CN114685580A - Flavonoid glycoside compound extracted and separated from herba Aconiti Bonga and using quercetin as aglycone, and method and application thereof - Google Patents

Flavonoid glycoside compound extracted and separated from herba Aconiti Bonga and using quercetin as aglycone, and method and application thereof Download PDF

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CN114685580A
CN114685580A CN202011564143.7A CN202011564143A CN114685580A CN 114685580 A CN114685580 A CN 114685580A CN 202011564143 A CN202011564143 A CN 202011564143A CN 114685580 A CN114685580 A CN 114685580A
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flavonoid glycoside
quercetin
bonga
herba aconiti
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李波
彭成
曹治兴
闫婕
郭大乐
钟钰
肖斌
黄龙
张巨琼
刘丁
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Chengdu Push Bio Technology Co ltd
Chengdu University of Traditional Chinese Medicine
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Abstract

The invention discloses a flavonoid glycoside compound which is extracted and separated from herba aconiti tangutici and takes quercetin as aglycone, a method and application thereof, wherein the structural formula is shown as (I). The flavonoid glycoside compound is obtained by taking dry herba Aconiti Bonga as a raw material and performing extraction and separation through the steps of methanol ultrasonic extraction, reduced pressure concentration, AB-8 resin column chromatography, C18 reversed phase chromatographic separation and the like, is a novel flavonoid compound with novel structure and pharmacological activity, can be applied to the preparation of anti-inflammatory drugs, and provides reliable basis for the preparation of new Tibetan drugs, the pharmaceutical research of flavonoid substances and the like.
Figure 100004_DEST_PATH_IMAGE002

Description

Flavonoid glycoside compound extracted and separated from herba Aconiti Bonga and using quercetin as aglycone, and method and application thereof
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to a flavonoid glycoside compound which is extracted and separated from herba aconiti tangutici and takes quercetin as aglycone, and a method and application thereof.
Background
Herba Aconiti Bonga is a common Tibetan medicine, and is derived from dried whole plant of Stapf of Aconitum kusnezoffii (Maxim.) belonging to Aconitum of Ranunculaceae (another source of herba Bonga is dried whole plant of Stapf of Aconitum naviculare (Bruhl.) belonging to Aconitum of Ranunculaceae). Originally recorded in the diagnosis of drugs by the King of the moon, it is cool in nature, bitter in taste, and slightly toxic, has the efficacy of clearing heat and removing toxicity, and is mostly used in the formulation of Tibetan medicine for infectious diseases
Treating diseases such as heat, influenza, liver and gallbladder heat diseases, lung heat, intestinal heat and food poisoning.
Aconitum plants are about 350 species throughout the world, and are mainly distributed in Asia and Europe and North America. China is the distribution center of Aconitum plants, and has 167 species, and is mainly distributed in Tibet, Yunnan, Qinghai, Sichuan and other places. Wherein the aconitum genus is about 60 varieties of 15 varieties of Tibetan medicine. Earlier summary found that the plants of Aconitum plant (aconitum carmichaeli A. pendula), Aconitum brachypodum A. brachypodum, Aconitum meidum A. pulchellum, etc. have been extracted from the Tibetan medicine plants of Aconitum
330 compounds such as alkaloid, flavone, phenolic acid and the like have been separated and identified, and modern pharmacological activity researches show that the plants have activity in the aspects of anti-inflammatory analgesia, anti-tumor, blood pressure reduction, immunoregulation and the like.
Herba nga mainly contains diterpene alkaloids, flavonoids, phenolic acids and other components, and the conventional research reports on the chemical components of Tibetan medicine herba nga mainly include: 1) the research progresses of the chemical components and the pharmacological action of Tibetan drug bonga are summarized in the text (Chinese journal of Experimental and prescribing, 6.2012, vol.18, 12 th, P298-302). Up to now, 22 alkaloids and 3 flavonoid glycosides compounds have been separated from bonga, and 35 chemical components of the volatile oil thereof are identified by GC-MS. At present, diterpene alkaloid is considered as the main active component of herba aconiti tangutici anti-inflammatory analgesic. 2) "separation of chemical components of Tibetan herba Aconiti Bonga and its thin-layer chromatography qualitative identification research" (2017 Master thesis, Chinese academy of traditional Chinese medicine) "the separation and identification of chemical components of a basic source of herba Bonga medicinal material (Aconitum Tanbergii) Stapf in the text. The medicinal materials are respectively processed by different extraction and separation methods, and the aconitum sinomontanum medicinal material is divided into an acidic separation part and an alkaline separation part. And repeatedly performing system separation on acidic and alkaline parts of aconitum sinomontanum nakai by using separation and purification means such as macroporous adsorption resin column chromatography, silica gel column chromatography, Sephadex LH-20 gel column chromatography, reversed-phase ODS column chromatography, preparative HPLC, semi-preparative HPLC and recrystallization, and performing total separation to obtain 30 compounds. A total of 22 compounds were identified by physical and chemical properties of the material, modern spectroscopic methods (UV, MS, 1H-NMR, 13C-NMR, HMBC, HSQC, 1H-1HCOSY, NOESY, etc.). Wherein, 7 compounds are separated and identified from the acidic part of aconitum tanguticum nakai, which are respectively: beta-sitosterol, 5-methoxy-N-salicylindrinic acid methyl ester, 5-hydroxy-3',4',5', 7-tetramethoxyflavone, ergosterol peroxide, benzoic acid, o-hydroxybenzyl alcohol and ferulic acid; wherein, 1 is a new compound, and 4 is a compound obtained by first separation. 15 compounds are separated and identified from the basic part of aconitum calophyllum which are respectively: salidroside, guanfu-base H, dihydroatenine, syringin, benzyl alcohol-O-beta-D-glucoside, p-hydroxybenzoic acid, vanillic acid, p-hydroxyphenylethanol, hydroxytyrosol, hepophylline, robinin, 6-acetyl isoaconitine, 2 beta, 9 beta, 11 alpha, 13 alpha-tetrahydroxy-2-O-2-methylutyryl-13-O-acetyl hexasaponin, 2 beta, 9 beta, 11 alpha, 13 alpha-tetrahydroxy-2-O-2-methylutyryl-hexasaponin; of these, 2 are new compounds and 3 are compounds isolated for the first time.
From the above, the existing research on the chemical components of herba aconiti tangutici is mainly focused on alkaloid components, the research on other structural types of compounds is very little, meanwhile, the research on the pharmacological aspect is mainly focused on the aspects of compression resistance, virus resistance and tumor resistance, the action mechanism is not deep, and further research needs to be carried out. Based on the method, the new flavonoid glycoside compounds are extracted and separated from the aconitum songaricum, the pharmacological action of the flavonoid glycoside compounds is clear, and the flavonoid glycoside compounds have certain reference value for the systematic research of chemical components of the aconitum songaricum, and further provide basis for the research of new Tibetan medicine medicaments which have exact curative effect and small side effect and can be widely applied to clinic.
Disclosure of Invention
One of the purposes of the invention is to provide a flavonoid glycoside compound which is extracted and separated from herba aconiti tangutici and takes quercetin as aglycon, and the flavonoid glycoside compound with novel structure and pharmacological activity is obtained by extraction and separation from herba aconiti tangutici medicinal materials, so that the pharmacological action of herba aconiti tangutici is further proved, and a certain reference value is provided for the systematic research of chemical components of herba aconiti tangutici.
The other purpose of the invention is to provide a method for extracting and separating the flavonoid glycoside compound taking quercetin as aglycon from the herba nga, which takes dry herba nga as a raw material and obtains the flavonoid glycoside compound with novel structure and pharmacological activity through the steps of methanol ultrasonic extraction, reduced pressure concentration, AB-8 resin column chromatography, C18 reversed phase chromatographic separation and the like.
The invention also aims to provide the application of the flavonoid glycoside compound which is extracted and separated from the herba aconiti tangutici and takes quercetin as aglycone in preparing anti-inflammatory drugs, and provide reliable basis for preparing new Tibetan drugs.
The purpose of the invention is realized by the following technical scheme: a flavonoid glycoside compound which takes quercetin as aglycon and is extracted and separated from herba Aconiti Bonga is provided, wherein the flavonoid glycoside compound which takes quercetin as aglycon has a structural formula shown in (I):
Figure 100002_DEST_PATH_IMAGE001
the flavonoid compound taking quercetin as aglycon is obtained by extracting and separating from dried herba aconiti tangutici, wherein the names are as follows: quercetin-3-O- [ beta-D-glucopyranosyl- (1 → 3) - (4-)O-trans-caffeoyl) - α -L-rhamnopyranosyl- (1 → 6) - β -D-galactopyranosyl]-7-O- β -D-glucopyranosyl- (1 → 3) - α -L-rhamnopyranoside, self-nominated as: herba Aconiti Bonga glycoside A.
The herba Aconiti Bonga is dry whole plant of Aconitum Nardostachyos or dry whole plant of Aconitum Caryophyllum nakai.
The herba Aconiti Bonga glycoside A has a molecular weight of 1242 and a molecular formula of C54H66O33。
A method for extracting and separating flavonoid glycoside compounds with quercetin as aglycon from herba Aconiti Bonga comprises the following steps:
A. ultrasonic extraction
Taking dried herba Aconiti Bonga as a raw material, crushing, and performing ultrasonic extraction by using 70-80% methanol to obtain an extracting solution;
B. concentrating under reduced pressure
B, decompressing the extracting solution obtained in the step A to-0.08-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain a water dispersion;
C. resin column chromatography
B, carrying out wet loading on the aqueous dispersion obtained in the step B, carrying out column chromatography separation by using AB-8 macroporous adsorption resin, collecting a chromatographic solution containing a total flavone component, and then carrying out reduced pressure concentration until no alcohol exists, thus obtaining a concentrated solution;
c18 reverse phase chromatography column separation
And C, filtering the concentrated solution obtained in the step C, and separating the filtrate by using C18 reverse phase chromatographic packing under high pressure: a: acetonitrile B: 0.1% V/V phosphoric acid water, mobile phase a: B = 18: 82V/V; the detection wavelength is 350 nm;
E. concentrating
And D, decompressing the collected liquid of the product obtained in the step D to-0.08 to-0.09 MPa at 50 ℃, concentrating to be dry, grinding the solid into powder, and then drying by air blow at 45 ℃ to obtain a light yellow powder, wherein the light yellow powder is the tetrangaside A which is a flavonoid glycoside compound with quercetin as aglycone and has the structural formula shown in the formula (I).
In the step A, the particle size of the crushed raw materials is 60-80 meshes.
In the step A, during ultrasonic extraction, the weight of the methanol is 8-10 times that of the raw material.
In the step A, the ultrasonic extraction is performed for 3-5 times, and each time lasts for 1 hour.
In the step B, water is added into the concentrated extracting solution according to the volume ratio of 1: 6-1: 10 for dispersing treatment.
In the step C, the mobile phase used for the AB-8 macroporous adsorption resin column chromatography separation is methanol-water =50: 50V/V.
The light yellow powder obtained by extraction and separation of the invention is positive in HCl-Mg reaction and Molish reaction, and is further proved to be a flavonoid glycoside compound.
On the basis, the further analysis results are as follows:
electrospray ionization mass spectrum ESI-MS of flavonoid glycoside compounds with the structural formula shown in (I) shows that: m/z 1241.37 [ M-H]-；1243.37 [M+H]+，1265.36 [M+Na]+The molecular weight of the compound is 1242, the molecular formula is C54H66O33。IRνmax（KBr，cm-1）：3384，2925，1653，1596，1517，1491，1271，1072，897，808。
1Delta 12.60 in H-NMR is the proton signal for the hydroxyl group in position 5.The aromatic proton signal can be seen in the low field part, and according to the coupling constant between protons, there are a group which is represented by the ABX system: δ 7.68 (1H, dd, J =8.4, 2.0 Hz), 6.84 (1H, d, J =8.4 Hz), 7.59 (1H, d, J =2.0 Hz), respectively from the flavone B-ring H-2 ', H-3 ', H-6 ' signals, suggesting that the B-ring is 1/4/5-trisubstitution pattern. The higher field set of doublets δ 6.80 (1H, d, J =2.0 Hz), 6.48 (1H, d, J =2.0 Hz) are the H-6 and H-8 signals of the flavone a-ring, suggesting a 1, 2, 3, 5-tetrasubstituted pattern for the a-ring. The compound is suggested to have the mother nucleus structure of quercetin. In addition, the low field region has a group of ABX systems delta 7.04 (1H, d, J =1.6 Hz), 7.00 (1H, dd, J =8.4, 1.6 Hz), 6.76 (1H, d, J =8.4 Hz), and a pair of proton signals delta 7.45 (1H, d, J =16.0 Hz), 6.21 (1H, d, J =16.0 Hz), which are characteristic signals of trans-caffeoyl H-8 and H-9, and indicate that the molecule contains a single source of trans-caffeoyl structure. δ 5.59 (1H, br s), 5.38 (1H, d, J =7.6 Hz), 4.56 (1H, br s), 4.49 (1H, d, J =7.6 Hz), 4.28 (1H, d, J =7.6 Hz), which is the terminal hydrogen signal of the saccharide, wherein δ 5.38 (1H, d, J =7.6 Hz), 4.49 (1H, d, J =7.6 Hz), 4.28 (1H, d, J =7.6 Hz) is δ 5.59 (1H, br s) in β configuration, and 4.56 (1H, br s) is in α configuration. In the high-field part, two methyl signals delta 1.14 (3H, d, J =6.0 Hz) and 0.98 (3H, d, J =6.0 Hz) are characteristic signal peaks of methyl hydrogen at the 6-position of rhamnose, and the compound is proved to have two rhamnose structure single sources. Quercetin reported in literature (allergic reaction, Hades, Yuhe, Qiaolian, etc., research on chemical components of semen Cuscutae, Chinese journal of Chinese medicine 2002, 27 (2): 115-1Compared with H-NMR data, two less hydroxyl signals are removed, the hydrogen signals of the B ring are basically consistent, the H-8 and H-6 of the A ring are slightly shifted to a low field, and the 3 and 7 positions are presumed to be connected with glyco-glycoside.
13C-NMR has a total carbon signal of 54, and the hydrocarbon is subjected to full attribution by DEPT135 degrees and HSQC, wherein the full attribution comprises 14 quaternary carbons, 35 tertiary carbons, 3 secondary carbons and 2 primary carbons. There are two carbonyl carbon signals delta 177.5, 166.0 in the low field portion, which are the quercetin 4-position and trans-caffeoyl 9-position carbon signals, respectively. Delta. for the preparation of a coatingC 104.5（δH 4.49 d，J=7.6 Hz），103.3（δH 4.28 d，J=7.6 Hz），101.9（δH 5.30 d，J=7.6 Hz），99.7（δH 4.56 br s），98.2（δH5.59 br s), respectively the terminal carbon signals of the sugars, according to 1) Bharat B.S. et al, New flavanoid glycosides fromAconitum naviculare(Bruhl) Stapf, a medical here from the trans-Himalayan region of New pal, Carbohydrate research 2006, 341, 2161-Aconitum anthoraL. (Ranuculaceae) phytochemistry 2008, 69(5):1220-1226, which presumably has a structural monosource of one galactose, two rhamnoses and two glucose. The mode of linkage of the groups is determined by HMBC, deltaH6.48 (H-6) and δC161.3 (C-7), 160.8 (C-5), ΔH6.48 (H-8) and δC161.3 (C-7), 155.8 (C-9); deltaH7.68 (H-2') and δC 156.7（C-2），148.6（C-4′），120.8（C-1′），115.1（C-2′）；δH 7.59 (H-6') and δC 156.7 (C-2), 148.6 (C-4 '), 144.8 (C-5'), further confirming that the compound has a mother-nucleus structure of quercetin. DeltaH5.38 (H-1') and deltaC 133.7 (C-3) correlation, δH5.59 (H-1 ""') and δC 161.3 (C-7) correlation, indicating that the sugar is attached to the 3,7 position of the mother nucleus. DeltaH4.56 (H-1') and δC 65.0 (C-6') relative; deltaH4.28 (H-1 "") and δC 76.3 (C-3'); deltaH5.00 (H-4') and ΔC 166.0 (Caffeoyl C-9), indicating that the Caffeoyl group is attached at the 4-position of rhamnose.
In combination with the above analysis, the compound was identified as quercetin-3-O- [ beta-D-glucopyranosyl- (1 → 3) - (4-)O-trans-caffeoyl) - α -L-rhamnopyranosyl- (1 → 6) - β -D-galactopyranosyl]-7-O- β -D-glucopyranosyl- (1 → 3) - α -L-rhamnopyranoside. The phase of the compound is not found by the search of the scifiniderReports that the compound is determined to be a neoflavonoid structure and is named as the tangga glycoside A.
1H-NMR and13the C-NMR data are shown in Table 1 below.
Figure DEST_PATH_IMAGE002
Figure DEST_PATH_IMAGE003
Figure DEST_PATH_IMAGE004
By passing1H-NMR、13C-NMR and DEPT135 ℃ and nuclear magnetism two-dimensional analysis technical means such as HSQC, HMBC, H-HCOSY, NOESY and the like determine that the compound is as follows: herba Aconiti Bonga glycoside A (flavonoid glycoside compound with quercetin as aglycone) has a structural formula shown in (I).
Meanwhile, pharmacological experiments prove that the flavonoid glycoside compound extracted and separated by the method has certain anti-inflammatory activity and can be applied to the preparation of anti-inflammatory drugs, wherein the structural formula of the flavonoid glycoside compound is shown as (I).
The invention has the beneficial technical effects that:
1. the flavonoid glycoside compound taking quercetin as aglycon provided by the invention is obtained by extracting and separating from dry herba aconiti, has the structural formula shown in (I), is determined in structure, and defines the relationship between the pharmacological activity and the herba aconiti effective components, thereby providing a certain reference value for the systematic research of herba aconiti chemical components.
2. The flavonoid glycoside compound is obtained by taking dry herba nga as a raw material and performing the technical steps of methanol ultrasonic extraction, AB-8 column chromatography separation and purification, C18 reversed phase chromatography packing high pressure preparation and separation and the like.
3. The invention reports the structure of the tetrandrine A for the first time, determines the relative configuration according to related data such as nuclear magnetism two-dimensional and the like, and is a novel brass glycoside compound; pharmacological research proves that the derivative has certain anti-inflammatory activity, can be used as an latent structure for development and utilization, and simultaneously provides reliable basis for large-scale tissue culture production of flavonoid glycoside substances, pharmaceutical research of flavonoid substances and the like and preparation of new Tibetan medicines.
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FIG. 1 is a graph showing the effect of bonggarin A on the release amount of IL-6, a LPS-induced RAW264.7 cytokine.
Detailed Description
The present invention will be described in further detail with reference to examples. It should be noted, however, that the following examples are not to be construed as limiting the scope of the present invention, and that many insubstantial modifications and variations of the invention can be made by those skilled in the art without departing from the spirit and scope of the invention as set forth herein.
Example 1
A flavonoid glycoside compound extracted from herba Aconiti Bonga and containing quercetin as aglycone is light yellow powder, has positive HCl-Mg reaction and Molish reaction, molecular weight of 1242, and molecular formula of C54H66O33Having the formula (I):
Figure DEST_PATH_IMAGE005
the flavonoid glycoside compound taking quercetin as aglycon is obtained by extracting and separating from dry herba aconiti tangutici, and the specific steps are as follows:
A. ultrasonic extraction
Taking dried herba Aconiti Bonga as a raw material, crushing, and performing ultrasonic extraction by using 70-80% methanol to obtain an extracting solution;
B. concentrating under reduced pressure
B, decompressing the extracting solution obtained in the step A to-0.08-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain a water dispersion;
C. resin column chromatography
B, carrying out wet loading on the aqueous dispersion obtained in the step B, carrying out column chromatography separation by using AB-8 macroporous adsorption resin, collecting a chromatographic solution containing a total flavone component, and then carrying out reduced pressure concentration until no alcohol exists, thus obtaining a concentrated solution;
c18 reverse phase chromatography column separation
And C, filtering the concentrated solution obtained in the step C, and separating the filtrate by using C18 reverse phase chromatographic packing under high pressure: a: acetonitrile B: 0.1% V/V phosphoric acid water, mobile phase a: B = 18: 82V/V; the detection wavelength is 350 nm;
E. concentrating
And D, concentrating the collected liquid of the product obtained in the step D to be dry after the pressure is reduced to-0.08 to-0.09 MPa at 50 ℃, grinding the solid into powder, and then drying by blowing air at 45 ℃ to obtain a light yellow powder, namely the flavonoid glycoside compound shown in the structural formula (I).
Example 2
Taking 10kg of dried herba Aconiti Bonga (dried whole plant of Aconitum Nardostachyos), pulverizing to 60 mesh particle size, adding 8 times of 70wt% methanol, ultrasonic extracting for 3 times, each for 1 hr, and mixing extractive solutions; decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:6 for dispersion treatment to obtain 20L of aqueous dispersion; wet loading the aqueous dispersion, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V as mobile phase), collecting chromatography liquid containing total flavone component, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 5L; filtering the concentrated solution, preparing and separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B: 0.1% V/V phosphoric acid water, A: B = 18: 82V/V is mobile phase; detection wavelength is 350 nm), collecting corresponding chromatographic peaks, concentrating the product collection solution to-0.08 to-0.09 MPa at 50 ℃ under reduced pressure until the product collection solution is dried, grinding the solid into powder, and drying by blowing at 45 ℃ to obtain 6g of a light yellow powder product, namely the flavonoid glycoside compound with the structural formula shown in (I).
The whole production process takes about 5 days;
the purity of the product was determined to be 99.36% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V phosphoric acid water, A: B = 40: 60V/V as the mobile phase; detection wavelength 350 nm).
Example 3
Taking 20kg of dried herba Aconiti Bonga (dried whole plant of Aconitum Carmichaeli Debx.), pulverizing to 70 mesh, adding 8.5 times weight of 75wt% methanol, ultrasonic extracting for 4 times, each for 1 hr, and mixing extractive solutions; decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:7 for dispersion treatment to obtain 36L of aqueous dispersion; wet loading the aqueous dispersion, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V as mobile phase), collecting chromatography liquid containing total flavone component, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 12L; filtering the concentrated solution, separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B: 0.1% V/V phosphoric acid water, A: B = 18: 82V/V as a mobile phase; detection wavelength is 350 nm), collecting corresponding chromatographic peaks, concentrating the product collected liquid to be dry at 50 ℃ under reduced pressure of-0.08 to-0.09 MPa, grinding the solid into powder, and drying by blowing at 45 ℃ to obtain 13g of light yellow powder product which is the flavonoid glycoside compound with the structural formula shown in (I).
The whole production process takes about 6 days;
the purity of the product was determined to be 99.08% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V phosphoric acid water, A: B = 40: 60V/V as the mobile phase; detection wavelength 350 nm).
Example 4
Collecting dried herba Aconiti Bonga (dried whole plant of Aconitum carmichaeli Debx) 30kg, pulverizing to 80 mesh, adding 9 times of 80wt% methanol, ultrasonic extracting for 5 times (each for 1 hr), and mixing extractive solutions; decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:8 for dispersion treatment to obtain 58L of aqueous dispersion; wet loading the aqueous dispersion, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V as mobile phase), collecting chromatography liquid containing total flavone component, and concentrating under reduced pressure until no alcohol exists to obtain 23L concentrated solution; filtering the concentrated solution, separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B: 0.1% V/V phosphoric acid water, A: B = 18: 82V/V as a mobile phase; detection wavelength is 350 nm), collecting corresponding chromatographic peaks, concentrating the product collected solution to be dry at 50 ℃ under reduced pressure of-0.08 to-0.09 MPa, grinding the solid into powder, and drying by blowing at 45 ℃ to obtain 20g of light yellow powder product which is the flavonoid glycoside compound with the structural formula shown in (I).
The whole production process takes about 7 days;
the purity of the product was determined to be 99.16% by rechecking the product purity using reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V phosphoric acid water, A: B = 40: 60V/V as the mobile phase; detection wavelength 350 nm).
Example 5
A. Taking 50kg of dried herba Aconiti Bonga (dried whole plant of Aconitum Nardostachyos), pulverizing to 60 mesh particle size, adding 8 times of 70wt% methanol, ultrasonic extracting for 3 times, each for 1 hr, mixing extractive solutions;
B. decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:6 for dispersion treatment to obtain 90L of aqueous dispersion;
C. wet loading the aqueous dispersion, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V as mobile phase), collecting chromatography liquid containing total flavone component, and concentrating under reduced pressure until no alcohol exists to obtain 30L concentrated solution;
D. filtering the concentrated solution, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: acetonitrile B: 0.1% V/V phosphoric acid water, A: B = 18: 82V/V as mobile phase; detection wavelength is 350 nm), and collecting corresponding chromatographic peak;
E. and (3) decompressing the collected liquid of the product to-0.08 to-0.09 MPa at 50 ℃, concentrating to be dry, grinding the solid into powder, and then drying by air blast at 45 ℃ to obtain 32g of light yellow powder product, namely the flavonoid glycoside compound with the structural formula shown in the formula (I).
The whole production process takes about 7 days;
the purity of the product was found to be 99.21% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V phosphoric acid water, A: B = 40: 60V/V as the mobile phase; detection wavelength 350 nm).
Example 6
A. Taking 50kg of dried herba Aconiti Bonga (dried whole plant of Aconitum carmichaeli (L.) Ramat.), pulverizing to 70 mesh particle size, adding 10 times of 75wt% methanol, ultrasonic extracting for 5 times, each for 1 hr, and mixing extractive solutions;
B. decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:10 for dispersion treatment to obtain 120L of aqueous dispersion;
C. wet loading the aqueous dispersion, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V is mobile phase), collecting chromatography liquid containing total flavone component, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 50L;
D. filtering the concentrated solution, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: acetonitrile B: 0.1% V/V phosphoric acid water, A: B = 18: 82V/V as mobile phase; detection wavelength is 350 nm), and collecting corresponding chromatographic peak;
E. and (3) decompressing the collected liquid of the collected liquid product at 50 ℃ to-0.08 to-0.09 MPa, concentrating to be dry, grinding the solid into powder, and then drying by air blast at 45 ℃ to obtain a light yellow powder product 38g, which is the flavonoid glycoside compound with the structural formula shown in the formula (I).
The whole production process takes about 9 days;
the purity of the product was determined to be 99.34% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V phosphoric acid water, A: B = 40: 60V/V as the mobile phase; detection wavelength 350 nm).
Example 7
A. Taking 50kg of dried herba Aconiti Bonga (dried whole plant of Aconitum carmichaeli (L.) Ramat.), pulverizing to 80 mesh particle size, adding 9 times of 80wt% methanol, ultrasonic extracting for 4 times (each for 1 hr), and mixing extractive solutions;
B. decompressing the extracting solution to-0.08 to-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:9 for dispersion treatment to obtain 100L of aqueous dispersion;
C. wet loading the aqueous dispersion, separating with AB-8 macroporous adsorbent resin column chromatography (methanol: water =50: 50V/V as mobile phase), collecting chromatography liquid containing total flavone component, and concentrating under reduced pressure until no alcohol exists to obtain 40L concentrated solution;
D. filtering the concentrated solution, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: acetonitrile B: 0.1% V/V phosphoric acid water, A: B = 18: 82V/V as mobile phase; detection wavelength is 350 nm), and collecting corresponding chromatographic peak;
E. and (3) decompressing the collected liquid of the collected liquid product at 50 ℃ to-0.08 to-0.09 MPa, concentrating to be dry, grinding the solid into powder, and drying by air blowing at 45 ℃ to obtain 35g of a light yellow powder product, namely the flavonoid glycoside compound with the structural formula shown in the formula (I).
The whole production process takes about 8 days;
the purity of the product was determined to be 99.19% by rechecking the product purity by reverse phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase components (A: methanol B: 0.1% V/V phosphoric acid water, A: B = 40: 60V/V as the mobile phase; detection wavelength 350 nm).
Example 8
The following experiment was carried out on a light yellow powder compound (i.e., the tango glycoside a referred to in the following experiment) having the structural formula shown in (i) extracted and separated in the above example 4:
compound anti-inflammatory activity assay:
(I) Experimental materials and instruments
Materials: mouse macrophage strain RAW264.7 (cell resource center of Shanghai Biotechnology institute of Chinese academy of sciences); DMEM medium, fetal bovine serum (Hyclone, usa); penicillin, streptomycin (beijing solibao limited); mouse IL-6 ELISA kit (Xinbo Sheng Biotechnology Co., Ltd., M200624-004 a); a nitric oxide test kit (Nanjing institute of bioengineering, A012-1-2).
The instrument comprises: BB15 CO2Cell culture chambers (Thermo corporation, usa); clean bench (SANYO corporation, japan); TDZ5-WS desk type low speed centrifuge (Hunan instrument laboratory development Co., Ltd.); multifunctional microplate reader SpectraMax (Molecular Devices, USA).
(II) Experimental method
1. Cell culture
Mouse macrophage strain RAW264.7 containing antibioticDMEM complete medium with elements (100U/mL penicillin, 100U/mL streptomycin) and 10% Fetal Bovine Serum (FBS) at 37 ℃ with 5% CO2In the incubator condition of (a).
2. ELISA method for detecting IL-6 release amount of RAW264.7 cells
Taking macrophage RAW264.7 in logarithmic phase, adjusting concentration, and adding 1 × 105Cell suspensions were plated at 24-well cell culture plates at individual/mL, 1mL of cell suspension was added per well, incubated for 24h, and the supernatant carefully aspirated. Adding 1mL of DMEM solution into the blank group; LPS model group added 1mL of DMEM solution containing LPS (100 ng/mL); LPS + Bongaside A group 1mL of DMEM solution containing LPS (100 ng/mL) and various concentrations (7.5, 15, 30, 60. mu.g/mL) of Bongaside A was added. Putting the mixture into a cell incubator to continue culturing for 24 hours. After performing the experimental procedures according to the ELISA kit instructions, each OD value was measured at 450nm, and the IL-6 cytokine release level was calculated from the OD values.
3. Statistical treatment
Data were processed using SPSS 26.0 statistical software and results were expressed as mean. + -. standard deviation (x. + -.s). One-way analysis of variance (One way-ANOVA) was used for comparisons between groups, and Least Significant Difference (LSD) was used for pairwise comparisons between groups. Plotting was performed using GraphPad Prism 7.
(III) results of the experiment
1. LPS + Bongaside A groups (Bongaside A concentrations of 7.5, 15, 30, 60. mu.g/mL, respectively), a blank group with addition of DMEM solution, and an LPS model group with addition of LPS (100 ng/mL) in DMEM solution, the IL-6 content (pg/mL) of each group is shown in Table 2:
Figure DEST_PATH_IMAGE007
2. the effect of bonggarin A on the release of LPS-induced RAW264.7 cytokine IL-6 is shown in FIG. 1. It can be seen that the LPS-induced production level of IL-6, an inflammatory factor of RAW264.7 macrophages, is significantly increased compared to the blank group (P< 0.001), indicating successful modeling of the LPS-induced inflammation model. In comparison with the model LPS group, Bonga A and LPS were incubated with RAW264.7 cellsThe expression of the inflammatory factor IL-6 is obviously reduced (P< 0.001), and the concentration range of 15-60. mu.g/mL is dose-dependent, and the expression level of IL-6 is lowest when the concentration of the compound is 60. mu.g/mL.
Experimental results show that the tangkunoside A can obviously reduce the release amount of an LPS-induced RAW264.7 macrophage inflammatory factor IL-6, has a good protective effect on LPS-induced RAW264.7 macrophage inflammatory reaction, and has anti-inflammatory activity.
Therefore, the flavonoid glycoside compound with the structural formula shown in (I) obtained by extraction and separation can be applied to the preparation of anti-inflammatory drugs.

Claims (8)

1. A flavonoid glycoside compound which is extracted and separated from herba Aconiti Bonga and takes quercetin as aglycon is characterized in that: the flavonoid glycoside compound has a structural formula shown in (I):
Figure DEST_PATH_IMAGE001
the names of the characters are: quercetin-3-O- [ beta-D-glucopyranosyl- (1 → 3) - (4-)O-trans-caffeoyl) - α -L-rhamnopyranosyl- (1 → 6) - β -D-galactopyranosyl]-7-O-beta-D-glucopyranosyl- (1 → 3) -alpha-L-rhamnopyranoside, molecular weight 1242, molecular formula C54H66O33。
2. A method for preparing the flavonoid glycoside with quercetin as aglycon according to claim 1, wherein the flavonoid glycoside comprises the following steps: the method comprises the following steps:
A. ultrasonic extraction
Taking dried herba Aconiti Bonga as a raw material, crushing, and performing ultrasonic extraction by using 70-80% methanol to obtain an extracting solution;
B. concentrating under reduced pressure
B, decompressing the extracting solution obtained in the step A to-0.08-0.09 MPa, concentrating until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain a water dispersion;
C. resin column chromatography
B, carrying out wet loading on the aqueous dispersion obtained in the step B, carrying out column chromatography separation by using AB-8 macroporous adsorption resin, collecting a chromatographic solution containing a total flavone component, and then carrying out reduced pressure concentration until no alcohol exists to obtain a concentrated solution;
c18 reverse phase chromatography column separation
And C, filtering the concentrated solution obtained in the step C, and separating the filtrate by using C18 reverse phase chromatographic packing under high pressure: a: acetonitrile B: 0.1% V/V phosphoric acid water, mobile phase a: B = 18: 82V/V; the detection wavelength is 350 nm;
E. concentrating
And D, decompressing the collected liquid of the product obtained in the step D to-0.08 to-0.09 MPa at 50 ℃, concentrating to be dry, grinding the solid into powder, and then drying by air blowing at 45 ℃ to obtain a light yellow powder, wherein the light yellow powder is a flavonoid glycoside compound taking quercetin as aglycone and has a structural formula shown in (I).
3. The method of claim 2 wherein the quercetin aglycon flavonoid glycoside is isolated from herba Aconiti Bonga as follows: in the step A, the particle size of the crushed raw materials is 60-80 meshes.
4. The method of claim 2 wherein the quercetin aglycon flavonoid glycoside is isolated from herba Aconiti Bonga as follows: in the step A, during ultrasonic extraction, the weight of the methanol is 8-10 times that of the raw material.
5. The method of claim 2 wherein the quercetin aglycon flavonoid glycoside is isolated from herba Aconiti Bonga as follows: in the step A, the ultrasonic extraction is performed for 3-5 times, and each time lasts for 1 hour.
6. The method of claim 2 wherein the quercetin aglycon flavonoid glycoside is isolated from herba Aconiti Bonga as follows: in the step B, water is added into the concentrated extracting solution according to the volume ratio of 1: 6-1: 10 for dispersing treatment.
7. The method of claim 2, wherein the method comprises the steps of: in the step C, the mobile phase used for the AB-8 macroporous adsorption resin column chromatography separation is methanol-water =50: 50V/V.
8. The use of the flavonoid glycoside extracted and separated from herba Aconiti Bonga of claim 1 and having quercetin as aglycone in the preparation of anti-inflammatory drugs.
CN202011564143.7A 2020-12-25 2020-12-25 Flavonoid glycoside compound extracted and separated from herba Aconiti Bonga and using quercetin as aglycone, and method and application thereof Withdrawn CN114685580A (en)

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