INTRODUCTION:

Kidney stone disease is a condition in which there are organic crystalline and matrix components in the calyx or renal pelvis¹. Many causes of kidney stones include increased body size. Urine pH is an essential factor in the formation of kidney stones². Prevention of kidney stone recurrence is mostly specific for any stone type (e.g., calcium oxalate, calcium phosphate, cystine, and uric acid stones). However, even when stones cannot be taken, urine pH and 24-hour urine assessment provide information about stone-forming factors that can guide the prevention of kidney stones³.

The Protein metabolism product are phosphate and oxalate, sodium uric acid, magnesium, and calcium. Ammonium phosphate and calcium phosphate predisposes to stone formation. The Cause of kidney stones can be occur of dietary habits, different nutritional status, and environmental factors such as humidity, temperature and other factors, vitamin A and vitamin B6 deficiency and hyperthyroidism, urinary tract infections, heredity⁴.

Treatment of kidney stones can be performed using various drugs through oral administration. However, a long-term use of the drugs is limited by side effects and lack of patient tolerance. Some drugs commonly used such as a thiazide diuretic group for those whose calcium content is increased in the urine, potassium citrate is useful for those who have low or average potassium citrate content and the formation of cystine and uric acid stones. Allopurinol can be used if the calcium oxalate stone-forming factor is caused by increasing uric acid in urine and normal urine calcium. It is not recommended to give allopurinol regularly to all uric acid stone formers⁵. In addition, surgery can make a large incision in the body but the risk of infection of post-operative wounds is quite high or surgery without hospitalization, such as Extracorporeal Shockwave Lithotripsy (ESWL) with limited treatment only for certain types and sizes of stones, so that each patient has different results.

The large cost of undergoing therapy is also a problem for most people⁶. Many alternative treatment options for kidney stone disease have arisen, one of which is by utilizing natural ingredients. The use of natural ingredients is more considered because it has smaller side effects, cheap and easy to obtain. One of them is apigenin which is derived from the celery plant (Apium graveolens L). Based on previous research, apigenin with a dose of 4.8mg/kg body weight against wistar rats was able to shed kidney stones. Many studies have been successfully using natural substances that have an effect as urolithiasis namely Lithout tablets are effective in reducing the crystallization of calcium oxalate in solution⁷, leaves extract of costus igneus has anti-urolithic activity⁸, leaves and seeds of Macrotyloma uniflorum can removal of kidney stones⁹, root bark can presence of calcium oxalate cristals of raphide bundles in the axial parenchyma¹⁰, aqueous extract of roots and seeds of Plectranthus tomentora (500 and 1000mg/kg) on hyperoxaluria rats shows the significant activity in decreasing kidney stones and serum levels (phosphorous, calcium, creatinine, urea)¹¹. In this study, the preparation was modified by the addition of polymers using hot-melt extrusion (HME) preparation techniques to increase the solubility of apigenin in the body, hence the effectiveness of apigenin as an anticalculi will increase.

HME is an essential technology for drug delivery applications in pharmaceutical research and manufacturing because of its process automation and low-scale upgrade properties, which reduce labor costs and capital investment¹² and more stable because it does not need solvents¹³. HME can improve solubility of a BCS Class II drug¹⁴. The use of Soluplus^® and Kollidon^® VA 64 polymers was based on the ability of both polymers in terms of increasing solubilization. It is suitable for HME because it has thermoplastic properties, appropriate glass temperature, low toxicity, and has a high degradation temperature¹⁵. One way to prepare drugs with HME method is solid dispersion, where many solid dispersions have succeeded in increasing solubility¹⁶.

The polymer is then passed through the die opening to obtain extrudates, continued processed into granules, tablets or beads. HME is a continuous, simple, and efficient process. No solvents or water are needed because liquid polymers can function as thermal binders¹⁷.

This study was designed to evaluate the ability of antinephrolithiasis activity of HME formulas of apigenin with Soluplus^® and Kollidon^® VA 64 polymers in rats. Soluplus^® and Kollidon^® VA 64 polymers are good polymer to increase solubility of BCS class II drug like lumefantrine¹⁸.

MATERIAL AND METHODS:

Chemicals and reagents:

The ingredients used were apigenin obtained from Hefei Dielegance Biotechnology Co., Ltd. China (98.21% purity), Ethylene glycol 0.75%, formaldehyde 10%, Ammonium chloride 2%, Pulvis Gummi Arabici (PGA), Formaldehyde 10%, soluplus^®, and Kollidon^® VA 64 (BASF-Megasetia), hematoxylin, eosin, ethanol 96%, liquid paraffin, and xylene.

Instrumentation:

The tools used were the metabolic cage, syringe, a set of surgical instruments, analytical scales, atomic absorption spectrophotometer (AAS), twin-screw extruder (Teach-Line ZK25T) in Polytechnique STMI Jakarta, microscope Olympus CX21.

Experimental Animals:

Healthy male rats of Wistar strain were used and fed 30g/rat/day and acclimatized for seven days before treatment. Rats received good lighting in the Laboratory of Pharmacology, Padjadjaran University. All procedures were recorded and have been approved by the research ethics commission with number 173/UN6/ KEP/EC/2019. Rats were divided to seven groups consisting of 4 mice per group. The groups consisted of normal, negative control, positive control, apigenin without HME, apigenin with HME soluplus^® polymer, and apigenin with HME Kollidon^® VA 64 polymer. All groups, except the normal group, were given 0.75% ethylene glycol and 2% ammonium chloride for seven consecutive days, and the 8th day treated according to their groups until the 14th day. Up to 24 hours, the rat urine was collected in a metabolic cage to check the calcium content in urine using AAS. Rats were sacrificed on day 15 for their kidneys to be taken. The right kidney was used for histological testing, and the left kidney was for further testing using AAS.

The division of groups is as follows:

Group 1: Normal control, mice were given PGA 2% from the first to the 14^th day.

Induction of kidney stones was given days 1 to 7 for groups 2 to 7, namely 0.75% v/v ethylene glycol and 2% w/v ammonium chloride and provided ad libitum 30g/ day feed.

Group 2: Negative control, mice were administered Soluplus^® polymers

Group 3: Negative control, mice were administered Kollidon^® VA 64 polymers

Group 4: Mice were administered apigenin without HME at a dose of 4.8mg/kg.

Group 5: Mice were administered apigenin with HME Soluplus^® polymer at a dose of 4.8mg/kg.

Group 6: Mice were administered apigenin with HME Kollidon^® VA 64 polymer at a dose of 4.8mg/kg.

Assessment of Antiurolithiatic:

Collection and analysis of urine:

All animals were stored in the metabolic cage and 24-hour urine samples were collected on the 15th day. One drop of concentrated HCl was added to the urine before storage at 4°C. Urine volume and pH were determined. The urine was analyzed for calcium content. Samples were also carried out microscopically for calcium oxalate crystals, and its confirmation of urolithiasis. The urine were centrifuged at 3000rpm for 7 minutes to unite the crystals, and observed under a simple microscope at 40X. The shape and number of crystals were found¹⁹.

Histology Test²⁰

The kidney organs were fixed in a 10% neutral formalin solution. After dehydration in multilevel ethanol sequences (70, 80, 90, 96%), tissue samples were cleaned in xylene and planted in paraffin. The 5μm tissue sections were stained with hematoxylin-eosin and each kidney slide was examined for necrosis and the presence of calcium oxalate crystals. Tissue slices were photographed using an optical microscope at 10X magnification.

Statistic analysis:

Data were analysed by analysis of Variants (ANOVA) and posthoc tests for parametric and a Kruskal Wallis test for non-parametric. If the value of p is <0.05, there is a significant difference between groups.

RESULTS:

Effect of Apigenin on Kidney Stones:

The shedding effect of apigenin on kidney stones is shown in Table 1. In rats treated with ammonium chloride 2% and ethylene glycol 0,75% as stone formation inducers (Groups II and III), high calcium oxalate (CaOx) crystals with a characteristic rectangular shape were observed in the urine samples. Crystals were found in all treatment groups. Apigenin given either without HME or with HME had good capacity of shedding kidney stones. However, apigenin with the HME Soluplus® (group V) had a higher capacity when compared to that without HME (group IV) and that with HME Kollidon^® VA 64 (group VI).

Table 1: Microscopic Results Of Calcium Oxalate Crystals in Urine

Group	Treatment	The average amount of calcium oxalate crystals
Group I (normal groups)	PGA (Pulvis gummi arabici) 2%	6,4 ± 1,34
Group II (negative groups 1)	Soluplus^® polymerand ammonium chloride 2% w/v and ethylene glycol 0,75% v/v	28,2 ± 3,96
Group III (negative groups 2)	Kollidon^® VA 64 Polymer and ammonium chloride 2% w/v and ethylene glycol 0,75% v/v	25,8 ± 1,92
Group IV	Ammonium chloride 2% w/v and ethylene glycol 0,75% v/v and apigenin at a dose of 4,8mg/kg	16,4 ± 2,30
Group V	Ammonium chloride 2% w/v and ethylene glycol 0,75% v/v and apigenin with HME Soluplus^® polymer at a dose of 4,8mg/kg	12,6 ± 1,67
Group VI	ammonium chloride 2% w/v and ethylene glycol 0,75% v/v and apigenin with HME Kollidon^® VA 64 polymer at a dose of 4,8mg/kg	18,4 ± 1,81

Effect of Apigenin  on Urine  pH and Urine Volume:

The urine pH and volume in rats after  treatment with apigenin are presented in Table 2.  Apigenin generally increased the urine pH as shown in groups V, VI, and VII, and apigenin with HME Soluplus^® polymers provided a higher effect. In other hand,  its effect on the urine volume looked decreasing the volume, except apigenin with HME Soluplus^® did not give any effect.

Table 2 Effects of apigenin on urine pH and urine volume

Group	Urine pH	Urine Volume
Group I	7,2±0,04	17,18±8,7
Group II	7,0 ± 0,025	15 ±7,3
Group III	8,7 ±0,33	20,62±5,9
Group IV	7,7 ±0,005	14 ±4,9
Group V	8,8 ±0,05	11,8 ±8,5
Group VI	8,85 ±0,41	17,18±11,29
Group VII	7,9 ± 0,66	12,18 ± 3,3

Values are mean ±SD for sex rats in each group (n=4). P<0.005 values are significantly different compared to the control group.

Effect of Apigenin on Kidney Weight and Calcium Levels in Kidney and Urine:

Table 3 or Figure 1 shows a ratio of kidney weight and amount of calcium in the kidney and urine. A comparison of the ratio of kidney weight to the bodyweight of rats did not show significant differences among groups. The amount of calcium in the kidneys showed that apigenin with HME Soluplus^® polymers significantly decreased the calcium level in the urine when compared to its negative control (group II). This effect was higher than the effect of apigenin with Kollidon® VA 64 polymers. Furthermore, the urine calcium level in rats treated with apigenin with HME Soluplus^® polymers was significantly increased as compared to its negative control (group II). The calculi parameters can be seen from the amount of calcium in the kidneys close to normal and less than the negative control.

Table 3 Ratio of kidney weight and amount of calcium in the kidney

Group	Ratio of Kidney Weight (g/100g)	Calcium level in the kidney (ppm)	Calcium level in urine (ppm)
Group I	0,72±0,05	151,19 ± 22,77	75,21 ± 77,81
Group II	0,76 ± 0,12	259,49 ±71	167,98 ± 144,01
Group III	0,67 ±0,05	535,77±170,84	10,19 ± 5,7
Group IV	0,76 ±0,10	219,95±102,44	8,57 ± 4,52
Group V	0,80 ±0,09	176,14±21,43	281,472±127,28
Group VI	0,68 ± 0,02	408,17 ± 29,51	9,77 ± 2,77

Values are mean ± SD for sex rats in each group (n=4). p <0.005 values are significantly different compared to the control group.

The following is a graphical description of the amount of calcium in the kidneys and urine for various groups:

Figure 1: Calcium level in kidney

Group 5 were apigenin groups with HME Soluplus^® which approached normal control but they were still under positive control. The amount of calcium decreased compared to negative controls respectively are positive control groups, the HME Soluplus^® group, the apigenin group without HME and the HME with Kollidon® VA 64.

Figure 2: Calcium level in urine

Histology Test

Histopathological testing of the kidney also supports the above results. The normal group showed a whole picture in the medullary and papillary tubules. The medulla and cortex are clearly visible. The negative control group of both Soluplus^® and HME Kollidon^® VA 64 polymers treated with ethylene glycol 0.75% and ammonium chloride 2% showed hydropic, fat, and cell necrosis degeneration and non-specific hemorrhagic due to kidney stone deposits showed. Figure 5, apigenin with Soluplus^®, shows histology approaches normal. This is in line with histopathological tests for administration of Macrotyloma uniflorum water extracts as anticalculi. The histopathological examination preventive and curative control groups showed the presence of calcium oxalate crystals in Lumina tubules accompanied by tubular congestion, edema and extensive bleeding cast formation. ²¹

DISCUSSION:

In this study, the formation of kidney stones was induced using ammonium chloride 2% and ethylene glycol 0.75%. This combination of inducing agents can produce crystalline precipitation of calcium oxalate in all rat kidneys (n = 4) within 7 days²². The formation of calcium oxalate from ethylen glycol is due to its metabolite. Ethylen glycol is metabolized in the body to be glycolic acid, which is responsible for acidosis, and oxalic acid, which induces the formation of calcium oxalate. The oxalic acid will settle in the form of oxalic acid monohydrate and stored in several tissues, especially the kidneys. Calcium ions will bind with dissolved oxalic acid to form insoluble calcium oxalate crystals²³. The use of ammonium chloride 2% is intended to increase the level of calcium oxalate²⁴.

In this study, calcium levels in the kidney of the negative control group which was administered inducing agents were increased, indicating the success of induction. The administration of apigenin, either epigenin without HME or with HME, decreased the elevated calcium concentration when compared to the negative control. This means that apigenin had shedding activity against kidney calcium oxalate stones. This activity is probably due to the binding of calcium in the stones by the hydroxyl group of apigenin to form a calcium-apigenin complex, which is more water-soluble. In addition, the alteration in the urine pH by apigenin from 7.2 to 7.9 - 8.85 indicated that the alkalization may influence the sheding capacity of apigenin¹⁹.

Figure 3: Ca Apigenin Complex Formation Reaction

Among the three formulas, the apigenin formulas with HME had better sheding activity against calcium oxalate stones than that without HME. This may be due to the difference of solubility among them, in which the apigenin with HME had higher solubility. Moreover, the apigenin HME with Soluplus® polymer was better than that with Kollidon® polymer.

The histopathological study supports this result. The kidney portions of the negative control mice showed extensive hypertrophy and inflammation in most of the kidneys, and those of mice treated with the apigenin formulas with HME changed the normal architecture but no hemorrhage was observed.

Apigenin formulation of HME application with soluplus® polymer succeeded in providing good sheding effects against the kidney stones. The HME is a method that can increase solubility of apigenin, hence will increase oral absorption and the effectiveness of apigenin as an anticalculi is expected to be increased.

CONCLUSION:

This study concludes that the apigenin formula produced by HME with Soluplus^® and Kollidon^® VA 64 polymers has the potential as anticalculi, but that with the HME Soluplus^®polymers is better than Kollidon^®.

ACKNOWLEDGEMENT:

Indonesian Ministry of Research, Technology, and Higher Education supported this research through a 2018 Doctoral Dissertation Research (PDD) grant and STMI Jakarta polytechnic supported for the use of twin-screw extruder tools.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interests.

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Received on 01.07.2020 Modified on 05.08.2020

Research J. Pharm. and Tech. 2021; 14(6):2931-2936.

DOI: 10.52711/0974-360X.2021.00514