CN118388575A - A deglycosylated derivative of Pulsatilla saponin B4 and its preparation method and application - Google Patents

Abstract

The invention discloses a pulsatilla saponin B4 deglycosylated derivative, a preparation method and application thereof, wherein the pulsatilla saponin B4 (AB 4) is used as a raw material, and the derivative is prepared by selectively deglycosylating the sugar chains of C-3 and C-28 through alpha-L-rhamnosidase and glucosidase, so that a compound with higher anti-inflammatory activity and better clinical application value than AB4 is obtained. The selective deglycosylated derivative disclosed by the invention does not show obvious cytotoxicity to human macrophages, can obviously reduce the p-IκBa and p-p65 protein levels in an NF- κB signal path, has obvious effects on colonitis and atopic dermatitis, has obviously better drug effects than AB4, and is better than commercially available mesalazine which is a common drug for treating colonitis and dexamethasone which is a common drug for treating atopic dermatitis. Compared with the traditional acid-base method, the preparation method disclosed by the invention has selectivity on glycosyl, can flexibly adjust and control different products to be enriched according to the needs, has mild and green reaction conditions, and is a technical method capable of realizing large-scale industrialization.

Description

A deglycosylated derivative of Pulsatilla saponin B4 and its preparation method and application

Technical Field

The present invention belongs to the field of biomedicine technology, and relates to a preparation method of a deglycosylated derivative of scutellaria scutellaria saponin B4 and its application in anti-inflammatory drugs. Scutellaria scutellaria saponin B4 is selectively deglycosylated by a bioenzymatic method to reduce the molecular weight and water solubility, thereby enhancing the transmembrane ability, increasing the binding with the target protein pyruvate carboxylase, and improving the anti-inflammatory activity.

Background technique

Pulsatilla chinensis is a perennial herbaceous plant of the genus Pulsatilla in the family Ranunculaceae. Its dried root is used as medicine and was first recorded in the "Shennong Bencao Jing". It is cold in nature, bitter in taste, and enters the stomach and large intestine meridians. Pulsatilla chinensis is rich in pentacyclic triterpenoid saponin compounds, which can be divided into two types according to the structure of their aglycones: oleanane type and lupine type. Among them, pulsatilla saponin B4 (AB4) is the main representative of lupine type saponins. According to relevant literature reports, it has anti-inflammatory, antiviral and immunomodulatory effects and can be used to treat diseases such as colitis and eczema. Previous molecular mechanism studies have found that AB4 exerts anti-inflammatory effects by targeting pyruvate carboxylase in the mitochondrial matrix. However, the AB4 structure contains 5 sugar units, the molecular weight is too large, and the water solubility is extremely strong, resulting in poor transmembrane ability and difficulty in binding to pyruvate carboxylase in mitochondria. The anti-inflammatory activity needs to be further improved.

By reducing the number of sugar groups in the AB4 molecule, the molecular weight can be reduced, the water solubility can be reduced, and the transmembrane ability can be improved. However, the changes in the pharmacological activity of AB4 deglycosylated derivatives are unpredictable, and the activity may be lost, reduced, increased, or even produce cytotoxicity. Although there are a variety of AB4 deglycosylated derivatives in nature, their content is extremely small, and extraction and purification are difficult. AB4 can also produce a variety of AB4 deglycosylated derivatives through human intestinal bacterial metabolism, but this method cannot be used for production or enrichment. Chemical hydrolysis deglycosylation is not selective, and the product cannot be controlled. At present, there is no method for selective deglycosylation of AB4, and it cannot be mass-produced. Therefore, the pharmacological activity of AB4 deglycosylated derivatives has not been studied so far.

Summary of the invention

Existing anti-inflammatory drugs all have obvious toxic side effects, and some have problems such as short half-life and low bioavailability. The present invention discloses a preparation method of a deglycosylated derivative of scutellaria scutellariae saponin B4 and some applications thereof in anti-inflammatory treatment, including colitis and atopic dermatitis. Compared with scutellaria scutellariae saponin B4, the five deglycosylated derivatives obtained by the bioenzyme selective hydrolysis method have reduced molecular weight and water solubility, and in particular, the anti-inflammatory activity of two derivatives is significantly higher than that of scutellaria scutellariae saponin B4. At the same time, the bioenzyme method has mild conditions and is environmentally friendly.

The present invention adopts the following technical solution:

A deglycosylated derivative of Pulsatilla saponin B4 has the following general chemical structure:

In the formula, _R1 includes 3-O- α -L-rhamnopyranosyl-(1→2) -α -L-arabinopyranosyl and 3-O- α -L-arabinopyranosyl; _R2 includes α-L-rhamnopyranosyl-(1→4)-D-glucopyranose-(1→6)-D-glucopyranose, D-glucopyranose-(1→6)-D-glucopyranose and D-glucopyranose.

As an example, the deglycosylated derivative of scutellaria saponin B4 is AB4-1, AB4-2, AB4-3, AB4-4 or AB4-5. These deglycosylated derivatives cannot be obtained in large quantities from nature and must be selectively deglycosylated by AB4. If a chemical method is used, a certain deglycosylated compound cannot be selectively obtained, and the chemical method requires the use of a large amount of chemical reagents, which pollutes the environment to a certain extent. The present invention discloses a method for preparing the deglycosylated derivative of scutellaria saponin B4, using compound AB4 as a raw material, and using α-L-rhamnosidase and glucosidase to prepare the deglycosylated derivative of scutellaria saponin B4.

The invention discloses a pharmaceutical composition, which takes the deglycosylated derivative of pulsatilla saponin B4 as an active ingredient.

The present invention discloses the use of the above-mentioned deglycosylated derivative of Pulsatilla saponin B4 or a pharmaceutical composition in the preparation of an anti-inflammatory drug. Preferably, the inflammation includes inflammation on the surface of the body and inflammation in the body. As an example, the present invention discloses the use of the above-mentioned deglycosylated derivative of Pulsatilla saponin B4 or a pharmaceutical composition in the preparation of a drug for treating atopic dermatitis and colitis.

The present invention also discloses the use of the deglycosylated derivative of Pulsatilla saponin B4 or the pharmaceutical composition in the preparation of immunomodulatory drugs.

In the present invention, the drug includes topical, oral, rectal or parenteral drugs. The drug is prepared into pharmaceutically acceptable dosage forms, such as pills, tablets, powders, capsules, granules (powders), ointments, solutions, gels or suppositories, and solutions include pills, drops, sprays, injections, and suspensions.

The present invention discloses a deglycosylated derivative of pulsatilla saponin B4, a preparation method and an application thereof. The compound pulsatilla saponin B4 (AB4) is used as a raw material, and selective deglycosylated derivatives are prepared by α-L-rhamnosidase and glucosidase for the sugar chains of C-3 and C-28, thereby obtaining a compound with better anti-inflammatory activity and higher bioavailability than AB4. The partially deglycosylated derivative disclosed in the present invention does not show obvious cytotoxicity to human macrophages, can significantly reduce the levels of p-IκBa and p-p65 proteins in the NF-κB signaling pathway, has significant effects in colitis and atopic dermatitis, and has significantly better efficacy than AB4, and is also better than mesalazine, a common drug used in the market for colitis, and dexamethasone, a common drug used in atopic dermatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the preparation scheme of the deglycosylated derivative of Pulsatilla saponin B4 of the present invention.

Figure 2 shows the toxicity of AB4 deglycosylated derivatives to THP-1 cells at a concentration of 100 μmol/mL.

FIG3 is a Western blotting diagram of the anti-inflammatory activity of AB4 derivatives and the statistical results.

Figure 4 shows the back skin condition of mice with atopic dermatitis induced by dinitrochlorobenzene (DNCB).

FIG5 is a schematic diagram of the scoring of the mouse back.

FIG6 is a schematic diagram of mouse ear weight difference.

FIG. 7 is a schematic diagram of the thickness difference of mouse ears.

FIG8 is a schematic diagram of mouse spleen index.

FIG. 9 is a photograph of mouse colon and a statistical graph of colon length.

FIG. 10 is a schematic diagram of the weight change rate of mice.

FIG11 is a schematic diagram of the disease activity index (DAI) of mice.

Detailed ways

As a pentasaccharide triterpenoid saponin, scutellaria baicalensis saponin B4 differs from scutellaria baicalensis saponin D, another component of scutellaria baicalensis, in that the only structural difference between the two is the sugar chains at the C-3 and C-28 positions, and scutellaria baicalensis saponin D has no anti-inflammatory activity. Therefore, the sugar chain of AB4 is crucial to its activity. The present invention uses α-L-rhamnosidase and glucosidase to prepare low-sugar derivatives of scutellaria baicalensis saponin B4, which are easy to penetrate the membrane and bind to the target and have better activity.

The present invention discloses a method for preparing the deglycosylated derivative of pulsatilla saponin B4. Compound AB4 is used as a raw material, and α-L-rhamnosidase and glucosidase are used to prepare the deglycosylated derivative of pulsatilla saponin B4, AB4-1, AB4-2, AB4-3, AB4-4 or AB4-5, and the chemical structural formula is as follows:

;

;

;

;

AB4-1: 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-3β, 23-dihydroxylupane-Δ ²⁰⁽²⁹⁾ ene-28-OD-glucopyranose-(1→6)-D-glucopyranoside; AB4-2: 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-3β, 23-dihydroxylupane-Δ ²⁰⁽²⁹⁾ ene-28-OD-glucopyranoside; AB4-3: 3-O-α-L-arabinopyranosyl-3β, 23-dihydroxylupane-Δ ²⁰⁽²⁹⁾ ene-28-O-α-L-rhamnopyranose-(1→4)-D-glucopyranose-(1→6)-D-glucopyranoside; AB4-4: 3-O-α-L-arabinopyranosyl-3β,23-dihydroxylupane-Δ ²⁰⁽²⁹⁾ ene-28-OD-glucopyranose-(1→6)-D-glucopyranoside; AB4-5: 3-O-α-L-arabinopyranosyl-3β,23-dihydroxylupane-Δ ²⁰⁽²⁹⁾ ene-28-OD-glucopyranoside.

The following scheme is used to modify and transform the structure of AB4, that is, the preparation method of the deglycosylated derivative of Pulsatilla saponin B4 is as follows:

(1) Using AB4 as raw material, prepare pulsatilla saponin B4 with ultrapure water, and then use acidic buffer to prepare rhamnosidase solution, mix the two and react, inactivate at high temperature after the reaction, add solvent, centrifuge and take supernatant. Concentrate the supernatant, filter it with a microporous filter membrane, and pass it through a preparative liquid phase. Separate and purify it with a preparative liquid phase to obtain a dry powder.

(2) Using AB4 as raw material, prepare pulsatilla saponin B4 with ultrapure water, and then use acidic buffer to prepare rhamnosidase solution. Mix the two and inactivate them at high temperature after reaction. Add glucosidase, inactivate them at high temperature after reaction, add solvent, centrifuge and take supernatant. Concentrate the supernatant, filter it with microporous membrane, pass it through preparative liquid phase, separate and purify it by preparative liquid phase, and obtain dry powder.

Those skilled in the art can obtain the product of the present invention (lupane-type pentacyclic triterpenoid saponin compound) according to the raw materials and reaction conditions of the present invention according to conventional techniques, or adopt other methods to obtain the product of the present invention.

The present invention discloses the use of the above-mentioned deglycosylated derivative of Pulsatillae Saponin B4 in the preparation of anti-inflammatory drugs. In particular, the present invention discloses the use of the above-mentioned deglycosylated derivative of Pulsatillae Saponin B4 in the preparation of drugs for treating colitis and atopic dermatitis.

The pharmaceutical composition disclosed in the present invention uses the above-mentioned deglycosylated derivative of Pulsatilla saponin B4 as an active ingredient and also includes a pharmaceutically acceptable carrier. The active ingredient and the pharmaceutical composition are used to prepare a drug for atopic dermatitis. For example, the drug contains a therapeutically effective amount of the deglycosylated derivative of Pulsatilla saponin B4 or its hydrochloride, perchlorate, mesylate, phosphate, citrate or sulfate and a pharmaceutically acceptable carrier.

In the present invention, pharmaceutically acceptable carriers refer to one or more compatible solid or liquid fillers or gel substances that can be used for medicine, have sufficient purity and low toxicity, and the components in the pharmaceutical composition and the active ingredients of the present invention are mutually blended without reducing the efficacy of the active ingredients. Pharmaceutically acceptable carriers include diluents, solubilizers, latent solvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, adhesives, absorbents, wetting agents, buffers, and cross-linking agents. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), cyclodextrins (such as hydroxypropyl cyclodextrin), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

In the present invention, the drug includes topical, oral, rectal or parenteral drugs. The drug is prepared into pharmaceutically acceptable dosage forms, such as pills, tablets, powders, capsules, granules (powders), ointments, liquids, gels or suppositories, and liquids include pills, drops, sprays, injections, and suspensions.

The present invention discloses an application of a deglycosylated derivative of pulsatilla saponin B4 in the preparation of an anti-inflammatory drug. The derivative of the present invention can be administered alone or in combination with other therapeutic drugs. The administration method of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include topical, oral, rectal, parenteral (such as intravenous, intramuscular or subcutaneous), etc. Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules; liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain a diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oil, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, sesame oil and sesame oil or a mixture of these substances. In addition to these inert diluents; the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances. In addition to the active ingredient, the suspension may contain a suspending agent, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or a mixture of these substances. Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions. And sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The prior art discloses that scutellaria saponin B4 (AB4) has anti-inflammatory applications, but scutellaria saponin B4 is highly water-soluble, has a short half-life, and has low oral availability, which limits its clinical application. The present invention structurally modifies AB4 to obtain a compound with better anti-inflammatory activity and very low toxicity. The preparation method of the deglycosylated derivative of scutellaria saponin B4 of the present invention is shown in Figure 1, and the in vivo and in vitro activity results are shown in Figures 2-11.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, percentages and parts are weight percentages and weight parts. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. In the following preparation examples, the reagents are existing products, mainly provided by Shanghai Chemical Reagent Company; analytical liquid chromatographs (LC-20A, SPD-M20A, CBM-20A) are produced by Shimadzu Corporation of Japan, and the preparative liquid phase used for compound purification is produced by Suzhou Huitong Separation and Purification Co., Ltd. NMR was recorded using a Varian Mercury 400M NMR instrument, and chemical shifts were expressed in δ (ppm); statistical analysis of all data was performed using GraphPad Prism 8 software, and the differences between statistically significant means were compared by GraphPad Prism 8. All data were analyzed using one-way analysis of variance or two-way analysis of variance, and if the P value was 0.05, there was a significant difference.

Embodiment 1

AB4-1: Using AB4 as raw material, a 20 mg/ml pulsatilla saponin B4 solution was prepared with ultrapure water; a pH=4 buffer (prepared with disodium hydrogen phosphate and citric acid) was used to prepare an 80 mg/ml rhamnosidase solution, and the rhamnosidase solution was filtered with a vacuum pump to obtain a clear solution; the two solutions were then mixed and reacted at 50°C for 3 h; after the reaction was completed, the solution was inactivated at 100°C for 30 min, an equal volume of methanol was added, and the solution was centrifuged at 5000 rpm for 15 min to obtain the supernatant; the supernatant was concentrated to one tenth of the original volume, filtered through a 0.22 μm microporous filter membrane, and purified by preparative liquid phase separation to obtain AB4-1. ¹ H NMR (400 MHz, MeOD) δ 5.48 (d, J = 8.1 Hz, 1H), 5.16 (d, J = 1.4 Hz, 1H), 4.74 (s, 1H), 4.61 (s, 1H), 4.56 (d, J = 5.0 Hz, 1H), 4.35 (d, J = 6.7 Hz, 1H), 1.71 (s,3H), 1.25 (d, J = 6.2 Hz, 3H), 1.02 (s, 3H), 0.96 (s, 3H), 0.90 (s, 3H), 0.68(s, 3H). ¹³ C NMR (101 MHz, MeOD) δ 176.24, 151. 78,110.34, 104.70, 104.28,101.86, 95.21, 82.29, 78.19, 77.97, 77.84, 76.64, 75.10, 73.98, 73.91, 73.63,72.11, 71.99, 71.50, 70.90, 70 .15, 69.52, 69.11, 64.74, 64.56, 62.71, 57.93,51.93, 50.56, 49.71, 49.50, 49.28, 44.03, 43.61, 41.94, 39.91, 39.36, 37.79,37.58, 34.89, 32.85, 31.50, 30.80, 26.86, 26.69, 22.08, 19.55, 18.78, 17.96,17.28, 16.72, 15.14, 13.51.

AB4-4: Preparation method 1: Use AB4 as raw material and prepare 20 mg/ml nephrolepis saponin B4 solution with ultrapure water; use pH=4 buffer (prepared by disodium hydrogen phosphate and citric acid) to prepare 80 mg/ml rhamnosidase solution, and filter the rhamnosidase solution with a vacuum pump to obtain a clear solution; then mix the two solutions and react at 50°C for 5 h; after the reaction, inactivate at 100°C for 30 min, add an equal volume of methanol, and centrifuge at 5000 rpm for 15 min to obtain the supernatant; concentrate the supernatant to one tenth of the original volume, filter through a 0.22 μm microporous filter membrane, and prepare the liquid phase, spin-dry the separated components to obtain dry powder AB4-4. ¹ H NMR (400 MHz, MeOD) δ 5.50 (d, J = 8.1 Hz, 1H), 4.76 (s, 1H), 4.62(s, 1H), 4.37 (d, J = 7.8 Hz, 1H), 4.34 (d, J = 6.5 Hz, 1H), 1.72 (s, 4H),1 .03 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.70 (s, 3H). ¹³ C NMR (101 MHz, MeOD)δ 176.40, 151.85, 110.23, 106.13, 104.79, 95.34, 83.47, 78 .35, 78.07, 77.99,77.91, 75.20, 74.55, 74.09, 73.02, 71.68, 71.21, 69.77, 69.63, 66.63, 65.08,62.85, 58.03, 52.03, 50.71, 49.71 , 49.50, 49.28, 44.00, 43.69, 42.08, 39.80,39.48, 37.95, 37.62, 35.06, 32.93, 31.61, 30.84, 26.95, 26.45, 22.13, 19.60,18.88, 17.29, 16.81, 15.15, 13.18.

Preparation method 2: AB4 was used as the raw material, and a 20 mg/ml solution of Pulsatilla saponin B4 was prepared with ultrapure water; a 120 mg/ml solution of rhamnosidase 2 (compared with the rhamnosidase in preparation method 1, it has stronger selectivity for the rhamnose bond at the C-3 position) was prepared with a buffer solution of pH=6 (prepared with disodium hydrogen phosphate and citric acid), and the rhamnosidase solution was filtered with a vacuum pump to obtain a clear solution; the two solutions were then mixed and reacted at 60°C for 12 h; after the reaction was completed, the solution was inactivated at 100°C for 30 min, an equal volume of methanol was added, and the solution was centrifuged at 5000 rpm for 15 min to obtain the supernatant; the supernatant was concentrated to one tenth of the original volume, filtered through a 0.22 μm microporous filter membrane, and then purified by preparative liquid phase separation to obtain AB4-4.

Embodiment 2

AB4-3: Using AB4 as raw material, a 20 mg/ml pulsatilla saponin B4 solution was prepared with ultrapure water; a pH=4 buffer (prepared with disodium hydrogen phosphate and citric acid) was used to prepare an 80 mg/ml rhamnosidase solution, and the rhamnosidase solution was filtered with a vacuum pump to obtain a clear solution; the two solutions were then mixed and reacted at 50°C for 2 h; after the reaction was completed, the solution was inactivated at 100°C for 30 min, an equal volume of methanol was added, and the solution was centrifuged at 5000 rpm for 15 min to obtain the supernatant; the supernatant was concentrated to one tenth of the original volume, filtered through a 0.22 μm microporous filter membrane, and then purified by preparative liquid phase separation to obtain AB4-4. ¹ H NMR (400MHz, Methanol- d ₄ ) δ 5.50 (d, J = 8.1 Hz, 1H), 4.77 (d, J = 2.4 Hz, 1H), 4.67– 4.62 (m, 1H) , 4.42 (d, J = 7.9 Hz, 1H), 4.38 – 4.28 (m, ^{1 13} C NMR (101 MHz, MeOD) δ 176.33, 151.77, 1 10.44, 106.32, 104.54, 102.94,95.27, 83.25, 79.57, 78.26, 78.04, 76.87, 76.71, 75.30, 74.52, 74.00, 73.74,72.96, 72.43, 72.20, 70 .97, 70.67, 69.74, 69.55, 66.79, 64.76, 61.91, 57.96,51.96, 50.55, 49.71, 49.49, 44.00, 43.64, 41.99, 39.76, 39.38, 37.87, 37.62,34.95, 32.85, 31.53, 30.82, 26.85, 26.50, 22.10, 19.53, 18.81, 17.84, 17.29,16.76, 15.12, 13.22.

AB4-2: AB4 was used as the raw material, and a 20 mg/ml solution of Pulsatilla saponin B4 was prepared with ultrapure water; a pH=4 buffer solution was used to prepare an 80 mg/ml rhamnosidase solution, and the rhamnosidase solution was filtered with a vacuum pump to obtain a clear solution; the two solutions were mixed, reacted at 50°C for 4 h, and then inactivated at 100°C for 30 min, and glucosidase was added to make the glucosidase concentration in the system 20 mg/ml, reacted at 100°C for 3 h, and then inactivated at 100°C for 30 min, an equal volume of methanol was added, and the supernatant was obtained by centrifugation at 5000 rpm for 15 min; the supernatant was concentrated to one tenth of the original volume, filtered through a 0.22 μm microporous filter membrane, and then purified by preparative liquid phase separation to obtain AB4-3. ¹ H NMR (400 MHz, Methanol- d ₄ ) δ 5.53 (d, J = 8.1 Hz, 1H), 5.18 (d, J = 1.7 Hz, 1H), 4.75 (d, J = 2.4 Hz, 1H), 4.66 – 4.62 (m, 1H), 4.59(d, J = 4.7 Hz , 1H), 1.73 (s, 5H), 1.27 (d, J = 6.2 Hz, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.92 (s, 4H), 0.70 (s, 3H). ¹³ C NMR (101 MHz, MeOD) δ 176.17,151.84 , 110.27, 104.28, 101.89, 95.22, 82.29, 78.78, 78.40, 76.67, 74.09,73.94, 73.65, 72.15, 72.03, 71.13, 70.17, 69.11, 64.72, 64.59, 62 .40, 57.92,51.97, 50.62, 49.71, 49.50, 44.05, 43.63, 41.96, 39.93, 39.37, 37.81, 37.50,34.92, 32.80, 31.45, 30.80, 26.87, 26.69, 22.08, 19.51, 18.78, 17.95, 17.20,16.64, 15.14, 13.50.

AB4-5: AB4 was used as the raw material, and a 20 mg/ml solution of Pulsatilla saponin B4 was prepared with ultrapure water; a pH=4 buffer solution was used to prepare an 80 mg/ml rhamnosidase solution, and the rhamnosidase solution was filtered with a vacuum pump to obtain a clear solution; the two solutions were mixed, reacted at 50°C for 4 h, and then inactivated at 100°C for 30 min, and glucosidase was added to make the glucosidase concentration in the system 20 mg/ml, reacted for 2 h, and then inactivated at 100°C for 30 min, an equal volume of methanol was added, and the supernatant was obtained by centrifugation at 5000 rpm for 15 min; the supernatant was concentrated to one tenth of the original volume, filtered through a 0.22 μm microporous filter membrane, and then purified by preparative liquid phase separation to obtain AB4-3. ¹ H NMR (400 MHz, Methanol- d ₄ ) δ 5.53 (d, J = 8.2 Hz, 1H), 4.75 (d, J = 2.4 Hz, 1H), 4.66 – 4.62 (m, 1H), 4.34 (d, J = 6.6 Hz, 1H), 1.73 (s, 4H), 1. 04 (s, 3H), 0.99 (s, 3H), 0.92 (s, 4H), 0.71 (s, 3H). ¹³ C NMR (101MHz, MeOD) δ 176.17, 151.84, 110.27, 106.32, 95.22, 83.25, 78.78, 78.4 0,74.52, 74.08, 72.96, 71.13, 69.74, 66.79, 64.75, 62.39, 57.92, 51.98, 50.61,49.71, 49.28, 43.99, 43.63, 41.97, 39.76, 39.37, 37.8 6, 37.49, 34.96, 32.80,31.45, 30.79, 26.86, 26.50, 22.08, 19.51, 18.80, 17.22, 16.67, 15.13, 13.20.

Embodiment 3

The following experiments were carried out using Pulsatilla saponin B4 or its deglycosylated derivative as the experimental group drugs.

Cytotoxicity experiment: THP-1 cells were seeded in 96-well plates at 1×10 ⁴ cells/well, 100 μL/well, and cultured conventionally. When the cells reached 80%, they were induced with 100 ng/mL phorbol-12-Myristate-13-Acetate (PMA) for 12 h, then the original culture medium was discarded and 100 μL of new complete culture medium was added. A medium zeroing well without cells and a normal group without drugs were set up; 1 μL of AB4 derivative was added to each well to make the final drug concentration 100 μmol/mL, and the cells were placed in an incubator for 24 h. After the incubation, 10 μL of CCK-8 was added to each well and incubated in the dark for 4 h. The absorbance of each well was measured at a wavelength of 450 nm using an enzyme reader, and the cell survival rate of each well was calculated to verify whether the derivative was cytotoxic.

Calculation formula: Cell survival rate (%) = [A (drug addition) - A (blank)] / [A (0 drug addition) - A (blank)] × 100

A (drug added): absorbance of wells with cells, CCK-8, and drug solution

A (blank): absorbance of wells with culture medium and CCK-8 but no cells

A(0 drug addition): absorbance of wells with cells and CCK-8 but no drug addition.

As shown in Figure 2, the cytotoxicity of B4 derivatives at a concentration of 100 μmol/mL. The statistical graph without the symbol "*" indicates that there is no significant difference compared with the normal control, that is, the B4 derivatives do not have obvious cytotoxicity at a dose of 100 μmol/mL. It can be seen from the figure that there are many non-cytotoxic derivatives.

Embodiment 4

Western blotting experiment: THP-1 cells were seeded in 6-well plates at 2×10 ⁶ cells/well, 2 ml per well, and cultured conventionally. When the cells reached 80%, they were induced with 100 ng/mL PMA for 12 h, then the original culture medium was discarded and new complete culture medium was added. 2 μL of AB4 or its deglycosylated derivative was added to the experimental group to make the final concentration 10 μmol/mL, and the control group was the blank group; 1 h later, the blank group was removed and 2 μL of 1 mg/mL LPS was added to the other groups for incubation for 2 h. Then the following operations were performed on ice: the supernatant of the six-well plate was removed, and PBS pre-cooled at 4°C was gently added along the edge and washed twice; after adding 1 mL of PBS, the cells were scraped off with a cell scraper, placed in a 1.5 mL centrifuge tube, centrifuged at 2000 g at 4°C for 3 min, the supernatant was discarded, and the cell pellet was collected. 100 μL of RIPA lysis buffer (protease inhibitors and phosphatase inhibitors were added before use) was added to each tube, pipetted and mixed, and lysed on ice for 10 min. After the cells were broken again by ultrasonic disruptor, centrifuged at 12000 g and 4 °C for 10 min, the cell supernatant was carefully collected in a new EP tube and stored on ice. According to the instructions, the total protein content was measured and calculated using the BCA protein quantification kit. After the protein sample was diluted with PBS, 5× SDS-PAGE loading buffer (50 μL of mercaptoethanol was added per mL) was added to make the final protein concentration 2 μg/μL; it was denatured by boiling at 100 °C for 10 min to prevent protein degradation. SDS-PAGE gels of different concentrations were prepared according to the required protein molecular weight, and the protein samples were loaded on the gel at a content of 20 μg/well for electrophoresis. The separated protein samples were transferred to polyvinylidene fluoride (PVDF) membranes, and the PVDF membranes were washed 3 times with Tris-HCl buffer solution (TBST buffer) for 10 min each time. After blocking with protein blocking solution at room temperature for 1 h, wash with TBST buffer several times until the blocking solution is washed away, and incubate the PVDF membrane with specific primary antibodies overnight in a 4°C refrigerator according to the instructions. The next day, wash the PVDF membrane thoroughly with TBST buffer, bind with the corresponding secondary antibody at room temperature for 1 h, wash it thoroughly with TBST buffer again, and finally refer to the instructions of the ultra-sensitive ECL chemiluminescence kit for protein exposure and color analysis.

AB4 and its derivatives can inhibit the activation of key proteins in the NF-κB signaling pathway, as shown in Figure 3. The numbers on the horizontal axis in the figure represent different deglycosylated derivatives of scutellaria saponin B4. Western blotting was used to show the levels of p-IκBa and p-p65 proteins in THP-1 macrophages. It was found that after 2 h of LPS stimulation of THP-1 cells, the p-IκBa protein level in the model group increased significantly ( P <0.001); compared with the LPS model group, the compounds of the present invention can reduce the P-IκBa protein level, and the P-IκBa protein level of multiple derivatives is significantly reduced compared with AB4 ( p < 0.05). These results suggest that these derivatives have better anti-inflammatory activity. It was found that the two compounds AB4-4 and AB4-5 had better activity.

Embodiment 5

Therapeutic effect of deglycosylated derivatives of scutellaria saponin B4 on DNCB-induced atopic dermatitis in mice: Existing relevant literature reports that AB4 has an improving effect on the DNCB (2,4-dinitrochlorobenzene)-induced atopic dermatitis model in mice; the low-sugar derivatives are now further verified through in vivo experiments. The following experiment was designed. Balb/c mice were raised normally for about one week, with free food and water. The mice were randomly divided into normal control group, model group, dexamethasone positive drug group (3.3 mg/kg), scutellaria saponin B4 (AB4) positive drug group (6.6 mg/kg), AB4-4 administration group (1.65 mg/kg), AB4-4 administration group (3.3 mg/kg), AB4-4 administration group (6.6 mg/kg), AB4-5 administration group (1.65 mg/kg), AB4-5 administration group (3.3 mg/kg), and AB4-5 administration group (6.6 mg/kg) according to body weight. One day before the experiment, the back skin of the mice was shaved with a disposable skin preparation knife, and an area of about 3 cmx3 cm was selected for standby use. On the first day, except for the normal control group, the other groups were sensitized by applying 50 μL of 5% DNCB on the back of the mice: the same method was applied once on the second day, and sensitized again: on the third day, 1% DNCB50 μL was applied externally to the inside and outside of the right ear shell of the mice with a pipette for stimulation, and continued to stimulate on the fourth and fifth days, and stimulated for three consecutive days, and the same amount of acetone matrix was applied to the left ear shell. The standard of model success is that the DNCB solution repeatedly stimulates the right ear and back skin of the mice to different degrees of flushing, papules, blisters, erosion, exudation, scabs, and desquamation. Day 1-7: Dexamethasone group: 0.08 g of dexamethasone cream was applied to the back and inside and outside of the right ear shell at 4 pm; AB4, AB4-4 low, medium and high dose groups, AB4-5 low, medium and high dose groups: 200 μL of 70% ethanol-water solution (0.66 mg/ml) was applied to the back and inside and outside of the right ear shell at 10 am, and once at 4 pm. The model group: 200 μL of 70% ethanol-water solution was applied to the back and inside and outside of the right auricle at 10 am, and once at 6 pm. The treatment was continued for 7 consecutive days. On the 8th day, the mice were killed, and the spleen and ears of the mice were taken to calculate the spleen index and ear weight difference (a 6 mm diameter puncher was used to punch the same part to obtain ear discs, which were weighed).

During the experiment, the eczema of the back skin of each group of mice was observed and photographed every day (obvious redness, swelling, macules, erosion and exudation on the skin). The eczema area and severity index (EASI) scoring standard was used to refer to the clinical symptoms of the skin of each group of mice. The four indicators of erythema, papules/pustules, scaling and scabs were evaluated, and the scores were scored from 0 to 3 points: 0 points = no symptoms, 1 point = mild, 2 points = moderate, 3 points = severe, and the total score was obtained by adding up the scores of each indicator. Two observers scored in a blind manner on the 1st, 3rd, 5th and 7th days of intervention, and recorded by digital photography. 24 hours after the last administration, the thickness of both ears was measured with a vernier caliper (the average value was taken after taking 3 points for measurement), and the thickness difference was calculated: thickness difference = right ear thickness - left ear thickness; the body weight was weighed and recorded every day.

Figure 4 shows the back skin condition of mice with DNCB-induced atopic dermatitis; Figure 5 is a schematic diagram of the back scoring of mice; Figure 6 is a schematic diagram of ear weight difference; Figure 7 is a schematic diagram of mouse ear thickness difference; and Figure 8 is a diagram of mouse spleen index.

When local tissues are stimulated by DNCB, cellular mediators such as histamine are released, which dilate the capillaries of the ear skin and mucous membranes and increase the permeability of the capillary wall through _H1 and _H2 receptors, leading to edema. The experimental results showed that after the first application of DNCB on the third day, the auricle skin of the model group was slightly red compared with the blank group. With the continuous action of the drug, the redness and swelling of the model group became increasingly serious from the fourth day, and desquamation occurred. On the fifth day, exudation, ulceration and scabs began to form, and the eczema mouse model was successfully established. Compared with the model group, the ear swelling symptoms of the auricles of the AB4 group and its derivatives were significantly reduced during the same period, and there was almost no ulceration in the ears; the effect of the dexamethasone positive drug group was slightly worse, with some exudation and scabs. Compared with the model group, the skin lesions of the skin of the AB4 and AB4 derivatives administration groups were significantly reduced during the same period, the skin was smoother, the exudation was less, the scabs were mild or fell off the earliest; the skin scabs of the positive drug group were more serious, the scabs almost did not fall off, and there were obvious scales and papules. Among them, AB4 was not as effective as the derivatives. In addition, the experimental results showed that after 7 days of administration, the spleen index of the model group mice increased significantly, the spleen index of the deglycosylated derivative of scutellaria baicalensis B4 showed a downward trend, and the spleen index of the dexamethasone group was significantly lower than that of the normal group compared with the model group, indicating that dexamethasone produced immunosuppression in mice, while the low-sugar derivative of scutellaria baicalensis B4 could improve the immunity of mice. All experimental results suggest that the low-sugar derivative of scutellaria baicalensis B4 has a protective effect on eczema lesions caused by DNCB, and the effect is better than that of scutellaria baicalensis B4 and glucocorticoids.

Embodiment 6

Therapeutic effect of the deglycosylated derivative of scutellaria saponin B4 on DSS-induced colitis in mice: Existing relevant literature reports that AB4 has an improving effect on the DSS (dextran sulfate sodium)-induced mouse atopic dermatitis model. The deglycosylated derivative is now further verified by in vivo experiments. The following experiment was designed: C57BL/6J mice were raised normally for about a week, with free food and water. The mice were randomly divided into a normal group, a model group (DSS group), an AB4 (100 mg/kg) group, a mesalazine (200 mg/kg) group, an AB4-4 administration group (5 mg/kg), an AB4-4 administration group (10 mg/kg), an AB4-4 administration group (20 mg/kg), an AB4-5 administration group (5 mg/kg), an AB4-5 administration group (10 mg/kg), and an AB4-5 administration group (20 mg/kg). The mice were weighed one day before modeling and pre-administered according to body weight. Then the drinking water of the model group and the administration group was replaced with 3% DSS. After modeling, mice were weighed every day, and drugs were administered according to their body weight, once a day. Mice in the normal group and model group were gavaged with drinking water once a day.

Colon length, body weight and disease activity index (DAI) score are important indicators for evaluating inflammatory bowel disease (IBD). After the 3% DSS model was successfully established, the daily body weight and DAI score of the mice were statistically analyzed. 24 hours after modeling, except for the normal control group mice, the other groups showed symptoms such as poor mental state, less smooth fur, and loss of appetite. The colon length is shown in Figure 9. After DSS modeling, the colon structure of the mice was destroyed, the mucosa was congested and adhered to the surrounding tissues, and the colon length was significantly shortened. The low, medium and high dose groups of AB4-4 and AB4-5 can increase the colon length of the modeled mice to varying degrees, among which the high dose group of AB4-5 was significantly different from the DSS group ( P <0.05). As shown in Figure 10, the weight of the mice in the model group continued to decrease during the development of the disease. The weight began to decrease compared with the normal group on the 5th day after modeling, and the weight on the 7th day was significantly different from the normal control group ( P <0.01). The AB4 and AB4 derivative treatment groups significantly improved the phenomenon of weight loss. The body weight of mice treated with AB4 derivatives began to recover from the 7th day after modeling compared with the model group.

The disease activity index (DAI) is a comprehensive score based on the percentage of weight loss, stool viscosity, and blood in the stool of mice. The scores of the three indicators are added together to obtain the DAI score. The higher the total DAI score, the more severe the inflammatory bowel disease. In order to evaluate the severity of the disease in each group of mice, the feces of each mouse were collected at the same time every day, the shape of the feces was observed, and the fecal occult blood was detected with reagents. After modeling, the mice in the 3% DSS group had loose stools, blood in the stool, or occult blood, while the conditions of the AB4 treatment group and the AB4 derivative treatment group improved, gradually changing from loose stools with blood to loose occult blood, and even normal; as shown in Figure 11, except for the mice in the normal control group, the DAI scores of the mice in the other groups increased significantly on the second day after modeling, decreased on the third day, and then increased. The DAI score of the DSS group was always higher than that of the drug-treated group. On the 4th day, the DAI scores of the AB4 treatment group, AB4 derivative and mesalazine group began to decline compared with the model group, and the situation improved. The AB4 derivative was better than the mesalazine group and AB4 group.

As a pentaglycosyl triterpenoid saponin, Pulsatilla saponin B4 differs from Pulsatilla saponin D, another component of Pulsatilla, in that the only difference in structure between the two is the sugar chains at C-3 and C-28, and Pulsatilla saponin D has no anti-inflammatory activity, so the sugar chain of AB4 is crucial to its activity. The present invention uses α-L-rhamnosidase and glucosidase to prepare the deglycosylated derivative of Pulsatilla saponin B4, which is easy to permeate the membrane and bind to the target, and has better activity. Compared with the chemical method, the enzymatic method has selectivity for sugar groups, mild conditions, adjustable reactions, environmental friendliness, high conversion efficiency, and can realize the industrial production of AB4 deglycosylated derivatives.

Claims (10)

1. A pulsatilla root saponin B4 deglycosylated derivative has the following chemical structural general formula:

；

wherein R ₁ comprises 3-O-alpha-L-rhamnopyranosyl- (1- > 2) -alpha-L-arabinopyranosyl or 3-O-alpha-L-arabinopyranosyl; r ₂ includes α -L-rhamnopyranose- (1.fwdarw.4) -D-glucopyranose- (1.fwdarw.6) -D-glucopyranosyl, D-glucopyranose- (1.fwdarw.6) -D-glucopyranosyl or D-glucopyranosyl.

2. The pulsatilla saponin B4 deglycosylated derivative according to claim 1, characterized in that the pulsatilla saponin B4 deglycosylated derivative is AB4-1, AB4-2, AB4-3, AB4-4 or AB4-5.

3. The method for preparing the pulsatilla chinensis saponin B4 deglycosylated derivative according to claim 1, which is characterized in that the pulsatilla chinensis saponin B4 deglycosylated derivative is prepared by using a compound AB4 as a raw material and adopting glycosidase hydrolysis.

4. A pharmaceutical composition comprising the deglycosylated derivative of pulsatilla chinensis saponin B4 of claim 1 as an active ingredient.

5. Use of the deglycosylated derivative of pulsatilla saponin B4 as claimed in claim 1 or the pharmaceutical composition as claimed in claim 4 for the preparation of an anti-inflammatory medicament.

6. The use according to claim 5, wherein the inflammation comprises body surface inflammation, in vivo inflammation.

7. The use according to claim 6, wherein the inflammation comprises colitis, atopic dermatitis.

8. Use of a deglycosylated derivative of pulsatilla saponin B4 according to claim 1 or a pharmaceutical composition according to claim 4 for the preparation of an immunomodulatory drug.

9. The use according to any one of claims 5 to 8, wherein the medicament comprises a topical, oral, rectal or parenteral medicament.

10. The use according to claim 9, wherein the pharmaceutical dosage form comprises a pill, tablet, powder, capsule, granule, paste, solution, injection, gel or suppository.