Study on the Preparation and Characterization of Glycyrrhizic Acid Liposomes ()
1. Introduction
Hepatology mainly includes the clinical diagnosis and treatment of viral hepatitis, chemical damage liver disease, autoimmune and cholestatic liver disease, genetic metabolic liver disease and vascular abnormal liver disease [1] [2]. The chemical injury liver disease has arisen from the stimulation of alcohol, drugs and poisons. There was still a gap in clinical diagnosis and treatment of non-communicable liver disease compared with developed countries. Viral hepatitis has always been the most common liver disease in China, including hepatitis A, B, C, D and E. Among them, hepatitis C and hepatitis B were the main liver diseases. Hepatitis B was a highly infectious disease with a high incidence rate and was seriously harmful to human health. China was the hardest hit area by hepatitis B. It was said that about 100 million people carried hepatitis B virus, accounting for about one-third of the global hepatitis B virus carriers. The number of people who die of viral hepatitis even accounted for half of the global number. Moreover, hepatitis B could cause a variety of acute and chronic liver diseases, such as liver failure, liver cirrhosis, liver cancer, etc. More than 1 million patients die of these complications every year [3] [4] [5]. Therefore, chronic hepatitis B was still the main liver disease endangering the health of Chinese people [6].
Glycyrrhizic acid was the most abundant triterpenoid saponin in Glycyrrhiza uralensis Fisch. It was also the main active component of Glycyrrhiza uralensis Fisch. It had anti-inflammatory, anti-tumor, anti-virus, anti-oxidation, immune regulation and other functions [7] [8]. The most widely used was the antiviral effect, which had the most significant effect on the liver. It could significantly reduce the necrosis of hepatocytes and the inflammatory reaction between hepatocytes, prevent hepatocyte steatosis, inhibit the proliferation of the liver fibrous layer and promote the regeneration of hepatocytes, which could effectively improve various kinds of liver diseases [9]. As a first-line drug for anti-inflammatory and liver protection treatment in the field of liver disease, the safety and effectiveness of glycyrrhizic acid have been confirmed in several clinical studies [10] [11].
However, Glycyrrhizic acid was difficult to dissolve in water and had low bioavailability, which led to its failure to exert its maximum efficacy in the treatment process. Moreover, if glycyrrhizic acid was directly used in clinical treatment, it had poor targeting and was easy to produce many adverse reactions [12] [13]. The main adverse reactions were followed: allergic reactions including skin allergic reactions, rashes, itching and anaphylactic shock; Digestive system adverse reactions such as nausea, vomiting, diarrhea and other symptoms; Endocrine system response: longer medication cycles can lead to a series of chronic diseases such as hypokalemia, hypertension, water and sodium retention [14] [15]. These problems greatly limited the practical application of glycyrrhizic acid in clinical practice, resulting in that glycyrrhizic acid could not play an important role in anti-inflammatory and liver protection.
In view of the above adverse reactions, the most effective way was to change the dosage form. Liposome was a kind of artificial nano-targeted preparation. By using the structural characteristics of its spherical phospholipid bilayer, drugs can be encapsulated in it and then delivered to the target organs to play the therapeutic role. Targeted preparation had the characteristics of location concentration, controlled drug release, low toxicity and side effects, improving the bioavailability of drug preparation [16]. Now there are many drugs that use these characteristics of targeted agents to improve the efficacy and reduce adverse reactions. All of these excellent characteristics would be attributed to the natural tendency of liposomes. After being encapsulated by liposomes, general drugs were mainly concentrated in liver, spleen, bone marrow and other organs. And the natural target organ of liposomes was mainly liver [17] [18]. Therefore, the liposome as a carrier to construct liver targeted glycyrrhizic acid nanoliposomes was helpful in achieving the targeted and precise treatment of glycyrrhizic acid, reducing the occurrence of adverse reactions, improving the bioavailability of glycyrrhizic acid and provides a new strategy for the clinical application of glycyrrhizic acid.
Therefore, this experiment would combine the anti-inflammatory, liver protective effects of glycyrrhizic acid and the natural targeting of liposomes to prepare liver targeted glycyrrhizic acid liposomes. The entrapment efficiency was selected as the index to optimize the best preparation conditions of glycyrrhizic acid nanoliposomes via orthogonal design. The evaluation indexes include encapsulation efficiency, morphology, particle size and stability. High-performance liquid chromatography was used to determine entrapment efficiency. The best preparation methods: Precision weighing glycyrrhizic acid 1 mg, lecithin 30 mg, cholesterol 30 mg, and the mixture of 2 mL trichloromethane methanol (1:2) mixed solution to dissolve. Transfer the mixed solution into the flask of the round bottom, and the uniform honeycomb film is formed by vacuum evaporation. Add 10 mL of the same temperature ultra-pure water into the round bottom flask, and the best liposome is obtained after emulsion ultrasonic 120 s. Finally, the entrapment efficiency of this batch of liposomes was about 90%.
2. Reagents and Apparatus
2.1. Reagents
Soybean lecithin and Cholesterol were offered by Xi’an Ruixi Biotechnology Co., Ltd. Glycyrrhizic acid standard (content ≥ 93.0%, Tokyo Chemical Industry Co., Ltd.); Acetonitrile (chromatographic purity, Merck Co., Ltd.); Methanol (chromatographic purity, Merck Co., Ltd.); Ultra pure water; Chloroform (analytically pure, Guangdong Guangshi reagent Technology Co., Ltd., batch No.: 2020040115); Phosphotungstic acid (content ≥ 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.).
2.2. Apparatus
LC-20a Shimadzu HPLC; Fresco 21 micro centrifuge; Ms204s electronic analytical balance; Kq-500de ultrasonic cleaning machine; Milli-q academic water purifier; MS-3 digital display vortex mixer; N-1100 rotary evaporator; A-1000s water flow ventilator; Ca-1111 cooling water circulating device; Q700 ultrasonic crusher; Seven Easy S20 pH meter; Dl-5-b low-speed large capacity centrifuge; Q700 ultrasonic crusher; Mastersizer micro Malvern laser particle sizer.
2.3. Chromatographic Conditions
Thermo acclaim TM 120 C18 (4.6 mm × 250 mm, 5 μm) Chromatographic column was selected for separation. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution. The flow rate was set as 0.8 mL·min−1. The detection wavelength was 254 nm with a column temperature 25˚C. According to the literature, the maximum absorption wavelength of glycyrrhizic acid was 249 nm, while the excipient had no obvious absorption at 254 nm, so the detection wavelength was 254 nm.
2.4. Preparation of Reference Solutions
Accurately weigh 1.5 mg of a glycyrrhizic acid reference substance, place it in a 2 mL centrifuge tube, add 1.5 mL of 30% methanol, and shake it evenly to prepare as the mother liquor of glycyrrhizic acid reference substance.
2.5. Preparation of Test Solution
Precisely extracted 1 mL of glycyrrhizic acid nanoliposome and placed it in a 5 mL volumetric flask, added methanol as demulsifier and solvent to demulsify and dilute it to 5 mL, shook it evenly and used it as the test solution of a glycyrrhizic acid liposome. The blank liposome was made into a blank liposome solution by the same method.
3. Methods and Results
3.1. Preparation of Glycyrrhizic Acid Nanoliposomes and Optimization of Preparation Conditions
3.1.1. Selection of Preparation Method
Liposomes were formed by the hydrophobic interaction of phospholipid molecules in the aqueous phase. The preparation methods were different. The particle size of liposomes can be from tens of nanometers to several microns, and the structure is not the same. There were many methods to prepare liposomes, such as multiple emulsion method, solvent injection method, reverse evaporation method and film dispersion method. Through the preliminary investigation, the particle size of liposomes prepared by the ultrasonic film dispersion method was more in line with the requirements and the operation was convenient, so the ultrasonic film dispersion method was selected to prepare glycyrrhizic acid nano liposomes.
3.1.2. Encapsulation Efficiency of Liposomes
The entrapment efficiency of liposomes refers to the percentage of the encapsulated substance (such as a certain drug) in the liposomal suspension of the total amount of drug. It was an important indicator of quality control of liposomes and nanoparticles, which would reflect the extent of drugs encapsulated by carriers [19]. Determination of liposome entrapment efficiency was usually by first separating drug-containing liposomes from free drugs and then calculating their concentration directly or indirectly by other methods [20]. Ultracentrifugation was chosen for this study, considering the nature of the liposomes containing glycyrrhizin and the operating conditions.
Determination method: 1 mL of glycyrrhizic acid liposome solution was added into an ultrafiltration centrifuge tube and centrifuged at 14,000 rpm for 10 min to collect the filtrate. After demulsification with methanol, the microporous membrane was filtered. In the same way, glycyrrhizic acid nanoliposomes were demulsified, centrifuged and diluted at the same time. The two diluted solutions were filtered by microporous membrane and then analyzed to calculate the content of glycyrrhizic acid. The formula of drug entrapment efficiency was as follows:
Encapsulation efficiency = (1 − Cf/Ct) × 100%.
where Cf is the amount encapsulated in the microparticle formulation and Ct is the total drug amount encapsulated versus unencapsulated in the microparticle formulation.
3.1.3. Prescription Optimization
By consulting the relevant literature, we found that the main factors influencing the encapsulation rate of liposomes were: drug to lipid ratio, mlecithin:mcholesterol, hydrating medium type, dosage of hydration medium, hydration time, ultrasonic time, etc. [21]. In this study, the drug encapsulation rate was used as the evaluation index, and three levels of three factors (drug lipid ratio, mlecithin:mcholesterol, hydrating medium type) were investigated. L9 (33) orthogonal design experiment was used to design and screen the prescription, as shown in Table 1. Nine batches of glycyrrhizic acid nanoliposomes were prepared and the encapsulation rate was determined according to the above methods. The results are shown in Table 2 and Table 3.
The results showed that the choice of hydration medium had a significant effect on the encapsulation rate of liposomes. According to the effect chart (Figure 1), the optimal technological conditions are drug to fat ratio = 1:30, lecithin: cholesterol = 1:1, hydration medium as pure water.
3.2. Methodological Validation of Chromatography for Glycyrrhizic Acid Determination
3.2.1. Specific Experiment
The blank methanol, glycyrrhizic acid reference solution and blank liposome test solution were detected. The spectrum results showed that the peak shape of glycyrrhizic acid was good under the chromatographic conditions, without the interference of miscellaneous peaks, and the retention time was 22.956 min. Compared with blank methanol and blank liposome, the chromatographic conditions were specific, and the results are shown in Figure 2.
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Table 1. Factor level of orthogonal test.
3.2.2. Limit of Detection
The mother liquor of glycyrrhizic acid reference substance was diluted to different concentrations and the liquid chromatogram was recorded. When S/N = 3, the concentration was the lowest detection limit. The results showed that the detection limit of glycyrrhizic acid in vitro was 0.78 μg·mL−1.
3.2.3. Linearity and Range
Accurately measure the appropriate amount of glycyrrhizic acid reference stock solution, and dilute with 30% methanol to glycyrrhizic acid test solution with the
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Figure 2. Typical chromatogram of glycyrrhizic acid.
concentration of 2.5, 5, 10, 20, 40, 80, 160 μg·mL−1 respectively. The regression equation of glycyrrhizic acid concentration (c) to absorption peak area (A) was obtained as follows: y = 7076.9x − 10633, R2 = 0.9999. The results were shown in Figure 3, which indicated that there was a good linear relationship between glycyrrhizic acid concentration and peak area in the range of 2.5 - 160 μg·mL−1.
3.2.4. Precision Test
Prepare glycyrrhizic acid reference solution with high (125 μg·mL−1), medium (25 μg·mL−1) and low (5 μg·mL−1) concentrations. Each concentration was determined 6 times a day for 3 days. The intra-day precision and inter-day precision were obtained in Table 4, Table 5. The results showed that the intra-day
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Figure 3. Standard curve of glycyrrhizic acid.
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Table 4. Intra-day precision (n = 6).
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Table 5. Inter-day precision (n = 6).
and inter day precision of high (125 μg·mL−1), medium (25 μg·mL−1) and low (5 μg·mL−1) glycyrrhizic acid were less than 1.0%, indicating that the precision of the test was good and met the requirements of methodology.
3.2.5 Stability Test
Take the prepared glycyrrhizic acid reference materials with high (125 μg·mL−1), medium (25 μg·mL−1) and low (5 μg·mL−1) concentrations to determine the peak area. The results were shown in Table 6, and the relative standard deviation was less than 1.0%, indicating that the test was good stability.
3.2.6. Repeatability Test
Take the prepared 125 μg·mL glycyrrhizic acid reference substance and operate six copies in parallel, then record the chromatographic peak area. The results are shown in Table 7. The relative standard deviation was within the allowable range, indicating that the test was good repeatability.
3.2.7. Sample Recovery Test
1 mL glycyrrhizic acid nanoliposome solution was used for testing recovery. The results are shown in Table 8. The average recoveries were above 90% and the
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Table 7. Repeatability test (n = 6).
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Table 8. Sample recovery rate test (n = 6).
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Figure 4. Glycyrrhizic acid nanoliposomes.
relative standard deviations were less than 1%, indicating that the excipients had no significant effect on the determination of glycyrrhizic acid in glycyrrhizic acid nanoliposomes.
3.3. Evaluation of Glycyrrhizic Acid Nanoliposomes
3.3.1. The Morphology and Distribution of the Liposomes
A batch of glycyrrhizic acid liposomes was prepared according to the optimized formula and uniform and transparent milky white suspension was observed (Figure 4). Take an appropriate amount of the glycyrrhizic acid liposome suspension, dilute it with ultrapure water, drop it on the special copper mesh, place it for two minutes, absorb the excess water with filter paper, dye it with phosphotungstic acid for 2 minutes, dry it naturally, observe it under the transmission electron microscope and take photos. The results are shown in Figure 5.
3.3.2. Particle Size and Zeta Potential of the Liposomes
Analyze the optimized glycyrrhizic acid nanoliposomes by Malvern laser particle size analyzer. The results were as follows: the hydrodynamic diameter was about 50 nm (Figure 6). The record of the particle size and Zeta potential were −28.9 mV (Figure 7).
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Figure 5. Electron micrograph of glycyrrhizic acid nanoliposomes.
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Figure 6. Particle size distribution of glycyrrhizic acid liposomes.
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Figure 7. Zeta potential diagram of glycyrrhizic acid liposome.
4. Summary and Discussion
In this project, glycyrrhizic acid liposomes were successfully prepared by the film dispersion method. The best preparation conditions were selected by an orthogonal design experiment. The best preparation method was as follows: accurately weigh 1 mg glycyrrhizic acid, 30 mg lecithin and 30 mg cholesterol, add 2 mL chloroform methanol (1:2) mixed solution to dissolve, transfer the mixed solution into a round bottom flask, A uniform honeycomb membrane was formed by vacuum evaporation, then O/W emulsion was added to 10 mL ultra pure water at the same temperature. The best liposomes were obtained after ultrasonic emulsion of 120 s. Finally, the entrapment efficiency of this batch of liposomes was about 90%.
The stability and morphological properties of liposomes were preliminarily investigated. The Glycyrrhizic nano liposomes were observed to be a homogeneous and transparent emulsion. Transmission electron microscopy showed that glycyrrhizic acid nano liposomes were spherical vesicles with a particle size of 100 nm. Zeta potential, also known as surface potential, is the characterization of the amount of charge on the surface of particles, which is related to the stability of the particle system. Zeta potential can be positive or negative, and it is related to the excipients used, such as the surface potential of lipid emulsion prepared with lecithin as emulsifier is negative. In general, the higher the absolute value of zeta potential, the greater the electrostatic repulsion between particles and the better the physical stability. Generally, the system is stable when the absolute value of zeta potential reaches 25 mV. The particle size was about 50 nm and the zeta potential was −28.8 mV, which indicated that the liposome was stable. The establishment of this method provides a new strategy for the further study of glycyrrhizic acid in the treatment of viral hepatitis.
NOTES
*These authors contributed equally to this work.
#Corresponding authors.