摘要
中药有效成分提取技术是中药生产质量控制的关键技术,传统的提取技术存在提取工艺复杂、提取效率低,溶剂消耗较大等不足之处,已经严重制约中药现代的进程。
常温超高压提取技术(High Hydrostatic Pressure Extraction,HPPE)是近年来发展起来的一种取得可喜进展的新提取技术,具有常温操作、高效节能、绿色环保的优点,已在食品和天然产物提取等领域获得广泛应用。
本文在重点考察单因素试验(提取压力、提取溶剂、料液比)基础上,正交优化得到了白花蛇舌草有效成分总黄酮的常温超高压提取工艺,考察了白花蛇舌草对香豆酸作为指标成分的可行性。将常温超高压提取液和回流提取液、超声提取液在pH稳定性、提取物中有效成分含量、提取物抗氧化活性、以及抗肿瘤活性方面进行了对比分析,还对超高压提取的强化传质机理进行了理论分析。同时还采用HPLC-TOF/MS方法对白花蛇舌草药材及注射液的化学成分进行了分析。最后以白花蛇舌草有效部位为试验材料,设计了常温超高压白花蛇舌草注射液的生产线。超高压提取新工艺结合先进的大孔树脂和膜分离技术,以及二次应用超高压技术进行超高压冷灭菌,保证整个生产工艺都在常温下完成。
Hedyotis diffusa Willd possesses the pharmacological effects of anti-tumor, enhancing immunity, antioxidation and clearing away heat and toxic material. The main task and direction for research and development of Hedyotis diffusa Willd is to extract and refine the components which occupies high medical value in Hedyotis diffusa Willd to relevant preparation by means of advanced extraction and separation.
This paper attempts to carry out the extraction technology research on bio-active components of Hedyotis diffusa Willd by high hydrostatic pressure extraction (HHPE) technique at ambient temperature, inspects the influences of extraction parameter on leaching rate of Hedyotis diffusa Willd flavones and p-coumaric acid, analyzes the components and antioxidant activity of the extract, and studies the mass transfer mechanism and dynamic model of high hydrostatic pressure extraction at ambient temperature. The main works and conclusions as follows:
1) Study and optimize high hydrostatic pressure extraction technology at ambient temperature for bio-active components of Hedyotis diffusa Willd, inspect the influences of pressure, acting time, extraction solvent, solid-liquid ratio etc. on extraction effects.
The leaching rates of flavones has obvious increase after treatment of high pressure. Pressure effect can accelerate the diffusion rate of solvent and solute to make it more thoroughly and more completely in very short time. In addition, because of the rapid change of cell’s internal and external pressure, cell tissue will be damaged, the contract area of solvent and tissue will be enlarged and mass transfer resistance of bio-active components will be reduced, the yield will be increased for the dissolution of bio-active components is more rapid and more complete. In the process of high hydrostatic pressure treatment, the pressure in system is equal, the speed of pressure transmission is fast and there is no pressure gradient, all of this makes the process of high hydrostatic pressure extraction simple and bio-active.
The analysis of optimum specimen results shows that the most remarkable factor influencing on flavones leaching rate is the treating pressure, then is the solid-liquid ratio and the solvent concentration. The optimum conditions as follows: 100Mpa, 60% ethanol, solid-liquid ratio: 60:1, extraction time: 3min, soak period: 1 hour. In the process of high hydrostatic pressure extraction for p-coumaric acid,the leaching rates of p-coumaric acid is similar to the leaching rates of flavones from Hedyotis diffusa Willd, we can draw a conclusion is tht p-coumaric acid as a component indicators can be used contol the quality of Hedyotis diffusa Willd,and the HPLC pattern of p-coumaric acid can be accurately reproduced.
Extract flavones from Hedyotis diffusa Willd under the condition of high hydrostatic pressure at ambient temperature has the characteristics of short extraction time, high extraction efficiency, simple operation and so on. Because the operation is carried out at ambient temperature and high hydrostatic pressure extraction is a physical process, it only has effects on secondary bonds with no influences on ionic bonds and covalent bonds, and can’t cause the damage for the bio-active components of small molecules; it is a safe and effective extraction method.
2) Extracting solution of Hedyotis diffusa Willd is obtained in the three ways: high hydrostatic pressure extraction at ambient temperature, circumfluence extraction and ultrasonic extraction, and investigations on pH stability of extracting solution gained by the three ways are carried out separately. The results indicate that the pH of extracting solution gained by means of circumfluence extraction and ultrasonic extraction decrease, pH variation of the extracting solution is under significant level ofα<0.05; pH of extracting solution gained by means of high hydrostatic pressure extraction basically has no variation, pH variation of the extracting solution is under significant level ofα<0.1. It can be seen that extracting solution of Hedyotis diffusa Willd gained by means of high hydrostatic pressure at ambient temperature has a good stability.
3) Contrast and analyze extracting effects of different extraction methods. Extraction is made for Hedyotis diffusa Willd iby means of ultrasonic, circumfluence and high hydrostatic pressure at ambient temperature. Analyze bio-active components contents, yield and antioxidant activity of the extract.
Clarity of extracting solution of Hedyotis diffusa Willd from high to low is high hydrostatic pressure extracting solution, ultrasonic extracting solution and circumfluence extracting solution. It shows that if extraction time is long, the degree of cell disruption is increased, the insoluble components in solution are grown, and the impurity level is also increased.
Leaching rate of flavones of Hedyotis diffusa Willd from high to low is high hydrostatic pressure method, ultrasonic method and circumfluence method. Ethanol solution with concentration too low or too high is not good for leaching of flavones series compounds, leaching rate of total bio-active components by high hydrostatic pressure extraction is higher than by other extracting methods which reflects that disruption effect of cell wall from pressure is bigger than ultrasonic and heat treatment. Extraction yield of p-coumaric acid from high to low is high hydrostatic pressure method, ultrasonic method and circumfluence method.
Scavenging activities of DPPH free radical and hydroxy radical of Hedyotis diffusa Willd extract from high to low is high hydrostatic pressure extracting solution, ultrasonic extracting solution and circumfluence extracting solution, therefore, antioxidant activity of extracting solution gained from different extracting methods has obvious differences. DPPH free radical scavenging activities of extracts gained from three methods are all above 68% (solution concentration is 0.044mg/mL) and hydroxy radical scavenging activities is all above 42% (solution concentration is 0.044mg/mL), it indicates that Hedyotis diffusa Willd has good scavenging activities of DPPH free radical and hydroxy radical and it is the source of natural antioxidant.
4 ) By the methods of HPLC-DAD-HPLC/TOFMS to analyze the chemical compositions of Chinese medicine injection and extracting solution of high hydrostatic pressure at ambient temperature of Hedyotis diffusa Willd. 37 chemical compositions of hedyotis diffusa Willd are identified by HPLC-DAD-TOF/MS analysis, composition analysis of commercial hedyotis diffusa Willd injection is carried out by establishment methods and chemical compositions are identified, analyze the extracting solution prepared by high hydrostatic pressure extraction (HHPE) technique, 37 chemical compositions are identified,and 6 chemical compositions are identified in the Hedyotis diffusa Willd injection self-made , 2 chemical compositions are identified in the commercial Hedyotis diffusa Willd injection saled.seen from the map, the injection of hedyotis diffusa Willd prepared by high hydrostatic pressure extraction (HHPE) technique has transparent appearance and simple and clear compositions. While commercial Hedyotis diffusa Willd injection adopts the technology included heating process which makes it appropriately produce some unknown compositions and increases unsafe factors. It indicates that high hydrostatic pressure extraction (HHPE) technique has various advantages.
Analyze the chemical compositions clearly by mass spectrometric analysis which could have an effective quality control and create a basis for further study of pharmacology and toxicology.
5) Theoretical analysis is carried out for reinforcement mass transfer mechanism in the process of high pressure extraction. Analysis results show that high pressure can obviously increase cell’s internal and external pressure difference so that the permeation rate of solvent can be increased which could reduce the time of penetration process, therefore, extraction time could be greatly reduced; in the stage of boosting, high pressure will lead to cell shape changed that causes cells of some friable materials to break down. Cell mass will be reduced and its shape will be changed from irregular to regular by high pressure, all the changes will cause cells of some friable materials to break down, as a result, cellular content escapes more completely, mass transport process of bio-active components will be immeasurably strengthened which makes the extraction yield of bio-active components higher. Violent cavitation will appeared with rapid pressure relief which causes cells to expand sharply. If the expansion exceeds cytomorphosis limits, the cells will break down, cellular content and solvent dissolved bio-active components can escape rapidly and completely, therefore, extraction yield of bio-active components is much higher.
6) A Chinese medicine (Hedyotis diffusa Willd) is taken as sample to put forward reaction course and extraction model by using theories of stochastic process,fractal geometry and dispersive kinetics. Extraction equations are fitted and obtained by computer programming and the correctness of the results is verified by means of autocorrelation of residues itself.
The analysis of autocorrelation functions of extraction series shows reaction-diffusion conforms to normal stationary Markov process, and the hypothesis of reaction mechanics which is in line with results simulated by computer that reaction-diffusion process undergoes different stable states was validated. In this paper, spectrum analysis which is closely related to autocorrelation functions is introduced to process the random series,and it reveals that the spectrum density of the series presents a decline trend with the increase of frequency and the spectrum density is rather affected by noise,and if activation energy of reaction-diffusion is same, the spectrum density of diffusion equation is also almost the same, it indicates that activation energy and spectrum density are closely connected with each other.
Simulated extraction equations reveal that diffusion rate mainly depends on initial matrix of a plant,which is relevant to pressure, solid-liquid ratio, solvent and temperature etc when it was extracted. The paper discusses that the change of extraction conditions has influences on the change of coefficients of extraction equations and further on the change of its extraction yield.
An important characteristic of condensed matter systems is that the reaction rate coefficient, obtained from diffusion equation of an extraction one, is not constant, but the function of time and fractal structure. The activation energy educed by reaction rate coefficient corresponds to the intermolecular force under other same conditions from 100 to 300 MPa,but when the pressure is less than 100 Mpa, the activation energy is much lower and extraction yield decreases.
Restructure of the phase space is introduced to calculate correlation dimensions of extraction series of flavones. Its dimension is below l,which shows that its reaction-diffusion is low-dimensional.
7)Research and analysis on vitro antitumor activity of human cervical adenocarcinoma HeLa cell. The test results showed the inhibitory effects of Hela cancer cells by the different processes extracts. Results indicated that probably the content and category of the total flavonoids are related to the anti-tumor mechanism, for the active anti-tumor of the extract of Hedyotis diffusa Willd is better, and the inhibitory proliferation effects of single-factor are close to the optimized results of total flavonoids extracted by HHPE, on the condition that HHPE to extract medicinal soak 1 hour ahead of time, pH value of 9, when the ethanol concentration of 40-60%, packing time 3min, pressure 100MPa. Compared with other agents, it can obtained the best anti-tumor activity of Hedyotis diffusa Willd eatract preparation, using the HHPE extraction technology (process elements: the concentration of 60% ethanol, liquid extract than 1:60,100 MPa UHP, 1000D membrane filtration, 400MPa ultra-high pressure sterilization).
8) Hedyotis diffusa Willd is taken as experimental material, production line of Chinese medicine injection adopted technique of high hydrostatic pressure extraction (HHPE) at ambient temperature is initially designed combined with previous research work : preprocessing (decontamination, cleaning, soaking etc.)→crushing (mechanical crushing)→preparation (mix the materials and extraction solvent in proportion)→loading into package→placing in high pressure vessel and airproofing the vessel→boosting→pressure maintaining→pressure relief→taking the mixture or the package from high pressure vessel→filtering, extracting solution obtained after removing medical slag→concentrating, separating and purifying→medicines, such as preparation, etc. depuration
The paper finished part of project named as“Research on Mechanism of Essential Components Extraction from Herb by High Hydrostatic Pressure at Ambient Temperature (30472135)”This paper proposes high hydrostatic pressure extraction (HHPE) technology used in bio-active components of Hedyotis diffusa Willd which provides a new method for extracting bio-active components of natural products and sets up theoretical basis for its development and large scale applications. The study suggests that bio-active components in Hedyotis diffusa Willd have good antioxidant activity and anti-cancer effect which have important significance for extraction and application. The research results of this paper have certain value to perfect and develop the extraction technique for bio-active components of natural products and have guiding significance for deep development and utilization of Hedyotis diffusa Willd.
引文
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