Enhanced oral bioavailability and in vivo antioxidant activity of chlorogenic acid via liposomal formulation

Enhanced oral bioavailability and in vivo antioxidant activity of chlorogenic acid via liposomal formulation

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Accepted Manuscript Title: Enhanced oral bioavailability and in vivo antioxidant activity of chlorogenic acid via liposomal formulation Author: Yingshu Feng Congyong Sun Yangyang Yuan Yuan Zhu Jinyi Wan Caleb Kesse Firempong Emmanuel Omari-Siaw Yang Xu Zunqin Pu Jiangnan Yu Ximing Xu PII: DOI: Reference:

S0378-5173(16)30083-7 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.01.081 IJP 15547

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

30-10-2015 30-1-2016 31-1-2016

Please cite this article as: Feng, Yingshu, Sun, Congyong, Yuan, Yangyang, Zhu, Yuan, Wan, Jinyi, Firempong, Caleb Kesse, Omari-Siaw, Emmanuel, Xu, Yang, Pu, Zunqin, Yu, Jiangnan, Xu, Ximing, Enhanced oral bioavailability and in vivo antioxidant activity of chlorogenic acid via liposomal formulation.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.01.081 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Enhanced oral bioavailability and in vivo antioxidant activity of chlorogenic acid via liposomal formulation

Yingshu Feng†, Congyong Sun†, Yangyang Yuan†, Yuan Zhu, Jinyi Wan, Caleb KesseFirempong, Emmanuel Omari-Siaw, Yang Xu, Zunqin Pu, JiangnanYu*, Ximing Xu*

Center for Nano Drug/Gene Delivery and Tissue Engineering, School of Pharmacy, Jiangsu University, Zhenjiang 212013, China


Correspondence to: X. Xu, Department of Pharmaceutics, School of Pharmacy, Center for Nano

Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Zhenjiang 212013, People’s Republic of China. E-mail: [email protected] *

Jiangnan Yu, Department of Pharmaceutics, School of Pharmacy, Center for Nano Drug/Gene

Delivery and Tissue Engineering, Jiangsu University, Zhenjiang, 212013, People’s Republic of China. E-mail:[email protected]

These authors contribute equally to this work.


Abstract In the present study, a formulation system consisting of cholesterol and phosphatidyl choline was used to prepare an effective chlorogenic acid-loaded liposome (CAL) with an improved oral bioavailability and an increasedantioxidant activity. The developed liposomal formulation produced








pharmacokinetic analysis of CAL compared withchlorogenic acid (CA), showed a higher value of Cmax(6.42 ± 1.49 min versus 3.97 ± 0.39 min)and a delayed Tmax(15 min versus 10 min), with

1.29-fold increase in relative oral bioavailability. The tissue distribution in mice also demonstrated that CAL predominantly accumulated in the liver which indicated hepatic targeting potential of the drug. The increased activities of antioxidant enzymes (Total Superoxide Dismutase (T-SOD) and Glutathione Peroxidase (GSH-Px)) and total antioxidant capacity (T-AOC), in addition to decreased level of malondialdehyde (MDA) in CCl4-induced hepatotoxicity study further revealed that CAL exhibited

significant hepatoprotective and antioxidant effects. Collectively, these

findings present a liposomal formulation with significantly improved oral bioavailability and an increasedin vivo antioxidant activity of CA. .

Key words Chlorogenic acid, liposome, bioavailability, antioxidant activity, hepatoprotective

1. Introduction Eucommiae Cortex,the dry bark of EucommiaulmoidesOliv., belongs to the Eucommiaceae Family. This medicinal plant has therapeutic effects on hypercholesterolemia, fatty liver, hypertension (Xirui et al 2014) and Alzheimer's disease (Butterfield 2002). Several studies have shown











anti-osteoporosis(Zhou et al 2009) and neuro protective(Bouayed et al 2007, Li et al 2008) activities. The Eucommiae Cortex also offersa very high antioxidant activities (Ae et al 2006, GC & CL 2000, Hsieh & Yen 2000, Zhang et al 2007), such as the inhibition of oxidative damage in

DNA, scavenging activities towards free radicals and reactive oxygen species (ROS). Chlorogenic acid (CA, 5-caffeoylquinic acid, Fig.1A) is one of the major components in Eucommiae Cortex.

The CA could exhibit significant biological activities such as

antioxidative(Mussatto et al 2011), antihypertensive(Zhao et al 2012), antibacterial(Zaixiang et al 2011), anti-tumor (AB et al 2007) and anti-inflammatory (Guo et al 2015, Shin et al 2015) properties. It can also be a promising precursor compound for the development of medical products that can resist HIV-1 RNase(Naso et al 2014). However, the hydrophilic nature of CA makes it difficult to traverse the lipophilic membrane barrier (Mcclements 2015), which leads to low bioavailability. According to recent in vivo studies, only 1/3 of CA reached blood circulation after ingestion and it was even rapidly metabolized (dos Santos et al 2005). These drawbacks undoubtedly limitedthe effective utilization and clinical application of this drug. Therefore, it is important to find a suitable delivery system which can successfully overcome these challenges so as to simultaneously improve the

bioavailability and antioxidant activity of the compound.

Several formulations have been reported to increase the bioavailability of CA, such as cyclodextrin inclusion compounds (Bhattacharyya et al 2014), phospholipid complex (Budryn et al 2015) and nanoparticles (Nallamuthu et al 2014).

However, none of these studies has

investigated the potential of liposomes as a drug delivery system. The liposome also offersan excellentcarrier system due to its high oral bioavailability, biocompatibility, biodegradability, stability, hepatic targetability, and high cell membrane permeability(Gómez-Hens


Fernández-Romero 2006, Slingerland et al 2012). Therefore, an attempt to investigate CA-loaded liposomesand its related pharmacological effectiveness could be helpful. In this report, chlorogenic acid-loaded liposomes(CAL) were successfully prepared and

characterized for in vivostudies. The bioavailability and tissue distribution studies of CAL were also evaluated in rats and mice respectively.. Additionally, the in vivo antioxidant activity of CAL was further studied through CCl4-induced hepatotoxicity in mice. 2. Materials and method 2.1 Materials Eucommiae Cortex was purchased from Anhui Xiehe Pharmaceutical Co., Ltd. (Anhui, China). Puerarin (98.0% purity) and ascorbic acid (99.0% purity) were purchased from Aladdin Industrial Corporation (Shanghai, China). Carbon tetrachloride, cholesterol, dichloromethane, ethanol, hydrochloric acid, castor oil and chromatographically pure methanol were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Soybean lecithin of analytical injection grade was purchased from Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Deionized water was obtained with a Millipore Milli-Q water system (Milford, MA, USA). All other chemicals were obtained commercially and of analytical grade. 2.2 Animals SD rats (280  10 g) and KM mice (22  1 g) were supplied by the Laboratory Animal Centre of Jiangsu University (Zhenjiang, China). Animals were kept under controlled conditions (room temperature 24 ± 0.5 °C, relative humidity 55 ± 5%) and an alternating 12 h day and night light cycles. The animals were housed and acclimatized to our laboratory conditions for 3 days with free access to water and fodder. The study

was conducted in accordance with the experimental

protocols reviewed and approved by the University Ethics Committee for the use of experimental animals and the Guide for Care and Use of Laboratory Animals. Prior to the experiment, animals were fasted for 12 h and provided with only water.

2.3 Extraction of chlorogenic acid The CA was isolated from Eucommiae Cortex. Briefly, dried Eucommiae Cortex powder (100 g) was triply extracted with 50% ethanol (1 L) at 50 °C for 2 h. The resulting solution was concentrated under vacuum and purified using D101macroporous resin (Nankai HECHENG S&T Co., Ltd., Tianjin, China) and C8 column (Beijing Green herbs Science and Technology Development Co., Ltd., Beijing, China), which yielded 10 mg white crystalline compound. The molecular structure and purity were determined using 1H and


C NMR spectral

data, which confirmed the product as chlorogenic acid.

2.4 Preparation of chlorogenic acid-loaded liposomes The CAL were prepared via the modified thin film drying method described in previous studies (Bhattacharyya et al 2014). In short, 250 mg of Soybean lecithin and 50 mg of cholesterol were dissolved in 15 mL of dichloromethane with ultrasonic treatment to form a clear and transparent solution. The product was evaporated using rotary evaporator (Heidolph Co., Germany) to remove dichloromethane. Subsequently, 50 mg of CA dissolved in 20 mL of ethanol was added to the product and the rotary evaporation repeated at 50 °C to remove traces of ethanol, which resulted in film-like complexes. The dried film was hydrated with water to give a 5 mg/mL CAL solution. The final formulation was stored at 4 °C until further analysis. 2.5 Characterization of chlorogenic acid-loaded liposomes 2.5.1 Transmission electron microscopy The morphology of CAL (5 mg/mL) was examined using Transmission Electron Microscopy (TEM, Tecnai 12, FEI, Amsterdam, Holland). A drop of diluted CAL (20 μL, 500 μg/mL) was placed on a copper grid and stained with phosphotungstic acid (2%). After drying at room

temperature, theprepared thin film was observed under TEM. 2.5.2Particle size and Zeta potential analysis The particle size and zeta potential of the CAL (5 mg/mL) were measured with a Brookhaven 90 Plus PALS instrument (Brookhaven Instruments Corp., Holtsville, NY, USA). The measured scattering intensities and zeta potential were then analyzed using software provided by Brookhaven. The sample (3 ml) was placed in a cuvette and analyzed at 25 °C with an angle of 90°. The particle size distribution of the nanoparticles was obtained from polydispersity index(PDI). All measurements were performed in triplicates. 2.5.3Entrapment efficiency Drug entrapment efficiency was determined according to previous studies (He et al 2013, Yang et al 2015). Briefly,

the CAL (1 mL) was added to Sephadex G50 column (1.6 cm × 20 cm)

and then eluted with water (200 mL), followed by NaCl solutions (200 mL) of different concentrations (0.05 mol/L, 0.10 mol/L, 0.30 mol/L and 0.50 mol/L). The encapsulated CA was first eluted because of its bigger particle size. Later,

the non-encapsulated CA was also eluted

due to the smaller particle sizenature. The filtrateswere collected with 10-mL tubes. The CA concentration was measured using an RP-HPLC method. The entrapment efficiency (EE) of CA in liposomes was calculated according to Eq.(1) EE %

100% Eq.(1)

Where Ci,refers to the concentration of tube i;Vi, the volume of tube i; n1, the tube index of minimum CA concentration; and n2, the total number of tubes. The HPLC system consisted of an LC-20AT pump and an SPD-m10Avp Diode array detector, with the detector linked to LC-solution Data Station Software (Shimadzu Corporation, Tokyo, Japan). Chromatographic

separation was performed using Waters ODS column (4.6×150 mm, 5μm particle size, USA) at 30 °C. The mobile phase contained 30% methanol and 70% phosphoric acid solution (0.1%), which was eluted at a flow rate of 1.0 mL/min. The detection wavelength was 327 nm and the injection volume was 20 μL. 2.5.4 Storage stability In the stability studies, the CAL samples(5 mL each) were stored at 4 °C for different times (1, 3, 6, 9, 12 and 15 days). The storage stability of CAL was evaluated using the particle size and entrapment efficiency. 2.6 Pharmacokinetic studies of chlorogenic acid-loaded liposomes2.6.1 Animal studies Twelve male SD rats (280  10 g) were randomly and equally divided into two groups. The free CA and CAL(each at a dose of 100 mg/kg body weight) were orally given to the first and second groups, respectively. Plasma samples were collected from the orbital vein of the rats at different time points (0, 0.08, 0.17, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6 and 11 h) after the oral administration. 2.6.2 Treatment of plasma samples Plasma samples (200 μL) were spiked with 200 μLof methanol and 20 μLof puerarin (internal standard (IS), 250 μg/mL, Fig. 1B) with uniform mixing, followed by vigorous vortexing for 1 min. The mixture was centrifuged at 10000 rpm for 10 min. An aliquot of the supernatant (20 μL) was injected into HPLC to determine the concentration of CA. The in vivo chromatographic conditions for bio-sample determination were similar to the in vitro HPLC studies (section 2.5.3). Pharmacokinetic parameters of the drug, namely the peak concentration of the plasma (Cmax), time to attain peak concentration (Tmax), mean residence time (MRT) and other related indices

were determined by BAPP 2.3 pharmacokinetic software (supplied by the Center of Drug Metabolism of China Pharmaceutical University, China). 2.7Biodistribution studies of chlorogenic acid-loaded liposomes2.7.1 Animal studies Thirty Kunming mice were randomly divided into two equal groups, namely CA group and CAL group. The CA and CAL samples (each withA dose of 100 mg/kg body weight) were given to the respective groups via oral administration. Plasma samples were subsequently collected from the orbit region of the mice at different times (0.25, 0.75 and 2 h) into heparinized tubes after the drug administration. Similarly, the heart, liver, spleen, lung, kidney, stomach and brain were excised, washed, weighed and pretreated as in the tissue treatment section (2.7.2). 2.7.2 Treatment of tissue samples Normal saline (3 mL) was added to each of the different samples (heart, liver (0.3 g homogenate), spleen, lung, kidney, stomach and brain) to homogenize them. Then, 20 μLof puerarin(250 μg/mL), aninternal standard, was added to each homogenate (2 mL) and uniformly mixed

by vigorous vortexing for 1 min. The mixture was centrifuged at 10000 rpm for 10 min,

and the supernatant (20 μL) was injected into HPLC to analyze the CA concentration. Drug targeting index (DTI) was calculated according to Eq.(2) DTI(tissue) = CCAL/CCA Eq.(2) Where CCAL refers to the drug concentration of tissues at different time points (0.25, 0.75 and 2h) after CAL oral administration; CCA refers to the drug concentration of tissues at different time points (0.25, 0.75 and 2h) following

CAoral administration.

2.8In vivoantioxidant activity 2.8.1 Animal grouping and experimental design The in vivo antioxidant activity was evaluated via CCl4-induced hepatotoxicity model (Gan et al 2012, Liu et al 2014,Vulić et al 2014).The mice were randomly divided into five groups (six mice each) including normal control group (I), model control group (II), positive control group (III), CA group (IV) and CAL group (V). Mice in group I and II were treated with physiological saline (50 mg/kg body weight per day) by gastric gavage over 15 consecutive days. Simultaneously, mice in group III, IV and V were respectively treated with Vitamin C (50 mg/kg bodyweight per day), free CA (50 mg/kg) and CAL (50 mg/kg) via the same drug administration. An acute liver injury was experimentally inducedon the 16th day. All the mice were orally administered with a single dose of CCl4 and castor oil mixture (0.3%, v/v, 5 mL/kg body weight), except the normal control group which were administered with equal amount of only castor oil. The mice were then weighed and sacrificed via cervical dislocation after 24 hours. Blood samples were collected by retro-orbital bleeding and immediately centrifuged at 4000 rpm for 10 min at 4 °C to obtain the sera. The livers were excised, washed, weighed and homogenized immediately in 0.1 g/mL of ice-cold physiological saline. The homogenate was centrifuged at 3000 rpm for 10 min and the supernatant collected for further studies. All the aforementioned treatments were conducted at 4 °C, and the samples were stored at - 80 °C for subsequent investigations.

2.8.2 Biochemical assays The protein content ofthe liver was determined by the Bradford method using test kits.


activities of Glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC), in addition to the levels of malondialdehyde (MDA) in liver and serum, were assayed using similar test kits. All the commercial reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All determinations were performed according to the manufacturer’s instructionsin triplicate. 2.9 Statistical analysis All the experimental data were expressed as mean ± standard deviation. The mean differences between the various groups were evaluated using Student’s t test. P-values less than 0.05 were considered as statistically significant. All computations were done by employing the statistical software of SPSS version 15.0(SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1 Characterization of chlorogenic acid-loadedliposomes 3.1.1










LiposomesTheliposomal formulation produced a spherical, homogeneous, smooth surface and multilamellar structurednanoparticles (Fig. 2A). The size and

distribution profile of CAL (Fig.

2B), revealed a mean particle diameter of 132.15 ± 3.03 nm and polydispersity index (PDI) of 0.18 ± 0.08. The low polydispersity index indicated a narrow range of particle size distribution. The Zeta potentialgave

-23.08 ± 2.41 mV.

These results indicated that the liposomal encapsulation approached a monodispersed stable system and could effectively deliver the drug. Homogeneity and small particle sizes could affect

the uptake by reticuloendothelial system, leading to passive accumulation in certain tissues which can directly affect the in vivo circulation time and bio-distribution (Li et al 2015). A negative zeta potential is also advantageous as nanoparticles with negatively charged surfaces have reduced plasma protein bio-adhesion and a low rate of non-specific cellular uptake (Chuangnian et al 2012). Moreover, the nanosized and well-distributed liposomes are likely to increase transmembrane activities, thereby improving the CA bioavailability(Chang et al 2011a). 3.1.2Encapsulation efficiency of chlorogenic acid-loaded liposomes Based on the established in vitro HPLC method, the content of CA was calculated using


standard curve, with Y = 59126C + 18058 (n = 5, r2 = 0.9999) and a linear range of 1 - 100 μg/mL, where C is the concentration of CA in CAL and Y is the peak area of CA. The encapsulation efficiency (EE) of CAL was estimated as53.08 ± 0.92%. The EE, regarded as a quality assessment index of liposomes, can be a critical factor for the clinical curative effect of CA. Lipophilic bioactive compounds possess higher EE due to their accumulation in lipid membranes. However, hydrophilic bioactive compounds are incorporated into the aqueous core of liposomes (Mcclements 2015). Thissituation is associated with some challenges such as the creation of high internal aqueous phase volumes, which usually lead to low EE (<50%). The relatively high EE of CAL (53.08 ± 0.92%) might be due to some important mechanisms such as

electrostatic interaction, encapsulation and adsorption(Eloy et al 2014).

3.1.3Stability of chlorogenic acid-loaded liposomesNo crystal formation and layer separations in the liposomal formulation were observed during the storage period (15 days) at 4 °C. The CAL had desirable and acceptable stability which indicated that the liposome could serve as a carrier for CA although there was a slight decrease in EE (Table 1).

3.2 Pharmacokinetic analysis The plasma CA was calculated by HPLC method using standard curve, with Y = 0.1538C + 0.0264 (n = 5, r2 = 0.9999) and a linear range of 0.5 - 50 μg/mL, where C is the concentration of CA in plasma and Y is peak area ratios of CA to IS. The plasma concentrations of CA after the oral administration of free CA increased rapidly to a Cmax at 10 min while

theCAL was at 15 min, Fig. 3. A second absorption peak was also

observed at approximately 120 min in the two plasma concentration–time curves, Fig. 3.Thissecond peak observation could suggest a reabsorption that emanates from hepatoenteral circulation as previously reported(Qi et al 2011). Compared with free CA (p < 0.01), the CAL significantly increased the level of Cmax and AUC0-11h, which led to a relative oral bioavailability of 129.38%, Table 2. The t1/2 of CAL was insignificant compared with CA (p ˃ 0.05), although the CAL significantly improved the Cmax and AUC0-11h (p < 0.01), Table 2. These findings were in agreement with other related studies in which the t1/2 of liposomal formulations could not be prolonged compared with the free drug (Chang et al 2011b, Chengxue et al 2013, Kumar et al 2014). Ideally, a prolonged t1/2 of a drug within an acceptable range could have some pharmaceutical and pharmacological significance;however, in certain drugs it may not be applicable as observed in this report and other related studies.These results clearly revealed that CAL could enhance oral bioavailability of CA due to their ability to improve in vivo absorption. Liposomal formulations are expected to selectively deliver payloads at the target sites and to maintain theirhigh concentrations in the blood(Dahmani et al 2012). The liposomes could also protect drugs from enzymatic metabolism because a major portion of the drug stays within liposomal vesicles, thus avoiding esophageal contact which leads to higher oral bioavailability (Yan et al 2006). Additionally, the liposomes are formed by phospholipid bilayers which result in improved membrane permeability and cellular absorption. The inclusion of cholesterol in the liposomal formulation could further enhance the in vivo stability of (Arouri et al 2013).


3.3 Biodistribution studies The tissue distribution of both CAL and free CA was evaluated in sevenmajor organs and their accumulations at different time points (0.25, 0.75 and 2 h) after oral administrationare as shown in Fig. 4A, B and C, respectively. A quick accumulation of CA in the liver and spleen was observed during the first 0.75 h (Fig 4A and 4B).

The CA in both organs and the others gradually reduced after the 0.75-h period.

Over the 2-h period, the CA content of CAL in the liver and spleen were significantly higher (p < 0.05) compared with free CA (Fig 4C). The highest CA content was observed in the spleen at 0.75 h The DTI(liver) (1.79±0.10,2.05±0.19, and 1.95±0.49 at 0.25, 0.75 and 2 h respectively) and DTI(spleen) (1.30±0.14, and 2.06±1.51 at 0.75 and 2 h respectively) also demonstrated that the formulated CA effectively targeted the liver and spleen. The accumulation of CA in the liver and spleen could be ascribed to passive trapping of nanoparticles by reticuloendothelium(Park et al 2010). These results clearly showed that the CAL significantly enhanced the liver and spleen accumulation as compared to the free drug. Additionally, the distribution of free CA in kidney was higher (p < 0.05) compared with CAL at 0.25 h and 0.75h (Fig 4). Conversely, a higher (p < 0.05) distribution of CAL in kidney was observed at 2 h which suggested that CAL delayed the excretion of CA content. These findings indicated that the predominant accumulation of CAL in the liver and spleen might be the reason for the enhanced antioxidant activity (Vora et al 2015). 3.4 In vivo antioxidant activity 3.4.1 Effect of chlorogenic acid-loaded liposomes on body and relative organ weights The body weight and relative weight of the liver, used to evaluate the effects of free CA and CAL are presented in Table 3. There were no significant differences in the body weights of mice

among the treated groups (p ˃0.05), Table 3. These results indicated that the average body weight of mice was not affected by the different treatments(CCl4, CA or CAL). However, significant increases were observed in the relative weights of the liver for the CCl4 control group compared with the normal control group (p < 0.05). Thesedata demonstrated that CCl4 successfully induced liver hypertrophy in mice. Conversely, Vitamin C group compared with the model control group, showed a remarkable decrease in the elevated relative organ weights of the liver. Similarly, the administration of free CA and CAL significantly decreased the relative organ weight of the liver. These findings also confirmed that Vitamin C and CA could reduce the increased liver weight caused by CCl4. 3.4.2 Effect of chlorogenic acid-loaded liposomes on activities of hepatic antioxidant enzymes GSH-Px and T-SOD are the main antioxidant enzymes in sera and livers(Yao et al 2005). In the present study, there were significant decreases in the activities of GSH-Px and T-SOD in these samples for the CCl4 treated group compared with the normal control group (p < 0.05), Table 4. However, pretreatment with free CA and CAL significantly increased the activities of these hepatic antioxidant enzymes compared with CCl4 treated group (p < 0.05). Similarly, the activities of GSH-Px and T-SOD in CAL group were also higher than the CA group (p < 0.05). 3.4.3 Effect of chlorogenic acid-loaded liposomes on the levels of hepatic MDA and T-AOC The MDA and T-AOC have been employed to evaluate theantioxidant activities of several substances (Bagchi et al 1995). In the present study, the hepatic MDA content in CCl4 treated group was significantly higher than the normal control (p < 0.05) for the sera and livers (Table 4). The CCl4 treatment also showed significant decrease in the levels of hepatic T-AOC


with normal control (p < 0.05). In contrast, pretreatment with free CA and CAL significantly

decreased the MDA content, but increased the levels of T-AOC when compared with the CCl4 treated group (p < 0.05).Similarly,the levels of hepatic MDA in CAL group were also lower than the CA group (p < 0.05). CCl4 is a well-knownhepatotoxin which generates free radicals in experimental study of liver diseases. In liver, the CCl4 is converted by the cytochrome P450 system into a trichloromethyl radical. This radical reacts with oxygen to form a trichloromethylperoxyl radical which further interacts with cell macromolecules to induce free radical-mediated lipid peroxidation and to provoke hepatocyte membrane breakdown (Kalegari et al 2014). The T-SOD, GSH-Px, T-AOC and MDA are therefore widely used to evaluate the antioxidant activity of a given sample. The increase in T-SOD, GSH-Px and T-AOC, as well as the decrease in MDA level could demonstrate good antioxidant activity against CCl4-induced liver injury. In the present study, the indexes of GSH-Px, T-SOD, T-AOC and MDA were adopted to reflect the antioxidant activities of free CA and CAL. The

free-radical scavenging enzymes, such as T-SOD and GSH-Px, are key

components of the antioxidant defense system to protect the cells and extracellular matrix from the oxidative injury (Yao et al 2005). The activities of T-SOD and GSH-Px were very low in the model control group; however, they were elevated

inthe free CA and CAL treatment, which

restored the normal enzyme activities (Table 4). Similarly, the CAL treated group exhibited significant increases

in T-SOD and GSH-Px activities than that of the CA group. T-AOC is

another index which indicates the capacity of the non-enzymatic antioxidant defense system. The pretreatment with free CA and CAL could effectively increase the level of T-AOC in the livers and sera which showed that the non-enzymatic antioxidant defense system of mice was enhanced (Table 4).

The CAL treatment also exhibited higher T-AOC levels compared with the CA group.


lower levels of MDA(an oxidative stress marker) suggested a reduced lipid peroxidation

and a weaker oxidant stress (Bagchi et al 1995). In the present study, the MDA level was remarkably increased in the model control group, but pretreatment with CA and CAL effectively inhibited the release of MDA. Moreover, compared with the CA group, CAL exhibited significant decreases in MDA levels. In summary, the free CA and CAL had very good hepatoprotective effects against CCl4-induced liver injury, and the liposomal encapsulation also enhanced the hepatoprotective effects of CA. The improved antioxidant activity of CA can be attributed to the increased oral bioavailability, high CA accumulation in the liver and delayed excretion of the liposomal formulation. 4. Conclusions Chlorogenic acid-loaded liposome was successfully developed for oral administration with an enhanced oral bioavailability and anincreased antioxidant activity. A regular, spherical and multilamellar-shaped distribution

nanoparticle was produced. The

pharmacokinetic and

biodistribution studies demonstrated that the CAL improved the bioavailability and accumulated in the liver, hence enhancing the in vivo antioxidant activity. The in vivo antioxidant activity also revealed that the CAL obviously increased the activities of antioxidant enzymes (T-SOD, T-AOC, and GSH-Px), and reduced MDA levels in the sera and livers of the mice. Therefore, the liposomal nanoformulation could serve as a promising carrier for chlorogenic acid to improve oral bioavailability and antioxidant activities. Acknowledgements This work was supported by the National Natural Science Foundation of China ( grant

30973677, 81373371), the Doctoral Fund of Ministry of Education of China ( grant 20113227110012),










Industry-University-Research Institution Cooperation ( JHB2012-37, GY2012049, GY2013055) in Jiangsu province and Zhenjiang, Program for Scientific research innovation team in Colleges and Universities of Jiangsu Province and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Figure captions    Fig.1 Structures of chlorogenic acid (A) and puerarin (B). 

        Fig.2 TEM image of CAL. 


Fig.3 Particle size distribution of CAL. 



Fig.4  Mean  plasma  concentration—time  curve  of  free  CA  and  CAL  after  single  oral  administration in rats (mean ± SD, 50 mg/kg, n=5). 

            Fig.5 Biodistribution profile of CA in mice tissues at 0.25 h (A), 0.75 h (B) and 2 h (C)  after oral administration of CA and CAL. Data were expressed as content of CA/μg in  organ. CA and CAL freshly prepared are administered at dose of 100 mg/kg. Values  presented represent mean ± SD, n=5, *p < 0.05, ** p < 0.01. 


Table 1. Storage stability of CAL (n=3) Time(day) 




132.15 ± 3.03 

132.82 ± 3.71 

133.28 ± 5.28 

134.07 ± 6.18 

135.21 ± 4.91    135.57 ± 7.92 


0.18 ± 0.08 

0.19 ± 0.10 

0.19 ± 0.03 

0.20 ± 0.02 

0.18 ± 0.04 

0.21 ± 0.02 


53.08 ± 0.92 

52.56 ± 1.07 

51.89 ± 0.53 

51.17 ± 1.39 

50.93 ± 0.59 

50.45 ± 0.84 

Table 2. The in vivo pharmacokinetic parameters of free CA and CAL after single oral administration in rats (mean ± SD, 100 mg/kg, n=5)


Parameters (units) 



AUC 0‐660(mg/L∙min) 

1730.66 ± 33.50 

2239.12 ± 107.00** 


769.66 ± 59.61 

640.48 ± 196.17 


10 ± 0 

15 ± 0 


3.97 ± 0.39 

6.42 ± 1.49** 


1181.31 ± 249.28 

1008.86 ± 115.72 

Values are significantly different from the CA group at the level of P < 0.01

Table 3. Effects of CA and CAL pretreatment on CCl4-induced alteration in body weight and relative liver weight in mice Relative weight(g/100g body weight)  Group 

Body weight(g)  Liver 














Group I, II and III represent the group of normal control, model control and positive control, respectively. Group IV were treated with 50 mg/kg of free CA daily, and Group V were treated with 50 mg/kg of CAL daily. Each value is presented as mean ± SD (n = 6). * Values are significantly different from the model group at the level of P < 0.05.

Table 4. Effects of CA and CAL on the activities of GSH-Px, T-SOD, T-AOC and MDA levels in serums and livers GSH‐Px(U/mL) 




Group  In serums 

In livers 

In serums 

In livers 

In serums  In livers 

In serums  In livers 















14.7±0.7*  43.8±21.4* 
















12.7±4.2†  41.5±9.2† 

1130.6±298.5†,#  1305.7±128.5†,#  263.1±58.6†,#  142.9±17.7†b,#  8.6±2.7†,#  4.9±0.5†,#  9.8±1.9†,#  36.0±4.1†,#  Group I, II and III represent the group of normal control, model control and positive control, respectively. Group IV were treated with 50 mg/kg of free CA, and Group V were treated with 50 mg/kg of CAL. Each value is presented as mean ± SD (n = 6). *

Values are significantly different from the normal group at the level of P < 0.05.

Values are significantly different from the control group at the level of P < 0.05.


Values are significantly different from the CA group at the level of P < 0.05.