CN113336808B - Glycoside compound extracted and separated from lily, and method and application thereof - Google Patents
Glycoside compound extracted and separated from lily, and method and application thereof Download PDFInfo
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- CN113336808B CN113336808B CN202110830176.XA CN202110830176A CN113336808B CN 113336808 B CN113336808 B CN 113336808B CN 202110830176 A CN202110830176 A CN 202110830176A CN 113336808 B CN113336808 B CN 113336808B
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- 229930182470 glycoside Natural products 0.000 title claims abstract description 62
- 241000234435 Lilium Species 0.000 title claims abstract description 51
- -1 Glycoside compound Chemical class 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 150000002338 glycosides Chemical class 0.000 claims abstract description 22
- 238000000605 extraction Methods 0.000 claims abstract description 17
- 239000011347 resin Substances 0.000 claims abstract description 16
- 229920005989 resin Polymers 0.000 claims abstract description 16
- 238000004440 column chromatography Methods 0.000 claims abstract description 12
- 238000010992 reflux Methods 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 12
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 51
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- 239000006185 dispersion Substances 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 23
- 239000000843 powder Substances 0.000 claims description 22
- 239000000047 product Substances 0.000 claims description 22
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- 238000001514 detection method Methods 0.000 claims description 15
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- HOVAGTYPODGVJG-VEIUFWFVSA-N methyl alpha-D-mannoside Chemical compound CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O HOVAGTYPODGVJG-VEIUFWFVSA-N 0.000 description 2
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- QXRAHTFDPBQKIM-HWKANZROSA-N 1-feruloyl-sn-glycerol Chemical compound COC1=CC(\C=C\C(=O)OCC(O)CO)=CC=C1O QXRAHTFDPBQKIM-HWKANZROSA-N 0.000 description 1
- PBUWCVBYMDVPCL-UHFFFAOYSA-N 3-anilinophthalic acid Chemical compound OC(=O)C1=CC=CC(NC=2C=CC=CC=2)=C1C(O)=O PBUWCVBYMDVPCL-UHFFFAOYSA-N 0.000 description 1
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- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
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- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 description 1
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- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
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- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
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- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 description 1
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 1
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- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
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- 230000003345 hyperglycaemic effect Effects 0.000 description 1
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- WABPQHHGFIMREM-UHFFFAOYSA-N lead(0) Chemical compound [Pb] WABPQHHGFIMREM-UHFFFAOYSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- HOVAGTYPODGVJG-UHFFFAOYSA-N methyl beta-galactoside Natural products COC1OC(CO)C(O)C(O)C1O HOVAGTYPODGVJG-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 238000000746 purification Methods 0.000 description 1
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- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000005856 steroid saponins Chemical class 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
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- 239000000341 volatile oil Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Steroid Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a glycoside compound extracted and separated from lily, a method and application thereof, and a structural formula of the glycoside compound is shown as (I). The glycoside compound is obtained by taking dried lily as a raw material, extracting and separating through steps of ethanol reflux extraction, decompression concentration, AB-8 resin column chromatography, C18 reversed phase chromatography separation and the like, is a novel glycoside compound with novel structure and pharmacological activity, can be used for preparing anti-lung cancer drugs, and provides reliable basis for preparing anti-cancer drugs, researching glycoside drugs and the like.
Description
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to a glycoside compound with pharmacological activity extracted and separated from lily, and a preparation method and application thereof.
Background
The plants of Liliaceae Lilium are about 115 species in the whole world, are mainly distributed in the North temperate region and subtropical mountain region, and are produced in 39 species and 26 varieties in China, wherein 25 species and 19 varieties are special products in China. The bulb of the lily plant contains starch, so that the lily plant can be eaten, and some kinds of lily plant can be used as medicines; the fresh flower contains volatile oil, and can be used as perfume. The lily medicinal material is derived from volume pill in 2020 edition of Chinese pharmacopoeiaLilium lancifoliumThunder and lilyL． browniiF. E. Brown var.viridulumBaker and lilium tenuifoliumL．PumilumDC dry bulb has effects of nourishing yin, moistening lung, clearing heart fire, and tranquilizing, and is used for treating symptoms such as chronic cough due to yin deficiency, cough due to fatigue, dysphoria, palpitation, insomnia, dreaminess, and absentmindedness. The lily is commonly used for dietary therapy, and has the health care effects of relieving cough and asthma, reducing blood sugar, resisting tumor, improving sleep, improving immunity, preventing senile dementia and the like, and has higher nutritional value.
A great deal of researches show that the liliaceae plant mainly contains polysaccharides, flavonoids, polyphenols, saponins, amino acids, alkaloids, steroids and other components, and the existing literature for the study of the chemical components of the liliaceae plant mainly comprises:
(one) week neutral, "research on chemical components of volume red lead" (university of Chinese medicine, report of Beijing, 1 month, 33 st volume, 1 st phase Chinese medicine chemistry) reported that 15 compounds were isolated from volume red lead bulb and identified as (1) beta-D-glucosyl- (1- > 4) -beta-D-glucopyranoside, (2) beta-D-fructofuranosyl-alpha-D-glucopyranoside, (3) methyl-alpha-D-glucopyranoside, (4) methyl-alpha-D-mannopyranoside, (5) adenine nucleoside, (6) carrot glycoside, (7)1-O-p-coumaroyl glyceride, (8) diosgenin 3-O- { O-alpha-L-rhamnosyl- (1- > 2) -O- [ beta-D-xylosyl (1- > 3) ] -beta-D-glucoside }, (9) (25R) -3 beta, 17 alpha-dihydroxy-5 alpha-spirostan-6-one-3-O-alpha-L-rhamnosyl- (1- > 2) -beta-D-glucopyranosyl- (1- > 2) -beta-D-xylosyl (3- > 3-O- { beta-D-xylosyl (1- > 3- > beta-D-xylosyl (3- > 3-), (11) (25R) -spirostan-5-ene-3 beta-O-alpha-L-rhamnopyranose- (1- > 2) - [ beta-D-glucopyranose- (1- > 6) ] beta-D-glucopyranoside, (12) eicosanol, (13) n-docosanoic acid, (14) stigmasterol, and (15) beta-sitosterol.
(II) Zhao Guohua, et al, "chemical structure and antitumor Activity of Lily polysaccharide" (food and biotechnology, month 1, vol.21, stage 1), reported studies on separation and purification, chemical structure and antitumor Activity of the major component LBPS-I of Lily polysaccharide. 1) A homogeneous LBPS-I polysaccharide is obtained by water extraction, ethanol precipitation and column chromatography, wherein the polysaccharide is pure non-starch glucose, and is glucose with alpha-D- (1, 4) -Glcp and alpha-D- (1, 3) -Glcp alternately forming a main chain in a ratio of 2:1 and having alpha-D- (1, 6) -Glcp side chains. 2) The research of the murine transplantable solid tumor shows that LBPS-I has a strong inhibiting effect on transplantable melanin B16 and Lewis lung cancer.
(III) Jiang Ru and the like adopt a hot water extraction ethanol precipitation method to separate water-soluble polysaccharide BHP from lily decoction pieces for the first time. Molecular weight 75000, acid hydrolysis. The thin layer was developed for polysaccharide component analysis. And (5) color development of the aniline phthalic acid. Spots of D-galactose, L-arabinose, D-mannose, D-glucose, L-rhamnose and the like are presented. The polysaccharide acts on the immune system of the body. Has obvious conditioning effect on the immune function of mice.
Fourthly, separating the mature plums and the like from the scale leaves of fresh longya lily produced by Sichuan, and obtaining two polysaccharides LP1 and LP 2. Accounting for 0.55 percent and 0.25 percent of the fresh weight respectively. In the component analysis of polysaccharide, LP1 consists of glucose and mannose in the ratio of 1:2.46, the molecular weight is 79400, and LP2 consists of glucose, mannose, arabinose and galacturonic acid. The ratio of the two polysaccharides is 1:0.73:2.61:1.8:0.84, the molecular weight is 181500, and the two polysaccharides have obvious blood sugar reducing function on hyperglycemic mice caused by tetraoxypyrimidine and are positively correlated with the concentration.
As can be seen from the content reported in the above documents, the existing research on the chemical components of liliaceae plants mainly focuses on polysaccharides and steroid saponins, other structural typesRelatively few compounds are reported. The inventor is on the pair of rolling pillsL. lancifoliumWhen the active ingredients are researched, a novel medicinal compound is found and separated, and the compound is a chlorophenyl glycoside compound, and pharmacological activity research shows that the compound has certain anti-lung cancer activity, and provides scientific basis for researching and developing novel anti-lung cancer medicaments.
Disclosure of Invention
The invention aims at providing a glycoside compound extracted and separated from lily. The novel compound with novel structure and pharmacological activity is obtained by extracting and separating from the lily medicinal material, so that the systematic research of lily chemical components is further advanced, and a material basis is provided for the pharmacological effect research of lily.
The second object of the present invention is to provide a method for extracting and separating glycoside compounds from lily. The method is characterized in that dried lily is used as a raw material, and the glucoside compound with novel structure and pharmacological activity is obtained through steps of ethanol reflux extraction, reduced pressure concentration, AB-8 resin column chromatography, C18 reversed phase chromatography separation and the like.
The invention further aims to provide an application of glycoside compounds extracted and separated from lily in preparing anti-lung cancer drugs, which provides a reliable basis for preparing new anti-lung cancer drugs.
The aim of the invention is achieved by the following technical scheme: a glycoside compound extracted and separated from lily, wherein the glycoside compound has a structural formula shown in (I):
the glycoside compound with the structural formula shown in the formula (I) is obtained by extracting and separating dried lily, wherein the name is: 2, 4-dichloro-3, 5-dimethoxy-benzyl-1-O-beta-D-glucopyranosyl- (1→6) -beta-D-glucopyranoside, belonging to the class of chlorophenyl glycosides, which are self-denominated: lily novel glycoside I.
The Bulbus Lilii is Liliaceae plant Plumbum PreparatiumLilium lancifoliumDry bulb of thunder.
The molecular weight of the lily neoglycoside I is 561, and the molecular formula is C 21 H 30 O 13 Cl 2 。
A method for extracting an isolated glycoside compound from lily, comprising the steps of:
A. reflux extraction
Taking dried lily as a raw material, crushing, and carrying out reflux extraction by using 80-90% ethanol to obtain an extracting solution;
B. concentrating under reduced pressure
Concentrating the extracting solution obtained in the step A to minus 0.08 to minus 0.09MPa until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain an aqueous dispersion;
C. resin column chromatography
B, loading the aqueous dispersion obtained in the step B by a wet method, separating by using an AB-8 macroporous adsorption resin column for chromatography, collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution;
d.c18 reverse phase chromatography column separation
Filtering the concentrated solution obtained in the step C, and separating the filtrate by using a C18 reverse phase chromatographic packing high-pressure preparation: a: acetonitrile B:0.1% V/V formic acid water, a: b=22:78V/V being the mobile phase; detection wavelength 215nm;
E. concentrating
And D, concentrating the product collection liquid obtained in the step D to minus 0.08 to minus 0.09MPa until acetonitrile is not present, freeze-drying the concentrated liquid, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain white powder, namely lily neoglycoside I which is a glycoside compound and has a structural formula shown in (I).
In the step A, the particle size of the crushed raw materials is 60-80 meshes.
In the step A, the weight of the ethanol is 8-10 times of that of the raw material during the reflux extraction.
In the step A, the reflux extraction is carried out 3 to 5 times for 1 hour each time.
In the step B, the concentrated extracting solution is added with water according to the volume ratio of 1:6-1:10 for dispersion treatment.
In the step C, the mobile phase used for the AB-8 macroporous adsorption resin column chromatographic separation is methanol/water=50:50V/V.
The white powder obtained by extraction and separation is positive in Molish reaction, and further proves that the white powder is a glycoside compound.
On this basis, the further analysis results are as follows:
electrospray ionization mass spectrometry ESI-MS of glycoside compounds of formula (I) shows: m/z 560.09 [ M-H ]] - ；562.33 [M+H] + ，584.36 [M+Na] + The molecular weight of the compound is 561, and the molecular formula is C 21 H 30 O 13 Cl 2 。IRν max （KBr，cm -1 ）：3400.6，1649.6，1026.0，999.7， 8272，766.2 cm -1 ；UV λ max 201 (3.72) nm。
The hydrogen spectrum has 2 methoxy groupsδ H 3.82, 3.88 (each 3H, s). The low field part shows two sugar end group hydrogen signalsδ H 4.29, d=7.6 hz,1h;4.26, d=7.6 hz,1 h). Through HSQC to hydrocarbon total attribution, delta is found by HMBC H 4.26 (H-1') and delta C 66.8 (C-6') is related, indicating that the sugar unit is attached to C-6, delta H 4.29 (H-1') and delta C 68.7 (C-6 ') is related, indicating that the sugar unit is attached to C-6'. Combining literature nuclear magnetic data, the compound is determined as follows:
2,4-dichloro-3,5-dimethoxyl-benzyl-1-O-β-D-glucopyranosyl-(1→6)-βd-glucopyranoside, chinese name: 2, 4-dichloro-3, 5-dimethoxy-benzyl-1-O- β -D-glucopyranosyl- (1→6) - β -D-glucopyranoside. Through scintinder search, no related report of the compound is found, and the compound is determined to be of a novel glycoside structure, and is named as lily novel glycoside I.
1 H-NMR 13 The C-NMR data are shown in Table 1 below.
By passing through 1 H-NMR、 13 The analysis technical means such as C-NMR, DEPT135 degrees and nuclear magnetism two-dimensional HSQC, HMBC, H-HCOSY, NOESY and the like confirm that the compound is: lily new glycoside I (new glycoside compound) has a structural formula shown in (I).
Meanwhile, pharmacological experiments prove that the glycoside compound with the structural formula shown as the formula (I) obtained by extraction and separation has certain anti-lung cancer activity and can be applied to the preparation of anti-lung cancer medicaments.
The beneficial technical effects of the invention are as follows:
1. the novel glycoside compound is obtained by extracting and separating dried lily, has a structural formula shown in the formula (I), has a definite structure and is used for researching the pharmacological activity of lung cancer resistance, and scientific basis is provided for systematic research of chemical components and pharmacological actions of lily.
2. The invention uses lily plant roll-up pillL. lancifoliumThe preparation method is simple and easy to control, can ensure that the purity of the lily new glycoside I reaches more than 99%, and has short time consumption in the whole production flow, and is suitable for industrial production.
3. The invention reports the structure of the lily new glycoside I for the first time, determines the relative configuration according to related data such as nuclear magnetism two-dimension and the like, and is a novel glycoside compound; pharmacological research proves that the compound has certain anti-lung cancer activity, can be used as a submerged structure for development and utilization, and provides reliable basis for large-scale tissue culture production of glycoside substances, activity research of glycoside substances, research and development of new anticancer drugs and the like.
Detailed Description
The present invention will be described in further detail with reference to examples. It is to be understood that the following examples are not to be construed as limiting the scope of the invention but are intended to cover all modifications and adaptations of the invention which, however, are within the scope of the invention as defined by the appended claims.
Example 1:
a glycoside compound extracted and separated from Bulbus Lilii is white powder, positive in Molish reaction, has molecular weight of 561, and has molecular formula of C 21 H 30 O 13 Cl 2 Has a structural formula shown in (I):
the glycoside compound is from PadanL. lancifoliumThe dry bulb is extracted and separated, and the specific steps are as follows:
A. reflux extraction
Taking dried lily as a raw material, crushing, and carrying out reflux extraction by using 80-90% ethanol to obtain an extracting solution;
B. concentrating under reduced pressure
Concentrating the extracting solution obtained in the step A to minus 0.08 to minus 0.09MPa until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain an aqueous dispersion;
C. resin column chromatography
B, loading the aqueous dispersion obtained in the step B by a wet method, separating by using an AB-8 macroporous adsorption resin column for chromatography, collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution;
d.c18 reverse phase chromatography column separation
Filtering the concentrated solution obtained in the step C, and separating the filtrate by using a C18 reverse phase chromatographic packing high-pressure preparation: a: acetonitrile B:0.1% V/V formic acid water, a: b=22:78V/V being the mobile phase; detection wavelength 215nm;
E. concentrating
And D, concentrating the product collection liquid obtained in the step D to be at 50 ℃ below zero to 0.08-0.09 MPa till no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain white powder, namely the glycoside compound with the structural formula shown in (I).
Example 2:
taking dried Bulbus Lilii (Rolling pill)L. lancifoliumPulverizing 10kg of dried bulb of (2) to 60 mesh particle size, reflux-extracting with 8 times of 80wt% ethanol for 3 times each for 1 hr, and mixing the extractive solutions; concentrating the extracting solution under reduced pressure to-0.08 to-0.09 MPa until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:6 to perform dispersion treatment to obtain an aqueous dispersion 20L; wet loading the aqueous dispersion, separating by AB-8 macroporous adsorbent resin column chromatography (methanol: water=50v/V is mobile phase), collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 5L; filtering the concentrated solution, preparing and separating filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B:0.1% V/V formic acid water, A: B=22:78V/V is a mobile phase, and detection wavelength is 215 nm), collecting corresponding chromatographic peaks, decompressing a product collection liquid to minus 0.08-minus 0.09MPa until no acetonitrile exists, freeze-drying the concentrated solution, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain 6g of white powder product, namely the glycoside compound with the structural formula shown in (I).
The whole production process takes about 5 days;
the purity of the product was rechecked by reverse phase analytical liquid chromatography (RP-HPLC) by changing the mobile phase component (A: methanol B:0.1% V/V formic acid water, A: B=46:54V/V as mobile phase; detection wavelength 215 nm), and the purity of the product was found to be 99.36%.
Example 3:
taking dried Bulbus Lilii (Rolling pill)L. lancifoliumPulverizing 20kg of dried bulb of (2) to 70 mesh particle size, reflux-extracting with 8.5 times of 85wt% ethanol for 4 times each for 1 hr, and mixing the extractive solutions; concentrating the extracting solution under reduced pressure to-0.08 to-0.09 MPa until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:7 to perform dispersion treatment to obtain an aqueous dispersion 36L; wet loading the aqueous dispersion, separating by AB-8 macroporous adsorbent resin column chromatography (methanol: water=50v/V is mobile phase), collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 12L; filtering the concentrated solution, and filling the filtrate with C18 reversed phase chromatographyHigh-pressure preparation and separation (A: acetonitrile B:0.1% V/V formic acid water, A: B=22:78V/V is a mobile phase, detection wavelength is 215 nm), collecting corresponding chromatographic peaks, decompressing a product collection liquid to minus 0.08-minus 0.09MPa at 50 ℃ until no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain 13g of white powder product, namely the glycoside compound with the structural formula shown in (I).
The whole production process takes about 6 days;
the purity of the product was checked again by reverse phase analytical liquid chromatography (RP-HPLC) by changing the mobile phase component (A: methanol B:0.1% V/V formic acid water, A: B=46:54V/V as mobile phase; detection wavelength 215 nm) and found to be 99.08%.
Example 4:
taking dried Bulbus Lilii (Rolling pill)L. lancifoliumPulverizing 30kg of dried bulb of (2) to 80 mesh particle size, reflux-extracting with 9 times of 90wt% ethanol for 5 times each for 1 hr, and mixing the extractive solutions; concentrating the extracting solution under reduced pressure to-0.08 to-0.09 MPa until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:8 to perform dispersion treatment to obtain an aqueous dispersion 58L; wet loading the aqueous dispersion, separating by AB-8 macroporous adsorbent resin column chromatography (methanol: water=50v/V is mobile phase), collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 23L; filtering the concentrated solution, preparing and separating filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B:0.1% V/V formic acid water, A: B=22:78V/V is a mobile phase, and detection wavelength is 215 nm), collecting corresponding chromatographic peaks, decompressing a product collection liquid to minus 0.08-minus 0.09MPa until no acetonitrile exists, freeze-drying the concentrated solution, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain 20g of white powder product, namely the glycoside compound with the structural formula shown in (I).
The whole production process takes about 7 days;
the purity of the product was rechecked by reverse phase analytical liquid chromatography (RP-HPLC) by changing the mobile phase component (A: methanol B:0.1% V/V formic acid water, A: B=46:54V/V as mobile phase; detection wavelength 215 nm), and was measured to be 99.16%.
Example 5:
A. taking dried Bulbus Lilii (Rolling pill)L. lancifoliumPulverizing to 60 mesh particle size, reflux-extracting with 8 times of 80wt% ethanol for 3 times each for 1 hr, and mixing extractive solutions;
B. concentrating the extracting solution to minus 0.08 to minus 0.09MPa until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:6 to perform dispersion treatment to obtain an aqueous dispersion 90L;
C. wet loading the aqueous dispersion, separating by AB-8 macroporous adsorbent resin column chromatography (methanol: water=50v/V is mobile phase), collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain 30L concentrated solution;
D. filtering the concentrated solution, separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B:0.1% V/V formic acid water, A: B=22:78V/V as mobile phase; detection wavelength 215 nm), and collecting corresponding chromatographic peaks;
E. and (3) decompressing the product collection liquid to minus 0.08 to minus 0.09MPa, concentrating to be dry, grinding the solid, and drying by blowing at 45 ℃ to obtain 32g of white powder product which is the glycoside compound with the structural formula shown in (I).
The whole production process takes about 7 days;
the purity of the product was 99.21% by changing the mobile phase component (A: methanol B:0.1% V/V formic acid water, A: B=46:54V/V as mobile phase; detection wavelength 215 nm), and rechecking the purity of the product by reverse phase analytical liquid chromatography (RP-HPLC).
Example 6:
A. taking dried Bulbus Lilii (Rolling pill)L. lancifoliumPulverizing to 70 mesh particle size, reflux-extracting with 10 times of 85wt% ethanol for 5 times each for 1 hr, and mixing extractive solutions;
B. concentrating the extracting solution to minus 0.08 to minus 0.09MPa until no alcohol exists, and then adding water into the concentrated extracting solution according to the volume ratio of 1:10 times for dispersion treatment to obtain an aqueous dispersion 120L;
C. wet loading the aqueous dispersion, separating by AB-8 macroporous adsorbent resin column chromatography (methanol: water=50v/V is mobile phase), collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution 50L;
D. filtering the concentrated solution, separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B:0.1% V/V formic acid water, A: B=22:78V/V as mobile phase; detection wavelength 215 nm), and collecting corresponding chromatographic peaks;
E. and (3) concentrating the collected liquid product to minus 0.08 to minus 0.09MPa at 50 ℃ until no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain 38g of white powder product, namely the glycoside compound with the structural formula shown in (I).
The whole production process takes about 9 days;
the purity of the product was checked again by reverse phase analytical liquid chromatography (RP-HPLC) by changing the mobile phase component (A: methanol B:0.1% V/V formic acid water, A: B=46:54V/V as mobile phase; detection wavelength 215 nm) and found to be 99.34%.
Example 7:
A. taking dried Bulbus Lilii (Rolling pill)L. lancifoliumPulverizing to 80 mesh particle size, reflux-extracting with 9 times of 90wt% ethanol for 4 times each for 1 hr, and mixing extractive solutions;
B. concentrating the extracting solution under reduced pressure to-0.08 to-0.09 MPa 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 water dispersion;
C. wet loading the water dispersion, separating by AB-8 macroporous adsorbent resin column chromatography (methanol: water=50v/V is mobile phase), collecting chromatographic liquid containing total flavone component, and concentrating under reduced pressure until no alcohol is present to obtain 40L concentrate;
D. filtering the concentrated solution, separating the filtrate by using C18 reversed phase chromatographic packing under high pressure (A: acetonitrile B:0.1% V/V formic acid water, A: B=22:78V/V as mobile phase; detection wavelength 215 nm), and collecting corresponding chromatographic peaks;
E. and (3) concentrating the collected liquid product to be no acetonitrile under the condition that the temperature of the collected liquid is reduced to between-0.08 and-0.09 MPa, freeze-drying the concentrated liquid, grinding the dried solid into powder, and vacuum-drying at the temperature of 45 ℃ to obtain 35g of white powder product, namely the glycoside compound with the structural formula shown in (I).
The whole production process takes about 8 days;
the purity of the product was checked again by reverse phase analytical liquid chromatography (RP-HPLC) by changing the mobile phase component (A: methanol B:0.1% V/V formic acid water, A: B=46:54V/V as mobile phase; detection wavelength 215 nm) and found to be 99.19%.
Example 8:
the white powder compound having the structural formula shown in (I) and obtained by extraction and separation in example 4 (namely, lily neoglycoside I called in the following experiment) was arbitrarily selected to carry out the following experiment:
compound anti-lung cancer activity assay:
(1) Experimental materials and instruments
And (3) cells: a is that 549 Cell (Wohunocel life technology Co., ltd.)
Drug and reagent: lily new glycoside I (purity > 99.0%, homemade); the DMSO solution is dissolved, the concentration of prepared mother solution is 200 mu mol/L, 1 mu L of each mother solution is diluted to 1ml by serum-free culture medium, and the concentration is 1: 4. RPMI-1640 medium (Gibco, lot number 8120139, U.S.A.), FBS (Procell, lot number SA201126, U.S.A.), PBS buffer powder (Biofix, lot number EZ6688C 183), penicillin-streptomycin solution (biosharp, lot number 1334GR 005), MTT (biosharp, lot number EZ6688D 183), DMSO (Chengdu Colon Chemicals, inc., lot number 67-68-5)
Instrument: thermo Varlosks microplate reader (U.S. Thermo Fisher Scientific Co.), allegraX-30R centrifuge (Beckman Coulter Co., U.S. Co., ltd.), ultra clean bench (Changhong Meishi, inc.), inverted microscope (Zeiss, germany), CO 2 Constant temperature incubator (Suzhou Jiemei electronics Co., ltd.).
(2) Experimental method
10% 1640 complete medium was used at 37℃with 5% CO 2 Culturing under concentration conditionAnd (5) nourishing. Digesting cells in logarithmic growth phase to obtain cell suspension, and mixing with the extract of 1×10 4 Inoculating the cells/ml into 96-well plate, and inoculating the cells into CO 2 Culturing in a constant temperature incubator for 24 hours. Discarding the culture medium, washing with PBS for 1 time, then adding lily new glycoside I (3 compound holes are arranged in each group) with different concentrations, and adding 3-hole blank culture medium as a normal control group; put it back into CO 2 Culturing in a constant temperature incubator for 24 hours. Then, 20. Mu.L of MTT solution per well was cultured in a medium for 4 hours, the culture solution was discarded, 150. Mu.L/well of DMSO solution was added, and after shaking in an microplate reader for 5 minutes, absorbance (OD) was measured at 490 nm.
Data were processed using SPSS 26.0 statistical software and results are expressed as mean.+ -. Standard deviation. Comparison between groups used One-way analysis of variance (One way-ANOVA).
(3) Experimental results
As shown in Table 2 below, the lilium novel glycoside I has inhibitory activity on the proliferation of A549 cells cultured in vitro, the maximum inhibition rate in the study dosage is 65.10%, and the IC50 is 0.029 (0.012-0.102) mu mol/L.
Therefore, the glycoside compound with the structural formula shown as (I) obtained by extraction and separation can be applied to the preparation of anti-lung cancer drugs.
Claims (6)
1. A method for extracting and separating glycoside compounds from lily, which is characterized by mainly comprising the following steps:
A. reflux extraction
Taking dried lily as a raw material, crushing, and carrying out reflux extraction by using 80-90% ethanol to obtain an extracting solution;
the lily is a roll pillLilium lancifoliumDried bulb of thunder;
B. concentrating under reduced pressure
Concentrating the extracting solution obtained in the step A to minus 0.08 to minus 0.09MPa until no alcohol exists, and then adding water into the concentrated extracting solution for dispersion treatment to obtain an aqueous dispersion;
C. resin column chromatography
B, loading the aqueous dispersion obtained in the step B by a wet method, separating by using an AB-8 macroporous adsorption resin column for chromatography, collecting chromatographic liquid containing glycoside components, and concentrating under reduced pressure until no alcohol exists to obtain concentrated solution;
d.c18 reverse phase chromatography column separation
Filtering the concentrated solution obtained in the step C, and separating the filtrate by using a C18 reverse phase chromatographic packing high-pressure preparation: a: acetonitrile B:0.1% V/V formic acid water, a: b=22:78V/V being the mobile phase; detection wavelength 215nm;
E. concentrating
Decompressing the product collection liquid obtained in the step D to minus 0.08 to minus 0.09MPa till no acetonitrile exists, freeze-drying the concentrated liquid, grinding the dried solid into powder, and vacuum-drying at 45 ℃ to obtain white powder, namely the glycoside compound with the structural formula shown in (I);
the glycoside compound of the structural formula shown in the formula (I) is a coiled pill prepared from plants of the family LiliaceaeLilium lancifoliumThe dry bulb of thunder god is obtained by extraction and separation, wherein the name is: 2, 4-dichloro-3, 5-dimethoxy-benzyl-1-O-beta-D-glucopyranosyl- (1- & gt 6) -beta-D-glucopyranoside, has a molecular weight of 561 and a molecular formula of C 21 H 30 O 13 Cl 2 The self-designation is: lily novel glycoside I.
2. The method for extracting isolated glycoside compounds from lily according to claim 1, wherein: in the step A, the particle size of the crushed raw materials is 60-80 meshes.
3. The method for extracting isolated glycoside compounds from lily according to claim 1, wherein: in the step A, the weight of the ethanol is 8-10 times of that of the raw material during the reflux extraction.
4. The method for extracting isolated glycoside compounds from lily according to claim 1, wherein: in the step A, the reflux extraction is carried out for 2 to 3 times, each time for 1 hour.
5. The method for extracting isolated glycoside compounds from lily according to claim 1, wherein: in the step B, the concentrated extracting solution is added with water according to the volume ratio of 1:6-1:10 for dispersion treatment.
6. The method for extracting isolated glycoside compounds from lily according to claim 1, wherein: in the step C, the mobile phase used for the AB-8 macroporous adsorption resin column chromatographic separation is methanol/water=50:50V/V.
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| JPS5498339A (en) * | 1978-01-18 | 1979-08-03 | Rikagaku Kenkyusho | Carcinostatic agent |
| JP2003113088A (en) * | 2001-10-09 | 2003-04-18 | Pola Chem Ind Inc | Carcinogenesis promoter-suppressant and composition containing the same |
| CN101029073A (en) * | 2007-03-29 | 2007-09-05 | 复旦大学 | Steroid saponin compound and its use in preparation antineoplastic |
-
2021
- 2021-07-22 CN CN202110830176.XA patent/CN113336808B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5498339A (en) * | 1978-01-18 | 1979-08-03 | Rikagaku Kenkyusho | Carcinostatic agent |
| JP2003113088A (en) * | 2001-10-09 | 2003-04-18 | Pola Chem Ind Inc | Carcinogenesis promoter-suppressant and composition containing the same |
| CN101029073A (en) * | 2007-03-29 | 2007-09-05 | 复旦大学 | Steroid saponin compound and its use in preparation antineoplastic |
Non-Patent Citations (2)
| Title |
|---|
| Anti-tumor promoting effect of glycosides from Prunus persica seeds;Fukuda, Toshiyuki等;《Biological & Pharmaceutical Bulletin》;第26卷(第2期);271-273 * |
| 黄柏和兰州百合的水溶性化学成分研究;章聚宝;《兰州理工大学硕士学位论文》;1-62 * |
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