Enhanced oral bioavailability and anti-gout activity of [6]-shogaol-loaded solid lipid nanoparticles

Accepted Manuscript Enhanced oral bioavailability and anti-gout activity of [6]-shogaol-loaded solid lipid nanoparticles Qilong Wang, Qiuxuan Yang, Xia Cao, Qiuyu Wei, Caleb K Firempong, Min Guo, Feng Shi, Ximing Xu, Wenwen Deng, Jiangnan Yu PII: DOI: Reference:

S0378-5173(18)30606-9 https://doi.org/10.1016/j.ijpharm.2018.08.028 IJP 17713

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International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

22 June 2018 29 July 2018 14 August 2018

Please cite this article as: Q. Wang, Q. Yang, X. Cao, Q. Wei, C.K. Firempong, M. Guo, F. Shi, X. Xu, W. Deng, J. Yu, Enhanced oral bioavailability and anti-gout activity of [6]-shogaol-loaded solid lipid nanoparticles, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.08.028

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Enhanced oral bioavailability and anti-gout activity of [6]-shogaol-loaded solid lipid nanoparticles Qilong Wang1, Qiuxuan Yang1, Xia Cao1, Qiuyu Wei, Caleb K Firempong, Min Guo, Feng Shi, Ximing Xu*, Wenwen Deng* and Jiangnan Yu* Department of Pharmaceutics, School of Pharmacy, Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Zhenjiang 212013, P.R. China

*Corresponding Authors: Ximing Xu, E-mail: [email protected]; Wenwen Deng, E-mail: [email protected]; Jiangnan Yu, E-mail: [email protected] Tel.: +86 511 85038451; Fax: +86 511 85038451. 1 These authors contributed equally to this work.

Abbreviations: SLNs, solid lipid nanoparticles; SSLNs, [6]-shogaol-loaded solid lipid nanoparticles; PO, potassium oxonate; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; PDI, polydispersity index; TEM, transmission electron microscopy; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor; XOD, xanthine oxidase; MSU, monosodium urate 1

Abstract: [6]-shogaol, an alkylphenol compound purified from the root and stem of ginger (Zingiber officinale), has attracted considerable interest due to its potential anticancer, antioxidative and antirheumatic properties. However, the oral bioavailability of [6]-shogaol has been severely limited because of its poor solubility. In this study, a significant quantity of high-purity [6]-shogaol (yield: 3.6%; purity: 98.65%) was extracted and encapsulated in solid lipid nanoparticles (SLNs) via highpressure homogenization (encapsulation efficiency: 87.67%) to improve its solubility and oral bioavailability. The resulting [6]-shogaol-loaded solid lipid nanoparticles (SSLNs) were stable, homogeneous and well-dispersed. Its mean particle size and zeta potential were 73.56 ± 5.62 nm and −15.2 ± 1.3 mV, respectively. Importantly, the in vitro release profile and in vivo oral bioavailability of SSLNs were significantly improved compared with the free drug. Furthermore, the SSLNs could remarkably lower the uric acid level via inhibiting the activity of xanthine oxidase and reduce the production of interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) in the hyperuricemia/gouty arthritis rat model, when compared to the free [6]-shogaol. Collectively, SLNs could serve as a promising drug delivery system to improve the oral bioavailability of [6]-shogaol for effective treatment of gouty arthritis. Keywords: [6]-shogaol; solid-lipid nanoparticles; hypouricemic; anti-inflammatory; bioavailability 1. Introduction For thousands of years, ginger (Zingiber officinale) has been extensively consumed as a spice and medicine worldwide. It has multiple medicinal properties such as antiinflammation (Moon et al., 2014), antivirus (Chang, Wang, Yeh, Shieh, & Chiang, 2013), anticancer (Ishiguro et al., 2007), antioxidation (Na, Song, Lee, Kim, & Kwon, 2016), antirheumatic (Defang et al., 2015), antimicrobial (Lin, Chen, Chung, & Yen, 2010), antiallergic (Park, Oh, Lee, Lee, & Kim, 2016), carminative (Wadikar, Nanjappa, Premavalli, & Bawa, 2010), anti-emetic (Marx, Isenring, & Lohning, 2017), and peripheral circulatory stimulant action (Mathew & Subramanian, 2014) due to its various active compounds including diarylheptanoid, gingerol, shogaol, paradol and gingerdiol (Wei, Ma, Cai, Yang, & Liu, 2005). Shogaols, the dehydrated form of gingerols, are a group of the active constituents of ginger that are enriched in dried and thermally treated ginger roots. [6]-shogaol [1-(4-hydroxy-methoxyphenyl)-4-decenone] is the most predominant ingredient among the shogaols. It has been widely investigated owing to its potent pharmacological activities including anti-allergy (Makchuchit, Rattarom, & Itharat, 2017), anti-oxidation (Xu et al., 2018), anti-inflammation (Annamalai & Suresh, 2018), anti-cancer (Li & Chiang, 2017) and anti-hypertension (Ghayur, Gilani, Afridi, & Houghton, 2005). Gout is the most prevalent form of inflammatory arthritis that is triggered by the deposition of monosodium urate (MSU) crystals within the joints and tissues (Montagna et al., 2016). It is a common metabolic disorder that is often associated with 2

hyperuricemia (Ruggeri, Basile, Drago, Rolli, & Cicchetti, 2018). Xanthine oxidase (XOD) which plays a crucial role in gout is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid (Liu et al., 2018). The uric acid accumulates in the body when the renal excretion is impaired, leading to hyperuricemia and further to deposition of MSU crystals in the joints and tissues (Han et al., 2017). The MSU crystals can activate the innate immune cells, resulting in the secretion of proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) (Zhang et al., 2018). To date, the first-line agents to treat acute gout are nonsteroidal anti-inflammatory drugs (NSAID) or corticosteroids (Billy, Lim, Ruospo, Palmer, & Strippoli, 2018). Combined administration of allopurinol (a XOD inhibitor) with diuretic acid medications is an optimal strategy for the treatment of chronic gout (Gaffo & Saag, 2009). The management of gout requires medication for a long period of time. However, long-term administration of the current medicines often causes severe adverse effects, such as NSAID-induced gastroduodenal injuries (Satoh & Urushidani, 2016), corticosteroids-caused physical side effects (Hendriksen et al., 2017), and allopurinol-induced severe allergic reaction and hypersensitivity syndromes (Day, Hung, & Chung, 2016). Hence, it is crucial to develop a safe and effective alternative for the treatment of gout. [6]-shogaol, known for its potent antioxidant and anti-inflammatory properties, has demonstrated great potential for the treatment of gouty arthritis via inhibiting MSU crystal-induced inflammation (Sabina, Rasool, Mathew, Ezilrani, & Indu, 2010). No serious adverse effects were reported. Nevertheless, the poor water solubility, low bioavailability and high cost of [6]-shogaol severely hampered its application. Therefore, it is imperative to develop an efficient drug delivery system to enhance the water solubility and improve the bioavailability of [6]-shogaol. Currently, a variety of preparations such as nanoparticles (Peng et al., 2015; Shen et al., 2015), microspheres (Wang et al., 2013) , (Liu et al., 2012), microcapsules (Cao et al., 2014; Shi, Feng, & Omari-Siaw, 2015), microemulsions (Liu et al., 2016; Yi et al., 2012) and liposomes (Wang et al., 2017; Zhu et al., 2015), have been developed for enhancing water solubility and improving bioavailability of the poorly water soluble drugs. Solid lipid nanoparticles (SLNs) have been regarded as a promising nanocarrier to improve solubility and oral bioavailability of hydrophobic drugs (Wang et al., 2014). SLNs enjoy clear advantages such as good biocompatibility (Silva et al., 2012), high physical stability, controlled release (Elbahwy, Ibrahim, Ismael, & Kasem, 2017), high targeting efficiency (Dal Magro et al., 2017), long-acting effects (Cao et al., 2012; Cao et al., 2013; Gastaldi et al., 2014), and cost-effective large-scale production by by high pressure homogenization (Mehnert & Mäder, 2001). In the present study, for the first time, [6]-shogaol-loaded solid lipid nanoparticles (SSLNs) were prepared via the high-pressure homogenization method, with the formulation consisting of a mixture of medium chain triglyceride and glyceryl monostearate as the lipid core, and the mixture of span 80 and tween 80 as the emulsifier. Moreover, due to the high cost of the pure [6]-shogaol, ginger extract powder (10% gingerol) was adopted to isolate high-purity [6]-shogaol for SSLNs preparation. The 3

optimized formulation was characterized in vitro and evaluated in vivo. The antihyperuricemic activities of SSLNs were investigated in the gouty arthritis rat model that was established by intraperitoneal injection of oxonic acid potassium emulsion together with intragastric administration of hypoxanthine suspension. The possible antigout mechanism of SSLNs was also explored. Collectively, solid lipid nanoparticles could be a promising dosage form to improve the solubility and bioavailability of [6]shogaol for the effective treatment of gouty arthritis. 2 Materials and methods 2.1 Materials [6]-shogaol (98% purity, analytical-grade reagent), potassium oxonate (PO), allopurinol, sodium urate, xanthine oxidase assay kit, uric acid standard and C-18 silica gel for column chromatography were provided by Aladdin Industrial Corporation (Shanghai, China). Ginger extract powder (10% gingerol) was obtained from Nanjing Zelang Biological Technology Co., Ltd. (Nanjing, China). Medium chain triglyceride, glyceryl monostearate, span 80 and tween 80 were purchased from Macklin (Shanghai, China). Acetic acid (chromatographically pure), thin layer chromatography (TLC), methylene chloride and the other pure reagents were bought from Sinopharm Chemical Reagent Co., Ltd, China. Double distilled water was purified via filtration in the Millipore system (Milli-Q). 2.2 Animals Male Sprague Dawley (SD) rats weighing 200 ± 10 g were obtained from the Laboratory Animal Center of Jiangsu University (Zhenjiang, China) and raised under specific pathogenic free (SPF) conditions. The animal experiment was conducted according to the regulations and guidelines for the care of laboratory animals, and it was approved by the Ethics Committee on Animal Experiments of Jiangsu University. 2.3 Purification of [6]-shogaol The ginger-extract powder (10% gingerol, 100 g) was extracted twice with ethyl acetate (500 mL each) via refluxing in a round bottom flask on water bath (75C) for 1 h. The resulting solution was exposed to an ultrasound for 30 min. After filtration, the filtrate was collected, concentrated, and dried to obtain the crude [6]-shogaol extract. The crude sample was dissolved in 50% methanol aqueous solution (50:50, v/v). Subsequently, the sample was applied to a C-18 silica gel column and eluted with serial solutions (100 mL each) of methanol/water mixtures (50:50, 60:40, 70:30, 80:20, 90:10 v/v), respectively. The eluates were collected using Eppendorf tubes (10 mL). High performance liquid chromatography (HPLC) and TLC were used to analyze the resulting eluate samples qualitatively and quantitatively. The HPLC system was equipped with a Symmetry®C18 column (4.6 mm×150 mm, 5 μm; Waters, USA). The mobile phase was methanol/water solution (70:30, v/v). The flow rate was 1.0 mL/min at 30C. The injection volume was 20 L, with the detection wavelength at 231 nm. The samples with high concentration of [6]-shogaol were combined and lyophilized to 4

obtain a high purity of [6]-shogaol product. 2.4 Preparation of solid lipid nanoparticles The high-pressure homogenization method was employed to prepare SSLNs (Severino et al., 2015). Briefly, [6]-shogaol (500 mg), medium chain triglyceride (2500 mg) and glyceryl monostearate (2500 mg) were weighed and put in a round-bottomed flask (100 mL). The flask was placed in water bath at 80C to yield a clear and homogenous lipid solution. An aqueous phase was also prepared by dissolving tween 80 (1500 mg, critical micelle concentration, CMC=0.014 mol/L) and span 80 (1500 mg, CMC=1.8 × 10-5 mol/L) in 90 mL of double distilled water at 80 °C. The aqueous phase was then slowly added to the agitated lipid phase. The prepared suspension was stirred continuously at 80C for 1 h to form pre-emulsion. The pre-emulsion solution was injected into a highpressure homogenizer (APV-2000, Beijing Udare Technology Co., Ltd, Beijing, China) and homogenized under 1000 bar for 10 cycles. After cooling to the room temperature, the SSLNs suspension was obtained. The colloidal dispersion was frozen at -86 °C for 4 h and lyophilized for 24 h. Finally, the powdered SSLNs was obtained and stored at 4 °C for further investigations. 2.5 Optimization of formulation The optimization of SSLNs was conducted through orthogonal experimental design (L9(3)4) based on a previous report (Kumar, Kharb, & Chaudhary, 2016). The four (4) factors were chosen as variables included: (A) the ratio of [6]-shogaol to lipids (w/w), (B) the ratio of medium chain triglyceride to glyceryl monostearate (w/w), (C) the ratio of tween 80 to span 80 (w/w), and (D) the ratio of [6]-shogaol to surfactant (w/w) (Table 1). The particle size was selected as an evaluation index of the optimized formulation. 2.6 Characterization of SSLNs 2.6.1 Particle size, polydispersity index, and zeta potential The mean particle size, polydispersity index (PDI) and zeta potential of SSLNs were measured using a Brookhaven 90 PlusPALS instrument (Brookhaven Instruments Corp., Holtsville, NY, USA). All samples were diluted to 100 g/mL of [6]-shogaol with doubled distilled water before measurement. The results were represented by mean ± standard deviation of three repetitions. All measurements were performed at 25°C. 2.6.2 Transmission electron microscopy (Elbahwy, Ibrahim, Ismael, & Kasem) The morphological characterization of SSLNs was analyzed using a transmission electron microscopy (TEM, JEM-2100, JEOL, Japan microscope). A drop of SSLNs suspension with 2 mg/mL of [6]-shogaol was dispersed on a copper grid of 200 meshes and dried for 5 min. A drop of phosphotungstic acid solution (1% w/v) was added to the SSLNs-loaded copper grid for the negative staining. The samples were dried and examined under the TEM. 2.6.3 Encapsulation efficiency 5

The efficiency of [6]-shogaol entrapped in SLNs was calculated using the ultracentrifugation method (Shi, Zhang, Yang, Guo, & Feng, 2015). The SSLNs suspension (1 mL) was put in ultrafiltration tubes and centrifuged in a high-speed cooling centrifuge (Biofuge Stratos, Heraeus, Germany) at 15557 g for 30 min at 4°C. The supernatant was collected and the amount of [6]-shogaol measured via the HPLC method. The encapsulation efficiency (EE%) for each preparation of SSLNs was calculated using the following equation: EE%= Entrapped[6]-shogaol / Total[6]-shogaol × 100% Equation (I) 2.7 In vitro release profile of SSLNs The dialysis method was used to investigate the in vitro release of SSLNs (Sun, Liu, Zhao, He, & Pan, 2013; Zhu et al., 2015). Dialysis membrane (MV 3500 Da, 35 mm × 5 m, Shanghai Green Bird Science and Technology Development, China) was soaked in Milli-Q water for 24 h before used. Six dialysis bags were used to check the in vitro release in three different media: water, pH 7.0; HCl, pH 1.2; and phosphate buffer saline (PBS), pH 7.4. The dialysis bags were equally filled with [6]-shogaol solution (2.0 mg/mL, 1 mL) or SSLNs (equivalent drug content). Then the dialysis bags were soaked in 2RS-G dissolution testers (Tianda Tian Technology Co., Ltd.) containing 100 mL of each dissolution cup at 100 rpm. Samples (1 mL each) were taken from each diffusion cup at various time points (0.1, 0.15, 0.25, 0.5, 0.75,1, 2, 4, 6, 8, 10, 12, 24 h). An equal volume of fresh medium was immediately added to each cup. The collected samples were filtered with the 0.45-μm microporous membrane and analyzed using HPLC in triplicates. 2.8 Pharmacokinetics of SSLNs in vivo Healthy male SD rats were used to evaluate the oral bioavailability of [6]-shogaol and SSLNs. Prior to the experiment, the rats were acclimatized to the animal house facilities for 7 days. They were fed on standard rat diet with free access to water and kept under constant environmental conditions. All animals were administered with a single oral dose of [6]-shogaol (100 mg/kg). The rats were randomly divided into two groups (n = 6), namely group I: [6]-shogaol (suspended in sodium carboxymethylcellulose to obtain a stable solution), and group II: SSLNs (dissolved in water). Blood samples (0.6 mL each) were collected from the capillaries in the eye-orbit area at different times (5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480 and 720 min). All blood samples were collected into heparinized tubes and kept in water bath at 37°C for 30 min. Afterward, all the samples were centrifuged at 1844 g for 10 min at 4°C to obtain the plasma. 1Naphthol (50 μL), serving as the internal standard, was added to each plasma (200 μL) sample. Methanol (0.8 mL) was also added to the plasma samples to precipitate the proteins. The mixtures were then vortexed for 1 min and centrifuged at 1844 g for 10 min. The supernate was collected for HPLC analysis. Pharmacokinetic parameters were analyzed using BAPP pharmacokinetic software (Centre of Drug Metabolism of China Pharmaceutical University, China). The main pharmacokinetic parameters including the maximum peak concentrations (CMAX), the time to achieve the maximum concentration (TMAX), half-life (T1/2), mean residue time (MRT0-t) and the area under 6

the concentration-time curve (AUC0-α) were obtained. 2.9 Anti-hyperuricemic activities of SSLNs in rats 2.9.1 Establishment of the hyperuricemia/gouty arthritis rat model Male SD rats weighing 200 ± 10 g were intraperitoneally injected with oxonic acid potassium emulsion (25 mg/mL, 10 mL/kg), intragastrically administered with hypoxanthine suspension (100 mg/mL, 10 mL/kg) (Xu et al., 2013), and injected with the urate crystal suspension (50 μL, 80 mg/mL) into the sub-plantar region of the right hind paw of the rats successively to establish the hyperuricemia/gouty arthritis rat model. The contents of uric acid, interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) were determined to evaluate the hyperuricemia/gouty arthritis model. 2.9.2 HPLC condition of serum uric acid The serum uric acid in rats was detected using HPLC equipped with a Symmetry®C18 column (4.6mm×250mm, 5μm; Waters, USA). The mobile phase was methanol/0.2% acetic acid-water solution at a volume ratio of 6:94. The flow rate was 1.0 mL/min at 30C. The sample (20 L) was injected into the HPLC and analyzed at the wavelength of 288 nm. The uric acid standard curve was established by dissolving the uric acid standard (10 mg) and sodium carbonate (10 mg) in double distilled water to yield 1 mg/mL standard stock solution. A series of the standard solutions (1, 2.5, 5, 10, 15, 20 g/mL respectively) were prepared. Regression analysis was carried out using the uric acid concentration as the abscissa, while the peak area served as the ordinate. 2.9.3 In vivo anti-gout activities of SSLNs Sixty male SD rats weighing 200 ± 10 g were randomly divided into ten groups (n = 6): normal control group (C); model group (M); positive control group (P, 40 mg/kg of allopurinol); [6]-shogaol low-dose group (S-L, 30 mg/kg); [6]-shogaol medium-dose group (S-M, 60 mg/kg); [6]-shogaol high-dose group (S-H, 120 mg/kg); SSLNs lowdose group (SSLNs-L, 30 mg/kg); SSLNs medium-dose group (SSLNs-M, 60 mg/kg); SSLNs high-dose group (SSLNs-H, 120 mg/kg), and vehicle control group SLNs (SLNs, 2000 mg SLNs/kg, equivalent to the SLNs in SSLNs-H). One hour after the establishment of the hyperuricemia/gouty arthritis rat model, each animal in groups C and M was administered intragastrically with 2 mL of physiological saline (0.9%), and each rat in the other eight groups was administered intragastrically with 2 mL of the corresponding sample described above. Three hours later, blood samples (1 mL each) were taken from the eye-orbit area using capillary tubes. The serum uric acid levels were measured via HPLC, while serum levels of IL1β and TNF-α were determined using enzyme-linked immunosorbent assay (ELISA) kits. 2.9.4 Determination of the XOD activity XOD can catalyze the oxidation of hypoxanthine to xanthine and then to uric acid, thus inhibiting XOD activity would be an effective strategy to treat hyperuricemia/gouty 7

arthritis. In this study, the XOD activity (both in serum and liver) in each group was determined to reveal the possible mechanism of the anti-gout effect of [6]-shogaol. Blood samples were prepared as described in the section “In vivo anti-gout activities of SSLNs”. The liver samples were prepared as follows. Briefly, the livers were immediately and carefully excised after the blood collection. The samples were then washed with 0.9% physiological saline and rapidly stored in liquid nitrogen. Half of the liver samples were sufficiently homogenized in 9 volumes of 80 mmol/L sodium phosphate buffer (pH 7.4) on ice. After that, the homogenate was centrifuged at 7378 g for 10 min at 4 °C. The supernate was collected for XOD activity assay. The XOD activities were determined using the colorimetric method with commercially available ELISA kits. 2.9.5 Histopathological examination All the rats were sacrificed, and different organs (liver, kidneys, lungs, heart, and spleen) were collected and fixed with 4% paraformaldehyde at 4 °C for 24 h. After washing with the PBS (pH 7.4), the samples were embedded in paraffin and sectioned at 5 μm for staining using hematoxylin and eosin. The prepared samples were visualized under a microscope (Nikon, Japan) at 200x magnifications. 2.10 Statistical analysis All the experimental results were expressed as the mean ± standard deviation (SD). Statistical significance was determined using Student’s t-test, with p < 0.05 being considered statistically significant. Statistical comparisons of data were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test with SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). 3 Results and discussion 3.1 Isolation and purification of [6]-shogaol The [6]-shogaol isolated from the ginger extract powder (10% gingerol) had a yield of 3.6%. The purity and chemical structure of [6]-shogaol were determined using TLC and HPLC, in which the [6]-shogaol was used as the standard. As shown in Fig. 1A, B, the laboratory-purified [6]-shogaol showed a single peak in the place in consistent with that of the [6]-shogaol standard. The TLC result (Fig. 1C) was in agreement with that of the HPLC. Moreover, according to the calibration curve of HPLC, the [6]-shogaol isolated in this study had a purity of 98.65% which was comparable to that of the commercially available [6]-shogaol standard (98%). Moreover, the ESI-MS of the purified [6]-shogaol in the negative model produced m/z 299.1624 deprotonated molecular ion [M+Na]− (Fig. 2A). Compared with the previous report (Vijendra Kumar, Murthy, Manjunatha, & Bettadaiah, 2014), the 1H NMR (400 MHz, CDCl3, TMS) spectrum demonstrated typical peaks of [6]-shogaol: δ 0.88 (t, 3H, C10), 1.31 (m, 4H, C8, C9), 1.48 (m, 2H, C7), 2.25 (m, 2H, C6), 2.84 (m, 4H, C2, C1), 3.87 (s, 3H, -OMe), 6.12 (d, 1H, C4), 6.65–6.82 (m, 4H, C2’, C3’, C5, C6’) (Fig. 2B). Collectively, the laboratory-purified [6]-shogaol possessed a high yield 8

and purity (3.6 g of the 98.65%-purity [6]-shogaol was obtained from 100 g of the ginger extract powder). Given the high cost of the commercial standard [6]-shogaol (1200 RMB for 20 mg of the standard [6]-shogaol), the laboratory-purified [6]-shogaol would be an abundant, cost-effective alternative of [6]-shogaol (18 g of the laboratorypurified [6]-shogaol cost around 200 RMB) for the subsequent experiments. 3.2 Preparation of SSLNs The formulation factors affecting the particle size were determined through orthogonal experimental design with K1, K2, and K3 representing the sum of each level (Table 2). The order of particle size was A > D > C >B, thus the ratio of [6]-shogaol to lipids > the ratio of [6]-shogaol to surfactant > the ratio of tween 80 to span 80 > the ratio of medium chain triglyceride to glyceryl monostearate, respectively. The optimal level of [6]-shogaol to lipids was 1:10. The particle size increased with increase in the proportion of [6]-shogaol. The optimal levels of the ratio of [6]-shogaol to surfactant, tween 80 to span 80 and medium chain triglyceride to glyceryl monostearate were 1: 6, 1: 1 and 1:1, respectively. Therefore, the optimal conditions for preparing SSLNs were as follows: [6]-shogaol (500 mg), medium chain triglyceride (2500 mg), glyceryl monostearate (2500 mg), tween 80 (1500 mg), and span 80 (1500 mg). A previous study (Asasutjarit & Sorrachaitawatwong, 2013) reported that using tween 80 or span 80 solely often resulted in non-uniform size distribution, abnormal morphology, and low physical stability. The combination usage of tween 80 and span 80 could bring a hydrophilic-lipophilic balance (HLB) value ranging from 4 to 15 by matching the mass ratio of the two surfactants to meet multiple requirements at the same time (Boonme et al., 2016; Liang et al., 2018; Wuttikul and Boonme, 2016). Therefore, tween 80 mixed with span 80 at the optimal ratio of 1:1 was applied as the surfactant to obtain nanoparticles with smaller particle size and uniform size distribution in the present study. Further optimization of different surfactant combinations will be investigated in our future study. 3.3 Characterization of SSLNs When dispersed in water, the SSLNs were homogeneous, stable and translucent slightly blueish suspension. The mean particle size of SSLNs was 73.56 ± 5.62 nm with PDI of 0.224 ± 0.031 (Fig. 3A). The small particle size was partly accounted for the increased [6]-shogaol solubility and enhanced intestinal absorption (Patel, Patel, Bhatt, & Patel, 2013). The zeta potential of SSLNs was −15.2 ± 1.3 mV. The negative charge was favorable for the formation of monodispersed nanoparticles and could enhance the stability of the SSLNs suspension (MacFarlane, Liu, & Solomon, 2015). The TEM image showed that SSLNs were of a uniform mono-dispersion, spherical shape, and small size (< 100 nm) (Fig. 3B), which was in consistent with the findings of the particle size distribution (Fig. 3A). The encapsulation efficiency of SSLNs was determined based on the equation: A=26846.23C+31748.2 (n = 5, r2 = 0.998), with a linear range of 0.00205–0.205 μg/mL, where C is the concentrations of [6]-shogaol in SLNs, and A represents the peak areas obtained from the HPLC. As a result, the encapsulation efficiency of SSLNs was 9

87.67%, indicating that most of the [6]-shogaol was encapsulated in SLNs. 3.4 In vitro release profile of SSLNs The accumulated release profile of SSLNs was monitored for 24 h at 37 ± 1 °C in three different media: water (pH 7.0), HCl (pH 1.2), and PBS (pH 7.4). As shown in Fig. 5A, the SSLNs had an obviously enhanced accumulative release of [6]-shogaol from the dialysis bag in the three media (water, 91.3%; HCl, 93.1%; PBS, 95.7%), when compared to that of the free [6]-shogaol (water, 62.3% ; HCl, 70.6 % ; PBS, 73.8%). Generally, this trend could be attributed to the small droplet size of SSLNs, which implied that the SLNs system could solubilize [6]-shogaol and significantly enhanced it's in vitro release. In addition, the solubility of the drug in the different pH conditions varies with the maximum solubility occurring in the PBS (pH 7.4). The SSLNs exhibited a biphasic drug-release pattern with an initial burst release being followed by a sustained release of [6]-shogaol. The initial burst release could be attributed to the adsorbed free [6]-shogaol molecules on the SLNs surface, while the sustained release could be the continuous diffusion of [6]-shogaol from the core of the lipid matrix (Shah, Malherbe, Eldridge, Palombo, & Harding, 2014). The biphasic behavior of [6]-shogaol could also be associated with the physicochemical nature of the phenylpropanoid-derived compound ([6]-shogaol) and its concomitant interaction with the lipid nanoparticles (Pegi, Julijana, Slavko, Janez, & Marjeta, 2003). 3.5 Pharmacokinetic analysis of SSLNs. The pharmacokinetic indices including CMAX, TMAX, T1/2, MRT0-t and AUC0-72 h were presented in Table 3. The HPLC spectra of the serum samples showed that a peak appeared in the same place as that of the standard [6]-shogaol after drug (free [6]shogaol and SSLNs) administration (Fig. 4). In comparison with the free [6]-shogaol, the CMAX and AUC0-∞ of SSLNs were significantly (P < 0.05) increased, with 5.88 folds for CMAX and 9.99 folds for AUC0-∞. Moreover, the T1/2, MRT0-t, and TMAX of SSLNs (329.17±2.14, 223.13±1.68, and 120.00 min for T1/2, MRT0-t, and TMAX, respectively) were significantly prolonged compared to that of the free [6]-shogaol (200.67±1.62, 150.09±1.35, and 45.00 min for T1/2, MRT0-t, and TMAX, respectively). In general, the half-life (t1/2) was less than MRT0-t. However, previous study (Abu-Basha et al., 2006), showed that T1/2 could be increased in the end for the non-compartmental model, suggesting that T1/2 could be greater than MRT0-t during the terminal phase of the noncompartmental model. In this study, our data showed that the value of MRT0-t was smaller than that of T1/2. It is probable that in this study, the pharmacokinetic model is the compartmental model, which will be investigated in our future work. Furthermore, drug concentration-time curve showed that a higher plasma concentration of [6]shogaol (>1.0 μg/mL) was sustained for a significantly longer duration (~ 8 h) compared with that of the free [6]-shogaol (~ 1 h) (Fig. 5B). These data indicated that SSLNs could significantly increase the absorption, prolong the acting time, and improve the bioavailability of [6]-shogaol in vivo. The enhanced oral absorption of [6]shogaol in SSLNs was likely due to the fact that the lipid layer of SSLN could overcome the absorption barrier of the intestinal mucosa and enhance drug bioavailability via 10

lymphatic absorption, which could reduce hepatic first pass metabolism and increase the total systemic bioavailability (Zhao et al., 2018). In this study, [6]-shogaol SLNs, composed of a lipid core of solid physiological lipids (medium chain triglyceride and glyceryl monostearate) along with surfactants (tween 80 and span 80) as the stabilizer, has a serial of advantages over other formulations. A previous review reported that SLNs enjoyed a greater stability when compared to microemulsions and liposomes, and a lower toxicity as compared to some polymeric nanoparticles due to the use of physiological and biocompatible lipids (Yadav et al., 2016). Moreover, SLNs could effectively protect the [6]-shogaol from chemical photochemical or oxidative degradation because of the immobilization of the [6]-shogaol molecules by solid lipids (Reddy and Venkateswarlu, 2004); and reduce drug leakage that is commonly observed in liposomes (Geng et al., 2014). Additionally, SLN possesses good biocompatibility (Doktorovova et al., 2009), feasibility of incorporating both hydrophilic and hydrophobic drugs (Weber et al., 2014), multiple routes of administration (Battaglia and Gallarate, 2012; de Mendoza et al., 2012; Gainza et al., 2014), and excellent reproducibility with a cost effective high-pressure homogenization method as the preparation procedure. Overall, because of the multiple advantages, SLNs could be the optimal formulation to improve the solubility, bioavailability, and anti-hyperuricemic activities of [6]-shogaol. 3.6 Effect of SSLNs on uric acid levels and gouty arthritis in rats The levels of the serum uric acid and the inflammation markers (IL-1β and TNF- α) in the M group were significantly (P <0.05) increased compared with those of the C group (Fig. 6), indicating the successful establishment of the hyperuricemia/gouty arthritis rat model. As shown in Fig. 6 and Fig. 7, the vehicle control group (SLNs) showed similar levels of uric acid, IL-1β, and TNF- α compared to the M group; by contrast, the SSLNs and free [6]-shogaol reduced the serum uric acid levels in a dose-dependent manner (Fig. 6). Specifically, the SSLNs with three different doses (30, 60, 120 mg/kg) remarkably (P<0.05) reduced the serum uric acid level by approximately 57.1%, 70.6%, and 75.3%, respectively, compared with the M group; however, the same doses of free [6]-shogaol lowered the serum uric acid level to a significantly less degree (P <0.05) by about 42.4%, 57.1%, and 66.7%, respectively. Moreover, the SSLNs (30, 60, 120 mg/kg) lowered the levels of IL-1β and TNF-α to an obviously greater extent (P <0.05) than the free [6]-shogaol compared to the M group (Fig. 7). Importantly, the SSLNs could lower the levels of IL-1β and TNF-α close to those of the P group, indicating that SSLNs had an anti-inflammation effect comparable to the positive control allopurinol. These results demonstrated that the SLNs system could greatly enhance the antihyperuricemic and anti-gouty arthritis activities of [6]-shogaol. 3.7 [6]-shogaol lowers uric acid via inhibition of XOD activity To preliminarily explore the possible mechanism of the anti-hyperuricemic/gouty arthritis effect of [6]-shogaol, the XOD activity both in serum and in liver was detected. As a result, the SSLNs could effectively inhibit the XOD activity (both in serum and in liver) in a dose-dependent manner, with 54.0% and 52.8% reduction of the XOD 11

activity in the serum and liver, respectively, in the hyperuricemia/gouty arthritis rats treated with the highest dose of SSLNs (120 mg/kg) compared with the M group (Fig. 8). Although the inhibition of the XOD activity by the free [6]-shogaol was also dose dependent, the degree of inhibition was less than that of the SSLNs (Fig. 8). Importantly, the XOD activities in the animals of the SSLNs-H group were comparable to that of the C group (Fig. 8), indicating that SSLNs had the capacity to lower the XOD activities close to the normal control level in the hyperuricemia/gouty arthritis rats. 3.8 Organ protection effect of SSLNs It is recognized that hyperuricemia/gouty arthritis is associated with severe multiple organ damage (Lan et al., 2015). To further investigate the effects of SSLNs on multiple organs, the histological examination was performed. As shown in Fig. 9A and 9B, notable pathologic changes such as edema, degeneration, and necrosis were observed in multiple organs including liver, kidney, lung, heart, and spleen in the M group compared with the C group, suggesting that multiple organ damage occurred in the hyperuricemia/gouty arthritis rats. After treatment, no improvement was observed in the rats administered with the SLNs (Fig. 9J) compared to the M group (Fig. 9B); by contrast, the organs in the rats treated with free [6]-shogaol and SSLNs exhibited varying degrees of restoration on histologic morphology (Fig. 9D-I) in a dosedependent manner. It is noteworthy that, in the SSLNs-H group (Fig. 9I), all the tissue morphologies were close to normal (Fig. 9A), indicating that the SSLNs had better organ restoration than the free [6]-shogaol. These results showed that the SLNs drug delivery system could effectively improve the organ protection effects of [6]-shogaol in the hyperuricemia/gouty arthritis rats. 4 Conclusion In this study, the formulation of [6]-shogaol-loaded solid lipid nanoparticles (SSLNs) was optimized by orthogonal design and successfully produced via high-pressure homogenization. The resulting SSLNs had small particle size (<100 nm), acceptable PDI and negative zeta potential. Morphological studies showed uniformly spherical nanoparticles with smooth surfaces. The encapsulation efficiency was around 87.67%. Importantly, the SSLNs remarkably increased the solubility and oral bioavailability of [6]-shogaol. Moreover, the SSLNs could lower the levels of serum uric acid, IL-1β and TNF-α to an obviously greater extent than those of the free [6]-shogaol in the hyperuricemia/gouty arthritis rats. It was revealed that the [6]-shogaol reduced the uric acid level by inhibiting the XOD activities. Furthermore, the SSLNs showed better protection effects on multiple organs compared with the free [6]-shogaol. Taken together, the SLNs could be a promising drug delivery system to increase the solubility and oral bioavailability of [6]-shogaol for improving its anti-gout properties. Author contributions Qilong Wang, Qiuxuan Yang, and Ximing Xu designed experiments and acquisitioned data, Qilong Wang, Xia Cao, and Qiuyu Wei analyzed data, Qilong 12

Wang, Wenwen Deng, Min Guo, and Feng Shi drafted manuscript, Caleb K Firempong, and Xia Cao contributed to intellectual content, Jiangnan Yu and Ximing Xu contributed to final approval of the version. All authors reviewed the manuscript. Acknowledgments This work was supported by National Natural Science Foundation of China (81473172, 81503025, 81720108030 and 81773695), National “Twelfth Five-Year” Plan for Science & Technology Support (Grant 2013BAD16B07-1), Special Funds for 333 and 331 projects (BRA2013198), China Postdoctoral Science Foundation (2017M621658 and 2017M621659), Program for Scientific Research Innovation Team in Colleges and Universities of Jiangsu Province (SJK-2015-4), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors also thank the University Ethics Committee for the kind guidance in the animal experiments. Conflict of interest The authors declare that they have no conflict of interest.

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Figure legends Figure 1 HPLC analysis of [6]-shogaol. a) Reference standard of [6]-shogaol; b) Extracted [6]-shogaol obtained from ginger; c) TLC analysis of [6]-shogaol: 1: standard of [6]-shogaol, 2: standard of [6]-shogaol and extracted [6]-shogaol obtained from ginger, 3: Extracted [6]-shogaol obtained from ginger. Figure 2 Identification of [6]-shogaol. a) MS of [6]-shogaol; b) 1H NMR of [6]-shogaol. Figure 3 Characterization of SSLNs. a) Particle size distribution of SSLNs; b) A TEM image of SSLNs. Figure 4 HPLC analysis of [6]-shogaol. a) [6]-shogaol obtained from blood sample; b) SSLNs obtained from blood sample Figure 5 [6]-shogaol released from SSLNs. a) In vitro release profile of SSLNs; b) In 18

vivo release profile of SSLNs. Figure 6 The XOD expression in Serum and Hepatic in ten Groups. normal control group (C); model group (M); positive control group (P, 40 mg/kg of allopurinol); [6]shogaol low-dose group (S-L, 30 mg/kg); [6]-shogaol medium-dose group (S-M, 60 mg/kg); [6]-shogaol high-dose group (S-H, 120 mg/kg); SSLNs low-dose group (SSLNs-L, 30 mg/kg); SSLNs medium-dose group (SSLNs-M, 60 mg/kg); SSLNs high-dose group (SSLNs-H, 120 mg/kg), and vehicle control group SLNs. *P＜0.05, Figure 7 The levels of the serum uric acid. *P＜0.05, SSLNs compared with free drug. Figure 8 The inflammatory factors expression of IL-1β and TNF-α. Figure 9 Histopathological analysis of organs (liver, kidney, lung, heart and spleen) from rats with different treatments: a) C; b) M; c) P; d) S-L; e) S-M; f) S-H; g) SSLNs-L; h) SSLNs-M; i) SSLNs-H; j) SLNs.

Table 1 Prescription composition of SSLNs preparation

A [6]shogaol: lipids

19

L e v B emedium l chain s triglycerid e: glyceryl monostear

F a c t o r C tween 80: span 80

D [6]shogaol: surfacta nt

1：10 1：8 1：6

ate 1 2：1 2 1：1 3 1：2

2：1 1：1 1：2

1：6 1：12 1：18

Table 2 Data obtained from orthogonal experimental design of SSLNs formulation e f x a p c e t r o i r m s e A B C D n t s 1 1 1 11 1 2 2 22 1 3 3 33 2 1 2 34 2 2 3 15 2 3 1 26 3 1 3 27 3 2 1 38 3 3 2 19 232.6 331. 326.4 278.9 K 8 17 2 81 336.9 273. 271.0 352.4 K 8 78 6 92 370.5 335. 342.7 308.7 K 4 25 2 33 K 110. 108.8 92.99 1 77.56 39 0667 333 / 3 K 112.3 91.2 90.35 117.4 2 2667 6 333 9667 / 3 20

76.35 63.27 93.06 102.58 97.42 136.98 152.24 113.09 105.21

K 123.5 111. 114.2 102.9 3 1333 75 4 1/ 3 45.95 20.4 23.88 24.50 R 333 9 667 334

Table 3 Plasma pharmacokinetic parameters of orally administered SSLNs in rats. A U C 0 7 2 0

C M A X

( μ g / m l )

( m i n μ g / m l ) F r e e

135.70±1.71

0.98±0.02

21

[ 6 ] s h o g a o l

200.67±1.62

150.09±1.35

45.00

1356.72±6.32

5.76±0.11

22

S S L N s

329.17±2.14

223.13±1.68

120.00

23

24

25

26

27

28

29

30

Graphical abstract ss

31