, 189, 7662. [12]Grimont PAD, Grimont F, Annual Review of Microbiology, 1978, 32, 248. [13]Ohno Y, Yano I, Journal of Biochemistry, 1979 85, 421. [14]Zheng SP, Ponder MA, Shih JY et al, Electrophoresis, 2007, 28, 488. [15]Rhodes ME, Fitz-Gibbon ST, Oren A et al, Environmental Microbiology, 2010, 12, 2623. [16]Mert HH, Ekmekçi v, Mycopathologia, 1987, 100, 89. [17]Imhoff JF, Advances in Space Research, 1986, 6, 306. [18]Buchanan RE, Gibbons NE. Bergey's Manual of Determinative Bacteriology, Williams and Wilkins Co. Baltimore; MA, USA, 1974. [19]Tiedje JM, Rodrigues DF, FEMS Microbiology and Ecology, 2007 59, 499. [20]Vishnivetskaya TA, Kathariou S.,Tiedje JM, The joint international symposia for subsurface microbiology (ISSM 2005) and environmental biogeochemistry (ISEB XVII),2005. [21]Vishnivetskaya TA, Siletzky R, Jefferies N et al, Cryobiology, 2007, 54, 240. [22]Kumar A, Singh VP, Kumar R, In: International Conference on Extremophiles, 2006, France. [23]López L et al, Ecotoxicology, 2005, 14, 312. [24]Anderson CR,Cook GM, Current Microbiology, 2004, 48, 347. [25]Usuda Y, Kavasaki H, Shimaoka M et al, Biochimica Biophysica Acta-Gene Structure and Expression, 1998, 1442, 379. [26]Suga S,Koyama N, Archieves of Microbiology, 2000, 173, 205. [27]Wada M, Yoshizumi A, Furukava Y et al, Bioscience Biotechnology Biochemistry, 2004, 68, 1488. [28]Hwang BY, Kim JH, Kim J et al, Biotechnol Bioprocess Engineering, 2005, 10, 371. [29]Hara I, Ichise N, Kojima K et al, Biochemistry, 2007, 46, 22. [30]Kasana RC,Yadav SK, Current Microbiology, 2007, 54,229. [31]Ponder MA, Gilmour SJ, Bergholz PW et al, FEMS Microbiology Ecology, 2005, 53, 115. [32]Grimont F, Grimont PAD, A handbook on biology of bacteria. Springer, 1992, 4126. [33]Alström S, Journal of Phytopathology, 2001, 149, 64. [34]Llanco LA, Nakano V, Ferreira CM et al, Brazilian Journal of Microbiology, 2011, 42, 1084. [35]Kalbe C, Marten P, Berg G, Microbiological Research, 1996, 151, 439. [36]Zhang F, Dashti N, Hynes RK et al, Annals of Botany, 1996, 77, 459. [37]Zhang F, Dashti N, Hynes RK et al, Annals of Botany, 1997, 79, 249. [38]Queiroz BPVd, Melo ISd, Brazilian Journal of Microbiology, 2006, 37, 450. [39]Ajithkumar B, Ajithkumar VP, Lriya R et al, International Journal of Systematic and Evolutionary Microbiology, 2003, 53, 258. [40]Bhadra B, Roy P,Chakraborty R, International Journal of Systematic and Evolutionary Microbiology, 2005, 55, 2158. [41]Hall-Stoodley L, Costerton JW, Stoodley P, Nature Review Microbiology, 2004, 2, 108. [42]Weingand-Ziadé A, Gerber-Décombaz C, Affolter v, Enzyme and Microbial Technology, 2003, 32, 867. [43]Deutch CE, Letters in Applied Microbiology, 2002, 35, 84. [44]Su NW, Lee MH, Journal of Industrial Microbiology and Biotechnology, 2001, 26, 258. [45]Lakshmanane P, Bageshwar UK, Gokhman I et al, Protein Expression and Purification, 2003, 28, 157. [46]Inouye K, Kuzuya K,Tonomura BI, Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, 1998, 1388, 214. [47]Tiwari P, Upadhyay MK, European Journal of Experimental Biology, 2012, 2, 467.

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[48]Oren A, Saline Systems, 2008, 4, 13. [49] Kondepudi KK, Chandra TS, European Journal of Experimental Biology,2011,1,121. [50]Karan R, Capes MD, Sarma VD, Aquatic Biosystems, 2012, 8, 15.

Toxicology Reports 1 (2014) 46–52

Contents lists available at ScienceDirect

Toxicology Reports

j ourna l h o mepa ge: www.elsev ier .com/ locate / toxrep

Evaluation for the anti-urolithiatic activity of Launaeaprocumbens against ethylene glycol-induced renal calculi inrats

Atul Makasanaa,∗, Vishavas Ranpariyab, Dishant Desaia, Jaymin Mendparaa,Vivek Parekha

a Department of Biochemistry, Saurashtra University, Rajkot 360 005, Gujarat, Indiab Purple Remedies Pvt. Ltd., Ahmedabad, Gujarat, India

a r t i c l e i n f o

Article history:Received 2 January 2014Received in revised form 19 March 2014Accepted 19 March 2014Available online 22 April 2014

Keywords:Calcium oxalateEthylene glycolLaunaea procumbensNephrolithiasisUrolithiasis

a b s t r a c t

Launaea procumbens Linn. is a plant commonly found in the west India and has beenreported to decrease the renal calculi. This study investigated the anti-urolithiatic activ-ity of L. procumbens against ethylene glycol-induced urolithiasis and its possible underlyingmechanisms. The crude methanolic extract of L. procumbens leaves was studied using ethyl-ene glycol-induced renal calculi in rat model. Results indicate that ethylene glycol feedingto rats resulted in to hyper oxaluria, hypercalciuria, as well as increased renal excretionof phosphate. Supplementation with methanolic extract of L. procumbens leaves (MELP)significantly prevented changes in urinary calcium, oxalate and phosphate excretion dose-dependently. The increased calcium and oxalate level and number of calcium oxalate crystalin the kidney tissue of calculogenic rats were significantly reverted by supplementationwith MELP. The MELP supplementation also prevents the impairment of renal functions.The mechanism underlying this effect is mediated possibly through antioxidant nephro-protection and its effect on urinary concentration of stone forming constituents and riskfactor.Conclusion: These results indicate that methanolic extracts of L. procumbens leaves areeffective against the urolithiasis.© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under

the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Kidney stone disease is a multi-factorial disorder result-ing from the combined influence of epidemiological,biochemical and genetic risk factors. It occurs both in menand women but the risk is generally higher in men and is

∗ Corresponding author. Tel.: +91 9727423031.E-mail addresses: atulmksn2008@yahoo.com (A. Makasana),

vishwash811@yahoo.co.in (V. Ranpariya), desai dishant21@yahoo.com(D. Desai), j.p.mendpara@gmail.com (J. Mendpara),vivek parekh1988@yahoo.in (V. Parekh).

becoming more common in young women [16]. Surgicaloperation, lithotripsy and local calculus disruption usinghigh-power laser are widely used to remove the calculi.However, these procedures are expensive and recurrenceis also common [14]. The recurrence rate without preven-tive treatment is approximately 10% at 1 year, 33% at 5 yearsand 50% at 10 years [4]. Various therapies including thiazidediuretics and alkali-citrate are being used in attempt to pre-vent recurrence but scientific evidence for their efficacy isless convincing [2].

In the traditional systems of medicine includingAyurveda, most of the remedies were taken from plantsand they were proved to be useful though the rationale

http://dx.doi.org/10.1016/j.toxrep.2014.03.0062214-7500/© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).

A. Makasana et al. / Toxicology Reports 1 (2014) 46–52 47

behind their use is not well established through sys-tematic pharmacological and clinical studies except forsome composite herbal drugs and plants. These plantproducts are reported to be effective in decreasing therecurrence rate of renal calculi with no side effects[14].

As per the indigenous system of medicine, the leavesof Launaea procumbens Linn. were reported to be use-ful in the treatment of a wide range of alimentsincluding urinary stones [11,18]. However, so far noscientific study has been reported regarding the anti-urolithiatic activity of methanolic extract of L. procumbensleaves (MELP). In this study, we investigated evalua-tion for the anti-urolithiatic activity of L. procumbensagainst ethylene glycol-induced renal calculi and its pos-sible underlying mechanisms using male Wistar albinorats.

2. Materials and methods

2.1. Plant material and preparation of extract

The leaves of L. procumbens were collected from thelocal area of Saurashtra University, Rajkot in the monthof August and shade dried. The plant was authenticatedby Dr. A.S. Reddy, Sardar Patel University, Vallabh Vid-hyanagar, Gujarat and a voucher specimen was depositedin the herbarium of the Department of PharmaceuticalSciences, Saurashtra University, Rajkot for future refer-ence. The dried leaves were coarsely powdered (passedthrough sieve no. 40) and packed in to the Soxhlet col-umn and extracted with 95% (v/v) methanol at 40–45 ◦Cfor 24 h. The extract obtained was evaporated at 45 ◦C,then dried and stored in airtight container (yield 27.69%,w/w).

2.2. Chemicals and apparatus

Ethylene glycol was purchased from Merck Ltd., Mum-bai, India. Various kits for biochemical estimation ofurine and serum were purchased from Span Diagnos-tics, Mumbai, India. All other chemicals and reagentsused were analytical grade and procured from approvedchemical suppliers. Apparatus such as the metabolic cage(INCO, Ambala, India), semiautoanalyzer (model Microlab-300, Merck, Germany), cold centrifuge (Remi, compufuge,Korea), UV-spectrometer (model UV 1800, Shimadzu,Japan) were used in the study.

2.3. Animals

Male Wistar albino rats (8 weeks old having bodyweight 150–200 g) were used for the present study. Theywere housed in polypropylene cages (five per cage) andmaintained under temperature (27 ± 2 ◦C) and light (12 hlight/dark cycles; lights on at 0700 h) controlled envi-ronment. The animals were given standard diet (Amrut,Pranav Agro Ind Ltd., Vadodara, India). The study proto-col was approved (approval no. SU/DPS/IAEC/2011/13) bythe Institutional Animal Ethics Committee constituted inaccordance with the rules and guidelines of the Committee

for the Purpose of Control and Supervision of Experi-ments on Animals, Ministry of Environment and Forest,India.

2.4. Ethylene glycol-induced urolithiasis model in rats

Urolithiasis was induced by ethylene glycol and ammo-nium chloride in experimental animals [5,9]. Briefly, thirtyanimals were randomly divided into five groups (n = 6) asfollow:

Group I: Vehicle Control group; received 0.5% (w/v) gumacacia solution (5 ml/kg p.o.) and maintained on regularrat food and drinking water ad libitum for 21 daysGroup II: Ethylene glycol (EG) group; received 0.75% (v/v)EG in drinking water alone for 21 daysGroup III: Cystone group; received standard drug cystone(750 mg/kg, p.o.) for 21 daysGroups IV and V: Treatment group; fed orally withmethanolic extract of L. procumbens leaves (MELP) 150 and300 mg/kg, respectively, for 21 daysGroups II–V received urolithiasis inducing treatment(0.75%, v/v ethylene glycol in drinking water) for 21 days.Further, ammonium chloride (1%, w/v in drinking water)was co-administered with EG for the first 3 days in orderto augment lithiasis effect of EG.

2.5. Collection and analysis of urine

All animals were kept in individual metabolic cages and24 h urine samples were collected on 0, 7, 14, 21st day ofcalculi induction treatment period. The volume of urinewas measured and calcium content estimated by diagnostickit (Span Diagnostics Pvt. Ltd., India) in clinical semiau-toanalyser. Various urine parameters such as oxalate [8],magnesium [7,12], phosphate [6], uric acid [20], and totalprotein [21] were performed as per the manuals providedwith various kits.

2.6. Serum analysis

The blood was collected from the retro-orbital sinusunder anaesthetic condition (diethyl ether used for anaes-thesia) and serum was separated by centrifugation at10,000 × g for 10 min and analyzed for creatinine and uricacid. The creatinine kit (Span Diagnostics Pvt. Ltd., India)and uric acid diagnostic kit (Span Diagnostics Ltd., India)were used to estimate serum creatinine and uric acid levelsrespectively.

2.7. Kidney histopathological study

Histopathological study of kidney was followed as perthe method of K. Divakar et al. (2010). Briefly, the abdomenwas cut open to isolate both kidneys from each animal. Iso-lated kidneys were then cleaned off extraneous tissue andrinsed in ice-cold physiological saline. The right kidney wasfixed in 10% neutral buffered formalin, processed in a seriesof graded alcohol and xylene, embedded in paraffin wax,sectioned at 5 �m and stained with haematoxylin (H) andeosin (E) for histopathological examination. The slides were

48 A. Makasana et al. / Toxicology Reports 1 (2014) 46–52

examined under light microscope (magnification 10×) tostudy light microscopic architecture of the kidney and cal-cium oxalate deposits. The left kidney was finely mincedand 20% homogenate was prepared in Tris–HCl buffer(0.02 mol/l, pH 7.4). Total kidney homogenate was used forassaying tissue calcium, oxalate [8] and lipid peroxidationactivity [17].

2.8. Statistical analysis

The results are expressed as mean ± SEM. The statisticalsignificance was assessed using One-way Analysis of Vari-ance (ANOVA) followed by Dunnett’s multiple comparisontest and value of p < 0.05 was considered significant.

3. Results

3.1. Urine analysis

The urinary output was increased significantly (p < 0.01)in calculi-induced rats. The urinary output of the vehi-cle treated group was 5.68 ± 0.91 ml/day/rat on the 21stday, which was increased to about 212.32% in renal stoneinduced Group II. In the prophylactic groups, the urine out-put was lower than that of the calculi-induced rats butsignificantly higher than vehicle treated rats (Table A1).

Urinary calcium, and magnesium excretion wasdecreased by stone inducing treatment group. However,supplementation with MELP significantly prevented thesechanges (Table A2).

In the present study, renal stone inducing treatmentto male Wistar rats resulted in hyperoxaluria. There wasan increase in oxalate phosphate and uric acid excre-tion in Group II. However, supplementation with MELPsignificantly prevented these changes in urinary oxalate,phosphate and uric acid excretion dose-dependently inGroups IV and V (Table A3).

3.2. Kidney homogenate analysis

Renal stone induction caused significant increase(p < 0.01) in lipid peroxidation of kidney tissue of the GroupII (Table A4), which was dose-dependently prevented inthe animals receiving 150 and 300 mg/kg treatment withMELP.

The deposition of the calcium in the renal tissuewas increased in the Group II (Table A4). However, 150and 300 mg/kg doses of methanolic extract significantly(p < 0.01) reduced the increase of calcium in renal tissue inprophylactic groups, whereas, only 300 mg/kg of methano-lic extract in prophylactic group significantly (p < 0.01)reduced the elevation of renal calcium content.

3.3. Serum analysis

Renal stone induction caused impairment of renal func-tion of the untreated rats as was evident from the markersof glomerular and tubular damage: elevated serum creati-nine, uric acid and also urinary protein loss (p < 0.01), whichwere dose-dependently prevented in the animals receiving

a simultaneous treatment with MELP at dose of 150 mg/kgand 300 mg/kg (Tables A4 and A5).

3.4. Kidney histopathology

Histopathological analysis revealed no calcium oxalatedeposits or other abnormalities in the nephron segmentsof vehicle treated group. On the other hand, many calciumoxalate deposits inside the renal tubules and dilation ofthe proximal tubules along with interstitial inflammationwere observed in the renal tissue of urolithiatic rats. Thenumber of calcium oxalate deposits in the renal tubules ofGroups IV and V rats was significantly less than the GroupII (Fig. B1).

4. Discussion

As traditional medicines are usually taken by the oralroute, same route of administration was used for evaluationof antiurolithiatic activity of L. procumbens against ethyleneglycol induced renal calculi in rats.

Male rats were selected to induce urolithiasis becausethe urinary system of male rats resembles that of humansand earlier studies have shown that kidney stone for-mation in female rats was significantly less than malerats [9] The phytochemical studies of MELP showedthe presence of alkaloids, glycosides, flavonoids, tannins,anthraquinones, fixed oils and fats, carbohydrates, ter-penoids and steroids.

Urinary super saturation with respect to stone-formingconstituents is generally considered to be one of thecausative factors in calculogenesis. Previous studies indi-cated that upon 14 days administration of ethylene glycolto young albino rats resulted into the formation of renalcalculi composed mainly of calcium oxalate. The biochem-ical mechanism for this process is related to an increasein the urinary concentration of oxalate. Stone formationin ethylene glycol fed rats is caused by hyperoxaluria,which causes increased renal retention and excretionof oxalate [9]. Renal calcium oxalate deposition by EG(ethylene glycol) and ammonium chloride in rats is fre-quently used to mimic the urinary stone formation inhumans. Ammonium chloride reported to accelerate thelithiaisis [1,5]. Therefore, this model was used to evaluatethe protective effect of L. procumbens against urolithia-sis.

Urinary chemistry is one of the important factors indetermining the type of crystal formed and the nature ofmacromolecules included on the surface of the crystals.Hence, the study of the urinary chemistry related to thecalculi forming minerals will provide a good indication ofthe extent of stone formation.

Consistent with some previous reports [2], stone induc-tion by EG caused an increase in oxalate and decrease incalcium urinary excretion in the Group II. Calcium excre-tion was reduced by MELP treatment in a dose-dependentmanner compared with Group II.

Normal urine contains many inorganic and organicinhibitors of crystallization, magnesium is one such well-known inhibitors. Low levels of magnesium are alsoencountered in stone formers as well as in stone-forming

A. Makasana et al. / Toxicology Reports 1 (2014) 46–52 49

rats. The magnesium levels return to normal on drug treat-ment [16]. Diets enriched with high magnesium have beenfound to protect against deposition of calcium oxalate inthe kidneys of vitamin B6 deficient rats. Promising resultsin preventing recurrence have been shown in patientstreated with potassium magnesium citrate. Magnesiumcomplexes with oxalate and reduce the super saturationof calcium oxalate by reducing the saturation of calciumoxalate and as a consequence reduces the growth andnucleation rate of calcium oxalate crystals [16,19]. Urinarymagnesium was significantly diminished in ethylene gly-col induced urolithic rats. The MELP treatment restoredthe magnesium excretion compared to EG treatmentgroup.

An increase in urinary phosphorus excretion wasobserved in ethylene glycol induced urolithic rats.Increased excretion of phosphorus has been reported instone formers [19]. Increased urinary phosphorus excre-tion along with oxalate stress seems to provide anenvironment appropriate for stone formation by formingcalcium phosphate crystals, which epitaxially induces cal-cium oxalate deposition [9,16,19].

The increase in urinary uric acid excretion was observedin urolithic rats. Increased excretion of uric acid has beenreported in stone formers and hyper oxaluric rats. Uric acidinterferes with calcium oxalate solubility and it binds andreduces the inhibitory activity of glycosaminoglycans [16].The predominance of uric acid crystals in calcium oxalatestones and the observation that uric acid binding proteinsare capable of binding to calcium oxalate and modulateits crystallization also suggests its primary role in stoneformation [16,19].

The present observation showed increased proteinexcretion in ethylene glycol induced urolithic rats [16,19].Proteinuria reflects proximal tubular dysfunction. Super-saturation of urinary proteins results in precipitation ascrystal initiation particle which when trapped acts as anidus leading to subsequent crystal growth. This is asso-ciated with proteinuria [16]. Administration of MELP hadprofound effects on minimizing the excretion of protein inGroup IV and thus might have prevented the nidus forma-tion for crystal nucleation.

In urolithiasis, the glomerular filtration rate decreasesdue to the obstruction to the flow of urine by stones inurinary system. So, the waste products particularly nitroge-nous substances such as urea, creatinine and uric acidaccumulate in blood. Also, increased lipid peroxidationand decreased levels of antioxidant potential have beenreported in the kidneys of rats supplemented with a calculi-producing diet. Elevated oxalate concentration in urine hasbeen reported to induce lipid peroxidation and cause renaldamage by reacting with polyunsaturated fatty acids in cellmembrane [9]. In calculi-induced rats, marked renal dam-age was seen as indicated by the elevated serum levels ofcreatinine and uric acid which are markers of glomerularand tubular damage. Treatment of MELP showed to preventthe elevation of serum levels of these markers and inhibitsthe lipid peroxidation.

Increase in calcium levels in the renal tissue of EGtreated rats was observed. The MELP treatment sup-presses this increase in intracellular calcium. The exact

reason of this effect is not clear, however it might bedue to the increased bioavailability of NO (nitric oxide)which in turns activates cGMP (3′,5′ cyclic guanosinemonophosphate) that controls the increase in intracel-lular calcium levels. Previous studies reported that NOdonors have the capacity to control the intracellular risein calcium levels. Thus, plant extract could effectively con-trol the levels of both the salts by the mechanism suchas inhibiting the oxalate or increasing the bioavailabilityof NO to sequester calcium through the cGMP pathway[15].

Microscopic examination of kidney sections derivedfrom ethylene glycol induced-urolithic rats showed poly-morphic irregular crystal deposits inside the tubules whichcause dilation of the proximal tubules along with inter-stitial inflammation that might be attributed to oxalate.Co-treatment with the MELP decreased the number andsize of calcium oxalate deposits in different parts of therenal tubules and also prevented damages to the tubulesand calyxes.

5. Conclusion

The current study was able to show the antiurolithi-atic effect of leaf extract of L. procumbens in ethyleneglycol induced renal calculi model. Therefore, MELP mayprevent calcium oxalate crystal deposition in the kid-ney by preventing hyperoxaluria-induced peroxidativedamage to the renal tubular membrane surface (lipid per-oxidation), which in turn can prevent calcium oxalatecrystal attachment and subsequent development of kidneystones.

Also, these results indicate administration of leaf extractof L. procumbens reduced and prevented the growth ofurinary stones. Earlier study reported that L. procumbensis useful in renal disorder amelioration (RA. et al., 2010).Therefore, the leaf extract of L. procumbens is helpful to pre-vent the recurrence of the disease as it showed its effect onearly stages of stone development.

The mechanism underlying this effect is possiblymediated through antioxidant nephroprotective prop-erties and also lowering the concentration of urinarystone-forming constituents. However, it requires moreinvestigation to clarify the exact mechanism of thisaction.

Conflict of interest statement

The authors declare that there are no conflicts of inter-est.

Acknowledgements

The authors are thankful to the Head, Departmentof Pharmaceutical Sciences, Saurashtra University, Rajkotfor providing research facilities to carry out presentwork.

50 A. Makasana et al. / Toxicology Reports 1 (2014) 46–52

Appendix A.

Fig. B1. Histopathology of kidney tissue of (A) vehicle control, (B) calculi induced group, crystal of calcium oxalate shown in the box (C) cystone treated,(D) prophylactic treatment with MELP at the dose 150 mg/kg, (E) prophylactic treatment with MELP at the dose 300 mg/kg. Microscopic magnification:40×.

Table A1Effect of methanolic extract of Launaea procumbens leaves on urinary output (ml/24 h) of different groups of rat.

Days Group I Group II Group III Group IV Group V

0 7.38 ± 0.87 7.50 ± 0.34 4.67 ± 0.44a**b** 7.83 ± 0.48 8.00 ± 0.377 9.31 ± 0.11b** 12.36 ± 0.42a** 10.42 ± 0.40 9.00 ± 0.52b** 13.17 ± 0.70a***

14 7.723 ± 0.39a*** 17.56 ± 1.77a*** 17.69 ± 0.50a*** 10.66 ± 0.50 13.56 ± 0.76a***

21 8.77 ± 0.65b*** 17.74 ± 1.13a*** 17.98 ± 0.86a*** 11.65 ± 0.67b*** 13.74 ± 0.54a***b**

Values are expressed in ml/24 h urine volume as mean ± SEM (n = 6). One-way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett test.Group I: Vehicle control, Group II: EG group, Group III: Cystone group, Group IV: 150 mg/kg MELP treatment group, Group V: 300 mg/kg MELP treatmentgroup. Comparisons are made against Group I (vehicle control)a and Group II (lithiatic control)b.

** p < 0.01.*** p < 0.001.

A. Makasana et al. / Toxicology Reports 1 (2014) 46–52 51

Table A2Effect of methanolic extract of Launaea procumbens leaves on urinary calcium and magnesium excretion rate (mg/24 h) of different groups of rat.

Days Group I Group II Group III Group IV Group V

Calcium (mg/24 h)0 0.06 ± 0.005b*** 0.18 ± 0.0a*** 0.11 ± 0.01a*b*** 0.06 ± 003b*** 0.04 ± 0.004b***

7 0.22 ± 0.004b* 0.17 ± 0.01a* 0.22 ± 0.02b* 0.03 ± 0.005a***b*** 0.02 ± 0.005a***b***

14 0.16 ± 0.003b* 0.24 ± 0.03a* 0.34 ± 0.03a***b** 0.02 ± 0.004a***b*** 0.03 ± 0.004a***b***

21 0.14 ± 0.005b* 0.09 ± 0.01a* 0.30 ± 0.02a***b*** 0.02 ± 0.004a***b*** 0.02 ± 0.003a***b***

Magnesium (mg/24 h)0 0.33 ± 0.05b*** 0.77 ± 0.06a*** 0.86 ± 0.05a*** 0.05 ± 0.002a***b*** 0.03 ± 0.003a***b***

7 1.29 ± 0.004b*** 0.17 ± 0.008a*** 0.22 ± 0.02a***b* 0.03 ± 0.005a***b*** 0.03 ± 0.005a***b***

14 0.79 ± 0.04b** 0.52 ± 0.06a** 0.94 ± 0.07b*** 0.24 ± 0.02a***b** 0.43 ± 0.01a***

21 0.54 ± 0.01b** 0.34 ± 0.03a** 0.69 ± 0.08b*** 0.23 ± 0.02a*** 0.34 ± 0.03a***

Values are expressed in mg/24 h urine sample as mean ± SEM. One-way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett test.Number of animals (N) = 6. Group I: Vehicle control, Group II: EG group, Group III: Cystone group, Group IV: 150 mg/kg MELP treatment group, Group V:300 mg/kg MELP treatment group. Comparisons are made against Group I (vehicle control)a and Group II (lithiatic control)b.

* p < 0.05.** p < 0.01.

*** p < 0.001.

Table A3Effect of methanolic extract of Launaea procumbens leaves on urinary oxalate, phosphate and uric acid excretion rate (mg/24 h) on different groups of rat.

Days Group I Group II Group III Group IV Group V

Oxlate (mg/24 h)0 0.56 ± 0.11 0.42 ± 0.04 0.18 ± 0.03a***b* 0.30 ± 0.01a* 0.38 ± 0.037 0.67 ± 0.17b*** 1.09 ± 0.03a*** 0.63 ± 0.05b*** 0.46 ± 0.06a**b*** 0.80 ± 0.04b***

14 0.32 ± 0.05b*** 1.63 ± 0.16a*** 1.20 ± 0.06a***b** 1.38 ± 0.04a*** 1.37 ± 0.03a***

21 0.20 ± 0.04b*** 2.05 ± 0.16a*** 1.26 ± 0.07a***b*** 1.38 ± 0.04a***b*** 1.55 ± 0.05a***b**

Phosphate (mg/24 h)0 0.15 ± 0.08 0.2 ± 0.02 0.19 ± 0.02 1.30 ± 0.02a***b*** 0.75 ± 0.11a***b***

7 0.24 ± 0.05 0.35 ± 0.04 0.25 ± 0.02 1.34 ± 0.04a***b*** 0.81 ± 0.05a***b***

14 0.16 ± 0.03b** 0.55 ± 0.08a** 0.28 ± 0.03 1.45 ± 0.05a***b*** 1.35 ± 0.15a***b***

21 0.15 ± 0.01b** 0.58 ± 0.07 a** 0.32 ± 0.01 1.53 ± 0.05a***b*** 1.42 ± 0.15 a***b***

Uric acid (mg/24 h)0 0.11 ± 0.02b*** 0.40 ± 0.06a*** 0.41 ± 0.02a*** 0.29 ± 0.02a** 0.31 ± 0.02a**

7 0.45 ± 0.1 0.71 ± 0.06 0.43 ± 0.01 0.46 ± 0.03 1.06 ± 0.17a**b*

14 0.36 ± 0.05b*** 1.36 ± 0.15a*** 0.77 ± 0.08a* 1.27 ± 0.10 a***b*** 1.38 ± 0.02a***

21 0.31 ± 0.01b*** 1.42 ± 0.09a**** 0.77 ± 0.06a***b*** 1.58 ± 0.09 a*** 1.50 ± 0.02a***

Values are expressed in mg/24 h urine sample as mean ± SEM. One-way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett test.Number of animals (N) = 6. Group I: Vehicle control, Group II: EG group, Group III: Cystone group, Group IV: 150 mg/kg MELP treatment group, Group V:300 mg/kg MELP treatment group. Comparisons are made against Group I (vehicle control)a and Group II (lithiatic control)b.

* p < 0.05.** p < 0.01.

*** p < 0.001.

Table A4Effect of methanolic extract of Launaea procumbens leaves on serum and kidney parameters on different groups of rat.

Parameter Group I Group II Group III Group IV Group V

Kidney (mg/g)Calcium 0.11 ± 0.01b** 0.65 ± 0.22a** 0.16 ± 0.05b** 0.35 ± 0.02 0.32 ± 0.03% Lipid peroxidation 24.43 ± 1.43b*** 100 ± 0.00a*** 45.22 ± 2.77a***b*** 69.00 ± 3.87a***b*** 59.07 ± 4.38a***b***

Serum (mg/dl)Creatinine 0.91 ± 0.02b*** 1.87 ± 0.04a*** 1.38 ± 0.02a***b*** 1.87 ± 0.08a*** 1.12 ± 0.09b***

Uric acid 3.23 ± 0.58b*** 8.50 ± 0.55a*** 5.77 ± 0.75a**b** 2.52 ± 0.28b*** 1.49 ± 0.24b***

Values are expressed in mg/g in kidney parameter and mg/dl in serum sample as mean ± SEM. One-way Analysis of Variance (ANOVA) followed by multiplecomparison Dunnett test. Number of animals (N) = 6. Group I: Vehicle control, Group II: EG group, Group III: Cystone group, Group IV: 150 mg/kg MELPtreatment group, Group V: 300 mg/kg MELP treatment group. Comparisons are made against Group I (vehicle control)a and Group II (lithiatic control)b.

** p < 0.01.*** p < 0.001.

52 A. Makasana et al. / Toxicology Reports 1 (2014) 46–52

Table A5Effect of methanolic extract of Launaea procumbens leaves on urinary total protein excretion rate (mg/24 h) on different groups of rat.

Days Group I Group II Group III Group IV Group V

0 0.21 ± 0.01 0.93 ± 0.06a** 0.87 ± 0.06a** 0.96 ± 0.19a*** 0.77 ± 0.17a*

7 1.19 ± 0.03b*** 1.85 ± 0.06a** 0.96 ± 0.07b*** 1.52 ± 0.11 1.30 ± 0.14b***

14 0.67 ± 0.04b** 3.33 ± 0.34a*** 1.14 ± 0.06a*** 1.60 ± 0.12 0.78 ± 0.25a**

21 0.75 ± 0.11b*** 3.66 ± 0.24a*** 2.60 ± 0.16a***b*** 3.01 ± 0.20a***b* 4.58 ± 0.06a***b**

Values are expressed in mg/24 h urine sample as mean ± SEM (n = 6). One-way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett test.Group I: Vehicle control, Group II: EG group, Group III: Cystone group, Group IV: 150 mg/kg MELP treatment group, Group V: 300 mg/kg MELP treatmentgroup. Comparisons are made against Group I (vehicle control)a and Group II (lithiatic control)b.

* p < 0.05.** p < 0.01.

*** p < 0.001.

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