XRD patterns of CaOxa crystals (a) and (c) without any additives, (b) and (d) with the 0.15% (w/v) C. igneus extract obtained using the gel and liquid methods. * denotes COD crystals 

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... changes in the morphology of CaOxa crystals obtained from SEM studies in the absence and presence of the C. igneus stem extract using both the gel and liquid methods (Fig.3). In the gel method, the CaOxa crystals that were formed in the absence of any additive were elongated hexagonal ( Fig. 3(a)) with an average size of 1874.1 x 857.8 μm. In the presence of the C. igneus stem extract, the CaOxa crystals were elongated tetragonal bipyramidal (Fig. 3(b)) with an average size of 1075.5 x 990.5 μm. In the liquid method (Fig. 3(c)), the CaOxa crystals The COM formed crystals in grown the displayed absence of many any different additive morphologies, were elongated but hexagonal the most prominent with an average faces were size (100), of 0.524 (001), μm (101), and (021). 0.563 COD μm. crystals In the presence usually appeared of the as C. bipyramids igneus stem with extract, (101) being the CaOxa the dominant crystals face were as elongated was previously tetragonal reported bipyramidal 21,22 . with an average size of 0.291 x 0.239 μm and 0.295 x 0.272 μm (Fig. 3(d)). The COM crystals grown displayed many different morphologies, but the most prominent faces were (100), (001), (101), (021). COD crystals usually appeared as bipyramids with (101) being the dominant face as was previously reported 21,22 . The FTIR spectra of CaOxa crystals obtained in the presence and absence of the C. igneus extract, using both the gel and liquid methods (Fig. 4 and 5). In Fig. 4(a), the peaks at 1642 and 1330 cm-1 are the main antisymmetric carbonyl stretching bands, and the band at 1031 cm-1 shows C-O stretching. The band at 886 cm-1 is due to C-C stretching, which reveals the presence of two carboxylate anions. This confirms the existence of the oxalate group in COM crystals. In Fig. 4(b), shifts from 1642 cm-1 for COM to 1635 cm-1 for COD as well as from 1330 cm-1 for COM to 1386 cm-1 for COD were observed, and the band at 1007 cm-1 is due to C-O stretching. The band at 877 cm-1 demonstrates a C-C stretch, which reveals the existence of an oxalate group in the COD crystals. In Fig. 5(a), the peaks at 1636 cm-1 and 1326 cm-1 were the main antisymmetric carbonyl stretching bands, and the band at 1024 cm-1 is due to the C-O stretching. This result indicates the presence of a carboxylate anion in COM crystals. In Fig. 5(b), shifts from 1636 cm-1 for COM to 1639 cm-1 for COD as well as from 1326 cm-1 for COM to 1366 cm-1 for COD were observed. In addition, The peaks two at 1618 bands cm-1 located and at 1318 925 cm-1 cm-1 are and the 654 main cm-1 antisymmetric were also assigned carbonyl to stretching COD. Figs. bands 4(c) and specific 5(c) to display the oxalate the FTIR family spectra and of the the metal raw stem carboxylate powder stretch of C. for igneus COM, , which respectively. is rich The in protein, peaks shifted iron, antioxidant from 1618 cm-1 components to 1652 and cm-1 phytochemicals or 1647 cm-1 (i.e., and from flavonoids, 1318 cm-1 alkaloids, to 1328 terpenoids, cm-1 or 1327 steroids, cm-1 saponins for COD, and as previously phenolics). reported Figs. 4(d) 7,10,11,21 and . 5(d) The shifting show the further FTIR supports spectra of the the aqueous hypothesis extract that powder C. igneus of C. stem igneus extracts containing favor water- the nucleation soluble phytochemicals, and or transformation which of were COM separated into COD. according to the polarity of the solvent 16 . There were no impurities found in the C. igneus -treated COD crystals (Figs. 2(b) and 3(b)). The peaks at 1618 cm-1 and 1318 cm-1 are the main antisymmetric carbonyl stretching bands specific to the oxalate family and the metal carboxylate stretch for COM, respectively. The peaks shifted from 1618 cm-1 to 1652 cm-1 or 1647 cm-1 and from 1318 cm-1 to 1328 cm-1 or 1327 cm-1 for COD, as previously reported 7,10,11,21 . The shifting further supports the hypothesis that C. igneus stem extracts favor the nucleation and or transformation of COM into COD. Fig. 6 shows the XRD patterns of CaOxa crystals obtained in the presence and absence of the C. igneus extract using both the gel (Fig. 6(a) and 6(b)) and liquid methods (Fig. 6(c) and 6(d)). The diffraction peaks obtained in both methods correlate well to the (hkl) indices of the COM phase (JCPDS card number 20-231) and the COD phase (JCPDS card number 17-541). As reported previously 3,11 , the diffraction peaks 14.95, 24.39, 30.12 and 33.13 for COM as well as 14.26, 20.01, 24.15, 32.17, 37.21 and 40.00 for COD were assigned. It is inferred from the above results that the C. igneus stem extract affected the nucleation and growth of COD crystals. HPTLC fingerprint patterns have been therefore evolved for extracts of Costus igneus . Lupeol standard was quantitated accurately using silica gel F 254 HPTLC pre-coated plates with the mobile phase for n- Hexane : Ethyl acetate (80:20v/v),the Rf value for Lupeol was about 0.55. The chromatographs of Lupeol and ethanol extract of costus igneus are shown in (Fig. 7(a)). The Rf value of Lupeol was matched with the Rf value of Costus igneus extract was about 0.55. Stigmasterol standard was quantitated accurately using silica gel F 254 HPTLC pre-coated plates with the mobile phase Toluene: Acetone: Acetic acid (8.9: 0.9 : 0.2 v/v/v), the Rf value was about 0.58. The chromatographs of stigmasterol and ethanol acetate of Costus igneus are shown in (Fig. 7(b)). The Rf value of stigmasterol was matched with the Rf value of extract was about 0.58. The amount of Lupeol (0.473 mg/100 mg) and Stigmasterol (1.913 mg/100 mg) in stem of A pentacyclic triterpenoid compound Lupeol and a steroid compound Stigmasterol were identified and isolated by HPTLC techniques for the first time. Previous study has reported that Lupeol, a triterpene compound has been isolated from Crataeva nurvala showed antioxaluric and anticalciuric effects in rats against hydroxyproline- induced hyperoxaluria 24 . The earlier investigators isolated lupeol from the methanol extract of stem bark of Grewia titiaefolia and evaluated the cytotoxic properties on in vitro cell lines 25 . The calibration curve was prepared by plotting peak area versus concentration (ng/spot) corresponding to each spot. The regression equation and correlation curves for Lupeol in Costus igneu were, regression via height y=149.076+32.745X and r=0.99794 sdv=0.72, regression via area y=213.109+1731.406X and r=0.99914 sdv=0.72. Stigmasterol in Costus igneus were, regression via height y=116.129+0.052X and r=0.99956 sdv=1.78, regression via area y=1732.776+2.151X and r=0.99999 sdv=0.08. COM crystals were grown using both gel and liquid growth methods and were characterized by SEM, FTIR and Powder XRD techniques. The COM crystal growth was reduced, and the morphology of the crystals changed from a hexagonal (COM) to a bipyramidal (COD) form due to the inhibitory action of the aqueous extracts of C. igneus under in vitro conditions. FTIR and Powder XRD techniques confirmed the functional groups and crystalline phases of the COM and COD crystals. SEM studies confirmed the morphology of the changed crystals. It also confirmed that the average size of the crystals was reduced from1874.1 x 857.8 μm to 1075.5 x 990.5 μm and from 0.524 μm to 0.291 μm for the gel and liquid methods, respectively. One-way ANOVA performed with treated and untreated crystal growth data obtained from both the gel and liquid methods showed significant differences (p <0.05). This inhibitory effect may be due to the presence of a terpenoid compound lupeol and a steroid compound stigmasterol from the stem of Costus igneus were identified by HPTLC techniques. This study confirmed that aqueous C. igneus extracts promote the formation of COD crystals and may possibly treat urinary stones by inhibiting the formation of COM crystals, which are a major component of urinary stones. This study was focused on finding a new alternative medicine for the treatment of calcium oxalate urinary stone. KS acknowledges DBT, New Delhi (BT/PR10018/NNT/28/95/2007) for providing financial support. KM acknowledges DBT for a Junior Research Fellowship and Dr. S. John Britto for identifying the plants. CDK thanks the Canada Research Chairs program, Cape Breton University and the Natural Sciences and Engineering Research Council Canada (NSERC) for their financial support of this work. KR thanks Cape Breton University, Canada for a visiting fellowship during this ...