CN114644676A

CN114644676A - Method for separating luffa glycoside C from ovate leaf holly bark

Info

Publication number: CN114644676A
Application number: CN202011517277.3A
Authority: CN
Inventors: 司红彬; 柴贝贝; 王莉贞; 夏娟; 张歌音; 李崇; 赖重波
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2022-06-21
Anticipated expiration: 2040-12-21
Also published as: CN114644676B

Abstract

The invention discloses a method for separating luffa glycoside C from ovate leaf holly bark, which comprises the following steps: sequentially soaking cortex Ilicis Rotundae in ethanol and water, mixing filtrates, and concentrating to obtain extract; extracting the extract, concentrating and drying; dissolving the extract, and performing gradient elution on the sample in macroporous adsorption resin to obtain four fractions; sequentially loading the target fraction of the four fractions onto a chromatographic column I and a chromatographic column II, performing gradient elution, respectively dropping the obtained samples on a thin-layer plate, respectively collecting the fraction I and the fraction II according to a specific displacement value, preparing the fraction II by using semi-preparative high performance liquid chromatography, and collecting the fraction III, namely the luffa glycoside C. The invention can separate the luffa glycoside C from the ovate leaf holly bark, provides reference basis for the basic research of the ovate leaf holly bark substance, the bacteriostasis mechanism of the bacteriostasis part and the clinical application research, and opens up a new path for the preparation source of the luffa glycoside C.

Description

Method for separating luffa glycoside C from ovate leaf holly bark

Technical Field

The invention belongs to the technical field of Chinese herbal medicine separation and extraction, and particularly relates to a method for separating luffa glycoside C from ovate leaf holly bark.

Background

The cortex Ilicis Rotunda Thunb is dry bark of Ilex rotunda Thunb of Aquifoliaceae, mainly produced in two regions, and is obtained by peeling trunk of Ilex rotunda in summer and autumn, removing impurities, cleaning, moistening, slicing, and sun drying. It is cold in nature and bitter in taste, and enters lung, stomach, large intestine and liver meridians. Has effects of clearing away heat and toxic materials, promoting diuresis, and relieving pain, and can be used for treating common cold, fever, tonsillitis, sore throat, acute and chronic pharyngitis, gastric and duodenal ulcer, rheumatic arthralgia, traumatic injury, and scald caused by hot water and fire.

With the recent intensive research on the traditional Chinese medicine ovate leaf holly bark, the chemical components thereof are being separated, and can be roughly classified into triterpenes, phenolic glycosides, flavonoids, aromatics, steroids, aldehydes and the like.

Triterpenoids separated from the ovateleaf holly bark medicinal material mainly comprise the following components: oleanolic acid (oleanolic acid), 3-acetyl oleanolic acid (3-O-acetyl oleanolic acid), 19 alpha-dihydroxyurs-12-en-3-one-28-oic acid, 3 beta, 19 alpha-dihydroxyurs-12-en-24, 28-dioic acid, Friedelin, 28-hydroxy-Friedelin, 3-hydroxy oleanane (3 beta-OH-oleanane), iron wintergreen (Rotundic acid), iron wintergreen isopropenylketal (Rotundic acid), long ridges of wintergreen (Pendulcoside), 3-O-alpha-L-rhamnopyranose- (1-2) -beta-D-glucopyranosyl-56- (1-2) -beta-D-glucopyranosyl-1-beta-2-D-arabinopyranose-28-D-381-beta-25-beta-O-anhydroxanthene-25-D-28-D-beta-linolenic acid, 3-O- [ alpha-L-rhamnopyranosy 1- (1 → 2) -beta-D-glucopyranosy-pyranosy 1(1 → 2) -beta-D-arabinopyranosyl 1] -3 beta, 19 alpha-dihydrours-12-en-28-oic-O-beta-D-glucopyranosy 1 ester.

In the fifties of the twenty century, the aromatic compounds separated from the ovate leaf holly bark are separated into the ovate leaf holly bark glucoside A of carbohydrate ligand, namely the later discovered Syringin (Syringin) is most representative, in the nineties, Sinapaldehyde glucoside (Sinapaldehyde glucoside) is separated from the acetone extract of the ethanol extract of the dry coarse powder of the ovate leaf holly bark, and then two phenolic glycoside compounds are separated from the ovate leaf holly bark medicinal material, namely, the ovate leaf holly bark alcohol (rotundaol) and the new compound of bissyringin ether (Disingin ether). Other aromatic compounds found in ovate leaf holly bark include sinapildehyde (sinapaldehyde), syringaldehyde (syringaldehyde), caffeic acid 4-O-beta-D-glucopyranoside, vanillic acid 4-O-beta-D-glucopyranoside, diisobutyl phthalate (Di-isobutryl phthalate), phloroglucinol, Cis-methyl isobutyl alcohol (Cis-methyl.

Researches show that two lignin compounds, syringaresinol 4' -O-beta-D-glucopyranoside and syringaresinol 4', 4' -O-beta-D-glucopyranoside, are separated from the ovateleaf holly bark medicinal material.

In addition to the above-mentioned types of compounds, the rotundine also contains steroid compounds: beta-sitosterol (beta-sitosterol), beta-daucosterol (beta-daucosterol) and fatty acid compounds: stearic acid (stearic acid), nonadecanoic acid (nonadecanoic acid), etc., Glucose (Glucose), Inositol (Inositol), Mellein (Mellein), palmitic acid (Hexadecanoic acid), 9,12-Octadecadienoic acid methyl ester (9, 12-octadienoic acid methyl ester), etc.

At present, researches find that the ovate leaf holly bark ethanol extract and the water extract have strong inhibition effects on staphylococcus aureus and beta hemolytic streptococcus; the ethanol extraction part of the ovateleaf holly bark can effectively inhibit the mouse ear swelling induced by dimethylbenzene, which shows that the ovateleaf holly bark has a remarkable anti-inflammatory effect. The ovateleaf holly bark total flavone extracted by soaking in 60% ethanol solution has antibacterial effect on escherichia coli (MIC 80mg/mL), streptococcus (MIC 80mg/mL), pseudomonas aeruginosa (MIC 80mg/mL), enterococcus faecalis (MIC 40mg/mL), proteus vulgaris (MIC 40mg/mL), staphylococcus aureus (MIC 20mg/mL) and acinetobacter baumannii (MIC 10 mg/mL). Researches show that after dry products and wet products of the ovate leaf holly bark are respectively soaked and extracted, the extracting solution is respectively acted on escherichia coli, typhoid bacillus and staphylococcus aureus, and the result shows that the bacteriostatic effect of the fresh extracting solution is superior to that of the dry extracting solution. In the aspect of the research of the anti-bacteria mechanism of the ovate leaf holly bark, the total flavonoids of the ovate leaf holly bark and the water extract of the ovate leaf holly bark are respectively found to increase the cell wall cell membrane permeability of escherichia coli producing ESBLs, reduce the DNA synthesis amount, increase the apoptosis proportion and reduce the total protein synthesis, and the research preliminarily discloses the anti-bacteria mechanism of the ovate leaf holly bark extract. In the aspect of research on anti-inflammatory mechanism, the purified ovateleaf holly bark extract extracted by an optimal process can obviously inhibit the activities of glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) in the serum of a mouse, can obviously inhibit the generation of Malondialdehyde (MDA), and can also reduce the expression level of a key enzyme cyclooxygenase-2 of an inflammatory mediator, so that the synthesis level of the inflammatory mediator is reduced to play an anti-inflammatory role. In addition, research finds that the component Rotendaprene in ovate leaf holly bark can inhibit the activity of TLR-2 in Akt and NF-kB channels to reduce the generation of inflammatory mediators, namely active oxygen, in keratinocytes, thereby playing an anti-inflammatory role.

Because the chemical components of the ovate leaf holly bark are very complex, the separation method of each compound is also relatively complex, so far, more compound components which are not separated and identified are still available, the bacteriostatic mechanism and the bacteriostatic component of the ovate leaf holly bark are not completely disclosed, and related reports of separating the luffa glycoside C from the ovate leaf holly bark are not seen.

Disclosure of Invention

Aiming at the problems, the invention provides a method for separating the luffa glycoside C from the ovate leaf holly bark, which can separate the luffa glycoside C from the ovate leaf holly bark and provides a reference basis for the basic research of the materials of the ovate leaf holly bark, the bacteriostatic mechanism of bacteriostatic parts and the clinical application research.

The technical scheme of the invention is as follows:

a method for separating luffa glycoside C from cortex Ilicis Rotundae comprises the following steps:

s1: soaking the ovateleaf holly bark in 75-85% ethanol solution, extracting at normal temperature for 2-4 days, filtering to obtain filter residue, soaking in 45-55% ethanol solution, extracting at normal temperature for 2-4 days, filtering again to obtain filter residue, soaking in purified water, extracting at normal temperature for 2-4 days, combining the three filtrates, and concentrating to obtain extract;

s2: sequentially extracting the extract with petroleum ether, ethyl acetate, n-butanol and water, and concentrating and drying each extraction layer;

s3: dissolving the extract of the water extractant layer in water, then loading the extract on macroporous adsorption resin, and carrying out gradient elution to obtain four fractions;

s4: dissolving a target fraction in the four fractions in water, loading the solution into a chromatographic column I for chromatographic separation, spotting the obtained sample on a thin-layer plate, and collecting the fraction I;

s5: dissolving fraction I, loading the sample into a chromatographic column II, performing gradient elution, spotting the obtained sample on a thin-layer plate, and collecting fraction II; and (3) preparing the fraction II by using a semi-preparative high performance liquid chromatography, and collecting the fraction III to obtain the luffa glycoside C.

Further, in step S3, the macroporous adsorbent resin is eluted with 0-90% methanol solution with a gradient of 30%.

Further, in step S4, the target fraction is a fraction eluted with a 60% methanol solution.

Further, in step S4, the column i is an MCI column, and pure water is used as an eluent.

Furthermore, the fraction I is a fraction with a specific displacement value of 0.26-0.70 in 20% methanol solution eluent.

Further, in the step S5, the chromatographic column ii is a C-18 chromatographic column, a 25-100% methanol solution is used as an eluent, and 5% is a gradient.

Furthermore, the fraction II is a fraction with a specific displacement value of 0.11-0.23 in 60-90% methanol solution eluent.

Further, in the step S5, the semi-preparative hplc is performed under conditions of a detection wavelength of 210nm and a mobile phase of 30% to 55% acetonitrile.

Further, the flow part III is the flow part of the third peak.

The concept of the ratio shift value described herein is: the ratio of the distance from the center of the compound spot to the origin to the distance from the solvent front to the origin is the specific shift value of the compound. After unfolding separation, the position of the compounds on the lamella plates is indicated by the specific shift value (Rf value).

The percentage amounts of the solutions described herein are all volume percentages.

The invention has the beneficial effects that:

1. the method can confirm the bacteriostatic part of the ovate leaf holly bark, sequentially extracts different polar parts of the ovate leaf holly bark by adopting petroleum ether, ethyl acetate, n-butyl alcohol and water, performs chromatographic separation on a water extraction layer through macroporous adsorption resin, combines multiple times of chromatographic analysis and point plate operation by selecting a proper chromatographic column, can separate the luffa glycoside C from the water extraction layer, provides a reference basis for basic research of the ovate leaf holly bark substance, bacteriostatic mechanism and clinical application research of the bacteriostatic part, and opens up a new path for preparing the luffa glycoside C.

2. The invention identifies the separated luffa glycoside C, as shown in example 4, the structure is determined, and the luffa glycoside C is confirmed; antibacterial tests are also carried out on the extract of different polarity parts of the ovateleaf holly bark, as shown in example 5, the antibacterial component is confirmed to be positioned in the water extraction layer, and the luffa glycoside C is positioned in 60% methanol eluent of the water extraction layer, and the eluent also has antibacterial activity, so that the luffa glycoside C is positioned in the antibacterial component.

3. The method has the advantages of simple operation, better continuity, stability and universality, good separation effect, mild condition and low cost, and can be suitable for separating and extracting various compounds.

Detailed Description

The present invention will be described in detail below with reference to specific examples.

Example 1

s1: soaking cortex Ilicis Rotundae in 75% ethanol solution, extracting at room temperature for 2 days, filtering to obtain residue, soaking in 45% ethanol solution, extracting at room temperature for 2 days, filtering to obtain residue, soaking in purified water, extracting at room temperature for 2 days, mixing the three filtrates, and concentrating to obtain extract;

s3: dissolving the extract of the water extraction layer in water, loading the extract on macroporous adsorption resin, and performing gradient elution by using 0-90% methanol solution, wherein 30% is a gradient to obtain four fractions;

s4: dissolving fractions eluted by a 60% methanol solution in the gradient elution in water, loading the fractions into an MCI chromatographic column, performing gradient elution by using a 10% -100% methanol solution, wherein 10% is a gradient, spotting a sample eluted by a 20% methanol solution on a thin-layer plate, and collecting fractions I with a specific displacement value of 0.26-0.70;

s5: dissolving the fraction I, loading the solution into a C-18 chromatographic column, performing gradient elution by adopting a methanol solution with the concentration of 25-100%, wherein 5% is a gradient, eluting a sample obtained by using a methanol solution with the concentration of 60-90% on a thin-layer plate, and collecting the fraction II with the specific displacement value of 0.11-0.23; and (3) preparing the fraction II by using a semi-preparative high performance liquid chromatography under the conditions that the detection wavelength is 210nm and the mobile phase is 30-55% of acetonitrile, and collecting the fraction III of the third peak, namely the luffa glycoside C.

Example 2

s1: soaking cortex Ilicis Rotundae in 80% ethanol solution, extracting at room temperature for 3 days, filtering to obtain residue, soaking in 50% ethanol solution, extracting at room temperature for 3 days, filtering to obtain residue, soaking in purified water, extracting at room temperature for 3 days, mixing the three filtrates, and concentrating to obtain extract;

s2 to S5 are the same as in example 1.

Example 3

s1: soaking cortex Ilicis Rotundae in 85% ethanol solution, extracting at room temperature for 4 days, filtering to obtain filter residue, soaking in 55% ethanol solution, extracting at room temperature for 4 days, filtering to obtain filter residue, soaking in purified water, extracting at room temperature for 4 days, mixing the filtrates, and concentrating to obtain extract;

s2 to S5 are the same as in example 1.

Example 4 identification experiment

The structure of the luffa glycoside C isolated in examples 1-3 was determined by physicochemical properties and spectral characteristics.

The luffa glycoside C separated in examples 1-3 is identified to be a light yellow crystal, a cluster peak exists between delta H0.60-1.50, and the luffa glycoside C contains 7 methyl proton signals, and the signals from low field to high field are respectively 1.30, 1.25, 1.20, 1.13, 1.01, 0.98 and 0.86, which indicates that the compound may be a sterol or terpenoid compound. Hydrogen spectrum data δ: 5.02(1H, d, J ═ 8Hz, H-1 '), 6.33(1H, d, J ═ 8.2Hz, H-1'), carbon spectrum data δ:96.0(C-1 '), 74.4 (C-2'), 79.6(C-3 '), 71.3 (C-4'), 79.1(C-5 '), 62.4 (C-6'), 105.6(C-1 '), 77.3 (C-2'), 78.6(C-3 '), 71.9 (C-4'), 78.2(C-5 '), 62.9 (C-6') indicate that the structure contains two sets of sugar segments, and the carbon spectrum data show 1 carboxyl carbon signal delta.7, 1 pair carbon double bond signal delta 144.1 and delta 122.7.¹H NMR(500MHz,MeOD)δ5.02(1H，d,J＝8Hz，H-1′,glc at C-3)，5.49(1H，dd,J＝5.4,Hz，H-12)，6.33(1H，d,J＝8.2Hz,H-1′，glc at C-28),1.30(3H，s),1.25(3H，s),1.20(3H，s),1.13(3H，s),1.01(3H，s),0.98(3H，s),0.86(3H，s)。¹³C NMR (125MHz, MeOD): 38.9(C-1), 26.9(C-2), 89.6(C-3), 39.8(C-4), 56.0(C-5), 19.8(C-6), 33.4(C-7), 40.1(C-8), 47.2(C-9), 37.1(C-10), 23.9(C-11), 123.1(C-12), 144.0(C-13), 42.4(C-14), 28.5(C-15), 23.6(C-16), 48.2(C-17), 42.0(C-18), 46.5(C-19), 36.0(C-20), 73.4(C-21), 41.5(C-22), 28.4(C-23), 15.8(C-24), 14.5(C-25), 17.7(C-26), 26.4(C-27), 176.7 (C-29), 31.31), 17.0(C-30), glc at C-28:96.0(C-1 '), 74.4 (C-2'), 79.6(C-3 '), 71.3 (C-4'), 79.1(C-5 '), 62.4 (C-6'), glc at C-3:105.6(C-1 '), 77.3 (C-2'), 78.6(C-3 '), 71.9 (C-4'), 78.2(C-5 '), 62.9 (C-6'). The above data are found in the literature "Lianglong, Liuchang yoga, Liguanyu, et al Lucyoside Q, a Triterpenoid Saponin from Leaves of Luffa cylindrica (L.). M.Roem [ J. ]]Journal of Chinese Pharmaceutical Sciences,1997(4):225-227 ", was identical and was identified as luffa glycoside C, which was first isolated from the plant and has the structure

Example 5 determination of bacteriostatic Activity of the bacteriostatic parts of Ovateleaf Holly bark

1. Experimental methods

Taking multi-drug-resistant Escherichia coli as a test bacterium, carrying out bacteriostatic activity determination on fractions, carrying out an experiment by a two-fold microdilution method, and referring to the description of CLSI for operation method and result judgment.

2. Preparation of samples

Experimental group 1: under the aseptic condition, dissolving each extraction layer of the step S2 in the example 2 by using 5 percent DMSO and sterilized water to respectively prepare mother liquor with the concentration of 1 g/mL;

experimental group 2: under the aseptic condition, dissolving the four fractions eluted by the macroporous adsorption resin of the step S3 in the example 2 by using 5 percent DMSO and sterilized water, and respectively preparing mother liquor with the concentration of 80 mg/mL;

blank control: sterile water containing 5% DMSO.

3. Preparation of E.coli suspension

Inoculating Escherichia coli colony on Macconkey agar culture medium under aseptic condition, inoculating into NB broth, culturing at 37 deg.C and 220rpm for 2-6 hr, adjusting bacteria solution to 0.5 McLeod turbidity with sterile NB broth, and diluting with bacteria concentration of about 10: 1000⁵CFU/mL, diluted bacterial liquid was used immediately.

4. Results of the experiment

(1) The results of the determination of the bacteriostatic activity of different polarity portions of ovateleaf holly bark in step S2 of example 2 are shown in Table 1.

TABLE 1 minimal inhibitory concentration of different polarity sites of cortex Ilicis Rotundae

Sites of different polarity	Blank space	Petroleum ether layer	Ethyl acetate layer	N-butanol layer	Aqueous layer
						MIC	+	+	+	+	0.5(g/mL)

Note: "+" indicates no bacteriostatic effect.

As can be seen from Table 1, only the water layer had inhibitory effect on the multi-drug resistant Escherichia coli, and the petroleum ether layer, ethyl acetate layer and n-butanol layer had no inhibitory effect on the multi-drug resistant Escherichia coli.

(2) The results of the determination of the bacteriostatic activity of each component of the water extraction layer of cortex Ilicis Rotundae in example 2 of the present invention are shown in Table 2.

TABLE 2 minimum inhibitory concentration of the fractions of the water extraction layer of Ilicis Rotundae

Run out	Blank space	0% methanol eluent	30% methanol eluent	Eluent of 60% methanol	90% methanol eluent
						MIC	+	+	+	40(mg/mL)	+

Note: "+" indicates no bacteriostatic effect

As can be seen from Table 1, the fractions eluted with 60% methanol all have inhibitory effect on multi-drug resistant Escherichia coli, and the other fractions have no inhibitory effect on the multi-drug resistant Escherichia coli.

Therefore, the luffa glycoside C separated and extracted by the method exists in the water extraction layer with bacteriostatic activity of the ovate leaf holly bark and 60% methanol eluent of the water extraction layer.

Claims

1. A method for separating luffa glycoside C from ovate leaf holly bark is characterized by comprising the following steps:

s3: dissolving the extract of the water extraction layer in water, then loading the extract on macroporous adsorption resin, and carrying out gradient elution to obtain four fractions;

s4: dissolving a target fraction in the four fractions in water, loading the solution into a chromatographic column I, carrying out chromatographic separation, spotting the obtained sample on a thin-layer plate, and collecting the fraction I;

s5: dissolving fraction I, loading the solution into a chromatographic column II, performing gradient elution, spotting the obtained sample on a thin-layer plate, and collecting fraction II; and (3) preparing the fraction II by using a semi-preparative high performance liquid chromatography, and collecting the fraction III to obtain the luffa glycoside C.

2. The method for isolating luffa glycoside C from ovate leaf holly bark of claim 1, wherein: in the step S3, the macroporous adsorption resin adopts 0-90% methanol solution as eluent, and 30% is a gradient.

3. The method of claim 2, wherein the step of isolating luffa glycoside C from ovate leaf holly bark comprises: in step S4, the target fraction is a fraction eluted with a 60% methanol solution.

4. The method of claim 1, wherein the step of isolating luffa glycoside C from ovate leaf holly bark comprises: in step S4, the column i is an MCI column, and pure water is used as the eluent.

5. The method of claim 4, wherein the step of isolating luffa glycoside C from ovateleaf holly bark comprises: the fraction I is a fraction with a specific displacement value of 0.26-0.70 in the water eluent.

6. The method for isolating luffa glycoside C from ovate leaf holly bark of claim 1, wherein: in the step S5, the chromatographic column II is a C-18 chromatographic column, a 25-100% methanol solution is used as an eluent, and a gradient is formed by 5%.

7. The method for isolating luffa glycoside C from ovateleaf holly bark of claim 6, wherein: the fraction II is the fraction with the specific displacement value of 0.11-0.23 in the eluent of 60-90% methanol solution.

8. The method for isolating luffa glycoside C from ovate leaf holly bark of claim 1, wherein: in the step S5, the semi-preparative high performance liquid chromatography is performed under the detection wavelength of 210nm and the mobile phase of 30-55% acetonitrile.

9. The method of claim 8, wherein the step of isolating luffa glycoside C from ovate leaf holly bark comprises: and the flow part III is the flow part of the third peak.