KR101783295B1 - Manufacturing method for mass-production of ginsenoside Rg4 and Rg6 - Google Patents

Abstract

The present invention relates to a method for preparing at least one ginsenoside selected from ginsenosides Rg4 and Rg6 from ginsenoside Re. Since there is a slight amount of the ginsenosides Rg4 and Rg6, it is not easy to use them but prepare ginsenosides Rg4 and Rg6 using only ginsenoside Re easily separated from the leaves of ginseng. The present invention prevents the incorporation of ginsenosides, which may be generated from the major ginsenosides such as ginsenosides Rc, Rd, Rf, etc. by comparing with when using extracts of ginseng, red ginseng and black ginseng, etc. The present invention easily purifies ginsenosides Rg4 and Rg6 and ic ginsenosides Rg4 and Rg6 and is easily capable of mass production.

Description

Manufacturing method for mass-production of ginsenoside Rg4 and Rg6 [

The present invention relates to a method for mass production of at least one ginsenoside of ginsenosides Rg4 and Rg6 from ginsenoside Re.

Ginseng ( Panax ginseng ) is a perennial plant belonging to the genus Ginseng ( Araliaceae ). It has been traditionally used in the prevention and treatment of various diseases in Korea and China. It has excellent therapeutic effect and has no side effect It is known.

Ginseng saponin, which is the main effect of ginseng, is known as ginsenoside and is known to have a unique chemical structure and pharmacological effect unlike saponin found in other plants.

Ginsenoside is a neutral glycoside conjugated with glucose, arabinose, xylose, rhamnose, and the like on a triterpenoid-based dammarane skeleton. At present, more than 30 chemical structures have been identified. Depending on the nature of the chemical structure, protopanaxadiol (PPD) (19 species), protopanaxatriol (PPT) (10 species) Oleanane system (one species).

The major ginsenosides present in fresh ginseng and white ginseng are Rg1, Re, Rf, Rh1, Rb1, Rb2, Rc, and Rd, accounting for 80-90% or more of the total ginsenosides. On the other hand, in the case of the specific ginsenoside, a part of the sugar is separated from the ginsenoside present in a large amount, or a dehydration reaction occurs, and when ginseng is extracted with water and alcohol, it is present in a trace amount.

The pharmacological activity by the ginsenoside is indicated by the specific ginsenoside originating from the above-mentioned ginsenosides present in large quantities, and it is considered that the pharmacological activity by the ginsenoside is enhanced by the immunosuppressive effect, anti-inflammatory, antiallergic, anticancer, hypotensive, anti-cholesterol, (Kim, YS et al., Arch Pharm Res., 2000; Tachikawa, E. et al., Biochem Pharmacol., 2003; Tsai, SC et al , Chin J Physiol., 2003, Shibata, S., J Korean Med Sci., 2001). In addition, in the case of specific ginsenosides, unlike ginsenosides present in large quantities, the intestinal absorption rate is excellent and is suitable for use as a pharmaceutical composition for various diseases. Therefore, research on a method for mass production of a specific ginsenoside not contained in ginseng or contained in a trace amount is required.

Regarding the method for producing ginsenosides Rg4 and Rg6, Korean Patent Registration No. 10-0564383 discloses a method for preparing a mixture of F4 (Rg4), Rg6, Rk3 and Rh4 using citric acid as the ginsenoside Re and Rg1 Korean Patent No. 10-1625474 discloses a method for mass-producing ginsenoside Rh4 by mixing an organic acid with ginsenoside Re and then subjecting it to high temperature and high pressure treatment. In Korean Patent Laid-Open No. 10-2012-0134166 Discloses a method for producing ginseng extract containing a trace amount of ginsenosides at a high concentration using lactic acid bacteria. However, the method for producing ginsenoside Rg4 and Rg6 by treating ginsenoside Re with high-temperature and high- It has not been disclosed.

Korean Patent No. 10-0564383 (Method for producing ginsenoside derivative, registered on March 20, 2006) Korean Patent No. 10-1625474 (Mass Production Method of Ginsenosides Al HQ 4, registered on May 24, 2016) Korean Patent Laid-Open No. 10-2012-0134166 (Method for producing ginseng extract containing trace amount of ginsenosides, December 12, 2012)

Kim, YS et al., Differential expression of protein kinase C subtypes during ginsenoside Rh2-Induced apoptosis in SK-N-BE (2) and C6Bu-1 cells, Arch Pharm Res., 23 (5), 518-524, 2000 . Shibata, S., Chemistry and cancer prevention activities of ginseng saponins and some related triterpenoid compounds, J Korean Med Sci., 16 (suppl), S28-37, 2001. Tachikawa, E. et al., In vitro inhibition of adrenal catecholamine secretion by steroidal metabolites of ginseng saponins, Biochem Pharmacol., 66 (11), 2213-2221, 2003. Tsai, S. C. et al., Stimulation of the secretion of luteinizing hormone by ginsenoside-Rb1 in male rats, Chin J Physiol., 46 (1), 1-7, 2003.

It is an object of the present invention to provide a method for mass production of at least one of ginsenosides Rg4 and Rg6 from ginsenoside Re.

The present invention relates to a process for the preparation of at least one ginsenoside of ginsenosides Rg4 and Rg6 from ginsenoside Re,

(Step 1) Separation of ginsenoside Re from ginseng;

(2 step) Step of cutting off the contact of external moisture with ginsenoside Re produced in step 1, and then treating at high temperature and high pressure; And

Chromatography of the high-temperature and high-pressure ginsenoside products in the (3 step) 2 step separating at least one ginsenoside of ginsenosides Rg4 and Rg6; .

Also, ginsenosides Rg4 and Rg6 are produced from ginsenoside Re through the process of Reaction Scheme 1 below.

[Reaction Scheme 1]

The ginsenoside Re obtained by separation and production from the ginseng in the above step is obtained from a ginseng extract. The ginseng extract can be prepared by using all the ginseng-derived roots, fruits, leaves, stems and the like derived from ginseng A large amount of ginsenoside Re can be obtained from leaves of ginseng.

Ginseng used in the extraction of the ginsenoside Re is a perennial plant belonging to the genus Panax (Panax), ginseng (Panax ginseng), hwagisam (Panax quinquefolia), jeonchilsam (thirty-seven, Panax notoginseng), jukjeol three (Panax japonicus), Panaxa pseudoginseng . And one species selected from Panax vietnamensis , Panax elegatior , Panax wangianus and Panax bipinratifidus , Panax angustifolium , It is most preferable to use Korean ginseng, which is known to have a high saponin content, but the type of ginseng is not particularly limited thereto.

The ginseng extract may be extracted with water, C1-C4 alcohol or a mixture thereof as a solvent. The C1-C4 alcohol may be extracted with methanol, ethanol, propanol, isopropanol, butanol and isobutanol Can be selected from the group consisting of. The water, the C1 to C4 alcohol, or the mixed solution thereof used in the preparation of the ginseng extract may be used in an amount of 1 to 40 times the volume of the ginseng used (1 to 40 L based on 1 kg of ginseng) 40 times, and the process can be repeated from 1 to 4 times.

In order to extract ginsenoside Re from the ginseng extract, ginseng water, C1-C4 alcohol, or a mixed solution thereof is dissolved in water and extracted with n-hexane, methylene chloride, acetone, chloroform, Ethyl acetate, diethyl ether, water-saturated butanol, and n-butanol, or sequentially fractionated by combining these solvents and then purified using column chromatography . The extraction time of the extract or the fraction thereof is not particularly limited, but it is preferably extracted within 10 minutes to 1 day. As the extraction apparatus, a conventional extraction apparatus, an ultrasonic pulverization extractor, or a fractionator may be used.

On the other hand, the ginseng extract may be isolated or isolated by known methods used for the separation and extraction of plant components, such as extraction with an organic solvent (alcohol, ether, acetone, etc.), distribution with hexane and water, Ginsenoside Re can be obtained by fractionation or purification using a suitable combination method. The chromatography can be carried out using silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

In the above two steps, ginsenoside Re produced in one step is placed in a closed container to block the contact of external moisture, and then the mixture is heated for 2 to 10 hours in a temperature range of 110 to 140 占 폚 and a temperature of 0.11 to 0.16 MPa It is more preferable to carry out the high-temperature and high-pressure treatment at a high temperature and a high pressure under a pressure condition for 4 to 10 hours at a temperature range of 110 to 140 ° C and a pressure condition of 0.11 to 0.16 MPa. However, when the ginsenoside Re is added with water or organic acid, the ginsenosides Rg4 and Rg6, which are the desired ginsenosides to be mass produced in the present invention, are not converted to other ginsenosides Senoside Rh4, etc.), or conversion to ginsenosides Rg4 and Rg6 is lowered, resulting in a loss of production cost. Particularly, when the ginsenosides Rg4 and Rg6 are prepared by treating an organic acid, the separation resin is contaminated during separation and purification, which makes it difficult to use for a long period of time, which is economically disadvantageous.

Further, it is preferable that the high-temperature high-pressure treatment time is less than 2 hours, the pressure is less than 0.11 MPa, or the pressure is less than 0.15 MPa, In the case of producing ginsenosides Rg4 and Rg6 under the condition that the temperature is lower than 110 占 폚, the yield for obtaining ginsenosides Rg4 and Rg6 is remarkably lowered due to the remaining ginsenoside Re not converted to the specific ginsenoside, It is not desirable. Further, when the high-temperature and high-pressure treatment time exceeds 10 hours, or when the pressure exceeds 0.16 MPa, or when the temperature exceeds 140 ° C, the conversion rate from ginsenoside Re to ginsenoside Rg4 and Rg6 is The increase in the effect of increasing the effect is not large and can be rather reduced, leading to a loss of manufacturing cost, which is not preferable.

It is preferable to use reversed-phase column chromatography (C18) or HPLC for the above-mentioned chromatography in the above-mentioned three steps, and the purified ginsenoside through the chromatography preferably has a purity of 90% or more, more preferably 90 to 99 %, And most preferably 95 to 99%.

Further, ginsenosides Rg4 and Rg6 separated and produced by the above two steps and three steps using 100 parts by weight of ginsenoside Re prepared in the above step are obtained in 40 to 60 parts by weight. However, in the process of obtaining less than 40 parts by weight of ginsenosides Rg4 and Rg6 using 100 parts by weight of ginsenoside Re, economic loss is undesirable.

The present invention relates to a method for producing at least one of ginsenosides Rg4 and Rg6 from ginsenoside Re, wherein the ginsenosides Rg4 and Rg6 are present in a minor amount in the ginseng extract, It is not easy to use it. However, since ginsenosides Rg4 and Rg6 are produced using only ginsenoside Re, which can be easily separated from leaves of ginseng through the present invention, extracts of ginseng, red ginseng, and black ginseng It is possible to prevent the incorporation of ginsenoside which can be produced relatively from the ginsenosides Rc, Rd, Rf and the like as compared with the case where the ginsenosides Rg4 and Rg6 can be easily purified, Lt; RTI ID = 0.0 > Rg4 < / RTI > and Rg6.

1 shows the results of HPLC analysis for conversion of ginsenoside Re to ginsenoside Rg4 and Rg6 when ginsenoside Re was subjected to high temperature and high pressure treatment under the conditions of Examples 1-2 to 1-5.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Preparation of Rg4 and Rg6 from ginsenoside Re >

10 kg of a 70% (v / v) aqueous methanol solution was added to 1 kg of dried ginseng leaves collected from Geumsan in September, 2013, and leached for one day, followed by ultrasonic shaking for 3 hours and filtration. After filtration, the remaining ginseng leaves were added with 8 liters of 70% (v / v) methanol, and the leaching and ultrasonic shaking procedures were repeated two more times. The filtrates obtained in the above procedure were combined and concentrated under reduced pressure to obtain a completely dried methanol extract 416 g was obtained. Then, the methanol extract was completely dissolved in 3 L of water by shaking ultrasonically, and then 2 L of diethyl ether was added thereto, followed by stirring at room temperature for 2 days. After the stirring, the solution was transferred to a separatory funnel to remove the diethyl ether layer, and 5 liters of water saturated butanol was added to the remaining water layer, followed by stirring in a stirrer for 2 days. The stirred solution was transferred to a separatory funnel and separated for more than one day. Only the butanol layer was collected and concentrated under reduced pressure to obtain 157 g of completely dried butanol extract. Thereafter, 157 g of the butanol fraction was dissolved in methanol, mixed with 500 g of silica gel (250 to 400 mesh), kneaded and concentrated under reduced pressure to prepare fine powder. In addition, the silica gel was kneaded in chloroform, poured into an open column of 12 x 50 cm, filled up to a height of 25 cm, and then separated on a sea sand (silica sand: silica gel, Used) was raised by 2 cm, and then the powder was raised. The mixture was mixed with chloroform: methanol: water at a ratio of 10: 2: 0.2 (1500 ml: 300 ml: 30 ml) in a separatory funnel, and the lower layer was taken and separated and purified using silica gel column chromatography as a developing solvent, A column fraction containing a large amount of side Re was obtained, which was concentrated under reduced pressure. The column fraction containing the ginsenoside Re was subjected to C18 reverse phase column chromatography. A C18 reversed phase packing material kneaded with a 40% (v / v) aqueous methanol solution was filled in a 3.5 x 60 cm flash column to a height of 30 cm, (V: v), 3: 1 (v / v) methanol: water in a 1: 1 (v: v) (v: v). Through the above procedure, 11.5 g of pure ginsenoside Re was finally obtained.

20 mg of the ginsenoside Re obtained in the above procedure was placed in each of five vials of 1.5 ml, and then the lid was closed without any treatment to block the contact of external moisture, and then the mixture was added to a 125 ml vial bottle. Thereafter, the mixture was put into a sterilizer (SANYO autoclave, MLS-3780 model) and steamed at 120 DEG C under a pressure of 0.13 to 0.15 MPa for 2 hours. After 2 hours, only one mixture out of the five mixtures was taken out, And pressure condition. By repeating the above procedure, the boiled water according to each time (2 hours, 4 hours, 6 hours, 8 hours and 10 hours) was obtained and each of the boiled water was dried in a Centrifan PE. The concentrated powders were firstly separated by C18 reverse phase column chromatography. At this time, the C18 reversed phase filler kneaded with a 40% (v / v) methanol aqueous solution was filled up to a height of 30 cm on a 3.5 x 60 cm flash column, and the concentrated powdery state water contents were added to 25% (v / v) methanol (V: v), 3: 1 (v: v), and purified by using methanol / water as a developing solvent. .

The first-purified fractions CFr01 and CFr02 from the C18 reverse phase column chromatography method were subjected to secondary purification by multiple preparative HPLC using YMC's LC-forte / R. The column used was YMC-Dispopack AT ODS-25 (40 g), which was separated and purified by connecting it to three multiples. Water (H ₂ O) and acetonitrile (CH ₃ CN) were used as the solvents used for separation. After sufficiently filling the YMC-Dispopack AT ODS-25 with an aqueous 15% (v / v) acetonitrile solution , And the sample was dissolved in a 35% (v / v) methanol aqueous solution and loaded. (20% B), 25-90 minutes (40% B), 90-90 minutes (40% B) and water (H ₂ O, A solvent) and acetonitrile (CH ₃ CN, B solvent) A concentration gradient of 120 minutes (50% B), 120-135 minutes (65% B), 135-155 minutes (85% B), 155-160 minutes (100% System (gradient elution system) to obtain ginsenosides Rg4 and Rg6. Further, prior to the separation and purification of the high-temperature and high-pressure ginsenoside water-containing product, a part of the concentrated powder in the rotary concentrator was subjected to HPLC analysis to confirm the conversion to ginsenosides Rg4 and Rg6, Lt; / RTI >

<Comparative Example 1> Preparation of comparative ginsenosides Rg4 and Rg6 (1)

Ginsenosides Rg4 and Rg6 were prepared from ginsenoside Re in the same manner as in Example 1-3 except that they were matured at 100 DEG C or 150 DEG C for 6 hours instead of being steamed at 120 DEG C for 6 hours in a sterilizer, The conversion of ginsenoside rinsed product to ginsenoside Rg4 and Rg6 was confirmed by HPLC analysis, which is shown in Table 2 of Experimental Example 1 as Comparative Examples 1-1 and 1-2.

< Comparative Example  2. Comparison with Gencenoside Rg4  And Of Rg6  Manufacturing ②>

Ginsenosides Rg4 and Rg6 were prepared from ginsenoside Re in the same manner as in Example 1-3 except that 50 μl of distilled water was mixed with ginsenoside Re and then ginsenoside rinsed at 120 ° C for 6 hours The conversion to ginsenosides Rg4 and Rg6 was confirmed by HPLC analysis, and this was shown in Comparative Example 2 in Table 2 of Experimental Example 1.

< Comparative Example  3. Compared to Gencenoside Rg4  And Of Rg6  Manufacturing ③>

Ginsenosides Rg4 and Rg6 were prepared from ginsenoside Re in the same manner as in Example 1-3 except that 50 μl of apple vinegar was mixed with ginsenoside Re, Was analyzed by HPLC to confirm the conversion to ginsenosides Rg4 and Rg6, which is shown in Comparative Example 3 in Table 2 of Experimental Example 1.

< Comparative Example  4. Comparison with Gencenoside Rg4  And Of Rg6  Manufacturing ④>

20 mg of ginsenoside Re obtained in the same manner as in Example 1 was taken in a 50 ml round-bottomed flask and dissolved in 20 ml of distilled water. A reflux device and a heating mantle were placed in the sample, and the mixture was heated to reflux at 100 ° C for 30 days. After the reaction was completed, the flask was cooled to room temperature, and distilled water was removed from the reduced pressure concentrator. Part of the dried reaction product was partially analyzed by HPLC for conversion from ginsenoside Re to ginsenoside Rg4 and Rg6. 1 in Table 2, and Comparative Example 4 in Table 2.

EXPERIMENTAL EXAMPLE 1 Confirmation of Conversion of Ginsenoside Rg4 and Rg6:

In order to measure the change of ginsenoside Re, the above-mentioned steaming products or mixtures of Example 1 and Comparative Examples 1 to 4 were dried in a Centrifan PE to remove a small amount of water contained in the steaming process, 1 mg of each sample was dissolved in 1 ml of methanol for HPLC, and the filtrate was filtered through a 0.50 μm filter and used for HPLC analysis.

HPLC performance conditions column ACE 5-C ₁₈ (250 x 4.6 mm) Flow rate 1.0 ml / min Detector UV detector, 205nm Solvent A; H ₂ O, B; CH ₃ CN
(35% B), 85- 105 minutes (50% B), 105-135 minutes (65% B), 135-145 minutes (85% B) 145-155 minutes (100% B), 155-160 minutes (100% B), 160-163 minutes (20% B), 163-165 minutes (20% B) Temperature 40 ℃

Condition Ginsenoside (占 퐂 / mg) Re Rg4 Rg6 Rh1-S Rh1-R Rh4 total Example 1-1 At 120 캜 for 2 hours, 68.8 280.0 198.4 63.1 19.4 11.4 641.1 Examples 1-2 At a temperature of 120 캜 for 4 hours, 0 306.0 215.2 63.9 35.8 25.2 646.1 Example 1-3 120 &lt; 0 &gt; C for 6 hours, 0 323.5 228.0 70.0 38.1 28.1 687.7 Examples 1-4 At 120 DEG C for 8 hours, 0 316.2 192.8 73.1 37.4 63.6 683.1 Examples 1-5 At 120 캜 for 10 hours, 0 303.8 184.8 70.3 32.3 95.2 686.4 Comparative Example 1-1 100 &lt; 0 &gt; C for 6 hours, 254.0 207.3 174.1 59.7 21.4 8.5 725.0 Comparative Example 1-2 Steamed at 150 ° C for 6 hours 0 231.1 104.2 59.5 44.2 114.7 553.7 Comparative Example 2 50 [mu] l distilled water, 120 [deg.] C, 6 hours 85.3 200.6 147.0 122.0 95.3 9.2 659.4 Comparative Example 3 After 50 쨉 l acid treatment,
120 &lt; 0 &gt; C for 6 hours, 0 32.3 21.6 43.6 25.3 284.7 407.5 Comparative Example 4 Boiling 30 days 0 3.4 20.6 62.1 26.4 4.1 116.6

Referring to Table 2 and FIG. 1, in the case of Examples 1-1 to 1-5 in which ginsenoside Re was treated at high temperature and high pressure while blocking the contact of external moisture, most of the ginsenosides Re were hydrolysable It was confirmed that there was almost no residual amount of decomposition, and in particular, 400 to 600 μg of ginsenoside Rg4 and Rg6 was produced based on 1 mg of ginsenoside Re. It was confirmed that most of the specific ginsenosides converted from ginsenoside Re, in particular, ginsenosides Rg4 and Rg6 were mostly converted.

However, unlike the present invention, in the case of Comparative Examples 1-1 and 1-2, in which the temperature for steaming exceeds 110-140 ° C, the conversion ratio to ginsenoside Rg4 or Rg6 is remarkable due to remaining ginsenoside Re , Respectively.

Further, in the case of Comparative Example 2 or 3 in which ginsenoside Re was treated with distilled water or organic acid, conversion to ginsenosides Rg4 and Rg6 was remarkably lower than that in Examples, and the conversion of ginsenoside Gin It was found that most of them were converted to the ginsenoside Rh4, which is another specific ginsenoside, without being converted to the senosides Rg4 and Rg6.

On the other hand, in the case of Comparative Example 4 heated to a high temperature of 100 ° C only under the condition that no pressure was given, the conversion speed to ginsenoside Rg4 and Rg6 was slow, It was confirmed that the ginsenoside Re completely disappeared only at the beginning and the conversion rate from ginsenoside Re to ginsenosides Rg4 and Rg6 was as very low as less than 10%.

Therefore, as in the present invention, the method of enclosing ginsenoside Re in a hermetically sealed container to block the contact of external moisture and subjecting it to high-temperature and high-pressure treatment remarkably increases the conversion rate to ginsenosides Rg4 and Rg6, Able to know.

Claims (3)

(Step 1) Separation of ginsenoside Re from ginseng;
(2 step) Step of cutting off the contact of external moisture with ginsenoside Re produced in step 1, and then treating at high temperature and high pressure; And
Chromatography of the high-temperature and high-pressure ginsenoside products in the (3 step) 2 step separating at least one ginsenoside of ginsenosides Rg4 and Rg6;
Wherein at least one of the ginsenosides Rg4 and Rg6 is prepared from ginsenoside Re. The method according to claim 1,
The high-temperature and high-pressure treatment in the two steps is carried out for 2 to 10 hours at a temperature of 110 to 140 ° C and a pressure of 0.11 to 0.16 MPa. From the ginsenoside Re, one of ginsenosides Rg4 and Rg6 Or more of ginsenosides. The method according to claim 1,
The ginsenoside Re of the above three processes is subjected to chromatography to obtain at least one of ginsenosides Rg4 and Rg6 having a purity of 90% or more. The ginsenoside Re is reacted with one of ginsenosides Rg4 and Rg6 Or more of ginsenosides.

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