Optimization of Extraction, Purification and Identification Process of Bullatine G

Jian LIU, Jinglong CAO, Hui XUE, Yannan LI, Wenshuang HOU, Yinghua LUO, Chenghao JIN*

1. College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China; 2. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Abstract The purification processes of bullatine G, such as acid water extraction, ammonia alkalization ether cold soaking method, ultrasonic assisted extraction and cellulase extraction, and the determination technologies of bullatine G content, such as UV-spectrophotometry, chromatography, near infrared spectroscopy (NLR), thin layer chromatography (TLC), high performance liquid chromatography-mass spectrometry (HPLC-MS) were reviewed, in order to provide a theoretical basis for the development and application of bullatine G.

Key words Bullatine G; Ditepenoid alkaloid; Purification process; Content determination

1 Introduction

Aconitumcarmichaelii, Aconiti Radix, is the dry maternal root of aconite belonging to Ranunculaceae, Angiospermae[1].A.carmichaeliiis an annual herb originally recorded inShengNong’sHerbalClassic, and it has been used medically for a long history, with the effects of dispelling wind and dampness, warming meridians and relieving pain,etc., and is mainly distributed in Sichuan Province and the middle and lower reaches of the Yangtze River in China[2]. In 1963, thePharmacopoeiaofthePeople’sRepublicofChinaincludedA.carmichaeliias a traditional Chinese medicinal plant, mainly used for the treatment of rheumatic diseases, cold pain in the heart and abdomen and joint pain[3].A.carmichaeliimainly contains alkaloids, flavonoids, polysaccharides, glycosides and other effective components, among which alkaloid is the main pharmacodynamic and toxic component ofA.carmichaeliiand the research focus of its chemical composition[4]. Bullatine G, also known as songorine, is a C20-ditepenoid alkaloid extracted fromA.carmichaeliiand otherAconitumplants, which is a major active substance inA.carmichaelii. Pharmacological studies have shown that bullatine G has anti-inflammatory, anti-tumor and other pharmacological activities, and is a natural drug with good development prospects[5]. In this paper, the purification processes and content determination technologies of bullatine G were reviewed, in order to provide the ideas and basis for further development and application of bullatine G.

2 Purification processes

Purification of traditional Chinese medicine is a complicated technological process and the core link of drug production. With the development of modern industrial technology and research, the purification process of traditional Chinese medicine has been improved, and different purification processes have their advantages and disadvantages. At present, the main purification methods of bullatine G include acid water extraction, ammonia alkalization ether cold soaking method, ultrasonic assisted extraction, cellulase extraction,etc.

2.1 Acid water extractionAcid water extraction is a relatively primitive extraction method, which mainly extracts by impregnation, osmose process and cooking process, with hydrochloric acid, acetic acid and other acid water as extraction solvents. Using acid water extraction method, Luo Chunmeietal.[6]decocted steamed slices of Aconiti Lateralis Radix Praeparata in acid water, purified merged water decoction by macroporous resin adsorption method, and finally obtained the extract. Further study showed that the pH of acid water may be the major factor affecting the total amount of alkaloids extracted from Aconiti Lateralis Radix Praeparata. In order to screen the optimal extraction scheme, the total extraction amount of 14 kinds of alkaloids was used as the evaluation indicator. Through the design of three-factor and three-level orthogonal test, the optimal extraction condition was determined to be decocted in 10 times of acid water (pH=3.5) for 3 times, 2 h each time. Compared with traditional acid water extraction methods, the improved acid water extraction method is simple, it not only has the advantages of saving the amount of acid water, shortening the extraction time, lowering production cost, and reducing environmental pollution, but also improves the extraction efficiency by nearly 20%. However, due to large volume of extraction liquid, difficulties in concentration and more water-soluble impurities, cation exchange resin method or other methods must be used for purification. In addition, since free alkaloids and their salts are easily soluble in methanol or ethanol solution, ethanol can be used to extract medicinal materials instead of water or acid water[7]. The extraction method is featured by wide applicability, easy filtration, convenient concentration, and less water-soluble impurities. However, there are pungent odors and fat soluble impurities in the extraction process, and the substances need to be further purified.

2.2 Ammonia alkalization ether cold soaking methodIn ammonia alkalization ether cold soaking method, medicinal materials are alkalized with ammonia water to make alkaloid components in a free state, and then soaked with ethyl ether, while solvents such as anhydrous ethanol, methanol and ethyl acetate can take the place of ethyl ether[8]. Li Yanjunetal.[9]crushed dry tubers ofA.soongaricumand soaked in hydrochloric acid solution overnight; after percolation extraction, the acid solution was merged and alkalized to pH > 9 with stronger ammonia water, and extracted by ethyl acetate to get ethyl acetate extract; the extract was separated by silica gel column chromatography, and finally bullatine G was obtained. Ammonia alkalization ether cold soaking method has the advantages of high extraction efficiency, simple operation and stable product, but has too much consumption of organic solvents, which may cause pollution to the environment.

2.3 Ultrasonic assisted extractionUltrasound assisted extraction is a modern purification process which has been applied to extract the active components of traditional Chinese medicine earlier. The energy is converted into high-frequency oscillation by ultrasonic transducer, and the cell wall of medicinal materials is broken by ultrasonic cavitation and mechanical action, so as to improve the extraction efficiency of medicinal materials. Using ultrasound assisted extraction, Wang Kunetal.[10]mixedA.carmichaeliiwith acid water for ultrasonic oscillation, and analyzed and predicted the optimal extraction conditions of aconitines such as bullatine G through Box-Behnken design; the optimal solvent, ultrasonic power, liquid dosage, extraction times and time were 1.4% hydrochloric acid aqueous solution, 600 W, 12 times liquid dosage, 2 times, and 1.5 h each time, respectively. Compared with traditional extraction methods, ultrasound assisted extraction has the advantages of wide applicability, low energy consumption, simpleness and reliability, good repeatability, high extraction yield and short extraction time. However, the method is restricted by noisy extraction process, poor product stability and high economic cost, and the extraction efficiency is limited by ultrasonic attenuation, so it is not suitable for large-scale industrial production.

2.4 Cellulase extractionWhen extracting active ingredients of natural medicines, the pharmaceutical components are usually wrapped in plant fibers, and limited contact with extraction medium leads to the problems of low extraction efficiency, waste of medicinal materials and environmental pollution. In order to solve these problems, cellulase extraction has been widely used in the extraction of active components of natural medicine in recent years. Using cellulase extraction, Li Yongshengetal.[11]dissolvedA.carmichaeliipowder in water and added with cellulose to extract aconitines such as bullatine G; the optimum pH, temperature, enzyme dosage, enzymolysis time were 4.5, 45 ℃, 8 mg/g, and 4 h, respectively. Cellulase extraction has the advantages of simple process, convenient operation, mild conditions, short action time and high extraction efficiency, and has good application potential. However, the method also has limitations. As proteins, enzymes are easily affected by environmental conditions. When the temperature, pH and other environmental factors are not suitable, the enzyme activity will reduce or lose, which seriously affects the extraction effect.

3 Content determination

The content of active component or indicative component is an important index in the quality control of traditional Chinese medicine, and an important means to evaluate the quality of medicine, ensure the efficacy of medicine and guarantee the safety of clinical medication. Content determination methods can be divided into two categories: chemical analysis methods and instrumental analysis methods. However, traditional Chinese medicine consists of many complex chemical components, and there may be many similar substances[12]. Chemical analysis methods can not meet the purpose of determining the content of a single chemical component, so instrumental analysis methods are more commonly used. Compared with chemical analysis methods, instrumental analysis methods have the advantages of simple operation and good repeatability, and improve the sensitivity and accuracy of content determination. At present, UV-spectrophotometry and chromatography are the most common instrumental analysis methods.

3.1 UV-spectrophotometryLiu Shuminetal.[13]determined the content of alkaloids such as bullatine G in differentAconitumplants in Xinjiang via UV-spectrophotometry; the plants such asA.leucostomumwere ground into power and soaked in acetate overnight, and the sample solution was obtained after being extracted by trichloromethane and filtered; the mass concentrations and absorbance of samples and the reference were determined at the wavelength of 415 nm, and the regression equation was drawn to obtain the content of samples. The results showed that the drug content ofA.leucostomumandA.soongaricumwas significantly higher than that of otherAconitumplants, which was in line with the national regulations. The method is characterized by simple operation, accurate results and good repeatability. Long Peixiaetal.[14]determined the content of alkaloids such as bullatine G in Aconiti Lateralis Radix Praeparata by first derivative-ultraviolet spectrophotometry; the powder of Aconiti Lateralis Radix Praeparata was prepared and dissolved in ammonia and ethanol solutions, and the sample solution was obtained by ultrasonic extraction and filtration; the ultraviolet absorption spectra and first derivative spectra of sample solution, reference solution and negative solution were drawn at the wavelength of 241 nm. The results showed that the method had similar results with UV-spectrophotometry, but the content was measured directly without separation, with less sampling volume and easier operation[14].

3.2 ChromatographyCompared with UV-spectrophotometry, chromatography is featured by high separation efficiency, wide application range and high sensitivity. According to the state of mobile phase, chromatography can be divided into four categories: gas chromatography, liquid chromatography, electrochromatography and supercritical fluid chromatography. At present, liquid chromatography is often used to determine the content of bullatine G. Wei Yingetal.[15]determined the content of bullatine G in different parts ofA.soongaricumby high performance liquid chromatography (HPLC); the roots ofA.soongaricumwere crushed and immersed in a mixed solution of water and cyclohexane to remove impurities, and the solution was successively extracted and concentrated with ethyl acetate solution and 90% ethanol to obtain the sample solution; the sample solution and mixed reference solution were determined by chromatograph at the wavelength of 235 nm; the results showed that the content of bullatine G in ethanol extract was 54.868 mg/g, significantly higher than that in ethyl acetate extract. These results indicate that the chemical components ofA.soongaricumvary among different parts, and ethanol extraction from the root ofA.soongaricumcould improve the extraction efficiency and drug utilization rate of bullatine G.

3.3 Near infrared spectroscopy (NLR)With the development of industrial technology and the deepening of research, researchers have incorporated many new technologies into the methods for content determination of traditional Chinese medicine, which improve the efficiency and reliability of determination results. Deng Fangetal.[16]detected the content of diester alkaloids such as bullatine G in Aconiti Lateralis Radix Praeparata and its processed products by NLR; the processed products of Aconiti Lateralis Radix Praeparata were ground into power, treated by ultrasonic sound and filtered to get the sample solution; the sample solution and reference solution were collected to draw NLR spectrum, and a quantitative model was established to determine the drug content; the results showed that there was no significant difference between the drug content determined by NIR and the actual drug content determined by HPLC. The method can effectively shorten the detection time and facilitate rapid analysis of a large number of samples. As a novel detection method, NIR has the advantages of high accuracy, good repeatability and small pollution, and can effectively reduce the detection cost and improve the detection efficiency.

3.4 Thin layer chromatography (TLC)Zhang Fanetal.[17]determined the content of bullatine G inA.soongaricumby TLC; the root, stem and leaf powder ofA.soongaricumwere dissolved in ethanol, made into extract and dissolved in distilled water before being extracted by chloroform; the chloroformic solution was combined and chloroform was recovered by a rotary evaporator until dry; a small amount of chloroform was dissolved to obtain the sample solution; the content of sample solution was determined by using the proportioning system of petroleum ether-acetone-aqueous ammonia and chloroform-methanol-aqueous ammonia on TLC; the results showed that the content of bullatine G in the root ofA.soongaricumreached up to 3.95 mg/g, with good separation effect and no trailing phenomenon. TLC has the advantages of simple operation, convenient and accurate results, and can effectively improve the detection efficiency.

3.5 High performance liquid chromatography-mass spectrometry (HPLC-MS)Jiang Dangetal.[18]determined the content of bullatine G in Aconiti Lateralis Radix Praeparata from different producing areas by HPLC-MS; Aconiti Lateralis Radix Praeparata was dried and crushed, dissolved in methanol solution and then filtered to obtain the sample solution of Aconiti Lateralis Radix Praeparata; the sample solution and mixed reference solution were determined by chromatograph; the results showed that there were significant differences in the content of bullatine G in Aconiti Lateralis Radix Praeparata from different producing areas, among which the content of bullatine G in Aconiti Lateralis Radix Praeparata from Sichuan Jiangyou producing area was the highest, more than twice of that in Aconiti Lateralis Radix Praeparata from Shaanxi Chenggu producing area. In order to improve drug utilization rate and extraction efficiency, Aconiti Lateralis Radix Praeparata from Sichuan Province can be selected for future extraction of bullatine G. HPLC-MS effectively combines the strong separation performance of high performance liquid chromatography with the strong identification performance of mass spectrometry, with good separation effect, simple method and good repeatability, providing a new idea for the development of content determination technology of bullatine G.

4 Conclusions and prospect

There is still a large space for the research of purification process and content determination technology of bullatine G. In order to ensure the extraction efficiency and product quality, it is necessary to carry out in-depth research on the purification process and content determination technology of bullatine G with the help of modern technologies and equipments. Only by establishing a perfect theoretical method and mature technical system can the current technical bottleneck be overcome, so as to further improve the application value of bullatine G.