European Journal of Pharmaceutical Sciences 93 (2016) 224–232

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

The preparation of Cistanche phenylethanoid glycosides liquid

proliposomes: Optimized formulation, characterization and proliposome
dripping pills in vitro and in vivo evaluation
Meng Li, Yunjing Li, Weiwei Liu, Rongli Li, Cuiying Qin, Nan Liu, Jing Han ⁎
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, China

a r t i c l e i n f o a b s t r a c t

Article history: Water-soluble Cistanche phenylethanoid glycosides (CPhGs) have poor permeability and low bioavailability.
Received 26 January 2016 However, liposomes can improve the permeability of such drugs and their poor stability, and proliposomes
Received in revised form 13 July 2016 have been used to overcome these problems. Based on this, Cistanche phenylethanoid glycoside liquid
Accepted 30 July 2016
proliposomes (CPhGsP) and dripping(?) pills were prepared and optimized using response surface methodology.
Available online 1 August 2016
The properties of CPhGsP were evaluated in terms of their encapsulation efficiency, particle size, zeta potential,
Keywords:
and morphology. The results obtained showed that the optimal formulation was drug/soybean phospholipid/
Liquid proliposomes poloxamer-188/sodium deoxycholate/propylene glycol 1:22.38:3.52:0.84:80 (w/w/w/w/v). This resulted in an
Cistanche phenylethanoid glycosides encapsulation efficiency, particle size, and zeta potential of hydrated proliposomes with phosphate buffer solu-
Stability tion (pH 7.4) of 51.97%, 671.7 nm, and −25.49 mV, respectively. Stability testing of CPhGsP and CPhGs ordinary
Dripping pills liposomes was carried out for 3 months at 4 ± 2 °C, 25 ± 2 °C, 40 ± 2 °C, 75 ± 5% RH. The results obtained
In vitro release showed that the stability of the proliposomes was better than that of ordinary liposomes at the same tempera-
ture, while a lower temperature of 4 °C is ideal for storage. Cistanche phenylethanoid glycoside liquid
proliposomes dripping pills (CPhGsPD) are efficiently released in gastrointestinal solution as shown by in vitro
release experiments and the structure of the liposomes does not destroy the proliposome dripping pills by hydra-
tion. In vivo experiments showed that the areas under the plasma level-time curves and peak concentrations of
CPhGsPD and hydrated proliposomes were higher than those of CPhGs. Moreover, with CPhGsPD, the pharmaco-
kinetic parameters were similar to those with hydrated proliposomes. These results showed that CPhGsPD offer a
good way to improve the oral delivery of CPhGs.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction hepatoprptective effects (Morikawa et al., 2010), anti-inflammatory ac-

The stem of Cistanche deserticola Y.C. Ma is a common traditional
Chinese medicine which is widely used in the treatment of kidney defi-
ciency, body weakness and constipation. It is known as “desert ginseng”,
because it can grow in arid deserts. The main bioactive ingredient is
phenylethanoid glycosides (PhGs), which is a group of water-soluble
natural products. Currently, over 34 kinds of PhGs have been found in
Cistanche (Jiang and Tu, 2009), and echinacoside (Fig. 1a) and acteoside
(Fig. 1b) are the representative and major active ingredients exhibiting
a variety of bioactivities, especially echinacoside. Modern pharmacolog-
ical studies have shown that PhGs from Cistanche have many medicinal
activities such as neuroprotective effects (Geng et al., 2004; Chen et al.,
2007; Zhang et al., 2015), antifatigue (Cai et al., 2010), and
⁎ Corresponding author at: Shenyang Pharmaceutical University, Wenhua Road No.
103, Shenyang 110016, China.
E-mail address: huagonglou314@163.com (J. Han).
tivity (Nan et al., 2013; Lin et al., 2002) and the ability to increase anti-
body production (Maruyama et al., 2008). However, PhGs have poor
permeability and low in vivo bioavailability (Li et al., 2015; Jia et al.,
2006; Cui et al., 2016; Jia et al., 2009).
Liposomes are colloidal particles that are almost spherical micro-
scopic vesicles and are composed of one or more lipid bilayers arranged
in a concentric fashion enclosing a number of aqueous compartments
(Bangham et al., 1965). These phospholipid vesicles formed by hydro-
philic and hydrophobic molecules have the potential to encapsulate li-
pophilic and hydrophilic substances. The structure of liposome
vesicles is similar to that of biofilms, which can improve drug perme-
ability and promote drug absorption. The location of a drug in the vesi-
cles depends on its physicochemical characteristics and the types of
lipids used in preparation of the liposomes (Gregoriadis, 1976). Lipo-
somes are composed of phospholipids which are biodegradable, non-
toxic and free from any antigenic, pyrogenic or allergic reactions.
Accordingly, liposomes have been extensively investigated for drug de-
livery, drug targeting, controlled release and to increase solubility

http://dx.doi.org/10.1016/j.ejps.2016.07.020
0928-0987/© 2016 Elsevier B.V. All rights reserved.
 M. Li et al. / European Journal of Pharmaceutical Sciences 93 (2016) 224–232
a O
HO
O
RhaO
HO
b O
HO
O
RhaO
HO
Glc=glucose
Fig. 1. Chemical structures of echinacoside (a) and acteoside (b).
(Yadav et al., 2011). In recent years, liposomes have attracted much at-
tention as drug carriers to improve the effect of treatment, reduce side
effects, and improve stability by protecting drugs from degradation or
transformation (Mi and Burke, 1994; Gabizon et al., 1982). However, li-
posomes have a number of physicochemical stability issues such as
phospholipid degradation by hydrolysis or oxidation and aggregation,
sedimentation and leakage of encapsulated drug in aqueous dispersions
which limits their use for oral drug delivery (Ariën et al., 1993; Grit et al.,
1989; Hunt and Tsang, 1981; Kumar et al., 2001).
In order to solve these problems and help in the development of a
new drug delivery system, proliposomes can be used. The concept of
proliposomes was first proposed by Payne et.al in 1986, who defined
proliposomes as dry, free flowing powders (Payne et al., 1986).
Proliposomes are capable of forming liposomal suspensions on
being added to the water phase or under in vivo conditions (Payne
et al., 1986). In recent years, liquid proliposomes have become a
“hot topic” for research. According to a number of reports,
proliposomes are a kind of transparent solution which form drug-
loaded liposomes when blank proliposomes are mixed with a physi-
ological saline solution containing the drug (Junping et al., 2000).
Compared with solid proliposomes, the preparation process for
liquid proliposomes is simpler and requires no special equipment.
In addition, the liquid proliposomes can automatically and rapidly
produce a liposome suspension and dissolve completely in the
water phase. The mean particle size of liposomes hydrated from
liquid proliposomes is smaller and uniformly distributed. Currently,
less-relevant studies about liquid proliposomes for oral administra-
tion are being reported.
Response surface methodology (RSM) is a technique used to
optimize multifactor experiments and empirically derive a func-
tional relationship between one or more experimental responses
and a set of input variables (Hamsaveni et al., 2001; Chiang et al.,
2003; Zhang et al., 2007). RSM can be easily applied in response
to the value of each factor level through the process of regression,
response surface and contour lines. On the basis of the response
value of each factor on the level, it can predict the optimum re-
sponse values and the corresponding experimental conditions,
and describe the relationship between the response and the inde-
pendent variables, and the impression between the response
225
OGlc
O
O
OH
OH
OH
OH
O O
OH
OH
OH
Rha=rhamnose
values and the interaction of the variables (Vicente et al., 1998;
Xiong et al., 2009). In addition, RSM saves time and effort com-
pared with other approaches, because it reduces the number of
experimental trials needed (Liyana-Pathirana and Shahidi, 2005;
Lee et al., 2006). Therefore, RSM is widely used to optimize process
parameters.
The aim of this study was to develop liquid proliposomes for the
water-soluble drug Cistanche phenylethanoid glycosides (CPhGs) and
further improve its bioavailability and increase the liposomal stability.
In addition, RSM was used to optimize the preparation of CPhGs liquid
proliposomes. Dripping pills were selected as containers for Cistanche
phenylethanoid glycoside proliposomes (CPhGsP). During dissolution
of the dripping pills, proliposomes were released and hydrated to
form Cistanche phenylethanoid glycoside liposomes (CPhGsL). The
proliposomes were characterized by their particle size, drug encapsula-
tion efficiency (EE %), zeta potential and morphology after hydration.
The drug dissolution rate of Cistanche phenylethanoid glycoside liquid
proliposomes dripping pills (CPhGsPD) were evaluated in phosphate
buffer solution (pH 7.4) and hydrochloric acid buffer solution
(pH 1.2). Finally, the accelerated stability testing of CPhGsP and CPhGs
ordinary liposomes were also studied for 3 months at 4 ± 2 °C, 25 ±
2 °C, 40 ± 2 °C, 75 ± 5% RH.
2. Materials and methods
2.1. Materials
CPhGs (purity N 80%) were obtained from the Hetian Emperor Chen
Medicine Biological Technology Co., Ltd. (Xinjiang, China). Echinacoside
was obtained from the Chengdu Yi Te Mu Biological Technology Co., Ltd.
(Chengdu, China). Chlorogenic acid (CA, purity ≥ 96.2%) was obtained
from the China Food and Drug Inspection Institute (Beijing, China).
Tween 80 and Span 20 were obtained from Sinopharm Chemical Re-
agent Co., Ltd. (Shenyang, China) and sodium deoxycholate was from
Shanghai Suo Lai Bao Biological Technology Co., Ltd. (Shanghai, China).
Poloxamer-188 (P-188) was obtained from BASF Co., Ltd.
(Ludwigshafen, Germany) and soybean phospholipid (SP) was a gener-
ous gift from Jiangsu Man Shi Biological Technology Co., Ltd. (Jiangsu,
China). All reagents used were of analytical grade or better.
226 M. Li et al. / European Journal of Pharmaceutical Sciences 93 (2016) 224–232
Fig. 2. Proliposomes, hydrated liposomes, and dropping pills preparation process.
2.2. Preparation of CPhGsP, CPhGsPD and hydrated liposomes
The preparation of proliposomes, hydrated liposomes, and dripping
pills are shown in Fig. 2. A mixture of CPhGs, soybean phospholipids,
poloxamer-188 and sodium deoxycholate were dissolved in propylene
glycol by sonication(ultrasound 3 s, interval 2 s, 160 W, 15 min)
(JY92-2D, Ningbo, China), and then filled into ampoules and sealed
after the removal of oxygen using sterile nitrogen gas. It was a red
brown transparent liquid. The dripping pills were made in our laborato-
ry and consisted of proliposome/P-1881:4 (v/w). The coolant was poly-
dimethylsiloxane, the cooling temperature was 2 ~ 10 °C, the melting
temperature was 80 °C, and a drip rate of 45 drops per minute was
used when preparing the dropping pills. These dropping pills were
dried for 12 h at room temperature.
Hydrated liposomes were formed automatically by dropping phos-
phate buffer solution (pH 7.4) on to CPhGsP and shaking the mixture
manually for 2 min.
2.3. Box-Behnken design of the preparation conditions
Base on the single factor experiments, we found that the ratio of
soybean phospholipids to drug (A), the ratio of poloxamer-188 to
drug (B) and the ratio of sodium deoxycholate to drug (C) were
the main factors influencing the entrapment rate of CPhGsP and
particle size. Therefore, the three factors mentioned above at three
levels were used and seventeen experiments were carried out
according to the Box-Behnken design (BBD). The range and values
of the experimental variable investigated in this study are presented
in Table 1. The response surfaces that described the interaction
between each factor and index were drawn according to the fitting
equation. Experimental data were analyzed by Design-Expert trial
software version 8.0.6.
2.4. Determination of Cistanche phenylethanoid glycosides
The content of CPhGs in proliposomes was determined by ultraviolet
(UV)-visible detection, and 333 nm was selected as detection wave-
length. Echinacoside was used as a reference. Eight milligrams of
echinacoside was placed in a 50 mL volumetric flask and made up to
the mark with methanol. Volumes of 0.5, 1.0, 1.5, 2.0, and 2.5 mL were
pipetted into a 10 mL volumetric flask and made up to the mark with
methanol and the absorbances were measured at 333 nm, with metha-
nol as a blank control. The standard curve was constructed by plotting
the concentration of echinacoside versus the absorbance: A =
0.0233C-0.0167 (r = 0.9997). Thelinearity was good over the range
8 ~ 40 μg·mL−1. All the recoveries were 97 ~ 102%, with relative stan-
dard deviations b 2%. The intra- and inter-day relative standard devia-
tions were b2%.
2.5. Characterization of CPhGsP
2.5.1. Measurement of size and zeta potential
The particle size, zeta potential and distribution of the hydrated lipo-
somes were measured using a laser particle size analyzer (PSS
Nicomp380ZLS, USA). Proliposomes were hydrated in phosphate buffer
(pH 7.4) for size measurements. The measurements were carried out in
triplicate and the results were expressed as mean ± standard deviation.
Table 1
Levels of factors used in BBD.
Range and levels
Factors Code
−1 0 1
Ratio of soybean phospholipids to drug (w/w) A 15:1 20:1 25:1
Ratio of poloxamer-188 to drug (w/w) B 3:1 4:1 5:1
Ratio of sodium deoxycholate to drug (w/w) C 0.5:1 1:1 1.5:1
 M. Li et al. / European Journal of Pharmaceutical Sciences 93 (2016) 224–232
2.5.2. Transmission electron microscopy
The morphology of the hydrated CPhGsL was examined by Trans-
mission Electron Microscopy (TEM) with negative staining (Dayan
and Touitou, 2000). Samples were dyed using 2% phosphotungstic
acid, and deposited on a carbon-coated copper grid to form a carbon
film. The samples were the examined by TEM after drying at room
temperature.
2.5.3. Evaluation of entrapment efficiency
Proliposomes were hydrated to form liposomal suspensions with 10
volumes of phosphate buffer (pH 7.4). Then 0.5 mL of the liposomes was
diluted with 10 mL methanol followed by ultraviolet (UV)-visible detec-
tion at 333 nm, the absorbance was `, and the calculated concentration
was C1. One milliliter samples of liposomes were placed in 1.5 mL cen-
trifuge Tubes (Eppendorf Tubes, EP Tubes) and placed in a TGL-16G cen-
trifuge (Shanghai, China) and centrifuged at 16,000 rpm for 35 min. A
0.5 ml sample of the supernatant was diluted with methanol to 10 mL,
and the absorbance A2 was measured, and the concentration of C2 was
calculated. The encapsulation efficiency (EE %) was calculated using
the following formula:
EE% ¼ ðC1−C2 Þ=C1  100%
Where C1 is the amount of drug in the liposome suspension and C2 is
the amount of drug in the eluate; their units are μg·mL−1.
2.6. Storage stability of CPhGsP and CPhGs ordinary liposomes
Liposomal suspensions are thermodynamically unstable and so they
can flocculate and leak after a long storage period. Hence, temperature
plays a key role in liposome stability (Laridi et al., 2003; Nguyen et al.,
2013; Park et al., 2013). Therefore, the stability of CPhGsP and CPhGs or-
dinary liposomes were studied at 4 ± 2 °C, 25 ± 2 °C, 40 ± 2 °C. The en-
capsulation efficiency was measured after 0, 7, 15, 30, 45, 60, 75 and
90 days and determinations were carried out in triplicate.
2.7. In vitro CPhGsPD release study
The in vitro release studies of CPhGsPD were carried out at 37 ± 0.5 °
C using phosphate buffer solution (pH 7.4) and hydrochloric acid buffer
solution (pH 1.2). The total amount of drug was determined at 0, 5, 10,
20, 30, 45, 60, 90 and 120 min. Samples were withdrawn at specific
times, and all samples were passed through a 0.45 μm membrane filter.
Also, the same amount of corresponding fresh dissolution medium was
added to the stripping cup. The CPhGs content was determined from the
standard curve of echinacoside. At the same time, the particle size, zeta
potential and entrapment efficiency were measured after hydration of
the dropping pills. All the experiments were carried out in triplicate.
2.8. Pharmacokinetic studies in rabbits
2.8.1. High performance liquid chromatography (HPLC)
The content of echinacoside was measured using an HPLC system
(SHIMADZU, Japan) equipped with a binary pump, and an ultraviolet
(UV) detector at 330 nm. A C18 analytical column (150 mm × 4.6 mm
I.D. 5 μm) was used and the column was maintained at 30 °C. The mobile
phase consisted of acetonitrile-methanol-0.1% formic acid (10:15:75, v/
v) and was used throughout the analysis at a flow rate of 1.0 mL·min−1
and the sample injection volume was 20 μL. The solutions were all
passed through a 0.22 μm membrane before injection. Echinacoside
was the reference. A standard curve was constructed by plotting the
concentration of echinacoside versus the absorbance: Y =
0.88364X + 0.00173 (r = 0.998). Where Y is the echinacoside and CA
concentration, and A is the echinacoside and CA peak area. The standard
curve ranged from 22.4–4480 ng·mL−1 and was linear. All recoveries
227
were above 80%, which was within the acceptable limits to meet the
guidelines for bioanalytical methods.
2.8.2. Pharmacokinetic design
Eighteen male New Zealand rabbits with an average weight of 2.1 ±
0.42 kg were used in this study. The rabbits were obtained from the Lab-
oratory Animal Center of Shenyang Pharmaceutical University (Shen-
yang, China). They were kept in an environmentally controlled
breeding room before starting the experiments and fed with standard
laboratory food and water ad libitum and fasted overnight before the
test. The rabbits were divided into three groups and each animal re-
ceived a dose in one of the following dosage forms: (1) the CPhGs
group was given CPhGs at a dose of 25 mg·kg− 1; (2) the hydrated
proliposome group was given hydrated proliposomes at a dose of
25 mg·kg−1; (3) the CPhGsPD group was given CPhGsPD at a dose of
25 mg·kg−1.
After oral administration of CPhGs, hydrated proliposomes, and
CPhGsPD, 2 mL blood samples were collected from the marginal ear
vein at 5, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240 and 360 min from
the three groups. Plasma was separated by centrifugation at 4000 rpm
for 15 min and stored at −20 °C until analysis.
2.8.3. Plasma sample preparation
Chlorogenic acid was used as the internal standard. When the plas-
ma had thawed, 100 μL plasma, 20 μL chlorogenic acid (CA,
0.2 mg·mL− 1) and 20 μL menthol were added to 1.5 mL centrifuge
tubes, and then vortex-mixed for 60 s. After adding 80 μL 10% trichloro-
acetic acid, each mixture was shaken for 120 s and then centrifuged
(12,000 rpm, 10 min) at 4 °C. The upper layer (160 μL) was transferred
to a clean centrifuge tube and dried under nitrogen at 50 °C. The residue
was reconstituted in methanol (100 μL) and aliquots (20 μL) of the su-
pernatant were passed through a 0.22 μm membrane filter and then
injected into the HPLC system for analysis.
3. Results and discussion
3.1. Effect of surfactants on the encapsulation efficiency and particle size
The ratio of drug to propylene glycol was fixed a 1:80 for the test.
The results of the effects of surfactants on the encapsulation efficiency
and particle size are shown in Table 2. It was found that the hydrated li-
posome encapsulation efficiency was inversely proportional to the par-
ticle size compared with liposomes without surfactant. The
encapsulation efficiency of samples with a single component of
poloxamer-188 was higher than that of samples with added sodium
deoxycholate, but in terms of particle size, the former was higher than
the latter. For the samples with added poloxamer-188 and sodium
deoxycholate, the encapsulation efficiency and the particle size were
somewhere in the middle. It has been reported that the smaller the par-
ticle size, the faster the particles will infiltrate plasma (Stuart and Allen,
2000). Therefore, the method of mixing surfactants was selected during
the experiments. In addition, with an increase in surfactant contents,
the particle size of the liposomes was reduced and the encapsulation
Table 2
Surfactants of the characteristics of CPhGsP in terms of their particle size, and encapsula-
tion efficiency CPhGsP (n = 3).
Encapsulation Particle
Formulation Proportion(w/w) efficiency (%) size(nm)
D + SP 1:20:1 35.43 ± 0.67 900.7 ± 15
D + SP + Tween 80 1:20:1 37.54 ± 0.86 881.3 ± 23
D + SP + Span 20 1:20:1 43.57 ± 0.95 806.3 ± 20
D + SP + SD 1:20:1 36.97 ± 1.04 643.5 ± 26
D + SP + P-188 1:20:1 48.73 ± 0.79 739.0 ± 22
D + SP + SD + P-188 1:20:0.5:0.5 46.81 ± 1.13 693.1 ± 19
D, drug; SD, sodium deoxycholate; SP, soybean phospholipid; P-188, Poloxamer-188.
228 M. Li et al. / European Journal of Pharmaceutical Sciences 93 (2016) 224–232

Table 3 Table 5
Results of response surface experiments. The results of fitting second-order equations.

Levels of independent factors Response EE (%) Std. Dev. 1.03 R-Squared 0.9938
Run Mean 44.97 Adj R-Squared 0.9859
A B C
C.V. % 2.28 Pred R-Squared 0.9596
1 15 4 1.5 34.54 PRESS 48.21 Adeq Precision 32.914
2 20 4 1 51.12
3 20 3 1.5 47.34
4 15 4 0.5 33.33
5 20 4 1 52.73 negative correlation in the above regression equation. The analysis of
6 20 5 0.5 44.58 the fitting result is presented in Table 5. The adjusted R2 and predicted
7 20 4 1 53.63
8 25 3 1 48.33
R2 for the predictive model was 0.9859 and 0.9596, respectively, and
9 15 5 1 26.58 the statistical test results of equation parameters, showed that the
10 20 4 1 52.45
11 20 3 0.5 48.33
12 20 5 1.5 42.57
13 25 4 1.5 50.29
14 20 4 1 53.90
15 15 3 1 30.54
16 25 5 1 43.73
17 25 4 0.5 50.48

efficiency can be reduced if it is excessive. Hence, in response to the sur-

face experiments, the content of poloxamer-188 and sodium
deoxycholate were investigated as factors.

3.2. Analysis of response surface

There were seventeen experimental runs for optimizing three indi-

vidual parameters in the Box-Behnken design (BBD) using Design-Ex-
pert 8.0.6 software, and the experimental conditions and relevant
results are shown in Table 3. The results obtained showed that the en-
capsulation efficiency ranged from 26.58 to 53.90%.
The statistical significance of the regression model was checked by
the F-test and p-value, and the analysis of variance for the experimental
results is presented in Table 4. The small p-value for the model
(b0.0001) shows that the model is significant, and a p-value b 0.05
shows that the model term is significant. The p-value for the ‘lack of
fit’ test was 0.5996, indicating the model term was not significant and
the quadratic model was adequate.
Using statistical processing and fitting, a multiple second-order
equation was obtained as follows:
Final equation in terms of coded factors:
EE = + 52.77 + 8.48A − 2.14B − 0.25C − 0.16AB − 0.35AC −
0.26BC − 9.51A2 − 5.96B2 − 1.10C2The above regression equation de-
scribes the effects of three independent variables on the index and
their correlation. The positive and negative coefficients relate to the var-
iable and response representative of the correlation, in which a positive
coefficient represents a positive correlation, otherwise there was a

Table 4
Statistical analysis of variance for the encapsulation efficiency of experiment results.

Sum of Mean F
Source dƒ p value Prob N F
squares squares value

Model 1186.35 9 131.82 124.97 b0.0001 Significant

A 575.28 1 575.28 545.41 b0.0001
B 36.47 1 36.47 34.57 0.0006
C 0.49 1 0.49 0.46 0.5174
AB 0.1 1 0.1 0.097 0.7644
AC 0.49 1 0.49 0.46 0.5174
BC 0.26 1 0.26 0.25 0.6347
Fig. 3. Response surface of soybean phosphatide to the CPhGs ratio, the poloxamer-188 to
A2 380.64 1 380.64 360.88 b0.0001
CPhGs ratio, and the sodium deoxycholate to CPhGs ratio on the encapsulation efficiency
B2 149.72 1 149.72 141.94 b0.0001
A: the 3-D response surface plot at different soybean phosphatide to CPhGs ratios,
C2 5.08 1 5.08 4.81 0.0643
poloxamer-188 to CPhGs ratios at fixed sodium deoxycholate to CPhGs ratios; B: the 3-D
Residual 7.38 7 1.05
response surface plot at different soybean phosphatide to CPhGs ratios and sodium
Lack of fit 2.54 3 0.85 0.7 0.5996 Not significant
deoxycholate to CPhGs ratios at a fixed poloxamer-188 to CPhGs ratio; C: the 3-D
Pure error 4.84 4 1.21
response surface plot at different poloxamer-188 to CPhGs ratios, and sodium
Cor total 1193.73 16
deoxycholate to CPhGs ratios at a fixed soybean phosphatide to CPhGs ratio.
 M. Li et al. / European Journal of Pharmaceutical Sciences 93 (2016) 224–232
experimental results adequately fit the equation and the selected
model. So, it can be used to predict a response and obtain a random for-
mula within the range of the designed factor and level by regression
equations.
In order to better comprehend the predictive models of the results
three-dimensional and variable interaction graphs of the models, the re-
sponse surface diagram of the encapsulation efficiency of CPhGs and the
Fig. 4. Factor interaction graph showing the effect of the ratio of soybean phospholipids to
CPhGs, the ratio of poloxamer-188 to CPhGs, and the ratio of sodium deoxycholate to
CPhGs A: Interaction between the ratio of soybean phospholipids to CPhGs and the ratio
of poloxamer-188 to CPhGs; B: Interaction between the ratio of soybean phospholipids
to CPhGs and the ratio of sodium deoxycholate to CPhGs; C: Interaction between the
ratio of poloxamer-188 to CPhGs and the ratio of sodium deoxycholate to CPhGs.
229
variable interaction graphs are shown in Fig. 3 and Fig. 4, respectively.
With an increase in the ratio of soybean phospholipids to drug the en-
capsulation efficiency increases then finally changes little as shown in
Fig. 3A, and the factor was the most significant (p b 0.0001). With an in-
crease in the ratio of poloxamer-188 to drug, the encapsulation efficien-
cy initially increases then falls as shown in Fig. 3B, and the factor was
significant (p b 0.0006). In addition, as shown in Fig. 3C, with an increase
in the ratio of sodium deoxycholate to drug, the encapsulation efficiency
does not change significantly, and the factor was not significant
(p b 0.5174). As shown in Fig. 4, the interaction of the ratio of soybean
phospholipids to drug (A) and the ratio of poloxamer-188 to drug (B)
is the weakest, while the ratio of soybean phospholipids to drug (A)
and the ratio of sodium deoxycholate to drug (C) is the strongest.
The feasibility of the model equation for predicting the optimum re-
sponse values was tested under optimum conditions. The set of opti-
mum conditions, determined by using the RSM optimization
approach, was tested experimentally and parallel tests were carried
out in triplicate according to the optimized formulation. The mean
Fig. 5. Intensity weighting particle size distribution a.CPhGsP hydrated liposomes
b.CPhGsPD hydrated liposomes with phosphate buffer solution (pH 7.4) c.CPhGsPD
hydrated liposomes with hydrochloric acid buffer solution (pH 1.2).
230 M. Li et al. / European Journal of Pharmaceutical Sciences 93 (2016) 224–232
Fig. 6. Transmission electron micrographs of CPhGsP hydrated liposomes with phosphate
buffer solution (pH 7.4).
experimental encapsulation efficiency was 51.97% ± 1.07%, which was
close to the predicted value of 54.30% (the ratio of soybean phospho-
lipids to drug of 22.38, the ratio of poloxamer-188 to drug of 3.52, and
the ratio of sodium deoxycholate to drug of 0.84).
3.3. Characterization of CPhGsP
3.3.1. Measurement of particle size and zeta potential
The particle size distribution of hydrated CPhGsP is presented in Fig.
5a. The average particle size and the average zeta potential were
671.7 nm and − 25.49 mV, respectively. The zeta potential showed
that the liposomes obtained have sufficient charge to inhibit the aggre-
gation of vesicles (Xiong et al., 2009). The zeta potential of CPhGsL was
negative, because the SP gave some ingredients a negative charge, such
as phosphatidylinositol and phosphatidylserine.
Fig. 7. Stability curve of CPhGs ordinary liposomes and CPhGsP at different temperatures.
Fig. 8. Release curve of CPhGsPD in phosphate buffer (pH 7.4) and hydrochloric acid buffer
(pH 1.2).
3.3.2. Transmission electron microscopy images of proliposomes
TEM images of the proliposomes after hydration are shown in Fig. 6,
which shows that the liposome vesicles were adjacent to the spherical
particles with distinct boundaries, which confirms the formation of lipo-
somes from proliposomes. The liposome vesicles as shown under TEM
have multilamellar vesicle walls, which confirms the formation of
multilamellar vesicles (MLVs) from proliposomes.
3.4. Storage stability results of CPhGsP and CPhGs ordinary liposomes
The stability curves of CPhGs ordinary liposomes and CPhGsP at dif-
ferent temperatures are shown in Fig. 7. Compared with CPhGs ordinary
liposomes, the CPhGsP encapsulation efficiency was much higher. The
encapsulation efficiency of proliposomes did not change markedly,
while the encapsulation efficiency of CPhGs ordinary liposomes and
CPhGsP at 4 °C and 25 °C, especially at 4 °C, were basically unchanged.
However, with an increase in time, the encapsulation efficiency of
CPhGs ordinary liposomes was significantly reduced, and the encapsu-
lation efficiency of CPhGsP groups was slightly reduced. These results
show that CPhGs ordinary liposomes and CPhGsP should be stored at
low temperatures, and the stability of proliposomes is better than that
of ordinary liposomes.
Fig. 9. Mean plasma concentration-time profile of echinacoside following oral
administration of CPhGs, hydrated proliposomes, and CPhGsPD in rabbits (n = 6).
 M. Li et al. / European Journal of Pharmaceutical Sciences 93 (2016) 224–232
Table 6
The main pharmacokinetic parameters of CPhGs, hydrated proliposomes, and CPhGsPD in rabbits (n = 6).
Parameter CPhGs
Tmax (min) 15.00 ± 0.00
Cmax (ng·mL−1) 193.17 ± 9.30
AUC0 − ∞ (ng·mL−1·min) 30,275.39 ± 4765.95
AUC0 − t (ng·mL−1·min) 15,494.70 ± 1183.66
MRT0 − ∞ (min) 301.289 ± 38.833
MRT0 − t (min) 80.790 ± 3.400
CL (L·h−1·kg−1) 0.001 ± 0.000
Vd (L·kg−1) 0.284 ± 0.028
Data are expressed as mean ± SD; Tmax time of peak concentration, Cmax peak concentration, AUC area under the curve, CL clearance rate, Vd volume of distribution, MRT mean residence
time.
3.5. Analysis of CPhGsPD in vitro release
The release curves of CPhGsPD in phosphate buffer (pH 7.4) and hy-
drochloric acid buffer (pH 1.2) are shown in Fig. 8. In phosphate buffer
(pH 7.4) and hydrochloric acid buffer (pH 1.2), dripping pills can be al-
most completely released in 30 min. However, the release is faster and
higher in phosphate buffer (pH 7.4). These results indicated that
CPhGsPD is efficiently released in gastrointestinal solution in theory.
In addition, the CPhGsPD hydrated particle size is shown in Fig. 5b and
Fig. 5c hydrated by phosphate buffer (pH 7.4) and hydrochloric acid
buffer (pH 1.2). The particle size was 671.7 nm, 645.7 nm and
532.8 nm for hydrated CPhGsP, and CPhGsPD in phosphate buffer
(pH 7.4) and hydrochloric acid buffer (pH 1.2) in Fig. 5, respectively.
The particle size showed less difference between hydrated CPhGsP
and CPhGsPD in phosphate buffer (pH 7.4). The size in hydrochloric
acid buffer was a little smaller than that in hydrochloric acid buffer.
The reason for this might be that some liposomes were disrupted in
the acidic environment (Brocks and Betageri, 2002). In addition, the
EE% and zeta potential, were 49.56 ± 0.75% and − 20.71 ± 2.1 mV in
phosphate buffer (pH 7.4), and 47.89 ± 0.84% and − 21.71 ± 1.7 mV
in hydrochloric acid buffer (pH 1.2), respectively, close the hydrated
proliposomes. These results showed that the structure of the liposomes
did not destroy the proliposome dropping pills following hydration. For
research involving water-soluble drug proliposomes and CPhGsPD the
oral route of administration provides a theoretical basis.
3.6. Pharmacokinetic studies in rabbits
The mean plasma concentration-time profiles of CPhGs, hydrated
proliposomes and CPhGsPD in rabbits are shown in Fig. 9. The plasma
echinacoside concentrations were higher in rabbits given hydrated
proliposomes and CPhGsPD than in those given CPhGs, at all time
points. The peak concentration (Cmax) and time to reach the peak con-
centration (Tmax) were obtained directly from the individual plasma
echinacoside vs. time profiles. The Cmax values of the CPhGsPD group
and the hydrated proliposomes group were higher than that obtained
with CPhGs. Using DAS 2.0 pharmacokinetic software, according to the
second chamber model, the statistical moment calculations of the
main pharmacokinetic parameters are summarized in Table 6. CPhGsPD
and hydrated proliposomes gave a Tmax of 30 min, compared with
15 min for CPhGs, the delayed Tmax with CPhGsPD and hydrated
proliposomes, were possibly due to the slow release of drug from the
hydrated liposomes.
When formulations containing liposomal CPhGs were administered,
some pharmacokinetic parameters were completely different from
CPhGs. Compared with CPhGs, the area under the plasma level-time
curve (AUC0 − ∞) for the CPhGsPD group and hydrated proliposomes
group was higher, confirming slower CPhGs distribution and elimina-
tion from the plasma of CPhGs encapsulated liposomes. The CPhGs
group mean residence time (MRT0 − ∞) was higher than that of the
other two, possibly because of the CPhGs low absorption in vivo and a
231
Hydrated proliposomes CPhGsPD
30 ± 0.00 30 ± 0.00
324.23 ± 11.38 315.17 ± 12.7
41,687.38 ± 8081.50 38,331.51 ± 4881.45
26,322.50 ± 2133.74 24,552.14 ± 1400.94
130.514 ± 40.062 149.553 ± 30.774
74.466 ± 2.579 72.039 ± 3.338
0.001 ± 0.000 0.001 ± 0.000
0.159 ± 0.041 0.126 ± 0.013
long residence time. Moreover, with CPhGsPD, the pharmacokinetic pa-
rameters were similar to those of hydrated proliposomes, showing that
the CPhGsPD absorption properties of liposomes confirmed they had
dissolved in vivo.
4. Conclusions
In this study, a form of liquid CPhGsP for oral administration was
prepared using a more efficient and simpler method. The preparation
of proliposomes was found to be well suited to obtain an optimal
proliposomal formulation of CPhGs by RSM. The optimal formulation
obtained was drug/soybean phospholipid/poloxamer-188/sodium
deoxycholate/propylene glycol.
1:22.38:3.52:0.84:80 (w/w/w/w/v). The proliposome encapsulation
efficiency reached 51.97%. In addition, the stability study indicates that
the proliposome stability is better than that of ordinary liposomes at
the same temperature, and a lower temperature of 4 °C is preferred
for storage. The CPhGsPD release is good in gastrointestinal solution in
theory as shown by in vitro release experiments and the structure of li-
posomes did not destroy the prolipospme dropping pills by hydration.
This liquid proliposome dripping pills formulation improve the oral bio-
availability of CPhGs in New Zealand rabbits and offers a new approach
to preparing liposomes for oral administration.
Acknowledgements
The authors would like to thank Dr. J. Han and Dr. N. Liu for valuable
discussions and interest in the work. Thanks also to Shenyang Science
and Technology Bureau (F16-205-1-45) for supporting this work.
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