TWI453215B - Method of seperating triterpenoid compounds from fungi and use of molecularly imprinted polymer - Google Patents

Description

Method and use of molecular typographic polymer for separating triterpenoids in fungi

The invention relates to a method for separating triterpenoids in fungi and the use of the molecular printing polymer, in particular to a method and a use for separating triterpenoids in fungi by molecular printing polymers.

Antrodia cinnamomea is a unique medicinal fungus in Taiwan. It is only parasitic on the old burdock of Taiwanese mountains at an altitude of 450-1500 meters. It is also known as the "ruby of Taiwan forest". The active constituents of Antrodia camphorata include polysaccharides, triterpenoids, steroids, superoxide dismutase (SOD), and other important nutrients such as adenosine and small Molecular proteins (immunoglobulins), vitamins (such as vitamin B, niacin, ergosterol), trace elements (calcium, phosphorus, strontium, selenium, iron, etc.), nucleic acids, etc., are extremely promising.

In the study, it was found that the triterpenoids are the most important chemical constituents of Antrodia camphorata, which is also the source of bitter ingredients in the extract of Antrodia camphorata. However, the fruiting bodies produced by the mycelium grown in different wood species also contain different levels and types of triterpenoids. Also in many fungi, such as Ganoderma lucidum, Brazilian mushrooms, Triterpenoids are also contained in mulberry yellow or the like, but the content and type of triterpenoids are different. In general, triterpenoids are roughly classified into ergostane and lanostane due to their basic structure, and different types of triterpenoids have metabolic pathways and effects in humans. Different.

In the past studies, different extraction methods, such as thermal reflux, microwave-assisted extraction, supercritical fluid extraction or other similar methods, were used to obtain the extract of Antrodia camphorata, and the content of triterpenoids was analyzed by HPLC. However, these methods do not separate specific types of triterpenoids. Therefore, the development of a method for highly isolating various triterpenoids can contribute to the study of the physiological mechanism of triterpenoids.

In view of the above-mentioned problems of the prior art, it is an object of the present invention to provide a method for isolating a triterpenoid in a fungus comprising: freeze-drying and grinding the fruiting body of the fungus; and powdery fruiting body Adding 10%~100% of the first solvent, and extracting at a temperature of 10 ° C ~ 100 ° C for a first predetermined time to obtain an extract, wherein the solid-liquid ratio of the powdered fruit body to the first solvent is 1/10~1/200; adding a triterpenoid molecularly imprinted polymer to the extract for a second predetermined time, and then adsorbing the triterpenoids in the extract at an adsorption rate of 40% to 90% a compound; and a second solvent added to separate the triterpenoid and the triterpenoid molecularly imprinted polymer.

Preferably, the fungus comprises Antrodia camphorata, Ganoderma lucidum, Brassica oleracea, mulberry yellow or any fungus comprising a triterpenoid.

Preferably, the triterpenoid compound may comprise an ergostane triterpenoid such as squalene, lanostane triterpenoids such as glycyrrhetinic acid (18- --glycyrrhetinic acid) or a compound thereof.

Preferably, the triterpenoid compound may further comprise antinc C, antcin K, zhankuic acid A, zhankuic acid B, 樟Japanese acid C (zhankuic acid C), sulphurenic acid, dehydroeburicoic acid, dehydrosulphurenic acid, or eburicoic acid.

Preferably, the extraction method may include ultrasonics-assisted extraction (UAE), heating reflux extruction (HRE), microwave-assisted extraction (WAE), supercritical fluid extraction Supercritical fluid (SFE) or the like.

Preferably, the first predetermined time may be from 0.05 to 1.5 hours, and the second predetermined time may be from 0.5 to 2.5 hours.

Preferably, the triterpenoid molecularly-printed polymer can adsorb the triterpenoid by non-covalent bonding, and the non-covalent bond can comprise a hydrogen bond, an ionic bond, a hydrophobic force, a van der Waals force or It is similar to a bond.

Preferably, the first solvent used in the process may be ethanol and the second solvent may be methanol, chloroform, or acetone.

According to another embodiment of the present invention, there is provided a use of a molecular printing polymer It can be used to isolate triterpenoids from fungal extracts.

Preferably, the molecularly-printed polymer adsorbs the triterpenoids in the fungal extract at an adsorption rate of 40% to 90%.

Preferably, the use further comprises a solvent to separate the triterpenoid compound adsorbed on the molecular rubbing polymer, wherein the solvent may be methanol, chloroform, acetone or any of the triterpenoids and has high polarity. Solvent.

Preferably, the fungal extract can be extracted from Antrodia camphorata, Ganoderma lucidum, Brassica oleracea L., Mulberry yellow or any fungus comprising the triterpenoid compound.

According to the method of the present invention for separating a triterpenoid in a fungus, it may have one or more of the following advantages:

(1) A method for separating a triterpenoid in a fungus according to the present invention, which provides an optimum extraction condition, and which can obtain an extract having a high triterpenoid content.

(2) A method for separating a triterpenoid in a fungus according to the present invention, wherein the molecular rubbing polymer is produced by using a triterpenoid as a template, and the concept is similar to a lock and a key, and thus has a high degree of specificity .

(3) A method for separating a triterpenoid in a fungus according to the present invention, wherein the molecular typographic polymer and the triterpenoid are adsorbed by non-covalent bonding, and thus can be easily adsorbed by a solvent The target compound is isolated.

S10~S40‧‧‧Steps

Figure 1 is a flow diagram of a method of isolating triterpenoids in fungi according to an embodiment of the present invention.

Figure 2 shows the triterpenoids extracted at different concentrations of ethanol at different times. The result of the content.

Figure 3 is a schematic diagram showing the content of triterpenoids in the residue of Antrodia camphorata after extraction with different concentrations of ethanol.

4A to 4D are HPLC chromatograms of extracts extracted under different conditions.

Figure 5 is the result of the ability to remove DPPH by extracts of different concentrations of ethanol.

Fig. 6A to Fig. 6C are graphs showing the influence of the content of triterpenoids in the extract extracted under different conditions.

Figure 7 is a bar graph of the adsorption of triterpenoids in different extracts of different molecularly imprinted polymers.

The invention will be further described in detail by the following preferred embodiments and the accompanying drawings. It should be noted that the experimental data disclosed in the following embodiments are for explaining the technical features of the present invention, and are not intended to limit the manner in which they can be implemented.

Please refer to Fig. 1, which is a flow chart of a method for isolating triterpenoids in fungi according to an embodiment of the present invention. First, the body body of Antrodia camphorata is freeze-dried and ground into a powder (step S10). In the present embodiment, the experiment is carried out using the example of Antrodia camphorata. However, the embodiment of the present invention is not limited thereto, and may include any fungus containing a triterpenoid compound such as ganoderma lucidum, Brazilian mushroom, and mulberry yellow.

Next, the extraction is carried out with a fixed concentration of an organic solvent. Here, ethanol is taken as an example, but the embodiment is not limited thereto, and may include various organic substances having the same efficacy. Solvent. In order to test the optimal extraction conditions, one aspect of the present invention is extracted with 0%, 50%, 95% ethanol for 10, 20, 30, 40, 50, 60 minutes, respectively, and then tested. The content of crude triterpenoids in the extract of the conditional extraction, wherein 0% ethanol is replaced by pure water, and the test results are shown in Fig. 2 and Fig. 3.

Figure 2 shows the results of extracting triterpenoids at different times for different concentrations of ethanol. Figure 3 is a schematic diagram showing the content of triterpenoids in the residue of Antrodia camphorata after extraction with different concentrations of ethanol. It can be seen from Fig. 2 that after 10 minutes of extraction, the concentration of triterpene extracted by 0% ethanol is about 0.83%, and as the concentration of ethanol increases, the content of extracted triterpenoids increases to 2.16. % and 3.09%. After 60 minutes of extraction, the triterpenoids extracted by 0% ethanol were about 1.93%, and the triterpenoids obtained after 50 minutes of extraction of 50% and 95% ethanol were about 3.27% and 4.19%.

Referring also to Fig. 3, the content of triterpenoids in the residue after extraction with 0% ethanol is about 2.93%, and the content of triterpenoids in the residue after extraction with 50% and 95% ethanol is about 1.47% and 0.97%. The higher the concentration of ethanol, the greater the amount of triterpenoids that can be extracted over the longer period of time.

However, the types of triterpenoids contained in the extract extracted by the above conditions may differ. Therefore, in the following, high pressure liquid chromatography (HPLC) will be carried out for extracts extracted with different concentrations of ethanol at different times, thereby discussing different concentrations of ethanol and different extraction times for inclusion therein. The effects of the triterpenoids and the species are shown in Figures 4A through 4D.

Figures 4A to 4D are HPLC chromatograms of extracts extracted under different conditions. Figure 4A is a schematic diagram of HPLC chromatogram analysis of extracts extracted with 0% ethanol for 10, 30, and 60 minutes, respectively. Figure 4B is a schematic diagram of HPLC chromatogram analysis of extracts extracted with 50% ethanol for 10, 30, and 60 minutes, respectively. Figure 4C is a schematic diagram of HPLC chromatogram analysis of extracts extracted with 95% ethanol for 10, 30, and 60 minutes, respectively. Fig. 4D is a schematic diagram of HPLC chromatogram analysis of extracts extracted with 0, 50, and 95% ethanol for 60 minutes, respectively.

From Fig. 4A to Fig. 4C, it can be seen that the extraction of the triterpenoids in the extract increases with time, regardless of the concentration of ethanol. And as shown in Fig. 4D, the triterpenoids detected in the extract extracted with 0% ethanol were less than those extracted with 50 or 95% ethanol under extraction for 60 minutes. liquid. From the above results, it is known that triterpenoids are easily extracted by high concentration of ethanol and may be affected by the extraction time.

However, in order to investigate whether the physiological activities of triterpenoids extracted by different concentrations are the same, in this embodiment, the ability of triterpenoids to scavenge free radical DPPH (α,α-diphenyl-β-picrylhydrazyl) is taken as an example. Test, the results are shown in Figure 5. However, the present embodiment is not limited thereto, and it can be tested by the physiological effects of various triterpenoids.

Figure 5 is the result of the ability of extracts extracted with different concentrations of ethanol to remove DPPH. Among them, the extracts extracted with different concentrations of ethanol were concentrated under reduced pressure to a paste and diluted to a concentration of 0, 0.01, 0.05, 0.1, 0.5, 1 mg/ml, respectively. Then, 4 mL each was added to 1 mL of freshly prepared 10 mM DPPH solution, shake-mixed, allowed to stand at room temperature for 30 minutes, and the absorbance at 517 nm was measured using a spectrophotometer. Among them, the group in which vitamin C was added was used as a positive control group. It can be seen from Fig. 5 that at a concentration of 1 mg/ml, the extract of Antrodia camphorata extracted from 0% ethanol has the ability to remove DPPH only. 50.13%, and the DPPH ability of the extract of Antrodia camphorata extract extracted with 50% and 95% ethanol was 88.51% and 91.05%, respectively.

From the above results, it was found that the extraction of the higher concentration of ethanol over a longer period of time can obtain an extract having a higher triterpenoid content and type, and the extracted extract does have physiological activity such as scavenging DPPH. Next, the optimum conditions for the ultrasonic extraction method will be discussed by combining different concentrations of extraction solvent, solid-liquid ratio, and temperature.

Common extraction methods include heat reflux extraction (HRE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), and ultrasonic extraction (Ultrassonics-Assisted). Extraction, UAE) or a similar method. In the aspect of the present embodiment, the ultrasonic extraction method will be exemplified, but it is not limited thereto, and it may include any of the above extraction methods.

The principle of the ultrasonic extraction method is to use the vibration generated by the ultrasonic wave to cause the liquid to crack and generate a cavitation phenomenon, which affects various physical or chemical actions in the liquid to cause the cell to rupture, thereby releasing the components in the cell. In order to achieve the extraction effect.

In the present embodiment, the three-layer three-variable Box-Behnken experimental design was carried out with different alcohol concentration, solid-liquid ratio and temperature, and the results showed that the maximum triterpenoid content extract (extraction rate: 3.26±0.19) %) was obtained at a solid-liquid ratio of 1/100 g/ml, a temperature of 75 ° C, and an ethanol concentration of 75%. The minimum triterpenoid content extract (extraction rate: 0.48 ± 0.19%) was obtained at a solid-liquid ratio of 1/100 g/ml, a temperature of 75 ° C, and an ethanol concentration of 25%.

Then bring the above 15 experimental results into the statistical analysis software (SAS) for the reaction surface Regression analysis, the results are shown in Figures 6A to 6C. Figure 6A shows the effect of 0% solid-liquid ratio and temperature on the content of triterpenoids in the extract. Figure 6B is a graph showing the effect of solid-liquid ratio and ethanol concentration on the content of triterpenoids in the extract at a fixed temperature of 50 °C. Figure 6B is a graph showing the effect of ethanol concentration and temperature on the content of triterpenoids in the extract at a fixed solid-liquid ratio of 1/100 (g/ml). Among them, in the 6A to 6C, the darker red color indicates the higher the content of the triterpenoid compound, and the greener the color, the lower the triterpenoid content. The values of the triterpenoids corresponding to the colors in the figure are shown in the figure.

As shown in Figure 6A, the increase in solid-liquid ratio is not necessarily proportional to the extraction effect. Too high a ratio will result in a decrease in the content of the extracted triterpenoids, and a low ratio will result in a triterpenoid due to too little fruiting body powder. The content is reduced. However, the extraction temperature will affect the extraction effect, and the excessive extraction temperature will reduce the content of the triterpenoids. As shown in Figure 6B, as the concentration of ethanol increases, the extraction rate of triterpenoids also increases, mainly because triterpenoids are fat-soluble compounds, so as the concentration of ethanol increases, the better The fat-soluble extraction effect. It can also be seen in Figure 6C that the higher the ethanol concentration, the better the extraction effect, but the too high extraction temperature will result in a decrease in the content of triterpenoids.

In summary, the ethanol concentration plays an important role in the extraction of triterpenoids. The high or low solid-liquid ratio and temperature connection have a negative effect on the extraction of triterpenoids. The optimum extraction conditions analyzed by the above reaction surface map are shown in Table 1.

As can be seen from Table 1, the optimum ultrasonic extraction conditions are solid-liquid ratio: 1/104.81 g/ml, temperature: 60.41 ° C, and ethanol concentration: 74.38%.

Next, referring back to FIG. 1, in the method for separating a triterpenoid in a fungus according to the present invention, the step S20 is to add 10% to 100% of ethanol at a solid-liquid ratio of 1/10 to 1/200 g/ml. The ultrasonic extraction method is performed at a temperature of 10 ° C to 100 ° C for 60 minutes, wherein the solid-liquid ratio is preferably 1/104.81 g/ml, the ethanol concentration is preferably 74.38%, and the temperature is preferably 60.41 ° C.

An extract is obtained by the step S20, and then a triterpenoid molecularly imprinted polymer (MIP) is added to the extract for 0.5 hour to 2.5 hours, preferably 1 hour (step S30). Then, a solvent is added to separate the triterpenoid compound from the triterpenoid molecularly-printed polymer (step S40).

The triterpenoid molecularly-printed polymer used in the present embodiment is synthesized by using methacrylic acid (MAA) as a monomeric functional group and a target compound as a mismatch, wherein The target compound used herein is 18-β-glycyrrhetinic acid or squalene. However, the target compound is not limited thereto, and may contain various triterpenoids such as antinc C, antcin K, zhankuic acid A, oyster mushroom B (zhankuic acid B), zhankuic acid C, sulphurenic acid, go Dehydroeburicoic acid, dehydrosulphurenic acid, eburicoic acid, and the like. Next, ethylene glycol dimethacrylate (EDMA) was used as a crosslinking agent to polymerize into a polymer. Finally, the target substance is washed out using a suitable solvent, thereby forming a polymer having a target compound shape pore and a specific functional group recognition position.

The above-mentioned triterpenoid molecular typographic polymer and the target compound are adsorbed by non-covalent bonding, such as hydrogen bonding, ionic bonding, hydrophobic interaction, van der Waals force or the like, so The target compound can be easily separated from the molecular typographic polymer by using a solvent, whether in the manufacture of a triterpenoid molecularly imprinted polymer or after adsorption of the target compound.

In the present embodiment, the triterpenoid molecularly-printed polymer produced by the above method and the non-imprited polymer (NIP) used as a control group were separately subjected to optimization conditions. After the extracted extract was extracted and extracted for 1 hour, the adsorption results are shown in Fig. 7.

Figure 7 shows the adsorption effect of different molecularly rubbed polymers in the extract. It can be seen from Fig. 7 that the molecular printing polymer prepared by using squalene can adsorb about 42.75%±2.19 squalene in the extract, and the molecular printing polymer prepared by glycyrrhetinic acid can adsorb. About 79.35% ± 3.04 of glycyrrhetinic acid in the extract. This result may be due to the fact that squalene is a macromolecular heterocyclic triterpenoid, and therefore the adsorption process may be affected by other cyclic structures in the adsorption process. However, since glycyrrhetinic acid is a single compound, the molecularly-printed polymer produced is captured by a specific substance due to the structure of the target compound, and thus has a better separation effect.

In addition, the adsorption rate of the non-printing polymer for squalene and glycyrrhetinic acid is about 5.19%±1.31 and 3.81%±0.68, indicating that the molecular squalene and glycyrrhetinic acid molecularly-printed polymer do have an adsorption effect.

In summary, the method for separating a triterpenoid in a fungus according to the present invention and the use of the molecular typographic polymer for isolating a triterpenoid in a fungal extract can obtain a higher content of triterpenes under optimum conditions. An extract of a compound-like compound, and the triterpenoid compound can be easily separated by using a molecularly-printed polymer having highly specific properties. The triterpenoids adsorbed on the molecularly imprinted polymer by non-covalent bonding can also be easily separated. Therefore, the method for isolating triterpenoids in fungi disclosed by the present invention can be effectively applied to the research and development of fungal active ingredients.

The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

S10~S40‧‧‧Steps

Claims (14)

A method for isolating a triterpenoid in a fungus, comprising: freeze-drying one of the fungi of a fungus and grinding into a powder; adding the powdered fruiting body to one of 74.38% of the first solvent, and at 60.41 ° C An extraction method is performed at a temperature for a first predetermined time to obtain an extract, wherein a solid-liquid ratio of the powdery fruit body to the first solvent is 1/104.81 g/ml, wherein the first solvent is ethanol. Adding a triterpenoid compound molecularly imprinted polymer to the extract for a second predetermined time, and then adsorbing one of the triterpenoids in the extract at an adsorption rate of 40% to 90%; And adding a second solvent to separate the triterpenoid compound from the triterpenoid molecularly imprinted polymer. The method of claim 1, wherein the fungus comprises Antrodia camphorata, Ganoderma lucidum, Brazilian mushroom, Mulberry or any fungus comprising the triterpenoid. The method of claim 1, wherein the triterpenoid comprises an ergostane triterpenoid, a lanostane triterpenoid. The method of claim 1, wherein the triterpenoid compound comprises 18-β-glycyrrhetinic acid, squalene, antcin C, and ricinic acid. K (antcin K), zhankuic acid A, zhankuic acid B, zhankuic acid C, sulphurenic acid, dehydrogenated perforation Acid (dehydroeburicoic acid), dehydrogenated polyporic acid ( Dehydrosulphurenic acid) or eburicoic acid. The method of claim 1, wherein the extraction method comprises ultrasonics-assisted extraction (UAE), heating reflux extruction (HRE), microwave-assisted extraction (microwave-assisted) Extraction, WAE), or supercritical fluid (SFE). The method of claim 1, wherein the first predetermined time is from 0.05 hours to 1.5 hours. The method of claim 1, wherein the second predetermined time is from 0.5 hours to 2.5 hours. The method of claim 1, wherein the triterpenoid molecular typographic polymer adsorbs the triterpenoid by a non-covalent bond. The method of claim 8, wherein the non-covalent bond comprises a hydrogen bond, an ionic bond, a hydrophobic force, or a van der Waals force. The method of claim 1, wherein the second solvent comprises methanol, chloroform or acetone. Use of a molecularly-printed polymer for the separation of one of the triterpenoids in a fungal extract using the method of claim 1 of the patent application. The use according to claim 11, wherein the molecular rubbing polymer adsorbs the triterpenoid in the fungal extract at an adsorption rate of 40% to 90%. The use of claim 12, further comprising separating the triterpenoid adsorbed onto the molecularly-printed polymer by a solvent, wherein the solvent comprises methanol, chloroform or acetone. The use according to claim 11, wherein the fungal extract is extracted from Antrodia camphorata, Ganoderma lucidum, Brassica oleracea, mulberry yellow or any true containing the triterpenoid bacteria.