The Feasibility of Novel Liposome Consisted of Sphingomyelin and β-sitosterol for Gypenosides Delivery

Abstract

The limited stability of liposome and high dose of cholesterol severely restricts the application of liposome as an effective formulation in food and drug fields. The feasibility of novel liposomes consisting of sphingomyelin and β-sitosterol, with high stability and lowering cholesterol function, was investigated. The result of molecular modeling indicated that liposome consisting of sphingomyelin and β-sitosterol was practicable. Gypenosides loaded liposome consisting of sphingomyelin and β-sitosterol (GSSL) was successfully prepared attaining a 91.3% entrapment efficiency, exhibiting an average particle size of 205 nm; −33.1 mV zeta potential with smooth continuous surface was observed by AFM. The results of stability experiments indicated that GSSL was more stable than that ordinary gypenosides loaded liposome composed of lecithin and cholesterol (GLCL). Furthermore, GSSL demonstrated much better lipid lowering effects than ordinary GLCL liposome. Therefore, replacing lecithin and cholesterol with sphingomyelin and β-sitosterol in preparation of liposomes is feasible and recommended.

Introduction

Liposome was first described as early as the mid 60s by Bangham (Bangham et al., 1965). The biocompatibility, biodegradability and low toxicity make them very suitable for application as food and drug delivery system. This efficient delivery system is regarded as an advantageous candidate food and drug carrier (Torchilin, 2005) and has been a central theme of research in food and drug field (Abra and Hunt, 1981). In spite of its merits, the poor stability and high dose of cholesterol severely restricts its application. Hence this formulation is not suitable for certain people, especially for patients with cardiovascular disease.

Sphingomyelin is an abundant component of the outer leaflet of eukaryotic cell membranes (Simons and Ikonen, 1997). Since the aliphatic chain of sphingomyelin, which is amide linked significantly less susceptible to acid hydrolysis than distearoylphosphatidylcholine (DSPC) which is ester linked (Webb et al., 1995). In this membrane lipids, high affinity between sphingomyelin and cholesterol or cholesterol analogues has been evidenced by numerous studies using different techniques (Xu et al., 2001). It has been suggested that the association between sphingomyelin and cholesterol or cholesterol analogues is stabilized by hydrogen bonding and the existence of sphingomyelin/cholesterol condensed complexes has been proposed. On the other hand, a steric shielding of cholesterol from aqueous medium by the sphingomyelin headgroup together with the van der Waals interactions between long-saturated sphingomyelin chains and cholesterol can also explain this condensation effect (Göran et al., 2006). Furthermore, because the headgroup moiety of sphingomyelin is identical to that of DSPC, it could be expected that liposomes composed of sphingomyelin and cholesterol would have circulation and drug retention characteristics similar to those of the DSPC/cholesterol formulation. Zant, et al. verified that liposomes consisting of sphingomyelin and cholesterol are highly resistant to destabilization by plasma lipoproteins and showed relatively long circulation times in blood for both intermediate size (100 200 nm) and small vesicles (Zant et al., 2010). Moreover, sphingomyelin has an antioxidant function and inhibits the oxidation of membrane lipids (Subbaiah et al., 1999). Therefore, sphingomyelin could replace DSPC to improve the physicochemical characteristics of liposome.

Cholesterol has a steroid backbone and its derivatives are included in liposome preparing (Chien et al., 2010). Normally, the cholesterol in a liposome serves as a component providing rigidity to the membranes and improving the properties of the bilayer membranes (Frans et al., 2005; New ERC, 1990). Cholesterol enhances the fluidity of the bilayer membrane, reduces the permeability of water soluble molecules through the membrane, and improves the stability of bilayer membrane in the presence biological fluids such as blood (Sriram and Rhodes, 1995). However, studies indicated that incidence of cardiovascular diseases is in proportion to the cholesterol concentration in blood. If the cholesterol concentration rose 10 mg/dl in blood, the incidence of cardiovascular diseases also will increase 10% (Scandinavian Simvastatin Survival Study Group, 1994; Hwang et al., 2010). Patients with hypercholesterolemia should severely restrict their intake of cholesterol even at low concentrations (Jogchum and Ronald, 2005; Laura et al., 2009). Therefore, the cholesterol in liposome severely limited the application of liposome in cardiovascular diseases.

β-sitosterol, distributed ubiquitously in nature, with similar conformation to cholesterol. It has been widely studied in reducing cholesterol since 1950 (Tsuge et al., 2004). In 2000, the US Food and Drug Administration (FDA) announced the effectiveness of β-sitosterol in decreasing the incidence of cardiovascular diseases and lowering cholesterol.

In summary, sphingomyelin is more stable than lecithin, β-sitosterol have similar structure to cholesterol but not induce cardiovascular diseases. Therefore, it is worthwhile to study the feasibility of using sphingomyelin and β-sitosterol as substitute for lecithin and cholesterol in liposome, explore whether the new liposome is more stable than ordinary liposome and research whether this novel formulation possess lipid-lowering function as we expect.

Gypenosides is an edible and medicinal food, the main chemical ingredient of Gynostemma pentaphyllum Makino (Cui et al., 1999), also have a lot of health functions, such as relieving cough, chronic bronchitis, diuretic, antipyretic and antiinflammatory (Li et al., 1993; Shang et al., 2006). In recent years, gypenosides has been reported to be effective in the treatment of cancer (Han et al., 1995; Chen et al., 2009; Lu et al., 2008; Wang et al., 2002) and cardiovascular diseases, especially in hyperlipidemia and ischemic heart diseases (Megalli et al., 2006). In this study, the feasibility of liposome composed of sphingomyelin and β-sitosterol as substitute for lecithin and cholesterol was explored. Gypenosides was loaded as active ingredient in liposome composed of sphingomyelin and β-sitosterol (GSSL), Molecular modeling, characterization, stability and antihyperlipidemic effects were compared with gypenosides loaded liposome composed of lecithin and cholesterol (GLCL).

Materials and Methods

Materials    Sphingomyelin was purchased from Avanti Polar Lipids ( > 90%, USA). Lecithin, gypenosides standard and cholesterol were purchased from Aladdin Reagent (Shanghai, China). β-sitosterol was purchased from Hangzhou Dayangchem Co.Limited (Hangzhou, China). Mice, experimental diets, and sawdust bedding were purchased from Qilongshan Animal Co., Ltd. (Nanjing, China). Total cholesterol (TC) enzymatic kits, triacetylglyceride (TAG) enzymatic kits, HDL-C kits, and LDL-C kits were purchased from FengHui Medical Science & Tech. Co. Ltd. (Shanghai, China). Gypenosides was obtained from Shanxi Undersun Biomedtech Co., Ltd. ( > 90%, Xi’an, China,).

Molecular modeling studie    In order to provide theoretical basis to guide experiment, prediction of the interaction positions between sphingomyelin and β-sitosterol, molecular mechanics was performed by simulations on various liposome by using Gaussian 03W (Gaussian Inc., USA).

Gypenosides Analysis

(1)Preparation of the standard solutions    Gypenosides standard was dried and accurately weighed and then dissolved in water and diluted to 0.01, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL separately, 2 mL 5% vanillin-acetic acid and perchloric acid (2:8) were added into gypenosides standard solutions mentioned above, respectively, heated in 60°C water bath for 15 min, cooled down to room temperature by water before 10 mL acetic acid added. The solutions were then analysed by UV/Vis Spectrophotometer at 555 nm (Lu et al., 2007).

(2)Preparation of sample solutions    0.5 mL protamine (10 mg/mL) was added into 2 mL gypenosides liposome solution keep stirring. 3 mL saline was added after 3 min, then the solution centrifuged for 2 min at room temperature (2000 rpm), 1 mL supernatant was taken to measure the absorbance at 555 nm to determine the amount of free gypenosides. The equations for the encapsulation efficiency (EE) index was as Equation 1:   

Preparation of various liposomes    30 mg gypenosides, 300 mg sphingomyelin (731 g/mol) and predetermined amount of β-sitosterol (414.7 g/mol) were dissolved in 50 mL ethanol. The solution was injected into a 50 mL 45°C phosphate buffered saline (7.0) before stirring continuously 2 hours to evaporate ethanol, obtaining GSSL. The blank liposome composed of sphingomyelin and β-sitosterol (BSSL) were prepared according to the precedure mentioned above without the addition of gypenosides.

30 mg gypenosides, 300 mg lecithin (760 g/mol) and predetermined amount of cholesterol (386.7 g/mol) were dissolved in 50 mL ethanol. The solution was injected into a 50 mL 45°C phosphate buffered saline (7.0) before stirring continuously 2 hours to evaporate ethanol, obtaining GLCL. The blank liposome composed of lecithin and cholesterol (BLCL) were prepared according to the precedure mentioned above without the addition of gypenosides.

Encapsulation efficiency of substituted β-sitosterol for cholesterol    Four kinds of gypenosides loaded liposomes were prepared according to the process mentioned above, including the mole ratio of sphingomyelin to β-sitosterol group was 4 to 1 group (GSSL (4/1) group), the same prescription without addition of β-sitosterol group (GSSL (4/0) group), mole ratio of lecithin to cholesterol was 4 to 1 group (GLCL (4/1) group), the same prescription without addition of cholesterol group (GLCL (4/0) group), and the encapsulation process was evaluated by EE according Equation 1.

Characterization of    GSSL liposome The particle size and zeta potential were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 (Malvern instruments, UK). All of the DLS measurements were performed at 25°C and at a scattering angle 90°. The morphology was observed by atomic force microscopy (AFM, Nano Scope III a, Veeco, USA) which was operated in tapping mode.

Stabilities of various type of liposome systems

(1)Effect of pHon stability    Various above mentioned formulated liposomes were immersed in universal buffer (pH 3, 4, 5, 6, 7, 8, 9, 10) separately. Residual percentage of entrapped gypenosides was determined at 0, 7, 14, 21, 28, 35, 42, 49, 56, 63 days. The equations for residual percentage of gypenosides was as Equation 2.

  

 (2)Effect of oxidation on stability    Various formulated liposomes above mentioned were stored at 25°C, respectively. TBARS (thiobarbituric acid reactive substances) was determined at 0, 7, 14, 21, 28, 35, 42, 49, 56, 63 days, following the method of Buege and Aust (1978).

Antihyperlipidemic effect various type of liposome systems

(1)Animals and Diets    Seventy male 6-week-old Kun Ming mice (Qinglongshan Animal Co., Ltd., Nanjing, China), weighing 18–22 g, were housed in polypropylene cages (5 per cage) in a room controlled at 25 ± 1°C and 60% humidity with a 12-h light/ dark cycle. The animal use and experimental procedures complied with the International Guiding Principles for Biomedical Research Involving Animals (1985), and the protocols were approved by the Animal Use and Care Committee of Yancheng teachers’ university. After 3 days of adaptation, the mice were weighed and randomly divided into 7 groups (n = 10/group). In the control group (CG), mice were given a regular rodent chow diet. The ingredient composition of rodent chow diet were 26% corn powder, 34% flour, 24.5% soybean meal, 5% fish meal, 1.5% wheat bran, 3% clover fodder, 2% plant oil, 4% premix. The nutrient composition of rodent chow diet were 20.5% protein, 4.6% fat, 52.5% nitrogen free extract, 4.4% fiber, < 0.1% cholesterol and < 0.1 plant sterols, the calories was 345.0 kcal/100 g. In the negative control group (NCG), GLCL (4/0) group, GLCL (4/1) group, GSSL (4/0) group, GSSL (4/1) group and gypenosides group mice were fed with a high-cholesterol-high-fat diet, which was prepared from regular rodent chow supplemented with 1% (w/w) cholesterol, 10% (w/w) pork oil, and 10% (w/w) egg yolk powder. The nutrient composition of high-cholesterol-high-fat diet were 19.4% protein, 18.0% fat, 42.6% nitrogen free extract, 3.5% fiber, 1.0% cholesterol and < 0.1 plant sterols, the calories was 418.0 kcal/100 g. Data were taken from product specification sheets provided by the manufacturer (Slac Animal Co., Ltd., Shanghai, China). In GLCL (4/0) group, GLCL (4/1) group, GSSL (4/0) group, GSSL (4/1) group and gypenosides group were administered by ig. according to the gypenosides dose was 80 mg/kg.

(2)Animal Experiment and Sample Collection    During the 4-week experimental period, mice were given free access to drinking water. The food was available ad libitum, unless mice were fasted for four hours prior to dosing, and experimental diets and drinking water were replaced every day. After 4 weeks, mice were kept fasted overnight (16 h), then weighed and sacrificed. Blood was collected from the retro-orbital sinus. Serum was separated by centrifugation (8000 rpm) for 15 min at 4°C and stored at −20°C for further measurement of serum TC, TAG, HDL-C and LDL-C.

(3)Analysis of Serum Lipids    Serum TC, HDL-C and LDL-C were measured by an enzymatic CHOD-PAP (cholesterol oxidase- peroxidase aminophenazone) method with corresponding test kit (FengHui Co. Ltd., Shanghai, China) using an automatic FH-400 biochemical analyzer (FengHui Co. Ltd., Shanghai, China). Serum TAG was analyzed according to the fully enzymatic GPO (glycerol phosphate oxidase)-PAP method. The presence of the sterols or stanols even at the same concentration as that of cholesterol did not influence the accuracy of cholesterol determination (He et al., 2011).

(4)Statistical analysis    The statistical analyses were performed using SPSS 16.0. One-way ANOVA was used to analyze the overall treatment effects on serum lipids. When a statistically significant effect was obtained, the Student-Newman-Keuls test was performed to determine the differences between treatment groups. Significance level was set at p < 0.05. All data are presented as means ± SD.

Results and Discussion

Molecular modeling studies    Molecular mechanics uses classical mechanics to model molecular systems. Molecular mechanics is an effective method, which can be used to predict the interaction between molecules, and judge the system is stable or not. In this study, molecular mechanics also was adopted to predict the interaction between sphingomyelin and β-sitosterol, compared with the interaction between lecithin and cholesterol. According to the modeling results, the values of the energy of lecithin, cholesterol, sphingomyelin, β-sitosterol were 195, 89.1, 65.9 and 107 kcal.mol⁻¹, respectively. Before formulation, the total energy of materials (two phospholipid molecules and one sterol molecular) in BLCL liposome and BSSL liposome were 479.1 and 239.3 kcal. mol ⁻¹. After formulation, the energy of BLCL and BSSL were decreased to 380.7 and 142.0, which indicated whatever kind of liposome system, the liposome systems were more stable than their original materials. Compared with BLCL liposome, BSSL liposome possessed even lower energy which presented that BSSL liposome was more stable than BLCL liposome in the theory. The binding energy of BLCL liposome, BSSL liposome were −98.5, −97.3 kcal.mol⁻¹, negative values indicated combination of lecithin and cholesterol, sphingomyelin and β-sitosterol were spontaneous, high absolute value presented the interaction between phospholipid and sterol was strong, it was beneficial to form a stable liposome system. A small segment of liposome presented in Fig. 1 illustrates that β-sitosterol would be located between two phospholipid molecules like cholesterol. Thus, β-sitosterol could play an important role as cholesterol in stablizing the liposome.

Fig. 1.

Molecular modeling image. A: a segment of GLCL liposome B: a segment of GSSL liposome.

Encapsulation efficiency of β-sitosterol for cholesterol substituted liposome    Experiments were carried out to encapsulate gypenosides in liposomes prepared with different formulations, including GLCL (4/0), GLCL (4/1), GSSL (4/0) and GSSL (4/1) (molar ratio). Fig. 2 depicts the relation between liposomes formulation and EE of gypenosides. Experimental results revealed that EE of GLCL (4/1) and GSSL (4/1) were 87.8 ±5.4% and 91.3 ± 3.5%. Two groups exhibited similar EE, which might be due to their similar molecular structure. In addition, EE of GSSL was slightly higher than that of GLCL, but there was no significant differences between two groups on EE. This was consistent with the result from Shimizu (Shimizu et al., 1993), who reported that liposomes prepared with sphingomyelin and cholesterol (1:0.25, molar ratio) showed the highest EE. Compared with Shimizu’s report, our data also demonstrated that sterol containing liposomes possess higher EE than those prepared without addition of cholesterol or β-sitosterol (GSSL (4/0) group was 61.3 ± 4.6% and GLCL (4/0) group was 58.7 ± 3.3%). According to previous report (Sriram and Rhodes, 1995), cholesterol improves the bilayer characteristics of the liposomes, reduces the permeability of water soluble molecules through the membrane, and improves the stability of the bilayer membrane in the presence biological fluids. Apart from this report, Shuler et al. also indicated the alkylated side chain of phytosterols exhibit higher effect than cholesterol for arranging a fatty acyl chain to reduce the fluidity of the bilayer membrane (Schuler et al., 1990). All the results demonstrated it was feasible to substitute sphingomyelin and β-sitosterol for lecithin and cholesterol in preparation of liposome.

Fig. 2.

EE of various liposomes.

Characterization of GSSL liposome    DLS revealed that the average particle size of sphingomyelin/β-sitosterol liposome increased from 85 to 205 nm (n = 3) after the gypenosides encapsulation, suggested that more compact hydrophobic inner core was formed due to gypenosides encapsulateion. The zeta potential of BSSL liposome and GSSL liposome were −36.3 mV and −33.1 mV, the absolute potential value were higher than 30 mV, which could result in a moderate repelling force between the liposomes while increasing the stability of the nano-system according to previous researches: a zeta potential of more than 61 mV usually indicates excellent stability, 41–60 mV good stability, 31 40 mV moderate stability, and 10 30 mV incipient instability (Plessis et al., 1996; Dong and Feng, 2004; Yu et al., 2013; Sergio et al., 2010). The representative AFM image of BSSL and GSSL (Fig. 3.) also showed that the two kind liposomes were almost spherical in shape. The size and hardness of GSSL increased after gypenosides loading.

Fig. 3.

AFM image. A: BSSL liposome B: GSSL liposome.

Stabilities of liposomes    Hydrolysis and oxidation were the main reasons of liposome instability during storage, commonly leading to drug leakage. The stability of liposome prepared with different formulations was evaluated according to the residual of gypenosides after storage at different hydrolysis and oxidation conditions created by different buffers and temperature.

(l)Tolerance of liposomes in different pH    Different formulated liposomes were immersed in different pH buffers (pH 5, 6, 7, 8, 9, 10) separately to investigate the changes in residual gypenosides. After 1, 3, 5, 7, 14, and 28 days the residual percentage of gypenosides was quantified. All the liposomes had lower released percentage of gypenosides in pH 6 or pH 7 for 14 days or 28 days storage. The reason might be that hydrolysis and oxidation of phospholipid could alter the membrane structure of liposome (Yin and Faustman, 1993), the slower hydrolysis rate of phospholipid in neutral conditions than in acidic or basic conditions (Sulkowski et al., 2005). Furthermore, this experiment also demonstrated that liposomes without sterols exhibited higher released percentages than those with sterols (Fig. 4.), the addition of sterols could improve the resistance of liposome, the liposome composed of different phospholipid had different tolerance to harsh pH environment. In this experiment, sphingomyelin had stronger resistance than lecithin in harsh pH environment. Interestingly, compared with liposome containing lecithin group, whether GSSL (4/1) or GSSL (4/0) group, the released gypenosides was lower than their counterpart, and had significant difference between two counterpart groups. This might be due to the slower hydrolysis rate of sphingomyelin than lecithin (Grit and Crommelin, 1993). According to the structure of lecithin and sphingomyelin, there are two carbonyl group- containing esters in one lecithin molecular. However, one sphingomyelin molecular only containing one amido bond. Theoretically, amido bond is more stable than ester bond, therefore, sphingomyelin is more stable than lecithin, so that the liposome consisted of sphingomyelin was more stable than liposome composed of lecithin.

Fig. 4.

Released gypenosides from different formulas in different pH for 14 days or 28 days.

(2)Stability of liposomes to oxidation    The stability to oxidation was evaluated by measuring the level of lipid oxidation in different formulated liposomes. The methods commonly used for assessing secondary oxidation products of lipid are thiobarbituric acid (TBARS) test, oil stability index (OSI) method and fluorescence assay (Fereidoon, 2005). Among these methods, TBARS test is the most widely used method to detect the oxidation of lipids due to its simplicity and accuracy. In this study, various formulated liposomes were stored at 25°C to explore the relationship between oxidation and residual gypenosides by TBARS test. TBARS values were measured at 7, 14, 21, 28, 35, 42, 49, 56 and 63 days of storage. Data (Fig. 5.) revealed that TBARS values of liposomes increased rapidly with storage days at 25°C. Results also indicated that β-sitosterol delayed TBARS production during storage period. This might be due to the intercept of oxygen by β-sitosterol, then becoming a stable free radical and interrupting the oxidation injury to the phospholipid (González-Larena et al., 2011; Winkler and Warner, 2008).

Fig. 5.

Change in TBARS of liposomes prepared with different formulations.

Interestingly, compared with liposome containing lecithin group, whether GSSL (4/1) or GSSL (4/0) group, the extend of lipids oxidation level was lower than their counterparts GLCL (4/1) or GLCL (4/0). The reason might be attributed to malonaldehyde, which is a main decomposition product of fatty acids. This usually reacts with TBA to form a stable pink chromophore with maximal absorbance at 532 nm (TBARS₅₃₂) (Dahle et al., 1962; Rosmini et al., 1996). Malonaldehyde is derived from carbonyl group, therefore, in the same condition; the number of carbonyl group in a compound probably represented the stability of oxidation in this experiment. There are two carbonyl groups in the structure of lecithin, while just one carbonyl group in sphingomyelin. Fewer number of carbonyl groups meant fewer malonaldehyde generated during storage, in turn less malonaldehyde reacted with TBA to form chromophore. Therefore, the liposome composed of sphingomyelin had higher stability of oxidation than that of lecithin.

Antihyperlipidemic effect of various formulated liposomes    Serum lipid profiles are shown in Table 1. Obviously, mice in NCG displayed a significant increase in levels of serum TC, LDL-C, and LDL-C/HDL-C after 4 weeks of feeding a high-cholesterol-high-fat diet in comparison with those in CG, indicating that the high-cholesterol-high-fat diet had induced hypercholesterolemia in test animals. Meanwhile, GSSL (4/1) group, GSSL (4/0) group and GLCL (4/0) group significantly decreased serum TC levels as compared with that in NCG. GSSL (4/1) group and GSSL (4/0) group also remarkably lowered serum TC contents than those in their counterparts. These results indicated that gypenosides liposome without cholesterol could effectively decrease serum TC. Compared with GSSL (4/0) group, GSSL (4/1) group was more effective in decreasing TC, this result was consistent with Ostlund’s: even a small amount of sterols in a micelle form is very effective in reducing cholesterol absorption (Ostlund et al., 1999). Compared with NCG group, the TAG of gypenosides group decreased greatly, which indicated that gypenosides had strong effect on decrease TAG. The TAG of other administered groups also significantly decreased compared with NCG. Especially, the TAG of GSSL (4/1) group and GLCL (4/1) group was very significantly decreased, which revealed that liposome containing sterol (either β-sitosterol or cholesterol) had better effect than liposome without sterol on decreasing TAthe stability of liposomes (G, the reason might be sterol can effectively maintain Ishikawa et al., 2004).

Table 1. Serum lipid profiles in different groups of test animals^a

Group	TC	TAG	HDL-C	LDL-C	LDL-C/HDL-C
CG	3.54 ± 0.28cd	0.67 ± 0.07c	1.88 ± 0.22a	1.42 ± 0.29d	0.78 ± 0.23e
NCG	4.79 ± 0.41a	1.03 ± 0.09a	1.19 ± 0.16b	3.41 ± 0.24a	2.90 ± 0.36a
GLCL (4/0) group	3.97 ± 0.32bc	0.85 ± 0.08b	1.32 ± 0.13b	2.45 ± 0.32bc	1.87 ± 0.30c
GSSL (4/0) group	3.87 ± 0.26bcd	0.83 ± 0.08b	1.45 ± 0.08b	2.22 ± 0.37c	1.53 ± 0.26d
GLCL (4/1) group	4.53 ± 0.29ab	0.79 ± 0.09bc	1.30 ± 0.18b	3.18 ± 0.27a	2.46 ± 0.19ab
GSSL (4/1) group	3.24 ± 0.27d	0.72 ± 0.08bc	1.53 ± 0.11b	1.52 ± 0.11d	1.00 ± 0.15e
Gypenosides group	4.24 ± 0.28abc	0.84 ± 0.08b	1.32 ± 0.11b	2.78 ± 0.32b	2.12 ± 0.37bc

^a  Data were analyzed using one-way ANOVA. Differences between treatment groups were further analyzed using the Student-Newman-Keuls test after a significant effect was detected. Results are the means ± SD (n = 10). Values bearing different lowercase letters (a, b, c, d and e) are significantly different (p < 0.05).

Compared with NCG, although HDL-C level of the treatment group was no difference, the LDL-C level of the treatment group decreased significantly except GLCL (4/1) group, which resulted in the value of atherogenicity index (LDL-C/HDL-C) decrease significantly. The less value of LDL-C/HDL-C meant lower incidence of cardiovascular diseases.

There were no significant differences between GSSL (4/1) group and CG in terms of the level of TC, TAG, LDL-C and LDL-C/HDL-C, suggesting that GSSL (4/1) group have obvious antihyperlipidemic effect.

Conclusions

According to the study, characterization clearly showed the EE, particle size, zeta potential and morphology of GSSL were similar with those of the ordinary GLCL liposome. Moreover, stabilities of GSSL stored in different pH and oxidation conditions were much better than that of GLCL. In addition, GSSL liposomes had better antihyperlipidemic effects. Therefore, the liposome consisting of sphingomyelin and β-sitosterol is practicable, this novel liposome expanded the application of liposome as a formulation in the field of lowering blood lipid.

Abbreviations

BLCL; blank liposome composed of lecithin and cholesterol, BSSL; blank liposome composed of sphingomyelin and β-sitosterol, CG; control group, EE; encapsulation efficiency, GLCL; Gypenosides loaded liposome composed of lecithin and cholesterol, GSSL; Gypenosides loaded liposome consisted of sphingomyelin and β-sitosterol, GSSL(4/1); the mole ratio of sphingomyelin to β-sitosterol in GSSL liposome was 4 to 1, and so on; NCG, negative control group, TAG; triacetylglyceride, TBARS; thiobarbituric acid reactive substances, TC; total cholesterol

Acknowledgment

Acknowledgments    We acknowledge the project supported by the National Natural Science Foundation of China (Grant No. 81102817), Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection of China (JLC-BE09021), Yancheng Teachers University Doctoral Scientific Research Foundation, we also acknoledge the financial support of Natural Science Foundation (09YCKL010) and teaching research project (13YCTCJY046) of Yancheng Teachers University.

References

•  Abra,  R. M. and  Hunt,  C. A. (1981). Liposome disposition in vitro. III. Dose and vesicle size effects. Biochemistry Biophysics, 666, 493-501.
•  Bangham,  A. D.,  Standish,  M. M., and  Watkins,  J. C. (1965). Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol., 13, 238-247.
•  Chen,  J.C.,  Lu,  K.W.,  Tsai,  M.L.,  Hsu,  S.C.,  Kuo,  C.L.,  Yang,  J.S.,  Hsia,  T.C.,  Yu,  C.S.,  Chou,  S.T.,  Kao,  M.C.,  Chung,  J.G., and  Gibson Wood,  W. (2009). Gypenosides induced G0/ G1 arrest via CHk2 and apoptosis through endoplasmic reticulum stress and mitochondria-dependent pathways in human tongue cancer SCC-4 cells. Oral Oncol., 45, 273-283.
•  Chien,  Y. L.,  Wu,  L. Y.,  Lee,  T. C., and  Hwang,  L. S. (2010). Cholesterol-lowering effect of phytosterol-containing lactic-fermented milk powder in hamsters. Food Chem. 119, 1121-1126.
•  Cui,  J.,  Eneroth,  P., and  Bruhn,  J.G. (1999). Gynostemma pentaphyllum: identification of major sapogenins and differentiation from Panax species. European Journal of Pharmaceutical Sciences, 8, 187-191.
•  Dahle LK,  Hill EG, and  Holman  RT. (1962). The thiobarbituric acid reaction and the autoxidations of polyunsaturated fatty acid methyl esters. Arch. Biochem. Biophys., 98, 253-261.
•  Dong  Y and  Feng  SS. (2004). Methoxy poly (ethylene glycol) -poly (lactide ) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials, 25, 2843-2849.
•  Fereidoon,  S. (2005). Bailey’s industrial oil and fat products (6^th ed.). New York: A John Wiley and Sons, Inc.
•  Frans,  H.,  Sanders,  T., and  Thompson,  G.R. (2005). Comparison of efficacy of plant stanol ester and sterol ester: short-term and longer-term studies. The American Journal of Cardiology, 96, 29-36.
•  González-Larena  M,  García-Llatas  G,  Vidal  MC,  Sánchez-Siles  LM,  Bar-berá  R, and  Lagarda  MJ. (2011). Stability of plant sterols in ingredients used in functional foods. J. Agric. Food Chem., 27, 3624-3631.
•  Göran Lindblom,  Greger Orädd, and  Andrey  Filippov. (2006). Lipid lateral diffusion in bilayers with phosphatidylcholine, sphingomyelin and cholesterol. An NMR study of dynamics and lateral phase separation. Chemistry and Physics of Lipids, 141, 179-184.
•  Grit,  M. and  Crommelin,  D.J. (1993). Chemical stability of liposomes: Implications for their physical stability. Chem Phys Lipids, 64, 3-18.
•  Han,  M.O.,  Liu,  J.X., and  Gao,  H. (1995). Effects of 24 Chinese medicinal herbs on nucleic acid, protein and cell cycle of human lung adenocarcinoma cell. Zhongguo Zhong Xi Yi Jie He Za Zhi, 15, 147-149.
•  He Wensen,  Jia Chengsheng,  Yang  Yebo,  Ma  Yuan,  Zhang  Xiaoming,  Feng  Biao, and  Jin  Jian. (2011). Cholesterol-Lowering Effects of Plant Steryl and Stanyl Laurate by Oral Administration in Mice. Agricultural and food chemistry, 59, 5093-5099.
•  Hwang  Jyh-Sheng,  Tsai  Yu-Ling, and  Hsu  Kuo-Chiang. (2010). The feasibility of antihypertensive oligopeptides encapsulated in liposomes prepared with phytosterols-b-sitosterol or stigmasterol. Food Research International, 43, 133-139.
•  Ishikawa  H,  Shimoda  Y, and  Matsumoto  K. (2004). Preparation and characterization of liposomal microencapsulated poly-gamma-glutamic acid for prevention of Ca-phosphate precipitation under intestinal environment. Food Sci. Technol. Res., 10, 227-231.
•  Jogchum,  P. and  Ronald,  P.M. (2005). Plant stanol and sterol esters in the control of blood cholesterol levels: mechanism and safety aspects. The American Journal of Cardiology, 96, 15-22.
•  Laura,  C.B.,  Joan,  C.E., and  Francisco,  B.V. (2009). New insights into the molecular actions of plant sterols and stanols in cholesterol metabolism. Atherosclerosis, 203, 18-21.
•  Li,  L.,  Jiao,  L., and  Lau,  B.H. (1993). Protective effect of gypenosides against oxidative stress in phagocytes. vascular endothelial cells and liver microsomes. Cancer Biother, 8, 263-272.
•  Lu,  H.F.,  Chen,  Y.S.,  Yang,  J.S.,  Chen,  J.C.,  Lu,  K.W.,  Chiu,  T.H.,  Liu,  K.C.,  Yeh,  C.C.,  Chen,  G.W.,  Lin,  H.J., and  Chung,  J.G. (2008). Gypenoside induced G0/G1 arrest via inhibition of cyclin E and induction of apoptosis via activation of caspase-3 and -9 in human lung cancer A-549 cells. In Vivo, 22, 215-221.
•  Lu  Jinqing,  Yu  Fan,  Chen  Li,  Xiao  Bo,  Tian  Yiaoping,  Xu  Yuting,  Zhan  Xiaolian, and  Wan  Wei. (2007). Determination of rutin and quercetin in Gynostemma pentaphyllum(Thunb.) Makino. by RP-HPLC. Chinese Traditional Patent Medicine, 29, 1193-1195.
•  Megalli,  S.,  Davies,  N. M., and  Roufogalis,  B. D. (2006). Anti-hyperlipidemicand hypoglycemic effects of Gynostemma pentaphyllum in the Zucker fatty rat. J Pharm Pharm Sci., 9, 281-291.
•  New,  R.R.C. (1990). Liposomes—APractical Approach. Oxford: Oxford University Press.
•  Ostlund RE JR,  Spilburg CA, and  Stenson  WF. (1999). Sitostanol administered in lecithin micelles potently reduces cholesterol absorption in human subjects. Am J Clin Nutr., 70, 826-831.
•  Plessis,  J.,  Ramachandran,  C.,  Weiner,  N., and  Muller,  D. G. (1996). The influence of lipid composition and lamellarity of liposomes on the physical stability of liposomes upon storage. Int. J. Pharm., 127, 273-278.
•  Rosmini,  M. R.,  Perlo,  F., and  Perez-Alvarez,  J. A. (1996). TBA test by an extractive method applied to “pate”. Meat Science, 42, 103-110.
• Scandinavian Simvastatin Survival Study Group. (1994). Randomized trial of cholesterol lowering in 4444 patients with CHD. Lancet, 344, 138-141.
•  Schuler  I,  Duportail  G,  Glasser  N,  Benveniste  P, and  Hartmann  MA. (1990). Soybean phosphatidylcholine vesicles containing plant sterols: a fluorescence anisotropy study. Biochim. Biophys. Acta., 1028, 82-88.
•  Sergio  M.C.,  Seokwon  L.,  Gerardo  R.,  Amparo  O., V.,  Christian  G. K.,  Sundaram  G., and  Jess  D. R. (2010). Biopolymer coating of soybean lecithin liposomes via layer-by-layer self-assembly as novel delivery system for ellagic acid. Journal of functional foods, 2, 99-106.
•  Simons,  K. and  Ikonen,  E. (1997). Functional rafts in cell membranes. Nature, 387, 569-572.
•  Shang,  L.,  Liu,  J.,  Zhu,  Q.,  Zhao,  L.,  Feng,  Y.,  Wang,  X.,  Cao,  W., and  Xin,  H. (2006). Gypenosides protect primary cultures of rat cortical cells against oxidative neurotoxicity. Brain Res., 1102, 163-174.
•  Shimizu,  M.,  Miwa,  Y.,  Hashimoto,  K., and  Goto,  A. (1993). Encapsulation of chicken egg yolk immunoglobulin G (IgY) by liposomes. Biosci. Biotechnol. Biochem., 57, 1445-1449.
•  Sriram and  Rhodes. (1995). Preparation and characterization of liposomes as therapeutic delivery systems: A review. Pharmaceutic Acta Helvetiae, 1, 95-111.
•  Sułkowski,  W.W.,  Pentak,  D.,  Nowaka,  K., and  Sułkowska,  A. (2005). The influence of temperature, cholesterol content and pH on liposome stability. Journal of Molecular Structure, 744, 737-747.
•  Torchilin  V.P. (2005). Recent advances with liposomes as pharmaceutical carriers. Nature Rev. Drug Discov., 4, 145-160.
•  Tsuge K,  Okabe M., and  Yoshimura  T. (2004). Dietary effects of porphyran from Porphyra yezoensis on growth and lipid metabolism of Sprague-Dawley rats. Food Sci. Technol. Res., 10, 147-151.
•  Wang,  Q.F.,  Chen,  J.C.,  Hsieh,  S.J.,  Cheng,  C.C.,  Hsu., and  S.L. (2002). Regulation of Bcl-2 family molecules and activation of caspase cascade involved in gypenosides- induced apoptosis in human hepatoma cells. Cancer Lett., 183, 169-178.
•  Webb  MS,  Harasym  TO,  Masin  D,  Bally  MB., and  Mayer  LD. (1995). Sphingomyelin-cholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models. Br J Cancer, 72, 896-904.
•  Winkler  JK and  Warner,  K. (2008). Effect of phytosterol structure on thermal polymerization of heated soybean oil. European Journal of Lipid Science and Technology, 110, 1068-1077.
•  Xu,  X.,  Bittman,  R., and  E.  London. (2001). Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/ sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide. J. Biol. Chem., 276, 33540-33546.
•  Yin,  M. C. and  Faustman,  C. (1993). Influence of temperature, pH, and phospholipid composition upon the stability of myoglobin and phospholipid: A liposome model. J. Agric. Food Chem., 41, 853-857.
•  Yu  Fan,  He  Chenghua,  Ayman  Y. Waddad,  Munyendo  Were LL.,  Lv  Huixia., and  Zhou  Jianping. (2013). N-octyl- N-arginine-chitosan (OACS) micelles for gambogic acid oral delivery: preparation, characterization and its study on in situ intestinal perfusion. Drug Development and Industrial Pharmacy, DOI: 10.3109/03639045.2013.786723, in press.
•  Zant  P., E.,  Stasiuk,  M.,  Gubernator,  J., and  Kozubek,  A. (2010). Resorcinolic lipids improve the properties of sphingomyelin-cholesterol liposomes. Chemistry and Physics of Lipids, 163, 648-654.