Urolithiasis

ANTIUROLITHIASIC EFFECT OF A PLANT FORMULATION OF HERNIARIA GLABRA, AGROPYRON REPENS, EQUISETUM ARVENSE AND SAMBUCUS NIGRA (HERBENSURINA®) IN THE PREVENTION OF EXPERIMENTALLY INDUCED NEPHROLITHIASIS IN RATS.

Anna Crescenti1, Francesc Puiggròs1, Arnau Colomé2, Josep Antón Poch2, Antoni Caimari1, Josep Maria del Bas1, Noemí Boqué1 and Lluís Arola3,4.

11Grup de Recerca en Nutrició i Salut (GRNS). Centre Tecnològic de Nutrició i Salut (CTNS), TECNIO, CEICS, Reus, Spain.2Laboratorios Deiters S.L. Badalona, Barcelona, Spain.3Centre Tecnològic de Nutrició i Salut (CTNS), TECNIO, CEICS, Reus, Spain.4Departament de Bioquímica i Biotecnologia, Nutrigenomics Research Group, Universitat Rovira i Virgili, Tarragona, Spain.

@ CORRESPONDENCE

Anna Crescenti SavallCentre Tecnològic de Nutrició i Salut (CTNS), TECNIO, CEICSAvenida Universitat, 143204 Reus (Spain)

anna.crescenti@ctns.cat

Accepted for publication: November 4th, 2015

Arch. Esp. Urol. 2015; 68 (10): 739-749

Summary.- OBJECTIVE: To determine the effect of a botanical formulation of Herniaria glabra, Agropyron repens, Equisetum arvense, and Sambucus nigra as a preventive agent in an experimentally induced nefrolithiasis model in rats.

METHODS: Six groups of six Wistar male rats each were induced for nefrolithiasis by treatment with 0.75% ethylene glycol (EG) and 1% ammonium chloride for three days and then EG only for 15 days. One group was treated with placebo (control group) and the other groups (treated groups) were treated with

30 mg/Kg, 60 mg/Kg, 125 mg/Kg, 250 mg/Kg and 500 mg/Kg of the plant extract formulation (PEF). 24-h urine and water samples were collected one day before EG administration and at 7, 13 and 18 days to determine diuresis, crystalluria and urine biochemistry. The kidneys were removed for histological analysis. The phytochemical characterization of PEF and each of its component plant extracts was performed using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

RESULTS: Animals treated with 125 mg/Kg of the PEF had statistically significantly lower calcium oxalate crystals deposits content compared to the control group. All PEF doses statistically significantly decreased the number of microcalcifications compared to the control group. Furthermore, the number of kidneys affected by subcapsular fibrosis was statistically significantly higher in control group than in treated groups with the PEF. The diuresis of the 125 mg/Kg and 500 mg/Kg PEF-treated groups was statistically significantly higher than that of the control group. A phytochemical analysis demonstrated the presence of flavonoids, dicarboxylic acids and saponins.

CONCLUSION: Treatment with PEF prevents deposits of calcium oxalate crystals formation and of microcalcifications in the kidney, and reduces the risk of fibrosis subcapsular. 125 mg/Kg of PEF is the dose that has a greater effect on the studied parameters.

A. Crescenti, F. Puiggròs, A. Colomé, et al.

INTRODUCTION

Kidney stone formation or nefrolithiasis is a common disorder with a worldwide prevalence greater than 10% and increasing annually. Approximately 80% of all kidney stones are predominately composed of calcium oxalate (CaOx) (1-2). One of the major

740

problems with kidney stones is the high rate of recurrence, which is approximately 50% in 5-10 years and 75% in 20 years. Once afflicted, the subsequent relapse rate is increased, and the recurrence interval is shortened (2-3).

Although the treatment and prevention of kidney stones have considerably improved in the last two decades through a combination of dietary procedures, surgical treatments and medicaments, the side effects of these methods and recurrence remain challenges to overcome. Therefore, it is worthwhile to search for alternative strategies for the prevention and treatment of nefrolithiasis (1). Plants have been used throughout the ages to treat urinary stones (4), and most of them have been demonstrated as be useful although the rationale behind their use is not well established (1,4). In the present study it has been evaluated for the first time in an experimentally induced nefrolithiasis model the potential beneficial effect in preventing nephrolithiasis of a plant formulation of Herniaria glabra, Agropyron repens, Equisetum arvense, and Sambucus nigra extracts that has been used in folk treatment of diverse affections of the urinaty system, including renal lithiasis. Furthermore, it has been assessed different doses of that plant extract formulation (PEF) to determine whether the currently recommended dose is suitable for the studied effect.

Herniaria glabra, Agropyron repens, Equisetum arvense and Sambucus nigra have been traditionally used for their diuretic, lithotriptic, antihypertensive, depurative, bacteriostatic and remineralizing effects, as well as to increase the elasticity of the urinary tissues (5-6). Furthermore, several studies have demonstrated that these plants have activities that are described as mechanisms of action of the antiurolithiatic effect of plants (5), such us antioxidant, diuretic and antimicrobial effect (7-12).

The aim of the present study was to determine whether a four PEF (Herniaria glabra, Agropyron repens, Equisetum arvense, and Sambucus nigra) prevents renal lithiasis in an experimentally induced nefrolithiasis model in rats. The specific objectives of the suty were: 1) To determine whether the PEF decrease the number of renal CaOx deposits; 2) To determine whether the PEF reduces the number of renal microcalcifications; 3) To determine wheter the PEF descreases the renal pielonefritis and subcapsular fibrosis; 4) To determine whether the PEF has a diuretic effect; 5) To determine whether the PEF changes urinay cristalluria and biochemistry; 6) To determine the effective dose of the PEF; 7) To determine the compunds in the PEF and each of its component plant extracts with described activities that may be beneficial against nephrolithiasis.

Resumen.- OBJETIVO: Determinar el efecto preven-tivo sobre la litiasis renal de una formulación botánica formada por Herniaria glabra, Agropyron repens, Equi-setum arvense y Sambucus nigra en un modelo experi-mental de nefrolitiasis en ratas.

MÉTODOS: Seis grupos de animales con seis ratas Wistar macho cada uno fueron inducidos a nefrolitiasis mediante el tratamiento con etilenglicol (EG) 0,75% y cloruro de amonio 1% durante tres días y posteriormen-te con EG durante 15 días más. Un grupo fue tratado con placebo (grupo control) y los otros grupos (grupos tratados) fueron tratados con 30 mg/Kg, 60 mg/Kg, 125 mg/Kg, 250 mg/kg y 500 mg/Kg de la formula-ción de extractos de plantas (FEP). Se midió el volumen de agua ingerida y de orina excretada durante 24 h en diferentes días del experimento y se determinó la diuresis, cristaluria y bioquímica. Se realizó el análisis histológico del riñón. La caracterización fitoquímica de la FEP se realizó mediante técnicas cromatográficas.

RESULTADO: La cantidad de depósitos de cristales de oxalato de calcio (OxCa) de los animales tratados con 125 mg/Kg de la FEP y el número de microcalcifica-ciones en todos los grupos tratados con la FEP fue menor comparado con el grupo control, siendo las diferencias estadísticamente significativas (d. e. s.). La presencia de fibrosis subcapsular fue mayor en el grupo control que en los grupos tratados (d. e. s.). La diuresis de los grupos tratados con 125 mg/Kg y 500 mg/Kg de la FEP fue mayor que la del grupo control (d. e. s.). El aná-lisis fitoquímico demostró la presencia de flavonoides, ácidos dicarboxílicos y saponinas.

CONCLUSIONES: La administración de la FEP previe-ne la formación de cristales de OxCa y de microcalci-ficaciones en el riñón y disminuye el riesgo de fibrosis subcapsular renal. La dosis de 125 mg/Kg de la FEP es la que presenta un mayor efecto sobre los parámetros estudiados.

Palabras clave: Extracto de plantas. Oxalato de calcio. Nefrolitiasis. Cálculos renales. Ratas.

Keywords: Plant extract. Calcium oxalate. Nefro-lithiasis. Kidney calculi. Rats.

ANTIUROLITHIASIC EFFECT OF A PLANT FORMULATION OF HERNIARIA GLABRA, AGROPYRON REPENS, EQUISETUM ARVENSE....

MATERIALS AND METHODS

This is a study to determine the preventive effect of PEF using five diferent doses of the PEF and 36 male Wistar rats induced to nephrolithiasis with ethylene glycol EG and ammonium chloride.

Plant material

Herbensurina® tablets, a botanical product that is traditionally used to manage urolithiasis in humans, were supplied by Laboratorios Deiters S.L. Each tablet (890 mg) of the PEF contains four medicinal plant extracts: Herniaria glabra (470 mg), Agropyron repens (235 mg), Equisetum arvense (56.4 mg) and Sambucus nigra (47 mg).

Animals and treatments

The animal protocol that was followed in this study was reviewed and approved by the Animal Ethics Committee of the University Rovira i Virgili (Tarragona, Spain) (4827) and was carried out in accordance to the European Parliament and of the Council Directive of 22 September 2010 on the protection of animals used for scientific purposes (2010/63/UE).

36 male Wistar rats weighing 250 to 275g (Harlan Laboratories, Barcelona, Spain) were housed singly at 22°C with a light/dark period of 12 h (lights on at 09:00) and throughout the study were fed a chow diet and had free access to food and tap water. The rats were acclimated for one week in metabolic cages before starting the experiment. After acclimatization, the animals were randomly divided into six groups of six rats each depending on the treatment that was received. To induce CaOx crystal formation, the animals were exposed to EG in the drinking water to a final concentration of 0.75% with 1% ammonium chloride for three days. The rats were then given only 0.75% EG for 15 days (13-14). One group (control group) was given the inducers of CaOx crystals and treated with the vehicle (low-fat condensed milk). The other groups (treated groups) were given the inducers of CaOx crystals and treated with 30 mg/Kg,60 mg/Kg, 125 mg/Kg, 250 mg/Kg and 500 mg/Kg of body weight of the PEF. The dose of PEF that was administered to the rats was equivalent to the human dose and was calculated from the amount in one Herbensurina® tablet using the formulas that were proposed by Reagan-Shaw S. et al. (15). The tested doses were the equivalent to the human dose (60 mg/Kg of body weight) and four additional doses (30 mg/Kg, 125 mg/Kg, 250 mg/Kg and 500 mg/Kg of body weight). All of the doses of

plant extracts were administered daily with a syringe and dissolved in low-fat condensed milk.

The animals were weighed weekly. 24-h water volume intake and urinary volume were determined one day before EG administration and then on days 7, 13 and 18 of treatment. At the end of the experimental study, blood samples were collected and animals were humanely euthanized.

Histological procedures and kidney microscopy

Immediately after sacrifice the kidneys were removed and weighed, and the right kidney was fixed in 4% formaldehyde for histological analysis by microscopy (Nikon Eclipse Ti Series, Japan). The number of renal tubules with some precipitate and the number of microcalcifications was randomly counted in 10 microscopic fields of 777.04 µmx621.02 µm. The amount of precipitate in the renal tubules was considered by multiplying by a factor of 1, 1.5 or 2 the number of affected tubules depending on whether the ratio of the tubules that were affected by precipitates occupying more than 50% of the surveyed area was between 0-39%, between 40-69% or between 70-100%, respectively.

Furthermore, two parameters that were related to the grade of degeneration of the kidney, pyelonephritis and subcapsular fibrosis, were also evaluated (16).

Microscopy and biochemistry of urine

The levels of optical density (OD), calcium (Ref. ab102505, Abcam, Cambridge, UK), oxalate (Ref. 591D, Trinity Biotech, Wicklow, Ireland) and citrate (Ref. ab83396, Abcam, Cambridge, UK) were determined in the urine at 18 days.

2mL of each urine specimen were filtered (0.22µm) and the crystal number, morphology and size were examined in four microscopic fields at a magnification of 3000x using a JEOL JSM-6400 scanning electron microscope (JEOL USA, MA, USA). Two different types of CaOx crystals were considered: CaOx monohydrate (COM) and CaOx dihydrate (COD). A semi-quantitative analysis was performed using a score from 1 to 3 according to the crystal size, from 1 to 4 according to the number of crystals counted, and from 1 to 3 according to the number of COM.

Biochemical assay in serum

Serum samples were used to determine the glutamic oxaloacetic transaminase (GOT) enzyme

741

A. Crescenti, F. Puiggròs, A. Colomé, et al.

(Ref. 990823, QCA, Barcelona, Spain) and glutamic pyruvic transaminase (GPT) (Ref. 998437, QCA, Barcelona, Spain) levels.

Phytochemical characterization

The presence of different phytochemical groups was screened in the aqueous plant extracts of Herniaria glabra, Agropyron repens, Equisetum arvense, Sambucus nigra and PEF using Gas chromatography-Mass spectrometry (GC-MS) and Liquid Chromatography-Mass spectrometry (LC-MS). GC-MS analysis was performed in a 7890A Series gas chromatograph that was coupled to a 7000 Series GCQqQ/MS and the chromatographic column J&W Scientific HP5-MS (30x0.25 mm i.d., 0.25 µm film) (all from Agilent Technologies). LC-MS analysis was performed in a 1290 Series UHPLC system that was coupled to a 6550 i-Funnel Q-TOF/MS, both from Agilent Technologies, and the chromatographic column Acquity UPLC HSS T3 C18, 1.8 µm, 2.1x100 mm from Waters. Compound identification was performed using Agilent Mass Hunter Qualitative Analysis software.

Statistical analysis

The results are expressed as means ± standard deviation (SD). The differences between the groups were analysed using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, repeated measures ANOVA followed by the DMS multiple comparison test or Kruskal-Wallis and U of Mann Whitney non-parametric tests, for quantitative variables. Qualitative variables were analysed with Chi-square test. The statistical package that was used was SPSS version 19.00. P values less than 0.05 were considered significant.

RESULTS

Effect of PEF treatment on kidney histology (CaOx crystals deposits, microcalcifications, pyelonephritis and subcapsular fibrosis)

Figure 1 shows the number of CaOx crystals deposits of the kidneys in the untreated and PEF-treated groups. Animals treated with 125 mg/Kg of the PEF had statistically significantly lower CaOx crystals deposits content compared to the control group (p=0.031). Figure 2 shows a representative example of kidney calculi from a control (Figure 2A) and a 125 mg/Kg PEF-treated animal (Figure 2B).

Table I shows the number of renal microcalcifications in the untreated and PEF-treated groups. All PEF doses statistically significantly decreased the number of microcalcifications compared to the control group (p=0.01 for 30 mg/Kg, 60 mg/Kg, 125 mg/Kg and 250 mg/Kg of the PEF; p=0.014 for 500 mg/Kg of the PEF). Figure 2C shows a representative example of microcalcifications from a control animal.

In the control group and in the groups that were treated with 30 mg/Kg and 60 mg/Kg PEF, all of animals except for one were affected by pyelonephritis, whereas in the 125 mg/Kg, 250 mg/Kg and 500 mg/Kg PEF-treated groups, only four animals were affected by pyelonephritis. However, non statistically significant differences were observed between untreated and PEF-treated groups. Regarding subcapsular fibrosis, all of the animals in the control group were affected, whereas only half of the animals were affected in the PEF-treated groups, with an estimated risk of 2 (CI: 0.899-4,452; p=0.046). Figure 2 shows a representative example of kidney degenerative parameters observed, pyelonephritis (Figure 2D) and subcapsular fibrosis (Figure 2E).

Effect of PEF treatment on urine parameters (diuretic effect, urine crystalluria and biochemistry)

Table II shows that the 24-h volumes of water consumption and urine were higher in the rats that were treated with 125 mg/Kg and 500 mg/Kg PEF at 7 and 13 days, although no statistically significant difference compared with those of the untreated group was observed due to the high variability between animals. However, the 24-h relative urine volume was statistically significantly higher at 7 and 13 days in the rats that were treated with 125 mg/Kg (p=0.043 and 0.046, respectively) and 500 mg/Kg (p=0.036 and 0.046, respectively) PEF.

742

Figura 1. Number of total CaOx crystals deposits in the control group and in the five groups that

were treated with different doses of the plant extract formulation (PEF). The data are mean ± SD (n = 6). *p<0.05 (ANOVA followed by Dunnett’s multiple

comparison test). CaOx: calcium oxalate.

ANTIUROLITHIASIC EFFECT OF A PLANT FORMULATION OF HERNIARIA GLABRA, AGROPYRON REPENS, EQUISETUM ARVENSE....

The analysis of crystalluria revealed that there were no differences in the number or size of urine crystals between any of the groups that were treated with PEF and the control group (Table I).

Furthermore, althought the number of COM crystals in 125 mg/Kg PEF-treated group was smaller compared with that of the control group, no statistically significant differences were observed between both groups. Figure 3 shows a representative example of crystals as observed in the control group and in the 125 mg/Kg PEF-treated group.

The urinary biochemical parameters that were obtained at the end of the experiment in each group are shown in Table III. No statistically significant differences were observed in the urinary OD and in the oxalate, calcium or citrate excretion levels between the PEF-treated groups and the control group.

Effect of PEF treatment on the body and kidney weight and on the plasma parameters in rats

As shown in Table IV, there was no statistically significant difference in the body weight or gain in body weight after 18 days among the control and PEF-treated groups. Furthermore, the relative weight of both kidneys was similar among all of the groups.Regarding the plasma GOT and GPT determinations, no statistically significant differences were found between the control and treated groups.

Phytochemical characterization

Table V shows results of the phytochemical analysis of the PEF and each of its component plant extracts.

743

Figura 2. Micrographs of paraffin-stained kidney sections. Representative example of kidney calculi from an untreated rat (A) and a 125 mg/Kg PEF-treated rat (B). Representative example of kidney microcalcifications from an

untreated rat (C). Example of the observed degenerative structures: pyelonephritis (D) and fibrosis subcapsular (E). Arrows indicate microcalcifications (2C), pyelonephritis (2D) and fibrosis subcapsular (2E).

PEF: Plant extract formulation. All magnifications are x10.

A. Crescenti, F. Puiggròs, A. Colomé, et al.744

Figura 3. Micrographs of urine samples. Representative example of urine crystals that were observed in an untreated rat (A) and a 125 mg/Kg PEF-treated rat (B). PEF: Plant extract formulation.

Examples of COM (Thin arrow) and COD (Thick arrow) crystals are indicated.3,000x magnification.

Table I. Microcalcifications number and number and size of urinary crystals.

Group

Control

30 mg/Kg

60 mg/Kg

125 mg/Kg

250 mg/Kg

500 mg/Kg

N

microcalcifications

28.75

(9.19)

6.83*

(1.35)

5.67*

(1.02)

3.50*

(0.99)

7.33*

(0.88)

4.50*

(1.80)

N

OxCa crystals

2.13

(0.31)

1.88

(0.37)

2.35

(0.19)

1.83

(0.31)

2.21

(0.40)

2.17

(0.27)

Size OxCa

crystals

3.00

(0.27)

2.92

(0.33)

2.88

(0.13)

2.75

(0.26)

2.92

(0.27)

3.42

(0.19)

N

COM

2.05

(0.32)

2.04

(0.50)

2.04

(0.44)

1.38

(0.29)

2.15

(0.51)

2.21

(0.39)

N: Number; OxCa: Calcium oxalate; COM: Calcium oxalate monohydrate crystals.Data are expressed as mean (standard deviation) (n=6). * p<0.05: significant difference compared with the control group(Kruskal-Wallis y U de Mann-Whitney tests).

DISCUSSION

This study has determined the preventive effect on the formation of kidney stones of an PEF with Herniaria glabra, Agropyron repens,

Equisetum arvense, and Sambucus nigra extracts in an experimentally induced nephrolithiasis model in rats. Although the PEF studied in this study has been used in traditionally for kidney health, there are no studies that have evaluated its effect in humans or in

ANTIUROLITHIASIC EFFECT OF A PLANT FORMULATION OF HERNIARIA GLABRA, AGROPYRON REPENS, EQUISETUM ARVENSE....

animals. Therefore, the study presented is the first to experimentally evaluate the effect of the PEF in animals. The main finding of the present study was that the treatment with 125 mg/Kg PEF decreased significantly the number of CaOx crystals deposits and the number of microcalcifications in the kidney. The usual recommended dose of the PEF studied is two Herbensurina® tablets equivalent to a dose of 120 mg/Kg of the PEF. Thus, the results of our study suggest that the higher recommended dose of the PEF may prevent the kidney stone formation. Although the results of our study provide scientific evidence of the use of PEF to prevent the formation of kidney stones, more studies are needed with an higher number of animals and determinations to corroborate the results obtained. It is also necessary to conduct toxicity and clinical studies with the PEF to determine effectiveness in humans.

The supersaturation of the urine with stone forming calcium salts is a prerequisite for crystal formation in the urinary tract (17). Diuretics increase urine volume, resulting in the reduced supersaturation of crystal-forming salts and aiding in the expulsion of already formed crystals (3). Interestingly, in our study, the urinary output markedly increased in the rats that were treated with PEF. Different authors have reported diuretic effects for Sambucus nigra (9), Herniaria glabra (7), Equisetum arvense (8) and Agropyron repens (10), and similar results were also reported for other plant extracts with suggested antiurolithiatic effects, such as Herniaria hirsuta (18), Bergenia ligulata (19) and Piper amalago (20). Therefore, it is hypothesized that PEF could prevent the formation of CaOx calculi through its diuretic effect.

It is important to point out that urine is normally a supersaturated solution with common stone-forming minerals and that only some individuals are prone to form urinary stones. One reason for this pattern is the presence of inhibitors of lithogenesis in urine, such as citrate. Urinary citrate decreases the saturation of CaOx by forming soluble complexes with calcium and

745

Tabl

e II.

The

24-

h w

ater

con

sum

ptio

n, u

rine

volu

me

and

rela

tive

urin

e vo

lum

e fro

m th

e co

ntro

l gro

up a

nd th

e pl

ant e

xtrac

t for

mul

atio

n-tre

ated

gro

ups.

Day

Con

trol

30 m

g/Kg

60 m

g/Kg

125

mg/

Kg

250

mg/

Kg

500

mg/

Kg

0

33.4

4

(4.1

7)

39.1

6

(4.7

4)

32.4

9

(2.3

4)

36.6

0

(3.0

4)

35.9

2

(1.5

0)

33.6

7

(2.8

2)

7

50.1

9

(6.8

4)

54.5

9

(7.6

9)

57.9

8

(5.3

6)

59.9

4

(14.

04)

59.9

7

(7.2

6)

61.4

9

(10.

51)

13

50.7

0

(5.8

4)

57.8

3

(4.5

5)

57.3

0

(3.8

0)

65.0

5

(9.5

7)

62.3

3

(6.5

8)

65.5

0

(6.9

2)

18

50.8

0

(4.8

5)

50.2

5

(3.1

9)

54.1

0

(3.6

3)

48.4

0

(7.0

2)

52.0

3

(6.8

8)

55.3

2

(4.6

4)

0

7.28

(1.2

0)

14.3

8

(3.6

4)

9.37

(1.9

9)

13.9

2

(3.0

2)

11.5

0

(1.7

7)

13.9

0

(3.3

1)

7

22.5

2

(4.2

1)

27.5

3

(4.0

4)

28.6

2

(1.6

2)

31.8

7

(5.6

8)

30.9

3

(5.0

6)

33.6

7

(4.5

1)

13

24.6

7

(4.8

8)

31.7

8

(3.7

8)

31.1

7

(2.5

3)

38.0

0

(6.4

9)

35.3

8

(6.8

3)

38.6

3

(6.1

3)

18

29.4

2

(4.9

5)

28.8

2

(3.4

5)

32.3

0

(3.5

9)

26.8

8

(4.9

5)

30.9

8

(5.9

7)

38.1

7

(3.9

0)

0

0.29

9

(0.0

59)

0.34

3

(0.0

49)

0.27

6

(0.0

46)

0.36

4

(0.0

54)

0.37

0

(0.0

64)

0.38

6

(0.0

66)

7

0.43

4

(0.0

58)

0.50

7

(0.0

35)

0.50

7

(0.0

36)

0.55

8*

(0.0

31)

0.51

1

(0.0

42)

0.56

3*

(0.0

42)

13

0.46

3

(0.0

54)

0.54

2

(0.0

27)

0.54

2

(0.0

11)

0.57

6*

(0.0

34)

0.54

8

(0.0

53)

0.57

6*

(0.0

34)

18

0.62

0

(0.0

28)

0.57

1

(0.0

50)

0.59

0

(0.0

29)

0.58

3

(0.1

16)

0.57

9

(0.0

38)

0.68

7

(0.0

26)

Wat

er in

take

(ml)

Urin

ary

volu

me

(ml)

Urin

e vo

lum

e/w

ater

inta

ke

Data

are

exp

ress

ed a

s m

ean

(stan

dard

dev

iatio

n) (n

=6).

* p<

0.05

: sig

nific

ant d

iffer

ence

com

pare

d w

ith th

e co

ntro

l gro

up (r

epea

ted

mea

sure

s AN

OVA

follo

wed

by

the

DMS

múl

tiple

co

mpa

rison

test)

.

A. Crescenti, F. Puiggròs, A. Colomé, et al.

by the direct inhibition of CaOx nucleation, growth and aggregation (1, 3). However, in our study, we did not observe any effect of PEF treatment on the citrate urinary levels. These results indicate that the antiurolithiatic effect of the PEF was not primarily mediated by this inhibitor of lithogenesis. In addition, in our study, treatment with PEF did not modify the urinary excretion of calcium or oxalate, elements that act as calculus promoters, suggesting that the treatment effect of PEF on calculus formation was independent of alterations in the urinary concentration of these lithogenic elements. It is important to point out that several previous studies with plant extracts also found no changes in the citrate, calcium or oxalate urinary levels (18, 21).

Epithelial cell injury promotes CaOx crystal adherence and retention in renal tubules, which are crucial for subsequent stone development. Furthermore, several in vivo and in vitro studies have demonstrated that both oxalate and CaOx crystals directly induce renal epithelial cell injury mediated by membrane lipid peroxidation through intracellular oxygen free radical generation (22). These results suggest that a

746

Table III. Urinary parameters from control group and plant extract formulation-treated groups.

Group

Control

30 mg/Kg

60 mg/Kg

125 mg/Kg

250 mg/Kg

500 mg/Kg

Oxalate

(mmol/24 h)

0.036

(0.007)

0.054

(0.004)

0.061

(0.006)

0.031

(0.007)

0.047

(0.007)

0.049

(0.007)

Calcium

(mg/24 h)

3.25

(0.24)

3.16

(0.30)

3.38

(0.16)

3.33

(0.12)

3.35

(0.41)

2.64

(0.28)

Citrate

(mmol/24 h)

0.027

(0.008)

0.027

(0.005)

0.018

(0.003)

0.027

(0.008)

0.018

(0.004)

0.034

(0.003)

D.O.

0.39

(0.07)

0.34

(0.04)

0.32

(0.04)

0.35

(0.07)

0.38

(0.03)

0.29

(0.03)

DO: Optical density.Data are expressed as mean (standard deviation) (n=6). No significant differences were observed in the urinary optical density and in the oxalate, calcium or citrate excretion levels between the control group and treated groups with plant extract formulation (ANOVA followed by Dunnett’s multiple comparison test, p<0.05).

reduction of renal oxidative stress could be an effective therapeutic approach. In fact, antioxidants, such as vitamin E, catechin and selenium, protect against renal oxidative injury by oxalate and CaOx crystal deposition (22-23). Interestingly, antioxidant effects have been reported for Sambucus nigra (11) and Equisetum arvense (12). Therefore, the antioxidant action of PEF could be important in explaining its antilithiatic action.

In our study, treatment with PEF decreased the number of COM crystals, although no statistically significant difference compared with untreated group was observed. This effect may be important in preventing kidney stone formation because the type of CaOx crystals plays a role in urinary oxalate retention. COM crystals are considered to be more harmful than COD because they are more likely than COD to attach to the kidney epithelial cells, resulting in the formation of kidney stones (24). These results suggest that PEF may contain substances that inhibit the nucleation of COM crystals, resulting in the reduced growth and aggregation of CaOx crystals.

ANTIUROLITHIASIC EFFECT OF A PLANT FORMULATION OF HERNIARIA GLABRA, AGROPYRON REPENS, EQUISETUM ARVENSE....9

Interestingly, the phytochemical characterization of PEF revealed the presence of phenolic compounds, including chlorogenic acid, quercitrin, apigenin derivates, p-coumaric acid, rutin, genistein and epicatechin gallate. Several studies have reported that phenolic compounds have antioxidant (21) and diuretic (25) activities. Furthermore, the administration of some phenolic compounds reduces renal stone formation (21). In addition to phenolic compounds, two other compounds with described antioxidant activity and antiurolithiatic activity were found, arginine (26) and mannitol (27). Our analysis revealed the presence of the saponin PEFogenin. Saponins possess an anti-crystallization property by disaggregating the suspension of mucoproteins, which promote crystallization (28). Finally, our phytochemical analysis revealed the presence of some dicarboxylic acids, including malic acid, succinic acid and glutamic acid. Interestingly, these compounds can dissolve CaOx crystals (29). Furthermore, Deng SP. et al. (30) demonstrated that some dicarboxylic acids, including succinic acid and malic acid, can inhibit the aggregation of COM crystals and induce the formation of COD crystals. Therefore, we suggest that phenolic compounds and some antioxidant agents, saponins, and dicarboxylic acids could be responsible for the observed effect of PEF in preventing the formation of kidney stones through antioxidant, diuretic, and disaggregation mechanisms.

We propose to do a further experiment with a greater number of animals and with biochemical analysis informing us of kidney function to confirm the results observed. To corroborate the effectiveness of the PEF in human it would be necessary to do toxicity and clinical studies.

CONCLUSIONS

Overall, our data presented in this study indicate that the administration of 125 mg/Kg PEF to experimentally-CaOx-induced nephrolithiatic rats significantly reduces the number of

Tabl

e IV.

Bod

y w

eigh

t, re

lativ

e ki

dney

wei

ght a

nd p

lasm

a G

OT

and

GPT

leve

ls in

con

trol g

roup

and

pla

nt e

xtrac

t for

mul

atio

n-tre

ated

gro

ups.

Gro

up

Con

trol

30 m

g/Kg

60 m

g/Kg

125

mg/

Kg

250

mg/

Kg

500

mg/

Kg

Initi

al b

ody

wei

ght (

g)

322.

33

(19.

40)

300.

83

(5.4

1)

296.

83

(6.4

2)

300.

50

(6.1

3)

296.

17

(4.7

0)

295.

17

(5.3

4)G

OT:

Glu

tam

ic O

xalo

acet

ic T

rans

amin

ase;

GPT

: Glu

tam

ic P

yruv

ic T

rans

amin

ase.

Data

are

exp

ress

ed a

s m

ean

(stan

dard

dev

iatio

n) (n

=6).

No

signi

fican

t diff

eren

ces

in th

e bo

dy w

eigh

t. in

the

rela

tive

wei

ght o

f bot

h ki

dney

s or

in th

e pl

asm

a G

OT

and

GPT

leve

ls w

ere

foun

d be

twee

n th

e co

ntro

l gro

up a

nd tr

eate

d gr

oups

with

her

bal e

xtrac

t com

bina

tion

(AN

OVA

follo

wed

by

Dunn

ett’s

mul

tiple

com

paris

on te

st. p

<0.0

5).

Fina

l bod

y w

eigh

t (g)

362.

83

(20.

58)

347.

67

(6.1

7)

345.

83

(12.

94)

341.

50

(20.

03)

353.

67

(7.2

7)

344.

00

(9.8

8)

Body

wei

ght g

ain

(g)

40.5

0

(7.1

5)

46.8

3

(2.7

8)

49.0

0

(3.9

9)

41.0

0

(7.6

6)

57.5

0

(1.7

2)

55.8

0

(1.0

8)

Righ

t kid

ney

wei

ght

0.53

(0.0

7)

0.54

(0.0

4)

0.60

(0.0

5)

0.59

(0.0

6)

0.60

(0.0

4)

0.69

(0.0

8)

Left

kidn

ey w

eigh

t

0.52

(0.0

8)

0.51

(0.0

4)

0.63

(0.0

7)

0.56

(0.0

5)

0.62

(0.0

4)

0.70

(0.1

0)

GO

T (U

/L)

6.93

(0.5

3)

8.09

(0.8

6)

5.09

(0.6

7)

7.51

(1.1

3)

6.25

(0.2

3)

7.61

(0.6

8)

GPT

(U/L

)

6.17

(0.9

9)

6.40

(1.0

4)

4.02

(0.6

1)

4.41

(0.8

5)

7.08

(2.2

5)

3.59

(0.3

9)

A. Crescenti, F. Puiggròs, A. Colomé, et al.

calcium oxalate crystals deposits and the number of microcalcifications in the kidney. Furthermore, the PEF significantly reduces the risk of subcapsular fibrosis and has an evident diuretic effect, but have no effect on renal pielonefritis or urinary biochemical parameters. 125 mg/Kg of PEF the usually recommended dose for Herbensurina®, is the dose that has a greater effect dose on the studied parameters. Phytochemical analysis of the PEF revealed the presence of compounds for which antioxidant, diuretic and disaggregation activities have been described.

ACKNOWLEDGEMENTS

We gratefully acknowledge the aid of Silvia Pijuan, the laboratory technician.

CONFLICT OF INTEREST

The authors declare no conflicts of interest. Arnau Colomé and Josep Antón Poch are employees of Laboratorios Deiters S.L. This does not alter the author’s adherence to all the Archivos Españoles de Urología policies on sharing data and materials.

Tabl

e V.

Cha

ract

eriz

atio

n of

the

phyt

oche

mic

al p

rofil

e of

Her

niar

ia g

labr

a, A

grop

yron

repe

ns, E

quise

tum

arv

ense

and

Sam

bucu

s ni

gra

and

the

form

ulat

ion

of th

ese

extra

cts

as a

naly

sed

by G

C-M

S an

d LC

-MS.

Com

poun

dSu

ccin

ic a

cid

Mal

ic a

cid

Glu

tam

ic a

cid

Citr

ic a

cid

D-M

anni

tol

Hec

ogen

in a

ceta

teC

hlor

ogen

ic a

cid

p-co

umar

ic a

cid

Que

rcitr

inG

enist

ein

Api

geni

n de

rivat

esA

rgin

ine

3-hy

drox

ycou

mar

inRu

tinEp

icat

echi

n ga

llate

PEF:

Pla

nt E

xtrac

t For

mul

atio

n; -;

no

pres

ence

of t

he c

ompo

und;

+, +

+, +

++, +

+++,

+++

++: r

elat

ive

distr

ibut

ion

of c

ompo

unds

in s

ampl

es.

Her

niar

ia g

labr

a++

++++

+++

++ ++++

+++ + ++ ++ + ++ ++ ++++ - - -

Agr

opyr

on r

epen

s- ++ - ++ ++++ - + - - - - + - - -

Equi

setu

m a

rven

se+ + - + + - - + - - - ++ ++ - -

Sam

bucu

s ni

gra

++ +++ -

+++

++ -++

++++

++ - - + +++ + ++ +

HEC

+++

++++ +

++++

+++

+++

++ +++

+++

++ + +++

++++

+++

+ + ++

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