JPS62207292A - Separation of purification of glucoside - Google Patents

Abstract

PURPOSE:To separate and purify a glucoside such as saponin, sesquiterpene glucoside, diterpenoid glucoside, etc., by liquid chromatography in high efficiency, by using hydroxyapatite as a separation agent and a water-containing organic solvent as an elutant. CONSTITUTION:Glucoside is separated and purified by liquid chromatography such as column chromatography, high-performance liquid chromatography, etc., using hydroxyapatite [Ca10(PO4)6(OH)2)] as a separation agent and a hydrated organic solvent or salt-containing hydrated organic solvent as an elutant (examples of the organic solvent are methanol, ethanol, acetonitrile, etc., and the examples of salt are sodium chloride, calcium chloride, etc.).

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a method for separating and purifying glycosides by liquid chromatography. More specifically, the present invention uses hydroxyapatite as a separating agent in combination with a water-containing solvent or a salt-containing water-containing organic solvent as an eluent to efficiently separate glycosides. It relates to methods for effective separation and purification.

Prior Art Glycosides are a general term for substances with glycoside bonds, and are composed of sugars bonded together (horosytes) and sugars and non-sugar components (aglycones) (heterosytes). There is. Glycoside in a narrow sense refers to the latter. These glycosides include saponins, which exist in the plant kingdom and have polycyclic compounds as aglycones.

Due to the difficulty in purifying and isolating this saponin and the complexity of its structure, research on this saponin has only made progress in the last few years.

The characteristics of saponins are represented by their foaming properties in aqueous solutions, red blood cell destruction activity (hemolytic activity), toxicity to fish, and the ability to form complexes with cholesterol (steroids). Central depressant, anti-fatigue, anti-i
! In recent years, pharmacological effects have been discovered, such as preventing cancer, promoting cholesterol metabolism, promoting lipid metabolism, and promoting the synthesis of nucleic acids and proteins, as well as preventing infection and antitumor.

As is well known, saponins are composed of aglycones and sugars.
Depending on the aglycon structure, they are classified into triterpenoid saponins and steroid saponins.

The former saponins have a wide distribution, and most of the herbal saponins belong to triterpenoid saponins.

For example, Akebia (Akebia guinata De
cne, ) (ChemPharn+, Bull,
24.1021 (1976)), Ruiyou Button (C
aulophyllum robustum Maxi
m,) (Chemical Abstracts (hereinafter referred to as C, A)
85.106644h (1976)), Yatsude (Fa
tsia japonica Decne, ) (P
hytochem-iStry 15.781 (197
6) ), Sapindus mukuro
ssi Gaertn, ) (C, A, 73.7754
4.110062m, 110071p (1970) and C,A, 74.13384f (1971)), the plant of Sapindium H
nioidesPlanch, ex Benth)
(Phytochemistry 20.1939
(1981)). On the other hand, the latter steroid saponins have been discovered in several types of plants and some marine animals (see Japanese Patent Application Laid-Open No. 59-231022).

Incidentally, liquid chromatography is frequently used for separation and purification of glycosides.

Typical separating agents include silica gel, alumina, Sephadex 1, and Celite. A multicomponent system is used as an eluent when separating and purifying glycosides using these separation agents. For example, when silica gel is used as a separating agent, the eluent is a water-containing multi-component mixed solvent, typically chloroform/methanol/water, and there are also systems in which these are combined with ethyl acetate, acetone, etc. Therefore, in order to separate and purify the desired glycoside, it is necessary to investigate the optimal combination of eluents in preliminary experiments, but this is time-consuming because the eluents are multi-component.

In addition, it is common to use an ultraviolet (UV) detector to detect the target substance (glycoside), but since ethyl acetate and acetone have their own absorption in the UV absorption region, they are usually used. In this case, it becomes impossible to detect the target substance.

Furthermore, there are also problems with the disposal of particularly hazardous substances such as chloroform. In addition, when separating and purifying glycosides, especially saponins, by combining the above separation agent and solvent system, saponins can be separated and purified by utilizing differences in their fine structures (for example, the sugar moieties are the same but the aglycone moieties are slightly different). However, such separation is generally difficult (see the book "Experimental Methods of Natural Products and Organic Compounds J, p. 352").
1977) Published by Kodansha, Journal or Chro
matography, 258, 135-146 (1
983) etc.].

Therefore, it is desired to establish a method for separating and purifying glycosides in order to solve the above problems.

[Summary of the invention]

The present invention aims to solve the above-mentioned problems by providing a liquid chromatography method using hydroxyapatite as a separating agent and combining this with a simplified eluent, thereby efficiently separating glycosides. It is something that we often try to separate and purify.

Therefore, the method for separating or purifying glycosides according to the present invention is a method for separating or purifying glycosides by liquid chromatography, using hydroxyapatite as a separating agent and using a water-containing organic solvent or a salt-containing water-containing organic solvent as an eluent. It is characterized in that it can be used as a liquid.

Effects The method of the present invention has the following advantages in order to solve the above problems.

i) Target glycosides can be efficiently separated and purified.

According to the method of the present invention, since hydroxyapatite, which is a highly polar compound, is used as a separating agent, the eluent used must also have a correspondingly high polarity. Therefore, it is possible to use a water-containing organic solvent system and not a multi-component system as the eluent. Therefore, when separating the target substance, since the components of the eluent are simplified, preliminary experiments to determine this combination are easy. Furthermore, as shown in the Examples below, the present invention allows target substances to be efficiently separated and purified even if they have different microstructures. That is, it is possible to efficiently separate and purify glycosides composed of saponin and acidic sugars as sugar moieties, which was particularly difficult in the prior art described above.

ii) The eluent used in the above-mentioned prior art often contains solvents with UV absorption such as chloroform, ethyl acetate, and acetone, which poses a problem of disposal. (2) If the absorption region is low, separation using a U (2) detector is impossible, but these problems can be solved by using hydroxyapatite as an adsorbent.

These effects of the present invention are mainly due to the use of hydroxyapatite as a separating agent in liquid chromatography. Hydroxyapatite has the property of adsorbing proteins very well, so it is currently used for protein,
Widely used for the separation and purification of enzymes, nucleic acids, etc. However, the finding that this compound is useful as a column packing material when separating glycosides by liquid chromatography was unexpected even for those skilled in the art.

[Detailed description of the invention]

Target Glycosides The substances targeted for separation and purification in the present invention are glycosides. As mentioned above, glycosides include holocyto and heterocyto, and the present invention is directed to glycosides in the narrow sense of these, ie, heterocyto.

Specifically, glycosides in this narrow sense include, for example, nabonine, sesquiterpene glycosides, diterbenoid hypoglycosides, and others.

An example of a glycoside targeted by the present invention is saponin. The facultative properties of saponins have been described above, and saponins can be broadly classified into triterpenoid saponins and steroid saponins depending on their chemical structures. These saponins can also be broadly classified into acidic glycosides and neutral glycosides. There are various types of saponins in the plant world, but the most representative one is Spanidus mukuros.
siGaertn, ), carrot (Panax gi
nseng C,A.

Meyer), Nye:] (Bupleurum
falcatum L, ), dice (Glycine
max), spinach (Spinacia o
leracea L,) (all of the above are sources of triterpenoid terpenes); Lily of the valley (C, keisukei)
Hiq, ), digitalis (D, purpurea)
) (All of the above are steroid saponin sources) There are others.

The glycoside sample provided to the method of the present invention is one prepared in advance as a glycoside-containing fraction from a glycoside-containing plant or the like, or a solution containing a plurality of glycosides. For example, when separating triterpenoid saponins from the plant Enmei, the sample is first extracted from the pericarp of Enmei without degreasing or using an organic solvent such as benzene, acetone, chloroborum, etc. A fraction obtained by extraction with an alkaline solution of an aliphatic alcohol (usually monohydric alcohol) (of about 1 to 4 carbon atoms) or with the above hydrous or non-aqueous lower aliphatic alcohol in the absence of an alkali. The process consists of treating the water with an alkaline solution (details can be found in Japanese Patent Application No. 1986-24).
6756).

In addition, when separating and purifying saponins from carrots, the sample is obtained by treating carrot roots with 50% methanol, centrifuging to obtain a supernatant, and applying this to a pretreatment column (details). (See Example 4 below).

In the present invention, liquid chromatography refers to ordinary column chromatography or high performance liquid chromatography.

Each of these methods is publicly known, and various books, documents, patent publications, etc. can be referred to. For example, regarding column chromatography, Narita "Practical Chromatography" ■, II (1964) published by Koyo Shoten, "Introduction to Chromatography" (1968) published by Koyo Shoten, r Chr.
omatoaraphyJ (3rd, CD,) (19
75) There are publications such as Re1nhoid, and regarding high-performance liquid chromatography, there are Narita "High-Performance Liquid Chromatography J (1972)" published by IG, "Liquid Chromatography and its Applications" (1974) published by Kodansha, etc.

Therefore, when performing chromatography, separating agents,
In other words, each chromatography can be performed with reference to these documents, except for using hydroxyapatite as the column packing material and using a water-containing organic solvent or a salt-containing water-containing organic solvent as the eluent. . For example, regarding the separation and purification of saponins, see Narita "Natural Product Organic Compound Experimental Methods JP 329-345, p. 34.
6-361 (1977) published by Kodansha.

1 μμ In the present invention, the separation agent is a column packing material, and refers to HydroVsiabatai 1 to 1. Hydroxyapatite as used herein is a compound represented by the chemical formula [Ca(PO4)6(OH)2], and is a chemically stable crystal.

Hydroxyapatite is part of the Narita “Experimental Chemistry Course” Part 2-
Separation and Purification-P305-(1967) It may be prepared as appropriate according to the literature of the Chemical Society of Japan, etc., or commercially available products may be used. It is convenient to use commercially available products. Then, a column using this can be filled according to a conventional method. In addition, in the column chromatography method,
There are wet methods and dry methods for filling the column with the separation agent, but the wet method is preferable from the viewpoint of separation efficiency of the target substance.

11 Differences In the present invention, the eluent used for oxidation mudgraphy is a water-containing organic solvent or a salt-containing water-containing organic solvent. Here, the water-containing organic solvent is a mixed solvent of water and an organic solvent miscible with water, and examples of the organic solvent include lower alcohols (methanol, ethanol, etc.), acetonitrile, and the like. The eluent used to separate and purify glycosides, particularly saponins, is basically a system of acetate to nitrile/water. The water-containing organic solvent containing salt is one obtained by adding an appropriate amount of common salt, calcium chloride, etc. to the above-mentioned solvent. Regarding the addition of salt G, for example, when neutral glycosides and acidic glycosides are mixed when separating saponin, it is preferable to add at least about 0.01 M of "common salt" to the water-containing organic solvent. The purpose of adding salt is to improve the separation ability of the target substance.It goes without saying that if the above separation operation is performed with salt added, a salting operation is required during the purification process of the target substance. Nor.

In the present invention, "separation or purification" and "separation/purification" mean separation and/or purification.

Experimental Example 1. Sample Preparation: 20 g of Enmeihi was torn into small pieces by hand, cold soaked and degreased with benzene (100 dX2), and then heated and extracted with a 2% potassium hydroxide methanol solution at 70°C for 100 d.
X3). Next, this methanol extract was evaporated under reduced pressure,
Add water (20d") to the residue and dilute with 2N hydrochloric acid (1)+14
Adjusted to. Then, about this aqueous solution, porous resin (M
Column chromatography using CI gel CHP20P (Mitsubishi Kasei) was performed to obtain a saponin mixture (7.1 g) from the fraction eluted with methanol.

2. Preparation of Column Hydroxyapatite 109 having a particle size of 46 to 49 μm was placed in a beaker, an appropriate amount of 80% methanol was added, and the mixture was stirred by gentle rocking to form a suspension. Next, this suspension has an inner diameter of 2 cttt and a length of 50
The hydroxyapatite was packed through a cII glass column.

3. Column chromatography The above sample (1100 Irr) was applied to the above column, and column chromatography was performed using 85% methanol as an eluent. Thirty fractions were collected with each eluted fraction being about 15 days. Next, after concentrating both of these fractions, 'R
E-chromatography (TLC) (conditions: TLC plate: silica gel G (Merck), developing solvent: ethyl acetate/methanol/water (8: 2: 1%), 5% sulfuric acid spray, color development by heating at 105°C) The components contained in each elution fraction were confirmed.As a result, the glycosides [Saponin A, B, C (Japanese Patent Application No. 60-2467
56, Mei III)] are liponin C and B.

It was eluted quickly in the order of A, and the separation power was higher than when using silica gel as an adsorbent (however, in this case,
The composition of the eluent used for each chromatography is different).

In addition, each glycoside was identified by giving the same Rf value as the standard sample. Saponins A, B and C are monodesmosides (see Japanese Patent Application Laid-open No. 59-2029'1), and are as follows.

1) Saponin A 3-〇-[α-shi-arabinopyranosyl-(1→3)-
α-Rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl]-hederagenin 2) Saponin B 5-0- (β-D-xylopyranosyl-(1→3)-
α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl]-hederagenin 3) Saponin C 3-0-(α-rhamnopyranosyl-(1→3)-
α-L-rhamnopyranosyl-(1→2)-α-shi-arabinopyranosyl]-hederagenin Example 2 Filled with plate-shaped hydroxyapatite consisting of 2 to 3 μm piece length, 4 to 5 μm, and 1 μm thickness Using a stainless steel column (inner diameter: 0.6 CIR, length: 101 mm), an appropriate amount of methanol solution of the triterpenoid saponin fraction obtained from Enmeich described in Example 1 was used as a sample, and high performance liquid chromatography was performed under the following conditions. Graphy (HP
LC) was performed. The chromatogram at this time is shown in FIG.

Conditions: Eluent: 87.5% acetonitrile, flow rate: 1.
Om/min, detection wavelength: 210 m + 1 PLc: TRI ROTAR-II [(JASCO) In Fig. 1, A-C are 5aponin-A,
5apindo-side-8 and 5apon i
n-C, (A) is the standard, (B) is A,
B' and C represent triterpenoid saponins (described above) separated by the method of the present invention.

Example 3 Panax ginseng C, A, Hey
Six kinds of saponins (20(S)-p
Using ginsessides -Rb1, -Rb -Rc, -Rd, which have rotopanaxadiol as an aglycone, and 20(S)-pro-2゛ginssides -Re, -RQ-, which have topanaxatriol as an aglycone, these were dissolved in an appropriate amount of methanol. Using the resulting mixture as a sample, high performance liquid chromatography was performed under the following conditions.

However, the column was the same as that used in Example 2.

The chromatogram at this time is shown in FIG.

Conditions: Eluent: (Δ) 85% acetonyl 1 hel, (B)
80% acetonitrile, linear gradient (A) → (B) (40 minutes), flow rate: 1.0 d/min, detection wavelength: 02 nm HPLC: TRIROTAR-III (JASCO) In the figure, Rq, Rf, Re , Rd, Rc, Rb R
b and Rb, 1 are each ginseno3゜
2 Indicates side peak.

Panax ginseng C, A, He
yer root) was washed with water and dried, 1.8 g of freshly dried carrots was placed in a 50 d centrifuge tube, and 20 d of 50% methanol was added.
Extraction was carried out by shaking in room 4 for 30 minutes. Then, centrifugation (
3000 rpm for 5 minutes), and 10 m of the supernatant obtained was taken into a 20 volume syringe and injected into a commercially available pretreatment column ("Seppac-C18": Waters Co.).

Then, after washing with an appropriate amount of water, the sample was eluted with methanol to a size of 10 m. Next, liquid chromatography was performed using the column used in Example 2 under the following conditions.

The chromac drum at this time is shown in FIG.

Conditions: Eluent: 0.1 mol of sodium chloride dissolved in 0% acetonitrile, flow rate: 1.0 d/min,
Detection wavelength: 210 m In the figure, (A) shows a chromatogram when a sample prepared from dried ginseng (produced in China) was separated, and (B) shows a chromatogram when a standard was separated.
c, mRb2 and m-Rb1 are respectively malonylginsenonide (m)-RC, m-Rb2 and m-Rb1
(Chem, Ph-artBull, 31.3353
(1983)).

[Brief explanation of drawings]

In Figure 1, (A>) is a reproduction of the chromatogram obtained when the standard of saponin in Enmeihi was separated, and (B) is a reproduction of the chromatogram obtained when the sample was separated. Figure 3 is a reproduction of a chromatogram obtained when a standard mixture of saponins obtained from carrots was separated. (△) in Figure 3 is a reproduction of a chromatogram of saponins separated from insect-dried carrots. , (B) is a reproduction of the chromatogram when the saponin standard was separated. Applicant's agent Sato - Male retention vf closed (ge) (A) 1st retention @ open (Example (B) Figure retention Hours PP] (minutes) (A) 3rd maintenance (ke) (B) Figure

Claims (1)

[Claims] 1. A method for separating or purifying glycosides by liquid chromatography, characterized in that hydroxyapatite is used as a separating agent and a water-containing organic solvent or a salt-containing water-containing organic solvent is used as an eluent. Separation or purification method.