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Prophylactic effects of Orthosiphon stamineus Benth. extracts on experimental induction of calcium oxalate nephrolithiasis in rats
Journal of Ethnopharmacology 144 (2012) 761–767
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Prophylactic effects of Orthosiphon stamineus Benth. extracts on experimental induction of calcium oxalate nephrolithiasis in rats Yu-Sen Zhong a, Chen-Huan Yu a, Hua-Zhong Ying a, Zhi-Yuan Wang a, Hua-Fang Cai b,n a b
Experimental Animal centre, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China
a r t i c l e i n f o
abstract
Article history: Received 26 April 2012 Received in revised form 10 September 2012 Accepted 16 September 2012 Available online 30 October 2012
Ethnopharmacological relevance: Orthosiphon stamineus (OS) popularly known as ‘‘diuretic agent’’ are traditionally used in folk medicine in the treatment of hyperuricemia, rheumatism, gout, nephritis, nephrolithiasis, urethritis and cystitis. Aim of the study: To evaluate prophylactic potentials of total flavonoids, total phenolics and polysaccharides from OS on experimental induction of calcium oxalate (CaOx) nephrolithiasis in rats. Materials and methods: Nephrolithic rats were induced by treating with 1.0% ethylene glycol and 1.0% ammonium chloride for 7 days. Rats in the treated groups were also given OS extracts at the doses of 80 mg/kg and 160 mg/kg. Urine samples (4 h) and serum samples were collected at 7th day for biochemical analysis. Kidney tissues were stained with H.E. and analyzed by light microscopy. Expressions of OPN protein were detected by immunohistochemistry. Rates of nucleation and aggregation of calcium oxalate crystals were derived from 20-min time-course measurements of optic density at 620 nm after mixing solutions containing calcium chloride, sodium oxalate and OS extracts at 37 1C, pH 5.7. Results: Polysaccharides exhibited the most significant prophylactic effects by reversing BUN and Scr levels, ameliorating histopathological changes, increasing urine C2O24 and Ca2 þ excretion and downregulating OPN protein expression of kidney in the model rats in comparison with those effects of total flavonoids and total phenolics. Polysaccharides could also significantly inhibit both nucleation and aggregation of CaOx crystals. Conclusions: Polysaccharides were the main therapeutic materials in OS. It had impressive prophylactic effects on CaOx stones in nephrolithic rats, playing a role as a regulator of OPN protein expression to increase urine C2O24 and Ca2 þ excretion and also as an inhibitor of CaOx crystallization. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Orthosiphon stamineus Nephrolithiasis Polysaccharides Osteopontin Calcium oxalate crystallization
1. Introduction Urolithiasis is a common, multifactorial and urological disorder in which calcium oxalate has a major role. The prevalence of urolithiasis is more than 10% in the world and increases annually (Tsujihata, 2008; Chandrashekar et al., 2012). Although it has been known from ancient times, the mechanism of renal stone formation remains unclear and few substances useful for the prevention of urolithiasis are available.
Abbreviations: OS, Orthosiphon stamineus Benth; CaOx, Calcium oxalate; OSEF, Total flavonoids extracted from OS ethanol extract; OSEP, Total phenolics extracted from OS ethanol extract; OSAP, Polysaccharides extracted from OS aqueous extract; PG, Paishi granules; PC, Positive control group, nephrolithiasis rats treated with PG; UP, Urine protein; BUN, Blood urea nitrogen; SCr, Serum creatinine; OPN, Osteopontin n Corresponding author. Tel.: þ86 571 88215498; fax: þ 86 571 8821 5497. E-mail address: [email protected] (H.-F. Cai). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.09.052
Orthosiphon stamineus Benth. (OS), belonging to Labiatae, as a popular medicinal herb is widely used in Southeast Asia for the treatments of hyperuricemia, rheumatism, gout, jaundice, nephritis, nephrolithiasis, urethritis and cystitis. The main polyphenolic compounds, such as sinensetin, eupatorin, 30 -hydroxy-5,6,7,40 tetramethoxyflavone, rosmarinic acid, cichoric acid and caffeic acid, had been isolated and identified from this species (Olah et al., 2003; Akowuah et al., 2005). Increasingly considerable attention has been given to its antioxidant, anti-inflammatory, diuretic, antihyperglycemic, hypouricemic, hepatoprotective and anti-fungal properties (Sriplang et al., 2007; Yam et al., 2007; Yam et al., 2009; Yam et al., 2010; Mohamed et al., 2011a; Pan et al., 2011). OS extracts also exhibited diuretic activity via increase of urine volume and electrolytes (Na þ and K þ ) excretion in hyperuricemic rats (Arafat et al., 2008; Adam et al., 2009). Moreover, aqueous and 50% ethanol extracts of OS are low toxicity (Mohamed et al., 2011b; Muhammad et al., 2011). Although many interesting results have been obtained from their
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potential therapeutic usefulness, no studies evaluating antinephrolithiasic mechanism of this herb have been reported yet. In this work, prophylactic effects of different fractions extracted from OS were evaluated on the growth and retention of calcium oxalate (CaOx) nephrolithiasis induced by administration of ethylene glycol (EG) and ammonium chloride (AC) in rats.
2. Materials and methods 2.1. Reagents Paishi granules (Lot. 040943), the commercially available herbal preparation for the treatment of nephrolithiasis, were purchased from Tongrentang Pharmaceutical Co., Nanjing, China, and were used as the positive control in this study. The granules were Glechomae herba, Plantagimis semen, Akebiae caulis and Glycyrrhizae Radix. Other reagents and chemicals used were of analytical grade and obtained from Juhua Chemical Reagent Co. Ltd. (Quzhou, China). 2.2. Preparation and quality control of herbal extract The aerial parts of Orthosiphon stamineus Benth. (Voucher Ref. No. 2009-0630) used in the study were all commercially available dry matter, which were purchased from Zhejiang herbal Pharmaceutical Co., and identified by associate professor Bing Yu, Zhejiang Chinese Medical University, China. To keep the consistency of the herbal chemical ingredients, all of the herbal components were originally obtained from the standard sources as stated with Chinese GAP grade and the drugs were extracted with standard methods according to Chinese Pharmacopoeia. Five hundred grams of the herb were soaked in 12 volumes of 75% ethanol (v/w) for 30 min and then boiled for 1 h. The suspension was then centrifuged (4000 rpm, 20 min) and then supernatant was decompressed and concentrated until it was dry. Then, the dry powders of OS ethanol extract were extracted with 3 volumes of petroleum ether, EtOAc and n-BuOH, successively, to obtain 3 fractions. By phytochemical screening (Basma et al., 2011; Vital and Rivera, 2011), EtOAc fraction (named OSEF) mainly consisted of flavonoids and the aglycones, and n-BuOH fraction (named OSEP) mainly consisted of phenolic acids (data not shown). By colorimetric method, the content of total flavonoids in OSEF was detected to be 85.3% using rutin as the standard (Li et al., 2011) while the content of total phenolics in the extract of OSEP was 93.2% using gallic acid as the standard (Yu et al., 2012). Other dried powders of this herb (500 g) were pretreated with 100% ethanol twice to remove pigments, monosaccharides and liposoluble materials (Yan et al., 2011). Then the pretreated samples were extracted with hot water by Heat-reflux extraction. After filtration, proteins in the supernatant were removed by using trichloroacetic acid. The raffinate phase was precipitated by the addition of ethanol to a final concentration of 80% (v/v). Precipitates were collected by centrifugation (3000 rpm, 15 min), washed successively with ethanol, acetone and diethyl ether, to obtain the purified polysaccharides (named OSAP). By colorimetric method, the carbohydrate content in OSAP was 95.6% using D-glucose as the standard (Wu et al., 2012). 2.3. Preparation of model treatment SPF Sprague-Dawley male rats (7–8 weeks old) weighting 200–220 g were purchased from Experimental Animal Center, Zhejiang Academy of Medical Sciences, China. Animals were housed in groups of five per standard cage, on 12 h light/dark cycle. The temperature in the laboratory was maintained at 2272 1C. These experiments were carried out in accordance with
local guidelines for the care of laboratory animals, and were approved by the ethics committee for research on laboratory animal use of the institution. Except the rats in the normal control group (NC), other rats received 1% EG and 1% AC in drinking water for 7 days to induce a chronic hyperoxaluria and generate calcium oxalate deposition in kidneys according to previously reports (Yamaguchi et al., 2005). At the same time, OS extract or paishi granule was intragastrically administered to experimental groups (group III–group IX) daily for 7 days, while the rats in normal control group (NC, group I) and the model control group (MC, group II) were only given saline. Each group had 10 rats. Group I Group II Group III Group IV
Normal rats treated with saline. Normal rats treated with 1% EG þ1% AC. Normal rats treated with 1% EGþ1% ACþPG (1200 mg/kg). Normal rats treated with 1% EGþ1% ACþ OSEF (160 mg/kg). Group V Normal rats treated with 1% EG þ1% ACþOSEF (80 mg/kg). Group VI Normal rats treated with 1% EG þ1% ACþOSEP (160 mg/kg). Group VII Normal rats treated with 1% EGþ 1% ACþOSEP (80 mg/kg). Group VIII Normal rats treated with 1% EG þ1% ACþOSAP (160 mg/kg). Group IX Normal rats treated with 1% EGþ1% ACþ OSAP (80 mg/kg).
2.4. Biochemical analysis On the 8th day of the experimental period, all the animals were placed in metabolic cages to obtain a 4-h urine collection. After that, the rats were sacrificed under anaesthesia. The levels of Mg2 þ , Ca2 þ and C2O24 in urine were evaluated by 9180 Electrolytes analyzer, while levels of urine protein were determined by Bradford method. Moreover, blood samples were collected from the jugular vein of each animal and submitted to biochemical tests. Serum levels of urea nitrogen and creatinine were estimated by commercially available kits (purchased from Nanjing Jiancheng bioengineering Institute, China). 2.5. Histopathological analysis Kidney tissue samples were subsequently fixed in a 10% solution of buffered formalin (pH 7.4) and enclosed in paraffin. Six to ten 4-mm-thick sections were prepared in a noncontiguous way and dyed with hematoxylin–eosin for evaluation under optical micrOSEPope. We note , þ, þþ and þþþ for normal feature, little, appreciable and severe necrosis, respectively. 2.6. Immunohistochemistry analysis Protein expressions of osteopontin (OPN) were detected using corresponding immunohistochemistry detection kit and the enhanced kit (Boster Bioengineering Institute, Wuhan, China) following the manufacturer’s instructions. The positive cells showed brown articles or clumps in the cytoplasm. Using Image pro plus software version 6.0 (Media Cybernetics, USA), the optical density values were measured under 200 objective lens of each slice for semiquantitative analysis. 2.7. Kinetics of calcium oxalate crystal growth Calcium oxalate crystals were synthesized without or with OS extracts by employing a single diffusion gel technique as
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described in the literatures (Kulaksizoglu et al., 2007; Pinales et al., 2011). All experiments were performed at 37 1C, using a circulating water bath (Jiangnan Instrument CO., LTD., China). The pH of the solution was adjusted to 5.7 by titration with small volumes of buffer solution (containing 250 mmol/L NaCl and 12.5 mmol/L CH3COONa). The pH value was monitored with a calibrated pH meter (Leinuo Co., Shanghai, China). For crystallization experiments, 1.0 mL of oxalate solution (12.5 mmol/L CaC12) was transferred into a 10-mm light path quartz cuvette. One milliliter of an additional calcium solution (12.5 mmol/L CaC12) was added to obtain final concentrations of 5 mmol/L calcium and 0.5 mmol/L oxalate. The optical density of the crystals formed was measured at 5-s intervals over 20 min at 620 nm using a spectrophotometer (SPECTUM UV 721, Shanghai lengguang Instrument CO., LTD., China). The increase in optic density reflects an increase in particle number in the function of time. The time from addition of calcium until the first detectable increment of OD620, named induction time Ti, reflects the time required for the formation of CaOx crystal nuclei. The maximum increase of optic density with time, namely SN, mainly reflects maximum rate of formation of new particles and thus crystal nucleation. After equilibrium has been reached, optic density decreases. No new particles were formed due to crystal aggregation. SA (the maximum slope of decrease of optic density at 620 nm with time, representing crystal aggregation) is derived from the maximum decrease in optic density. At various times, crystals were observed by polarized light micrOSEPopy and photographed at a magnification of 400. All experiments with inhibitors of calcium oxalate crystallization were compared with assay concentrations of 4 mmol/L calcium and 0.5 mmol/L oxalate. Inhibition ratio was calculated as (1-[SNi/SNc]) 100 for rate of nucleation (IN) and (1-[SAi/ SAc]) 100 for the rate of aggregation (IA) where i stands for inhibitors (OS extracts or trisodium citrate) and c for control.
2.8. Statistical analysis All parameters were recorded for individuals within all groups. All data were presented as the mean 7SD. Statistical analysis was performed with two-tailed indirect Student tests, using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). And statistical analysis for multiple comparisons was performed by a one-way ANOVA test. A value of Po0.05 was considered significant.
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3. Results 3.1. Effects of OS extracts on biochemical parameters in nephrolithiasis rats After 7 days’ induction, prolonged administration of 1% EG and 1% AC in drinking water to rats could accelerate the synthesis of CaOx, inhibit the metabolism of oxalic acid and decrease the excretion of CaOx from the kidney to urine as reported before (Atmani et al., 2003; Moriyama et al., 2009). As shown in Table 1, compared with the NC rats, the levels of UP, C2O24 , Ca2 þ and Mg2 þ in the urine of nephrolithiasis rats increased significantly (all Po0.01), indicating that the model was successful for inducing metabolic disorder of CaOx. Compared with the MC group, the levels of UP, urine C2O24 and urine Ca2 þ were suppressed to some extent by treatments of OS extracts at the doses of 80 mg/kg and 160 mg/kg. There were also no significant differences in the excretion of urine Ca2 þ levels among NC and OSAP-treated groups (treated with 80 mg/kg or 160 mg/kg). Furthermore, administration of OSAP at the dose of 160 mg/kg could increase the levels of urinary excretion 4 h after treatment. Comparison with MC group, there was a significant increase in the levels of urine volumes in the rats treated with OSAP at the dose of 160 mg/kg (Po0.05). But after treatment with OSEF and OSEP at the doses of 80–160 mg/kg for 7 days, it did not show any significant changes in the levels of urine volumes, compared with MC group (all P4 0.05). However, administration of OS extracts (OSEF, OSEP and OSAP) could not increase the excretion of urine Mg2 þ levels. On the other hand, only administration of OSAP at the doses of 80–160 mg/kg (both Po0.01) and OSEF at 80 mg/kg (Po0.05) could significantly decrease the levels of BUN and Scr (Fig. 1). However, compared with MC, treatment with OSEP at 80–160 mg/ kg and OSEF at 160 mg/kg could not decrease the levels of BUN (all P40.05); and the levels of Scr were also not significantly decreased by OSEF and OSEP (all P40.05) as shown in Fig. 1C.
3.2. Effects of OS extracts on histopathological features The degrees of kidney injury and histopathology listed in Table 2. The rats treated with OS extracts revealed little necrosis and moderate alterations in the structure of renal tubules, suggesting that OS extracts had potent CaOx lowering effects in
Table 1 Effects of OS extracts and PG on levels of 4-h urine volumes, UP, urine C2O24 , urine Ca2 þ and urine Mg2 þ in nephrolithiasis rats. Group
Dose (mg/kg)
Urine volumes (mL)
UP (mg/L)
Ca2 þ (mmol/L)
C2O24 (mmol/L)
Mg2 þ (mmol/L)
NC MC PC OSEF OSEF OSEP OSEP OSAP OSAP
– – 1200 160 80 160 80 160 80
3.10 7 0.58n 2.40 7 0.71a 2.50 7 0.71a 2.75 7 0.52a 2.29 7 0.47b 2.33 7 0.76b 2.35 7 0.61b 3.46 7 0.55n 2.60 7 0.39b
4.207 0.74nn 6.79 7 0.56b 4.43 7 0.60nn 4.057 0.52nn 6.037 0.67nb 4.16 7 0.53nn 4.23 7 0.40nn 4.077 0.94nn 5.23 7 0.50nnb
5.49 7 0.68nn 6.54 7 0.74b 6.30 7 0.69a 5.79 7 0.48n 5.96 7 0.85 5.76 7 0.71n 6.14 7 0.89a 5.56 7 0.52nn 5.89 7 0.49n
0.47 70.20nn 1.31 70.57b 1.13 70.42b 0.85 70.16nb 0.88 70.42nb 0.81 70.40nb 0.94 70.42b 0.68 70.12nna 0.88 70.09nb
24.63 7 4.29nn 21.21 7 4.27b 22.97 7 5.49a 22.69 7 3.19a 22.88 7 5.33a 22.78 7 6.46a 20.377 5.57b 21.42 7 4.23b 21.84 7 2.12b
OSEF, total flavonoids extracted from OS ethanol extract; OSEP, total phenolics extracted from OS ethanol extract; OSAP, polysaccharides extracted from OS aqueous extract; NC, normal control group; MC, model control group; PC, positive control group, nephrolithiasis rats treated with pspaishi granules; UP, urine protein. Data denoted were means7 SD, n¼ 10. Compared with MC. n
Po 0.05. P o 0.01; compared with NC. Po 0.05. P o0.01.
nn
a b
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the nephrolithiasis rats. However, administration of OS extracts could not decrease the levels of kidney indexes (all P40.05) as shown in Fig. 1A.
20 16
3.3. Effects of OS extracts on expressions of OPN protein
**
8 4 80 SE P1 60 O SE P8 0 O SA P1 60 O SA P8 0 O
SE F
16 0 O
O
SE F
PC
M C
36
24
*
**
18
3.4. Inhibition of OS extracts on CaOx crystallization
0 P8 SA
O
P1
60
80 SA O
O
SE F
0
C
** N
Fig. 1. Effects of OS extracts and PG on levels of kidney index (A), BUN (B) and SCr (C). Data denoted are means7 S.D., n¼10; Compared with MC, nP o0.05, nn P o 0.01.
**
**
16
0 SA
P8
60 O
SA P1
O
SE P8 0 O
O
SE P
16 0
80 SE F O
F1 60
PC O
SE
M C
N
C
0
*
SE P
40
**
O
**
80
4.8 4.2 3.6 3.0 2.4 1.8 1.2 0.6 0.0
80
*
**
120
SE P
SCr (mmol/L)
160
O
200
The growth and agglomeration of CaOx crystals were differently modulated by various OS extracts (Table 3). Among the OS extracts tested, OSAP had the most significant inhibitory effect on the nucleation of CaOx crystals. In the presence of OSAP, Ti increased and the slopes of calcium oxalate crystal growth (SN and SA) decreased (Po0.01 and Po0.05, respectively). At the
0
16 0 O SE F8 0 O SE P1 60 O SE P8 0 O SA P1 60 O SA P8 0
PC
O SE F
M C
N C
0
SE F
**
6
16
12
**
**
O
BUN (mmol/L)
30
PC
N
C
0
OPN protein expression was slightly detected in the kidney tissue of NC rats, while a strong expression was shown in the MC group (Fig. 2). Compared with that in the MC group, administration of OSAP at the doses of 80–160 mg/kg had significant decrease in the expressions of OPN protein in the kidney tissue (both P o0.01). However, administration of OSEP at the doses of 80–160 mg/kg and OSEF at 80 mg/kg could not decrease the levels of OPN protein expressions (all P40.05). These results were also consistent with the morphological observations by using strong magnified microscopy of kidney crystal deposition.
M C
12
Optical density of OPN
Kidney index (mg/g)
24
Fig. 2. Optical density of OPN protein expressions of renal tissues in nephrolithiasis rats
Table 2 Effect of OS extracts on the kidney histopathology in nephrolithiasis rats. Group
Dose (mg/kg)
Renal cortex
Renal medulla
Renal papillae
Renal pelvis
NC MC PC OSEF OSEF OSEP OSEP OSAP OSAP
– – 1200 160 80 160 80 160 80
0.07 0.0nn 3.2 7 0.7b 3.0 7 0.7b 3.0 7 0.5b 2.9 7 0.6b 3.1 7 0.5b 3.2 7 0.4b 2.7 7 0.6b 3.0 7 0.4b
0.0 70.0nn 3.3 70.8b 2.6 70.8nb 2.8 70.4b 2.9 70.3b 2.9 70.5b 3.0 70.4b 2.6 70.7nb 3.0 70.8b
0.07 0.0nn 3.5 7 0.8b 2.7 7 1.1nb 2.6 7 0.5nb 3.2 7 1.0b 2.4 7 0.8nnb 2.5 7 0.7nb 2.2 7 1.0nnb 2.6 7 0.7nb
0.07 0.0nn 3.7 7 1.3b 2.4 7 1.2nb 2.5 7 1.2nb 2.2 7 1.0nnb 1.6 7 0.8nnb 1.7 7 0.8nnb 2.1 7 1.8nb 2.2 7 1.0nnb
OSEF, total flavonoids extracted from OS ethanol extract; OSEP, total phenolics extracted from OS ethanol extract; OSAP, polysaccharides extracted from OS aqueous extract; NC, normal control group; MC, model control group; PC, positive control group, nephrolithiasis rats treated with pspaishi granules; UP, urine protein. Data denoted were means7 SD, n¼10. Compared with MC. WPo 0.05. n
P o0.05. Po 0.01; compared with NC. Po 0.01.
nn
b
Table 3 Effect of OS extracts on kinetic parameters of calcium oxalate crystal growth in vitro. Group Final concentrations (mg/mL)
Ti (s)
SN ( 10 3/s)
IN (%)
SA ( 10 3/s)
IA (%)
MC OSEF OSEF OSEP OSEP OSAP OSAP
33.0 7 5.7 49.0 7 9.6n 44.0 7 22.7 10.07 11.2 28.0 7 21.4 191.0 7 47.9nn 116.0 7 62.4nn
2.267 0.70 1.387 0.40 1.327 0.41 0.907 0.37nn 1.787 0.29 0.547 0.11nn 0.927 0.29nn
– 38.9 41.6 62.2 21.2 76.1 59.3
0.88 7 0.11 0.88 7 0.16 0.88 7 0.13 0.66 7 0.22 0.64 7 0.27 0.58 7 0.14n 0.607 0.16n
– 0 0 25.0 27.3 34.1 31.8
– 0.5 0.05 0.5 0.05 0.5 0.05
OSEF, total flavonoids extracted from OS ethanol extract; OSEP, total phenolics extracted from OS ethanol extract; OSAP, polysaccharides extracted from OS aqueous extract; MC, model control group; PC, positive control group, nephrolithiasis rats treated with pspaishi granules; UP, urine protein; Ti, induction time; SN, the maximum increase of optic density with time; SA, the maximum slope of decrease of optic density at 620 nm with time; IN, the rate of nucleation (IN ¼ 100 [SNi/SNc] 100); IA, the rate of aggregation (100 [SAi/SAc] 100). Data denoted were means 7SD, n ¼5. Compared with MC. n
Po 0.05. Po 0.01.
nn
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24 h after reaction
48 h after reaction
765
72 h after reaction
Model control
+ sodium citrate (0.24 µg/mL)
+ OSHF (1.20 µg/mL)
+ OSHF (0.24 µg/mL)
+ OSHP (1.20 µg/mL)
+ OSHP (0.24 µg/mL)
+ OSAP (1.20 µg/mL)
+ OSAP (0.24 µg/mL)
Fig. 3. Inhibition of OS extracts on calcium oxalate crystallization in vitro ( 400). Normal control experiment was performed with 0.6 mmol/L calcium and 0.6 mmol/L oxalate. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
higher final concentration (0.5 mg/mL) of OSAP, nucleation percentage inhibition ratio IN was 76.1% and crystal aggregation IA was inhibited by approximately 34.1%. And it appeared in a dosedependent manner. The photographs at 400 magnification depicted particle sizes and densities of CaOx crystals during maximum rates of aggregation (Fig. 3). A visible decrease in calcium oxalate crystal
growth with increasing amounts of OSAP could be observed in Fig. 3. Not only is there a reduction in crystal size (from 1 mm average size for pure crystals to 0.5 mm average size for crystals grown with OSAP), but there is also a color change from light orange–brown for pure crystals to white-transparent for crystals with the inhibitor. Pure calcium oxalate crystals showed spherulites with smooth surfaces. However, after treated with
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0.24–1.20 mg/mL of OSAP, calcium and oxalate in the solution failed to produce massy crystals but lots of needlelike ones. Compared with standard condition (contained 5 mmol/L calcium and 0.5 mmol/L oxalate in the solution), particle sizes and densities were not significantly affected by OSEF and OSEP.
4. Discussion This study provided evidences that OS extracts acted as an anti-nephrolithiasic agent in mouse model of CaOx nephrolithiasis and as an inhibitor of CaOx crystallization. In both in vivo and in vitro experiments, OS extracts (especially OSAP) could markedly reduce CaOx crystal formation. Oral administration of OSAP at the doses of 80–160 mg/kg could significantly decrease the levels of UP and BUN, ameliorate CaOx crystal deposition induced histopathological features and diminish epithelial cell necrosis. Therefore, OSAP showed the highest therapeutic effects in comparison with the other two fractions OSEF and OSEP. These finding suggested that OSAP, i.e., the purified polysaccharides from OS were the main therapeutic materials. The important process in the kidney stone formation is the conversion of retentive crystals in renal tubules to concrete stones. Mg2 þ and OPN play essential roles in the CaOx crystalcell interaction (Konya et al., 2003). Mg2 þ could act as an inhibitor of crystal formation (Lieske, Farell and Deganello, 2004) while the possible role of OPN in kidney stone formation is inhibition of calcium oxalate monohydrate (COM) nucleation and reduction of the growth and aggregation of CaOx crystals (Khan et al., 2002; Yasui et al., 2002; Prat et al., 2011). Prolonged administration of EG, a precursor of oxalate, to rats could make a dramatic increase in the expression of OPN protein and mRNA was found in renal epithelial cells as reported previously (Khan, 1995; Okada et al., 2008). After 7-day treatment, OSEF, OSEP and OSAP at the doses of 80–160 mg/kg could significantly decrease the expression of OPN protein compared with that in MC group while all of them could not change the levels of urine Mg2 þ , well matched with amelioration of several histopathological features and reduction of CaOx crystal sizes. It indicated that OS extracts could reduce deposition of CaOx crystals to renal epithelial cells by down-regulating expression of OPN protein in the kidneys. It is well known that the type of CaOx crystals plays a role in urinary oxalate excretion. Urinary crystal has a potency to transform from calcium oxalate dihydrate (COD) or calcium oxalate trihydrate (COT) to calcium oxalate monohydrate (COM) (Yamaguchi et al., 2005). In the standard condition, there were many long hexagon crystals COM, which were increased as time extended. Citrate could inhibit calcium oxalate crystallization at the beginning of the reaction but there was no significant difference 48–72 h after the reaction compared with that in the standard condition. However, in the presence of OSAP, the main crystals in solution were COT, the acicular and needle-like crystals. OSAP could significantly affect both nucleation and aggregation, i.e., produce fewer, smaller and less aggregated crystals. The absorbance at 620 nm, which reflected the presence of crystallized CaOx, decreased spontaneously in the presence of OSAP, indicating that OSAP could inhibit crystal formation in solution and decreased in both SN and SA. OSEF and OSEP did not seem to be mediated by morphological changes of crystals. Since monosaccharide composition and molecular weight are crucial to the bioactivities of polysaccharides, the characteristics of OSAP had not been studied and thus further investigations about the structural analysis and evaluation of the bioactivities of OSAP should be done. In conclusion, polysaccharides were the main therapeutic materials in OS, playing a role as a regulator of OPN protein
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Prophylactic effects of Orthosiphon stamineus Benth. extracts on experimental induction of calcium oxalate nephrolithiasis in rats Yu-Sen Zhong a, Chen-Huan Yu a, Hua-Zhong Ying a, Zhi-Yuan Wang a, Hua-Fang Cai b,n a b
Experimental Animal centre, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China
a r t i c l e i n f o
abstract
Article history: Received 26 April 2012 Received in revised form 10 September 2012 Accepted 16 September 2012 Available online 30 October 2012
Ethnopharmacological relevance: Orthosiphon stamineus (OS) popularly known as ‘‘diuretic agent’’ are traditionally used in folk medicine in the treatment of hyperuricemia, rheumatism, gout, nephritis, nephrolithiasis, urethritis and cystitis. Aim of the study: To evaluate prophylactic potentials of total flavonoids, total phenolics and polysaccharides from OS on experimental induction of calcium oxalate (CaOx) nephrolithiasis in rats. Materials and methods: Nephrolithic rats were induced by treating with 1.0% ethylene glycol and 1.0% ammonium chloride for 7 days. Rats in the treated groups were also given OS extracts at the doses of 80 mg/kg and 160 mg/kg. Urine samples (4 h) and serum samples were collected at 7th day for biochemical analysis. Kidney tissues were stained with H.E. and analyzed by light microscopy. Expressions of OPN protein were detected by immunohistochemistry. Rates of nucleation and aggregation of calcium oxalate crystals were derived from 20-min time-course measurements of optic density at 620 nm after mixing solutions containing calcium chloride, sodium oxalate and OS extracts at 37 1C, pH 5.7. Results: Polysaccharides exhibited the most significant prophylactic effects by reversing BUN and Scr levels, ameliorating histopathological changes, increasing urine C2O24 and Ca2 þ excretion and downregulating OPN protein expression of kidney in the model rats in comparison with those effects of total flavonoids and total phenolics. Polysaccharides could also significantly inhibit both nucleation and aggregation of CaOx crystals. Conclusions: Polysaccharides were the main therapeutic materials in OS. It had impressive prophylactic effects on CaOx stones in nephrolithic rats, playing a role as a regulator of OPN protein expression to increase urine C2O24 and Ca2 þ excretion and also as an inhibitor of CaOx crystallization. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Orthosiphon stamineus Nephrolithiasis Polysaccharides Osteopontin Calcium oxalate crystallization
1. Introduction Urolithiasis is a common, multifactorial and urological disorder in which calcium oxalate has a major role. The prevalence of urolithiasis is more than 10% in the world and increases annually (Tsujihata, 2008; Chandrashekar et al., 2012). Although it has been known from ancient times, the mechanism of renal stone formation remains unclear and few substances useful for the prevention of urolithiasis are available.
Abbreviations: OS, Orthosiphon stamineus Benth; CaOx, Calcium oxalate; OSEF, Total flavonoids extracted from OS ethanol extract; OSEP, Total phenolics extracted from OS ethanol extract; OSAP, Polysaccharides extracted from OS aqueous extract; PG, Paishi granules; PC, Positive control group, nephrolithiasis rats treated with PG; UP, Urine protein; BUN, Blood urea nitrogen; SCr, Serum creatinine; OPN, Osteopontin n Corresponding author. Tel.: þ86 571 88215498; fax: þ 86 571 8821 5497. E-mail address: [email protected] (H.-F. Cai). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.09.052
Orthosiphon stamineus Benth. (OS), belonging to Labiatae, as a popular medicinal herb is widely used in Southeast Asia for the treatments of hyperuricemia, rheumatism, gout, jaundice, nephritis, nephrolithiasis, urethritis and cystitis. The main polyphenolic compounds, such as sinensetin, eupatorin, 30 -hydroxy-5,6,7,40 tetramethoxyflavone, rosmarinic acid, cichoric acid and caffeic acid, had been isolated and identified from this species (Olah et al., 2003; Akowuah et al., 2005). Increasingly considerable attention has been given to its antioxidant, anti-inflammatory, diuretic, antihyperglycemic, hypouricemic, hepatoprotective and anti-fungal properties (Sriplang et al., 2007; Yam et al., 2007; Yam et al., 2009; Yam et al., 2010; Mohamed et al., 2011a; Pan et al., 2011). OS extracts also exhibited diuretic activity via increase of urine volume and electrolytes (Na þ and K þ ) excretion in hyperuricemic rats (Arafat et al., 2008; Adam et al., 2009). Moreover, aqueous and 50% ethanol extracts of OS are low toxicity (Mohamed et al., 2011b; Muhammad et al., 2011). Although many interesting results have been obtained from their
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potential therapeutic usefulness, no studies evaluating antinephrolithiasic mechanism of this herb have been reported yet. In this work, prophylactic effects of different fractions extracted from OS were evaluated on the growth and retention of calcium oxalate (CaOx) nephrolithiasis induced by administration of ethylene glycol (EG) and ammonium chloride (AC) in rats.
2. Materials and methods 2.1. Reagents Paishi granules (Lot. 040943), the commercially available herbal preparation for the treatment of nephrolithiasis, were purchased from Tongrentang Pharmaceutical Co., Nanjing, China, and were used as the positive control in this study. The granules were Glechomae herba, Plantagimis semen, Akebiae caulis and Glycyrrhizae Radix. Other reagents and chemicals used were of analytical grade and obtained from Juhua Chemical Reagent Co. Ltd. (Quzhou, China). 2.2. Preparation and quality control of herbal extract The aerial parts of Orthosiphon stamineus Benth. (Voucher Ref. No. 2009-0630) used in the study were all commercially available dry matter, which were purchased from Zhejiang herbal Pharmaceutical Co., and identified by associate professor Bing Yu, Zhejiang Chinese Medical University, China. To keep the consistency of the herbal chemical ingredients, all of the herbal components were originally obtained from the standard sources as stated with Chinese GAP grade and the drugs were extracted with standard methods according to Chinese Pharmacopoeia. Five hundred grams of the herb were soaked in 12 volumes of 75% ethanol (v/w) for 30 min and then boiled for 1 h. The suspension was then centrifuged (4000 rpm, 20 min) and then supernatant was decompressed and concentrated until it was dry. Then, the dry powders of OS ethanol extract were extracted with 3 volumes of petroleum ether, EtOAc and n-BuOH, successively, to obtain 3 fractions. By phytochemical screening (Basma et al., 2011; Vital and Rivera, 2011), EtOAc fraction (named OSEF) mainly consisted of flavonoids and the aglycones, and n-BuOH fraction (named OSEP) mainly consisted of phenolic acids (data not shown). By colorimetric method, the content of total flavonoids in OSEF was detected to be 85.3% using rutin as the standard (Li et al., 2011) while the content of total phenolics in the extract of OSEP was 93.2% using gallic acid as the standard (Yu et al., 2012). Other dried powders of this herb (500 g) were pretreated with 100% ethanol twice to remove pigments, monosaccharides and liposoluble materials (Yan et al., 2011). Then the pretreated samples were extracted with hot water by Heat-reflux extraction. After filtration, proteins in the supernatant were removed by using trichloroacetic acid. The raffinate phase was precipitated by the addition of ethanol to a final concentration of 80% (v/v). Precipitates were collected by centrifugation (3000 rpm, 15 min), washed successively with ethanol, acetone and diethyl ether, to obtain the purified polysaccharides (named OSAP). By colorimetric method, the carbohydrate content in OSAP was 95.6% using D-glucose as the standard (Wu et al., 2012). 2.3. Preparation of model treatment SPF Sprague-Dawley male rats (7–8 weeks old) weighting 200–220 g were purchased from Experimental Animal Center, Zhejiang Academy of Medical Sciences, China. Animals were housed in groups of five per standard cage, on 12 h light/dark cycle. The temperature in the laboratory was maintained at 2272 1C. These experiments were carried out in accordance with
local guidelines for the care of laboratory animals, and were approved by the ethics committee for research on laboratory animal use of the institution. Except the rats in the normal control group (NC), other rats received 1% EG and 1% AC in drinking water for 7 days to induce a chronic hyperoxaluria and generate calcium oxalate deposition in kidneys according to previously reports (Yamaguchi et al., 2005). At the same time, OS extract or paishi granule was intragastrically administered to experimental groups (group III–group IX) daily for 7 days, while the rats in normal control group (NC, group I) and the model control group (MC, group II) were only given saline. Each group had 10 rats. Group I Group II Group III Group IV
Normal rats treated with saline. Normal rats treated with 1% EG þ1% AC. Normal rats treated with 1% EGþ1% ACþPG (1200 mg/kg). Normal rats treated with 1% EGþ1% ACþ OSEF (160 mg/kg). Group V Normal rats treated with 1% EG þ1% ACþOSEF (80 mg/kg). Group VI Normal rats treated with 1% EG þ1% ACþOSEP (160 mg/kg). Group VII Normal rats treated with 1% EGþ 1% ACþOSEP (80 mg/kg). Group VIII Normal rats treated with 1% EG þ1% ACþOSAP (160 mg/kg). Group IX Normal rats treated with 1% EGþ1% ACþ OSAP (80 mg/kg).
2.4. Biochemical analysis On the 8th day of the experimental period, all the animals were placed in metabolic cages to obtain a 4-h urine collection. After that, the rats were sacrificed under anaesthesia. The levels of Mg2 þ , Ca2 þ and C2O24 in urine were evaluated by 9180 Electrolytes analyzer, while levels of urine protein were determined by Bradford method. Moreover, blood samples were collected from the jugular vein of each animal and submitted to biochemical tests. Serum levels of urea nitrogen and creatinine were estimated by commercially available kits (purchased from Nanjing Jiancheng bioengineering Institute, China). 2.5. Histopathological analysis Kidney tissue samples were subsequently fixed in a 10% solution of buffered formalin (pH 7.4) and enclosed in paraffin. Six to ten 4-mm-thick sections were prepared in a noncontiguous way and dyed with hematoxylin–eosin for evaluation under optical micrOSEPope. We note , þ, þþ and þþþ for normal feature, little, appreciable and severe necrosis, respectively. 2.6. Immunohistochemistry analysis Protein expressions of osteopontin (OPN) were detected using corresponding immunohistochemistry detection kit and the enhanced kit (Boster Bioengineering Institute, Wuhan, China) following the manufacturer’s instructions. The positive cells showed brown articles or clumps in the cytoplasm. Using Image pro plus software version 6.0 (Media Cybernetics, USA), the optical density values were measured under 200 objective lens of each slice for semiquantitative analysis. 2.7. Kinetics of calcium oxalate crystal growth Calcium oxalate crystals were synthesized without or with OS extracts by employing a single diffusion gel technique as
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described in the literatures (Kulaksizoglu et al., 2007; Pinales et al., 2011). All experiments were performed at 37 1C, using a circulating water bath (Jiangnan Instrument CO., LTD., China). The pH of the solution was adjusted to 5.7 by titration with small volumes of buffer solution (containing 250 mmol/L NaCl and 12.5 mmol/L CH3COONa). The pH value was monitored with a calibrated pH meter (Leinuo Co., Shanghai, China). For crystallization experiments, 1.0 mL of oxalate solution (12.5 mmol/L CaC12) was transferred into a 10-mm light path quartz cuvette. One milliliter of an additional calcium solution (12.5 mmol/L CaC12) was added to obtain final concentrations of 5 mmol/L calcium and 0.5 mmol/L oxalate. The optical density of the crystals formed was measured at 5-s intervals over 20 min at 620 nm using a spectrophotometer (SPECTUM UV 721, Shanghai lengguang Instrument CO., LTD., China). The increase in optic density reflects an increase in particle number in the function of time. The time from addition of calcium until the first detectable increment of OD620, named induction time Ti, reflects the time required for the formation of CaOx crystal nuclei. The maximum increase of optic density with time, namely SN, mainly reflects maximum rate of formation of new particles and thus crystal nucleation. After equilibrium has been reached, optic density decreases. No new particles were formed due to crystal aggregation. SA (the maximum slope of decrease of optic density at 620 nm with time, representing crystal aggregation) is derived from the maximum decrease in optic density. At various times, crystals were observed by polarized light micrOSEPopy and photographed at a magnification of 400. All experiments with inhibitors of calcium oxalate crystallization were compared with assay concentrations of 4 mmol/L calcium and 0.5 mmol/L oxalate. Inhibition ratio was calculated as (1-[SNi/SNc]) 100 for rate of nucleation (IN) and (1-[SAi/ SAc]) 100 for the rate of aggregation (IA) where i stands for inhibitors (OS extracts or trisodium citrate) and c for control.
2.8. Statistical analysis All parameters were recorded for individuals within all groups. All data were presented as the mean 7SD. Statistical analysis was performed with two-tailed indirect Student tests, using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). And statistical analysis for multiple comparisons was performed by a one-way ANOVA test. A value of Po0.05 was considered significant.
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3. Results 3.1. Effects of OS extracts on biochemical parameters in nephrolithiasis rats After 7 days’ induction, prolonged administration of 1% EG and 1% AC in drinking water to rats could accelerate the synthesis of CaOx, inhibit the metabolism of oxalic acid and decrease the excretion of CaOx from the kidney to urine as reported before (Atmani et al., 2003; Moriyama et al., 2009). As shown in Table 1, compared with the NC rats, the levels of UP, C2O24 , Ca2 þ and Mg2 þ in the urine of nephrolithiasis rats increased significantly (all Po0.01), indicating that the model was successful for inducing metabolic disorder of CaOx. Compared with the MC group, the levels of UP, urine C2O24 and urine Ca2 þ were suppressed to some extent by treatments of OS extracts at the doses of 80 mg/kg and 160 mg/kg. There were also no significant differences in the excretion of urine Ca2 þ levels among NC and OSAP-treated groups (treated with 80 mg/kg or 160 mg/kg). Furthermore, administration of OSAP at the dose of 160 mg/kg could increase the levels of urinary excretion 4 h after treatment. Comparison with MC group, there was a significant increase in the levels of urine volumes in the rats treated with OSAP at the dose of 160 mg/kg (Po0.05). But after treatment with OSEF and OSEP at the doses of 80–160 mg/kg for 7 days, it did not show any significant changes in the levels of urine volumes, compared with MC group (all P4 0.05). However, administration of OS extracts (OSEF, OSEP and OSAP) could not increase the excretion of urine Mg2 þ levels. On the other hand, only administration of OSAP at the doses of 80–160 mg/kg (both Po0.01) and OSEF at 80 mg/kg (Po0.05) could significantly decrease the levels of BUN and Scr (Fig. 1). However, compared with MC, treatment with OSEP at 80–160 mg/ kg and OSEF at 160 mg/kg could not decrease the levels of BUN (all P40.05); and the levels of Scr were also not significantly decreased by OSEF and OSEP (all P40.05) as shown in Fig. 1C.
3.2. Effects of OS extracts on histopathological features The degrees of kidney injury and histopathology listed in Table 2. The rats treated with OS extracts revealed little necrosis and moderate alterations in the structure of renal tubules, suggesting that OS extracts had potent CaOx lowering effects in
Table 1 Effects of OS extracts and PG on levels of 4-h urine volumes, UP, urine C2O24 , urine Ca2 þ and urine Mg2 þ in nephrolithiasis rats. Group
Dose (mg/kg)
Urine volumes (mL)
UP (mg/L)
Ca2 þ (mmol/L)
C2O24 (mmol/L)
Mg2 þ (mmol/L)
NC MC PC OSEF OSEF OSEP OSEP OSAP OSAP
– – 1200 160 80 160 80 160 80
3.10 7 0.58n 2.40 7 0.71a 2.50 7 0.71a 2.75 7 0.52a 2.29 7 0.47b 2.33 7 0.76b 2.35 7 0.61b 3.46 7 0.55n 2.60 7 0.39b
4.207 0.74nn 6.79 7 0.56b 4.43 7 0.60nn 4.057 0.52nn 6.037 0.67nb 4.16 7 0.53nn 4.23 7 0.40nn 4.077 0.94nn 5.23 7 0.50nnb
5.49 7 0.68nn 6.54 7 0.74b 6.30 7 0.69a 5.79 7 0.48n 5.96 7 0.85 5.76 7 0.71n 6.14 7 0.89a 5.56 7 0.52nn 5.89 7 0.49n
0.47 70.20nn 1.31 70.57b 1.13 70.42b 0.85 70.16nb 0.88 70.42nb 0.81 70.40nb 0.94 70.42b 0.68 70.12nna 0.88 70.09nb
24.63 7 4.29nn 21.21 7 4.27b 22.97 7 5.49a 22.69 7 3.19a 22.88 7 5.33a 22.78 7 6.46a 20.377 5.57b 21.42 7 4.23b 21.84 7 2.12b
OSEF, total flavonoids extracted from OS ethanol extract; OSEP, total phenolics extracted from OS ethanol extract; OSAP, polysaccharides extracted from OS aqueous extract; NC, normal control group; MC, model control group; PC, positive control group, nephrolithiasis rats treated with pspaishi granules; UP, urine protein. Data denoted were means7 SD, n¼ 10. Compared with MC. n
Po 0.05. P o 0.01; compared with NC. Po 0.05. P o0.01.
nn
a b
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the nephrolithiasis rats. However, administration of OS extracts could not decrease the levels of kidney indexes (all P40.05) as shown in Fig. 1A.
20 16
3.3. Effects of OS extracts on expressions of OPN protein
**
8 4 80 SE P1 60 O SE P8 0 O SA P1 60 O SA P8 0 O
SE F
16 0 O
O
SE F
PC
M C
36
24
*
**
18
3.4. Inhibition of OS extracts on CaOx crystallization
0 P8 SA
O
P1
60
80 SA O
O
SE F
0
C
** N
Fig. 1. Effects of OS extracts and PG on levels of kidney index (A), BUN (B) and SCr (C). Data denoted are means7 S.D., n¼10; Compared with MC, nP o0.05, nn P o 0.01.
**
**
16
0 SA
P8
60 O
SA P1
O
SE P8 0 O
O
SE P
16 0
80 SE F O
F1 60
PC O
SE
M C
N
C
0
*
SE P
40
**
O
**
80
4.8 4.2 3.6 3.0 2.4 1.8 1.2 0.6 0.0
80
*
**
120
SE P
SCr (mmol/L)
160
O
200
The growth and agglomeration of CaOx crystals were differently modulated by various OS extracts (Table 3). Among the OS extracts tested, OSAP had the most significant inhibitory effect on the nucleation of CaOx crystals. In the presence of OSAP, Ti increased and the slopes of calcium oxalate crystal growth (SN and SA) decreased (Po0.01 and Po0.05, respectively). At the
0
16 0 O SE F8 0 O SE P1 60 O SE P8 0 O SA P1 60 O SA P8 0
PC
O SE F
M C
N C
0
SE F
**
6
16
12
**
**
O
BUN (mmol/L)
30
PC
N
C
0
OPN protein expression was slightly detected in the kidney tissue of NC rats, while a strong expression was shown in the MC group (Fig. 2). Compared with that in the MC group, administration of OSAP at the doses of 80–160 mg/kg had significant decrease in the expressions of OPN protein in the kidney tissue (both P o0.01). However, administration of OSEP at the doses of 80–160 mg/kg and OSEF at 80 mg/kg could not decrease the levels of OPN protein expressions (all P40.05). These results were also consistent with the morphological observations by using strong magnified microscopy of kidney crystal deposition.
M C
12
Optical density of OPN
Kidney index (mg/g)
24
Fig. 2. Optical density of OPN protein expressions of renal tissues in nephrolithiasis rats
Table 2 Effect of OS extracts on the kidney histopathology in nephrolithiasis rats. Group
Dose (mg/kg)
Renal cortex
Renal medulla
Renal papillae
Renal pelvis
NC MC PC OSEF OSEF OSEP OSEP OSAP OSAP
– – 1200 160 80 160 80 160 80
0.07 0.0nn 3.2 7 0.7b 3.0 7 0.7b 3.0 7 0.5b 2.9 7 0.6b 3.1 7 0.5b 3.2 7 0.4b 2.7 7 0.6b 3.0 7 0.4b
0.0 70.0nn 3.3 70.8b 2.6 70.8nb 2.8 70.4b 2.9 70.3b 2.9 70.5b 3.0 70.4b 2.6 70.7nb 3.0 70.8b
0.07 0.0nn 3.5 7 0.8b 2.7 7 1.1nb 2.6 7 0.5nb 3.2 7 1.0b 2.4 7 0.8nnb 2.5 7 0.7nb 2.2 7 1.0nnb 2.6 7 0.7nb
0.07 0.0nn 3.7 7 1.3b 2.4 7 1.2nb 2.5 7 1.2nb 2.2 7 1.0nnb 1.6 7 0.8nnb 1.7 7 0.8nnb 2.1 7 1.8nb 2.2 7 1.0nnb
OSEF, total flavonoids extracted from OS ethanol extract; OSEP, total phenolics extracted from OS ethanol extract; OSAP, polysaccharides extracted from OS aqueous extract; NC, normal control group; MC, model control group; PC, positive control group, nephrolithiasis rats treated with pspaishi granules; UP, urine protein. Data denoted were means7 SD, n¼10. Compared with MC. WPo 0.05. n
P o0.05. Po 0.01; compared with NC. Po 0.01.
nn
b
Table 3 Effect of OS extracts on kinetic parameters of calcium oxalate crystal growth in vitro. Group Final concentrations (mg/mL)
Ti (s)
SN ( 10 3/s)
IN (%)
SA ( 10 3/s)
IA (%)
MC OSEF OSEF OSEP OSEP OSAP OSAP
33.0 7 5.7 49.0 7 9.6n 44.0 7 22.7 10.07 11.2 28.0 7 21.4 191.0 7 47.9nn 116.0 7 62.4nn
2.267 0.70 1.387 0.40 1.327 0.41 0.907 0.37nn 1.787 0.29 0.547 0.11nn 0.927 0.29nn
– 38.9 41.6 62.2 21.2 76.1 59.3
0.88 7 0.11 0.88 7 0.16 0.88 7 0.13 0.66 7 0.22 0.64 7 0.27 0.58 7 0.14n 0.607 0.16n
– 0 0 25.0 27.3 34.1 31.8
– 0.5 0.05 0.5 0.05 0.5 0.05
OSEF, total flavonoids extracted from OS ethanol extract; OSEP, total phenolics extracted from OS ethanol extract; OSAP, polysaccharides extracted from OS aqueous extract; MC, model control group; PC, positive control group, nephrolithiasis rats treated with pspaishi granules; UP, urine protein; Ti, induction time; SN, the maximum increase of optic density with time; SA, the maximum slope of decrease of optic density at 620 nm with time; IN, the rate of nucleation (IN ¼ 100 [SNi/SNc] 100); IA, the rate of aggregation (100 [SAi/SAc] 100). Data denoted were means 7SD, n ¼5. Compared with MC. n
Po 0.05. Po 0.01.
nn
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24 h after reaction
48 h after reaction
765
72 h after reaction
Model control
+ sodium citrate (0.24 µg/mL)
+ OSHF (1.20 µg/mL)
+ OSHF (0.24 µg/mL)
+ OSHP (1.20 µg/mL)
+ OSHP (0.24 µg/mL)
+ OSAP (1.20 µg/mL)
+ OSAP (0.24 µg/mL)
Fig. 3. Inhibition of OS extracts on calcium oxalate crystallization in vitro ( 400). Normal control experiment was performed with 0.6 mmol/L calcium and 0.6 mmol/L oxalate. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
higher final concentration (0.5 mg/mL) of OSAP, nucleation percentage inhibition ratio IN was 76.1% and crystal aggregation IA was inhibited by approximately 34.1%. And it appeared in a dosedependent manner. The photographs at 400 magnification depicted particle sizes and densities of CaOx crystals during maximum rates of aggregation (Fig. 3). A visible decrease in calcium oxalate crystal
growth with increasing amounts of OSAP could be observed in Fig. 3. Not only is there a reduction in crystal size (from 1 mm average size for pure crystals to 0.5 mm average size for crystals grown with OSAP), but there is also a color change from light orange–brown for pure crystals to white-transparent for crystals with the inhibitor. Pure calcium oxalate crystals showed spherulites with smooth surfaces. However, after treated with
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0.24–1.20 mg/mL of OSAP, calcium and oxalate in the solution failed to produce massy crystals but lots of needlelike ones. Compared with standard condition (contained 5 mmol/L calcium and 0.5 mmol/L oxalate in the solution), particle sizes and densities were not significantly affected by OSEF and OSEP.
4. Discussion This study provided evidences that OS extracts acted as an anti-nephrolithiasic agent in mouse model of CaOx nephrolithiasis and as an inhibitor of CaOx crystallization. In both in vivo and in vitro experiments, OS extracts (especially OSAP) could markedly reduce CaOx crystal formation. Oral administration of OSAP at the doses of 80–160 mg/kg could significantly decrease the levels of UP and BUN, ameliorate CaOx crystal deposition induced histopathological features and diminish epithelial cell necrosis. Therefore, OSAP showed the highest therapeutic effects in comparison with the other two fractions OSEF and OSEP. These finding suggested that OSAP, i.e., the purified polysaccharides from OS were the main therapeutic materials. The important process in the kidney stone formation is the conversion of retentive crystals in renal tubules to concrete stones. Mg2 þ and OPN play essential roles in the CaOx crystalcell interaction (Konya et al., 2003). Mg2 þ could act as an inhibitor of crystal formation (Lieske, Farell and Deganello, 2004) while the possible role of OPN in kidney stone formation is inhibition of calcium oxalate monohydrate (COM) nucleation and reduction of the growth and aggregation of CaOx crystals (Khan et al., 2002; Yasui et al., 2002; Prat et al., 2011). Prolonged administration of EG, a precursor of oxalate, to rats could make a dramatic increase in the expression of OPN protein and mRNA was found in renal epithelial cells as reported previously (Khan, 1995; Okada et al., 2008). After 7-day treatment, OSEF, OSEP and OSAP at the doses of 80–160 mg/kg could significantly decrease the expression of OPN protein compared with that in MC group while all of them could not change the levels of urine Mg2 þ , well matched with amelioration of several histopathological features and reduction of CaOx crystal sizes. It indicated that OS extracts could reduce deposition of CaOx crystals to renal epithelial cells by down-regulating expression of OPN protein in the kidneys. It is well known that the type of CaOx crystals plays a role in urinary oxalate excretion. Urinary crystal has a potency to transform from calcium oxalate dihydrate (COD) or calcium oxalate trihydrate (COT) to calcium oxalate monohydrate (COM) (Yamaguchi et al., 2005). In the standard condition, there were many long hexagon crystals COM, which were increased as time extended. Citrate could inhibit calcium oxalate crystallization at the beginning of the reaction but there was no significant difference 48–72 h after the reaction compared with that in the standard condition. However, in the presence of OSAP, the main crystals in solution were COT, the acicular and needle-like crystals. OSAP could significantly affect both nucleation and aggregation, i.e., produce fewer, smaller and less aggregated crystals. The absorbance at 620 nm, which reflected the presence of crystallized CaOx, decreased spontaneously in the presence of OSAP, indicating that OSAP could inhibit crystal formation in solution and decreased in both SN and SA. OSEF and OSEP did not seem to be mediated by morphological changes of crystals. Since monosaccharide composition and molecular weight are crucial to the bioactivities of polysaccharides, the characteristics of OSAP had not been studied and thus further investigations about the structural analysis and evaluation of the bioactivities of OSAP should be done. In conclusion, polysaccharides were the main therapeutic materials in OS, playing a role as a regulator of OPN protein
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