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Effect of Avocado (Persea gratissima) Leaf Extract on Calcium Oxalate Crystallization

Kidney stones or nephrolithiasis is one of the most common urologic diseases worldwide and places a heavy burden on human health. The annual prevalence of kidney stones is estimated at 3–5% and the approximate lifetime prevalence is 15–25% worldwide1. Kidney stone formation is a biological process involving the crystallization of several urinary minerals. These urinary stones develop when the minerals in urine aggregate and grow instead of being dissolved and passed out of the body2. Kidney stones are predominantly (approximately 80%) composed of calcium oxalates and calcium phosphates, of which 70% are calcium oxalate stones3. Calcium oxalate exists in three forms—monohydrate (COM, CaC2H4•H2O), dihydrate (COD, CaC2H4•2H2O), and trihydrate (COT, ABSTRACT

CaC 2 H 4 •3H 2 O). The most stable form under normal atmospheric conditions is calcium oxalate monohydrate. Specific solution conditions (e.g., supersaturation) and temperatures are required for the other forms [4][5][6] . Calcium oxalate monohydrate crystals are known to be the main component of stone formation in the urinary tract and kidneys. Kidney stones are a significant clinical problem so a large body of research is focused on their biomineralization, especially on preventing kidney stone formation by the use of various crystal growth modifiers [7][8][9][10][11][12] . As well as using chemical additives to reduce kidney stone formation, long-term treatment of kidney stones using herbs and plants is becoming more popular 6 . In this study, avocado leaf extract, a traditional, natural, harmless treatment was investigated as a crystal growth modifier. Avocado leaves are a natural ingredient used in traditional medicine as a diuretic, anti-inflammatory, antihypertensive, and antioxidant 13 . They contain a range of active compounds, including alkaloids, tannins, saponins, triterpenoids, and flavonoids. Avocado leaves have been demonstrated to effectively prevent calcium oxalate crystallization 14,15 . Thus, in this work, the researcher investigated the effect of avocado leaf extract on calcium oxalate crystallization. The crystalline structure and morphology of crystals prepared in solutions with and without avocado leaf extract were determined and compared via in vitro experiments.

METHODOLOGY
Analytical-grade calcium chloride (CaCl 2 •2H 2 O) and sodium oxalate (Na 2 C 2 O 4 ) were obtained from Merck Company. Distilled water was used throughout the experiments.
The crystallization experiments were done in a cylindrical jacketed crystallizer with a working capacity of 0.5 L. The experimental set-up included a thermostat, pH control system, stirrer, peristaltic pump, and syringe pump. The temperature was controlled using a thermostat. During the crystallization experiments, the temperature and pH of the suspension were kept at 37 °C and 7.4. A three-blade stirrer was installed in the center of the crystallizer to ensure the desired stirring rate (400 rpm). In each experiment, 200 mL of CaCl 2 •2H 2 O was placed in the crystallizer and then left to reach thermal equilibrium. Afterwards, 200 ml of Na 2 C 2 O 4 solution was fed into the crystallizer using a peristaltic pump to produce calcium oxalate monohydrate crystals. During the crystallization process, the crystallizer was kept closed.
To evaluate the effect of avocado leaf extract on calcium oxalate crystals in vitro, a 5 wt% aqueous extract was used. Firstly, dried avocado leaves were ground to a fine powder, 2.5 g of the powder was added to 50 mL of boiling distilled water, and the mixture was boiled for 5 min. The solution was allowed to cool to room temperature and then filtered. The prepared extracts were then utilized in the experiments and the extracts were added to the crystallizer via a syringe pump. At the end of the experiments, the suspension was removed from the crystallizer and a portion of the sample was dried. The remaining portion of the sample was subjected to aging at 37 °C at pH 7.4 for 2 months.
To characterize the calcium oxalate end-products, various techniques were used. X-ray diffraction measurements (XRD) were performed using a Bruker D2 Phaser tabletop diffractometer in the diffraction angle range of 2θ = 10-60°. The surface morphology of the calcium oxalate was explored using scanning electron microscopy (SEM; Zeiss EVO LS 10) and transmission electron microscopy (TEM, JEOL JEM 1220). To determine the functional groups in the crystals, a Fourier-transform infrared spectrometer (FTIR; Shimadzu IR Affinity-1) was used. The zeta potential of the sample was measured using a Malvern Zeta Sizer Nano Series Nano-ZS. Thermal analysis was performed under a nitrogen atmosphere using a Setaram LABSYS Evo thermogravimetric (TG) analyzer. The sample (15 ± 0.1 mg) was placed in a crucible and heated from 30 °C to 800 °C at a constant heating rate of 10 °C/min.

Characterization of the Avocado Leaves
Prior to the crystallization experiments, the general properties of the avocado leaves were investigated by FTIR, XRD, SEM, and TG analysis. The FTIR spectrum shown in Figure 1 was collected in the range of 600-4000 cm -1 to identify the chemical structure of the avocado leaves.
The wide transmittance band at 3313.71 cm -1 represents the O-H stretching vibrations. The peaks at 2927.94 cm -1 and 2850.79 cm -1 were assigned to the asymmetric and symmetric vibrations of CH 2 , respectively.

Figure 1 . FTIR spectrum of the avocado leaves
The band at 1728.22 cm -1 was associated with the C=O stretching vibration in carboxylic acids, aldehydes, and ketones due to polyphenols. The strong band at 1614.42 cm -1 can be attributed to the C=C vibrations in the aromatics. The peaks shouldered at 1448.54 cm -1 and 1365.60 cm -1 are related to the CH 2 and CH 3 bending vibrations, respectively. A band at 1039.63 cm -1 indicated the presence of esters in the sample. The functional groups detected on the leaves indicated the presence of polyphenols and flavonoids 16 .
XRD analysis was used to identify the structure of the avocado leaves and XRD result is given in Figure 2. As seen from the patterns, the intense peaks of sample were identified at 2θ of 29.4, 38.4, 44.5, 64.9 and 77.9°.

Figure 2 . XRD spectrum of the avocado leaves
SEM analysis was used to determine the morphology of the avocado leaves. Figure 3 demonstrates that the surface of the leaves was heterogeneous with non-uniform sized and long cylindrical shaped particles. The thermal structural evolution of the leaves was investigated by using a thermogravimetric analyzer. The TG and differential thermogravimetric (DTG) curves of the sample are shown in Figure 4. The thermal degradation of the sample showed four weight-loss regions. The first stage in the temperature range from approximately 35 °C to 150 °C represents the evaporation of moisture content, whilst the second stage (165-395 °C) is related to the decomposition of hemicellulose, which is the main component of the leaves. The third (395-550 °C) and fourth (590-710 °C) steps were attributed to the decomposition of cellulose and lignin, respectively. Hemicellulose is thermally less stable than cellulose and lignin owing to its amorphous structure and so it starts decomposing readily in the lower temperature range. The cellulose decomposition requires higher temperature owing to the presence of strong hydrogen bonding. Lignin, being the most complex structure, is assumed to decompose slowly over a broad temperature range from ambient to 800 °C. Figure 5 depicts the XRD patterns of the calcium oxalate crystals precipitated with and without the avocado leaf extract media. The sample crystallized in the absence of the extract was monohydrate form with no other crystalline phases nor phase transformations, and the major diffraction peaks of the sample were in agreement with JCPDS card no: 00-020-0231. As can be seen from the patterns of the crystals, four dominant peaks at 2θ = 14.9, 24.5, 30.1, and 38.3° attributed to the (101), (020), (220), and (130) planes of the monoclinic structure were detected in each pattern, in agreement with the results of previous studies 17 . Materials Analysis Using Diffraction (MAUD) software was utilized to determine the unit cell parameters and detect the crystal system. The computed lattice parameters of the crystals precipitated in pure media were determined as a = 9.989 Å, b = 7.298 Å, c = 6.310 Å, and β = 107.0°, which were in agreement with the results of a previous study 18 . The crystal system was monoclinic with space group P21/n.

Structure Analysis
With avocado leaf extract added to crystallization media, the crystals were all in the monohydrate form as in the pure media. There was no change in the crystal structure of the calcium oxalate monohydrate crystals formed in the presence of the extract. However, differences in the intensities of the dominant peaks were observed in the extract media compared to those in pure media. The lattice parameters for the extract media were a = 10.004 Å, b = 7.309 Å, c = 6.307 Å, and β = 107.0°.

FTIR and Zeta Analysis
The FTIR spectra of the crystals precipitated with and without avocado leaf extract were collected for the determination of their functional groups. Figure  6 displays the FTIR spectra of the samples in the range of 600-4000 cm -1 .
The wide transmittance bands in the range of 3000-3500 cm −1 represent the asymmetric and symmetric O-H stretching of the water molecules. Two bands at approximately 1600 cm -1 and 1310 cm -1 denoted the presence of symmetrical and anti-symmetrical C=O stretching vibrations. The peaks at ~950 cm -1 and ~880 cm -1 were assigned to C-C stretching vibrations. The band at 659 cm -1 was associated with the bending and wagging modes of water 19,20 . Figure 6, it can be clearly seen that the extract introduced new peaks in the range of 1000-1150 cm -1 . Thus, the avocado leaf extract adsorbed on the calcium oxalate surface and affected the surface properties of the crystals. Therefore, in addition to FTIR analysis, zeta potential measurements were carried out to identify the surface charge of the samples.  The negativity of the zeta potential value increased significantly in the presence of the avocado leaf extract media with time, reaching -31.8 mV at the end of day 60.
The growth and agglomeration of urinary crystals are inhibited by a more negative crystal surface, in agreement with the literature 21 . Thus, with the increment of the surface charge negativity, the crystals aggregate less readily because of their high electrostatic repulsion and stability, which indicates the inhibitory effect of the avocado leaf extract on calcium oxalate and kidney stone formation. In other words, this extract shows potential for preventing kidney stone formation by inhibiting crystal formation. The results will make a useful contribution to advancing research efforts on kidney stone formation, a significant clinical issue and an important area of biomineralization research. This study provides guidance for future studies in this area as well as experimental evidence for the inhibition of pathological crystal formation in the urinary tract. Figure 8 shows SEM images of the calcium oxalate monohydrate crystals precipitated in pure media with the corresponding energy dispersive X-ray (EDX) spectra. The images were taken at different times. In Figure 8a, the crystals precipitated in pure media at the end of 1 day were in a hexagonal form and form robust aggregates on one another. The mean particle size of the crystals was 1.3 μm. In order to evaluate the morphological changes over time, the SEM image of the calcium oxalate monohydrate precipitated at the end of 1 day was compared to those of the crystals kept in solution for 2 months. The crystals preserved their aggregated form but grew bigger to a mean particle size of 2.1 μm. In both measurements, the surface of the crystals was smooth, and the crystals were similarly regular shaped and uniform sized. In addition, the elemental contents of the crystals were determined as Ca, C, and O, in good agreement with the literature 6 . Figure 9 displays the morphological features of the calcium oxalate crystals precipitated in the avocado leaf extract media at different times.

Morphology Analysis
As shown in Figure 9, the surface properties and morphology of the crystals were directly related to the duration of the sample exposure to the extract media. It was observed that the crystals were still in hexagonal form after being exposed to the extract for 30 days, but they started to lose their aggregation tendency. The mean particle size of the crystals was 1.6 μm. Moreover, the precipitated crystals lost their smooth surfaces, started to deform, and surface nucleation occurred in the extract media. After 45 days, the nuclei formation on the crystal surface increased and these nuclei grew to form new crystal shapes. Additionally, the longer precipitation time further enhanced the breaking and deformation tendencies of the crystal surface. The mean particle size of the crystals was approximately 850 nm. After being exposed to the extract for 60 days, the surface became rougher, fractured, and more layered. The mean particle size continued to fall with time, being measured as 720 nm. In order to detect these surface changes more precisely and to gain more insight into the effect of the extract on the crystal morphology, TEM images were obtained at the end of 60 days in pure media and in the presence of avocado leaf extract. The results are presented in Figure 10. It can be clearly seen in Figure 10a that the surfaces of the crystals precipitated in pure media were smooth, in parallel with the SEM results, and there was no surface nucleation or defects. The calcium oxalate crystals were in a regular hexagonal shape, in agreement with the literature 22 . On the other hand, the crystals precipitated in avocado leaf extract media underwent both morphological changes and surface nucleation (Figure 10b). Furthermore, they completely lost their evenly distributed appearance.
From the evaluation of the SEM and TEM images together, it can be concluded that the extract had a significant modifying effect on the calcium oxalate morphology. The regular hexagonal-shaped crystals with smooth surface precipitated in pure media transformed into weaker crystals with a deformed surface. It was also noted that the duration of exposure to the extract also had an effect on the morphology of the calcium oxalate crystals.

Thermal Analysis
The thermal decomposition behavior of the calcium oxalate crystals was investigated in this study. The TG and DTG curves of the crystals precipitated with and without the extract with a heating rate of 10 °C/min are shown in Figure  11. Thermal decomposition of the samples indicated three weight loss stepsdehydration, decarbonylation, and decarbonation-respectively. A similar decomposition profile was also detected for the extract media. The weight loss of the crystals precipitated in pure media was 61.5%, which was in agreement with the literature 23 . Increment of 0.3%, were observed for struvite crystals prepared in avocado extract media, respectively. The increase in the weight loss was attributed to the decomposition of the traces of the extract remaining the sample.