KR20190141433A - Ginsenosides and manufacturing method thereof - Google Patents

Abstract

Ginsenoside and a manufacturing method thereof are provided. The manufacturing method of ginsenoside includes a step of inducing a hydrothermal reaction of a ginsenoside-containing material, wherein carbon dioxide may be provided to the hydrothermal reaction. The ginsenoside may be manufactured by the method. It is also possible to manufacture ginsenoside in high yields with a short reaction time.

Description

Ginsenosides and its manufacturing method {GINSENOSIDES AND MANUFACTURING METHOD THEREOF}

The present invention relates to ginsenosides and a method of making the same.

Ginseng is widely used as a medicinal plant because of its biological activity such as immune system regulation. The bioactivity is caused by a group of compounds called ginsenosides, and the ginsenosides can be converted into converted ginsenosides having higher bioactivity. Several methods have been developed to increase the yield of the converted ginsenosides, the most typical of which is the steaming process.

Although the conversion ginsenoside yield and thus anticancer effect are improved by the steaming process, the process is operated under heterogeneous conditions, so that the reaction time is long and the yield is limited.

In order to solve the above problems, the present invention provides a method for producing high productivity ginsenosides.

The present invention provides a ginsenoside having a high bioactivity effect by the ginsenoside production method.

Other objects of the present invention will become apparent from the following detailed description and the accompanying drawings.

Ginsenoside manufacturing method according to embodiments of the present invention includes the step of hydrothermally reacting the ginsenoside containing material, carbon dioxide may be provided to the hydrothermal reaction.

Carbonic acid produced from the carbon dioxide may act as a catalyst of the hydrothermal reaction.

The hydrothermal reaction may be performed using subcritical water or supercritical water.

The ginsenoside-containing material may include ginseng extract extracted from ginseng.

It may further comprise the step of preparing the ginseng extract before the hydrothermal reaction.

The ginseng extract manufacturing step may include the step of drying the ginseng, grinding the dried ginseng into a powder, and extracting the ginseng extract from the powder.

The ginseng extract may be extracted using a volatile solvent.

Ginsenosides according to embodiments of the present invention may be prepared according to the above production method.

The ginsenoside may include ginsenosides in which intact ginsenosides are converted through at least one of deglycosylation or dehydration.

The converted ginsenosides may comprise one or more of (S) -Rg3, (R) -Rg3, Rk1, and Rg5.

According to embodiments of the present invention, ginsenosides can be prepared in high yield in a short reaction time. Ginsenosides having a large anticancer effect can be prepared according to the above production method. In addition, by introducing carbon dioxide into the hydrothermal reaction, the rate constants of the decomposition reaction of desugar and dehydrated converted ginsenosides (Rk1, Rg5) can be reduced while increasing their production rate constants. . As a result, the yield of converted saponins can be further increased. In the ginsenoside manufacturing method, ginsenoside-containing materials may be used in various forms such as ginseng extract as well as ginseng powder. In addition, the productivity of ginsenosides can be improved.

1 is a schematic diagram of the ginseng extract preparation step and hydrothermal reaction step and the conventional steaming process according to an embodiment of the present invention.
2 shows the structure of ginsenosides.
3 is a calculation of the solubility of carbon dioxide according to an embodiment of the present invention according to the temperature.
Figure 4 shows a chromatogram of the sample for each process according to an embodiment of the present invention.
FIG. 5 compares yields of samples according to processes according to the example of FIG. 4. FIG.
Figure 6 shows the yield according to the temperature of the converted ginsenosides according to an embodiment of the present invention.
Figure 7 shows the yield of ginsenosides by process according to an embodiment of the present invention.
FIG. 8 compares the characteristics of a hydrothermal process and a steaming process to which carbon dioxide is added according to the embodiment of FIG. 7.
FIG. 9 is an Arenius plot showing the decomposition of pure ginsenosides in the hydrothermal process and hydrothermal process with the addition of carbon dioxide.
10 shows the antiproliferative effect of each process according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail through examples. The objects, features and advantages of the present invention will be readily understood through the following examples. The invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments introduced herein are provided so that the disclosure may be made thorough and complete, and the spirit of the present invention may be sufficiently delivered to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

Although terms such as first and second are used herein to describe various elements, the elements should not be limited by such terms. These terms are only used to distinguish the elements from one another.

As used herein, the term "ginseng" may include not only ginseng in a narrow sense, but also red ginseng, ginseng, etc. The present invention may be applied to plants or crops including ginsenosides as well as ginseng, and the present invention. It should not be limited to this ginseng.

Ginsenoside manufacturing method according to embodiments of the present invention may include the step of hydrothermally reacting the ginsenoside containing material and carbon dioxide may be provided to the hydrothermal reaction.

The hydrothermal reaction is a reaction in the presence of hot water, especially high temperature and high pressure water, the hot water may be a good reaction medium for hydrolysis due to the high ion formation constant and low dielectric constant. Carbon dioxide provided in the hydrothermal reaction may be dissolved in water to produce carbonic acid. The solubility of the carbon dioxide can increase with pressure, and the resulting high concentration of carbonic acid can act as a catalyst. In the hydrothermal reaction provided with carbon dioxide, the carbonic acid is an environmentally friendly catalyst compared to other organic acids such as hydrochloric acid and sulfuric acid, and since the carbonic acid can be gradually removed by reduced pressure, there is no need to further neutralize with alkali. have.

The hydrothermal reaction may be performed using subcritical water or supercritical water. The critical temperature of the water used for the hydrothermal reaction is 374 ° C., the critical pressure is 22.1 MPa, and the subcritical water is a high temperature medium pressure steam (first subcritical water) above the critical temperature and below the critical pressure or below the critical temperature and saturated It may comprise a medium temperature medium pressure liquid (second subcritical water) of at least the water vapor pressure. The supercritical water may appear as a high temperature and high pressure deep steam above the critical temperature and above the critical pressure.

The supercritical water and the first subcritical water of 400 to 650 ° C. and 10 to 30 MPa may include both liquid and gaseous properties. For example, the density is about 0.03 to 0.4 times that of liquid water (1 g / cm ³ ) at room temperature, and is several tens to several hundred times larger than water vapor at 100 ° C. and 0.1 MPa. Viscosity is low at the gas level, and the self diffusion coefficient is between liquid and gas. That is, the supercritical water and the first subcritical water are highly active fluids having the same kinetic energy as gas molecules and having a density of about 1/10 of the liquid.

The second subcritical water at 100 to 350 ° C. and 0.1 to 25 MPa may have a large hydrolysis capacity as a liquid at high temperature and high pressure. The supercritical water and the subcritical water can control microscopic physical properties such as solvation structures of fluid molecules from macroscopic properties such as density and solubility by controlling temperature and pressure.

The ginsenoside-containing material may include ginseng extract extracted from ginseng. The ginseng is a representative material containing the ginsenoside, may include ginseng, dried ginseng, red ginseng. The ginseng is natural ginseng unprocessed with ginseng grown for 4 to 6 years, the dry ginseng is dried ginseng, and the red ginseng is light yellow brown or light red brown ginseng dried by steaming the ginseng in steam.

The ginsenoside manufacturing method may further include preparing the ginseng extract before the hydrothermal reaction.

1 is a schematic diagram of the ginseng extract preparation step and hydrothermal reaction step and the conventional steaming process according to an embodiment of the present invention.

Referring to Figure 1, the ginseng extract manufacturing step may include the step of drying the ginseng, grinding the dried ginseng into a powder, and extracting the ginseng extract from the powder. In addition, the extract preparation step may be performed without steaming the ginseng using steam, unlike the existing steaming process on the right.

The drying step of the ginseng may be carried out at 40 ℃ to 60 ℃, specifically, 50 ℃. The dried ginseng may be ground in a milling step and filtered through 30 to 50, specifically 40 mesh sieves. The ginseng extract may be extracted from the ground powder using a volatile solvent, for example, ethanol. For the extraction, a soxhlet method for extracting a nonvolatile component in a solid using the volatile solvent may be used. The extract may be diluted with water after the evaporation of more than two-thirds of the initial volume of the rotary evaporator to remove a volatile solvent such as ethanol, and the hydrothermal reaction step may be performed.

Ginsenosides according to embodiments of the present invention may be prepared according to the method for preparing ginsenosides. The ginsenoside is a major compound found in ginseng, a medicinal crop that has been found to have bioactivity such as immune system regulation and anticancer effects.

2 shows the structure of ginsenosides.

Referring to FIG. 2, the ginsenosides may include intact ginsenosides and converted ginsenosides. The pure ginsenosides such as Rb1, Rc, Rb2, and Rd can be present in raw ginseng and can be distinguished by the form to which the sugar moiety is attached. The pure ginsenosides can be converted to converted ginsenosides of (S) -Rg3, (R) -Rg3, Rk1, and Rg5 through deglycosylation and / or dehydration.

The (S) -Rg3 significantly lowers the growth of human gastric cancer cells compared to the pure ginsenoside, and the (R) -Rg3 has a high angiogenesis inhibitory (antiangiogenic) effect that prevents new blood vessel formation in tumors. Has In addition, Rk1 shows antitumor activity against human hepatocellular carcinoma (HepG2) cells through a decrease in telomerase activity and introduction of cell death, and Rg5 can effectively induce the death of breast cancer cells. By using the ginsenoside manufacturing method, a conversion ginsenoside having a high bioactivity effect can be prepared.

EXAMPLE

Crop Material Manufacturing

Four-year-old ginseng purchased at Gyeongdong Market (Seoul, Korea) was washed and chopped into pieces. The ginseng flakes were dried for 3 days in a 50 ℃ oven. The dried ginseng was ground and filtered through a 40 mesh sieve. The ginseng powder thus produced was stored at minus 4 ° C. In order to extract the compound from 10 g of ginseng powder, Soxhlet extraction was performed for 8 hours using 300 mL of 50% ethanol (Samjeon, Korea). The extract was concentrated to less than 30% of the initial volume of the extract using a rotary evaporator to remove the ethanol. The extract was diluted with water until a final concentration of 4% solids content was used as reactant in subsequent process steps.

[Hydrothermal reaction with added carbon dioxide (CO ₂ -assisted hydrothermal reaction]]

The hydrothermal reaction with added carbon dioxide of ginseng extract was carried out in an oil bath using a batch reactor equipped with a SUS line, a valve and a pressure gauge. After the ginseng extract was added to the reactor, liquefied carbon dioxide was injected into the reactor through the SUS line. The reaction was carried out at 100 ° C., 120 ° C., and 140 ° C. for 0.5 hours to 48 hours, 15 minutes to 6 hours and 8 minutes to 90 minutes, respectively. The reaction pressure was 20 MPa for all reactions.

To investigate the effect of carbon dioxide addition, a nitrogen-added hydrothermal reaction (N ₂ -assisted hydrothermal reaction) was performed. In the nitrogen-containing hydrothermal reaction, a high pressure gas regulator (Chiyoda Seiki Co., LTD., Kobe, Japan) was used to inject gas into the reactor. The hydrothermal reaction to which the carbon dioxide was added and the hydrothermal reaction to which the nitrogen was added were performed at 100 ° C. and 10 MPa for 1 hour.

[Control group manufacture]

As a method for pure ginsenoside conversion, a slightly modified steaming process was performed by the method of preparing red ginseng and ginseng. Freshly cut ginseng was dried in a 50 ° C. oven for 3 days. The dried ginseng was steamed at 100 ° C. and 120 ° C. to be made of red ginseng and ginseng, respectively, and then both samples were again dried in a 50 ° C. oven for 3 days. After drying, the resulting red and ginseng were ground and filtered through a 40 mesh sieve. The powder thus produced was extracted, concentrated and diluted as described previously.

[High Performance Liquid Chromatography (HPLC) Analysis Method]

For quantitative analysis, standard ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rd, and (S) -Rg3 were obtained from Chromadex (ChromaDex, Irvine, CA, USA). Rk1 and Rg5 were purchased from an embo engine (Ambo, Daejeon, Korea), and (R) -Rg3 was purchased from Sigma-Aldrich (St. Louis, MO, USA). To calculate yield of each ginsenoside, high performance liquid chromatography (HPLC) analysis was performed using an Agilent Zorbax SB-Aq column, 150 × 4.6 mm, 50 μm; Santa Clara, CA, USA. It became. Water for the high performance liquid chromatography (J.T. Baker, Center Valley, PA, USA) and acetonitrile for the high performance liquid chromatography (acetonitrile, Samjeon, Seoul, Korea) were used as the eluent. Compounds were separated at 25 ° C. using a linear gradient of water (A) and acetonitrile (B) according to the following protocol: 0-6 minutes (18-23% B), 6-48.5 minutes (23 To 40% B), 48.5 to 58 minutes (40% B), 58 to 60 minutes (40 to 100% B), 60 to 70 minutes (100% B), 70 to 71 minutes (100 to 18% B), 71 to 75 minutes (18% B), and 5 minutes (18% B) added. The flow rate was 1.0 mL / min and the injection volume was 20 μL. The compound was detected at 203 nm. Samples were filtered through 0.2 μm syringe filters (Advantec, Tokyo, Japan) before performing the high performance liquid chromatography analysis. The ginsenoside yield was calculated by the following equation (1).

Yield = ginsenoside weight (mg) / ginseng dry weight (g) (One)

[Cell Culture]

Human bone marrow-derived mesenchymal stem cells (MSCs) were purchased from Lonza, Allendale, NJ, USA. Fibrosarcoma cell line HT1080s and breast cancer cell line MDA-MB-231s were obtained from the Korea Cell Line Bank (Seoul, Korea). The cells were treated with 10% (v / v) fetal bovine serum (FBS, Gibco-BRL) and 1% (v / v) penicillin-streptomycin in a humid atmosphere at 37 ° C. with 5% carbon dioxide. Growing in high glucose Dulbecco's modified Eagle's medium (Dulbecco's modified Engle's Medium, DMEM; Gibco-BRL, Carlsbad, MD, USA) supplemented with penicillin-streptomycin, PS; Gibco-BRL. The cells were plated at 3 × 10 ³ cells / cm ² on a glass dish.

[Cell Viability and Proliferation]

Cell counting kit-8 assay (CCK-8, Sigma-Aldrich, St. Louis, MO, USA) was performed to determine the number of viable cells (n = 3 per groups) according to the protocol. Cells were treated with 0 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL of processed ginseng for 48 hours. Cell growth was compared to no untreated control using cell viability. Quantitative Real-Time PCR (qRTPCR) was performed to determine the expression level of caspase-3 in cells treated with 10 μg / mL for 48 hours as an indicator of cell death.

[result]

[Effect of Carbon Dioxide Addition on Hydrothermal Reaction]

3 is a calculation of the solubility of carbon dioxide according to an embodiment of the present invention according to the temperature.

When carbon dioxide is added to the reactor in hydrothermal reaction, it becomes carbonic acid according to the following equation (2).

CO ₂ + H ₂ O ↔ H ₂ CO ₃ ↔ H ⁺ + HCO ₃ ^- ↔ 2H ⁺ + CO ₃ ^2- (2)

The carbonic acid is a weak acid at ambient temperature and pressure. Since the solubility of the carbon dioxide increases as the pressure increases, the pH decreases and the carbon dioxide can act as a strong acid catalyst.

Referring to FIG. 3, the red line represents the solubility of the carbon dioxide in water calculated at a reaction temperature of 100 ° C., the blue line is 120 ° C., and the black line is 140 ° C., and the solubility is increased with increasing pressure. An increase was observed. During the reaction at 20MPa solubility is higher than 33 times greater than the solubility of about 0.03 molCO ₂ / kg (water) at atmospheric pressure and room temperature with 1molCO ₂ / kg (water) or more. Moreover, the carbon dioxide solubility increased with temperature rise because the hydrothermal reaction with the addition of carbon dioxide was carried out at 20 MPa after the crossover pressure. That is, reaction conversion increased based on both the temperature and the carbon dioxide solubility.

Figure 4 shows a chromatogram of the sample for each process according to an embodiment of the present invention, Figure 5 compares the yield of the sample for each process according to the embodiment of FIG.

In order to confirm the effect of carbon dioxide addition, hydrothermal reactions were carried out using carbon dioxide and nitrogen. The reaction was carried out at 100 ° C., 10 MPa for 1 hour, and the water / gas weight ratio was 4. In addition, the hydrothermal reaction was carried out at saturated vapor pressure (0.1 MPa) to determine whether the pressure is affected in a reaction involving only water.

Referring to Figure 4, (a) is a chromatogram of the raw sample, (b), (c), (d) is a hydrothermal sample using hydrothermal samples, nitrogen for 1 hour at 100 ℃, 0.1MPa respectively Chromatograms of treated samples, hydrothermally treated samples using carbon dioxide, the peaks of each chromatogram represent ginsenosides. The peak is (1) ginsenoside Rg1, (2) Re, (3) Rf, (4) Rb1, (5) Rc, (6) Rb2, (7) Rd, (8) (S) -Rg3, (9) (R) -Rg3, (10) Rk1, and (11) Rg5.

The area of the peak at the beginning of detection in the chromatogram of the raw sample was reduced after the hydrothermal reaction process, while the area of the peak at the late detection was increased significantly. This shows that during the hydrothermal reaction, the pure ginsenosides corresponding to (1) to (7) were converted to transformed ginsenosides corresponding to (8) to (11). Since the converted ginsenosides are generated through deglycosylation and / or dehydration of the pure ginsenosides, the converted ginsenosides are less polar than the pure ginsenosides. Thus, the converted ginsenosides dissolve relatively slowly and separate.

In addition, comparing the use of carbon dioxide and nitrogen, the peak area of the converted ginsenosides was greater under carbon dioxide conditions.

Referring to Figure 5, the hatched bar is a hydrothermal sample for 1 hour at 100 ℃, 0.1MPa, yellow bar is a hydrothermal sample with nitrogen for 1 hour at 100 ℃, 10MPa, green bar at 100 ℃, 10MPa The observed yield of hydrothermally treated samples with carbon dioxide for 1 hour. The yields of (S) -Rg3, (R) -Rg3, Rk1, and Rg5 of the carbon dioxide-added reaction were found to be almost two times higher than those in the nitrogen-added reaction. This means that carbon dioxide has been transformed into an acid catalyst. In contrast, the conversion ginsenoside yields were similar in the hydrothermal reaction with nothing added at 0.1 MPa and the hydrothermal reaction with nitrogen added at 10 MPa.

In the reaction according to the above embodiment, there was little effect of pressure in the hydrothermal reaction in which nothing was added. Since the reaction was carried out at a low temperature of 100 ° C to 140 ° C as compared to the critical temperature of 374 ° C, even when the pressure increased to 20MPa, the properties of water such as dielectric constant and ion generation constant did not change significantly. This is a small thing.

[Yield of Converted Ginsenosides in Hydrothermal Reaction Added Carbon Dioxide]

Figure 6 shows the yield according to the temperature of the converted ginsenosides according to an embodiment of the present invention.

Referring to Figure 6, A to C are (S) -Rg3, (R) -Rg3, Rk1, and by a hydrothermal reaction to which carbon dioxide is added at a temperature of 100 ℃, 120 ℃, 140 ℃ and a pressure of 20MPa, respectively, and Yields of the converted ginsenosides of Rg 5, D to F, show the yields of the converted ginsenosides by hydrothermal reaction with nothing added at temperatures of 100 ° C., 120 ° C., and 140 ° C. and pressures of 0.1 MPa, respectively. The (S) -Rg3 is a red circle, the (R) -Rg3 is a white circle, the Rk1 is a green triangle, and the Rg5 is a blue rectangle.

In the case of Graph A at 100 ° C., the hydrothermal reaction to which carbon dioxide was added was performed using 0.5, 1, 3, 5, 9, 17, 24, and 48 hours as reaction time, respectively. By the reaction, (S) -Rg3 is 6.02 mg / g dw (dw = dry weight) at 5 hours, (R) -Rg3 is 4.62 mg / g dw in 1 hour, and Rk1 is 4.15 mg in 9 hours / g dw, and Rg5 showed a maximum yield of each of the converted ginsenosides of 8.85 mg / g dw at 24 hours.

In the case of Graph B at 120 ° C., the hydrothermal reaction to which carbon dioxide was added was performed using 0.25, 0.5, 1.5, 3, 4.5, and 6 hours as reaction time, respectively. Samples reacted for 0.25 and 0.5 hours showed a maximum yield of (R) -Rg3 and (S) -Rg3 of 5.87 mg / g dw and 5.31 mg / g dw, respectively. The Rk1 showed a maximum yield of 4.37 mg / g dw at 3 hours and the Rg5 at 9.98 mg / g dw at 4.5 hours.

In the case of Graph C at 140 ° C., the hydrothermal reaction to which carbon dioxide was added was performed using 8, 10, 15, 30, 60, and 90 minutes as reaction time, respectively. The (S) -Rg3 and (R) -Rg3 obtained a maximum yield of 5.07 mg / g dw and 6.40 mg / g dw at 10 minutes, respectively, while the Rk1 and Rg5 were 5.33 mg / g dw at 50 minutes respectively. And a maximum yield of 11.7 mg / g dw was observed. The Rk1 and Rg5 were produced by E1 removal reaction from (S, R) -Rg3. According to Zaitsev's rule, the main product of the E1 removal reaction was a highly substituted alkenes, so the yield of Rg5 was higher than that of Rk1.

As the temperature was increased by 20 ° C., the maximum yield of (S) -Rg3 decreased to 6.02 mg / g dw, 5.31 mg / g dw, 5.07 mg / g dw, whereas of (R) -Rg3 The maximum yield is 4.62 mg / g dw, 5.87 mg / g dw, 6.40 mg / g dw, and the maximum yield of Rk1 is 4.15 mg / g dw, 4.37 mg / g dw, 5.33 mg / g dw, of Rg5 Maximum yield increased to 8.85 mg / g dw, 9.98 mg / g dw, 11.7 mg / g dw. This result also appeared in the hydrothermal reaction to which the carbon dioxide of D to F was not added.

When the reaction temperature increased, the rate of production and decomposition of the converted ginsenosides both increased, and the maximum yield of the converted ginsenosides can be determined from the two rates. In the case of (S) -Rg3, the increase in decomposition rate with increasing temperature is greater than the increase in production rate, thereby lowering the maximum yield. In contrast, the maximum yields of (R) -Rg3, Rk1, and Rg5 appeared to increase with increasing reaction temperature, because the increase in production rate was greater than the increase in decomposition rate. In particular, the yield of (R) -Rg3 at 100 ° C and 120 ° C was lower than the yield of (S) -Rg3, but at 140 ° C it was higher than the yield of (S) -Rg3. This is because the rate of formation of (R) -Rg3 increased significantly compared to the rate of decomposition as the reaction temperature increased. Therefore, relatively low temperatures in this process are advantageous for increasing the yield of (S) -Rg3, while higher temperatures are more advantageous for the maximum yields of (R) -Rg3, the Rk1 and the Rg5.

Compared to the normal hydrothermal process without the addition of carbon dioxide, the hydrothermal process with the added carbon dioxide greatly reduced the time required to obtain a similar yield of ginsenosides. For example, the maximum yields of (S) -Rg3, Rk1, and Rg5 in the general hydrothermal process at 100 ° C. were observed at 12 hours, while the carbon dioxide-added hydrothermal process achieved the same yield in 2 hours.

At the same reaction temperature the maximum yield of all converted ginsenosides increased with the introduction of carbon dioxide. More specifically, the maximum yield of (S) -Rg3 increased about 0.35 mg / g at 100 ° C, and the maximum yield of (R) -Rg3, Rk1, and Rg5 was about 0.59 at 140 ° C, respectively. 1.17, and 3.58 mg / g increased. The increase in Rk1 and Rg5 yields was greater than the increase in (S, R) -Rg3 yield.

The rate of production and decomposition of each converted ginsenoside was accelerated by the acid catalyst, carbonic acid. Among the converted ginsenosides, the rate of production of Rk1 and Rg5 was found to be particularly increased. In addition, the hydrothermal reaction with the addition of carbon dioxide at all temperatures achieved yields that were not obtained even with extended reaction times under hydrothermal reactions using only water. For example, in a hydrothermal reaction at 140 ° C. without carbon dioxide, the Rg5 yield reached a peak value of 8.03 mg / g after 75 minutes, after which the yield decreased with increasing reaction time. However, the yield of Rg5 in the hydrothermal reaction with carbon dioxide added was higher than 8.03 mg / g after only 8 minutes. Thus, higher yields of (S) -Rg3, (R) -Rg3, Rk1, and Rg5 were obtained in the hydrothermal reaction to which the carbon dioxide was added.

[Comparison of Yield of Ginsenosides from Hydrothermal and Steaming Processes with Carbon Dioxide]

For comparison with the existing steaming process, a control group comprising red ginseng and ginseng was prepared by steaming dried ginseng at 100 ° C. and 120 ° C. for 3 hours. It was then compared to a hydrothermally treated sample with carbon dioxide at 100 ° C. and 120 ° C. for 3 hours. Apart from these processes, to investigate the effect of the introduction of carbon dioxide in the hydrothermal reaction, a hydrothermal process without addition of carbon dioxide was also performed at the same reaction temperature and time.

Figure 7 shows the yield of ginsenosides by process according to an embodiment of the present invention.

Referring to FIG. 7, A is a comparison of the process yields of a steaming process, a hydrothermal process, and a hydrothermal process to which carbon dioxide is added at 100 ° C., and B is a comparison of the yields of each process at 120 ° C. The white bar is the yield obtained through the steaming process, the hatched bar is the yield obtained through the hydrothermal process and the green bar is the yield obtained through the hydrothermal process to which the carbon dioxide is added, and the yield of the converted ginsenoside is generally Carbon dioxide was highest after the hydrothermal reaction added and lowest after the steaming process.

However, for the yield of (S, R) -Rg 3 at 120 ° C., the hydrothermal process with carbon dioxide added was lower than the yield achieved during the normal hydrothermal process with no addition. The hydrothermal process to which the carbon dioxide was added maximized and decreased the yield of (S, R) -Rg 3 in 30 minutes because of the fast reaction rate due to carbonic acid. In contrast, the general hydrothermal process showed a maximum yield of (S, R) -Rg3 at 2 hours.

Comparing the steaming process at 100 ° C. with the hydrothermal process to which the carbon dioxide was added, the yields of (S) -Rg3, (R) -Rg3, Rk1, and Rg5 are 14.3, 9.07, 9.83, respectively, than those obtained from red ginseng. , And 9.27 times higher.

The yield of ginsenosides at 120 ° C. increased by 1.91, 1.47, 2.03, and 2.20 times compared to the yield achieved from sun ginseng. Despite the reactions carried out at the same reaction temperature and duration, there are two reasons for this dramatic increase in yield. First, the hydrothermal process to which carbon dioxide is added is a uniform condition, while the steaming process is a nonuniform condition. In homogeneous reactions the reactants are in the same phase, but in heterogeneous reactions the reactants are in different phases. The homogeneous reaction conditions increase the reaction rate, mass transfer rate and heat transfer rate faster than the heterogeneous reaction conditions.

FIG. 8 compares the characteristics of a hydrothermal process and a steaming process to which carbon dioxide is added according to the embodiment of FIG. 7.

Referring to FIG. 8, A is a hydrothermal process to which carbon dioxide is added, and B is a steaming process to show the distribution of pure hexagonal ginsenosides of green hexagons, water of blue circles, and carbon dioxide of yellow triangles.

Transform ginsenosides can be obtained through deglycosylation, which is a form of hydrolysis of pure ginsenosides. Therefore, the reactants are the pure ginsenosides and water. In the hydrothermal process to which carbon dioxide is added, there may be two layers, a supercritical carbon dioxide layer at a temperature of 100 ° C., 120 ° C., and 140 ° C. and a pressure of 20 MPa, and a hot water layer. In the aqueous layer, the pure ginsenoside and the water itself may be present together in the same phase because the water soluble ginseng extract is the reaction medium. Therefore, the hydrothermal process to which the carbon dioxide is added becomes a uniform condition. In contrast, the pure ginsenosides are present in solid ginseng, while the water is present in the vapor state during the steaming process, indicating heterogeneous conditions. Thus, the homogeneous conditions can be an advantage of the hydrothermal process leading to high yields within a short reaction time.

Second, an acid catalyst was added by in-situ formation of carbonic acid in a hydrothermal process to which carbon dioxide was added. The amount of dissolved carbon dioxide has increased due to the high pressure, which can form strong acids due to the high concentration of protons (H +) in water. In order to quantitatively measure the effect of the acid catalyst, the decomposition rate constants of the pure ginsenosides were calculated from hydrothermal and hydrothermal processes with the addition of carbon dioxide.

Among the pure ginsenosides, Rb1, Rc, Rb2, and Rd can be converted to (S) -Rg3, (R) -Rg3, Rk1, and Rg5. Thus, the decomposition rate constant was determined using the sum of the concentrations of the four pure ginsenosides, and the decomposition reaction of the pure ginsenosides was assumed to be a first order reaction. The reaction rate constants obtained are shown in Table 1.

T (℃) k in H ₂ O (s ^-1 ) k in H ₂ O / CO ₂ (s ^-1 ) 100 0.000070 0.000259 120 0.000488 0.001967 140 0.002666 0.005300

FIG. 9 is an Arenius plot showing the decomposition of pure ginsenosides in the hydrothermal process and hydrothermal process with the addition of carbon dioxide.

Referring to FIG. 9, the Arennius diagram was generated to determine the apparent activation energy of the hydrothermal process indicated by the circle and the carbon dioxide-added hydrothermal process indicated by the green triangle. The magnitude of the slope (11692, R ² = 0.9726) of the hydrothermal process to which carbon dioxide was added was smaller than the slope (14032, R ² = 0.99999) of the hydrothermal process. As a result, the introduction of carbon dioxide into the hydrothermal process lowers the apparent activation energy by 16.7%, resulting in faster reaction rates.

[Anticancer activity]

The anticancer activity of ginseng is mainly due to the conversion ginsenosides. Comparing the yield obtained under controlled conditions with the yield of converted ginsenosides, higher yields were obtained at 120 ° C. than at 100 ° C. Therefore, the anticancer activity of three different methods, steamed ginseng, hydrothermally treated ginseng without carbon dioxide addition, and hydrothermally treated ginseng added carbon dioxide, was compared for 3 hours at 120 ° C. Cell growth inhibition effects were evaluated in human bone marrow derived mesenchymal stem cells (MSCs), HT1080s, and MDA-MB-231s to determine the anticancer activity of the samples.

10 shows the antiproliferative effect of each process according to an embodiment of the present invention.

Referring to Figure 10, A in mesenchymal stem cells, B in HT1080s, C in MDAMB-231s ginseng extract obtained by the circular steaming process, ginseng extract obtained by hydrothermal process without addition of red triangle carbon dioxide, blue square When the ginseng extract obtained by the hydrothermal process to which carbon dioxide was added was treated, the relative cell growth rate for the cells that were not treated with any ginseng extract was compared.

The growth of the mesenchymal stem cells, considered normal stromal cells, was not inhibited in any sample at all concentrations, indicating that treated ginseng was not cytotoxic to normal cell lines. On the other hand, the sample has been shown to be cytotoxic to cancer cell lines. No significant change in growth was observed in the HT 1080 cells up to 5 μg / mL, but cells treated with 10 μg / mL of the sample induced growth inhibition of the HT 1080.

Percent inhibition is followed by hydrothermally treated ginseng (CHPG) with relative carbon growth (67.9%)> steamed ginseng (SG) (80.2%)> hydrotreated ginseng (HPG) (90.1%). appear. Of the three samples, the hydrothermally treated ginseng added with carbon dioxide contained the highest yield of converted ginsenosides and showed the highest antiproliferative effect. However, when comparing the steamed ginseng and the hydrothermal ginseng, the steamed ginseng has a higher anti-proliferative effect even though the yield of the converted ginsenoside is lower than that of the hydrothermal ginseng. appear. Therefore, other components as well as the transforming ginsenosides may be involved in the inhibition of growth of HT1080 cells.

For MDA-MB-231s, hydrothermal ginseng showed no antiproliferative effect up to 5 μg / mL, whereas hydrothermally treated ginseng (93.9%) with carbon dioxide at the same concentration and steamed ginseng ( 91.3%) had a slight effect. Growth inhibition at 10 μg / mL was in the order of hydrothermally treated ginseng (59.2%)> hydrotreated ginseng (83.4%)> steamed ginseng (86.4%). The antiproliferative effect was increased in a concentration-dependent manner in all cancer cell lines, the highest inhibitory effect of the hydrothermally treated ginseng added with carbon dioxide

Since the hydrothermal process with the addition of carbon dioxide specifically enhances the yield of converted ginsenosides, higher antiproliferative effects can be obtained with only a small amount of sample compared to other conventional techniques. Using ginseng extract steamed in MDA-MB-231 cells for 2 hours can inhibit cell proliferation from 100 μg / mL to 20%. SW-480 cells (human colon cancer cells) can be suppressed with steamed ginseng (notoginseng) extract. Treatment at a concentration of 50 μg / mL can inhibit cell growth by up to about 25%. However, according to embodiments of the present invention, hydrothermally treated ginseng added with carbon dioxide, using lower concentrations of the sample (10 μg / mL), was 32.1% and 40.8 in HT 1080 and MDA-MB-231 cells, respectively. % Can further inhibit cell growth.

In addition, D and E of Figure 10 shows the degree of gene expression of caspase-3 of HT 1080 and MDA-MB-231 for the ginseng treated by the process, respectively. To determine whether the antiproliferative effect of treated ginseng is due to apoptosis, gene expression of caspase-3, a measure of cell death, was treated with a sample of 10 μg / mL for 48 hours. The cells were evaluated.

In FIG. 10 D and E, white bars represent steamed ginseng, hatched bars represent hydrothermal ginseng without carbon dioxide addition, and green bars represent hydrothermally treated ginseng added carbon dioxide.

For HT 1080 cells, the relative genetic expression of caspase-3 with steamed ginseng, hydrotreated ginseng, and hydrotreated ginseng added with carbon dioxide was 1.46, 0.93, and 1.40, respectively. The hydrothermally treated ginseng with lower inhibitory effect also showed lower gene expression of the caspase-3 compared to the hydrothermally treated ginseng and steamed ginseng conditions to which the carbon dioxide was added.

For MDA-MB-231 cells, the gene expression levels of caspase-3 were 1.53, 1.65, and 1.79 in steamed ginseng, hydrotreated ginseng, and hydrotreated ginseng added carbon dioxide, respectively. The expression of caspase-3 was highest for hydrothermally treated ginseng added carbon dioxide, and the hydrothermally treated ginseng appeared higher than the steamed ginseng. This result is related to the antiproliferative effect of the sample. Since the gene expression level of caspase-3 is generally greater than 1, the cancer cell growth inhibitory effect of the treated ginseng may be due to the induction of apoptosis.

So far, specific embodiments of the present invention have been described. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (10)

Hydrothermally reacting the ginsenoside containing material,
Ginsenoside production method characterized in that the carbon dioxide is provided to the hydrothermal reaction. The method of claim 1,
The carbonic acid produced from the carbon dioxide acts as a catalyst of the hydrothermal reaction. The method of claim 1,
The hydrothermal reaction is ginsenoside production method characterized in that it is carried out using subcritical water or supercritical water. The method of claim 1,
The ginsenoside-containing material comprises ginseng extract extracted from ginseng. The method of claim 4, wherein
Ginsenoside production method comprising the step of preparing the ginseng extract before the hydrothermal reaction. The method of claim 5,
The ginseng extract manufacturing step,
Drying the ginseng,
Grinding the dried ginseng into powder, and
Ginsenoside manufacturing method comprising the step of extracting the ginseng extract from the powder. The method of claim 6,
The ginseng extract is ginsenoside manufacturing method characterized in that the extraction using a volatile solvent. Ginsenosides prepared according to the method of any one of claims 1 to 7. The method of claim 8,
The ginsenosides ginsenosides, characterized in that the ginsenosides are obtained by converting intact ginsenosides through at least one of deglycosylation or dehydration. The method of claim 9,
The converted ginsenosides include one or more of (S) -Rg3, (R) -Rg3, Rk1, and Rg5.

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