Beruflich Dokumente
Kultur Dokumente
EXPERIMENTAL STUDY
TOPIC
JTCM | www. journaltcm. com 205 April 15, 2016 | Volume 36 | Issue 21 |
Kou YY et al. / Experimental Study
(XOD) inhibitors, which inhibit the activity of XOD, ment of hyperuricemia and gout have not been com-
an enzyme involved in purine metabolism, of which al- pletely elucidated. Thus, the aim of the present study is
lopurinol is the most often prescribed; uricosuric to evaluate the anti-hyperuricemic effect of this com-
agents, which act on the proximal tubules in the kid- pound and identify the possible underlying mecha-
neys to interfere with the absorption of uric acid from nisms in animal models of hyperuricemia.
the urine back into the blood, and include probenecid,
benzbromarone and sulfinpyrazone; and the enzyme
urate oxidase, including the recombinant form rasburi- MATERIALS AND METHODS
case, which catalyzes the oxidation of uric acid to the
more soluble allantoin which is more readily excreted Reagents
via the kidneys. However, these existing anti-hyperuri- Potassium oxonate (PO) was purchased from Adamas
cemic agents possess undesirable effects. For example, Reagent Co., Ltd. (Shanghai, China). Yeast was ob-
allopurinol, which is the drug of choice, has side effects tained from Oxoid (Basingstoke, Hampshire, Eng-
including gastrointestinal irritation, bone marrow sup- land), and allopurinol tablets from the Haizhen phar-
pression, hypersensitivity syndromes, hepatitis and maceutical company (Zhejiang, China). Benzbroma-
worsening renal function, which are unable to be toler- rone capsules were from the Huashen pharmaceutical
ated by approximately 5% of patients. Uricosuric company (Chengdu, China). Uric acid test kits (phos-
agents are used in patients with allopurinol-allergic syn- photungstic acid method), creatinine assay kits (picric
dromes as well as in under excreters with normal renal acid method) and XOD test kits (colorimetric) were
function and no history of urolithiasis. However, urico- purchased from Jiancheng Biotech (Nanjing, China).
suric agents, such as benzbromarone, also have issues. All assays were carried out according to the manufac-
Though benzbromarone is still marketed in several turer's instructions. The total protein assay kit (BCA)
countries by other drug companies, it was withdrawn was from Pierce (Rockford, IL, USA).
by Sanofi-Synthélabo in 2003 after reports of serious
hepatotoxicity. Similarly, the uricosuric agents probene- Plant material and preparation
cid and sulfinpyrazone have been reported to be neph- The RPP15, a herbal mixture comprised of 15 ingredi-
rotoxic when used to treat hyperuricemia associated ents: Ruxiang (Olibanum), Kuanjinteng (Caulis
with moderate chronic renal insufficiency.5-11 Enzyme Tinosporae Sinesis), Juemingzi (Semen Cassiae Obtusifoli-
therapy using urate oxidase is only for treating severe ae), Zhaxungao (Brag zhun), Huangkuizi (Semen Abel-
hyperuricemia and is not widely used. Thus, anti-hyper- moschi), Tibetan Changpu (Bhizoma Acori Calami),
uricemic agents have been limited in their clinical use Baxiaga (Adhatoda vasica Nees), Ercha (Catechu), Hezi
due to their severe side effects. Therefore, it is impor- (Fructus Chebulae), Anxixiang (Benzoinum), Maohezi
tant to search for alternative anti-hyperuricemic agents (Fructus Terminaliae Billericae), Tiebangchui (Radix
with more favorable toxicological profiles, and in par- Aconiti Penduli), Muxiang (Radix Aucklandiae),
ticular from natural sources. Shexiang (Moschus), Yuganzi (Fructus Phyllanthi Embli-
Tibetan medicine, which has historic connections to cae) was procured from the Qinghai Provincial Tibetan
Traditional Chinese Medicine, is an integral part of Ti- Medical Hospital, the authority in the area on Tibetan
betan culture and has been developed over many centu- medicine. According to Tibetan doctors, the recom-
ries. It is beneficial for chronic diseases such as diges- mended dosage of RPP15 for adults is 2.4 g (total raw
tive problems, arthritis, gout, asthma, and problems re- materials/day). In rats and mice, equivalent doses are
lated to the liver, kidneys and heart.12 RuPeng15 pow- approximately 7 and 10 times the human dose, respec-
der (RPP15) is a herbal multi-compound remedy com- tively.18 Based on clinical observations of the safety of
prised of 15 specific herbs that originate from tradition- the drug, we chose 10, 20 and 30 times the human
al Tibetan medicine. It is produced by combining the dose as lower, middle and high dosage, respectively.
Ten Tastes Frankincense powder with the Five Musks Three doses of RPP15 were used at 0.4, 0.8 and 1.2 g/
pill as detailed in the classical recipe recorded in "The kg suspended in distilled water and administered by
Four Medical Tantras," compiled by Yuthok Yönten oral gavage for 6-10 days in the study. Allopurinol and
Gönpo in the ninth century. Both recipes have been benzbromarone were used as positive controls and simi-
continually improved by Tibetan doctors over the cen- larly prepared in distilled water.
turies. In 1813, according to Tibetan Notes, the two
recipes were combined together to comprise what is Animals
now called RPP15. RPP15 has been described to have Fifty six male Sprague-Dawley rats (8-10 weeks of age,
anti-gout, anti-inflammatory and anti-hyperuricemic weighing 180-200 g) and 72 male Kun-Ming strain
effects.13-15 Although RPP15 has been seen to be an ef- mice (24-27 g) were obtained from The Medical Col-
fective and safe prescription in the treatment of hyper- lege Experimental Animal Center of Lanzhou Universi-
uricemia and gout for centuries, Tibetan populations ty, China (Certificate of Quality: SCXK-2009-0004).
have used it primarily based on classical records and Both rats and mice were fed a commercial laboratory
clinical experience.16,17 Its functional roles in the treat- diet (Beijing Keao Xieli Feed Co., Ltd., China) and al-
JTCM | www. journaltcm. com 206 April 15, 2016 | Volume 36 | Issue 21 |
Kou YY et al. / Experimental Study
lowed ad libitum access to food and water. All animals tive days by oral gavage 2 h after hyperuricemia induc-
were maintained on a 12 h day-night cycle and the tion. Blood samples were obtained by removing the
temperature and humidity were kept at 22-24 ℃ and eyeballs at 2 h after the last administration. This proto-
50% , respectively. They were handled gently as much col was performed on the mice only under anesthesia.
as possible to minimize animal suffering and the num- Samples were then centrifuged and the serum uric acid
ber of animals used was reduced according to the rec- and creatinine levels determined. Before the measure-
ommendation of the local and national ethics commit- ment, mice were also required to fast for 12 h, but had
tees. All experimental protocols described in this study free access to water. After the animals were sacrificed,
were conducted in accordance with The Medical Col- the liver was removed, weighed, cut into pieces, and
lege Experimental Animal Center of Lanzhou University. the XOD activity determined using a commercially
available kit.
Induction of hyperuricemia and experimental
protocol Statistical analysis
Hyperuricemic rats were generated by intraperitoneal The data are expressed as the mean ± standard devia-
injection of 250 mg/kg PO dissolved in 0.9% saline so- tion ( xˉ ± s). The analyses were conducted using SPSS
lution 1 h before the last administration of the test Statistic17.0 software (SPSS, Inc., Chicago, IL, USA).
compound.19,20 Rats were randomly allocated to seven The statistical comparison of healthy controls with hy-
groups using a random number table method, with peruricemic controls was performed by independent
eight rats in each group. Group Ⅰ served as a healthy sample t test and each experimental group with hyper-
control treated with vehicle. In group Ⅱ, hyperurice- uricemic controls analyzed using one-way analysis of
mia was induced in rats by PO and treated with vehicle variance followed by Dunnett's multiple comparison
(hyperuricemic model group). Groups Ⅲ-Ⅴ were test to determine the level of significance. A P-value of
comprised of PO-induced hyperuricemia treated with P < 0.05 was considered statistically significant.
RPP15 (0.4, 0.8, 1.2 g/kg). Groups Ⅵ and Ⅶ hyper-
uricemic rats were treated with allopurinol (5 mg/kg)
and benzbromarone (10 mg/kg) respectively, which
RESULTS
served as the positive controls. RPP15 (0.4, 0.8, 1.2 g/ Effect of RPP15 on serum uric acid levels in healthy
kg), allopurinol (5 mg/kg) and benzbromarone (10 mg/ rats
kg) were administered orally once a day for 8 days. On
The serum uric acid levels after treatment with RPP15
the seventh day, 2 h after administration, blood sam-
in comparison with healthy controls is depicted in Ta-
ples were taken from the healthy control group and the
ble 1. As shown, statistically significant differences
groups treated with RPP15 (0.4, 0.8, 1.2 g/kg) by cut-
were observed in average serum uric acid levels be-
ting the tail tips to detect the serum uric acid levels af-
tween rats fed with RPP15 and the healthy control
ter centrifuging blood samples at 3500 rpm for 10 min,
group. Serum uric acid levels in groups treated with
at 4 ℃ for plasma separation. Animals were required to
RPP15 (0.4, 0.8, 1.2 g/kg) were significantly lowered
fast for 12 h before such measurements, but had free
compared with the healthy control group (P = 0.04,
access to water. Then, on the eighth day, 250 mg/kg
P = 0.02, and P < 0.0001 respectively).
PO suspended in 0.9% saline solution was adminis-
tered intraperitoneally to each animal at 1 h before the Table 1 Effect of RPP15 on serum uric acid in healthy rats
oral administration. Before the injection, the suspen- (μmol/L, xˉ ± s)
sions were sonicated at 4 ℃ for 20 min and vortexed Group n SUA
vigorously. Blood samples were taken as previously de-
Healthy control 8 75±16
scribed at 3 h after the injection of PO to measure se-
rum uric acid and creatinine level, and then the rats Healthy-0.4 g/kg RPP15-treated 8 57±9a
were anesthetized using an intraperitoneal injection of Healthy-0.8 g/kg RPP15-treated 8 48±13a
urethane (1.0 g/kg). Then, 5 h after hyperuricemia was
Healthy-1.2 g/kg RPP15-treated 8 37±8b
induced by PO, urine was taken from the bladder to
measure uric acid and creatinine. Notes: healthy rats were treated with vehicle, 0.4 g/kg RPP15,
0.8 g/kg RPP15, or 1.2 g/kg RPP15 orally daily at 8:00 am.
For the mouse experiments, mice were randomly divid-
RPP15: RuPeng15 powder; SUA: serum uric acid. aP < 0.05,
ed into six groups with twelve mice per group: Group b
P < 0.01, versus healthy control group.
I, healthy control group treated with vehicle; Group II,
hyperuricemia group, in which hyperuricemic mice Effect of RPP15 on levels of uric acid and creatinine
were generated by orally administering yeast extract in serum and urine of rats with hyperuricemia
paste (30 g/kg), twice daily for 8 days.21,22 Groups induced by potassium oxonate
Ⅲ-Ⅵ, mice were given RPP15 (0.4, 0.8, 1.2 g/kg) and Table 2 shows that rats treated with PO had serum uric
allopurinol (5 mg/kg) on the third day after hyperurice- acid levels significantly higher than that of the healthy
mia induction. The freshly prepared compounds were control group (P < 0.0001). In addition, the allopuri-
administered to the respective groups for six consecu- nol positive control group demonstrated significantly
JTCM | www. journaltcm. com 207 April 15, 2016 | Volume 36 | Issue 21 |
Kou YY et al. / Experimental Study
lower levels of both serum and urine uric acid (P < nine level in both serum and urine were not significant-
0.0001, P = 0.001 respectively) in this model. Howev- ly different in RPP15 treated rats compared with the
er, the benzbromarone-treated group did not show sta- hyperuricemic control (all P > 0.05).
tistically significant changes in the level of either serum
or urine uric acid (P = 0.082, P = 0.484). The Effect of RPP15 on uric acid and creatinine levels in
RPP15-treated groups demonstrated decreased serum serum and activity of XOD in the liver in yeast
uric acid levels at low, middle and high dosages (all P < extract paste-induced hyperuricemia in mice
0.0001), as well as significantly reduced urine uric acid After 30 g/kg yeast extract paste was administered daily
(P = 0.020, P = 0.002, and P = 0.002 respectively) for 8 days, the serum uric acid and XOD activity in
compared with the hyperuricemic control. The creati- the liver was significantly increased in the model group
Table 2 Effect of RPP15 on levels of uric acid and creatinine in the serum and urine in potassium oxonate-induced hyperuricemia
in rats (μmol/L, xˉ ± s)
Group n SUA UUA SCr UCr
Healthy control 8 60±11 908±314 50±8 1600±610
Hyperuricemic control 8 179±22 a
838±206 49±6 1759±283
Hyperuricemic-0.4 g/kg RPP15-treated 7 117±32c 432±158b 46±5 1229±272
Hyperuricemic-0.8 g/kg RPP15-treated 7 127±25 c
297±103 c
53±3 1220±246
Hyperuricemic-1.2 g/kg RPP15-treated 7 105±22 c
222±52 c
44±9 1173±358
Hyperuricemic-allopurinol-treated 7 64±27 c
148±77 c
51±4 2092±455
Hyperuricemic-benzbromarone-treated 7 155±23 1077±234 51±6 1731±898
Notes: healthy rats were only treated with vehicle, rats with hyperuricemia induced by intraperitoneal injection of 250 mg/kg PO one hour
before the last test compound administration were treated with vehicle, 0.4 g/kg RPP15, 0.8 g/kg RPP15, 1.2 g/kg RPP15, 5 mg/kg allo-
purinol, 10 mg/kg benzbromarone for 8 days. RPP15: RuPeng15 powder; PO: potassium oxonate; SUA: serum uric acid; UUA: urine uric
acid; SCr: serum creatinine; Ucr: urine creatinine. aP < 0.01 versus healthy control group; bP < 0.05, cP < 0.01 versus hyperuricemia control
group. Some Blood Samples had hemolysis that happened when taking blood that could interfere with plasma uric acid,and we removed
the hemolysis samples, so the number of animals had been reduced.
Table 3 Effect of RPP15 on uric acid and creatinine levels in serum in yeast extract paste-induced hyperuricemia in mice (μmol/L,
xˉ ± s)
Group n SUA SCr
Healthy control 9 77±11 36±5
Hyperuricemic control 10 118±23a 38±4
Hyperuricemic-0.4 g/kg RPP15-treated 11 96±15b 38±7
Hyperuricemic-0.8 g/kg RPP15-treated 11 75±14 b
40±6
Hyperuricemic-1.2 g/kg RPP15-treated 11 45±22 b
39±5
Hyperuricemic-allopurinol-treated 9 53±12b 42±6
Notes: healthy mice treated only with vehicle, hyperuricemic mice generated by orally administering yeast extract paste (30 g/kg) twice daily
for 8 days were treated with vehicle, 0.4 g/kg RPP15, 0.8 g/kg RPP15, 1.2 g/kg RPP15, 5 mg/kg allopurinol administrated on the third day
after hyperuricemia induction for six consecutive days. RPP15: RuPeng15 powder; SUA: serum uric acid; SCrL: serum creatinine. aP <
0.05, versus healthy control group; bP < 0.01, versus hyperuricemia control group induced by yeast. Some Blood Samples had hemolysis
that happened when taking blood that could interfere with plasma uric acid, and we removed the hemolysis samples, so the number of ani-
mals had been reduced.
Table 4 Effect of RPP15 on XOD activity in the liver in yeast extract paste-induced hyperuricemia in mice (U/g protein, xˉ ± s)
Group n XOD Activity
Healthy control 12 0.61±0.23
Hyperuricemic control 12 1.51±0.49a
Hyperuricemic-0.4 g/kg RPP15-treated 12 0.72±0.17b
Hyperuricemic-0.8 g/kg RPP15-treated 12 0.76±0.11b
Hyperuricemic-1.2 g/kg RPP15-treated 12 0.55±0.15b
Hyperuricemic-allopurinol-treated 12 0.49±0.23c
Notes: when the mice were sacrificed under anesthesia, activity of XOD in the liver was measured by test kits in the six groups that includ-
ed the healthy control group, hyperuricemic model group induced by yeast, and hyperuricemic groups receiving 0.4 g/kg RPP15, 0.8 g/kg
RPP15, 1.2 g/kg RPP15 and 5 mg/kg allopurinol by intragastric administration. RPP15: RuPeng15 powder; XOD: xanthine oxidase. aP <
0.01, versus healthy control group; bP < 0.05, cP < 0.01, versus hyperuricemia control group.
JTCM | www. journaltcm. com 208 April 15, 2016 | Volume 36 | Issue 21 |
Kou YY et al. / Experimental Study
(P = 0.028, P < 0.0001) compared with the healthy but this was not statistically significant,it might be the
control. The level of serum uric acid in the RPP15 reason elevated serum uric acid induced by potassium
(0.4, 0.8, 1.2 g/kg) and allopurinol-treated groups oxonate exceeded the antihyperuricemic effects of benz-
were all significantly decreased (P = 0.008, P < 0.0001, bromarone. Our test compound RPP15 exerted a uric
P < 0.0001, P < 0.0001 ) in a dose-dependent manner acid lowering effect in both yeast-induced hyperurice-
(Table 3). The level of XOD activity in the liver was al- mia in mice and PO-induced hyperuricemia in rats. It
so shown to be reduced compared with the hyperurice- was also shown that RPP15 (0.4, 0.8, 1.2 g/kg) re-
mic mice (P = 0.037, P = 0.048, P = 0.013, P = duced serum uric acid levels in healthy rats. Excretion
0.008) (Table 4). of urine uric acid was also demonstrated to be less in
the RPP15 (0.4, 0.8, 1.2 g/kg) treated hyperuricemic
rats when compared with the hyperuricemic controls
DISCUSSION where the further metabolism of uric acid was inhibit-
Uric acid is the fi nal oxidation product of purine me- ed by oxonate. Therefore, we could conclude that
tabolism and is excreted in urine in humans, who have RPP15 primarily inhibited uric acid synthesis. In addi-
lost hepatic uricase activity during evolution. As a con- tion, in the RPP15 treated hyperuricemic mice, attenu-
sequence, humans have higher serum uric acid levels ation of serum uric acid levels was consistent with
compared with most mammals. Several factors includ- XOD inhibitory activity in the liver. Therefore, the
ing high-protein diet, alcohol consumption, high cell uric acid lowering mechanism of action of RPP15
turnover, and renal failure, can result in elevated uric might be due to its XOD-inhibitory activity, thus hin-
acid levels. In recent decades, hyperuricemia has re- dering the enzymes involved in uric acid synthesis and
ceived increasing attention as a major public health reducing the formation of uric acid.
problem because of its high prevalence and the associat- In conclusion, the present study demonstrates that
ed increases in the risk of hypertension, cardiovascular RPP15 has a uric acid lowering effects in healthy and
disease, diabetes and chronic kidney disease.23-25 Patho- hyperuricemic animals, and its mechanism of action
genic mechanisms of hyperuricemia include uric acid may be related to inhibiting the activity of XOD. How-
overproduction or under-excretion caused by aberra- ever, further studies are required to confirm this in hu-
tions in renal uric acid handling, thus XOD and urate man subjects and to investigate the underlying mecha-
transporters play important roles in urate homeostasis. nisms involved in greater detail. These data would pro-
XOD is mostly expressed in the liver and is an impor- vide a scientific basis for the clinical application of
tant enzyme in humans that plays a crucial role in me- RPP15 in hyperuricemic patients.
diating purine metabolism. The major function of
XOD is to convert hypoxanthine to xanthine and xan-
thine to uric acid, with 70% of the daily output of uric REFERENCES
acid being excreted through the kidneys.26-29
Our animal models of hyperuricemia were induced by 1 Chizyński K, Rózycka M. "Hyperuricemia". Pol Merkur
yeast and PO administration. Yeast, containing abun- Lekarski 2005; 19(113): 693-696.
dant protein, nucleic acids, vitamin B constituents, 2 Yu J, Han J, Mao J, Guo L, Gao W. Association between
serum uric acid level and the severity of coronary artery
and so forth, can be completely hydrolyzed into organ-
disease in patients with obstructive coronary artery disease.
ic alkalis (including purine and pyrimidine) and phos-
Chin Med J (Engl) 2014; 127(6): 1039-1045.
phoric acid in vivo. Therefore, after yeast administra-
3 Kang DH, Chen W. Uric acid and chronic kidney disease:
tion, normal purine metabolism can be disturbed by in-
new understanding of an old problem. Semin Nephrol
creased XOD activity and the acceleration of uric acid
2011; 31(5): 447-452.
production, so that large quantities of uric acid will be 4 Akande AA, Jimoh AK, Akinyinka OA, Olarinoye GO.
generated. This model is similar to human hyperurice- Serum uric acid level as an independent component of the
mia induced by high-protein diets due to purine and metabolic syndrome in type 2 diabetic blacks. Niger J Clin
nucleic acid metabolic disturbances.21,22 In contrast, PO Pract 2007; 10(2): 137-142.
is an inhibitor of uricase, and an intraperitoneal injec- 5 Pacher P, Nivorozhkin A, Szabó C. Therapeutic effects of
tion of oxonate partially blocks the conversion of uric xanthine oxidase inhibitors: renaissance half a century af-
acid to allantoin and hence artificially elevates serum ter the discovery of allopurinol. Pharmacol Rev 2006; 58
uric acid levels in rats to provide a hyperuricemic ani- (1): 87-114.
mal model.19,20 6 Wang Y, Bao X. Effects of uric acid on endothelial dys-
The present study demonstrated that allopurinol signif- function in early chronic kidney disease and its mecha-
icantly decreased levels of serum and urine uric acid nisms. Eur J Med Res 2013; 18(1): 26.
consistent with its XOD inhibitory activity in hyperuri- 7 Cheng CL, Chao PH, Hsu JC, Weng MM, On AW, Yang
cemia models. In hyperuricemic rats, benzbromarone, YH. Utilization patterns of antihyperuricemic agents fol-
which acts on the urate anion transport pathway and lowing safety announcement on allopurinol and benzbro-
inhibits renal proximal tubule urate reabsorption, marone by Taiwan Food and Drug Administration. Phar-
showed a trend towards an uric acid lowering effect, macoepidemiol Drug Saf 2014; 23(3): 309-313.
JTCM | www. journaltcm. com 209 April 15, 2016 | Volume 36 | Issue 21 |
Kou YY et al. / Experimental Study
JTCM | www. journaltcm. com 210 April 15, 2016 | Volume 36 | Issue 21 |