How to Seperate Ginsenoside from Ginseng Extract?

Ginseng is the dried root of the plant Panax ginseng C.A. Mey, which is mainly produced in the Changbai Mountains of Jilin, Liaoning, Heilongjiang, Hebei, Shanxi and other places in China. It is a traditional and precious Chinese medicinal herb. Modern research has shown that more than 40 ginsenoside monomers have been isolated and identified in ginseng, followed by ginseng polysaccharides, amino acids, proteins, ginseng diol, ginsenoside, and other active ingredients. Among these, ginsenosides are one of the main active ingredients in ginseng, and have pharmacological activities such as protecting heart function, lowering blood sugar, anti-oxidation, anti-fatigue, and anti-tumor [1-3]. Selecting a reasonable extraction and separation method to obtain high-quality ginsenosides has become a research hotspot.

According to literature reports [4-5], traditional extraction and separation methods, such as decoction, percolation, Soxhlet extraction, and column chromatography, have played a significant role in the development of the traditional Chinese medicine pharmaceutical industry. However, these methods all have problems to varying degrees, such as long extraction cycles, high loss of effective ingredients, and low extraction efficiency. With the continuous development of modern science and technology, many new extraction and separation techniques have emerged, such as supercritical carbon dioxide extraction technology, microwave-assisted extraction technology, and ultrasonic extraction technology [6-7]. The use of these techniques not only reduces production costs, but also improves yields, providing technical guidance for the industrialization, precision, and automation of ginseng.

1 Extraction methods

In order to solve the many problems in the extraction of ginseng, in recent years, research on new techniques for extracting traditional Chinese medicine has been very active in China, and considerable progress has been made. These techniques not only extract the maximum amount of ginsenosides from ginseng, but also avoid problems such as the loss of ginsenosides and the dissolution of inactive ingredients.

1.1 Microwave extraction method

Microwave extraction has the advantages of simple equipment, time saving, high extraction rate, low investment, solvent saving and low pollution. Liu Yonglian [8] et al. used microwave extraction to extract ginsenosides from dried American ginseng roots, and found that the yield of ginsenosides was as high as 5.53%, which was 29% higher than that of ethanol reflux extraction, and the extraction time was 2% of that of ethanol reflux.

Another experiment [9] confirmed that the extraction rate of ginsenosides by microwave extraction was 8%, which was 2.67 times that of the conventional reflux method. Zhang Jing et al. [10] used microwave extraction to extract ginsenosides with an extraction rate of 5.25%, which was 1.67 times that of the conventional reflux method. Song Yahui [11] and others used microwave extraction to extract ginsenosides, and the results confirmed that the extraction rate of ginsenosides under this method was about 8%, while the reflux method was 3.27%. It is worth noting that microwave extraction is only suitable for heat-stable products. For heat-sensitive substances, microwave heating can lead to denaturation or even inactivation of these ingredients.

1.2 Ultrasonic extraction method

Ultrasonic extraction method uses a small amount of solvent, has high extraction efficiency, and does not affect the activity of ginsenosides. Ji Xiaohui [12] and others used ultrasonic extraction to extract ginsenoside Re from the stems and leaves of American ginseng, and the extraction rate was 2.77%, which is about 1.2 times that of the conventional water extraction method. Zheng Yi [13] and others used an ultrasonic method to extract ginsenosides, with an extraction rate of 8.13%, which is much higher than the 5.01% of the traditional extraction method. Jin Daming [14] and others used an ultrasonic method to extract ginsenosides, using a central composite design method to determine the optimal extraction conditions: ethanol concentration 64%, ultrasonic time 108min, solvent ratio 26mL / g. The total ginseng saponin extraction rate under these conditions was 5.23%, which confirmed that this method has the advantages of high extraction rate and low energy consumption compared to other traditional methods.

1.3 Supercritical fluid extraction method

Supercritical fluid extraction technology is a new extraction method that is non-toxic, does not use residual solvents, is low-cost, and saves energy. Zhang Le [15] used supercritical fluid extraction technology to extract ginsenosides due to their low polarity. The extraction rate of ginsenosides was found to be about 2.76%, which was slightly lower than the conventional reflux extraction method (3.26%). Although this method is more difficult to extract highly polar saponins, it has the advantages of less pollution and no solvent residue when extracting rare saponins with low polarity, which is environmentally friendly and incomparable to the conventional reflux method.

Jiang Xiaoqing [16] and others used supercritical fluid extraction technology to extract ginsenosides Rh1 and Rh2 from ginsenosides. The results showed that the yields of ginsenosides Rh1 and Rh2 were 7.33% and 14.69%, respectively, which were higher than those of the traditional reflux extraction method. Another experiment proved [17] that after a specific surfactant was introduced into the extraction system, the extraction rate of ginsenosides reached 15.9%, which is 13.3 times higher than that without the addition of surfactant. Although this technology has the advantages of low-temperature operation, rapidity and environmental protection, it has problems such as high equipment investment, high production costs and safety. Therefore, attention should be paid to these problems when promoting and applying the method.

1.4 Enzyme extraction method

Enzyme hydrolysis is a new technology used in recent years for the extraction of active ingredients from natural plants. The use of the right enzyme can gently break down plant tissue, accelerate the release of active ingredients, and thereby increase the extraction rate [18]. Zhang Ying [19] and others have demonstrated that the extraction of ginseng after treatment with laccase from the basidiomycete Trametes versicolor can significantly increase the extraction rate of total ginsenosides. This method improves the extraction rate by 65.31% compared to water extraction. Wang Ye [20] and others found that the enzymatic hydrolysis of lac enzyme increased the extraction rate of ginsenoside Re to 0.511%, which was 90.0% higher than the traditional heating reflux method. Wu Qing [21] and others used the cellulase method to extract ginsenosides from ginseng leaves and found that the ginsenoside extraction rate was as high as 6.29%. Although the enzymatic extraction method has the advantages of high catalytic efficiency and mild catalytic conditions, this technology has high requirements for enzymes and production conditions. Therefore, in future research work, it is necessary to strengthen the control of the generated products and establish the screening of special active enzymes.

1.5 Biomimetic extraction method

Bionic extraction [22] simulates the digestion and operation of the human gastrointestinal tract, using acidic water and alkaline water of different pH to extract in sequence to obtain a bionic extract. Since the extraction of ginsenosides is mainly based on the principle of “like dissolves like”, the extraction solvent and conditions are very different from the physiological conditions of the human digestive system, so that the saponin components are generally effective in vitro, but will become ineffective once they enter the human body. Based on this phenomenon, Chen Xin [23] used a biomimetic solvent and water as extraction solvents to extract ginsenosides, and confirmed that the yield of ginsenosides extracted by biomimicry was 61.31%, which was higher than the yield of 54.26% obtained by the water extraction method. Although this method has the characteristics of high extraction rate, short production cycle, and does not change the original function of traditional Chinese medicine, it is still a thermal extraction method at present, which has a certain impact on heat-sensitive active ingredients. Therefore, attention should be paid to the protection of some heat-sensitive active ingredients when using this technology.

1.6 Other methods

In recent years, due to the rapid development of ginsenoside extraction technology, many techniques have emerged in addition to the above extraction techniques. For example, the two-phase extraction method: it is a new extraction technique that uses the difference in the distribution of substances in two phases for extraction. Zhang Ru et al. [24] used the two-phase extraction method to extract ginsenosides from ginseng roots, and found that the recovery rate of ginsenosides in this system was higher than that of the traditional extraction method. Maceration method: Since ginsenosides are highly soluble in water, they can be extracted from all ginseng saponins. Zhang Chunhong [25] et al. used the maceration method to extract ginsenosides with an extraction rate of 8.33%.

Another experiment [26] proved that after ginseng was soaked for 48 hours using the maceration method, the extraction rates of ginsenosides Rb1, Rg1, and Re were 2.906%, 0.2450%, and 1.3420%, respectively. Reflux method: Wu Zhengzhong [27] and others used a reflux method to extract ginsenosides, with a total ginsenoside extraction rate of 5.52% and a total ginsenoside Rg1 and Re extraction rate of 0.2473%, which is higher than the traditional maceration method. High-pressure extraction method: Chen Ruizhan [28] and others used high-pressure extraction to extract ginsenosides from ginseng, and the results proved that the yield of ginsenosides extracted by high-pressure extraction was 7.76%, which was much higher than that of the traditional extraction method.

2. Methods for separating and purifying the monomers

Ginseng saponins are chemically unstable and are easily hydrolyzed in the presence of enzymes and acidic conditions. Currently, the methods for separating and purifying the ginseng saponin monomers include the separation and purification methods using macroporous adsorption resins, the high-speed countercurrent chromatography separation method, and the foam flotation separation method. These methods have the advantages of high separation and purification, good separation effect and fast speed, and have broad application prospects.

2.1 Macroporous adsorption resin separation and purification method

Xie Liling [29] et al. studied the purification process of ginseng total saponins through macroporous adsorption resin, and found that the total extraction rate of ginsenosides Rg1, Re and Rb1 obtained after separation and purification by macroporous resin was 0.989%. Another report [30] also demonstrated that the purity of ginsenosides extracted using weakly polar macroporous resin can reach more than 60%. Cai Xiong [31] and others confirmed that the elution rate of ginsenosides after enrichment and purification using macroporous resin was more than 90%. Liu Jihua [32] and others confirmed the use of macroporous adsorption resin to extract total ginsenosides from American ginseng pulp, and the total ginsenoside content exceeded 50%. Sun Chengpeng [33] and others used D101C macroporous adsorption resin to separate and purify total ginsenosides from ginseng roots, and the separation purity reached 94.62%. Although this method has a high separation purity, it also has certain limitations in application, and the separation targets are mainly concentrated on ingredients such as saponins and alkaloids. In application, a detection method for resin residues and cracking products should also be established, and reasonable limit standards should be formulated.

2.2 High-speed countercurrent chromatography separation method

High-speed countercurrent chromatography is a new separation technique that has been developed in recent years. It can separate more than 90% of the sample. It has the advantages of large preparation volume, good separation effect, and fast speed. Zhang Min [34] and others used high-speed countercurrent chromatography to separate Re, Rg1, and Rg3, three ginsenoside monomer compounds, and the purity was detected by HPLC to be more than 95%. In addition, according to a literature report [35], ginsenosides Rg1, Rf and Rd were prepared using the same method, and their purities were 96.2%, 94.3% and 95.1% respectively as determined by HPLC. This confirmed that high-speed counter-current chromatography is simpler and faster than conventional column chromatography, with better practical application value.

2.3 Foam flotation separation method

The foam flotation separation method is a technique that uses the difference in adsorption of substances on the surface of bubbles to separate and purify. It has the characteristics of high enrichment and no need for organic solvents. Wang Yutang [36] and others used dynamic foam flotation to separate and enrich diol-type ginsenosides in ginseng water extract. The results showed that the enrichment efficiency of dynamic foam flotation for ginsenosides Rb1, Rc, Rb2 and Rd was better than that of other methods, with recovery rates of 93.3%, 98.6%, 96.9% and 98.3%, respectively. The presence of surface-active ingredients in the solution is one of the necessary conditions for foam separation. Ginseng saponins have surface-active properties, and can produce stable foam when stirred or aerated, which makes ginseng water extract suitable for foam separation [37]. It can be seen that the use of foam flotation separation can also effectively increase the enrichment factor of ginseng saponins and improve the yield of ginseng saponins.

3. Combined use of multiple technologies

With the rapid development of modern extraction and separation technologies, the combined use of multiple technologies has gradually penetrated into the ginseng industry. The combined use of technologies can be targeted at the characteristics of ginseng extracts, and the combination of technologies can be carried out in an integrated manner to give full play to their respective advantages, complement each other's deficiencies, and expand their respective application scopes. This allows for the extraction of ginsenosides with higher yields in a shorter time, with lower energy consumption, and at a faster rate, and has broad application prospects.

3.1 Ultrasonic-enhanced supercritical fluid extraction method

Ultrasonic-enhanced supercritical fluid extraction technology is a combined technology that enhances the ability of supercritical fluid extraction to separate effective substances in traditional Chinese medicine through an ultrasonic field. This technology has the characteristics of reducing extraction pressure and temperature, shortening extraction time, reducing energy consumption, reducing fluid flow, and high extraction rate. Luo et al. [38] used ultrasonic-enhanced supercritical fluid extraction to extract ginsenosides and analyzed the extraction rate of ginsenosides before and after the addition of ultrasound. The results showed that the extraction rate of ginsenosides was 8.06% before the addition of ultrasound. Under the optimized conditions after the addition of ultrasound, the ginsenoside extraction rate reached 13.20%. It can be seen that the addition of ultrasound can significantly improve the extraction rate and production efficiency of supercritical CO2 extraction of ginsenosides.

3.2 Ultrasonic-silica gel column chromatography

Wang Lele [39] and others used a combined ultrasonic-silica gel column chromatography technique to separate and purify ginsenoside Rg1. The results confirmed that this method can be used to separate 50 g of ginseng to obtain about 9.91 g of ginsenoside Rg1 with a purity of 89.63%. This method is accurate, low-cost, and the purity of the product obtained is high. and can be used as an effective method for obtaining high-quality ginsenoside Rg1. This method not only retains the advantages of the ultrasonic method, such as being simple to operate, short in time, and high in yield, but also retains the advantages of the classical silica gel column chromatography technique for further separating and purifying ginsenoside monomers and improving their purity. Therefore, the combined ultrasonic-silica gel column chromatography technique is also an effective way to improve the yield and purity of ginsenosides.

3.3 Macroporous adsorption resin-silica gel column chromatography

In order to obtain ginsenoside Rd, Wang Yan [40] and others used macroporous adsorption resin technology to extract total ginsenosides from 1 g of ginseng, and then separated ginsenoside diol to obtain ginsenoside Rd. The ginsenoside diol was then separated using silica gel column chromatography to obtain relatively pure ginsenoside Rd. The result was relatively pure ginsenoside Rd 500 mg, with a yield of 50% and a purity of 98%. This method takes advantage of the characteristics of the two methods for ginseng extract, and combines the two methods in an integrated manner to give full play to their respective advantages, complement each other's deficiencies, expand their respective application scopes, and improve the purity of ginsenosides.

4 Conclusion

Ginsenosides are one of the main active ingredients in ginseng, with good pharmacological activity and clinical medicinal value. There is a large market demand for them, so there is increasing emphasis on how to efficiently extract high-quality ginsenosides. In recent years, with the continuous introduction and development of new technologies in the field of traditional Chinese medicine, some results have been achieved in the extraction and separation of ginsenosides. This paper compares the commonly used methods for extracting ginsenosides from ginseng. It shows that these new technologies have the advantages of being highly targeted, having high yields, causing little loss of ingredients and low energy consumption.

However, they also have their own limitations. Different extraction methods focus on the crude extraction of ginsenosides, and the purity of the extracted ginsenosides is not high. The method of separating and purifying the monomers can make up for the shortcomings of the above extraction methods. However, no matter which method is used to extract and separate ginsenosides, the purity cannot be maximized. Only by combining technologies in an integrated manner, giving full play to their respective advantages and complementing each other's deficiencies, can the scope of application and effect of each be expanded. From the current research, the combined use of multiple technologies is mostly at the laboratory research stage. There are still many technical issues to be resolved in order to apply it to the production of ginseng preparations. Research institutes and enterprises need to work together to improve the intrinsic quality of ginseng and continue to explore and develop new technologies so that they can be widely used in ginseng production and play a role in the modernization of the ginseng industry.

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