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In vitro and in vivo evaluation of curcumin loaded hollowmicrospheres prepared with ethyl cellulose and citric acid
Chao Pi, Jiyuan Yuan, Hao Liu, Ying Zuo, Ting Feng, ChenglinZhan, Jun Wu, Yun Ye, Ling Zhao, Yumeng Wei
PII: S0141-8130(18)30733-5DOI: doi:10.1016/j.ijbiomac.2018.04.171Reference: BIOMAC 9579
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Received date: 13 February 2018Revised date: 31 March 2018Accepted date: 30 April 2018
Please cite this article as: Chao Pi, Jiyuan Yuan, Hao Liu, Ying Zuo, Ting Feng, ChenglinZhan, Jun Wu, Yun Ye, Ling Zhao, Yumeng Wei , In vitro and in vivo evaluation ofcurcumin loaded hollow microspheres prepared with ethyl cellulose and citric acid. Theaddress for the corresponding author was captured as affiliation for all authors. Pleasecheck if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.04.171
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In vitro and in vivo evaluation of curcumin loaded hollow microspheres prepared
with ethyl cellulose and citric acid
Chao Pi1,#
,Jiyuan Yuan1,#
, Hao Liu1, Ying Zuo
2, Ting Feng
1,, Chenglin Zhan
1, Jun Wu
3, Yun Ye
1,4, Ling
Zhao1,*
, Yumeng Wei1,*
1. Department of Pharmaceutics, School of Pharmacy, Southwest Medical University, No. 3-5,
Zhongshan Road, Luzhou, Sichuan, 646000, P.R.China
2. Department of General Internal Medicine, the Affiliated Hospital of Traditional Chinese Medicine of
Southwest Medical University, No. 3-5, Zhongshan Road, Luzhou, Sichuan, 646000, P.R.China
3. Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of
Pharmacy, 307 N. Broad Street, Clinton, SC 29325, USA
4. Department of Pharmacy, The Affiliated Hospital, Southwest Medical University, No.25, Taiping
Street, Luzhou, Sichuan, 646000, China
#These authors contributed equally to this work.
*Address correspondence to:
Ling Zhao, School of Pharmacy, Southwest Medical University, No.3-5, Zhongshan Road, Jiangyang
District, Luzhou, Sichuan, 646000, P.R.China
Tel: +86 830 3162292; Fax: +868303162292
E-mail: [email protected]
Yumeng Wei, School of Pharmacy, Southwest Medical University, No.3-5, Zhongshan Road, Jiangyang
District, Luzhou, Sichuan, 646000, P.R.China
Tel: +86 830 3162291; Fax: +86 830 3162291
E-mail: [email protected]
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Abstract: Curcumin (CUR) demonstrates a variety of biological activities; however, the poor oral
bioavailability limits its clinical application. The objective of this study was to develop and evaluate
characteristics and bioavailability of hollow microspheres loading curcumin (CUR-HPs). CUR-HPs
were prepared by solvent diffusion and evaporation method. The effect of viscosity of ethyl cellulose
(EC), amount of EC, citric acid (CA) and CUR on physicochemical characteristics and in vitro release
profile of CUR-HPs were evaluated. Scanning electron microscopy (SEM) showed microspheres had
smooth surfaces with hollow structures. The yield of CUR-HPs was (96 ± 1.80) %. The floating rate at
24 h was (89.67 ± 4.91) % and the drug loading was (3.41 ± 0.21) %. Nearly 95% of CUR was released
from the HPs at 24h. In vitro release profiles of CUR-HPs fitted the Korsmeyer et al’s equation and
indicated that CUR was released through the combination of diffusion and erosion mechanisms. The
bioavailability of CUR-HPs was 12-fold higher than that of CUR. The peak time was delayed for 7.5 h
and peak concentration of CUR-HPs was 3.21 times than that of free CUR. The CUR-HPs might be a
promising strategy to achieve sustained release and increase oral bioavailability of CUR.
Keywords: Curcumin, hollow microspheres, sustained release, pharmacokinetics, oral bioavailability
Introduction
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As a multi-unit based drug delivery system, hollow microspheres have unique advantages in the
development of poorly soluble drugs [1]. Firstly, the hollow microspheres had good floating ability in
gastric solution due to its low density resulted from the internal hollow structure. The hollow
microspheres could increase the residence time of the drug in the stomach and were not affected by
gastric empting, which reduced remarkable individual difference in the drug absorption [2]. Secondly,
as an oral controlled release multi-unit dosage form, the hollow microspheres could control drug
release and improve aqueous solubility of poorly water-soluble drugs to increase oral bioavailability [1],
because drugs were dispersed in the polymer structure as an amorphous or microcrystals state. Thus,
hollow microspheres have been considered as a potential drug delivery system for poorly soluble drugs
[3-4].
Curcumin[(E,E)-1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione,CUR] extracted
from Zingiberaceae family is a 3,5-dione phenol effective compound and demonstrates a variety of
biological activities, such as anti-inflammation, anti-oxidation and anti-cancer[5-7]. Packard et al.
proposed that CUR may have anti-cancer activity in 1985 [8]. CUR has been listed as one of the third
generation cancer chemoprevention drug in the United States National Cancer Institute due to its safety
and effectiveness. CUR inhibited proliferation and induced apoptosis of various cancer cells including
MCF, HNSCC, and MDA-MB-231[9-11] through relating signaling pathways and regulating proteins
[12-16]. However, the poor oral bioavailability of CUR limited its clinical use [1]. According to the
Biopharmaceutics Classification System (BCS), CUR has been classified as Type IV due to its low
water solubility and intestinal permeability [17].Thus, the poor oral bioavailability and short half-life
time of CUR were the main challenges for oral administration.
This study aimed to develop hollow microspheres loading CUR (CUR-HPs) by solvent diffusion and
evaporation method and carry out in vitro and in vivo evaluation to improve oral availability of CUR.
Materials and methods
Materials
Curcumin (CUR, purity > 98%) was purchased from Must Bio-Technology Company Ltd. (Chengdu,
China). Methanol and acetonitrile of HPLC grade, Acetate, methanol and citric acid (CA) of analytical
grade were purchased from Luzhou SanrongJia experimental supplies Co., Ltd. (Luzhou, Sichuan,
China). Water used throughout the study was prepared by double distillation of water. Triethylamine
and ethanol, phosphoric acid was obtained by Luzhou Shuangjiang Ruilong Chemical Reagent Co. Ltd.
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(Luzhou, Sichuan, China). Ethyl cellolulose (EC 10, 45 and 100 cp) was provided by Shanghai Coloron
Coating Technology Com., Ltd. (Shanghai, China).
Preparation and optimization
CUR-HPs composed of curcumin (CUR), Ethyl cellolulose(EC) and citric acid (CA), were prepared
by solvent diffusion and evaporation method. EC, CA and CUR powder were dissolved in the mixture
of 3 ml ethanol and 1 ml ether at room temperature. Next, the solution was added to liquid paraffin
(containing 7% span) and constantly stirred by a magnetic stirring for about 4 h. The microspheres
were washed with hexane, and then put into a vacuum drying oven at 40℃away from light for 10 h.
Finally, the hollow microspheres were stored in desiccators at room temperature.
As shown in Table 1, a single factor experiment was designed. The effects of different molecular
weight of EC, amount of EC10 cp, CA and CUR on the quality of microspheres were measured. To
optimize the formulation, the effects of CUR, CA, and EC on the loading rate, accumulative release,
and floating rate were evaluated by orthogonal experiment. All factors were taken at three levels for L9
(33) Latin orthogonal design experiments. Yield (Y) and floating rate (FR) for 24 h, encapsulation
efficiency (EE) as the index score, comprehensive score X=Y+FR+EE, the higher the better.
Evaluation of formulation
Drug loading
A certain amount of CUR-HPs was crushed into powder. A portion of the powder samples (about 10
mg of CUR) was put into a 100 ml volumetric flask and dissolved in absolute ethanol. The
concentration was calculated by the standard curve prepared using the UV absorption. The drug
loading was calculated by Eq. (1).
WDrug loading (%) =(CSample ×100)/ MSample× 100% (1)
Where CSample(μg/ml) is the concentration of sample, MSample (g) is the mass of sample.
Floating test
The floating rate was calculated in 0 h,12 h and 24 h by dispersing CUR-HPs in hydrochloric
acid(0.1 mol/l) containing 0.25% sodium dodecyl sulfate (SDS). At predetermined time intervals, the
number of CUR-HPs was counted. The floating rate was described by Eq (2).
Floating rate (%)=(Number of floating microspheres)/ (Total numbers of microspheres)×100% (2)
In vitro release
In vitro release behavior was investigated by paddle stirring method using ZRS-8G type intelligent
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dissolution test instrument. Hydrochloric acid (0.1 mol/l) containing 0.25% SDS was selected as the
dissolution medium. Briefly, a certain amount of CUR-HPs (containing 10 mg of CUR) in the capsules
were put into 900 ml dissolution medium under 37℃and 120 rpm. Samples (5 ml) were collected at
predetermined time intervals and then filtered through a 0.45 μm hydrophilic Millipore membrane. The
equal volume of blank medium at 37℃ was supplemented immediately after sample collection. The
dissolved CUR in the sample solution was determined by UV-visible spectrophotometer
(UV1700-1800) at 425nm.
Inter-batch differences
Floating rate, accumulative release, drug loading, yield and particle size of CUR-HPs were carried
out for three batches (1, 2, and 3) samples, respectively. The formula Eq. (3) was used to determine
similarity of CUR-HPs by the in vitro accumulative release.
f2=50×log {[1+ (1/n) 2]-0.5×100} (3)
Scanning electron microscopy (SEM)
The morphologies of the freshly prepared CUR-HPs were examined by scanning electron
microscopy (SEM) (Hitachi S-3000N, Japan).
Power X-ray diffraction (XRD)
The diffraction behavior was obtained between 10-90 θ at room temperature. The X-ray diffraction
of powers behavior was measured by an X’ D/MAX-2500/PC diffract meter (Rigaku Corporation,
Tokyo, Japan) with a copper anode (Cu Ka radiation, 40 mA, 40 kV, k = 0.15405 nm).
Differential scanning calorimeter (DSC)
The crystal of CUR-HPs was measured by DSC (METTLER 1100LF RT-35, Switzerland). The
sample (about 3 mg) was heated in an open aluminum standard pan, and scanned from 25 ℃to 350℃at
the rate of 10 ℃/min, used argon gas (99.99%) as the purging gas.
Particle size, entrapment efficiency and yield
The mean particle size was measured by an optical microscope with the help of a calibrated ocular
micrometer. Entrapment efficiency and yield were calculated used the formula Eq. (4) and Eq. (5).
Entrapment efficiency (%) =(1－Cf/Ct)×100% (4)
Where Cfis the amount of free drug, Ct is total drug amount of CUR-HPs.
Yield (%) = [Dry microsphere weight / (CUR weight + EC weight)] × 100% (5)
Analysis of drug kinetics
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Four kinetic models including Higuchi’s equation, Korsmeyer et al’s equation, first-order release
equation, and zero-order release equation, as shown in Eqs. (6-9), were chosen to assess the in vitro
drug release behavior, respectively.
M t = Kh t1 / 2
+ b(6)
M t = K k tn( 7 )
Ln (100-Mt) = K1 + b (8)
M t = K 0 + b (9)
Stability study
The effects of high temperature, high humidity and bright light on the stability of CUR-HPs were
examined as follows: For high temperature test, CUR-HPs were placed at 60℃ for 10 d. On days 5 and
10, the drug loading, accumulative release and floating rate were measured, respectively. If the
temperature at 60℃ had significant impacts on the quality of CUR-HPs, the above operation would be
repeated at 40℃. For high humidity test, CUR-HPs were stored at 25℃the relative humidity of (90 ±
5)%for 10 d. On days 5 and 10, the drug loading, accumulative release and floating rate were measured.
If the relative humidity of (90 ± 5) % at 25℃ influences the quality of CUR-HPs, the above operation
would be repeated at (70 ± 5) % of humidity at 25℃. For bright light test, CUR-HPs capsules were
stored at (4500 ± 500) LX for 10 d. On the days 5 and 10, the drug loading, accumulative release and
floating rate were measured, respectively.
Pharmacokinetics
The healthy SD rats (250 ± 30 g) were obtained from the Laboratory Animal Center of Southwest
Medical University. Animals were allowed free access to food and water in this study. Animal
experiments were approved by the Southwest Medical University animal ethical experimentation
committee (Sichuan, China) (No 2015DW040).
Forty rats were randomly divided into two groups with 20ratsineachgroup. Free CUR solution and
CUR-HPs at a dose of 50 mg/kg body weight were orally administered to the two groups, respectively.
The blood samples (0.25 ml each) were collected from rats at predetermined time points (5 min, 10 min,
15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h) and centrifuged at 5000 rpm for 3 min immediately to
obtain the plasma. Citric acid buffer (pH 3.5, 25 μl) was added to the 100 μl of plasma sample and
mixed by vortex for 30 s, and the resultant mixture samples were extracted twice with 2 ml of
acetonitrile-methanol (9:1, v/v) by vigorous vortex for 3 min. The supernatant obtained at 5000 rpm for
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3 min was dried under a steam of nitrogen gas. Each sample was re-dissolved with 200 μl of mobile
phase and centrifuged at 10000 rpm for 10 min to obtained supernatant. Finally, 20 μl of supernatant
was injected into HPLC system for analysis.
CUR in the samples was determined by HPLC analysis using a reverse phase stainless steel
column (Inertsil ODS-SP C18, 4.6 × 250 mm). The mobile phase consisted of 1% (v/v) phosphate
buffer (pH 3.5) and acetonitrile (35%:65%, v/v) with a flow rate (1 ml/min). The column temperature
was 30℃ and the effluent was monitored at 425 nm.
Data analysis
The pharmacokinetic parameters including the maximum plasma drug concentration (Cmax
), the area
under the plasma drug concentration time curve up to 24 h post administration (AUC0–t
), the time to
reach the maximum plasma drug concentration (Tmax
), the mean residence time (MRT) and the
elimination half-life (T1/2z
) were estimated by DAS 2.0 pharmacokinetics software. All the values were
reported as mean ± SD and P<0.05 was considered the statistical significant difference.
Results and discussion
Preparation and optimization
CUR has good pharmacological effects, such as anti-tumor, anti-inflammatory and so on [18].
During the phase I clinical trial, the patients were given CUR 6 g daily but didn’t find any significant
toxic and side effects suggesting that CUR wasa safe candidate drug [19]. However, CUR has fast
metabolism, poor stability and poororal bioavailability, which seriously limits its clinical application
[20]. Hollow microspheres, as multiple unit drug delivery systems, not only control drug release and
reduce the frequency of administration, but also have good floating result in reducing gastric emptying
and improving the oral bioavailability of insoluble drug [1]. Therefore, CUR-HPs were developed in
the present study.
In a single factor experiment, the effect of different molecular weight of EC on the quality of
CUR-HPs was investigated. As shown in Table 1, the same amounts of EC100 cp(F1), EC45 cp (F2) and
EC10 cp(F3) were used to prepare CUR-HPs, respectively. However, CUR-HP was not obtained in the
case of EC100 cp, because the viscosity of EC100 cp is too large to disperse into small milk drops under the
same shearing force. Fig. 1(a) showed that the accumulative release for 24 h of F2 was slower than F3,
suggesting that with the increase in molecular weight of EC, the viscosity increased and the release of
drug was slower [1]. Additionally, compared with F2, F3 showed favorable appearance and uniform
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particle size. Therefore, EC10 cp was selected for further study. The effect of amount of EC10 cp on the
quality of CUR-HPs was investigated. As shown in Table 1, 0.52 g, 0.26 g, and 1.04 g of EC10 cp were
used to prepare CUR-HPs in F3, F4, F5, respectively. CUR-HPs were not formed in the case of EC10 cp
amounts 0.26 g and 1.04 g, because viscosity with 0.26 g EC10 cp was too small to form stable milk
drops and viscosity with 1.04 g EC10 cp was too large to disperse into small milk drops. Thus, EC10 cp
0.52 g was appropriate for further evaluations. CUR was stable at pH less than 7 [21]. Therefore,
amount of CA as a PH modulator played an important role to control the quality of CUR-HPs. CA
(0.075 g, 0.038 g and 0.15 g) were used to prepare CUR-HPs in F3, F6 and F7, respectively. Fig. 1(b)
indicated that the accumulative rate of CUR-HPs increased with the amount of CA. The accumulative
rates of F3, F6 and F7 were (89.49± 1.27) %, (77.50± 0.95) % and (86.55± 0.89) % at 8 h, respectively.
As shown in Table 2, floating rates at 2 h and 24 h of F3, F6 and F7 were (73.67 ± 2.52)% and (70.00 ±
1.00)%, (60.33 ± 1.53) % and (55.33 ± 1.53) %, (97.00 ± 1.00) % and (96.00 ± 1.73) %, respectively,
which suggested most of the microspheres could float well in the medium in the case of 0.15 g of CA.
In addition, drug loading of F3, F6 and F7 was (5.77 ± 0.71) %, (5.74 ± 0.27) % and (6.07 ± 2.01) %,
respectively. There is no significant difference in the drug loading among the three formulations. The
CUR-HPs in F7 showed the higher drug release, better floating properties and drug loading. Thus, 0.15
g of CA was selected for further study. In order to investigate the effect of amount of CUR on
microspheres, 0.07 g, 0.035 g and 0.14 g of CUR were used to prepare CUR-HPs in F7, F8 and F9,
respectively. Fig. 1(c) indicated that the accumulative rates were (89.49± 0.80) %, (44.26± 0.86) %,
(85.04± 0.87) % for F7, F8 and F9 at 8 h, respectively. As shown in Table 2, the floating rates at 2 h and
24 h of F7, F8 and F9 were (84.00 ± 2.00) % and (83.33 ± 1.15) %, (84.00 ± 2.00) % and (86.33 ±
1.15) %, (63.67 ± 2.52) % and (60.00 ± 1.00) %, respectively. When CUR reached 0.14 g, the floating
rate of microspheres was remarkable decreased. Drug loadings of F7, F8 and F9 were (5.77 ± 0.71) %,
(5.36 ± 0.17) % and (6.99 ± 0.66) %, respectively. Finally, 0.07 g of CUR was selected for further
study.
On the basis of pre-test and single factor test, the amounts of EC10 cp, CA and CUR were the most
important factors affecting the quality of microspheres. Based on the results of the orthogonal test and
the range analysis, Table 3 showed that the order of the effect on the quality of CUR-HPs was
CA>CUR> EC10 cp. According to the K value, the optimal formulation was EC10 cp (0.624 g), CA (0.15
g), and CUR (0.07 g).
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Validation and evaluation of formulation
Reproducibility of CUR-HPs
In order to investigate the reproducibility of the optimized formulation, three batches of CUR-HPs
were prepared with amplifying 50 times. As shown in Table 4 and Fig. 2, three batches of samples
prepared in this study showed no significant differences in yield, drug loading, floating rate, particle
size and accumulative release behavior (P >0.05), suggesting that the optimal formulation had a good
reproducibility. The accumulative release CUR from the CUR-HPs after 24 h mostly reached
99%-102%. Interestingly, most of the CUR-HPs remained satisfactory floating properties after 24 h.
The possible reason is that the channels of hollow microspheres were too small to penetrate into the
hollow cavities by the simulated gastric fluid or the channels were not connected to the cavities.
Similarity factors of three batches were 89.29 (1), 81.54 (2) and 83.60 (3), respectively and higher than
50, which indicated that the preparation of the three batches of samples had similar drug release
behavior [22].
Morphology and Phase analysis
SEM experiment was carried out to assess the morphology of CUR-HPs. The CUR-HPs was
spherical with homogeneous particle size, smooth surface and hollow cavity inside (Fig. 3). In addition,
CUR, massing mixing all materials made of CUR-HPs, blank hollow microspheres and CUR-HPs were
measured by XRD to evaluate the degree of CUR crystalline in hollow microspheres (Fig. 4). The
characteristic diffraction peaks for crystalline CUR was not observed in CUR-HPs, which indicated
that CUR in CUR-HPs existed as an amorphous form. In the DSC test (Fig. 5), the thermodynamic
curve of pure CUR displayed an endothermic peak at 187.04℃ [23], which corresponded to CUR
melting point. The endothermic peaks were not presented. These results suggested that CUR existed in
the HPs was amorphous state, consisted with XRD.
Release behavior of CUR-HPs
Different kinetics release models were used to reflect different drug release mechanisms [24]. In
order to explore the mechanism of CUR-HPs, Higuchi’s equation, Korsmeyer et al’s equation,
First-order release equation and Zero-order release equation were used to analyze in vitro released data
and the kinetic parameters were shown in Table 5. In vitro release profiles of CUR-HPs could be best
expressed by Korsmeyer et al’s equation, where the coefficient of correlation (r) was 0.9735 and the
slope (n) was 0.8402, suggesting that the diffusion was the dominant mechanism of drug release from
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CUR-HPs. In general, when n ranged from 0.45 to 0.89, the drug released from delivery system by the
combination of diffusion and erosion mechanisms [25].
Stability of CUR-HPs
High temperature, high humidity and bright light tests were used to investigate the stability of
CUR-HPs for 10 d and the results were shown in Table 6 and Table 7. Table 6 showed that the
accumulative release of three samples at 24 h after 10 d changed from (98.17 ± 1.89) % to (79.10 ±
1.38) % at 60℃and from (101.42 ± 2.06) % to (101.56 ± 1.67) % at 40℃, respectively. Drug loading of
the above three samples varied from (3.43 ± 0.11) % to (2.29 ± 0.19) % at 60 ℃ vs. (3.55 ± 0.23) % to
(3.51 ± 0.11) % at 40℃ , as well as floating rates at 24 h decreased from (87.00 ± 1.00) % to (65.00 ±
2.00) % at 60℃ vs. (89.00 ± 2.00) % to (86.00 ± 1.00) % at 40℃. The results suggested that
temperature played an important role in the stability of CUR-HPs. Namely, high temperature (60℃)
had a greater impact on the floating rate, drug loading, and drug release behavior of CUR-HPs than the
low temperature (40℃). Table 7 presented the effects of high humidity and bright light on
characteristics of CUR-HPs. Overall the drug loading and accumulative rates were similar in the above
conditions at days 0, 5 and 10. No marketable change was observed in physical properties in terms of
color, surface morphology and particle flow of the CUR-HPs. The results of test suggested that hollow
microsphere technology improved stability of CUR.
Pharmacokinetics of CUR-HPs
The aim of in vivo study in SD rats was to evaluate the pharmacokinetic behavior of CUR-HPs after
oral administration in comparison with CUR as the control. As shown in Fig. 6, the chromatograms of
CUR showed a stable baseline and good resolution between CUR and endogenous materials in plasma.
The limit of quantification (LOQ) for CUR in biological samples was 5 ng/ml. The standard curve with
CUR concentrations ranging from 5 to 2000 ng/ml exhibited good linearity for all measured samples.
The intra-day and inter-day precision values were less than 10%. The extraction recoveries of CUR in
the concentration of 20, 200 and 2000 ng/ml from biological samples were more than 95%. Therefore,
the method met the requirements of biological sample analysis.
The average plasma drug concentration-time curve was shown in Fig. 7. The pharmacokinetic
parameters were summarized in Table 8. After oral administration of CUR, the rapid drug absorption
was observed. The concentration of CUR in plasma presented a declining trend and the drug
concentration were undetectable after 12 h. Maximum peak value was 13.25 (μg/l) at 0.5 h and
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consistent with previous report [26]. The concentration in plasma after oral administration of CUR-HPs
presented double peaks, associated with the vitro drug release behavior of CUR-HPs. The drug
concentration reached maximum peak value (42.51 μg/l) at 8 h, which was 4 times that of free CUR.
The peak time of CUR-HPs was delayed for 7.5 h. Moreover, the MRT of CUR-HPs was 2.84-fold
larger than CUR. CUR-HPs increased CUR half-time by approximately 13 h, which was longer than
previous research [26]. Compared with CUP, CUR-HPs increased AUC (0-t) by 12.08 times than that of
free CUR as the control, showing a greater extent of oral absorption.
Conclusion
In this research, CUR-HPs were successfully developed with desirable characteristics. The hollow
microspheres improved CUR half time and oral bioavailability. CUR-HPs might be a promising drug
delivery system to improve clinic application.
Acknowledgments
This study was financially supported by the Science and Technology Fund for Distinguished Young
Scholars of Sichuan Province (No.2017JQ0013)，the Joint Fund of Luzhou City and Southwest Medical
University [No.2017LZXNYD-T02, 2015LZCYD-S09 (4/8)]，the scientific research Foundation of the
Education Department of Sichuan Province (No.17ZA0439, 18ZB0646), the scientific research
Foundation of Sichuan Provincial Human Resource and Social Security Department (No.2016-183),
the Joint Fund of Sichuan Province, Luzhou City and Southwest Medical University (No.14JC0134,
14ZC0026, 14ZC0066), the research grant from National and Sichuan province Innovative
Entrepreneurship Training Program For Undergraduates (No.201310632015. 201307010325, 2014051-
7330416), the scientific research Foundation of Sichuan Provincial Health Department (No.130270,
130269) and the scientific research Foundation of Southwest Medical University (No.2016-63).
Declaration of interest
The authors report no conflicts of interest.
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Table 1 Different formulations of CUR-HPs.
Formulation
Formulated amount (g)
F1 F2 F3 F4 F5 F6 F7 F8 F9
CUR 0.070 0.070 0.070 0.070 0.070 0.070 0.070 0.035 0.140
CA 0.075 0.075 0.075 0.150 0.150 0.038 0.150 0.150 0.150
EC100 cp 0.52 - - - - - - - -
EC45 cp - 0.52 - - - - - - -
EC10 cp - - 0.52 0.26 1.04 0.52 0.52 0.52 0.52
Abbreviations: CUR, curcumin; CA, citric acid; EC100 cp, ethyl cellulose with a viscosity of 100; EC45
cp, ethyl cellulose with a viscosity of 45; EC10 cp, ethyl cellulose with a viscosity of 10.
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Table 2 Amounts of CA and CUR effect on floating rate and drug loading of CUR-HPs (Mean ± SD,
n=3).
No. F3 F6 F7 F8 F9
Floating rate for
2 h (%)
73.67 ± 2.52 60.33 ± 1.53* 97.00 ± 1.00* 86.67 ± 1.53*
63.67 ± 2.52*
Floating rate for
24 h (%)
70.00 ± 1.00 55.33 ± 1.53* 96.00 ± 1.73* 86.33 ± 1.15* 60.00 ± 1.00*
Drug loading
(%)
5.77 ± 0.71 5.74 ± 0.27 6.07 ± 2.01 5.36 ± 0.17 6.99 ± 0.66*
Note: Compared with F3,*P < 0.05.
Abbreviations: CUR, curcumin; CA, citric acid;CUR-HPs, curcumin hollow microspheres.
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Table 3 Results of orthogonal experimental design.
No. EC10 cp CA CUR Y(%) FR (%) EE (%) X
1 0.416 0.120 0.056 89.310 30.000 47.760 167.070
2 0.416 0.150 0.070 97.440 80.000 53.660 231.100
3 0.416 0.180 0.084 70.600 58.000 43.460 172.060
4 0.520 0.120 0.070 97.70 66.000 48.870 212.570
5 0.520 0.150 0.084 95.820 70.000 48.530 214.350
6 0.520 0.180 0.056 83.880 46.000 51.000 180.880
7 0.624 0.120 0.084 91.180 73.000 47.690 211.870
8 0.624 0.150 0.056 96.960 87.000 51.440 235.400
9 0.624 0.180 0.070 91.260 81.000 36.030 208.290
K1/3 190.077 197.170 186.607 - - - -
K2/3 202.600 226.950 217.320 - - - -
K3/3 218.520 187.077 199.427 - - - -
R 28.443 39.873 30.713 - - - -
Abbreviations: FR, floating rate; EE, entrapment efficiency; Y, yield; X, X = FR + EE + Y; CUR,
curcumin; CA, citric acid; EC10 cp, ethyl cellulose with a viscosity of 10.
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Table 4 Drug loading, floating rate for 24 h, accumulative release for 24 h, yield and particle size of
three batches samples(Mean ± SD, n=3).
Parameters
Batches
Average
1 2 3
Drug loading (%) 3.39 ± 0.28 3.68 ± 0.31 3.43 ± 0.24 3.41 ± 0.21
Floating rate (%) 95.00 ± 5.20 85.33 ± 1.53 88.67 ± 2.52 89.67± 4.91
Accumulative release (%) 97.76 ± 5.13 97.66 ± 1.14 93.60 ± 2.19 96.34 ± 2.37
Yield (%) 96.19 ± 1.80 97.20 ± 1.58 94.85 ± 1.37 96.08 ± 1.18
Particle size (μm) 641.67 ± 14.43 666 ± 14.43 658.33 ± 14.43 655.56 ± 14.43
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Table 5 Kinetic parameters of CUR-HPs.
Higuchi’sequation Korsmeyeretal’s equation First order equation Zero order equation
r kh r n r k1 r k0
0.974 0.552 0.991 0.840 0.971 0.214 0.950 4.116
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Table 6 Changes of floating rate, drug loading and accumulative release of CUR-HPs at 60 ℃ and 40 ℃
(Mean ± SD, n=3).
Time (d)
60 ℃ 40 ℃
0 5 10 0 5 10
Floating rate
for 0 h (%)
92.33 ± 0.58 94.00 ± 1.00 81.67 ± 1.53** 92.00 ± 2.00 88.67 ± 1.53 87.33 ± 1.53
Floating rate
for 2 h (%)
92.33 ± 0.58 89.00 ± 1.00 76.33 ± 2.52** 91.67 ± 1.53 88.00 ± 1.00 85.67 ± 0.58
Floating rate
for 24 h (%)
87.00 ± 1.00 85.33 ± 1.53 65.00 ± 2.00** 89.00 ± 2.00 85.67 ± 1.53 86.00 ± 1.00
Drug loading
(%)
3.43 ± 0.11 2.64 ± 0.12** 2.29 ± 0.19** 3.55 ± 0.23 3.38 ± 0.08 3.51± 0.11
Accumulative
release (%)
98.17 ± 1.89 84.03 ± 1.83 79.10 ± 1.38** 101.42 ± 2.06 104.05 ± 3.23 101.56 ± 1.67
Note: Compared with 0 d,**
P<0.01 at 60℃.
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Table 7 Changes including floating rate, drug loading and accumulative release of CUR-HPs in the
condition of high humidity (92.5%) and bright light (4500 ± 500 LX) at 25℃(Mean ± SD, n=3).
Time (d)
High humidity (92.5%, 25℃) Bright light (4500 ± 500 LX)
0 5 10 0 5 10
Floating rate
for 0 h (%)
93.33 ± 1.53 91.00 ± 1.00 84.67 ± 1.53 92.00 ± 2.00 89.33 ± 0.58 82.33 ± 1.53
Floating rate
for 2 h (%)
91.33 ± 1.15 86.00 ± 1.73 84.67 ± 1.53 92.33 ± 2.51 86.67 ± 2.08 80.33 ± 1.53
Floating rate
for 24 h (%)
87.33 ± 1.53 86.33 ± 2.08 82.67 ± 2.51 88.00 ± 1.00 88.33 ± 1.15 80.00 ± 1.00
Drug loading
(%)
3.46 ± 0.13 3.51 ± 0.47 3.45 ± 0.20 3.43 ± 0.10 3.50 ± 0.09 3.54 ± 0.17
Accumulative
release (%)
100.56 ± 0.34 102.20 ± 0.75 101.12 ± 1.85 100.99 ± 0.86 101.06 ± 2.35 101.09 ± 1.70
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Table 8 Main pharmacokinetics parameters of CUR and CUR-HPs.
Parameter Units CUR CUR-HPs
AUC(0-t) μg/l*h 43.595 526.495
MRT(0-t) h 3.282 9.314
T1/2z h 6.462 19.601
Tmax h 0.5 8.000
Cmax μg/l 13.25 42.51
Abbreviations: AUC(0-t), the area under the plasma drug concentration time curve up to 24 h post
administration; MRT(0-t), the mean residence time of 24 h post administration; T1/2z, the elimination
half-life; the time to reach the maximum plasma drug concentration; Tmax, peak time; Cmax, the
maximum plasma drug concentration; CUR, curcumin; CUR-HPs, hollow microspheres of curcumin.
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Fig. 1 Three factors effect on prescription. (a) is amount of EC 45 cp(F2) and EC10 cp (F3) effect on release
of CUR-HPs for 24 h in simulate gastric fluid containing 0.1 mol/l HCL and 0.25% SDS (900 ml, 37℃).
(b) is amount of CA effect on release of CUR-HPs for 24 h in simulate gastric fluid containing 0.1
mol/l HCL and 0.25% SDS (900 ml, 37℃). (c) is amount of CUR effect on release of CUR-HPs release
for 24 h in simulate gastric fluid which containing 0.1 mol/l HCL and 0.25% SDS (900 ml, 37℃). The
values are expressed as mean ± SD (n=6).
Fig. 2 Values are expressed as mean ± SD (n=6). Accumulative release curves in vitro of three batches
CUR-HPs in simulate gastric fluid containing 0.1 mol/l HCL and 0.25% SDS (900 ml, 37℃).
Fig. 3 Scanning electron micrograph of CUR-HPs.
Fig. 4 XRDpatterns. (a) is free CUR, (b) is massing mixing all materials made of CUR-HPs, (c) is
blank hollow microspheres, and (d) is CUR-HPs.
Fig. 5 DSC patterns.(a) is pure CUR, (b) is massing mixing all materials made of CUR-HPs, and (c) is
CUR-HPs.
Fig. 6 Specific properties of CURin plasma sample.
Fig. 7 Pharmacokinetics of CUR-HPs compared with free CUR in rats.
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