Urinary stone formation: Efficacy of seed extract of Ensete superbum (Roxb.) Cheesman on growth inhibition of calcium hydrogen phosphate dihydrate crystals K.J. Diana a , K.V. George b,n a School of Environmental Sciences, M.G. University, Kottayam, Kerala, India b Department of Botany, C.M.S. College, Kottayam, Kerala, India article info Article history: Received 7 April 2012 Received in revised form 12 October 2012 Accepted 15 October 2012 Communicated by S. Veesler Available online 26 October 2012 Keywords: A1. Crystal morphology A1. Characterization A1. X-ray diffraction A2. Growth from solutions B1. Calcium compounds B1. Phosphates abstract The effect of aqueous seed extract of Ensete superbum (Roxb.) Cheesman on in vitro crystallization and growth patterns of calcium hydrogen phosphate dihydrate (CaHPO 4 2H 2 O, CHPD) crystals was studied using single diffusion gel growth technique. Reduction in growth of CHPD crystals was noticed with increasing concentrations of seed extract. The morphology of CHPD or brushite crystals was studied by microscopy. The structural changes of the treated crystals were assessed by SEM, FT-IR, XRD and TGA/DTA analysis. It is expected that this multidisciplinary approach for in vitro crystallization and characterization of CHPD crystals will provide a better explanation to develop novel strategies for prevention of urinary stones. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Urinary stone is a serious debilitating problem all over the world as it affects nearly 12% of the world population. The disease frequency is on the rise due to global warming, life style and dietary habits [1, 2]. The present day medical management of urinary stone includes lithotripsy and surgical procedures [3]. Regardless of these advances, recurrence rate seems to be high and without proper treatment and preventive measures, approximately 75% of these patients will have at least one recurrence [4]. The presence of amorphous calcium phosphate is a common finding in urinary sediments and is the most commonly encountered crystal material in urine [5, 6]. It is also reported that calcium phosphate in small quantities was seen in renal and urethral stones [7,8]. The presence of calcium phosphate, in small amount, was also detected in the assumed attachment part of the stone [9–11]. These observations suggest that kidney stones have their origin on a calcium phosphate precipitate and that the early stages of these stones are attached to the renal papilla [9, 12–14]. Even after extensive research in Urology, no satisfactory drugs have so far been developed in modern medicine to disintegrate/ dissolve the urinary stone and patients mostly rely on alternative systems of medicine for better relief [15]. In Ayurvedic system of medicine the pseudostem and seeds of Ensete superbum (Roxb.) Cheesman (Wild/Rock Banana) were used for the treatment of various human ailments like, kidney stone [16], Leucorrhea [17], Measles [18], Stomachache and easy delivery [19]. An investigation among the Ulladen tribes of Kerala further confirmed its potential for the treatment of urolithiasis [20]. However, there are no records of systematic pharmacological studies that support this claim. Since the gel method of crystal growth provides simulation of synovial cartilage and other biological fluids [21], a few multidisciplinary researches [16, 22–28] were initiated recently on in vitro gel growth of urinary stone constituents and the inhibitory role played by extracts or juices of natural products. In this context, the present study was undertaken to assess the efficacy of the aqueous extract of the seeds of E. superbum on growth inhibition of calcium hydrogen phosphate dihydrate (CaHPO 4 2H 2 O, CHPD) using single diffusion gel growth technique. 2. Materials and methods 2.1. Ensete superbum seed (Ess) extract and calcium chloride. The seeds of Ensete superbum were collected from the plant and authenticated in the Department of Botany, CMS College, Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/jcrysgro Journal of Crystal Growth 0022-0248/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcrysgro.2012.10.036 n Corresponding author. Tel./fax: þ91 9447409557. E-mail address: [email protected] (K.V. George). Journal of Crystal Growth 363 (2013) 164–170
Transcript
Journal of Crystal Growth 363 (2013) 164–170
Contents lists available at SciVerse ScienceDirect
Urinary stone formation: Efficacy of seed extract of Ensete superbum (Roxb.)Cheesman on growth inhibition of calcium hydrogen phosphatedihydrate crystals
K.J. Diana a, K.V. George b,n
a School of Environmental Sciences, M.G. University, Kottayam, Kerala, Indiab Department of Botany, C.M.S. College, Kottayam, Kerala, India
a r t i c l e i n f o
Article history:
Received 7 April 2012
Received in revised form
12 October 2012
Accepted 15 October 2012
Communicated by S. Veeslermicroscopy. The structural changes of the treated crystals were assessed by SEM, FT-IR, XRD and
The effect of aqueous seed extract of Ensete superbum (Roxb.) Cheesman on in vitro crystallization and
growth patterns of calcium hydrogen phosphate dihydrate (CaHPO4 �2H2O, CHPD) crystals was studied
using single diffusion gel growth technique. Reduction in growth of CHPD crystals was noticed with
increasing concentrations of seed extract. The morphology of CHPD or brushite crystals was studied by
TGA/DTA analysis. It is expected that this multidisciplinary approach for in vitro crystallization and
characterization of CHPD crystals will provide a better explanation to develop novel strategies for
prevention of urinary stones.
& 2012 Elsevier B.V. All rights reserved.
1. Introduction
Urinary stone is a serious debilitating problem all over the worldas it affects nearly 12% of the world population. The disease frequencyis on the rise due to global warming, life style and dietary habits [1,2].The present day medical management of urinary stone includeslithotripsy and surgical procedures [3]. Regardless of these advances,recurrence rate seems to be high and without proper treatment andpreventive measures, approximately 75% of these patients will haveat least one recurrence [4]. The presence of amorphous calciumphosphate is a common finding in urinary sediments and is the mostcommonly encountered crystal material in urine [5,6]. It is alsoreported that calcium phosphate in small quantities was seen inrenal and urethral stones [7,8]. The presence of calcium phosphate, insmall amount, was also detected in the assumed attachment part ofthe stone [9–11]. These observations suggest that kidney stones havetheir origin on a calcium phosphate precipitate and that the earlystages of these stones are attached to the renal papilla [9,12–14].
Even after extensive research in Urology, no satisfactory drugshave so far been developed in modern medicine to disintegrate/dissolve the urinary stone and patients mostly rely on alternative
ll rights reserved.
.
orge).
systems of medicine for better relief [15]. In Ayurvedic system ofmedicine the pseudostem and seeds of Ensete superbum (Roxb.)Cheesman (Wild/Rock Banana) were used for the treatment of varioushuman ailments like, kidney stone [16], Leucorrhea [17], Measles[18], Stomachache and easy delivery [19]. An investigation among theUlladen tribes of Kerala further confirmed its potential for thetreatment of urolithiasis [20]. However, there are no records ofsystematic pharmacological studies that support this claim. Sincethe gel method of crystal growth provides simulation of synovialcartilage and other biological fluids [21], a few multidisciplinaryresearches [16,22–28] were initiated recently on in vitro gel growth ofurinary stone constituents and the inhibitory role played by extractsor juices of natural products. In this context, the present study wasundertaken to assess the efficacy of the aqueous extract of the seedsof E. superbum on growth inhibition of calcium hydrogen phosphatedihydrate (CaHPO4 �2H2O, CHPD) using single diffusion gel growthtechnique.
2. Materials and methods
2.1. Ensete superbum seed (Ess) extract and calcium chloride.
The seeds of Ensete superbum were collected from the plantand authenticated in the Department of Botany, CMS College,
Table 1Composition of calcium chloride and Ess extract in different treatments.
Treatments Volume of
calcium chloride
(1 M) (ml)
Volume of
deionized
water (ml)
Volume of
additive
(ml)
Concentration
of additive in
%
CHPD
(control)
5 5 0 0
EN1 5 4 1 1
EN2 5 3 2 2
EN3 5 2 3 3
EN4 5 1 4 4
K.J. Diana, K.V. George / Journal of Crystal Growth 363 (2013) 164–170 165
Kottayam. The seeds were cleaned in tap water, shade dried andpowdered. The aqueous Ess extract was prepared by boilingpowdered seeds (40 g) in double distilled water (500 ml) andthen it was filtered, evaporated and dried to yield solid residue(4 g) with a yield of 10% w/w on dry weight basis. It was made upto 100 ml by adding double distilled water and was kept as thestock solution (100%). 1–4 ml (1–4%) of this solution were used asaddictives in treatments EN1–EN4.
One molar calcium chloride (5 ml) was used in all treatment.However, the final concentration of calcium chloride was main-tained constant (0.5 M) in all the treatments by adding adequatevolume of deionized water. The total volume of the supernatantsolution was maintained 10 ml in all the treatments (Table 1).
Note: Photograph taken after 5 minutes growth under compound microscope
at 40�magnification.
2.2. Single diffusion gel growth technique
For growing CHPD crystals, hydrogel method developed byHenisch et al. [29] was used with appropriate modifications. Thehydrogel was prepared from sodium metasilicate (Na2SiO3 �9H2O,molecular weight 284 g/mol) and specific gravity of the gel wasadjusted to 1.06 using deionized water. Freshly prepared CaCl2
solutions were used for growing CHPD crystals. The pH of the gelsolution was adjusted to 6.5 by adding one molar orthopho-sphoric acid. The gel thus prepared was used as a medium forgrowing crystals employing sterile Petri-plates and Test tubes.
The initial stages of crystal growth were studied by micro-scopic observation of the CHPD crystals grown in the micro-slides. A drop of solution prepared for gel was put at the middleof a sterile micro slide and it was kept inside a sterile Petri dish.The gel is then covered with cover slip and allowed to set slowly.Thereafter, suitable concentration of the calcium chloride solu-tion was poured up to the level of the cover slip. The pouredsolution was allowed to diffuse slowly through the gel to initiatenucleation, aggregation and growth of micro-crystals. Differentconcentrations of the Ess extract were then added to calciumchloride to study the growth inhibition and dissolution ofCHPD crystals. The slides were observed after 5 min using acompound microscope (Olympus); the photographs were takenand were subjected to morpho-metric analysis using imageanalyzer software.
CHPD crystal growth inhibition studies were also conducted insterile test tubes. For that, 10 ml of the gel solution was poured inand was allowed to set slowly at room temperature. Equal volumeof calcium chloride solution was added into each tube. Differentconcentrations of the Ess extract (I% to 4%) were then addedthrough the sides of the test tubes and the inhibitory effects wereanalyzed based on the changes noted with respect to number, sizeand morphology of crystals. After 3 weeks, the crystals wereharvested from the gel, washed in nanopure water, filtered andthen air dried. The dried crystals were characterized by SEM, FTIR,TG/DTA and XRD techniques.
2.3. Characterization techniques
2.3.1. SEM
The crystals were subjected to high-resolution surface imagingby scanning electron microscopy with a JEOL JSM-6390LV SEM.The samples were analyzed directly after gold coating using amagnetron sputtering unit.
2.3.2. FTIR studies
FTIR studies of the gel grown crystals were conducted to findout whether there is any structural changes in terms of thefunctional groups of the Ess treated CHPD crystals when com-pared to that of Control. Samples were prepared by grinding withKBr in a mortar and pestle. The spectra were recorded in ThermoNicolet, Avatar 370 model FTIR spectrophotometer (of Spectralrange: 4000–400 cm�1 and Resolution: 4 cm�1 with KBr beamsplitter DTGS Detector HATR Assembly).
2.3.3. X-ray powder diffractometry
X-Ray Powder Diffractometry of the CHPD as well as Esstreated CHPD crystals were conducted to provide additionalinformation about the structure of a material at the atomic level.The X-ray powder diffractogram (XRD) was recorded using aBruker AXS, X-ray powder diffractometer (Model D8) with copperX-ray source (radiation of wave length 1.5406 A) and Si(Li)PSDdetector.
2.3.4. Thermal analysis—TGA and DTA
For thermogravimetric analysis (TGA) and differential thermalanalysis (DTA), we used a model: Perkin-Elmer, Diamond TGA/DTA, Flexible axial and radial view instrument with high concen-tration capabilities. The thermogram was obtained by heating asample from 30–1010 1C in an atmosphere of nitrogen withheating rate of 20 1C/min, using a-Al2O3 as standard reference.TGA/DTA analysis was performed with a view to determine theweight changes as well as changes in temperature between thesample i.e., treatment crystals (Ess treated CHPD) and the refer-ence i.e., CHPD pure crystals (control), as a function of tempe-rature or time.
3. Results and discussion
The results of the present study revealed the synergetic actionof bioactive compounds of Ensete superbum on in vitro growth
K.J. Diana, K.V. George / Journal of Crystal Growth 363 (2013) 164–170166
inhibition of CHPD crystals (Figs. 1 and 2). A precipitate wasformed at the gel solution interface within 10–15 min in all thetreatments. Liesegang rings then started to appear just below theinterface within 6–10 h. The number of these rings graduallyincreased with time, and a total of about 10–12 rings withapproximate thickness of 0.5 mm was formed in the control testtubes. However, a gradual reduction in the number of Liesegangrings (approximately 6–10), was noted in the treatments,EN1–EN4 with increasing concentration of the seed extract. Inthe mean time, the first few Liesegang rings slowly get disap-peared leading to the formation of small crystals. Crystals werealso formed in between the Liesegang rings. The morphology ofthe crystals formed in the control was mostly star or swordshaped while that formed in the treatments (EN1–EN4) wasspindle, round or oval in appearance (Table 2).
The interference with crystal growth and aggregation is apossible therapeutic strategy for prevention of recurrent stonediseases [30,31]. It is suggested that, macro-molecules of highermolecular weight of plant extracts exert their action similar tonatural urinary inhibitors and inhibit crystal nucleation, growth andaggregation [32]. Besides, many chemicals of biological origin suchas nephrocalcin, a type of acidic glycoprotein of renal origin, act asimportant inhibitors of calcium oxalate nucleation, growth andaggregation [33,34]. There are similar reports about magnesiumand citrate, which mainly inhibit crystal aggregation. It was also
reported that phytochemical compounds like Glycosaminoglycans(GAGs) have the property to inhibit the crystallization of urinarystone compounds [35]. It is noted that the size of the crystalsformed is inversely proportional to the total number of nucleationsites. As it is evident from Figs. 1–4, in the Ess extract treatments(Figs. 2 and 4), the crystal morphology get transformed leading tothe formation of a large number of spherical/oval/grain like smallercrystals with less transparent and smooth surface. Moreover, thefrequency of the sharp edged and larger crystals or crystal aggre-gates were much reduced in treatments (EN1, EN2, and EN3&EN4).This finding is in agreement with the results of earlier studies basedon a glycoprotein inhibitor of calcium oxalate crystal growth[36,37]. As pointed out by Bharat et al. [38], change in morphologyof crystal is an important phenomenon because, if the painful startype or spiky, needle, irregular stones were converted into smoothspherical or oval grain like ones, then, their passage through theurethra is less painful.
Figs. 5 and 6 shows the SEM photographs of CHPD and EN4respectively. CHPD crystals shows complete sheet like morphology inSEM and its size is more than 10 micro meter, whereas EN4 crystalsshows rectangular shape and the size of each unit seems to be lesserthan one micro meter. Earlier researchers had studied the growthmorphology of CHPD crystals and stated it to be typical in form of
Fig. 5. SEM of (C) CHPD crystal.
Fig. 6. SEM of EN4.
0 500 1000 1500 2000 2500 3000 3500 4000 4500
0
20
40
60
80
100
3381.66
2382.00
2359.26
705.00
% T
rans
mitt
ance
Wave number (cm-1)
C EN4
Fig. 7. FTIR of control-CHPD (C) and treatment (EN4) crystals.
Table 3FT-IR wave numbers and vibrations assignment of CHPD and EN crystals.
K.J. Diana, K.V. George / Journal of Crystal Growth 363 (2013) 164–170 167
thin plates with prominent (010) and lateral (401) faces [39,40]. Themonoclinic unit cell of CHPD consists of alternating bi-layers orientedparallel to the (010) plane with one layer consisting of calcium andhydrogen phosphate ion while the other layer of water molecules. Inaqueous solution the bi-layers of water molecules are for most of thereal time exposed at the surface of (010) face, whereas the later faceshave a mixed ionic character with intercalated water molecules[40,41]. The surface bound water is supposed to form week bondswith molecules in the growth medium [42]. Consequently, there arechances for the surface bound water of the CHPD to form bonds withthe bioactive compounds of the Ess extract thereby weakening theionic character which leads to structural alteration of the treatedcrystals into smaller units.
Several workers [43–45] have reported IR spectroscopy analysisof calcium hydrogen phosphate dihydrate (CHPD) crystals. Accord-ing to Rajendran and Dale Keef, the FT-IR of the CHPD crystalsgrown by single diffusion gel method resembles with that ofcommercially available CHPD [46]. FT-IR spectra of the gel growncrystals (control-CHPD and treatment-EN4) are shown in Fig. 7 andthe respective assignment of the vibrations are given in Table 3.
In the control (CHPD), the presence of water of crystallizationwas referenced from absorptions at 3543.16, 3487.57 and 3282.79 cm�1, which are due to intermolecular and weakly H bondedOH. The absorption at 1650.29 cm�1 is due to H–O–H symmetricbending vibrations and PQO associated stretching vibrations wereobserved at wave numbers 1211.2, 1129.96 and 1064.34 cm�1.Likewise, the P–O–P asymmetric stretching vibrations wereobserved at 873.10 and 790 cm�1. The absorption at 665 cm�1 isdue to (H–O–) PQO and the strong absorption at 578 and525 cm�1are due to acid phosphates. Compared to the control(CHPD), in the treatment (EN4), all bands are present along withtwo additional bands of absorption one at 3381.66 and another at705.00 cm�1, which suggests the possibility of the incorporation ofbioactive compounds (impurities) of the Ess extract with CHPDaltering its morphology as well as structural entity. According to theprevious studies, the FTIR band at 705 cm�1 is due to O–C–O inplane bending and 3381 cm�1 is due to O–H stretching [47,48].A bond formation can take place between the OH (hydroxyl) groupof CHPD and O–C–O of Ess resulting in a shift of 3282 cm�1
vibration frequency to higher wave number 3482 cm�1.
K.J. Diana, K.V. George / Journal of Crystal Growth 363 (2013) 164–170168
The X-ray diffractogram of the gel grown CHPD crystal (Fig. 8)of the control matches with the JCPDS data (72-0713). The XRDpattern of Ess extract treated crystals (EN4), when compared toXRD curve of CHPD (control) given in Fig. 8, shows shift in thepeak positions, change in peak intensity and appearance of newpeeks. This finding also suggests the chances of incorporation ofbioactive compounds within the framework of CHPD.
Fig. 9 shows the TG-DSC curve of CHPD in which the weightloss occurs in two stages. The major weight loss of about 20%occurs between 123 1C and 191 1C which indicates the loss ofwater of hydration. The endothermic peak in DTA around 123 1Cwith the associated shoulders indicates the stepwise removal ofwater during this temperature range. Subsequently in the hightemperature range of 191–441 1C, two molecules of CaHPO4combine and result in the elimination of a water moleculeleading to the formation of calcium pyrophosphate and nearly74% of the sample is stable. The observed mass loss correspondswell with the DSC curve. The following chemical reactions are
0 10 20 30 40 50 60 70 80 90
0
2000
4000
6000
8000
47.9
49
39.3
890
41.0
136.4
241
35.5
0530
.703
229
.53
34.5
450
28.4
0625
.31623
.520
19.5
120
16.9
230
11.7
450
8.78
02
Lin
(cou
nts)
2-Theta-scale
CHPD EN4
Fig. 8. XRD of CHPD (C) and treatment (EN4) crystals.
Fig. 9. Thermogr
expected to occur during the dehydration and decompositionstages [49].
2CaHPO4 �2H2O-2CaHPO4þ4H2Om (1)
2CaHPO4-Ca2P2O7þH2Om (2)
Fig. 10 illustrates TG-DSC curve of EN4 crystals. The majorweight loss of about 18% occurs between 135 and 193 1C in theEN4 crystals. In the second stage nearly 6% weight loss occurredup to 441 1C. Subsequently about 2% loss occurred at 932 1C andthe rest of the sample (74%) was stable. Thus there is an increasein peak temperature which indicates the improved thermalstability of CHPD due to incorporation of bioactive compounds.The additional peak at 932 1C may also indicate the possibility ofincorporation of impurities coming from Ess extract.
The Brushite crystal has a plate-like morphology dominated by(010) faces and the structure within the (010) plane is composedof two corrugated rows of Ca2þ and HPO4
2� that are offset in the/010S direction. Between these calcium and phosphate contain-ing sheets, are layers of water molecules bound to the calciumions above and below the (010) plane [50]. It is assumed thatwhen the interconnecting layers of water molecules, that boundto the calcium ions above and below the (010) plane, is unavail-able for bonding due to incorporation of bioactive compounds inEss extract, further growth of CHPD crystals may get arrested.This explains one of the possible mechanisms for the efficacy ofthe seed extract of Ensete superbum on growth inhibition of CHPDcrystals under in vitro conditions. The FTIR and TGA/DTA dataprovide additional evidence that O–C–O group from Ess extractmight have formed a bond with –OH on the surface of (010) faceof CHPD. It is assumed that this incorporation of the bioactivecompounds within the framework of CHPD might have blockedfurther growth and aggregation Ess extract treated CHPD crystals.
4. Conclusion
The seed extract of Ensete superbum was evaluated for its anti-lithic activity employing single diffusion gel growth technique.This technique was utilized as a simplified screening static modelto study the growth, inhibition of urinary stones under in vitro
am of CHPD.
Fig. 10. Thermogram of EN4.
K.J. Diana, K.V. George / Journal of Crystal Growth 363 (2013) 164–170 169
conditions. The study revealed that the seed extract of Ensete
superbum is very effective to inhibit the growth of CHPD crystalgrowth under in vitro gel conditions. The SEM, FTIR, XRD, andTGA/DTA data provide evidence for the morphological and struc-tural changes of the treated crystals. It is expected that thismultidisciplinary approach for in vitro crystallization and char-acterization of CHPD crystals will provide a better explanation todevelop novel strategies for prevention or dissolution of urinarystones. The results also confirm the traditional use of the Ensete
superbum seed extract as an effective antiurolithiatic agent.Further studies are needed to isolate, characterize and screenthe biological activities of the antiurolithiatc compounds from theseed extract of the Ensete superbum.
Acknowledgments
The authors are thankful to the UGC, Bahadurshah Zafar Marg,New Delhi 1100029 Ref. No. 35-46/2008 (SR) dated 20 March2009 and M.G. University, Kottayam for the financial assistance.The authors also acknowledge the Principal, C.M.S. College andDirector, School of Environmental Sciences, M.G. University,Kottayam and STIC, Cochin University, for providing the researchand instrumentation facilities.
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