CN104945360A - Preparation method and application of phenylpropanoid compound in tobacco - Google Patents
Preparation method and application of phenylpropanoid compound in tobacco Download PDFInfo
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- CN104945360A CN104945360A CN201510357418.2A CN201510357418A CN104945360A CN 104945360 A CN104945360 A CN 104945360A CN 201510357418 A CN201510357418 A CN 201510357418A CN 104945360 A CN104945360 A CN 104945360A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/87—Benzo [c] furans; Hydrogenated benzo [c] furans
- C07D307/88—Benzo [c] furans; Hydrogenated benzo [c] furans with one oxygen atom directly attached in position 1 or 3
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Abstract
The invention discloses a of a phenylpropanoid compound showing as the first formula (Please see the formula in the instruction) and a preparation method and application thereof and belongs to the technical field of tobacco chemistry. Tobacco serves as a raw material, extraction is conducted with a high-concentration methanol aqueous solution or a high-concentration ethanol aqueous solution or a high-concentration acetone aqueous solution as an extraction solvent, and the extraction solutions are merged, filtered, and decompressed condensed to form extract; the extract is packed by the adoption of a silica gel to be conducted silica gel column chromatography; a mixing solvent of chloroform-acetone serves as an eluent to conduct gradient elution, and TLC monitors and merges same parts and condenses; separation and purification by use of high-pressure liquid chromatography are further conducted on the part obtained by eluting, collecting and condensing by the mixing solvent of the chloroform-acetone with a volume matching of 6:4, and the phenylpropanoid compound is obtained. The activity test indicates that the compound has a good inhibiting effect on rotavirus. The compound is simple in structure, good in activity and capable of being used as a guiding compound for resisting the rotavirus.
Description
Technical field
The invention belongs to technical field of tobacco chemistry, be specifically related to a kind ofly from tobacco, extract the phenylpropanoids obtained first.Meanwhile, the invention still further relates to the preparation method and application of this compound.
Background technology
Tobacco is the plant that chemical composition is the most complicated in the world, and secondary metabolite is very abundant, and through the research of decades, the monomer chemistries material that people identify out at present from tobacco just more than kind more than 3000, and also has many compositions not yet to identify out.Tobacco, except being mainly used in cigarette smoking purposes, also therefrom can extract the multiple chemical composition having utility value, therefrom finds that there is the guiding compound of value of exploiting and utilizing.
Phenylpropanoid Glycosides is the biologically active substance that a class occurring in nature extensively exists.Because plant Phenylpropanoid Glycosides constituent structure type is many, stereochemistry is complicated, has multiple biological activity, very active to the research in this field both at home and abroad; No matter be naturally occurring, or the phenylpropanoids that synthetic obtains, all cause the extensive concern of chemist.Have research to confirm, pharmacological action and the chemical structure of phenylpropanoids are closely related, can research and develop more phenylpropanoids further, therefrom find effective lead compound and active group.The present invention is separated and obtains a kind of phenylpropanoids with anti-rotavirus activity from tobacco, and this compound it is not yet seen relevant report.
Summary of the invention
The object of the present invention is to provide a kind of new phenylpropanoids.
Another object of the present invention is to provide a kind of method preparing described phenylpropanoids.
The present invention also aims to provide the application of described Phenylpropanoid Glycosides compound in the medicine preparing anti-rotavirus.
Except as otherwise noted, the percentage ratio adopted in the present invention is mass percent.
The present invention isolates a kind of new phenylpropanoids from tobacco, and its molecular formula is C
11h
10o
5, there is the structural formula as shown in formula I:
, formula I;
This Compound nomenclature is 5-hydroxyl-6-(3-hydroxypropanoyl)-isobenzofuran-1 (3
h)-one, English name is: 5-hydroxy-6-(3-hydroxypropanoyl)-isobenzofuran-1 (3
h)-one, be light yellow gum thing.
Prepare the method for above-mentioned phenylpropanoids, the method comprises the following steps:
Step (1), medicinal extract extracts: (the present invention is to the granularity not requirement after tobacco leaf pulverizing to pulverize tobacco sample or be cut into segment, the concrete length of the segment be cut into is not limited), with high concentration methanol (w%:80% ~ 100%) or high concentration ethanol (w%:80% ~ 100%) or high density acetone (w%:60% ~ 90%) for Extraction solvent carries out soak extraction, Extraction solvent: tobacco (weight ratio)=2 ~ 4:1, soak 24 h ~ 72 h, extract 3 ~ 5 times, united extraction liquid, filtering and concentrating becomes medicinal extract;
Step (2), silica gel column chromatography: the medicinal extract weight that step (1) obtains is after the pure methyl alcohol of its 1.5 ~ 3 times amount, straight alcohol or pure acetone dissolve, with 80 ~ 100 order silica gel mixed samples that weight is medicinal extract 0.8 ~ 1.2 times, simultaneously, with 160 ~ 300 order silica gel dry column-packings that weight is medicinal extract 2 ~ 4 times amount, then dry method loading, carries out silica gel column chromatography; The mixed solvent being followed successively by the chloroform-acetone of (1:0,20:1,9:1,8:2,7:3,6:4,1:1 and 1:2) using volume proportion carries out gradient elution as eluent, and thin layer chromatography is followed the tracks of, and merges identical part, collects each several part elutriant and concentrates; Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio;
Step (3), high pressure liquid chromatography separation and purification: the mixed solvent wash-out of the chloroform-acetone with volume proportion being 6:4 is collected the concentrated part obtained and namely obtains described phenylpropanoids with high pressure liquid chromatography separation and purification further.
High pressure liquid chromatography separation and purification adopts column length × internal diameter to be 21.2 mm × 250 mm, and inner film thickness is 5
μthe C of m
18chromatographic column, flow velocity is 20 mL/min, moving phase to be mass concentration be 28% methanol aqueous solution, UV-detector determined wavelength is 318 nm, each sample introduction 200
μl, collects the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness.
After described high performance liquid chromatography separation and purification, a preferred subsequent process scheme is that gained compound uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated with gel filtration chromatography, with further separation and purification.
The structure of the phenylpropanoids prepared with aforesaid method measures by the following method.The compounds of this invention is light yellow gum thing; UV spectrum (solvent is methyl alcohol),
l max(log
e) 318 (3.28), 286 (3.59), 215 (4.02) nm; Infrared spectra (pressing potassium bromide troche)
n max3460,2927,1730,1652,1608,1536,1487,1285,1121,1072,927,805 cm
-1; High resolution mass spectrum (HRESIMS) provides quasi-molecular ion peak
m/z[221.0457 M-H]
-(calculated value 221.0450).Shown in composition graphs 1 and Fig. 2
1h and
13c NMR spectrum provides a molecular formula C
11h
10o
5, degree of unsaturation is 7.From
1h and
13cNMR composes (attribution data is in table 1) signal can find out in compound have 1,2,4, a 5-quaternary phenyl ring, a 3-hydroxypropanoyl, an oxidation methylene radical, ester carbonyl group, a phenolic hydroxyl group.Lactone is constituted by Sauerstoffatom according to the HMBC of H-2' with C-3' relevant (Fig. 3) susceptible of proof C-2' and C-3'.Be connected to-1 of phenyl ring according to the HMBC of H-8 and C-1, H-6 and C-7 relevant susceptible of proof 3-hydroxypropanoyl, according to phenolic hydroxyl group hydrogen (
d h10.86 s) susceptible of proof phenolic hydroxyl group relevant with the HMBC of C-3 with C-1, C-2 be substituted in C-2 position.So far the structure of this compound is determined.
table 1. compound
1
h NMR and
13
c NMR data (C
5
d
5
n)
| No. | d C | d H (m, J, Hz) | No. | d C | d H (m, J, Hz) |
| 1 | 120.9 s | 7 | 198.4 s | ||
| 2 | 166.1 s | 8 | 42.1 t | 3.45 (t) 6.8 | |
| 3 | 112.0 d | 6.94 s | 9 | 58.7 t | 4.14 (t) 6.8 |
| 4 | 152.0 s | 1′ | 69.0 t | 5.40 s | |
| 5 | 116.3 s | 2′ | 169.1 s | ||
| 6 | 130.4 d | 8.32 s | Ar-OH | 10.86 s |
Described phenylpropanoids is applied to the medicine preparing anti-rotavirus.
compared with prior art, the present invention has following beneficial effect:the compounds of this invention is separated first, is determined as phenylpropanoids by above-mentioned nucleus magnetic resonance and measuring method of mass spectrum, and characterizes its concrete structure.Through the experiment to anti-rotavirus, its TC
50value is 206.7
μg/mL, IC
50value is 8.22
μg/mL, therapeutic index TI are 25.15; Its therapeutic index exceedes the therapeutic index 18.90 of contrast virazole; It is active that compound has good anti-rotavirus.Above result discloses compound of the present invention preparing in anti-rotavirus medicaments good application prospect.The compounds of this invention structure is simple better active, and the guiding compound that can be used as anti-rotavirus medicaments research and development is researched and developed for anti-rotavirus medicaments preparation.
Accompanying drawing explanation
Fig. 1 is the carbon-13 nmr spectra of phenylpropanoids of the present invention;
Fig. 2 is the proton nmr spectra of phenylpropanoids of the present invention;
Fig. 3 is that the main HMBC of phenylpropanoids of the present invention is correlated with.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or improvement, all fall into protection scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by buying the conventional products obtained.
If the solution in the present invention only gives solute, do not disclose solvent, then those skilled in the art should know solvent is water.In the present invention, pure methyl alcohol refers to 100% methyl alcohol, and straight alcohol refers to 100% ethanol, and pure acetone refers to 100% acetone.
embodiment 1
Prepare phenylpropanoids C
11h
10o
5, comprise medicinal extract extraction, silica gel column chromatography, high pressure liquid chromatography separation, specifically adopt following steps:
Step (1), medicinal extract extracts: get tobacco leaf and pulverize, be Extraction solvent with high concentration methanol (w%:95 %) or high concentration ethanol (w%:95%) or high density acetone (w%:70%), Extraction solvent: tobacco (weight ratio)=2:1, soak 54 h, extract 4 times, united extraction liquid, filtering and concentrating becomes medicinal extract.
Step (2), silica gel column chromatography: the medicinal extract weight that step (1) obtains is after the pure methyl alcohol of its 2.5 times amount, straight alcohol or pure acetone dissolve, with 80 ~ 100 order silica gel mixed samples that weight is medicinal extract 1.2 times, simultaneously, with the 250 order silica gel dry column-packings that weight is medicinal extract 3 times amount, then dry method loading, carries out silica gel column chromatography; The mixed solvent being followed successively by the chloroform-acetone of (1:0,20:1,9:1,8:2,7:3,6:4,1:1 and 1:2) using volume proportion carries out gradient elution as eluent, and TLC monitoring merges identical part, collects each several part elutriant and concentrates; Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio;
Step (3), high pressure liquid chromatography is separated: the mixed solvent wash-out of the chloroform-acetone with volume proportion being 6:4 is collected the concentrated part obtained and namely obtains described phenylpropanoids with high pressure liquid chromatography separation and purification further, high pressure liquid chromatography separation and purification is employing 21.2 mm × 250 mm, 5
μthe C of m
18chromatographic column, flow velocity is 20 mL/min, and moving phase is the methyl alcohol of 28%, and UV-detector determined wavelength is 318 nm, each sample introduction 200
μl, collects the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness.
Material after high pressure lipuid chromatography (HPLC) separation and purification, a preferred aftertreatment scheme is that gained compound uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated with gel filtration chromatography, with further separation and purification.
The present invention's raw tobacco material used does not limit by area and kind, all can realize the present invention, and below to derive from the raw tobacco material of cigarette industry limited liability company Different sources in Yunnan, the present invention will be further described:
embodiment 2
Tobacco sample derives from Yunnan Yuxi, and kind is Yuxi K326.Tobacco is sampled 2.0 kg and pulverize methyl alcohol soak extraction 5 times with 95%, extract 24 h, Extraction solvent: the weight ratio=2:1 of tobacco leaf at every turn, extracting solution merges, and filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 105 g.Medicinal extract weight is after the pure dissolve with methanol of its 2.0 times amount, with the thick silica gel mixed sample of 100 order of 120 g, the 160 order silica gel dress posts of 0.6 kg, carry out silica gel column chromatography, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5
mm) preparative column is stationary phase, flow velocity is 20 ml/min, and UV-detector determined wavelength is 318 nm, and each sample introduction 200 mL, collects the chromatographic peak of 23.8 min, evaporate to dryness after repeatedly cumulative, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 3
Tobacco sample derives from Dali, and kind is cloud and mist 200, and tobacco is sampled 3.5 kg and be cut into segment, the alcohol immersion with 95% extracts 4 times, each extraction 48 h, Extraction solvent: the weight ratio=4:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 250 g.Medicinal extract weight is after the pure dissolve with methanol of its 2.0 times amount, with the thick silica gel mixed sample of 80 order of 250 g, the 200 order silica gel dress posts of 0.8 kg carry out silica gel column chromatography, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, obtain this new compound.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 4
Tobacco sample derives from Kunming, Yunnan, and kind is the large gold dollar of safflower, tobacco is sampled 5 kg and pulverizes, the soak extraction 3 times of the acetone with 75%, each extraction 72h, Extraction solvent: the weight ratio=3:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 380 g.Medicinal extract weight is after the pure dissolve with methanol of its 1.6 times amount, with the thick silica gel mixed sample of 90 order of 400 g, the 180 order silica gel dress posts of 1.4 kg carry out silica gel column chromatography, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 5
Tobacco sample derives from Dali, and kind is cloud and mist 200, and tobacco is sampled 3.5 kg and be cut into segment, the alcohol immersion with 80% extracts 3 times, each extraction 72 h, Extraction solvent: the weight ratio=2:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 250 g.Medicinal extract weight is after the pure dissolve with methanol of its 1.5 times amount, with weight be medicinal extract 1.2 times the thick silica gel mixed sample of 100 order, silica gel column chromatography is carried out with the 300 order silica gel dress posts that weight is medicinal extract 2 times amount, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, obtain this new compound.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 6
Tobacco sample derives from Dali, and kind is cloud and mist 200, and tobacco is sampled 3.5 kg and pulverize, the alcohol immersion with 100% extracts 5 times, each extraction 24 h, Extraction solvent: the weight ratio=3:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 251 g.Medicinal extract weight is after the pure dissolve with methanol of its 3.0 times amount, with weight be medicinal extract 0.8 times the thick silica gel mixed sample of 90 order, silica gel column chromatography is carried out with the 160 order silica gel dress posts that weight is medicinal extract 4 times amount, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 7
Tobacco sample derives from Dali, and kind is cloud and mist 200, tobacco is sampled 3.5 kg and is cut into segment, the methyl alcohol soak extraction with 80% 5 times, each extraction 24h, Extraction solvent: the weight ratio=3.5:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 248 g.Medicinal extract weight is after the straight alcohol of its 1.5 times amount dissolves, with weight be medicinal extract 0.85 times the thick silica gel mixed sample of 100 order, silica gel column chromatography is carried out with the 160 order silica gel dress posts that weight is medicinal extract 4 times amount, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 8
Tobacco sample derives from Dali, and kind is cloud and mist 200, tobacco is sampled 3.5 kg and pulverizes, the methyl alcohol soak extraction with 90% 3 times, each extraction 72h, Extraction solvent: the weight ratio=2.5:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 252 g.Medicinal extract weight is after the straight alcohol of its 3.0 times amount dissolves, with weight be medicinal extract 1.2 times the thick silica gel mixed sample of 80 order, silica gel column chromatography is carried out with the 300 order silica gel dress posts that weight is medicinal extract 2 times amount, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, obtain this new compound.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 9
Tobacco sample derives from Dali, and kind is cloud and mist 200, tobacco is sampled 3.5 kg and pulverizes, the methyl alcohol soak extraction with 100% 4 times, each extraction 50 h, Extraction solvent: the weight ratio=3:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 251 g.Medicinal extract weight is after the straight alcohol of its 2.5 times amount dissolves, with weight be medicinal extract 0.8 times the thick silica gel mixed sample of 90 order, silica gel column chromatography is carried out with the 250 order silica gel dress posts that weight is medicinal extract 2.6 times amount, join successively than being 1:0 with volume, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 10
Tobacco sample derives from Dali, and kind is cloud and mist 200, tobacco is sampled 3.5 kg and pulverizes, the acetone soak extraction with 60% 5 times, each extraction 72 h, Extraction solvent: the weight ratio=2.4:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 250 g.Medicinal extract weight is after the pure acetone of its 2.8 times amount dissolves, with weight be medicinal extract 1.1 times the thick silica gel mixed sample of 80 order, silica gel column chromatography is carried out with the 160 order silica gel dress posts that weight is medicinal extract 2 times amount, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 11
Tobacco sample derives from Dali, and kind is cloud and mist 200, tobacco is sampled 3.5 kg and pulverizes, the acetone soak extraction with 90% 3 times, each extraction 24 h, Extraction solvent: the weight ratio=2:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 245 g.Medicinal extract weight is after the pure acetone of its 1.5 times amount dissolves, with weight be medicinal extract 1.2 times the thick silica gel mixed sample of 100 order, silica gel column chromatography is carried out with the 300 order silica gel dress posts that weight is medicinal extract 4 times amount, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
embodiment 12
Tobacco sample derives from Dali, and kind is cloud and mist 200, tobacco is sampled 3.5 kg and pulverizes, the acetone soak extraction with 70% 4 times, each extraction 60h, Extraction solvent: the weight ratio=4:1 of tobacco leaf, extracting solution merges, filter, concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 255 g.Medicinal extract weight is after the pure acetone of its 3.0 times amount dissolves, with weight be medicinal extract 0.8 times the thick silica gel mixed sample of 90 order, 1 carries out silica gel column chromatography with the 250 order silica gel dress posts that weight is medicinal extract 3.5 times amount, 1:0 is followed successively by with volume proportion, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, chloroform-acetone the gradient elution of 1:2, TLC monitoring merges identical part and concentrates, obtain 8 parts, wherein chloroform-acetone the wash-out with volume proportion being 6:4 is concentrated prompt logical sequence 1,100 half preparative high-performance liquid chromatographic of part peace obtained to be separated, methyl alcohol with 28% is moving phase, Zorbax SB-C18 (21.2 ' 250 mm, 5 mm) preparative column is stationary phase, flow velocity is 20 ml/min, UV-detector determined wavelength is 318 nm, each sample introduction 200 mL, collect the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness, products therefrom uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, is separated, obtains this new compound with Sephadex LH-20 gel filtration chromatography.
Wherein, during gradient elution, during the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
qualification example 1
The qualification of------compound structure
Compound prepared by Example 1, compound is light yellow gum thing; UV spectrum (solvent is methyl alcohol),
l max(log
e) 318 (3.28), 286 (3.59), 215 (4.02) nm; Infrared spectra (pressing potassium bromide troche)
n max3460,2927,1730,1652,1608,1536,1487,1285,1121,1072,927,805 cm
-1; High resolution mass spectrum (HRESIMS) provides quasi-molecular ion peak
m/z[221.0457 M-H]
-(calculated value 221.0450).Shown in composition graphs 1 and Fig. 2
1h and
13c NMR spectrum provides a molecular formula C
11h
10o
5, degree of unsaturation is 7.From
1h and
13cNMR composes (attribution data is in table 1) signal can find out in compound have 1,2,4, a 5-quaternary phenyl ring, a 3-hydroxypropanoyl, an oxidation methylene radical, ester carbonyl group, a phenolic hydroxyl group.Lactone is constituted by Sauerstoffatom according to the HMBC of H-2' with C-3' relevant (Fig. 3) susceptible of proof C-2' and C-3'.Be connected to-1 of phenyl ring according to the HMBC of H-8 and C-1, H-6 and C-7 relevant susceptible of proof 3-hydroxypropanoyl, according to phenolic hydroxyl group hydrogen (
d h10.86 s) susceptible of proof phenolic hydroxyl group relevant with the HMBC of C-3 with C-1, C-2 be substituted in C-2 position.So far the structure of this compound is determined.
qualification example 2
Compound prepared by Example 2-12 is yellow jelly.Measuring method is identical with embodiment 13, confirms that compound prepared by embodiment 2-12 is described phenylpropanoids---5-hydroxyl-6-(3-hydroxypropanoyl)-isobenzofuran-1 (3
h)-one.
application examples 1
Arbitrary phenylpropanoids prepared by Example 1 ~ 12 carries out anti-rotavirus activity test, and test situation is as follows:
Anti-rotavirus adopts cell in vitro method of testing, after namely sample and virus act on MA104 cell simultaneously, detect samples for viral and infect the provide protection causing necrocytosis, thus working sample is to the active function of HRV by Alarmablue method.
a the cytotoxicity of () medicine detects
After MA104 cell is cultivated and is formed individual layer in 96 porocyte culture plates, add the sample liquid of different concns, continue cultivation after 3 days, change the nutrient solution containing Alamarblue, continue cultivation detects its 530/590nm place fluorescent value after 2 ~ 3 hours, thus detect sample to the toxicity of MA104 cell, and calculate half cytotoxic concentration (TC
50).
b the effect of () medicine anti-rotavirus detects
After MA104 cell is cultivated and is formed individual layer in 96 porocyte culture plates, the virus liquid of 100TCID50 and be no more than 20% Cytotoxic gradient concentration drug solution and be added on MA104 cell simultaneously, continue to cultivate after 4-6 days, the nutrient solution changed containing Alamarblue continues cultivation detects its 530/590nm place fluorescent value after 2 ~ 3 hours, and calculation of half inhibitory concentration (IC
50).
(c) foundation TC
50
/ IC
50
the therapeutic index of computerized compound.
Result shows, the TC of the compounds of this invention
50value is 206.7
μg/mL, IC
50value is 8.22
μg/mL, therapeutic index TI are 25.15; Its therapeutic index exceedes the therapeutic index 18.90 of contrast virazole; It is active that compound has good anti-rotavirus.Above result discloses compound of the present invention preparing in anti-rotavirus medicaments good application prospect.The compounds of this invention structure is simple better active, and the guiding compound that can be used as anti-rotavirus medicaments research and development is researched and developed for anti-rotavirus medicaments preparation.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (7)
1. a kind of phenylpropanoids as shown in formula I,
, formula I;
The called after of this compound: 5-hydroxyl-6-(3-hydroxypropanoyl)-isobenzofuran-1 (3
h)-one [5-hydroxy-6-(3-hydroxypropanoyl)-isobenzofuran-1 (3H)-one].
2. the preparation method of phenylpropanoids according to claim 1, is characterized in that, comprises the following steps:
Step (1), medicinal extract extracts: take tobacco leaf as raw material, tobacco leaf is pulverized or is cut into segment, be that Extraction solvent carries out soak extraction with the acetone of the methyl alcohol of concentration expressed in percentage by weight 80% ~ 100%, the ethanol of concentration expressed in percentage by weight 80% ~ 100% or concentration expressed in percentage by weight 60% ~ 90%, Extraction solvent: the weight ratio=2 ~ 4:1 of tobacco leaf, soaks 24 h ~ 72 h, extracts 3 ~ 5 times, united extraction liquid, filtering and concentrating becomes medicinal extract;
Step (2), silica gel column chromatography: the medicinal extract weight that step (1) obtains is that 160 ~ 300 order silica gel dry column-packings of its 2 ~ 4 times amount carry out silica gel column chromatography; With the mixed solvent of chloroform-acetone for eluent carries out gradient elution, thin layer chromatography is followed the tracks of, and merges identical part, collects each several part elutriant and concentrates; During gradient elution, the mixed solvent volume proportion of chloroform-acetone gradually changes to 1:2 from 1:0;
Step (3), high pressure liquid chromatography separation and purification: the mixed solvent wash-out of the chloroform-acetone with volume proportion being 6:4 is collected the concentrated part obtained and namely obtains required Phenylpropanoid Glycosides compound with high pressure liquid chromatography separation and purification further.
3. the preparation method of phenylpropanoids according to claim 2, it is characterized in that: in step (3), compound after high pressure liquid chromatography separation and purification uses pure dissolve with methanol again, then with pure methyl alcohol for moving phase, with the further separation and purification of gel filtration chromatography.
4. the preparation method of phenylpropanoids according to claim 2, is characterized in that: in step (3), and described high pressure liquid chromatography separation and purification is employing 21.2 mm × 250 mm, 5
μthe C of m
18chromatographic column, flow velocity is 20 mL/min, moving phase to be mass concentration be 28% methanol aqueous solution, UV-detector determined wavelength is 318 nm, each sample introduction 200
μl, collects the chromatographic peak of 23.8 min, repeatedly cumulative rear evaporate to dryness.
5. the preparation method of phenylpropanoids according to claim 2, it is characterized in that: in step (2), described medicinal extract is before silica gel column chromatography, after medicinal extract dissolves by the pure methyl alcohol of medicinal extract 1.5 ~ 3 times amount, straight alcohol or pure acetone by weight, 80 ~ 100 order silica gel mixed samples of medicinal extract 0.8 ~ 1.2 times again by weight, after mixing, loading.
6. the preparation method of phenylpropanoids according to claim 2, it is characterized in that: in step (2), during described gradient elution, the mixed solvent volume proportion of chloroform-acetone is followed successively by 1:0,20:1,9:1,8:2,7:3,6:4,1:1 and 1:2; During the mixed solvent wash-out of each ratio, be eluted to till not having composition to wash down, then change the mixed solvent of next ratio.
7. phenylpropanoids according to claim 1 is preparing the application in anti-rotavirus medicaments.
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