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International Journal of Phytomedicine and Phytotherapy

Effect of Aegle marmelos on the growth of brushite crystals

Abstract

Background

The urinary stone formation is a very common health problem due to the prolonged medication. Calcium Hydrogen Phosphate Dihydrate (CaHPO4.2H2O, CHPD and Brushite) is one of the major constituent of urinary stone crystals. The formation and deposition of brushite crystals is the central cause of recurrent kidney stone disease among the global population. The present study aims to investigate the effect of Aegle marmelos leaves on the growth of brushite crystals to explicate the inhibitory effect of urinary stones from a different point of view.

Methods

The CHPD crystals were grown by the Single Diffusion Gel growth technique. In order to inhibit the formation of urinary stones, CHPD crystals are grown in the laboratory in the pure form and by adding the prolonged medicine A. marmelos taken especially by diabetic patients. A comparative study of the pure and A. marmelos leaves added crystals has been carried out using the Growth factor, Powder X-ray diffraction, Microstructural parameters, FTIR and SEM-EDAX.

Results

Total mass of the grown crystals is found to decrease with increasing concentrations of leaf extract of the A. marmelos. On comparing with undoped, crystalline size of the A. marmelos doped samples is reduced. Disappearance of absorption band and peak shift in the FTIR shows the incorporation of functional groups of A. marmelos. The morphology changes of the treated crystals are assessed in SEM.

Conclusions

The result shows that herbal extracts prepared from A. marmelos have good inhibitory effect on the growth of the brushite urinary stone crystals considered.

Introduction

Nephrolithiasis is a common health issue that affects at least once in a lifetime between 2 and 5% of the population worldwide. Because of this condition, the prevalence of urinary stones is increasing and about 12,000 patients are hospitalized each year. Many risk factors have been associated with this debilitating and costly illness including geographical [1] and weather conditions [2] physical traits [3] nutritional issues [4] and more recently, metabolic syndrome [5] are some of the causes of an increased risk of kidney stones. Approximately 85% of kidney stones are calcium stones [6] in combination with either oxalate or phosphate, or both in the form of apatite or brushite [7]. The first stage of kidney stone includes the nucleation, growth and accumulation of crystals. In the second stage, the crystals are remained in the kidneys and/or renal tubules, and eventually they move from the interior part of the kidney to the papillary surface of the kidney to form a stone nidus. Minerals especially calcium in the urine, then develop on the speck in a similar manner to that in which a pearl grows in an oyster shell. The formation of the nidus may be analogous to the first stage in the physiological calcification of the bone in which the calcium phosphate nucleus develops in the organic matrix [8, 9]. It is reported that calcium phosphate is the major solute component which crystallize rapidly in urine. Its presence is also reported in small quantities in renal and urethral stones [10]. Moreover, small amounts of calcium phosphate have been found in the assumed binding portion of the stone [11]. These observations suggest that other stones originate from the precipitation of calcium phosphate and that the renal papilla is bound to the early stages of these stones. Once a crystal nidus has been established, it develops into a kidney stone by precipitation or by the process of crystal growth. Initially, kidney stones often have no symptoms. The first symptom of kidney stone is extreme pain as stones block the flow of urine [12].

Calcium Hydrogen Phosphate Dihydrate (CHPD, Brushite, and CaHPO4.2H2O) plays a major role in the urinary calculi formation. Calcium phosphate stone formation is related to conditions such as hyperparathyroidism [13] and renal tubular acidosis [14]. Brushite is a unique form of CaP that can form large symptomatic stones in certain patients. The molecular structure of brushite is shown in Fig. 1. It can be difficult to treat brushite stones as they are resistant to shock waves and ultrasonic lithotripsy and also require ballistic fragmentation. Patients with brushite stone disease are less likely to become stone free after surgery and often experience repetition in the stone despite maximum medical intervention [16]. In order to evaluate the transportation of renal phosphate, Dhayat et al. [17] used the ratio of tubular maximum reabsorption of phosphate to glomerular filtration rate (TmP/GFR) showing that low TmP/GFR is correlated with increased excretion of calcium in the urine and increased prevalence of brushite stones. Xie et al. [18] showed that ACP plays a vital role in nucleating calcium oxalate stones by promoting the accumulation of amorphous calcium oxalate (ACO) precursors at early induction stages. As compared to other stones, phosphate stones were high in magnesium, strontium, zinc and chromium [19]. Moreira et al. [20] reported that the brushite stones were associated with higher urinary calcium excretion and higher urinary supersaturation CaP when compared to apatite stones. Aggressive measures to reduce urinary calcium can be especially helpful in preventing brushite stone formation.

Fig. 1
figure1

Molecular structure of brushite (Bindhu et al.) [15]

When any patient takes any drugs constantly and continuously, it ends up with some side effects. In some cases, the intake of drugs changes the biochemistry of the urine and it may induce the crystallization of metabolic compounds of unusual morphology, which indicates the possibility that there are peculiar conditions for the formation of urinary stones. In order to find out the formation of urinary stones as being the unwanted effect of the intake of diabetic medicines, the present work is carried out. In this study, the A. marmelos is taken for consideration. A. marmelos, known as Bael in India, is a famous Rutaceae plant, one of Ayurveda’s most important plants [21]. Different parts of this plant, such as leaves, roots, seed, bark and fruit are used by the Ayurvedic practitioners due to its various medicinal properties [22, 23]. Traditionally, fever, diabetes, diarrhea, abscesses and snake bites have been treated with the plant [24, 25]. Moreover, extracts of A. marmelos have good anti-diabetic properties [26]. Although several studies have been carried out with this plant, there is no documentation for the effect of diabetic drug A. marmelos on urinary stones. Hence, the present study is intended to investigate the effect of A. marmelos leaves on urinary stone formation.

Experimental

Chemicals

Sodium Meta Silicate (SMS) – {Na2SiO3.9H2O} and Calcium Acetate (Ca(C2H3O2)2) have been purchased from Loboma chemicals Ltd. All other chemicals such as Calcium Chloride (CaCl2) and Orthophosphoric acid (H3PO4) have been purchased from Merck (Mumbai, India). All the chemicals are of analytical grade and are used without any further purification or post-treatment. Double distilled water is used for cleaning the glass wares.

Plant materials and identification

Fresh leaves of A. marmelos were collected from the natural surroundings and authenticated at the Department of Botany, S.T. Hindu College, Nagercoil, Tamil Nadu, India. The collected leaves were washed with double distilled water followed by shade drying. The dried leaves were grounded into a fine powder and used for further experiments.

Growth of brushite crystals

The single diffusion gel growth method is employed to study the growth and inhibiting behavior of brushite crystals using A. marmelos [27, 28]. For crystallization, glass test tubes of size 140 mm length and 25 mm in diameter are used in this method. Sodium meta-silicate solution of specific gravity 1.03 g/cc has been impregnated with orthophosphoric acid according to the desired pH value. The solutions pH value is set to 6.5. The time required to set the gel firmly was dependent on pH value of the mixture. Within 48 h, good quality gels were set in the test tubes for the pH value chosen above. Once when the solution undergoes a gelation phase, an aqueous solution of calcium chloride and calcium acetate of a specific molarity was carefully poured over the gel using pipette to prevent any breakage of the gel. The test tubes were capped with airtight stopples after pouring supernatant solution. The following reaction is expected to take place leading to the formation of calcium hydrogen phosphate dihydrate crystals.

$$ {\mathrm{CaC}\mathrm{l}}_2.{2\mathrm{H}}_2\mathrm{O}+{\mathrm{CaC}}_4{\mathrm{H}}_6{\mathrm{O}}_4+{\mathrm{H}}_3{\mathrm{PO}}_4\to {\mathrm{CaHPO}}_4.{2\mathrm{H}}_2\mathrm{O}+{\mathrm{CaC}\mathrm{O}}_3+\mathrm{HCl} $$

To study the effect of A. marmelos leaf extract on the growth of brushite crystals, the following method was used. The finely powdered leaf extracts of different concentrations (10 mg to 50 mg) were separately added along with calcium chloride and calcium acetate and the crystals are grown as before. The Ca2+ ions and additional ions available in the A. marmelos extract are diffused into the gel and react with the phosphate ions in the gel column. This reaction leads to the formation of medicines added calcium hydrogen phosphate dihydrate crystals thereby showing its promotery or inhibitory effect on these crystals. The above experiment was conducted simultaneously with the control system to compare the growth and morphology of the undoped CHPD and A. marmelos doped CHPD crystals.

The formation of Liesegang rings followed by subsequent crystallization and aggregation of CHPD crystals were noted. In about 25 days, the crystal growth was completed. The grown crystals removed and collected carefully in a clean petri dish and then harvested using distilled water to remove the gel. Then the harvested crystals are dried by placing them at room temperature in a filter paper. The dried crystals are finely powdered and used for further characterization.

Characterization techniques

Powder X-ray diffraction

Powder X-ray diffraction (PXRD) is used to identify the crystal structure and collected on a XPERT-PRO diffractometer with CuKα radiation (λ = 1.54060 Å) over a range of 10–80° in 0.05 step sizes with an integration time of 10s.

Fourier transform infrared spectroscopy

The FTIR spectra were recorded in Shimadzu - FTIR 8400S with Spectral range: 4000–400 cm− 1 and Resolution: 4 cm− 1 with KBr beam splitter and DLATGS Detector.

Scanning Electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX)

The surface features of the crystals are studied using Scanning Electron Microscope (SEM Model ZEISS EVO 18). EDAX is recorded using dispersive spectrometer (AMETEK EDAX) attached to the scanning electron microscope for carrying out elemental analysis of the crystals.

Results and discussion

Growth kinetics

The silica gel is set and 1.5 M concentration of calcium chloride and calcium acetate was poured on it, while we understand the growth mechanism, it is found that the Ca2+ ions diffuse through the gel column and immediately react with phosphate and precipitated forming Liesegang rings in just 4–5 h. The number of Liesegang rings gradually increase with time and a total of about 8 rings are formed in the control test tubes (Fig. 2(a)). It is found that the Liesegang rings of CHPD are formed because of the diffusion of ions in the gel medium and shown that the diffusion kinetics is proved by studying the distance [29, 30]. In due course of time, the crystals are grown where the rings disappeared. The grown crystals of undoped CHPD are shown in Fig. 2(a). Figure 2(b) shows the grown crystals of CHPD by adding 50 mg of A. marmelos. Comparing the undoped crystals, the number of crystals grown with A. marmelos is less. The results infer the parameters such as the pH value, the concentration of reactants, and the column height of the solutions affect the formation of Liesegang rings [31, 32]. On the other hand when we add herbal extracts to the supernatant solution, we observe the less number of Liesegang rings and reduction in length of CHPD crystals grown. This may be due to the changes in the kinetics and diffusion processes and inhibitory effectiveness [33].

Fig. 2
figure2

CHPD (Brushite) crystals grown in gel medium (a) Without Inhibitor, (b) 50 mg of A. marmelos added CHPD crystals

The Promotery/Inhibitory effect is judged on the basis of number of crystals grown and total mass of the grown crystals. The same condition is maintained for controlled and test samples.

Figure 3 shows the harvested crystals. Figure 3 (a0) shows undoped CHPD crystals. The crystals are in thin platelet like structure with an average length of 1.2 cm. The rest (a1-a5) show the CHPD crystals added with 10 mg, 20 mg, 30 mg, 40 mg and 50 mg of A .marmelos respectively. The average size of the A. marmelos doped CHPD crystals are less when compare to the undoped CHPD crystals. When the concentration of A. marmelos is increased, the size of the crystals is decreased. At the same time it is reduced furthermore for 50 mg concentration.

Fig. 3
figure3

Harvested CHPD (Brushite) crystals grown without inhibitor (Undoped (a0)), with A. marmelos (a1-a5) respectively

Owing to its distinctive ionic structure, CHPD serves as a good model crystal for the study of interactions between additives and crystals. Skiric et al. [34] have summarized as follows:

  1. 1.

    The significance of the molecular structure and additive, i.e., tiny or very large molecule, number of chemical compounds in the molecule and its total charge in the growth of CHPD and other associated crystals. This may be useful for selecting molecules that inhibit the growth of CHPD crystals. One negative charge of glutamate and asparate ions, for instance, has little effect on the CHPD crystal faces, although it can be enhanced by adding more negative groups like OH

  2. 2.

    The amount of inhibition in a given crystalline face is specified by the structural fit between the organic molecule and the ionic structure of the particular crystal face. This can have an effect on the crystalline faces that are exposed to the solution. The negatively charged small molecules such as citrate ions, interact with the lateral surfaces of the CHPD crystals and hence the crystallization is slowed and the morphology of the crystals is modified.

  3. 3.

    The influence of the exposed hydration layer is seen on the surface of the crystal. In polyaspartic acid, such structural fit exist between the distances of carboxylic groups in the polyaspartic β and in the case of CHPD, the distances of the calcium ion across two adjacent layers make up a two-layer Ca-HPO4 below the hydrated layer parallel to the plane (010) [35].

Total mass of the grown CHPD crystals

Figure 4 shows the histogram depicting total mass of CHPD crystals grown with A. marmelos at 10 mg, 20 mg, 30 mg, 40 mg and 50 mg concentrations.

Fig. 4
figure4

Promotery/Inhibitory effect of Brushite crystals

For the cases of 10 mg to 50 mg doped samples, the total mass is decreased than that of undoped. While comparing with the prior concentrations, the total mass for 50 mg concentration is extremely low. The herbal extracts (A. marmelos) contains phytochemicals such as alkaloids, terpenoids, amino acids, carbohydrates, flavonoids and phenols [36] etc. These phytochemicals interact within themselves at low concentrations, and so the penetration decrease and so the total mass. At high concentrations, the each constituent of phytochemicals agglomerate forming an interface between the cations thereby decrease the formation of crystals.

The ANOVA statistical analysis is performed for total mass and listed in Table 1.

Table 1 ANOVA statistical analysis of CHPD crystals

The single factor analysis of variance (ANOVA) followed by Tukeys test is carried out using origin 9 software. It is done to compare the total mass of undoped with doped samples. ANOVA statistical analysis indicates that the variations in the total mass of CHPD with doped samples are highly significant at 0.05 level.

Powder X-ray diffraction

Powder X-ray diffraction study is performed on grown crystals to identify the phase formation and degree of crystal perfection. Powder X-ray diffraction patterns are recorded in 2θ steps of 0.05 between 10° - 80° using a XPERT-PRO diffractometer with CuKα radiation at a wavelength of 1.54060 Å operating at 30 mA and 40 kV. The powder X-ray diffractogramfor both undoped and various medicines added CHPD crystals are shown in Figs. 5 and 6 and the h k and l values in brackets are provided.

Fig. 5
figure5

Powder XRD spectrum of undoped CHPD crystal

Fig. 6
figure6

Powder XRD spectrum of 50 mg A. marmelos added CHPD crystal

The XRD pattern of undoped CHPD crystals (Fig. 5) show prominent peak at 2θ = 11.75° which corresponds to the (0 2 0) plane and the other peaks observed at 2θ = 21.00°, 23.47°, 29.32°, 30.53°, 34.19°, 35.60°, 37.10°, 39.69°, 41.47°, 42.27°, 45.42°, 47.90°, 48.53°, 50.24°, 53.59°, 56.97°, 60.92° and 74.97° belongs to (1 2 1), (0 3 1), (1 4–1), (1 2 1), (1 5 0), (0 6 0), (2 2–2), (1 6–1), (1 5–2), (2 4 0), (1 2–3), (0 8 0), (0 6 2), (2 6 0), (3 4–1), (0 9 1), (2 8–2) and (3 8–3) reflections, respectively, of monoclinic structure of CHPD (JCPDS No. 72–0713). Compared to undoped CHPD crystals, the intensity of (0 2 0) peak increases for the doped crystals. This results in a decrease of peak intensity of all the remaining peaks. The (0 2 0) peak appears with maximum intensity in undoped CHPD and medicines added CHPD crystals. The higher intensities of (0 2 0) plane is a feature of typical deviation from an isotropic orientation of crystallite grains. The higher the number of plate like grains in the sample, the higher the fraction of the (0 2 0) planes which are aligned along the specimen surface [34]. The highly resolved peaks at specific 2θ Bragg angles in the crystals indicate the crystalline nature of the grown crystals. The software CellCalc is used to calculate lattice parameters and is tabulated in Table 2. The crystallographic parameters obtained are well correlated with data available in the literature (JCPDS No [72–0713]). The changes in the lattice parameters and unit cell volume of doped samples may be attributed to the incorporation of added medicines in the host of CHPD crystal.

Table 2 Unit cell parameters, Unit cell volume (V) and Angle (β) for undoped and A. marmelos added CHPD crystals

Crystallite size

The crystallite size of the grown crystals is estimated from the Scherrer equation [37],

$$ \mathrm{D}=\frac{\mathrm{K}\uplambda}{\upbeta \mathrm{cos}\uptheta} $$

Where D is the crystallite size, K (=0.89) is the constant, λ is the wavelength of the X-rays (λ = 0.54060 Å for CuKα radiation), β is the full width at half maximum value and θ is the Bragg diffraction angle.

Microstrain

The microstrain is calculated using the following relation [38].

$$ \upvarepsilon =\frac{\upbeta\ \mathrm{cos}\uptheta}{4} $$

Dislocation density

A dislocation is an imperfection in a crystal associated with the misregistry of the lattice in one part of the crystal with that in another part [38, 39].

The dislocation density (δ) is evaluated by the formula [40],

$$ \updelta =\frac{1}{D^2} $$

Stacking fault probability (α)

The stacking fault probability α is the fraction of layers undergoing stacking sequence faults in a given crystal and hence one fault is expected to be found in \( \raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$\upalpha $}\right. \) layers. The stacking fault probabilities are calculated from the shift of the X-ray line of the crystal with reference (JCPDS No: 72–0713 & 77–2303), using the relation between the stacking fault probability α and the peak shift Δ (2θ) [38].

$$ \upalpha =\left[\frac{2{\pi}^2}{45\sqrt{3}\tan \theta}\right]\Delta\ \left(2\uptheta \right) $$

The Microstructural properties of the grown crystals calculated from the above formulas are given in Table 3.

Table 3 Microstructural parameters of undoped and A. marmelos added CHPD crystals

The crystallite size, microstrain, Dislocation density and stacking fault probability of undoped CHPD and A. marmelos added CHPD crystals are shown in Table 3. The crystallite size of the samples is determined by employing Debye Scherrer’s equation. The crystallite size of undoped CHPD is around 51 nm. On doping with A. marmelos, the crystallite sizes are found to decrease. This is probably due to the growth inhibition. The dislocation density of the doped crystals increases comparing with the undoped CHPD. This may be due to the piling up of atoms one over the other. This in turn deforms the crystal structure and result into fracture due to brittleness. Thus the crystal formation is inhibited. The stacking fault probability is found to increase on doping, and no major changes are observed. Also it is observed that the Microstructural parameters dislocation density and microstrain depend upon crystallite sizes.

Fourier transform infrared spectroscopy

The structural studies of Calcium Hydrogen Phosphate Dihydrate crystals by IR spectra show various absorption bands which are characteristics of different vibrational modes. The FTIR spectra of undoped CHPD and 50 mg A. marmelos are shown in Fig. 7 and 8 and functional groups are mentioned in Table 4.

Fig. 7
figure7

FTIR spectrum of undoped CHPD crystal

Fig. 8
figure8

FTIR spectrum of 50 mg A. marmelos added CHPD crystal

Table 4 Band Assignments and absorption bands of undoped and A. marmelos added CHPD crystals

FTIR spectrum of undoped Calcium Hydrogen Phosphate Dihydrate (Fig. 7) show the absorption peaks at wavenumbers range of 3162.08 cm− 1, 3277.8 cm− 1, 3488.99 cm− 1, 3544.92 cm− 1 and 3729.42 cm− 1 are attributed to intermolecular and weakly H bonded OH because of water of crystallization [41, 42]. The characteristic peak at 2925.11 cm− 1 is due to OH stretching vibrations of water [43] and the HPO42− weak absorption bands [30] is observed at 2384.82 cm− 1 and 1729.06 cm− 1. The band at 2348.48 cm− 1 is attributed to PO-H symmetric stretching vibrations [44]. Two bands are observed due to O=P-OH stretching vibrations [45] of HPO42− near 2124.93 cm− 1, 2304.76 cm 1. An absorption band at 1649.02 cm− 1is occurred due to H-O-H symmetric bending vibrations [46] The absorption peak near 1511.18 cm− 1, 1455.08 cm− 1 and 1061.74 cm− 1 are assigned due to P=O stretching vibrations [29, 47]. The absorption takes place around 1394.86 cm− 1 and 1337.94 cm− 1are due to P=O asymmetric stretching vibrations [44]. The peaks about at 1218.93 cm− 1 and 1136.96 cm− 1 are occurred due to P=O associated stretching vibrations of PO4, while peaks at wavenumbers 986.52 cm− 1, 873.69 cm− 1 and 794.62 cm− 1 are due to P-O-P asymmetric stretching vibrations [29, 42]. The strong absorption peaks of acid phosphates [29, 48] lie at different wavenumbers of 660.57 cm− 1, 577.64 cm− 1 and 527.49 cm− 1.

By comparing the FTIR spectra of undoped CHPD and A. marmelos added CHPD, we determined that the foremost distinction amongst them is the disappearance of the peak encountered at 935.38 cm− 1 in the A. marmelos added sample (Table 4) corresponding to P-OH stretching vibrations. The leaf extracts of A. marmelos contains phytochemicals like alkaloids, flavonoids, phenols, saponins, carbohydrates, protein, phytosterol, tannins, glycosides, phenolic compounds, cardiac glycosides [36]. These phytochemicals are rich in polar groups which may interact with phosphate ion in CHPD leading to the disappearance of P-O symmetric stretching vibrations respectively. Moreover, slight shifting in FTIR spectra of doped samples suggests the encapsulation of A. marmelos.

Scanning Electron microscopy

Particle size and Surface morphology of the grown crystals are revealed by scanning-electron microscopy (SEM Model ZEISS EVO 18). The scanning electron micrographs of the samples, undoped CHPD and 50 mg A. marmelos added CHPD crystals are shown in Figs. 9 and 10.

Fig. 9
figure9

SEM micrograph of undoped CHPD crystal

Fig. 10
figure10

SEM micrograph of 50 mg A. marmelos added CHPD crystal

Figure 9 illustrates SEM photograph of undoped CHPD crystals. Plate like structure is observed [15]. The particle size of undoped CHPD calculated using SEM picture is around 94 nm. The SEM image of the 50 mg A. marmelos added CHPD crystals is shown in Fig. 10. Particles agglomerate and are in spherical shape. Particle size is in the range of 30–50 nm.

The SEM images of the undoped and A. marmelos added CHPD crystals show difference in their morphologies. The undoped CHPD crystals shows needle and platelets shaped morphology, whereas 50 mg A. marmelos added CHPD, we have the particles agglomerated and are in spherical shape. The growth morphology of CHPD crystals is typically in the form of thin plates with prominent (0 2 0) plane. The monoclinic unit cell of CHPD consists alternating bi-layers of calcium, hydrogen phosphate ion and a layer of water molecules oriented parallel to the (0 2 0) plane. As the bi-layers of water molecules are exposed at the surface of (0 2 0) face, the surface bound water can form bonds with molecules in the growth medium [49, 50]. The thin layer of water molecules on the surface of the CHPD may form bonds with the phytochemicals of the A. marmelos extract, thus weakening the ionic nature of the CHPD and leading to the structural modification of the doped crystals into tiny units. The reduction in platelet shape can be attributed to the influence of added extracts onto the CHPD crystal formation altering the growth kinetics of the platelets [49, 51, 52]. The decrease in particle size of medicines added crystals may be due to the inhibition of the grain growth.

Energy dispersive X-ray analysis

In order to study the elemental composition of undoped and A. marmelos added CHPD crystals, qualitative and quantitative analysis is performed by energy dispersive X-ray analysis (EDAX) using dispersive spectrometer (AMETEK EDAX) attached to the scanning electron microscope. The spectrum obtained from EDAX analysis of undoped CHPD and 50 mg A. marmelos added CHPD crystals is shown in Figs. 11 and 12 and the average atomic and weight percentage of individual elements is given in Tables 5 and 6.

Fig. 11
figure11

EDAX spectrum of undoped CHPD crystal

Fig. 12
figure12

EDAX spectrum of 50 mg A. marmelos added CHPD crystal

Table 5 EDAX data for undoped CHPD crystal
Table 6 EDAX data for 50 mg A. marmelos added CHPD crystal

EDAX pattern of undoped CHPD (Fig. 11) shows the presence of elements such as Calcium, Phosphorus and Oxygen present in the grown crystal. This clearly indicates that the crystals are of Calcium Hydrogen Phosphate Dihydrate indeed. The higher peak of Ca, P and O shows that the more concentrated the element are in the specimen. The Ca/P ratio of undoped CHPD crystal if found as 1.08, which is closely associated with the actual value of 1 according to the chemical formula [53]. Similarly EDAX pattern of 50 mg A. marmelos added CHPD crystal is shown in Fig. 12. The spectra corresponding to 50 mg A. marmelos added CHPD also show peaks corresponding to all major elements Ca, P and O. The Ca/P ratio of crystals grown with 50 mg A. marmelos is found as 1.03.

Conclusion

The effect of A. marmelos on the growth of brushite crystals is examined in vitro. The present study reveals that the herbal extracts of A. marmelos inhibit the growth of brushite crystals. It is observed that the incorporation of A. marmelos caused a decrease in the number of grown brushite crystals and their average size. Powder X-ray diffraction exhibit monoclinic crystal structure and the lattice parameters are in good agreement with the reported values. Also it confirms the incorporation of additional ions in the A. marmelos leaf extracts into the host of CHPD. FTIR confirms the presence of functional groups. SEM shows the morphological changes in the treated crystals. The SEM micrograph of crystals grown in the presence of A. marmelos shows reduction in the grain size with addition of A. marmelos as compared with pure CHPD crystals. EDAX confirms the presence of required elements. From the above observed results, the intake of A. marmelos is good for diabetic patients as it reduces the growth of urolithiasis. However, further in vivo studies are needed to evaluate its potential antiurolithiatic activity.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article

Abbreviations

CHPD:

Calcium Hydrogen Phosphate Dihydrate

CaP:

Calcium Phosphate

GFR:

Glomerular Filtration Rate

ACP:

Amorphous Calcium Phosphate

ANOVA:

Analysis of Variance

JCPDS:

Joint Committee on Powder Diffraction Standards

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Acknowledgements

The authors are thankful to Department of Physics (DST-FIST), S.T. Hindu College, Nagercoil for providing facilities for the completion of this work.

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GA planned the study, designed the experiments and drafted the manuscript. RM participated in the preparation of herbal extracts and interpretation of results. THF supervised the project and reviewed the manuscript. RR and GR involved in discussing the concepts. All authors read and approved the final manuscript.

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Correspondence to G. Anushya.

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Anushya, G., Mahesh, R., Freeda, T.H. et al. Effect of Aegle marmelos on the growth of brushite crystals. Clin Phytosci 7, 43 (2021). https://doi.org/10.1186/s40816-021-00280-6

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Keywords

  • Brushite
  • Aegle marmelos
  • Gel growth
  • X-ray diffraction
  • Microstructural parameters