Physiochemical property and antibacterial activity of formulation containing polyprenol extracted from Ginkgo biloba leaves

Industrial Crops & Products 147 (2020) 112213

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Physiochemical property and antibacterial activity of formulation containing polyprenol extracted from Ginkgo biloba leaves

T

Chang-Wei Zhanga,*, Ming-Fei Lib, Ran Taoa, Mi-Jun Pengc, Zhi-Hong Wangc, Zhi-Wen Qia, Xing-Ying Xuea, Cheng-Zhang Wanga,* a

Institute of Chemical Industry of Forest Products, CAF, Nanjing, 210042, China Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, 100083, China c Guangdong Provincial Public Laboratory of Analysis and Testing Technology, Guangdong Institute of Analysis, Guangzhou, 510070, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Ginkgo biloba leaves Polyprenol Physiochemical property Purification Liposomal gel Antibacterial activity

Polyprenol is an important active ingredient in Ginkgo biloba leaves (GBL) with great industrial application potential. In this paper, a novel high energy ball milling extraction technology coupled with silver-thiolate material purification method was developed, with which a high purity of GBL polyprenol (GBP) was obtained. Results showed that this method had a high extraction efficiency, and GBP of a purity up to 99.8 % was produced. GBP liposomal gel (GLG) was prepared, and its stability, rheological property, release behavior and antibacterial activity were evaluated. According to the results, GLG had a small particle size and high homogeneity which was desirable for topical application, and it also manifested good physiochemical stability and suitable plastic flow property. In addition, GLG illustrated excellent inhibitory effect against Staphylococcus aureus, Escherichia coli, Candida albicans and Streptococcus pneumoniae, and the minimal inhibitory concentration was 25∼200 μg/mL. The systematic research provided a useful method for the biorefining of GBL.

1. Introduction Biorefinery is a charming process which produces various chemicals, fuels and bio-based materials with biomass as raw material. A unique and abundant biomass resource in China, Ginkgo has been used to make edible starch and oil. Ginkgo biloba leaves (GBL), widely used as industrial raw materials of medicinal extracts, mainly contains active ingredients such as flavonoid, lactone, polysaccharide, polyprenol, carotenoid and so on (Ni et al., 2018; Beek and Montoro, 2009). So far, the research on GBL has focused on flavonoid and lactone (Liu et al., 2015; Xie et al., 2014; Su et al., 2015), whereas the rest active ingredients of GBL were rarely used. Polyprenol exists in GBL lipids, and its chain consists of an ω-unit, two trans isoprene units, a large number (10–20) of cis-isoprene units and a terminal (α) cis-isoprene unit with the ester group (Zhang et al., 2019b). In recent years, GBL polyprenol (GBP) has attracted increasingly more attention due to its biological activities, such as anti-oxidation, immunomodulation, anti-tumor and so on (Tao et al., 2019). Traditionally, high-purity GBP was obtained by extracting with organic solvent, saponification and separation through silica gel column chromatography (Zhang et al., 2019a). However, the purification of GBP is impeded by two aspects: the difficulty of completely extracting GBP ⁎

bound to cell wall, and silica gel column chromatography separation is lack of selectivity for GBP, resulting in complicated steps and low separation efficiency. Due to the high cost of high-purity GBP, these issues limit the development and application of high-value GBP-based functional products. In recent years, cell disruption assisted extraction technologies have attracted much attention due to the high efficiency and complete extraction (Lee et al., 2017; Ansari et al., 2018). Bakhshabadi et al. (2017) applied microwave to pretreat black cumin seeds, and found that the extraction efficiency of the seeds oil increased from 46.97 % to 79.45 % as the microwave power increased from 180 W to 900 W. Heydari Majd et al. (2014) compared the extraction efficiency of phenolic compounds from bovine pennyroyal between ultrasound extraction and maceration extraction, and reported that ultrasound extraction was better for extracting phenolic compounds from bovine pennyroyal plant. Zhou et al. (2017) optimized conditions of total polyphenols from Ulmus pumila barks using enzymatic hydrolysis-assisted extraction, and a higher extraction yield (16.04 mg GAE/g DW) of total polyphenols and an excellent antioxidant capacity in vitro were achieved. As a novel cell disruption technology, high-energy ball milling technology has been widely applied in the lipids extraction (Safi et al., 2014). For example, Meullemiestre et al. (2016) applied the high-energy ball milling

Corresponding authors. E-mail addresses: [email protected] (C.-W. Zhang), [email protected] (C.-Z. Wang).

https://doi.org/10.1016/j.indcrop.2020.112213 Received 1 September 2019; Received in revised form 4 January 2020; Accepted 4 February 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.

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of GBP as index. Similarly, the optimal extraction time (1 h, 2 h, 3 h, 4 h and 5 h), extraction temperature (60℃, 70℃, 80℃, 90℃ and 100℃) and concentration of NaOH (15 %, 20 %, 25 %, 30 % and 40 %) were individually confirmed by following the above extraction method. Based on this, orthogonal experiments were carried out to obtain the optimum condition of GBP extraction. The control group was performed at the temperature of 70 ℃, with the petroleum ether as the extraction solvent, the extraction time 2 h and solid-to-liquid ratio of 1:10. In addition, since the mixture of chloroform and methanol (2:1, v/v) was considered as complete extraction solvent of lipids, GBP’s chloroform and methanol extraction experiment was also performed in the same condition with the control group. 1 kg of GBL was extracted according to the above optimal extraction method for extracting GBP, and then crude GBL lipids were obtained by saponification, water scrubbing and acetone dewaxing (Zhang et al., 2015). Crude GBL lipids were purified by the previously synthesized functional nano-silica containing silver ions material (Zhang et al., 2019a, 2019b). The obtained high-purity GBP was used as a raw material for preparing liposomal gel.

technology to extract lipids from oleaginous yarrowia lypolitica yeast for a biojet fuel. To our knowledge, there are no reports on high-energy ball milling assisted GBP extraction. Efficient and simple separation methods are of essence for the industrial production of high-purity GBP. Due to the high selectivity of silver ions on double bonds, the design and synthesis of silver-thiol chromatographic materials for the separation of unsaturated compounds has gradually become a focus of research in recent years (Aponte et al., 2012). GBP is a specific example of polyisoprenoid alcohols, which consists of a large number of unsaturated double bonds (Gawarecka and Swiezewska, 2014). Therefore, silver-thiol chromatographic materials have great potential for GBP separation. In our previous work, a nano-silica containing silver ions material was synthesized, which rapidly improved the purity of GBP (Zhang et al., 2019a, 2019b). Obviously, high-energy ball milling combined with silver-thiol material shows great potential in improving extraction and purification efficiency of GBP. Antioxidant activities of GBP make it become a potential cosmetic feedstock, but its strong hydrophobicity and large molecular weight result in its poor transdermal permeability (Dragicevic-Curic et al., 2009). Embedding GBP into liposomal gel is a very effective method to solve this problem, because liposomal gel plays a vital role in improving drugs penetration into the skin (Johnsen et al., 2018; Mathiyazhakan, 2018) and prolonging drugs release time to increase the therapeutic effect (Kaplan et al., 2018; Singh and Dhiman, 2016). Liposomal gel is mainly made up of liposome, gel and active ingredients, in which gel is a very important factor for the stability of entire system. Nowadays, carbomer is widely used as the raw material for liposomal gel due to its mutual compatibility with liposome, and the prepared liposomal gel possess small particle size, high homogeneity, excellent entrapment efficiency and suitable plastic flow property for transdermal delivery (Ahad et al., 2014; Zidan et al., 2017). Therefore, fabricating carbomerbased GBP liposomal gel (GLG) is a promising strategy for the efficient utilization of GBP in the cosmetic field. The main purpose of this research is to develop an efficient separation method for GBP and to prepare GLG. A high-energy ball milling coupled with silver-thiolate purification technology was applied to obtain high-purity GBP. Then, GLG was fabricated using film dispersion and mechanical stirring method. Its stability, rheological property, release behavior and antibacterial activity were systematically evaluated.

Equal quality of lecithin and cholesterol were dissolved in the mixed methanol and chloroform (2.5:1, v/v) solution, before the addition of GBP. Organic solvents were removed from the mixtures at 60℃ with a rotary evaporator until forming a thin film, and GBP liposome was obtained. The thin film was hydrated with phosphate buffer saline (PBS, pH = 7.4) for 40 min. at 25 ℃, and then GBP liposomal dispersion (GLD) was obtained. The GLD was extruded with 300 nm of polycarbonate membranes, and its pH value was determined at 25 ℃.

2. Materials and methods

2.5. Preparation of GBP liposomal gel

2.1. Materials

Carbomer resin was added to aqueous solution containing preservatives (Sepicide HB 0.28 %, w/w, and Sepicide CI 0.20 %, w/w), propylene glycol (10 %, w/w), and edetate disodium (0.20 %, w/w) for 20 h. The mixture was stirred at 400 rpm/min for 20 min. to obtain homogeneous and dispersed carbomer resin. Then the solution was neutralized by the addition of sodium-hydroxide (carbomer:sodiumhydroxide = 1:2.5, w/w) to produce gel containing carbomer. GLD was put into the carbomer gel and mixed by a homogenizer (CRS2000/4, Berlin, Germany) at 4000 rpm/min for 5 min. to produce GLG. In addition, carbomer gel containing GBP (GBP gel), Gentamicin sulfate and Miconazole nitrate were respectively prepared in the above-mentioned method.

2.3. Morphological characterization observation of GBL with SEM Scanning electron microscope (SEM) was applied to observe GBL before and after extraction. The dried GBL samples were fixed on an SEM stub using double-sided adhesive tape and coated with a gold layer of 50∼100 nm thickness. Granule morphologies of untreated GBL, GBL processed with high-energy ball milling, and GBL after extraction were observed with an SEM (S-3400 N, Tokyo, Japan) at an acceleration voltage of 20 keV and 100× magnification. 2.4. Preparation of GBP liposome dispersion

GBL provided by Jiangsu Ginaton Biotechnology Co., Ltd, was dried at 60 ℃ for 6 h, then getting processed with a planetary ball mill (QMQX4, Shanghai, China) to obtain powders in sizes of less than 76.89 μm. Methanol, n-hexane, chloroform, lecithin, cholesterol, phosphate buffer saline, polycarbonate membranes, carbomer resin, propylene glycol, edetate disodium, sodium hydroxide, cellulose nitrate membrane, gentamicin sulfate, miconazole nitrate, gentamicin and chloramphenicol were purchased from Aladdin Ltd. Escherichia coli CGMCC 1.797 (E. coli), Candida albicans CGMCC 2.538 (C. albicans), Staphylococcus aureus CGMCC 1.89 (S. aureus) and Streptococcus pneumoniae CGMCC 1.8722 (S. pneumoniae) were obtained from China General Microbiological Culture Collection Center.

2.6. Physical characterization of GBP liposome in dispersion and gel The polydispersity index (PDI) and particle size of GBP liposome in GLD and GLG were analyzed by a Laser particle size analyzer (Microtrac S3500, California, America). Before measurement, GLD solution was diluted fifty times with PBS (pH = 7.4). GLG solution was firstly diluted to five times using PBS (pH = 7.4), and then the supernatant was obtained by centrifuging at 4000 rpm/min for 5 min. At last, the supernatant was further diluted to ten times using PBS (pH = 7.4). All these measurements were performed in triplicate.

2.2. Optimization of GBP purification process 5.0 g GBL powders were weighed and put into a 500 mL erlenmeyer flask, and a certain amount of liquor was added at the solid-to-liquid ratio (mass of GBL powders: volume of 20 % NaOH solution) of 1:5, 1:10, 1:15, 1:20 and 1:25, respectively. The erlenmeyer flask was then placed into a water bath at 70℃ for 2 h. After the extraction, the optimal solid-to-liquid ratio was determined using the extracted amount 2

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2.7. Stability evaluation of GBP liposomal gel

2.11. Antibacterial activity assay

Firstly, GLG was respectively pre-treated at a low speed 4000 r/min, 15 min. and a high speed 10,000 r/min, 15 min. utilizing a centrifuge GL-16 M, Shanghai, China. In addition, GLG was stored at a bright place light intensity was 4500 ± 500 and darkness at 4℃ and 25℃ for 5 months, respectively. The particle size, pH value and the PDI were measured at one month intervals, and the GBP content in all samples was detected after the storage period. All these experiments were carried out in triplicate.

Antibacterial activity experiments were carried out according to the method proposed by Tao et al. (2016) with some modifications. A small piece of E. coli, C. albicans, S. aureus and S. pneumoniae were individually picked from bacterial tubes in the sterile environment with a sterile inoculating loop, and inoculated into 100 mL of nutrient broth (NA, HiMedia), then placed in a shaker at 30℃ and activated for 2 d. The final concentrations of E. coli, C. albicans, S. aureus and S. pneumoniae were 5.0 × 109, 1.5 × 109, 3.8 × 109 and 2.3 × 109 colony forming units (CFU/mL), respectively. 100 μL of suspensions of E. coli, C. albicans, S. aureus and S. pneumoniae were separately pipetted with a sterile pipette before coated on the Mueller-Hinton agar (MSA, HiMedia). After the bacterial suspensions were fully permeated into the media, three sterilized oxford cups were evenly placed in a petri dish with sterile forceps. Then, 100 μL of previously prepared GLD (concentration of GBP was 2.0 mg/mL) and GLG (concentration of GBP was 1.6 mg/mL) were directly added separately to oxford cups, and carbomer gel containing gentamicin sulfate (0.5 mg/mL) and carbomer gel containing miconazole nitrate (0.5 mg/mL) were served as a positive control. The culture dishes were placed in an incubator at 30 ℃ for a period of time so as to observe the antibacterial effect of each sample and measure the diameters of the inhibition zone with a vernier caliper.

2.8. Rheological evaluation of GBP liposomal gel The rheological property of GLG was analyzed by utilizing a rotational and oscillatory rheometer (MARS III Haake, Berlin, Germany). The GLG continuous flow tests was performed at 20 ± 0.1℃ for 48 h. The measurement was performed by increasing the shear rate from 0 s−1 to 200 s−1 and reducing it back to 0 s−1, and both of the two stages lasted for 200 s respectively. The viscoelastic property of GLG was analyzed using dynamic (oscillatory) measurements. Firstly, the linear viscoelastic area was defined, in which viscoelastic parameters were irrelevant to the strain amplitude. This strain sweep test was conducted at a fixed frequency of 1 Hz with the initial and final strains of separately 0.6 % and 100 %. The measurement was carried out at 20 ± 0.1 ℃ with a cone/plate-measuring device MK 24 at an angle of measuring cone 1° and a radius of measuring cone 75 nm.

2.12. Determination of MIC and MBC concentrations The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of GLG and GLD were detected with the modified method proposed by Araujo et al. (2017). 100 μL of nutrient broth was pipetted into every well of 96-well microtitration plate. Then 100 μL of GLD (concentration of GBP was 2.0 mg/mL) and GLG (concentration of GBP was 1.6 mg/mL), carbomer gel containing Gentamicin sulfate (concentration of gentamicin was 0.5 mg/mL) and carbomer gel containing Miconazole nitrate (concentration of chloramphenicol was 1.8 mg/mL) were transferred into the first row of micro-titre plates. These solutions were separately diluted (two-fold dilutions) in nutrient broth. At last, 100 μL of E. coli, C. albicans, S. aureus and S. pneumoniae suspension (adjusted to 0.5 McFarland, approximately 108 CFU/mL) were separately added in each well. All of the micro-titre plates were incubated at 37 ℃ for 24 h. The MIC values were determined as the lowest concentration inhibiting the growth of bacteria. The final concentrations of GBP in the diluted solution of GLD (ranging from 8.0 to 512 μg/mL) and GLG (ranging from 6.25 to 400 μg/mL) were detected by HPLC. Each sample was tested in triplicate. Carbomer gel containing Gentamycin sulfate (content of gentamicin ranged from 0.49 to 125 μg/mL) and carbomer gel containing Miconazole nitrate (content of chloramphenicol ranged from 0.49 to 125 μg/mL) were used as a positive control in the assay. After broth microdilution tests, a 10 μL of sample was pipetted from wells and subcultured on new agar nutrient plates under 37 ℃ for 24 h in order to observe possible microbial development and also determine MBC values of GLG and GLD.

2.9. Release behavior evaluation of GBP liposomal gel The release behavior evaluation of GLD: GBP release behavior from GLD was evaluated using Franz diffusion cells with a receiver compartment volume of 15.6 mL and an effective diffusion area of 2.18 cm2. The phosphate buffer (pH 4.0) in the receptor phase was continuously stirred at the temperature of 35 ± 0.5 ℃ for 24 h. 1 mL of GLD was added to the donor compartment at appropriate time, and 0.2 mL of the receptor medium was pipetted from the receiver compartment to analyze GBP content. Meanwhile, the same volume of fresh phosphate buffer (pH 4.0) was added to the receiver compartment. Each experiment was carried out in three independent cells. Release behavior assesstion of GLG: GBP release behavior from GLG was assessed according to the method proposed by Cutrignelli et al. (2014). Appropriate volumes of the GLG were added to a 10 mL vial, and 2 % (w/w) agarose gel was carefully stratified on the surface of GLG. The entire release medium was removed from each vial at different time intervals (1, 2, 4, 6, and 24 h), and then centrifuged at 3000 r/min for 5 min, and GBP content of GLG was detected at each time interval by HPLC.

2.10. Drug release kinetics of GBP liposomal gel The GBP release kinetics from GLG were investigated according to previous reports (Higuchi, 1961; Korsmeyer et al., 1983) and the equations were showed as follows:

2.13. Content and purity determination of GBP

Mt =k t M∞

(1)

Mt = Kt n M∞

(2)

The content and purity of GBP were detected on a Shimadzu LC20AT-DAD High performance liquid chromatography. The HPLC operating conditions were as follows: column (2.5 μm, 150 mm × 4.6 mm) model was Thermo BDS HYPERSIL, the column temperature was 30 ℃, detection wavenumber was 210 nm, the mobile phase was isopropanol and methanol (v/v, 0.32/0.18), and the flow rate was 0.5 mL/min.

Where Mt/M∞ refers to the fraction of drug released at each time point (t), k refers to the kinetics constant incorporating structural and geometric characteristics of the controlled release device, and n refers to the diffusional exponent. 3

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Fig. 1. Effect of single factor on the extraction amount of GBP.

3. Results and discussion

Table 1 Results and analysis of L9(4)3 orthogonal experiment.

3.1. Results for GBP purification Fig. 1(a) demonstrates that the extraction amount of GBP changs along with the increasement of NaOH concentration. When NaOH concentration exceeded 30 %, the extraction amount of GBP decreased. The reason was that high NaOH concentration was viscous, which caused a part of GBP to be entrapped into extraction solution, so it was hard to be extracted completely by organic solvent; when NaOH concentration reached 30 %, the extraction amount of GBP was the largest. As shown in Fig. 1(b), the effect of extraction is the best when solidliquid ratio reaches 1:15, which is also the selected ratio. According to Fig. 1(c), the extraction amount of GBP rises at first, then decreases with the prolongation of extraction time. This was because short extraction time resulted in incomplete extraction, and long extraction time caused structural modification in the strong alkali environment. When the extraction time lasted for 3 h, the extraction amount of GBP was the topmost. It is obvious in Fig. 1(d) that when at the extraction temperature of 90 ℃, extraction amount of GBP achieves the climax. The important factors in extraction efficiency for GBP from top to bottom are solid-liquid ratio, followed by extraction time, NaOH concentration, and extraction temperature (Table 1). By comparing k values, it can be confirmed that optimal levels of each factor were A2, B2, C3, and D3, respectively. Therefore, the optimal extraction combination of GBP was A2B2C3D3. NaOH concentration, extraction time and solidliquid ratio displays significant effects on the extraction amount of GBP, and extraction temperature shows the slightest effect on the extraction amount of GBP (Table 2). Thence, the optimal process combination was adjusted to A2B2C1D3, i. e., concentration of NaOH 30 %, solid-liquid ratio 1:15, extraction temperature 80℃, and extraction time 4 h. Under these conditions, the extraction amount of GBP reached 0.81 %, which was 12.8 % higher than control group (the extraction amount of GBP was 0.72 %). In addition, the amount of GBP extracted by chloroform and methanol extraction was also 7.2 % lower than the amount

No.

A Concentration of NaOH (%)

B Solidtoliquid ratio

C Extraction temperature (℃)

D Extraction time (h)

Quality of GBP (mg)

1 2 3 4 5 6 7 8 9 k1 k2 k3 R

1 1 1 2 2 2 3 3 3 81.20 92.93 89.43 11.73

1 2 3 1 2 3 1 2 3 80.73 94.40 88.43 13.67

1 2 3 2 3 1 3 1 2 87.73 86.60 89.23 2.630

1 2 3 3 1 2 2 3 1 86.80 81.97 94.80 12.83

72.9 ± 1.7 80.6 ± 1.7 90.1 ± 2.5 91.5 ± 2.3 99.8 ± 2.7 87.5 ± 1.3 77.8 ± 1.5 102.8 ± 2.6 87.7 ± 1.6

Note: k refers to the average values of experiment results at the corresponding level for each factor. Table 2 Variance analysis. Source of variance

Degree of freedom

Sum of squares

Means quare

F value

F0.05

F0.01

Significance

A B C D Error Total

2 2 2 2 2 8

218.0 281.6 10.50 252.4 10.50

72.68 93.86 3.501 84.12

20.76 26.81 1.000 24.03

19

99

* * *

extracted by the aforementioned optimal process. Therefore, processing GBL by ball milling before extraction could promote the complete

4

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Fig. 2. SEM photographs of GBL (a), GBL processed with high-energy ball milling (b) and GBL after extracting GBP (c).

3.2. Effect of cell disruption on the morphology of GBL

Table 3 Physico-chemical parameters of liposomes in GLG and GLD. Sample

Particle size (nm)

PDI

pH values

GLD GLG

96.4 ± 0.3 92.8 ± 0.2

0.178 ± 0.006 0.136 ± 0.005

6.39 ± 0.01 6.75 ± 0.02

As shown in Fig. 2, the cell wall of GBL after being extracted by high-energy ball milling is disrupted thoroughly, which also explains the larger amount of GBP extracted by high-energy ball milling compared with the traditional extraction method. 3.3. Particle size and PDI of liposomes in GLD and GLG

extraction of GBP. Purification experiment results indicated that the content of GBP in crude lipids can quickly increased from 54.6% to 99.8% by the purification of already synthesized silver-thiol material. Zhang et al. (2019a); (2019b) developed an enzymolysis-assisted ultrasound extraction combined with silver-thiol material separation method for GBP, the content of GBP in crude lipids can enhance from 43.9% to 80.8%. Therefore, high-energy ball milling assisted extraction coupled with silver-thiol material separation method possessed higher efficiency for the purification of GBP.

It is evident that liposomes in both GLG and GLD demonstrates a small particle size and high homogeneity (Table 3), which is suitable for topical application (Dragicevic-Curic et al., 2009). The PDI values of GLG and GLD were lower than 0.20, indicating that liposomes in both formulations had homogeneous particle distribution. In addition, GLG and GLD shows similarly mild acid pH values acceptable for topical preparations (Table 3). 3.4. Physicochemical stability of GLG According to physical stability experiments, the micromorphology of GLG did not change and there was no precipitation phenomenon

Fig. 3. Particle size (a), PDI (b) and pH (c) change of GLG at different times. 5

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Table 4 Korsmeyer-Peppas and Higuchi elaborations of GBP diffusion profiles from gel. Formulation

GBP gel GLG

Higuchi equation

Korsmeyer–Peppas elaboration

Equation

Equation

n

y=3.832x+6.420 (r2=0.94) y=0.330x+0.839 (r2=0.95)

y=0.324x+0.964 (r2=0.98) y=0.257x+0.034 (r2=0.98)

0.32 0.26

Fickian-type diffusion (n = 0.5). Actually, it was found an n value was lower than 0.5 (n values ranging from 0.26 to 0.39), whereas the values of r2 increased for GLG and GBP gel indicating a higher correlation of this equation with the experimental data.

3.6. Rheological characterization of GLG

Fig. 4. Release curves of GBP in different media.

As can be seen in Fig. 6(a), the flow curve of GLG shows a nonNewtonian plastic flow behavior. According to the yield value, the gel network was resistant against an external force before it began to flow (Dragicevic-Curic et al., 2009). The ascendant curve was analyzed by the measurement software, and the best fitting (R > 0.999) indicated the Herschel-Bulkley model. The yield stress represented the flow behavior at small shear rates, i.e. before and after the formulations’ application. Topical formulations needed to have the yield stress, due to the lower resistance against flow under high shear condition, whereas at rest the flow was zero, which was suitable for topical utilization (Bousmina, 1999). Thus, formulations possessing the yield stress did not drip from knife, spatula and finger, but kept its shape until sheared by spreading pressures which surpassed the yield value, before the flowing and spreading. The yield stress value could be an indicator of the stability of formulations since a good correlation was showed between the elastic parameters and the yield value, which were widely applied in the forecast of long-term stability of semisolid formulations (Gasperlin et al., 1998). Typical yield stress values of semisolid preparations are always between 20 Pa and 80 Pa. When yield stress value is less than 20 Pa, the formulations begin to flow at low external force. The stress values of GLG were calculated with the Herschel-Bulkley mathematical model, and the high stress value of GLG (141.85 ± 18.62 Pa) in the results indicated a higher resistance against an external force before the system began to flow. The magnitude of yield stress was relevant to the strength of the inter-particle interaction of the three-dimensional network micro-structure in creams (Adeyeye et al., 2002). As yield stress and elastic parameters were found to have correlation with each other in some formulations, high yield values may also indicate the domination of the elastic behavior. This correlation was also verified in this

following the processing respectively by low-speed centrifuge and highspeed centrifuge. Moreover, as shown in Fig. 3, whether in a dark or bright place, and at both a low temperature (4 ℃) and a room temperature (25 ℃), the particle size, PDI, pH and GBP content of GLG demonstrate little variation for 5 months. These stability experiment results proved the fabricated GLG showed excellent physical and chemical stability, and its outstanding development potential in the cosmetic field because of antioxidant capacity of GBP (Zhang et al., 2015). 3.5. Sustained release property of GLG As shown in Fig. 4, both GLD and GLG have a prolonging effect on GBP release. During the release test, the amounts of GBP in the release medium gradually increased with the time prolonged. In addition, the release speed of GBP in GLG was slower than GBP gel, which was relevant to the drug reservoirs property of GLG. Moreover, the total GBP release amount of GLG after 24 h was lower than GBP gel. Therefore, the diffusion of GBP had a dual mechanism: firstly passing through the phospholipid bilayer before through the gel matrix. Both processes slowed down the spread of GBP in the receptor phase. It was easy to confirm the diffusion kinetic of GBP from the release profiles of GLG. In this research, Higuchi equation and KorsmeyerPeppas elaboration equation were applied to interpret the diffusion kinetic of GBP from GLG. The diffusion kinetic of GBP is obviously in accordance with the linear kinetic of Higuchi (r2 ranging from 0.94 to 0.95) in GLG (Table 5), illustrating the diffusion-type process as the most important process for GBP release. But according to the diffusional exponents obtained by plotting the Korsmeyer-Peppas equation in the logarithmic form (Fig. 5 and Table 4), GBP in GLG and GBP gel are not

Fig. 5. Higuchi elaboration (a) and Korsmeyer-Peppas elaboration (b) of release profiles. 6

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Table 5 Values of antibacterial ring for GLG and GLD. Sample

Content of active ingredient (mg/mL)

GLG GLD Gentamycin sulfate Miconazole nitrate

1.60 2.00 0.50 0.50

Diameters of bacteriostatic ring (mm) S. aureus

C. albicans

E. coli

14.3 12.1 19.0 17.4

8.5 ± 0.1 8.2 ± 0.1 12.5 ± 0.3 11.3 ± 0.2

16.0 14.3 21.4 20.3

± ± ± ±

0.1 0.2 0.3 0.2

± ± ± ±

S. pneumoniae 0.2 0.1 0.3 0.1

11.2 ± 0.2 9.7 ± 0.1 12.0 ± 0.2 11.8 ± 0.1

Fig. 6. Flow curve (a) and frequency sweep (b) of GLG. Full symbols represent the EM values, while empty symbols represent the VM values.

Fig. 7. Inhibition zone pictures of GLD (a) and GLG (b) against four bacterial strains.

a measurement of the energy lost per cycle reflects the liquid-like component storage modulus. As opposed to the VM, EM refers to a measurement of the energy stored and recovered per cycle of deformation and indicates the solid-like component of viscoelastic behavior. The loss tangent is the ratio of the VM and the EM, which demonstrates overall viscoelasticity of the sample. It can be seen in Fig. 6(b) that EM value of GLG is higher than its VM value, which means a domination of elastic behavior is stronger than the viscous behavior. In addition, the tanδ value of GLG was less than 0.5, which proves that elastic properties dominate in GLG as well.

Table 6 Values of MIC for GLG and GLD against four bacterial strains. Values of MIC (μg/mL)

S. aureus C. albicans E. coli S. pneumoniae

GLG

GLD

Gentamycin sulfate

Miconazole nitrate

50 200 25 100

64 256 32 128

3.91 7.81 1.95 15.6

15.6 31.3 3.91 0.98

study by oscillatory measurements. The viscoelasticity of GLG was evaluated by monitoring the viscous modulus (VM), elastic modulus (EM) and loss tangent (tanδ). The VM as 7

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3.7. Antibacterial activity of GLG

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Polyprenol has antibacterial activity, and antibacterial effect could be enhanced when used together with other bacteriostatic active substances (Shruthi et al., 2013; Tao et al., 2016). Both GLG and GLD have remarkable inhibitory effect on the four bacterial strains, and the antibacterial ability follows the order: E. coli＞S. aureus＞S. pneumoniae ＞C. albicans (Fig. 7 and Table 5). GLG and GLD were not demonstrated to have bactericidal effect under their test concentrations after subculturing in agar nutrient. The inhibition zone values range of GLG is 8.5–16.0 mm, and MIC value range of GLG is 25−200 μg/mL; meanwhile, the inhibition zone values range of GLD is 8.2–14.3 mm, and MIC values range of GLD is 32−256 μg/mL. (Tables 5 and 6). In addition, the inhibitory effect of GLG was stronger than GLD, and the reasons will be further studied in the following research. 4. Conclusion Compared with the traditional extraction method, high-energy ball milling coupled with silver-thiol material separation method could not only increase extraction efficiency of GBP, but also selectively and quickly enhance the purity of GBP. This new extraction and separation method has great potential for GBL biorefinery and the industrial production of GBP. The prepared GLG possessed a small particle size, high homogeneity, good physiochemical stability, suitable plastic flow property, excellent GBP sustained release performance, with notable inhibitory effect against Staphylococcus aureus, Escherichia coli, Candida albicans and Streptococcus pneumonia. Overall, the liposomal gel formulation is a promising strategy for the development and application of GBP in cosmetic and medicine fields. CRediT authorship contribution statement Chang-Wei Zhang: Conceptualization, Methodology, Writing original draft, Project administration. Ming-Fei Li: Writing - review & editing, Visualization, Methodology. Ran Tao: Investigation, Methodology. Mi-Jun Peng: Methodology, Writing - review & editing. Zhi-Hong Wang: Visualization, Investigation. Zhi-Wen Qi: Methodology, Investigation. Xing-Ying Xue: Visualization, Investigation. Cheng-Zhang Wang: Supervision, Conceptualization, Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The support of this work by Jiangsu Province Foundation for Youths (BK20180152) and DAS’ Project of Science and Technology Development (2019GDASYL-0502003) are gratefully acknowledged. References Adeyeye, M.C., Jain, A.C., Ghorab, K.M., Reilly, W.J., 2002. Viscoelastic evaluation of topical creams containing microcrystalline cellulose/sodium carboxymethyl cellulose as stabilizer. AAPS Pharm. Sci. Technol. 2, 1–10. https://www.aapspharmscitech.org. Ahad, A., Raish, M., Al-Mohizea, A.M., Al-Jenoobi, F.I., Alam, M.A., 2014. Enhanced antiinflammatory activity of carbomer loaded meloxicam nanoethosomes gel. Int. J. Biol. Macromol. 67, 99–104. https://doi.org/10.1016/j.ijbiomac.2014.03.011. Ansari, F.A., Gupta, S.K., Nasr, M., Rawat, I., Bux, F., 2018. Evaluation of various cell drying and disruption techniques for sustainable metabolite extractions from microalga grown in wastewater: a multivariate approach. J. Clean. Prod. 182, 634–643. https://doi.org/10.1016/j.jclepro.2018.02.098. Aponte, J.C., Dillon, J.T., Tarozo, R., Huang, Y.S., 2012. Separation of unsaturated organic compounds using silver-thiolate chromatographic material. J. Chromatogr. A

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