CN101775061A - Ilex latifolia thunb saponin compound - Google Patents
Ilex latifolia thunb saponin compound Download PDFInfo
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Abstract
The invention relates to an ilex latifolia thunb saponin compound, in particular to a new triterpenoid separated from ilex latifolia thumb, a preparation process, a medical application and an analyzing method. The compound comprises fireweed butanone A, B, D, E, F, G, fireweed saponin LZ11, fireweed saponin LZ12 and derivate thereof, wherein the fireweed butanone A and the derivate thereof have structures shown in the formula one.
Description
Technical field:
The present invention relates to isolating effective constituent from Leaf of Chinese Holly, particularly relate to new triterpene compound and preparation technology, medicinal use and the analytical procedure of separation from Leaf of Chinese Holly with medicinal use.
Background technology:
Leaf of Chinese Holly is the medicinal plant of south China and west and south folk tradition, and many tender leaf or leaves with plant are used as medicine.The property bitter but sweet flavor has the effect of clearing heat and cooling blood, nourishing the liver and kidney, can loose wind-heat, refresh oneself, relieving thirst and restlessness.Be used for the treatment of diseases such as headache, dentalgia, hot eyes, pyreticosis polydipsia and dysentery more." Chinese medicine voluminous dictionary " record, Leaf of Chinese Holly also has the effect in " arteries and veins of invigorating blood circulation, cool uterus ".Application that China is among the people more than 2,000 years and modern medicine clinical trial show, Leaf of Chinese Holly has clearing heat and detoxicating, inhibiting bacteria and diminishing inflammation, relieving spasm to stop pain, the step-down fat-reducing, press down the cancer anti-cancer, produce refreshing effect to the mind, the benefit that makes eye bright is thought, slake thirst and help produce saliva, go greasy sobering up, be good for the stomach and keep fit, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve blood viscosity and microcirculation, vasodilation, remove free radical, strengthen pharmacological functions such as body immunity, long-term drinking has good medical care effect, so be described as health tea, beauty treatment tea, diet tea, the blood-pressure decreasing tea and the tea that lengthens one's life.
Forefathers only separate the chemical constitution study of Leaf of Chinese Holly and have obtained the minority triterpene compound.We are by carrying out system to Leaf of Chinese Holly, modern, the isolation technique of science, 95% ethanol extraction from Kudincha Holly (Ilex kudincha C.J.Tseng) dry leave, use silica gel column chromatography repeatedly, macroporous resin column chromatography, Sephadex LH-20, the preparation thin layer, separation means of purification such as preparation HPLC obtain 40 compounds altogether, the structure of having identified 36 compounds by the physico-chemical property and the spectral data of compound, be respectively α-amyrin (α-amyran, 1), ursolic acid (ursolic acid, 2), Oleanolic Acid (oleanic acid, 3), bitter leaves ketone A (kudinone A, 4), bitter leaves saponin(e LZ1 (kudinoside LZ1,5), bitter leaves ketone B (kudinone B, 6), bitter leaves saponin(e LZ3 (kudinoside LZ3,7), bitter leaves saponin(e LZ4 (kudinoside LZ4,8), bitter leaves saponin(e LZ5 (kudinoside LZ5,9), bitter leaves saponin(e LZ6 (kudinoside LZ6,10), bitter leaves saponin(e LZ7 (kudinoside LZ7,11), bitter leaves saponin(e LZ8 (kudinoside LZ8,12), bitter leaves saponin(e LZ9 (kudinoside LZ9,13), bitter leaves saponin(e LZ10 (kudinoside LZ10,14), bitter leaves ketone C (kudinone C, 15), bitter leaves saponin(e LZ11 (kudinoside LZ11,16), bitter leaves saponin(e LZ12 (kudinoside LZ12,17), bitter leaves saponin(e LZ13 (kudinoside LZ13,18), bitter leaves saponin(e LZ14 (kudinoside LZ14,19), bitter leaves saponin(e LZ15 (kudinoside LZ15,20), bitter leaves saponin(e LZ16 (kudinoside LZ16,21), bitter leaves saponin(e LZ17 (kudinoside LZ17,22), bitter leaves saponin(e LZ2 (kudinoside LZ2,23), bitter leaves saponin(e LZ18 (kudinosideLZ18,24), bitter leaves saponin(e LZ19 (kudinoside LZ19,25), bitter leaves saponin(e LZ20 (kudinoside LZ20,26), bitter leaves saponin(e LQ1 (kudinoside LQ1,27), gallic acid (gallic acid, 28), forulic acid (ferulic acid, 29), Vanillin (vanillin, 30), Methyl caffeoate (methyl caffeate, 31), coffic acid (caffeic acid, 32), p-Hydroxybenzaldehyde (p-hydroxybenzaldehyde, 33), β-Gu Zaichun (β-sitosterol, 34), daucosterol (daucosterol, 35), α-aromadendrol-3 β-cetylate (3 β-palmitoyl-α-amyrin, 36).
Table 2-1 has listed the structure and the main authentication method thereof of these 36 compounds.
Wherein, compound 4～14,16,17,23～26 (adding up to 17) is not for seeing the new compound of bibliographical information, and compound 27 is a new natural product, and compound 31,33 obtains for separating from holly first.Compound 1～27 is a triterpene compound, and wherein compound 1～4,6,15 is the aglycon of triterpene compound, and compound 5,7～14,16～27 is a triterpene saponin componds.Compound 28～33 is a phenolic acid compound, and compound 34,35 is a steroid compound, and compound 36 is the ester of triterpenoid.
Summary of the invention:
Purpose of the present invention: one group of Leaf of Chinese Holly triterpene compound is provided, comprises bitter leaves ketone A, B, D, E, F, G, bitter leaves saponin(e LZ
11, bitter leaves saponin(e LZ
12And derivative, wherein bitter leaves ketone A and derivative thereof have structure shown in the formula 1, wherein, and R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone C sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
Wherein bitter leaves ketone B and derivative thereof have structure shown in the formula 2, wherein, and R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone B sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
Bitter leaves ketone D and derivative thereof have structure shown in the formula 3, wherein, and R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone D sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
Bitter leaves ketone E and derivative thereof have structure shown in the formula 4, wherein, and R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone E sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
Bitter leaves ketone F and derivative thereof have structure shown in the formula 5, wherein, and R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone F sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
Bitter leaves ketone G and derivative thereof have structure shown in the formula 6, wherein, and R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, β-D-can draw uncle's glycosyl, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone G sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
Bitter leaves saponin(e LZ
11, have structure shown in the formula 7.
Bitter leaves saponin(e LZ
12, have structure shown in the formula 8.
The concrete compound of the present invention is as follows:
The present invention also comprises the preparation method of the above compound of the present invention, it is characterized in that, step is as follows: Leaf of Chinese Holly water or water-containing organic solvent extract, extract is used macroporous resin, ion exchange resin, polymeric amide, dextrane gel, reverse phase silica gel isochromatic spectrum means are refining separates, elder generation's water or lower concentration organic solvent wash-out are removed impurity, obtain product with the high levels of organic solvents wash-out again, adopt above repeatedly chromatographic separation to make with extra care purifying, or recrystallization purifying, wherein, extraction is selected from ethanol with organic solvent, methyl alcohol, acetone, macroporous resin, ion exchange resin comprises styrene type, the acrylic type macromolecule resin.
In addition, the present invention also provides the method for utilizing the Leaf of Chinese Holly crude extract acid hydrolysis contain The compounds of this invention to prepare The compounds of this invention, and wherein said acid is selected from mineral acid and organic acid as sulfuric acid, hydrochloric acid, formic acid, acetate, trifluoroacetic acid.
The present invention also comprises the structure determination and the detection method of above-claimed cpd: as: HPLC analyzes, and is detector with mass spectrum (MS) detection, ultraviolet (UV) detection, circular dichroism spectrum (CD) detection, light scattering detector etc.As following detection method:
The configuration of reference substance solution: triterpene compound chemical reference substance precision takes by weighing in right amount, is mixed with an amount of reference substance solution with methyl alcohol.
The configuration of sample solution: precision takes by weighing the Leaf of Chinese Holly sample, and each is an amount of, with 100ml chloroform (or methyl alcohol, ethyl acetate, acetone, acetonitrile, sherwood oil, organic solvent and solution in different concentration thereof such as hexanaphthene) extract, reclaim extracting solution, methyl alcohol (or chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, organic solvent and solution in different concentration thereof such as hexanaphthene) dissolving, the little chromatographic column pre-treatment of SPE through filling ODS, 95% acetonitrile (or methyl alcohol, ethyl acetate, acetone, chloroform, sherwood oil, organic solvent and solution in different concentration thereof such as hexanaphthene) washing, merge elutriant, methanol constant volume, filter, promptly.
Testing method: carrying out HPLC and analyze, is detector with mass spectrum (MS) detection, ultraviolet (UV) detection, circular dichroism spectrum (CD) detection, light scattering detector etc.With the chromatographic peak area of each compound in the sample, corresponding chromatographic peak area with the standard control sample according to calibration curve method (or 1 method of external standard, 2 methods of external standard etc.), carries out quantitative analysis, calculates, promptly.
The present invention also comprises the pharmaceutical composition that contains compound of the present invention.
The described composition pharmaceutical preparation that can be The compounds of this invention be mixed with the medicine acceptable carrier.
These preparations comprise that general pharmaceutical dosage form is as tablet, capsule, particle, oral liquid, injection etc.
Pharmaceutical composition of the present invention is determined usage and dosage according to patient's situation in use, but obeys every day three times, each 1-20 agent, as: 1-20 bag or grain or sheet, every dose of 1mg-1000mg.
The present invention also comprise compound of the present invention in clearing heat and detoxicating, the inhibiting bacteria and diminishing inflammation of preparation, relieving spasm to stop pain, step-down fat-reducing, press down the cancer benefit that give protection against cancer, produces refreshing effect to the mind, makes eye bright and think, slake thirst and help produce saliva, go to be bored with and sober up, be good for the stomach and keep fit, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve the application in the medicines such as blood viscosity and microcirculation, vasodilation, removing free radical, enhancing body immunity.
Below be the effect determination experiment of The compounds of this invention:
Test shows that The compounds of this invention has the Hydroxymethylglutaryl coenzyme A reductase enzymic activity of inhibition and anti-inflammatory activity, and it is as follows wherein to suppress Hydroxymethylglutaryl coenzyme A reductase enzymic activity result:
Below experiment is used to illustrate that The compounds of this invention is more superior than existing similar compound.
The mouse of The compounds of this invention is oral once to be given and maximum administration concentration 10g/Kg body weight, there is no toxic side effects, and blood parameters is all normal, therefore illustrates that The compounds of this invention and other compounds have bigger advantage.
Description of drawings:
The extraction schema of Fig. 1 process flow sheet Leaf of Chinese Holly
The separation process figure of Fig. 2 process flow sheet Leaf of Chinese Holly petroleum ether layer
The separation process figure of Fig. 3 process flow sheet Leaf of Chinese Holly chloroform layer
The separation process figure of Fig. 4 process flow sheet Leaf of Chinese Holly ethyl acetate layer
Fig. 5 process flow sheet Leaf of Chinese Holly n-butanol layer macroporous resin separation process figure
Fig. 6 process flow sheet Leaf of Chinese Holly n-butanol layer macroporous resin 70% ethanol elution thing separation process figure
The CD of Fig. 7 compound 7 and UV spectrum
The 3D model (sector rule) of Fig. 8 compound 7
Embodiment:
Embodiment 1: the separation of compound
Bitter leaves dry leave 6kg is with twice of 95% ethanol 60L refluxing extraction.Decompression recycling ethanol gets syrupy shape medicinal extract.Use the suitable quantity of water suspendible, use equivalent sherwood oil, chloroform, ethyl acetate, n-butanol extraction successively, get each layer extract and be respectively 354g, 30g, 85g, 640g.
1. petroleum ether layer
Petroleum ether layer extract (354g) is through silica gel column chromatography repeatedly, with sherwood oil/acetone gradient (100: 0～0: 100) wash-out.
Sherwood oil/acetone (100: 6) flow point recrystallization obtains compound 2 (150mg)
Sherwood oil/acetone (100: 20) flow point recrystallization obtains compound 34 (60mg)
Sherwood oil/acetone (100: 1) gets time flow point 15, gets compound 1 (2mg) through preparation thin layer (sherwood oil/acetone (10: 1)), recrystallization
Sherwood oil/acetone (100: 10) gets time flow point 16, gets compound 36 (2mg) through preparation thin layer (sherwood oil/acetone (5: 1)), recrystallization
2. chloroform layer
Chloroform layer extract (30g) is through silica gel column chromatography repeatedly, with sherwood oil/acetone gradient (100: 0～0: 100) wash-out.
Sherwood oil/acetone (100: 1) gets flow point 10, flow point 10 successively through silica gel column chromatography (sherwood oil/acetone (50: 1)), the preparation thin layer (sherwood oil/acetone (10: 1)), (the chloroform/methanol elution system, 70% chloroform/methanol flow point recrystallization gets compound 3 (8mg) to sephadex lh-20
Sherwood oil/acetone (100: 2) gets flow point 35, flow point 35 successively through silica gel column chromatography (sherwood oil/acetone (30: 1)), the preparation thin layer (sherwood oil/acetone (8: 1)), (the chloroform/methanol elution system, 50% chloroform/methanol flow point recrystallization gets compound 4 (15mg) to sephadex lh-20
Sherwood oil/acetone (100: 5) gets flow point 4, flow point 4 successively through silica gel column chromatography (sherwood oil/acetone elution system (20: 1)), the preparation thin layer (sherwood oil/acetone (8: 1)), (the chloroform/methanol elution system, 60% chloroform/methanol flow point recrystallization gets compound 6 (9mg) to sephadex lh-20
Sherwood oil/acetone (100: 8) gets flow point 15, and (the chloroform/methanol elution system, 50% chloroform/methanol flow point recrystallization gets compound 5 (60mg) to flow point 15 through silica gel column chromatography (sherwood oil/acetone elution system (10: 1)), sephadex lh-20 successively
3. ethyl acetate layer
Ethyl acetate extraction layer (85g) is through silica gel column chromatography repeatedly, with sherwood oil/acetone (100: 0～0: 100) gradient elution.
Sherwood oil/acetone (100: 4) flow point recrystallization gets compound 35 (5mg), compound 33 (3mg)
Sherwood oil/acetone (100: 6) flow point recrystallization gets compound 30 (2mg)
Sherwood oil/acetone (100: 8) flow point recrystallization gets compound 31 (15mg), compound 32 (35mg)
Sherwood oil/acetone (100: 10) flow point recrystallization gets compound 29 (3mg)
Sherwood oil/acetone (100: 15) flow point recrystallization gets compound 28 (5mg)
4. n-butanol layer
N-butanol extraction layer (640g) successively with water, 30% ethanol, 70% ethanol, 95% ethanol elution, obtains water layer (200g), 30% alcohol layer (40g), 70% alcohol layer (183g), 95% alcohol layer (5g) through macroporous resin PD101 column chromatography respectively.
Get 70% ethanol elution part 180g, through silica gel column chromatography, with chloroform/methanol (100: 0～0: 100) gradient elution.
Chloroform/methanol (100: 4) gets flow point 38, flow point 38 gets time cut 3-10 through dextrane gel separation and purification (chloroform/methanol 35%, 45%, 60%, 65%, 70%, 75%, 80%, 90% gradient), and inferior cut 3-10 is again through anti-phase preparative high-performance liquid chromatographic
(60% acetonitrile) connects the peak successively in different retention time, reclaims solvent recrystallization and gets compound 17 (14mg), compound 18 (5.6mg), compound 19 (6mg), compound 20 (8mg) respectively.
Chloroform/methanol (100: 5) gets flow point 40, and flow point 40 is through the dextrane gel separation and purification, and 50% chloroform/methanol wash-out gets compound 7 (60mg), 75% chloroform/methanol wash-out compound 8 (15mg).
Chloroform/methanol (100: 6) gets flow point 45, flow point 45 gets flow point 10-15 through dextrane gel separation and purification (chloroform/methanol 45%, 60%, 65%, 75%, 80%, 85%), flow point 10-15 is again through anti-phase preparative high-performance liquid chromatographic (78% acetonitrile), connect the peak successively in different retention time, reclaim solvent recrystallization and get compound 11 (3.8mg), compound 12 (12mg), compound 13 (5mg) respectively.
Chloroform/methanol (100: 7) gets flow point 52-55, flow point 52-55 gets time cut 10-14 through dextrane gel separation and purification (chloroform/methanol gradient), inferior cut 10-14 is again through anti-phase preparative high-performance liquid chromatographic (62% acetonitrile), connect the peak successively in different retention time, reclaim solvent recrystallization and get compound 26 (0.8mg), compound 27 (5.9mg) respectively.
Chloroform/methanol (100: 8) gets flow point 58-62, flow point 58-62 gets time cut 10-14 through dextrane gel separation and purification (chloroform/methanol 60%, 65%, 70%, 80% gradient), inferior cut 10-14 is again through anti-phase preparative high-performance liquid chromatographic (55% acetonitrile), connect the peak successively in different retention time, reclaim solvent recrystallization and get compound 14 (15mg), compound 15 (2.5mg), compound 16 (5.6mg) respectively.
Chloroform/methanol (100: 10) gets flow point 69-71, flow point 69-71 gets time cut 10-14 through dextrane gel separation and purification (chloroform/methanol 60%, 65%, 70%, 80% gradient), inferior cut 10-14 is equipped with phase high performance liquid chromatography (74% acetonitrile) through counter again, connect the peak successively in different retention time, reclaim solvent recrystallization and get compound 9 (120mg), compound 10 (25mg) respectively.
Chloroform/methanol (100: 12) gets flow point 71-86, flow point 71-86 gets time flow point 10-16 through dextrane gel separation and purification (chloroform/methanol 50%, 55%, 60%, 65%, 75%, 80%, 90% gradient), inferior flow point 10-16 is again through RPLC (84% acetonitrile), connect the peak successively in different retention time, the recovery solvent recrystallization gets recrystallization respectively and gets compound 21 (30mg), compound 22 (8mg), compound 23 (12mg), compound 24 (7mg), compound 25 (45mg).
Embodiment 2: chemical structure is identified
Utilize 1 dimension, 2 dimension nuclear magnetic resonance spectrums (1D, 2D-NMR), mass spectrum (MS), circular dichroism spectrum (CD) the spectrum means of etc.ing and other physico-chemical processes determined the chemical structure of 35 compounds that separation obtains.Wherein the chemical structure and the identification of means of triterpene and glycoside derivative (comprising new compound) thereof are as follows:
Embodiment 3:HPLC-TOF-MS analyzes
The configuration of reference substance solution: triterpene compound chemical reference substance precision takes by weighing in right amount, is mixed with an amount of reference substance solution with methyl alcohol.
The configuration of sample solution: precision takes by weighing the Leaf of Chinese Holly sample, and each is an amount of, with 100ml chloroform (or methyl alcohol, ethyl acetate, acetone, acetonitrile, sherwood oil, organic solvent and solution in different concentration thereof such as hexanaphthene) extract, reclaim extracting solution, methyl alcohol (or chloroform, ethyl acetate, acetone, acetonitrile, sherwood oil, organic solvent and solution in different concentration thereof such as hexanaphthene) dissolving, the little chromatographic column pre-treatment of SPE through filling ODS, 95% acetonitrile (or methyl alcohol, ethyl acetate, acetone, chloroform, sherwood oil, organic solvent and solution in different concentration thereof such as hexanaphthene) washing, merge elutriant, methanol constant volume, filter, promptly.
Testing method: carrying out HPLC and analyze, is detector with mass spectrum (MS) detection, ultraviolet (UV) detection, circular dichroism spectrum (CD) detection, light scattering detector etc.With the chromatographic peak area of each compound in the sample, corresponding chromatographic peak area with the standard control sample according to calibration curve method (or 1 method of external standard, 2 methods of external standard etc.), carries out quantitative analysis, calculates, promptly.
Embodiment 4: the saccharide residue three-dimensional arrangement is measured
Reaction principle: sugared thiazole front three silicon ether reaction
The standard sugar of different steric configurations is reacting the thiazole derivative chromatogram different in kind that the back produces, retention time t with chiral reactant L-acthiol-J hydrochloride
RFor the chromatographic peak of 3.75min is L-Rha, t
RFor the chromatographic peak of 4.65min is L-Ara, therefore can distinguish the steric configuration of saccharide residue.
4.1 the hydrolysis reaction of compound 16
Get the sample of 1mg respectively, be dissolved in the ampoule, envelope bottle, 110 ℃ of reaction 2h with 1ml 2NTFA.CHCl
3Extract water layer N three times
2Dry up, add methyl alcohol, use N again
2Dry up, there is not TFA in this process in product repeatedly.
4.2 sugared thiazole trimethylsilyl ethers is synthetic
The product of complete hydrolysis adds 1ml pyridine and 2ml L-acthiol-J hydrochloride (L-cysteine methy esterhydrochloride), and 60 ℃ of reaction 2h use N
2Dry up product, add again the trimethyl silane imidazoles (N-(trimethyl) imidazole, TMLI), 0.2ml, 60 ℃ the reaction 1h.
4.3 aftertreatment
Add 1ml water termination reaction, (3 * 1ml), extraction liquid is concentrated into 1ml, carries out gas-chromatography (GC) analysis with the hexanaphthene extraction.Standard sugar begins operation from " adding 1ml pyridine and 2ml L-acthiol-J hydrochloride ... ".
4.4GC condition
Capillary column: OV-17,30m * 0.32mm * 0.5um
Column temperature: 220 ℃ of vaporizers: 280 ℃ of 280 ℃ of detector: FID
Embodiment 5: the lactonic ring ring-opening reaction
Reaction principle: in alkaline environment, open the lactonic ring reaction.
Open lactonic ring by alkaline condition, feed liquor facies analysis respectively is by the variation of CD collection of illustrative plates before and after the contrast reaction, the steric configuration of checking lactonic ring.
5.1 the hydrolysis of compound 10
Get the 1mg sample, add 2mol/L sodium hydrate methanol solution 1ml, 70 ℃ of heating in water bath 3h, reactant is transferred pH value 6～7, and vacuum is spin-dried for, and with the small amount of methanol dissolving, is spin-dried for three times repeatedly.With the small amount of methanol dissolving, the laggard HPLC-CD of filtering membrane analyzes again.
5.2 liquid-phase condition
Chromatographic column: TEDAChrom YWG C 18 (5 μ m, 4.6 * 250mm)
Moving phase: methyl alcohol: water (100: 75)
Embodiment 6: acid hydrolysis
Get the sample of 1mg respectively, be dissolved in the ampoule, envelope bottle, 110 ℃ of reaction 2h with 1ml 2N trifluoroacetic acid (TFA).CHCl
3Extract water layer N three times
2Dry up, add methyl alcohol, use N again
2Dry up, there is not TFA in this process in product repeatedly, promptly.
Embodiment 7:: structure authentication method bitter leaves saponin(e LZ
3(kudinoside LZ
3) be example
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.High resolution mass spectrum provides [M+Na]
+Peak m/z 787.4230, calculated value 787.4245, error-1.5ppm, the combined carbon spectrum determines that molecular formula is C
41H
64O
13The UV maximum absorption wavelength is 228nm.
1H-NMR (600MHz, Pyr-d
5) provide in the spectrum seven methyl proton signal δ 1.68 (3H, s), 1.62 (3H, s), 1.49 (3H, s), 1.18 (3H, s), 1.08 (3H, s), 0.90 (3H, s), 0.87 (3H, s), two oxygen carbonaceous subsignal 3.26 (1H even, dd, J=12.0,4.2Hz, H-3), 5.92 (1H, br.s, H-12).
13C-NMR (150MHz, Pyr-d
5) in the spectrum, δ 175.4 is 28 carbonyl carbon signals, two olefinic carbon signal δ 146.4,137.5 are respectively 13,18 double key carbon signals, three oxygen carbon signal δ of company 88.8,85.7 and 74.1, wherein δ 74.1 is 19 even characteristic signals of oxygen carbon, δ 85.7 and the 175.4 feature carbon signals for F ring C-20 and C-28 lactonic ring
[4]In HMBC spectrum, H-12 (δ 5.92) and C-11 (δ 26.6), C-13 (δ 146.4) and C-14 (δ 43.9) have coherent signal, have confirmed that C-11,12,13,18 matrix tablet dislocation miss! Do not find Reference source.(seeing Fig.2-1).Equally, H-22 (δ 2.57) and C-17 (δ 44.1), C-18 (δ 137.5), C-21 (δ 28.0) and C-28 (δ 175.4) have coherent signal, confirm that lactonic ring links to each other with two keys.In the NOESY spectrum, H-12 (δ 5.92) and CH
3There is coherent signal-29 (δ 1.68), determine the β that is oriented to of 12 hydrogen, and hydroxyl is oriented to α (seeing Fig.2-12).In CD spectrum, compound 7 presents negative Cotton effect (seeing Fig.2-13) at the 232nm place, according to sector rule in conjunction with octant rule
[5]Observing lactonic ring (sees
The relevant spectrum of Fig.2-12 compound 7
Fig.2-14), can determine that the steric configuration of lactonic ring is for (17R 20S), can determine that in conjunction with the NOESY spectrum aglycon of compound 7 is bitter leaves ketone B.
1In the H-NMR spectrum, (1H, d J=5.4Hz) are the anomeric proton signal of pectinose to δ 4.91; (1H br.s) is the anomeric proton signal of rhamnosyl to δ 6.15, δ 1.63 (3H, d J=6.0Hz) are rhamnosyl 6 " position methyl feature proton signal.
1Provide the end group carbon J of rhamnosyl in the H-NMR spectrum
C1-H1Be 174.5Hz, judge the α-L that is configured as of rhamnosyl.
13Also provide pectinose (δ 104.9) and rhamnosyl (δ 101.8) end group carbon signal in the C-NMR spectrum.The chemical displacement value of aglycon portion C-3 is 88.8ppm, has compared to low field displacement about 10.7ppm with aglycon, shows that the C-3 position becomes glycosides.In the HMBC spectrum, (1H, d J=6.0Hz) have long-range relevantly with 3 carbon of aglycon (δ 88.8) the anomeric proton δ 4.91 of pectinose, and (1H is br.s) with 2 of pectinose ' position carbon (δ 75.9) has long-range relevant the anomeric proton δ 6.15 of rhamnosyl.Equally, in the NOESY spectrum, 3 proton δ of aglycon, 3.2 δ (1H, dd, J=12.0 is 4.2Hz) with the anomeric proton δ 4.91 (1H of pectinose, d, J=6.0Hz) coherent signal is arranged, (1H is br.s) with pectinose 2 ' proton δ 4.57 (1H for rhamnosyl anomeric proton δ 6.15, t, J=5.4Hz) coherent signal is arranged, can determine pectinose, link to each other with 3 of aglycons in the inboard; Rhamnosyl is connected in 2 of pectinose ' position in the outside.The absolute configuration of determining compound 7 thus is:
3-O-α-L-rhamnopyranosyl (1 → 2)-α-L-arabinopyranosyl-(3S)-3 β, 12 α, 19 α-trihydroxyurs-13 (18)-en-28,20 β-lactone are one not see the new compound of bibliographical information, called after bitter leaves saponin(e LZ
3(the carbon signal ownership sees Table 2-2).
Embodiment 8: part of compounds structure appraising datum
Compound 4: bitter leaves ketone A (kudinone A)
White solid, Liebermann-Burchard reacting positive, Molish reaction negative.UV(MeOH)：228nm。IR(KBr)：3500，3369，3233，2939，1715，1653，1461，1383，1368，1255，1183，1133，1075，1044，1001，946，927，848，805，773，699，662，551。HR-TOF-MS:m/z 995.6579 ([2M+Na]
+, C
60H
92NaO
10, calculated value 995.6588, error-0.9ppm).CD?spectrum(c＝1.08*10
-4，MeOH)：232nm(-29.8)。
1H-NMR(300MHz，Pyr-d
5)：δ4.93(1H，dd，J＝11.9，5.5Hz，H-12)，3.46(1H，dd，J＝10.5，5.4Hz，H-3)，1.80(3H，s，H-29)，1.54(3H，s，H-30)，1.23(3H，s，H-27)，1.22(3H，s，H-23)，0.99(3H，s，H-24)，0.93(3H，s，H-26)，0.89(3H，s，H-25)。
13The C-NMR data see Table 2-2.
Compound 5: bitter leaves saponin(e LZ
1(kudinoside LZ
1)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：228nm。HR-TOF-MS:m/z 949.4785 ([M+Na]
+, C
47H
74NaO
18, calculated value 949.4773, error is-1.2ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：234nm(-42.5)。
1H-NMR (300MHz, Pyr-d
5) aglycon: δ 4.93 (1H, dd-like, J=11.9,5.4Hz, H-12), 3.24 (1H, d, J=11.1,3.6Hz, H-3), 1.78 (3H, s, H-29), 1.53 (3H, s, H-30), 1.25 (3H, s, H-27), 1.18 (3H, s, H-23), 1.07 (3H, s, H-24), 0.86 (3H, s, H-26), 0.80 (3H, s, H-25).Pectinose: δ 4.83 (1H, d, J=5.9Hz, H-1 '), glucose: δ 5.10 (1H, d, J=7.8Hz, H-1 "), rhamnosyl: δ 6.18 (1H, br.s, H-1 " '), 1.60 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-2.
Compound 6: bitter leaves ketone B (kudinone B)
Colourless crystallization, Liebermann-Burchard reacting positive, Molish reaction negative.UV(MeOH)：226nm。HR-TOF-MS:m/z 995.6575 ([2M+Na]
+, C
60H
92NaO
10, calculated value 995.6588, error-1.3ppm).CDspectrum(c＝1.08*10
-4，MeOH)：230nm(-8.4)。
1H-NMR(600MHz，Pyr-d
5)：δ5.91(1H，br.s，H-12)，3.42(1H，dd，J＝10.5，5.4Hz，H-3)，1.66(3H，s，H-29)，1.59(3H，s，H-27)，1.50(3H，s，H-30)，1.24(3H，s，H-23)，1.04(3H，s，H-24)，0.94(3H，s，H-26)，0.93(3H，s，H-25)。
13The C-NMR data see Table 2-2.
Compound 7: bitter leaves saponin(e LZ
3(kudinoside LZ
3)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：228nm。HR-TOF-MS:m/z 787.4230 ([M+Na]
+, C
41H
64NaO
13, calculated value 787.4245, error-1.5ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：232nm(-62.5)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.92 (1H, br.s, H-12), 3.26 (1H, dd, J=12.0,4.2Hz, H-3), 1.68 (3H, s, H-29), 1.62 (3H, s, H-27), 1.49 (3H, s, H-30), 1.18 (3H, s, H-23), 1.08 (3H, s, H-24), 0.90 (3H, s, H-26), 0.87 (3H, s, H-25).Pectinose: δ 4.91 (1H, d, J=5.4Hz, H-1 '), 4.57 (1H, t, J=5.4Hz, H-2 '), 4.32 (m, H-3 '), 4.32 (m, H-4 '), 3.84 (1H, dd, J=11.4,1.8Hz, H-5 '), 4.32 (m, H-5 ').Rhamnosyl: δ 6.15 (1H, br.s, H-1 "), 4.64 (m, H-2 "), 4.74 (1H, d, J=1.8Hz, H-3 "), 4.32 (m, H-4 "), 4.60 (m, H-5 "), 1.63 (3H, d, J=6.0Hz, H-6 ").
13The C-NMR data see Table 2-2.
Compound 8: bitter leaves saponin(e LZ
4(kudinoside LZ
4)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：229nm。HR-TOF-MS:m/z 803.4207 ([M+Na]
+, C
41H
64NaO
14, calculated value 803.4194, error 1.3ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：232nm(-29.5)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.93 (1H, s, H-12), 3.35 (1H, dd, J=9.6,4.2Hz, H-3), 1.64 (3H, s, H-29), 1.63 (3H, s, H-27), 1.49 (3H, s, H-30), 1.32 (3H, s, H-23), 0.98 (3H, s, H-24), 0.90 (3H, s, H-26), 0.87 (3H, s, H-25).Pectinose: δ 4.74 (1H, d, J=7.2Hz, H-1 '), glucose: δ 5.40 (1H, d, J=7.8Hz, H-1 ").
13The C-NMR data see Table 2-2.
Compound 9: bitter leaves saponin(e LZ
5(kudinoside LZ
5)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：230nm。HR-TOF-MS:m/z 801.4424 ([M+Na]
+, C
42H
66NaO
13, calculated value 801.4401, error is-2.3ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：234nm(-85.0)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.32 (1H, br.s, H-12), 3.35 (3H, s, 12-OCH
3), 3.25 (1H, dd, J=12.0,4.2Hz, H-3), 1.58 (3H, s, H-29), 1.50 (3H, s, H-30), 1.44 (3H, s, H-27), 1.18 (3H, s, H-23), 1.07 (3H, s, H-24), 0.88 (3H, s, H-26), 0.84 (3H, s, H-25).Pectinose: δ 4.91 (1H, d, J=6.0Hz, H-1 '), rhamnosyl: δ 6.20 (1H, br.s, H-1 "), 1.63 (3H, d, J=6.6Hz, H-6 ").
13The C-NMR data see Table 2-2.
Compound 10: bitter leaves saponin(e LZ
6(kudinoside LZ
6)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：229nm。HR-TOF-MS:m/z 949.4778 ([M+Na]
+, C
47H
74NaO
18, calculated value 949.4773, error 0.5ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：232nm(-34.0)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.92 (1H, br.s, H-12), 3.30 (1H, dd, J=10.2,4.0Hz, H-3), 1.63 (6H, m, H-29/27), 1.49 (3H, s, H-30), 1.21 (3H, s, H-23), 1.12 (3H, s, H-24), 0.89 (3H, s, H-26), 0.86 (3H, s, H-25).Pectinose: δ 4.85 (1H, d, J=5.4Hz, H-1 ').Glucose: δ 5.09 (1H, d, J=7.8Hz, H-1 ").Rhamnosyl: δ 6.14 (1H, br.s, H-1 " '), 1.63 (3H, m, H-6 " ').
13The C-NMR data see Table 2-2.
Compound 11: bitter leaves saponin(e LZ
7(kudinoside LZ
7)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：227nm。HR-TOF-MS:m/z 963.4938 ([M+Na]
+, C
48H
76NaO
18, calculated value 963.4929, error is 0.8ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：230nm(-10.6)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.31 (1H, br.s, H-12), 3.35 (3H, s, 12 α-OCH
3), 3.30 (1H, dd, J=11.7,3.9Hz, H-3), 1.57 (3H, s, H-29), 1.50 (3H, s, H-30), 1.44 (3H, s, H-27), 1.21 (3H, s, H-23), 1.11 (3H, s, H-24), 0.87 (3H, s, H-26), 0.83 (3H, s, H-25).Pectinose: δ 4.86 (1H, d, J=5.7Hz, H-1 '), glucose: δ 5.12 (1H, d, J=7.8Hz, H-1 "), rhamnosyl: δ 6.19 (1H, s, H-1 " '), 1.63 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-2.
Compound 12: bitter leaves saponin(e LZ
8(kudinoside LZ
8)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：233nm。HR-TOF-MS:m/z 977.5078 ([M+Na]
+, C
49H
78NaO
18, calculated value 977.5086, error is-0.8ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：236nm(-98.0)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.42 (1H, br.s, H-12), 3.29 (1H, dd, J=12.0,4.5Hz, H-3), 1.63 (3H, s, H-29), 1.49 (3H, s, H-30), 1.48 (3H, s, H-27), 1.22 (3H, s, H-23), 1.11 (3H, s, H-24), (1.06 3H, t, J=6.0Hz, H-12 β), 0.89 (3H, s, H-26), 0.85 (3H, s, H-25).Pectinose: δ 4.86 (1H, d, J=5.7Hz, H-1 '), glucose: δ 5.11 (1H, d, J=7.8Hz, H-1 "), rhamnosyl: δ 6.16 (1H, s, H-1 " '), 1.65 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-2.
Compound 13: bitter leaves saponin(e LZ
9(kudinoside LZ
9)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：230nm。HR-TOF-MS:m/z 1111.5297 ([M+Na]
+, C
53H
82NaO
22, calculated value 1111.5301, error is-0.4ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：232nm(-35.0)。
1H-NMR (300MHz, Pyr-d
5) aglycon: δ 5.92 (1H, br.s, H-12), 3.27 (1H, dd, J=11.2,3.6Hz, H-3), 1.64 (3H, s, H-29), 1.61 (3H, s, H-27), 1.49 (3H, s, H-30), 1.23 (3H, s, H-23), 1.16 (3H, s, H-24), 0.88 (3H, s, H-26), 0.85 (3H, s, H-25).Pectinose: δ 4.73 (1H, d, J=6.9Hz, H-1 ').Glucose (inboard): δ 5.15 (1H, d, J=8.4Hz, H-1 "), glucose (outside): δ 5.26 (1H, d, J=7.8Hz, H-1 ").Rhamnosyl: δ 6.39 (1H, br.s, H-1 " '), 1.70 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-2.
Compound 14: bitter leaves saponin(e LZ
10(kudinoside LZ
10)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：227nm。HR-TOF-MS:m/z 1125.5472 ([M+Na]
+, C
54H
86NaO
23, calculated value 1125.5458, error is 1.4ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：246nm(-6.4)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.31 (1H, br.s, H-12), 3.35 (3H, s, 12 α-OCH
3), 3.27 (1H, dd, J=12.0,4.8Hz, H-3), 1.58 (3H, s, H-29), 1.50 (3H, s, H-30), 1.43 (3H, s, H-27), 1.23 (3H, s, H-23), 1.15 (3H, s, H-24), 0.88 (3H, s, H-26), 0.84 (3H, s, H-25).Pectinose: δ 4.78 (1H, d, J=6.6Hz, H-1 ').Glucose (inboard): δ 5.16 (1H, d, J=7.8Hz, H-1 "), glucose (outside): δ 5.26 (1H, d, J=7.8Hz, H-1 ").Rhamnosyl: δ 6.39 (1H, br.s, H-1 " '), 1.70 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-2.
Compound 15: bitter leaves ketone C (kudinone C)
White crystals, Liebermann-Burchard reacting positive, Molish reaction negative.HR-TOF-MS:m/z 959.6380 ([2M+Na]
+, C
60H
88NaO
8, calculated value 959.6377, error 0.3ppm).UV(MeOH)：260nm。CD?spectrum(c＝1.08*10
-4，MeOH)：248nm(-52.5)。
1H-NMR(300MHz，Pyr-d
5)：δ7.52(1H，d，J＝10.2Hz，H-12)，5.86(1H，d，J＝10.5Hz，H-11)，3.47(1H，dd，J＝9.9，5.4Hz，H-3)，1.69(3H，s，H-29)，1.52(3H，s，H-30)，1.22(3H，s，H-23)，1.03(3H，s，H-27)，1.01(3H，s，H-24)，0.94(3H，s，H-25)，0.87(3H，s，H-26)。
13The C-NMR data see Table 2-3.
Compound 16: bitter leaves saponin(e LZ
10(kudinoside LZ
10)
White crystals, the Liebermann-Burchard reacting positive, the Molish reacting positive, acid hydrolysis detects L-arabinose and L-rhamnosyl.IR(KBr)：3429，2938，1730，1634，1455，1384，1236，1130，1073，984，947，783，627。UV(MeOH)：262nm。HR-TOF-MS:m/z 769.4130 ([M+Na]
+, C
41H
62NaO
12, calculated value 769.4139, error-0.9ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：260nm(-22.5)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 7.52 (1H, dd, J=10.5,3.0Hz, H-12), 5.79 (1H, d, J=10.5Hz, H-11), 3.23 (1H, dd, J=9.9,5.4Hz, H-3), 1.69 (3H, s, H-29), 1.52 (3H, s, H-30), 1.16 (3H, s, H-23), 1.04 (3H, s, H-27), 1.04 (3H, s, H-24), 0.88 (3H, s, H-25), 0.82 (3H, s, H-26).Pectinose: δ 4.88 (1H, d, J=6.0Hz, H-1 '), 4.57 (m, H-2 '), 4.33 (m, H-3 '), 4.29 (m, H-4 '), 4.33 (m, H-5 '), 3.84 (1H, d, J=10.5Hz, H-5 ').Rhamnosyl: δ 6.16 (1H, br.s, H-1 "), 4.60 (m, H-2 "), 4.71 (1H, br.s, H-3 "), 4.29 (m, H-4 "), 4.57 (m, H-5 "), 1.64 (3H, d, J=6.3Hz, H-6 ").
13The C-NMR data see Table 2-3.
Compound 17: bitter leaves saponin(e LZ
12(kudinoside LZ
12)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：226，280nm。HR-TOF-MS:m/z 755.3972 ([M+Na]
+, C
40H
60NaO
12, calculated value 755.3982, error is-1.0ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：254nm(-1.12)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 7.51 (1H, dd, J=10.8,3.0Hz, 12-H), 5.76 (1H, d, J=10.8Hz, 11-H), 3.27 (1H, dd, J=11.4,4.2Hz, H-3), 1.67 (3H, s, H-29), 1.51 (3H, s, H-30), 1.25 (3H, s, H-23), 1.03 (3H, s, H-27), 0.95 (3H, s, H-24), 0.84 (3H, s, H-25), 0.81 (3H, s, H-25).Pectinose (pyranose form): δ 4.90 (1H, d, J=6.0, H-1 '), 4.28 (1H, m, H-5 '), (3.77 1H, dd, J=12.6,3.0Hz, H-5 '), pectinose (furan type): δ 6.18 (1H, br.s, H-1 "), 4.28 (1H, m, H-5 "), 4.22 (1H, m, H-5 ").
13The C-NMR data see Table 2-3.
Compound 18: bitter leaves saponin(e LZ
13(kudinoside LZ
13)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：255nm。HR-TOF-MS:m/z 799.4253 ([M+Na]
+, C
42H
64NaO
13, calculated value 799.4245, error 0.8ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：262nm(-24.2)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 7.50 (1H, d, J=10.8Hz, H-12), 5.72 (1H, d, J=10.8Hz, H-11), 3.34 (1H, dd, J=11.8,4.2Hz), 1.67 (3H, s, H-29), 1.51 (3H, s, H-30), 1.23 (3H, s, H-23), 1.15 (3H, s, H-24), 1.02 (3H, s, H-27), 0.84 (3H, s, H-25), 0.80 (3H, s, H-26).Glucose: δ 4.96 (H-1 '), rhamnosyl: δ 6.60 (1H, br.s, H-1 "), 1.71 (3H, d, J=6.0Hz, H-6 ').
13The C-NMR data see Table 2-3.
Compound 19: bitter leaves saponin(e LZ
14(kudinoside LZ
14)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：246nm。HR-TOF-MS:m/z 785.4098 ([M+Na]
+, C
41H
62NaO
13, calculated value 785.4088, error is 1.0ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：265nm(-13.8)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 7.49 (1H, d, J=10.8Hz, H-12), 5.76 (1H, d, J=10.8Hz, H-11), 3.22 (1H, dd, J=11.6,4.2Hz), 1.68 (3H, s, H-29), 1.51 (3H, s, H-30), 1.22 (3H, s, H-23), 1.02 (3H, s, H-27), 1.01 (3H, s, H-24), 0.86 (3H, s, H-25), 0.82 (3H, s, H-26).Pectinose: δ 4.94 (H-1 '), glucose: δ 5.18 (1H, d, J=7.8Hz, H-1 ").
Compound 20: bitter leaves saponin(e LZ
15(kudinoside LZ
15)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：261nm。HR-TOF-MS:m/z 785.4097 ([M+Na]
+, C
41H
62NaO
13, calculated value 785.4088, error is 0.9ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：256nm(-60.5)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 7.51 (1H, dd, J=10.2,3.0Hz, H-12), 5.77 (1H, d, J=10.8Hz, H-11), 3.34 (1H, dd, J=12.0,4.2Hz), 1.67 (3H, s, H-29), 1.51 (3H, s, H-30), 1.19 (3H, s, H-23), 1.08 (3H, s, H-27), 1.04 (3H, s, H-24), 0.87 (3H, s, H-25), 0.81 (3H, s, H-26).Pectinose: δ 4.74 (1H, d, J=7.2Hz, H-1 '), glucose: δ 5.41 (1H, d, J=7.8Hz, H-1 ").
13The C-NMR data see Table 2-3.
Compound 21: bitter leaves saponin(e LZ
16(kudinoside LZ
16)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：261nm。HR-TOF-MS:m/z 931.4666 ([M+Na]
+, C
47H
72NaO
17, calculated value 931.4667, error-0.1ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：254nm(-72.0)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 7.49 (1H, d, J=9.0Hz, H-12), 5.75 (1H, d, J=10.2Hz, H-11), 3.28 (1H, dd, J=10.8,3.6Hz), 1.67 (3H, s, H-29), 1.51 (3H, s, H-30), 1.19 (3H, s, H-23), 1.08 (3H, s, H-27), 1.04 (3H, s, H-24), 0.86 (3H, s, H-25), 0.81 (3H, s, H-26).Pectinose: δ 4.85 (1H, d, J=5.4Hz, H-1 '), glucose: δ 5.10 (1H, d, J=7.8Hz, H-1 "), rhamnosyl: δ 6.16 (1H, br.s, H-1 " '), 1.62 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-3.
Compound 22: bitter leaves saponin(e LZ
17(kudinoside LZ
17)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：262nm。HR-TOF-MS:m/z 1093.5211 ([M+Na]
+, C
53H
82NaO
22, calculated value 1093.5195, error is 1.6ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：258nm(-25.0)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 7.48 (1H, dd, J=10.5,2.6Hz, H-12), 5.74 (1H, d, J=10.5Hz, H-11), 3.25 (1H, dd, J=11.5,3.9Hz), 1.68 (3H, s, H-29), 1.51 (3H, s, H-30), 1.12 (3H, s) 1.12 (3H, s), 1.02 (3H, s), 0.85 (3H, s, H-24), 0.82 (3H, s).Pectinose: δ 4.73 (1H, d, J=6.6Hz, H-1 '), glucose (inboard): δ 5.16 (1H, d, J=7.5Hz, H-1 "), glucose (outside): δ 5.26 (1H, d; J=7.6Hz, H-1 " '), rhamnosyl: δ 6.16 (1H, br.s, H-1 " "), 1.71 (3H, d, J=6.3Hz, H-6 " ").
13The C-NMR data see Table 2-3.
Compound 23: bitter leaves saponin(e LZ
2(kudinoside LZ
2)
White crystals, Liebermann-Burchard reacting positive, Mo1ish reacting positive.UV(MeOH)：268nm。HR-TOF-MS:m/z 947.4616 ([M+Na]
+, C
47H
72NaO
18, calculated value 947.4616, error 0ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：268nm(-19.0)。
1H-NMR (300MHz, Pyr-d
5) aglycon: δ 3.25 (dd, J=11.9,4.5Hz, H-3), 2.59 (1H, dd, J=15.0,2.4Hz, H-11), 2.51 (1H, m, H-11), 2.51 (1H, m, H-21), 2.37 (1H, td, J=7.8,3.0Hz, H-16), 2.03 (1H, s, H-2), 1.90 (1H, s, H-15), 1.80 (1H, m, H-22), 1.79 (1H, s, H-2), 1.72 (1H, dd, J=6.0,2.4Hz, H-9), 1.50 (1H, s, H-6), 1.38 (3H, s, H-30), 1.37 (1H, s, H-1), 1.31 (1H, s, H-6), 1.30 (1H, s, H-7), 1.28 (3H, s, H-29), 1.24 (1H, s, H-15), 1.19 (3H, s, H-23), 1.09 (3H, s, H-24), 1.00 (3H, s, H-27), 0.82 (3H, s, H-26), 0.79 (3H, s, H-25), 0.77 (1H, s, H-1), 0.71 (1H, d, J=4.5Hz, H-5).Pectinose: δ 4.84 (1H, d, J=6.0Hz, H-1 '), 4.67 (1H, t, J=6.6Hz, H-2 '), 4.32 (1H, m, H-3 '), 4.54 (1H, br.s, H-4 '), 4.19 (m, H-5 '), 3.74 (1H, dd, J=10.2,2.0Hz, H-5 ').Glucose: δ 5.13 (1H, d, J=7.8Hz, H-1 "), 3.94 (1H, m, H-2 "), 4.19 (1H, m, H-3 "), 4.19 (1H, m, H-4 "), 3.94 (1H, m, H-5 "), 4.51 (1H, dd, J=10.8,1.8Hz, H-6 "), 4.32 (1H, m, H-6 ").Rhamnosyl: δ 6.20 (1H, br.s, H-1 " '), 4.75 (1H, br.s, H-2 " '), 4.60 (1H, m, H-3 " '), 4.32 (1H, m, H-4 " '), 4.60 (1H, m, H-5 " '), 1.63 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-3.
Compound 24: bitter leaves saponin(e LZ
18(kudinoside LZ
18)
White solid, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：238nm。HR-TOF-MS:m/z 905.4869 ([M+Na]
+, C
46H
74NaO
16, calculated value 905.4875, error is-0.6ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：250nm(+3.3)，267nm(-5.0)，386nm(1.5)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.89 (1H, s, H-18), 5.35 (1H, t, J=3.0Hz, H-12), 3.32 (1H, dd, J=12.0,4.2Hz, H-3), 2.40 (3H, s, H-29), 1.51 (3H, s, H-28), 1.22 (3h, s.H-23), 1.14 (3H, s, H-24), 1.06 (3H, s, H-27), 0.90 (3H, s, H-25), 0.86 (3H, s, H-26).Pectinose: δ 4.88 (1H, d, J=6.0Hz, H-1 '), glucose: δ 5.11 (1H, d, J=7.8Hz, H-1 "), rhamnosyl: δ 6.16 (1H, br.s, H-1 " '), 1.63 (3H, d, J=6.6Hz, H-6 " ').
13The C-NMR data see Table 2-3.
Compound 25: bitter leaves saponin(e LZ
19(kudinoside LZ
19)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：278nm。HR-TOF-MS:m/z 859.4455 ([M+Na]
+, C
44H
68NaO
15, calculated value 859.4456, error is-0.1ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：234nm(-7.8)，280nm(-37.5)，317nm(+24.3)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.64 (1H, t-like, 12-H), 2.55 (1H, dd, J=12.0,3.6Hz, H-21), 2.05 (1H, H-11), 2.00 (1H, dd, H-21), 1.97 (1H, H-11), 1.80 (3H, d, J=3.0Hz, H-27), 1.25 (3H, s, H-22), 1.16 (3H, s, H-22), 0.94 (3H, s, H-26), 0.93 (3H, s, H-25), 0.92 (3H, s, H-24).Pectinose: δ 4.88 (1H, d, J=5.4Hz, H-1 '), glucose: δ 5.11 (1H, d, J=7.8Hz, H-1 "), rhamnosyl: δ 6.17 (1H, br.s, H-1 " '), 1.64 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-3.
Compound 26: bitter leaves saponin(e LZ
20(kudinoside LZ
20)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：253nm。HR-TOF-MS:m/z 933.4821 ([M+Na]
+, C
47H
74NaO
17, calculated value 933.4824, error is-0.3ppm).CD?spectrum：(c＝1.05*10
-4，MeOH)：252nm(-57.0)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 3.29 (1H, dd, J=12.0,4.2Hz, H-3), 2.42 (3H, s, H-30), 1.21 (3H, s, H-23), 1.20 (3H, d, J=3.0Hz, H-28), 1.15 (3H, s, H-27), 1.12 (3H, s, H-26), 1.10 (3H, s, H-24), 0.78 (3H, s, H-25).Pectinose: δ 4.87 (1H, d, J=6.0Hz, H-1 '), glucose: δ 5.12 (1H, d, J=7.8Hz, H-1 "), rhamnosyl: δ 6.19 (1H, br.s, H-1 " '), 1.63 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-3.
Compound 27: bitter leaves saponin(e LQ
1(kudinoside LQ
1)
White crystals, Liebermann-Burchard reacting positive, Molish reacting positive.UV(MeOH)：246nm。HR-TOF-MS:m/z 919.5041 ([M+Na]
+, C
47H
76NaO
16, calculated value 919.5031, error is 1.0ppm).CDspectrum：(c＝1.05*10
-4，MeOH)：232nm(-18.0)，256nm(+6.0)，267nm(-2.4)。
1H-NMR (600MHz, Pyr-d
5) aglycon: δ 5.41 (1H, t, J=3.0Hz, H-12), 3.22 (1H, dd, J=12.0,4.2Hz, H-3), 1.25 (3H, s), 1.15 (3H, s), 1.08 (3H, s), 1.05 (3H, s), 0.90 (3H, s), 0.87 (3H, s), 0.85 (3H, s), 0.76 (1H, d, J=12.0Hz, H-5).Pectinose: δ 4.88 (1H, d, J=5.4Hz, H-1 '), glucose: δ 6.13 (1H, d, J=8.4Hz, H-1 "), rhamnosyl: δ 6.10 (1H, br.s, H-1 " '), 1.61 (3H, d, J=6.0Hz, H-6 " ').
13The C-NMR data see Table 2-3.
Claims (13)
1. one group of Leaf of Chinese Holly saponins compound comprises bitter leaves ketone A, B, D, E, F, G, bitter leaves saponin(e LZ
11, bitter leaves saponin(e LZ
12And derivative, wherein bitter leaves ketone A and derivative thereof have structure shown in the formula 1, wherein, and R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone A sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
2. the compound of claim 1, wherein bitter leaves ketone B and derivative thereof have structure shown in the formula 2, wherein, R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone C sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
3. the compound of claim 1, wherein bitter leaves ketone D and derivative thereof have structure shown in the formula 3, wherein, R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone C sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
4. the compound of claim 1, wherein bitter leaves ketone E and derivative thereof have structure shown in the formula 4, wherein, R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone C sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
5. the compound of claim 1, wherein bitter leaves ketone F and derivative thereof have structure shown in the formula 5, wherein, R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone C sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
6. the compound of claim 1, wherein bitter leaves ketone G and derivative thereof have structure shown in the formula 6, wherein, R and R
1Independently be selected from hydrogen, glycosyl separately, acyl group, alkyl or aromatic substituent, described glycosyl is selected from: β-D-glucosyl group, α-L-rhamanopyranosyl, β-D-xylosyl, β-D-xylosyl, the Arabic glycosyl of β-D-, α-L-arabinose base, alpha-beta-D-glucopyranosyl-(1 → 3)-[α-L-rhamanopyranosyl-(1 → 2)]-α-L-arabinose base; Acyl group is selected from: ethanoyl, benzoyl, phthalic acid monoacyl, Succinic Acid monoacyl, oxysuccinic acid monoacyl, Citric Acid monoacyl, and the various salts that become of polyacid acyl group, as: 3-diformate mono acyl group bitter leaves ketone C sodium salt, alkyl is selected from straight chain and side chain class alkyl; Aromatic substituent is selected from various aromatics substituting groups.
7. the compound of claim 1, wherein bitter leaves saponin(e LZ
11, have structure shown in the formula 7.
8. the compound of claim 1, wherein bitter leaves saponin(e LZ
12, have structure shown in the formula 8.
9. the preparation method of any one compound among the claim 1-8, it is characterized in that, step is as follows: Leaf of Chinese Holly water or water-containing organic solvent extract, extract is used macroporous resin, ion exchange resin, polymeric amide, dextrane gel, reverse phase silica gel isochromatic spectrum means are refining separates, elder generation's water or lower concentration organic solvent wash-out are removed impurity, obtain product with the high levels of organic solvents wash-out again, adopt above repeatedly chromatographic separation to make with extra care purifying, or recrystallization purifying, wherein, extraction is selected from ethanol with organic solvent, methyl alcohol, acetone, macroporous resin, ion exchange resin comprises styrene type, the acrylic type macromolecule resin.
10. the preparation method of any one compound among the claim 1-8 is characterized in that step is as follows: utilize the Leaf of Chinese Holly crude extract acid hydrolysis preparation that contains claim 1-8 compound, described acid is selected from sulfuric acid, hydrochloric acid, formic acid, acetate, trifluoroacetic acid.
11. any one compound of claim 1-8 in clearing heat and detoxicating, the inhibiting bacteria and diminishing inflammation of preparation, relieving spasm to stop pain, step-down fat-reducing, press down the cancer benefit that give protection against cancer, produces refreshing effect to the mind, makes eye bright and think, slake thirst and help produce saliva, go to be bored with and sober up, be good for the stomach and keep fit, improve looks and the arteries and veins of invigorating blood circulation, anti-ageing, reducing blood-fat, decreasing cholesterol, triglyceride reducing, improve medicine and Application in Food such as blood viscosity and microcirculation, vasodilation, removing free radical, enhancing body immunity.
12. any one compound of claim 1-8 is characterized in that, is following particular compound:
13. contain the pharmaceutical composition of any one compound of claim 1-8.
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