Étude de l’effet inhibiteur de la cristallisation de l’oxalate de calcium monohydrate par deux plantes médicinales et aromatiques : Ammi visnaga et Punica granatum

25 mars 2018

Auteurs : R. Kachkoul, T. Sqalli Houssaini, Y. Miyah, M. Mohim, R. El Habbani, A. Lahrichi
Référence : Prog Urol, 2018, 3, 28, 156-165




 




Introduction


The urinary lithiasis is a pathology characterized by the formation of calculi in the kidneys or in the urinary tract. Some forms are recurrent, particularly severe and can lead to kidney failure, causing pain and bleeding. The disease reflects sanitary conditions, dietary habits and living standards [1]. The study of the epidemiological profile reveals that the lithiasis constantly evolves during the last 50 years and affects 4-20% of the population, with an estimated recurrence rate of 50% during the first 5 years with the predominance of the calcium oxalate [2, 3, 4, 5, 6, 7].


Calcium oxalate crystals are mainly found in three different forms: calcium oxalate monohydrate (COM) or Whewellite, calcium oxalate dihydrate (COD) or Weddellite and calcium oxalate trihydrate (COT), [8, 9, 10]. "COM" is the most thermodynamically stable form and the most common in clinical calculations and has a greater affinity for renal tubular cells; therefore it is the initiator of the formation of stones in the kidney [11, 12]. Crystallization of calcium oxalate begins with increased urinary supersaturation, with subsequent formation of solid crystalline particles in the urinary tract [13]. This is followed by nucleation, whereby salts forming computations in a supersaturated urinary solution come together in clusters, which then are increased in size by the addition of new constituents to form aggregates [13, 14, 15]. These crystals are developed and accumulated with other crystals in solution and are finally retained and accumulated in the kidney [15, 16, 17]. Renal lesions promote crystal retention and development of a computing nest on the renal papillary surface owing to crystal nucleation at lower levels of supersaturation [18].


Additionally, one or more treatments are necessary, namely medical and/or surgical treatment. Unfortunately, these methods, in most cases, remain expensive and invasive having side effects. Therefore, the use of alternative methods, addressing medicinal and aromatic plants (PMA) is a very popular field in Morocco. In this case, ethnobotanical studies accomplished by various researchers, have revealed the efficacity of the latter in the treatment of lithiasis with the aim of preventing recurrence of stones as well as relieving renal colic. In this context, our aim was to test experimentally the efficiency of two plants against the crystallization of calcium oxalate.


Ammi visnaga is a medicinal and aromatic plant known by many common names, "bisnaga", "toothpicks" and "khella". It is an annual or biennial plant of the family Apiaceae (Umbelliferae). Besides, it has been used for a long time in the treatment of certain diseases thanks to the presence of bioactive molecules such as khelline, visnagine and visnadine [19].


The second studied plant is called Punica granatum known as "grenadier" and belongs to the family of Punicaceae . It has several biological activities, a great curative and preventive power against chronic diseases.


Materials and methods


Methanolic extract of Ammi visnaga and Punica granatum


Samples of Ammi visnaga and Punica granatum were harvested in the Taounate region between June and November. In this respect, the seeds of Ammi visnaga and the bark fruit of Punica granatum were dried in the open and then crushed. Further, 20g of the powder is introduced into the cellulosic cartridge and then placed in the soxhlet assembly which is surmounted by a refrigerant. A total of 170mL of methanol are introduced into the flask and then brought to a boil for 4h at a temperature in the order of 65°C. Hence, the solvent when heated condenses and accumulates in the siphon tank, which increases the contact time between the solvent and the product to be extracted. While the solvent reaches a certain level, it initiates the siphon and returns to the flask, causing the dissolved substance. At the end of the extraction, the solvent contained in the distillation flask with the extract is removed by a rotary vacuum evaporator [20, 21].


The inhibiting effect of the calcium oxalate's crystallization


The inhibition of the crystallization was studied according to the protocol described by Hess et al. [22]: stock solutions of calcium chloride dehydrate CaCl2 :2H2 O (15mm) and sodium oxalate Na2 C2 O4 (1.5mm) containing 200mm NaCl and 10mm sodium acetate were adjusted to pH 5.7. Before being used in crystallization experiments, the solutions were filtered through filters having a pore diameter of 0.22μm and reheated to 37°C.


For crystallization experiments without inhibitor, 1.5mL of the CaCl2 :2H2 O solution was transferred to a quartz cuvette optical path 10mm. Additionally, an identical volume of the Na2 C2 O4 solution was added to trigger the formation of the crystals and corresponds to "t" equal to zero. Final concentrations test are 5mm and 0.5mm calcium and oxalate respectively, with a calcium/oxalate concentration ratio equal to 10, which corresponds to the formation of the wewellites.


The temporal measurements of the optical density "OD" were recorded over a period of one hour: every 15s for the first ten minutes and every thirty seconds thereafter. All crystallization experiments were performed three times, with the assistance of a UV-Visible spectrophotometer with a wavelength &lgr; equal to 620nm which corresponds to the formation of the crystals.


The crystallization tests in the presence of the extract were achieved with several concentrations ranging from 0.25; 0.5; 1-2g/L. The experiments were carried out with 1mL of CaCl2 : 2H2 O and 1mL of the extract to which 1mL of Na2 C2 O4 is added, which promotes both the formation of the crystals and triggers the measurements.


The turbidity test system reveals the induction time "ti" which corresponds to the time required for the formation of the first stable crystalline core and then growing to a size which makes it detectable by the UV-Visible spectrophotometer called nucleation. To add the maximum slope of OD increases as a function of time, determined by linear regression analysis corresponding mainly to an increase in the number of particles as a function of time. Since OD is an accurate measure of particle concentration per unit volume, OD also reflects the particle size as well. Therefore, the maximum slope of OD increasing with time is the nucleation slope "SN" which essentially represents nucleation of the crystals. At equilibrium, the optical density is maximal "ODmax " and corresponds to the saturation and to the end of the nucleation process. Moreover, a gradual decrease in OD with time is observed and linked to the decrease in the number of crystals due to their aggregations. However, the maximum slope of the OD decrease as a function of time has a negative value. For convenience, a positive number for the aggregation slope "SA" will be used for all comparisons. Subsequently, the percentages of inhibitions were calculated by the relation of Hess and al. [22, 23].
Nucleation   inhibition   rate:1−SNiSNs×100Aggregation   inhibition   rate:1−SAiSAs×100where "i" and "s" represent tests with and without inhibitor respectively. The correlation coefficient (R) and the coefficient of variation (CV) are calculated to verify the validity of our results.


Crystal characterization


The characterization and the determination of the chemical composition were made by the Fourier transform infrared spectra (FT-IR) on synthesized crystals. Indeed, over a volume V of a solution of CaCl2 : 2H2 O (15mm), the same volume of Na2 C2 O4 (1.5mm) is added. The mixture is stirred continuously for half an hour and then the solution is left to stand for 24h at 37°C. Thereafter, the precipitate is then filtered by a microfiltration membrane 0.22μm in diameter and then dried [24].


Microscopic observation


The microscopic observation of crystals was carried out using a polarized optical microscope equipped with a digital camera and connected to a computer.


Statistical analysis


The results are expressed as mean±SD, statistically analyzed and linear regression was performed using ANOVA OneWay followed by the Tukey multiple comparison test using Minitab 17, the P -value<0.05 is considered as significant.


Results


The inhibiting effect of the calcium oxalate's crystallization


The values corresponding to the induction time (ti) and the maximum optical density (ODmax ) for the experiments in the absence and the presence of the extracts: Ammi visnaga (A.V) and Punica granatum (P.G) are given in Table 1.


The analysis of the values given in the Table 1 reveals that the induction time is greater in the presence of the extracts of the two plants and potassium citrate (Cit.K) taken as a reference, and passes from the value of 10 second (s) in absence of inhibitor (S.I) to 67s; 217s and 232s in the presence of 2g/L of A.V; Cit.K and P.G respectively. "ti" is more prolonged when the value of the concentration of the three inhibitors is increased. Indeed the latter passes from 30-67s (P <0.05) for A.V, from 105-217s (P <0.005) for the Cit.K and finally from 165 s-232s (P <0.005 for all comparisons vs SI) for the P.G when the concentration of the extract increases from 0.25g/L to 2g/L.


Further, the maximum optical density "ODmax " is a significant parameter which reflects the end of the nucleation and the beginning of the aggregation. On the other hand temporal OD without inhibitor and for different concentrations of the extracts is shown in Figure 1 for A.V and in Figure 2 for P.G that of the Cit.K taken as a reference is shown in Figure 3.


Figure 1
Figure 1. 

Evolution temporal of the variation the optical density (OD) in the presence of extract A.V.




Figure 2
Figure 2. 

Evolution temporal of the variation the optical density (OD) in the presence of extract P.G.




Figure 3
Figure 3. 

Evolution temporal of the variation the optical density (OD) in the presence of extract potassium citrate (Cit.K).




The analysis of the curves exhibits that ODmax reaches the value of 0.280 after 280s for the test without inhibitor and 0.214 to 0.195 (P <0.05) for concentrations of between 0.25 and 2g/L respectively for the extract A.V while ODmax was observed at more delayed times with values from 0.083-0.059 (P <0.001) for the P.G extract and from 0.130-0.056 (P <0.005 for all comparisons vs SI) for Cit.K for concentrations of 0.25-2g/L respectively.


The percents inhibition of nucleation and aggregation are calculated from the slopes of the curves and shown in Table 2 and Table 3 respectively, as well as the correlation coefficients and the coefficients of variation.


The percent inhibition of nucleation is relatively identical with both the extract of P.G and Cit.K. Indeed, they are in the range of 95.80±0.25 to 97.8±0.12% (P <0.05 vs Cit.K) and 95.55±0.35 to 97.37±0.16% for concentrations from 0.25-2g/L, with R >0.97 and CV lower 10% for both. On the other hand, the inhibition percentage of Ammi visnaga 's nucleation are slightly lower with values of 67.94±2.3 to 73.25±0.81% (P <0.005 for all comparisons vs. Cit.K) for concentrations of 0.25-2g/L respectively, with R >0.98 and CV less than 10%.


However, the maximum aggregation inhibition rate was observed in P.G with values of 74.90±1.62% at 83.46±1.34% (P <0.05 vs Cit.K), whereas the aggregation inhibition rate for Cit.K is in the order of 64.29±3.17% at 77.12±2.16% for concentrations of 0.25-2g/L respectively; As for A.V the values are 50.73±4.54 at 59.44±3.3% (P <0.005 for all comparisons vs. Cit.K).


The curves corresponding to the effect of concentration on the rate of nucleation and aggregation inhibition for both plants and the Cit.K solution are shown in Figure 4 and Figure 5, respectively.


Figure 4
Figure 4. 

The effect of the concentration on the rate of nucleation inhibition.




Figure 5
Figure 5. 

The effect of concentration on the rate of aggregation inhibition.




Our results reveal a small variation in the effect of concentrations in the range studied, the inhibition of crystallization both in the nucleation stage with (R =0.50; P <0.001) for A.V, (R =0.95; P <0.001) for P.G and (R =0.76; P <0.001) for Cit.K and in the aggregation stage with (R =0.82; P <0.005) for A.V, (R =0.97; P <0.001) for P.G and (R =0.96; P <0.001) for Cit.K.


The characterization of crystal


The crystals synthesized by a calcium/oxalate concentration ratio of 10 were characterized by Fourier transform infrared spectroscopy (FT-IR). In order to verify their chemical constitution, and the spectrum obtained is represented in Figure 6.


Figure 6
Figure 6. 

Infrared spectrum of the synthesized crystals.




According to Figure 6, multiple bands are observed between 3060 and 3600cm−1 critical to the OH stretching of the water and corresponding to crystals of COM. Although the COD crystals have only one absorption peak at 3445cm−1 [25]. In addition to an off-water bending band at 786cm−1 [24]. However, two absorption bands were observed at 1614cm−1 and 1320cm−1, the first is attributed to the stretching band antisymmetric carbonyl (vas [COO]) and the second is compatible with the metal-carboxylate symmetrical stretch band (vs [COO]), it's two bands attest the formation of COM [26].


Microscopic observation


In order to validate our results obtained by the spectrophotometric model, we followed the presence of crystals and aggregates using an optical microscope. For this reason, the images taken during the nucleation and aggregation phase in the absence and presence of 2g/L of the inhibitors are displayed in Figure 7 and Figure 8 respectively.


Figure 7
Figure 7. 

Crystals of calcium oxalate. A. Absence of inhibitor (S.I). B. Potassium citrate (Cit.K). C. Ammi visnaga (A.V). D. Punica granatum (P.G).




Figure 8
Figure 8. 

Aggregates of calcium oxalate crystals. A. Absence of inhibitor (S.I). B. Potassium citrate (Cit.K). C. Ammi visnaga (A.V). D. Punica granatum (P. G).




Analysis of the images taken during the nucleation phase by the optical microscope and shown in Figure 7 shows that the number of crystals is larger in the case of S.I (A) and becomes less important in the order: the A.V (C), Cit.K (B), and P.G (D). This means that the plant extracts have a significant nucleation inhibiting effect, in particular by the P.G extract, which apparently is even more effective than the Cit.K.


The analysis of the images taken during the aggregation phase shown in Figure 8, shows the presence of larger and more numerous aggregates in the S.I case, the size and number of these decreases for A.V (C), Cit.K (B) and P.G (D). On the other hand, the aggregation rate was determined every 5min up to 60min and the results obtained are shown in Table 4.


According to Table 4, the aggregate rate gradually increases with time and then decreases, except in the case of S.I. which continues to increase; this is probably due to the inhibition of both crystal growth and aggregation [13]. In addition, this percentage is not only decreased with the nature of the inhibitor, but also by adding the dissociation of the aggregates by bioactive molecules of the extracts S.I<A.V<Cit.K<P.G.


Discussion


In this work we tried to valorise and verify the efficacy of these two plants, in order to decrease the crystallization and avoid stone formation by precipitation of calcium oxalate, using a turbidity model based on the measurement of the change in absorbance in a saturated solution as a function of time and which allows us to determine the nucleation phase, the aggregation phase and the induction time. The latter remains a very important parameter, which gives an estimate on the start of the nucleation. Moreover, any prolongation of "ti" is related to the inhibition of nucleation [27]. This extension can not only represent a nucleation delay, but also mean an increased rate of nucleation of very small particles that are undetectable [22].


In this study, the induction time is well prolonged and depends on the concentration. This result clearly explains the delay in crystal formation due to the inhibitory effect of plant extracts. Moreover, the comparison of the ti values for the two plants at different concentrations shows that P.G is more effective and delays the formation of calculations. It is even more effective than the Cit.K used generally in the medical treatment of lithiasis.


In addition, our results are similar to those of Hess et al. [23] and Driouch et al. [28] who found values of 22 and 24s respectively for tests without inhibitor and of 109.9s with Cit.K (3.5mm) for Hess et al. The ti values reported by Abdelmalek et al. [29] on ionic inhibitors such as magnesium Mg2+ and fluoride ion F− are 240s and102 respectively.


The level of inhibition of nucleation and aggregation is very high in the two extracts but with little variation in the different concentrations studied and in particular the P.G extract which remains more effective compared to Cit.K. These results far outweigh the results of work by Saha and Verma [30, 31] who worked on Dolichos biflorus and Bergenia ciliata and found values of the nucleation inhibition rate for the 2g/L concentration equal to 37 and 48% respectively and an aggregation inhibition rate of 48% and 62% respectively,


Indeed, Ammi visnaga and Punica granatum are medicinal plants rich in bioactive molecules such as: saponins, flavonoids, furanochromone and their derivatives: khelline, visnagine and visnadine [19, 32] tannins and alkaloids [33]. The latter probably act through their ability to form soluble chemical species that will reduce the risk of crystallization or adsorption to the surface of crystals due to their numerous anionic charges. In this respect, their fixations on the crystallites lead to an alteration of electrical attraction's phenomena between the atoms situated on the surface of the crystal and the ions present in the solution, and consequently an inhibition of crystal growth and aggregation [13]. Thus the crystals can be evacuated more easily in the urine [13].


On the other hand, the interest of the determination of the aggregation rate lies in the estimation of the lithogenic potential and in the evaluation of the effectiveness of the therapeutic measures intended to reduce the risk of recurrence, adding that the presence of large aggregates creates an aggravated risk of crystal retention in the urinary tree and thus of computational nucleation [34]. Moreover, our results of the microscopic observation and the percentages of aggregation vary in correlation with the calculated inhibition rates and confirm the validity of our spectrophotometric method, and display that the extract of P.G can be used as an effective inhibitor to slow down or even prevent recurrence of the formation crystals of calcium oxalates.


Conclusion


The use of medicinal and aromatic plants for the prevention and treatment of urinary stones, especially in the case of recurrence which has a major problem, remains an alternative choice for medical methods in the lithiasic population. For this purpose, this study is devoted to the experimental verification of the efficacy of the plants: Ammi visnaga and Punica granatum against the crystallization of calcium oxalate. Our results reveal that the inhibition rate of nucleation in the order of 73 and 97.8% as well as the inhibition rate of aggregation is 59.44 and 83.46% respectively for Ammi visnaga and Punica granatum . Our comparison with a medical product which is potassium citrate concludes a comparable efficiency of the extract of Punica granatum and slightly lower for Ammi visnaga . So, microscopic observation confirms the validity and reproducibility of the spectrophotometric method.


Disclosure of interest


The authors declare that they have no competing interest.




Table 1 - Induction time, optical density max, in the presence and absence of inhibitors.
  Concentration g/L  With inhibitor 
Without inhibitor (S.I) 
  A.V  P.G  Cit.K 
Induction time
ti (s) 
0.25  30±2.88a  165±15.21d  105±14.14d  10±2.80 
0.5  52±8.66a  202±11.16d  165±7.07c 
60±9.60b  217±10.60d  188±10.60d 
67±7.63a  232±21.81c  217±10.60d 
Optical density max
(ODmax
0.25  0.214±0.008d  0.083±0.001d  0.130±0.061c  0.280±0.021 
0.5  0.204±0.019a  0.075±0.002d  0.108±0.024c 
0.198±0.002d  0.061±0.008d  0.065±0.008c 
0.195±0.025c  0.059±0.001d  0.056±0.02c 



Légende :
A.V: Ammi visnaga ; P.G: Punica granatum ; Cit.K: potassium citrate; S.I: absence of inhibitor.

[a] 
P <0.05 vs S.I.
[b] 
P <0.01 vs S.I.
[c] 
P <0.005 vs S.I.
[d] 
P <0.001 vs S.I.


Table 2 - Percent inhibition of nucleation in the presence of the extracts of A.V, P.G and the solution of Cit.K.
Concentration g/L  %Inhibition 
R  
CV 
A.V  Cit.K  P.G  A.V  Cit.K  P.G  A.V  Cit.K  P.G 
0.25  67.94±2.3b  95.55±0.35  95.80±0.25a  0.98  0.99  0.99  7.1  6.1 
0.5  72.32±0.18c  96.52±0.01  95.90±0.08c  0.98  0.97  0.99  0.6  0.3  2.1 
72.86±0.93c  97.01±0.006  96.90±0.28a  0.99  0.99  0.97  3.4  0.2  9.1 
73.25±0.81c  97.37±0.16  97.80±0.12a  0.99  0.99  0.99  5.5 



Légende :
A.V: Ammi visnaga ; P.G: Punica granatum ; Cit.K: potassium citrate; R: correlation coefficient.

[a] 
P <0.05 vs potassium citrate (Cit.K).
[b] 
P <0.005 vs potassium citrate (Cit.K).
[c] 
P <0.001 vs potassium citrate (Cit.K).


Table 3 - Percent inhibition of aggregation in the presence of the extracts of Ammi visnaga (A.V), Punica granatum (P.G) and the solution of potassium citrate (Cit.K).
Concentration g/l  %Inhibition 
R  
CV 
A.V  Cit.K  P.G  A.V  Cit.K.  P.G  A.V  Cit.K  P.G 
0.25  50.73±4.54a  64.29±3.17  74.90±1.62a  0.97  0.98  0.98  9.2  8.8  6.4 
0.5  51.99±3.34b  68.25±2.4  76.74±1.21a  0.98  0.97  0.97  6.9  7.9  5.2 
58.12±3.63a  69.70±2.85  77.60±1.96a  0.97  0.95  0.97  8.6  9.4  8.7 
59.44±3.31c  77.12±2.16  83.46±1.34a  0.98  0.95  0.95  8.1  9.4 



Légende :
A.V: Ammi visnaga ; P.G: Punica granatum ; Cit.K: potassium citrate; R: correlation coefficient.

[a] 
P <0.05 vs potassium citrate (Cit.K).
[b] 
P <0.01 vs potassium citrate (Cit.K).
[c] 
P <0.005 vs potassium citrate (Cit.K).


Table 4 - Percent of agrégats.
Time (min)  Percent of agrégats (%) 
S.I  A.V  P.G  Cit.K 
9.29±1.66  10.69±4.26  2.05±1.22d  0.51±1.02d 
10  21.46±2.19  14.51±4.52a  2.08±1.5d  5.66±2.33d 
15  27.95±5.08  13.04±4.14c  3.2±1.72d  7.84±3.03d 
20  29.29±5.79  14.95±3.16c  10.23±3.45d  10.94±2.32d 
25  27.34±4.65  16.49±3.92d  12.88±4.02d  9.21±3.89d 
30  30.18±6.01  16.64±3.93c  12.87±4.42c  12.87±3.82d 
35  30.46±4.6  17.48±4.37c  13.69±5.2c  14.61±2.8d 
40  31.40±4.11  22.18±5.31a  8.62±4.52d  23.60±3.36c 
45  27.47±7.53  20.83±4.06b  11.96±4.79d  22.07±3.12c 
50  32.77±5.19  22.76±5.82a  16.90±3.85b  19.29±5.05a 
55  32.70±5.23  22.02±4.56b  16.97±3.76b  19.52±3.42c 
60  31.75±3.17  18.46±4.82d  17.36±5.43c  17.64±3.72d 



Légende :
A.V: Ammi visnaga ; P.G: Punica granatum ; Cit.K: potassium citrate; S.I: absence of inhibitor.

[a] 
P <0.05 vs control.
[b] 
P <0.01 vs control.
[c] 
P <0.005 vs control.
[d] 
P <0.001 vs control.


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