Highly-acylated anthocyanins from purple sweet potato (Ipomoea

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Bioactive Constituents, Metabolites, and Functions

Highly-acylated anthocyanins from purple sweet potato (Ipomoea batatas L.) alleviate hyperuricemia and kidney inflammation in hyperuricemic mice: possible attenuation effects on allopurinol Jiu-liang Zhang, Zi-cheng Zhang, Qing Zhou, Yang Yang, and Yu Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01810 • Publication Date (Web): 16 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019

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Journal of Agricultural and Food Chemistry

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Highly-acylated anthocyanins from purple sweet potato (Ipomoea

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batatas L.) alleviate hyperuricemia and kidney inflammation in

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hyperuricemic mice: possible attenuation effects on allopurinol

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Zi-cheng Zhang †, Qing Zhou ‡, Yang Yang †, Yu Wang †, Jiu-liang Zhang †, §, *

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Address:

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†

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430070, China

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‡ Department

College of Food Science and Technology, Huazhong Agricultural University, Wuhan,

of Pharmacy, Wuhan City Central Hospital, Tongji Medical College, Huazhong

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University of Science and Technology, Wuhan, 430014, China

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§

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430070, China

Key Laboratory of Environment Correlative Dietology, Ministry of Education, Wuhan,

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*Corresponding author:

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E-mail: [email protected]

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Tel: +86-027-87282111, Fax: +86-027-87282111.

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1 ACS Paragon Plus Environment


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Abstract

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Allopurinol is the first-line medication for hyperuricemia treatment. However, severe drug-

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related adverse effects were often reported among patients who received allopurinol

21

administration. This study is aimed to evaluate the possible attenuation effects of highly-

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acylated anthocyanins from purple sweet potato (HAA-PSP) on hyperuricemia and kidney

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inflammation in hyperuricemic mice treated with allopurinol. Compared with 5 mg kg-1

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allopurinol used alone, the combination of 25 mg kg-1 HAA-PSP and 2.5 mg kg-1 allopurinol

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could not only reduce serum uric acid level in hyperuricemic mice, but also attenuate the

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kidney damage as indicated by the level of serum biomarkers as well as histopathological

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examination. Inflammatory response was partially mitigated by inhibiting the protein

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expression of typical cytokines in the kidney. Our findings provide new evidence for the

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supplementary therapeutic potential of HAA-PSP with allopurinol on hyperuricemia and

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inflammation-related syndromes. Moreover, this study provides theoretical basis for

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assessing potential of anthocyanin-rich foods in health.

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Keywords:

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Highly-acylated anthocyanins; Purple sweet potato (Ipomoea batatas L.); Hyperuricemia; Anti-inflammatory; Allopurinol

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Introduction

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Long-term hyperuricemia could cause the deposition of uric acid crystals in and around

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joints in human, leading to one of the extensive metabolic diseases, gout. The occurrence and

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development of hyperuricemia is relevant to the purine-metabolism disorder and persistent

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increase of serum urate concentrations

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of people were found suffering from hyperuricemia and gout. In China, the number of

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patients with hyperuricemia had grown to over 180 million by 2014, making it the second

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metabolic disease after diabetes

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linked with the development of chronic kidney diseases 6, 7. Once xanthine oxidase (XO) with

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increased catalytic activity produces excessive uric acid (UA) which is beyond the renal

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excretion capacity, the uric acid that cannot be excreted in vitro will deposits and crystallizes

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in the kidney, and directly causes renal injury. Secondly, the UA crystals deposited in the

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kidney will induce oxidative stress, inflammatory cell accumulation and release of

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inflammatory cytokines by regulating the nuclear factor-κB (NF-κB) pathway, which may

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together aggravate renal injury 8, 9.

4, 5.

1-3.

In the past decades worldwide, a growing number

In addition, hyperuricemia is believed to be closely

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As an inhibitor of XO, allopurinol can effectively suppress the conversion of xanthine to

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UA in the purine synthesis pathway, which will further reduce the UA level in human. For

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over 50 years, allopurinol has been widely used as the first-line medication for pharmacologic

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urate-lowering therapy of gout and hyperuricemia 10. However, the clinical use of allopurinol

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still has its limitations. First, previous research has revealed that the uric acid produced by

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normal human purine metabolism is the most abundant plasma antioxidant molecule

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Blindly pursuing the minimum level of uric acid may break the purine metabolic balance,

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resulting in a series of negative chain reactions, thereby increasing the incidence of other

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diseases. In particular, allopurinol may cause allopurinol hypersensitivity syndrome (AHS) in

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patients, a rare but life-threatening disease suspected to be immune-mediated and 3 ACS Paragon Plus Environment

11.


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characterized by Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) 12, 13.

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The patients of AHS may suffer from leukocytosis, eosinophilia, fever, acute liver injury and

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kidney damage, and can be fatal in up to 32% of cases

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spared from the death are often be accompanied with sequelae including renal insufficiency 15.

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Pharmacogenomic studies have indicated that individuals carrying human leukocyte antigen

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HLA-B*5801 are more susceptible to AHS, especially for the Asians (with a prevalence of

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7.4%) 16, 17. According to the guidelines of 2012 American College of Rheumatology, before

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administrating allopurinol, high-risk patients need to take the detection of HLA-B*5801, a

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testing marker of AHS, to assess the risk of drug use18, 19. However, due to the limitations of

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genetic screening and detection techniques in China, the complexity of current detection

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methods, and other influencing factors, the detection of HLA-B*5801 allele is still limited to

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wide clinical application

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potentially high-risk group of AHS when choosing allopurinol for the therapy. In addition,

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the dosage and side effects of allopurinol are more uncertain for the hyperuricemia patients

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who are detected to carry the HLA-B*5801 allele.

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20.

14,

and even those who have been

That means patients with hyperuricemia in China may be a

Sweet potato (Ipomoea batatas L.), the seventh largest crop in the world, is a root-derived 21.

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food that originated in Latin America

One of its varieties, purple sweet potato (PSP), is

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mainly planted in Japan and Korea, which was introduced into China in the 1980s 22. Because

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of its high contents of anthocyanins and other phytochemicals beneficial to human health,

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purple sweet potato is popular among consumers as a functional food. The composition of

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anthocyanins in PSP is complex, and acylated anthocyanins are the dominant component

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(93%) 23. Anthocyanins in PSP mainly belong to the families of cyanidin and peonidin, with

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several non-, mono- or di-acylated glucosides and acyl groups such as feruloyl, caffeoyl,

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hydroxybenzyl or coumaroyl groups 24. The functional activity and property of anthocyanins

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vary with their chemical structures. Acylated anthocyanins display higher pH stability 25 than


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anthocyanidins and various health benefits, such as cancer prevention 26, visual protection 27,

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anti-diabetes activity

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against oxidative stress damage 31. Our previous research has reported the anti-hyperuricemic

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effect of anthocyanin-rich purple sweet potato extracts (APSPE) on potassium oxonate-

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induced hyperuricemic mice 32. Furthermore, our in vitro experiment confirmed that highly-

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acylated anthocyanins from purple sweet potato (HAA-PSP) is the main active component in

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APSPE that inhibits XO

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inhibitor, which calls for further in vivo validation. Besides, it is not clear yet about the

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mechanism by which anthocyanins relieve organ inflammation caused by hyperuricemia and

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its supplementary therapeutic effect with allopurinol.

28,

hepatoprotective activity

33.

29,

antioxidant capacity

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and protection

However, HAA-PSP was only presumed to be a potential XO

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In this study, we evaluated the hypouricemic and renal protective effects of HAA-PSP,

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allopurinol and their combination on potassium oxonate-induced hyperuricemic mice.

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Important biochemical indicators in serum that reflect renal injury and oxidative stress level,

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such as creatinine (Cr), blood urea nitrogen (BUN), total superoxide dismutase (T-SOD)

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activity

and

malondialdehyde

(MDA)

level,

were

accurately

measured.

Renal

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histopathological examination and western blot analysis of inflammatory molecules such as

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cytokines TNF-α, TGF-β1, IL-6, IL-1β, ICAM-1, COX-2 and NF-κB in kidney were

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performed to explore the possible mechanisms. Our results provide important insights into

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the potential attenuation therapeutic effects of HAA-PSP on hyperuricemia and

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inflammation-related syndromes treated with allopurinol.

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Materials and Methods

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Plant Materials and Chemicals

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The dried purple sweet potato powder (Ipomoea batatas L. cultivar Eshu No.8) came from

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the donation by Puzetian Food Co. Limited (Wuhan, Hubei, China). Potassium oxonate and

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allopurinol were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Assay kits 5 ACS Paragon Plus Environment


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for determination of serum UA, Cr, BUN, T-SOD, and MDA were obtained from Nanjing

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Jiancheng Biotechnology Institute (Nanjing, Jiangsu, China). Bicinchoninic acid (BCA)

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protein assay kit was purchased from Beyotime Biotechnology Co. (Haimen, Jiangsu, China).

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Peonidin3-O-(6-O-(E)-caffeoyl-(2-O-(6-O-(E)-feruloyl)-β-D-glucopyranosyl)-β-D-

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glucopyranoside)-D-glucopyranoside (Peo 3-caffeoyl-feruloyl soph-5-glc, purity> 97%) was

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obtained from Luo in our lab

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Chemical Co. (Saint Louis, Missouri, USA). Formic acid in HPLC grade was purchased from

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Guangfu Fine Chemical Research Institute (Tianjin, China). Ultrapure water was purified by

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Milli-Q Direct 8 System from Millipore Co. (Billerica, Massachusetts, USA). All other

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regular reagents were of analytical grade and were purchased from Sinopharm Chemical

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Reagent Co., Ltd (Shanghai, China).

122 123 124

29.

Acetonitrile in HPLC grade was purchased from Sigma

Preparation and characterization of highly-acylated anthocyanins from purple sweet potato The extraction, enrichment and purification of anthocyanins were carried out in accordance 32.

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with our previously described method

Dried purple sweet potato powder was dissolved,

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whisked by magnetic force in 40% anhydrous ethanol with the temperature of 60°C,

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centrifuged and evaporated. Then, the crude anthocyanin extract, which is the concentrated

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supernatant, was loaded on an AB-8 macroporous resin column with weak polarity (particle

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size 0.3−1.25 mm; 25 mm × 100 mm; purchased from Nankai Hecheng Science &

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Technology Co., Tianjin, China) and washed with 70% acidic ethanol. The sample collected

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by elution was evaporated, freeze-dried, and finally named as Anthocyanin-rich Purple Sweet

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Potato Extract (APSPE). Next, APSPE received a secondary purification using a Daisogel SP-120-

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30/50-ODS-B column (DAΪSO Co., Osaka, Japan) to separate different enriched fractions.

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The primary purified product was loaded on the column and then sequentially washed with

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water and 10% methanol solution (v/v). Subsequently, Fraction 1, 2, 3 and 4 were collected


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by isocratic elution with 20%, 30%, 40% and 50% methanol (v/v) in sequence. All elution

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solvents were added with 0.1% TFA. Since fraction 3 was found to have a stronger inhibitory

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effect on XO in vitro according to our previous report 33, it was further evaporated, freeze-

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dried, named as HAA-PSP and used in this experiment.

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In our previous study, HAA-PSP was characterized by HPLC-DAD with C18 column and 33.

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11 individual anthocyanins were separated

Meanwhile, all structures of 11 individual

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anthocyanins were further compared and identified based on our UPLC-ESI-MS/MS database,

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and the results were displayed in Figure S1 (Supporting Information) and Table 1. Besides,

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the quantification of HAA-PSP was performed by HPLC-DAD at 525 nm using external

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standard method with Peo 3-caffeoyl-feruloyl soph-5-glc as the standard sample. The whole

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quantitative analysis was operated three times under the same conditions as the samples did.

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The anthocyanin content of HAA-PSP was finally determined as 599.1 ± 1.9 mg Peo 3-

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caffeoyl-feruloyl soph-5-glc equivalent g-1.

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Animals

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8-week male Kun-Ming strain mice with SPF grade used in this study were purchased from

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the Hubei Provincial Laboratory Animal Public Service Center (Certificate No. SCXK

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(Hubei) 2015-0019, Wuhan, China), which is under the Hubei Provincial Center for Disease

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Control and Prevention. The whole animal experiment was carried out at the Center for

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Laboratory Animals in Huazhong Agricultural University with the approval of the Scientific

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Ethics Committee (Permission No. HZAUMO-2017-031). All procedures involving animals

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throughout the experimental period were strictly followed the Chinese legislation on the use

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and care of laboratory animals. During the study, mice were housed with 8 per cage under

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external environmental conditions of 25 ± 1°C, 40 ± 10% humidity and normal light/dark (12

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h/12 h) cycle. The animals had free access to distilled water as well as standard laboratory



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pellet diet during whole experimental period, and they had one week of acclimation before

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the experiments beginning.

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Animal model of potassium oxonate induced-hyperuricemic mice

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At the beginning of the formal experiment, mice were randomly divided into 6 groups,

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including normal control group (NC group), hyperuricemia group (HUA group), allopurinol

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group (AP group), HAA-PSP group (HAA group), HAA-PSP with low allopurinol group

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(HAA-LAP group) and HAA-PSP with high allopurinol group (HAA-HAP group). To induce

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hyperuricemia, 5 groups of mice except for the NC group were orally administered with 250

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mg kg-1 bw-1 potassium oxonate solution (dispersed in 0.5% carboxymethyl cellulose sodium

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(CMC)-Na) once daily for 7 consecutive days. Mice in NC group were treated with solvent

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vehicle. The dosages of allopurinol (5 mg kg-1 bw-1) and HAA-PSP (25 mg kg-1 bw-1) used in

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AP group, HAA group, and HAA-HAP group were determined based on the conversion from

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human clinical practice reported in Chinese Pharmacopoeia Committee, our previous report

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and our preliminary studies 32. In order to determine whether the combination of low-dosage

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allopurinol and HAA-PSP can enhance the hypouricemic effect and alleviate kidney

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inflammation, the allopurinol dose in the HAA-LAP group was set as 2.5 mg kg-1 bw-1, which

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was half of the dose in AP group and HAA-HAP group. The test agents (allopurinol and

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HAA-PSP), alone or in combination, were dispersed in 0.5% CMC-Na solution and was

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administered by gavage 1 h after the administration of potassium oxonate. Food, but not

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water, was withdrawn from the cages 1 h before the oral administration. The volume of drug

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was calculated based on the body mass of the mice measured immediately before the

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administration. Mice received diet fasting for 12 h before the biological samples collection on

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the 7th day, with distilled water still available.

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Sample collection of blood and organ tissues


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The mice were sacrificed 1 h after the final administration on the seventh day. Whole

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blood sample was collected and kept clotted for 1 h and centrifuged at 2050 × g for 10 min.

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The serum supernatant was collected and store at –20°C until analysis. Kidney and liver

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tissues were separated on the ice plate rapidly and carefully. After cleaning with saline, they

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were weighed quickly. One of the two kidney tissue samples was immediately immersing in

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4% phosphate-buffered formalin (pH 7.1) for histopathological analysis, while the rest was

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quickly stored at –80°C for western blot analysis.

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Determination of organ indexes and serum biochemical indicators

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The liver and renal index on a fresh weight basis was calculated according to the following

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formulas:

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𝐿𝑖𝑣𝑒𝑟 𝐼𝑛𝑑𝑒𝑥 (𝑚𝑔/𝑔) =

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𝐾𝑖𝑑𝑛𝑒𝑦 𝐼𝑛𝑑𝑒𝑥 (𝑚𝑔/𝑔) =

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑙𝑖𝑣𝑒𝑟 (𝑚𝑔) 𝑏𝑜𝑑𝑦 𝑚𝑎𝑠𝑠 (𝑔) 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑘𝑖𝑑𝑛𝑒𝑦 (𝑚𝑔) 𝑏𝑜𝑑𝑦 𝑚𝑎𝑠𝑠 (𝑔)

196

The levels of UA, BUN, Cr, T-SOD activity and MDA in the serum were determined using

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commercially available kits based on the instructions of the manufacturers. Each assay was

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performed in triplicate.

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Histopathological examination of renal tissues

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Kidney samples fixed with formalin were gradually dehydrated in ethanol, clarified in

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xylene, embedded in paraffin, sectioned and stained with haematoxylin and eosin (H&E) for

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microscopy observation at a 400 × magnifications.

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Western blot analysis

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Kidney samples stored at –80°C were prepared for western blot analysis of TNF-α, TGF-

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β1, IL-6, IL-1β, ICAM-1, COX-2 and NF-κB p65 according to standard procedures. The

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whole procedure was carried out at 4°C. Briefly, kidney cortex was intermittently

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homogenized on ice in RIPA buffer including PMSF for 30 min and then centrifuged at

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13200 × g for 5 min. The supernatant was collected, sub-packed and stored at 20°C. BCA 9 ACS Paragon Plus Environment


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protein assay kit was used to determine the protein content of the supernatant and bovine

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serum albumin was used as standard sample. The total proteins were incubated in boiling

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water for 10 min.

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Electrophoresis plastic was composed of 5% concentration gel and 12% separation gel.

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Equal amount (40 μg) of different samples was separated on gels, respectively, until the

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target proteins were fully separated. The proteins were then electrophoretically transferred to

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polyvinylidene difluoride membrane. The membranes were blocked in TBST (Tris-buffered

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saline containing 0.1% Tween-20) containing 5% skimmed milk powder and incubated for 2

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h. Then, the membranes were treated individually with specific antibody diluted in TBST,

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including GAPDH (1:1000), TNF-α (1:2000), TGF-β1 (1:1000), IL-6 (1:1000), IL-1β

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(1:1000), ICAM-1 (1:200), COX-2 (1:200) and NF-κB p65 (1:1000) antibodies, respectively.

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The information about the primary antibodies was listed in Table 2.

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HRP-conjugated goat anti-rabbit and anti-mouse IgG (Table 2) diluted in TBST were used

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as the secondary antibody (1:50000) to detect the immunoreactive bands. They were made

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visible by the enhanced chemiluminescence and exposed to X-ray film. The contents of target

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proteins were analyzed using Glyko BandScan software (Glyko, Novato, CA, USA) and

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normalized by the respective blotting from mGAPDH.

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Statistical analysis

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All the results were presented as the average of three replications and expressed as mean ±

228

SD values. Using IBM SPSS® Statistics 22 and Origin version 9.0 for Windows to perform

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One-way analysis of variance and Duncan multiple tests in order to determine the significant

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difference at p < 0.05.

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Results

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Hypouricemic

effect

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hyperuricemic mice

of

HAA-PSP,

allopurinol


and

their

combination

in

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As shown in Fig.1, after 7 days of potassium oxonate administration, HUA group had a

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significantly (p < 0.05) higher level of serum UA compared with NC group, indicating that

236

hyperuricemia was effectively established in mice. After HAA-PSP treatment at 25 mg kg-1

237

bw-1, the UA level was significantly (p < 0.05) decreased from 91.56 μmol L-1 to 52.33 μmol

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L-1 (by nearly 43%). Meanwhile, the UA level in AP group showed a dramatic (p < 0.05)

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decrease and was below the detection limit, which was the same trend as in our previous

240

study

241

effect was more obvious (p < 0.05) than the use of HAA-PSP alone. With increasing

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proportion of allopurinol in the drug system, the UA levels in two cotreatment group were

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decreased in a dose-dependent manner by 69.69% in HAA-LAP group and by 94.31% in

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HAA-HAP group, respectively.

245 246

32.

When HAA-PSP and allopurinol were applied together, the anti-hyperuricemic

Effects of HAA-PSP, allopurinol and their combination on body and organ weight and serum biochemical parameters in hyperuricemic mice

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The body mass, liver weights, kidney weights and organ indexes of hyperuricemic mice

248

were presented in Table 3. All the mice in six groups showed no significant differences,

249

indicating that the combination use of HAA-PSP and allopurinol did not have severe toxic

250

side-effects on organs.

251

As the BUN and Cr level in serum are effective indicators of renal function 34, and there is

252

a close correlation between Cr level and UA synthesis in gout patients 35, levels of BUN and

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Cr were evaluated, and the results were displayed in Fig.2. As expected, compared with NC

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group, the serum Cr level in HUA group (Fig.2A) was significantly (p < 0.05) increased.

255

After treatment with allopurinol alone or in combination with HAA-PSP, the Cr levels were

256

significantly decreased (p < 0.05) to approximately the normal value. As shown in Fig.2B,

257

the BUN level of AP group was increased to 9.08 mmol L-1, while the cotreatment of HAA-

258

PSP and allopurinol could restore it to the normal level.



259

The T-SOD activity and MDA level in the serum were measured and illustrated

260

respectively. When the oxidative stress is developed, the T-SOD activity will be decreased

261

with an increase in MDA level observed, as the results of mice in HUA group (Fig. 2C and

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2D). What’s more, the individual usage of allopurinol was found to have little effect on both

263

T-SOD and MDA level in the serum. However, when anthocyanins were combined with

264

allopurinol, the T-SOD activity increased while the MDA level showed an obvious decrease.

265

These results suggested that the application of HAA-PSP alone or together with low-dosage

266

allopurinol could attenuate the oxidative stress caused by potassium oxonate through up-

267

regulating the T-SOD activity and down-regulating the MDA level in the serum.

268

Histopathological examination

269

Histopathological analysis of hyperuricemic mice’s renal tissues were displayed in Fig.3.

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Mice in NC group maintained normal kidney structure, without significant inflammatory

271

responses. The morphology of glomerulus, renal tubules and interstitium stayed normal with

272

a compact arrangement of cells. However, the kidneys of mice in HUA group displayed

273

significant morphological changes in glomerulus and focal infiltration of inflammatory cells

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(Fig. 3A-3B). What’s worse, the renal tissues of AP group showed an obvious proximal

275

convoluted tubular injury characterized by a vacuolar degeneration of tubular cells and

276

swelling of epithelial cells. The glomerular basement membrane was thickened and the

277

glomerular mesangial matrix was slightly proliferated (Fig. 3C) compared with those of the

278

NC group. When the same concentration of allopurinol was used in combination with HAA-

279

PSP (HAA-HAP group), the renal injury of mice was slightly attenuated. Although the

280

vacuolar degeneration of tubular cells and swelling of epithelial cells could still be observed,

281

the glomerular injury and inflammatory cell infiltration were alleviated significantly (Fig. 3F).

282

The examination results of renal tissues in HAA group (Fig. 3D) and HAA-LAP group (Fig.

283

3E) suggested that when the dosage of allopurinol decreased (2.5 mg kg-1 bw-1 in HAA-LAP


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group and none in HAA group, respectively), the renal injury of mice was alleviated to

285

varying degrees, as indicated by decreased inflammatory cell infiltration, restored

286

morphological structure of glomerulus and proximal convoluted tubules, and reduced

287

thickening of glomerular basement membrane and proliferation of glomerular mesangial

288

matrix. These results indicated that HAA-PSP can ameliorate the renal damage caused by

289

hyperuricemia or the application of allopurinol.

290 291

Effect of HAA-PSP on renal inflammation by suppressing renal inflammatory cytokines and inhibiting NF-κB activation

292

As shown in Fig.4, potassium oxonate significantly (p < 0.05) increased the protein levels

293

of renal inflammatory cytokines including TNF-α, TGF-β1, IL-6 and IL-1β in hyperuricemic

294

mice. Surprisingly, after the treatment with high dosage of allopurinol (AP group), the protein

295

levels of the above 4 inflammatory cytokines all significantly (p < 0.05) increased rather than

296

declined. Instead, HAA-PSP could significantly (p < 0.05) inhibit the expression of TNF-α,

297

TGF-β1, IL-6 and IL-1β protein in the kidney, whether used alone or in combination with

298

allopurinol. Particularly, although the dosage of allopurinol was different between HAA-LAP

299

and HAA-HAP group, the determination results were similar. Even so, the cotreatment with

300

HAA-PSP and low-dosage allopurinol still exhibited a good and stable effect in regulating

301

renal inflammatory cytokines levels, which was also observed in the protein levels of ICAM-

302

1, COX-2 and NF-κB p65 in renal tissues of mice. These results demonstrated that HAA-PSP

303

could suppress the expression of renal inflammatory cytokines, inhibit the activation of NF-

304

κB p65, alleviate the renal inflammation in hyperuricemic mice and reduce the adverse

305

effects of allopurinol.

306

Discussion

307

Allopurinol is the first-line clinical therapy for the treatment of gout and hyperuricemia.

308

However, severe drug-related adverse effects can be observed among patients who received 13 ACS Paragon Plus Environment


36.

309

allopurinol administration, including AHS, SJS and TEN

310

more likely to occur in Asian people than in people from other regions. What’s worse, the

311

usage of high-dose allopurinol will also reduce serum uric acid level to below normal levels,

312

and long-term use may easily trigger other diseases. Pharmacologically, it is possible to take

313

advantage of other compounds or natural products to decrease the dosage of allopurinol and

314

further reduce its dose-related adverse effects without sacrificing the therapeutic effect. In

315

previous studies, the hypouricemic effect of anthocyanin from purple sweet potato has been

316

reported and evaluated

317

complexity of the composition, the key active components in APSPE and their effects on

318

hyperuricemia remain unknown. In a subsequent study in vitro, we attempted to conduct a

319

secondary purification of APSPE on a ODS column to separate copigments, lowly-acylated

320

anthocyanins and highly-acylated anthocyanins. The results suggested that highly-acylated

321

anthocyanins have strong inhibitory activity on XO

322

HAA-PSP administration was significantly lower than that of APSPE used in the previous

323

study, but the anti-hyperuricemic bioactivity was much stronger. A comparison of the

324

differences in anthocyanin content and composition between HAA-PSP and APSPE showed

325

that highly-acylated anthocyanin in purple sweet potato is the key active component for the

326

anti-hyperuricemic effect. In addition, the combination of HAA-PSP and 2.5 mg kg-1 bw-1

327

allopurinol can not only reduce UA level to normal range, but also avoid the extreme low

328

level of UA caused by the individual usage of high-dose allopurinol. The above results

329

suggest that HAA-PSP has certain supplementary therapeutic potential in the treatment of

330

hyperuricemia.

331

32, 37.

These adverse effects are far

However, due to the incomplete sample purification and

33.

In the present study, the dosage of

Since it is widely acknowledged that hyperuricemia is a risk factor for the progression of 38,

332

chronic kidney diseases

this study aims to evaluate the potential of highly-acylated

333

anthocyanins as a supplement of allopurinol on renal protection of hyperuricemic mice.


Page 14 of 38

Page 15 of 38


334

Cotreatment with HAA-PSP and allopurinol could effectively regulate serum Cr level in

335

hyperuricemic mice, and restore the BUN level in serum to normal. Hence, the combination

336

of highly-acylated anthocyanins with allopurinol may have a better mitigation effect on

337

kidney damage in hyperuricemic mice. In addition, histopathological analysis results of renal

338

tissues demonstrated that HAA-PSP could attenuate renal injury characterized by

339

morphological change of glomerulus, inflammatory cell infiltration, proximal convoluted

340

tubular injury, thickening of glomerular basement membrane and mesangial matrix

341

proliferation. Thus, it can be speculated that HAA-PSP can reduce the renal injury in

342

hyperuricemic mice caused by hyperuricemia or the application of allopurinol.

343

Previous study has pointed out that hyperuricemia patients are more likely to suffer from

344

oxidative stress-induced renal injury, due to the long-lasting toxic effects of high serum uric

345

acid level 39. Kidney is one of the primary viscera affected by oxidative stress resulting from

346

excessive accumulation of UA 40. Potassium oxonate is believed to stimulate oxidants, such

347

as O2·− and the product of oxidative stress MDA, while reduce the activity of antioxidant

348

enzymes SOD 41. In the present study, T-SOD activity was significantly inhibited while the

349

MDA level was increased in hyperuricemic mice. Highly-acylated anthocyanins seemed to

350

have a better effect on oxidative stress compared with allopurinol, which is consistent with

351

our previous research findings 30. The cotreatment of HAA-PSP and low-dosage allopurinol

352

surprisingly displayed strong effects on serum T-SOD activity, MDA level and oxidative

353

stress, further demonstrating its good prospects to be applied in the treatment of

354

hyperuricemia.

355

Inflammatory response is known as the pathologic feature of hyperuricemia and

356

contributes to initiating and developing renal injury 42. The activation and release of various

357

inflammatory molecules in renal tissue, such as cytokines TNF-α, IL-6, IL-1β and TGF-β1 43,

358

adhesion molecule ICAM-1

44

and synthetase COX-2 45, has played an important role in the



359

progression of nephritis and hyperuricemia. As important inflammatory cytokines, TNF-α, as

360

well as IL-6 and IL-1β of the interleukin family could participate in the inflammatory

361

response and promote the proliferation of glomerular mesangial cells and mesangial matrix 46,

362

47.

363

regulate the secretion of excessive collagen fibers and promote the formation of renal

364

interstitial fibrosis

365

inflammatory cells and endothelial cells, enhance the recruitment of inflammatory leukocytes

366

and prolong the infiltration time of inflammatory cells 49. Over-expression of COX-2 in renal

367

cortex can promote inflammatory cell infiltration and accumulation, which will also stimulate

368

the synthesis of ROS

369

inflammatory response. The expression of renal inflammatory cytokines such as TNF-α,

370

TGF-β1, IL-6 and IL-1β was stimulated, and the protein levels of ICAM-1 and COX-2 were

371

increased in hyperuricemic mice. Surprisingly, although we proved that high-dose allopurinol

372

has a significant anti-hyperuricemic effect, its administration was found to aggravate the

373

renal inflammation of hyperuricemic mice, possibly due to the toxic side effects of

374

allopurinol, which will increase the burden of the kidney and thus enhance the inflammatory

375

response. However, HAA-PSP could effectively down-regulate the expression of renal

376

inflammatory molecules in hyperuricemic mice, reduce the additional damage caused by

377

allopurinol, and then alleviate a variety of renal injuries including glomerulosclerosis, renal

378

interstitial fibrosis and inflammatory cell infiltration.

379

Meanwhile, TGF-β can not only accelerate the process of glomerulosclerosis, but also

48.

The adhesion molecule ICAM-1 could increase the adhesion between

50.

In this study, hyperuricemic mice were found to suffer from

In addition, the activation of NF-κB is also one of the major causes of renal inflammation

380

51, 52.

381

induced by potassium oxonate administration, indicating its causal role in the pathogenesis of

382

renal inflammatory injury 53. As a rapid response transcription factor, activated NF-κB binds

383

to the specific DNA sequences in the nucleus to initiate and up-regulate the transcription of

Activation of NF-κB signaling pathway was frequently observed in kidney dysfunction


Page 16 of 38

Page 17 of 38


54.

384

inflammatory mediators and cytokines

As a result, proinflammatory cytokines, oxygen

385

free radicals and other inflammatory mediators are produced and infiltrated into renal tissues

386

in large quantities, further triggering inflammatory responses and kidney dysfunction. In

387

addition, the cytokine TNF-α and IL-1β induced by activated NF-κB could also further

388

activate the NF-κB signaling pathway

389

significantly activated in hyperuricemic mice, and high dose of allopurinol aggravated the

390

activation of NF-κB p65, which brought about a further increase in the release of

391

inflammatory cytokines in kidneys of hyperuricemic mice. On the contrary, application of

392

HAA-PSP alone or in combination with allopurinol could effectively hinder the activation

393

process of NF-κB p65, reducing the release of various inflammatory mediators and relieving

394

kidney damage.

55.

Our results showed that NF-κB p65 was

395

In this study, we investigated the possible attenuation effects of HAA-PSP on

396

hyperuricemia and kidney inflammation in potassium oxonate-induced hyperuricemic mice

397

treated with allopurinol. The combination of HAA-PSP at 25 mg kg-1 and allopurinol at 2.5

398

mg kg-1 showed a significantly better hypouricemic effect and toxicity reducing effects on the

399

kidney damage compared with the application of HAA-PSP or allopurinol alone. The

400

cotreatment with the two components could not only alleviate the oxidative stress by

401

regulating serum T-SOD activity and MDA level, but also down-regulate the protein

402

expression of typical cytokines by mediating the NF-κB pathway, thereby reducing the

403

infiltration of inflammatory cells and alleviating kidney damage. Our results provide new

404

evidence for the potential supplementary effect of HAA-PSP with allopurinol on

405

hyperuricemia and inflammation-related syndromes. Moreover, this study provides

406

theoretical basis for assessing potential of anthocyanin-rich foods in health.

407

Acknowledgments



408

This research is supported by the Fundamental Research Funds for the Central

409

Universities of China (No. 2662018PY022), the Natural Science Foundation of Hubei

410

Province (No. 2018CFB738) and the Clinical Research Project of Health and Family

411

Planning Commission of Wuhan Municipality (No. WX13A05). The authors would like to

412

thank Mr. Zuo-xiong Liu for his participation in the language modification process.

413

The authors declare that there are no conflicts of interest.

414

415

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580



581

Figure Legends:

582

Fig.1 Effects of HAA-PSP, allopurinol and their combination on UA level in serum of

583

hyperuricemic mice. Values were expressed as the means ± SD (n=8). Different letters

584

marked above the bar are significantly different by ANOVA multiple test (p < 0.05).

585

Fig.2 Effects of HAA-PSP, allopurinol and their combination on (A) Cr level, (B) BUN level,

586

(C) T-SOD activity and (D) MDA level in serum of hyperuricemic mice. Values were

587

expressed as the means ± SD (n=8). Different letters marked above the bar are

588

significantly different by ANOVA multiple test (p < 0.05).

589

Fig.3 Effects of Fraction 3, allopurinol and their combination on the renal histopathology of

590

hyperuricemic mice. (A) kidney section of normal control group; (B) kidney section of

591

hyperuricemia group; (C) kidney section of 2.5 mg kg-1 allopurinol group; (D) kidney

592

section of 25 mg kg-1 HAA-PSP group; (E) kidney section of 25 mg kg-1HAA-PSP

593

with 2.5 mg kg-1 allopurinol group; (F) kidney section of 25 mg kg-1 HAA-PSP with 5

594

mg kg-1 allopurinol group. Magnification 400×, Scale bar: 50.00 μm

595

Fig.4 Western blot of HAA-PSP, allopurinol and their combination on protein expression for

596

TNF-α, TGF-β1, IL-6, IL-1β, ICAM-1, COX-2 and NF-κB p65 in renal tissue of

597

hyperuricemic mice. The contents of target proteins were normalized to GAPDH.

598

Values were expressed as the means ± SD (n=8). Different letters marked above the bar

599

are significantly different by ANOVA multiple test (p < 0.05).


Page 26 of 38

Page 27 of 38

600


Table 1 Identification and quantification of HAA-PSP by HPLC at 525 nm Identification

Quantification (mg/g)

Cyanidin 3-(6’,6’’-dicaffeoyl sophoroside)-5-glucoside

34.8 ± 6.5

Cyanidin 3-(6’caffeoyl-6’’p-hydroxybenzoyl sophoroside)-5-glucoside

28.5 ± 1.6

Peonidin 3-caffeoyl sophoroside-5-glucoside

37.6 ± 3.5

Cyanidin 3-(6’caffeoyl-6’’feruloyl sophoroside)-5-glucoside

45.9 ± 1.1

Peonidin 3-(6’,6’’-dicaffeoyl sophoroside)-5-glucoside

102.9 ± 5.9

Peonidin 3-(6’caffeoyl-6’’p-hydroxybenzoyl sophoroside)-5-glucoside

160.8 ± 13.8

Peonidin 3-(6’caffeoyl-6’’feruloyl sophoroside)-5-glucoside

165.0 ± 8.9

Peonidin 3-(6’caffeoyl-6’’p-coumaryl sophoroside)-5-glucoside

3.1 ± 0.5

Peonidin 3-(6’feruloyl-6’’p-hydroxybenzoyl sophoroside)-5-glucoside

3.2 ± 0.2

Peonidin 3-(6’coumaryl-6’’p-hydroxybenzoyl sophoroside)-5-glucoside

6.8 ± 0.8

Peonidin 3-(6’,6’’-diferuloyl sophoroside)-5-glucoside

10.4 ± 0.1

Total

599.1 ± 1.9

601 602

* tR represents retention time.

603

* Peo 3-caffeoyl-feruloyl soph-5-glc was used as the standard sample in quantification.

604

* Values were expressed as the means ± SD (n=3). 27 ACS Paragon Plus Environment


605

Page 28 of 38

Table 2 Antibodies used for Western Blot analysis

Company

Description

Catalog Number

Goodhere Biotechnology

Rabbit mGAPDH antibody

AB-P-R 001

Proteintech Group

Mouse mTNF-α monoclonal antibody

60291-1-Ig

(Chicago, IL, USA)

Rabbit mIL-6 antibody

21865-1-AP

Rabbit mNF-κB P65 rela antibody

10745-1-AP

Affinity Biosciences

Rabbit mTGF-β1 antibody

AF1027

(Cincinnati, OH, USA)

Rabbit mIL-1β antibody

DF6251

Boster Biological Technology

Rabbit mCOX-2 antibody

BA0738

(Wuhan, PR China)

Rabbit mICAM-1 antibody

BA2189

Goat anti-mouse-IgG-HRP

BA1051

Goat anti-rabbit-IgG-HRP

BA1054

(Hangzhou, PR China)


Page 29 of 38

607


Table 3 Effects of HAA-PSP, allopurinol and their combination on body, liver and kidney weights in hyperuricemic mice. Group

Body Mass (g)

Liver Weight (g)

Kidney Weight (g)

Liver Index (mg/g)

Kidney Index (mg/g)

NC

33.10 ± 1.96

1.43 ± 0.12

0.46 ± 0.04

43.37 ± 3.45

14.02 ± 0.71

HUA

33.29 ± 2.04

1.51 ± 0.14

0.47 ± 0.04

45.28 ± 2.15

14.06 ± 0.88

AP

33.86 ± 1.51

1.47 ± 0.15

0.45 ± 0.06

43.48 ± 2.95

13.15 ± 1.61

HAA

33.46 ± 1.50

1.49 ± 0.11

0.46 ± 0.03

44.59 ± 2.25

13.76 ± 0.93

HAA-LAP

34.90 ± 1.28

1.61 ± 0.12

0.48 ± 0.05

46.00 ± 2.90

13.72 ± 1.27

HAA-HAP

33.75 ± 1.26

1.60 ± 0.10

0.48 ± 0.07

47.54 ± 2.78

14.26 ± 2.25

608 609

* NC: normal control group; HUA: hyperuricemia group; AP: allopurinol group; HAA: HAA-PSP group; HAA-LAP: HAA-PSP with low

610

allopurinol group; HAA-HAP: HAA-PSP with high allopurinol group.

611

* Values were expressed as the means ± SD (n=8).

612

* Different letters marked above the bar are significantly different by ANOVA multiple test (p < 0.05).

613



614

Fig.1

615 616

Fig.2

617

(A)

618


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Page 31 of 38

619


(B)

620 621

(C)

622



623

(D)

624 625

Fig.3 (A)

(B)

(C)

(D)

626

627


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Page 33 of 38


(E)

(F)

628 629

Fig.4

630



631

632


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Page 35 of 38


633

634



635

636


Page 36 of 38

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637



Purple Sweet Potato (Ipomoea batatas L.)

642

a

100 80 60

b

bc

40

c

20

d

d 0 -20

Vehicle

Vehicle

5

25

Allopurinol (AP) Normal

25 & AP 2.5 25 & AP 5 (mg/kg) HAA-PSP

Hyperuricemia

Serum Uric Acid Level

Kidney Injury

Hyperuricemic Mice

HAA-PSP

15

120

b

a

b

a ab

Low Dose Allopurinol

a b

b b 100

90

80

Vehicle

Vehicle

5

25


Hyperuricemia

25 & AP 2.5 25 & AP 5 (mg/kg)

a

10

Serum MDA Level (nmol/mL)

110

Serum T-SOD Activity （ U/mL)

641

120

Serum Uric Acid Level （ mol/L)

638 639 640

Page 38 of 38

5

0

Vehicle

Vehicle

HAA-PSP


5

25


Oxidative Stress Inflammation Response

a a

Hyperuricemia

25 & AP 2.5 25 & AP 5 (mg/kg) HAA-PSP

Highly-acylated anthocyanins from purple sweet potato (Ipomoea

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