CN113087606A - New diaryl nonane IV and III free radical inhibitor in saxifraga tangutica and separation preparation process and application thereof - Google Patents
New diaryl nonane IV and III free radical inhibitor in saxifraga tangutica and separation preparation process and application thereof Download PDFInfo
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- 239000003112 inhibitor Substances 0.000 title claims abstract description 101
- 150000003254 radicals Chemical class 0.000 title claims abstract description 91
- 238000002360 preparation method Methods 0.000 title claims abstract description 62
- -1 diaryl nonane Chemical compound 0.000 title claims abstract description 41
- BKIMMITUMNQMOS-UHFFFAOYSA-N normal nonane Natural products CCCCCCCCC BKIMMITUMNQMOS-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 241000284928 Saxifraga tangutica Species 0.000 title claims abstract description 30
- 238000000926 separation method Methods 0.000 title claims abstract description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 87
- 239000011347 resin Substances 0.000 claims abstract description 26
- 229920005989 resin Polymers 0.000 claims abstract description 26
- 238000012216 screening Methods 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000004480 active ingredient Substances 0.000 claims abstract description 7
- 238000013375 chromatographic separation Methods 0.000 claims abstract description 4
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 239000000825 pharmaceutical preparation Substances 0.000 claims abstract 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 63
- 239000000523 sample Substances 0.000 claims description 61
- 238000001035 drying Methods 0.000 claims description 49
- 239000000243 solution Substances 0.000 claims description 49
- 239000007788 liquid Substances 0.000 claims description 44
- 239000000706 filtrate Substances 0.000 claims description 40
- 238000001514 detection method Methods 0.000 claims description 37
- 241000205578 Thalictrum Species 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 238000004587 chromatography analysis Methods 0.000 claims description 26
- 238000002156 mixing Methods 0.000 claims description 26
- 241000468460 Thalictrum aquilegiifolium Species 0.000 claims description 24
- 241001647091 Saxifraga granulata Species 0.000 claims description 23
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 claims description 22
- 239000007864 aqueous solution Substances 0.000 claims description 21
- 239000000284 extract Substances 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 16
- 230000005526 G1 to G0 transition Effects 0.000 claims description 15
- 239000000945 filler Substances 0.000 claims description 15
- 238000002347 injection Methods 0.000 claims description 15
- 239000007924 injection Substances 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 12
- 241001596270 Aizoon Species 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 9
- 239000004952 Polyamide Substances 0.000 claims description 8
- 229920002647 polyamide Polymers 0.000 claims description 8
- 239000003937 drug carrier Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims 3
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims 3
- 235000013402 health food Nutrition 0.000 claims 2
- 238000004128 high performance liquid chromatography Methods 0.000 claims 2
- 235000013305 food Nutrition 0.000 abstract description 15
- 239000000463 material Substances 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 5
- 230000005764 inhibitory process Effects 0.000 abstract description 5
- 239000002904 solvent Substances 0.000 abstract description 5
- 239000000969 carrier Substances 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- 238000010586 diagram Methods 0.000 description 14
- 230000000694 effects Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 150000002989 phenols Chemical class 0.000 description 4
- 241000220156 Saxifraga Species 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000002211 ultraviolet spectrum Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 2
- 241000220151 Saxifragaceae Species 0.000 description 2
- 241000014078 Thomsonia Species 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 2
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- 239000012982 microporous membrane Substances 0.000 description 2
- GMVPRGQOIOIIMI-DODZYUBVSA-N 7-[(1R,2R,3R)-3-hydroxy-2-[(3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoic acid Chemical group CCCCC[C@H](O)C=C[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DODZYUBVSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 241000336847 Luda Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 201000001352 cholecystitis Diseases 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 239000002516 radical scavenger Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010567 reverse phase preparative liquid chromatography Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/20—Unsaturated compounds containing keto groups bound to acyclic carbon atoms
- C07C49/24—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups
- C07C49/245—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups containing six-membered aromatic rings
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Abstract
The invention discloses a new diaryl nonane IV and III free radical inhibitor in saxifraga tangutica, a separation preparation process and application thereof. The specific preparation process comprises the following steps: the method comprises six steps of extraction, coarse separation of a microporous resin column, on-line screening of free radical inhibitor components, high-pressure reverse phase chromatographic column separation, on-line screening of free radical inhibitors and high-pressure reverse phase column preparation. The prepared diaryl nonane SaxitansinA and Saxitansidide B can be applied to preparation of free radical inhibition drugs or health-care foods, and can be specifically used as active ingredients to be prepared into various pharmaceutical preparations or health-care foods according to any pharmaceutically or food scientifically acceptable carriers. The extraction solvent, the microporous resin column and the solvent and the separation material used for the separation of the high-pressure reversed-phase chromatographic column in the preparation process can be recycled; the raw material source is wide, the large-scale operation can be realized through the technological steps of cold soaking extraction of methanol at room temperature, rough separation of a microporous resin column and the like, and the purity of the product can be ensured to be more than 95% through high-pressure preparative chromatographic separation.
Description
Technical Field
The invention relates to the technical field of separation of new diaryl nonane free radical inhibitors in saxifraga tangutica, in particular to new diaryl nonane IV and III free radical inhibitors in saxifraga tangutica and a separation preparation process and application thereof.
Background
Saxifraga tangutica Engl, also known as Saxifraga glauca, is an annual evergreen herb of the genus Saxifraga (Saxifragaceae) of the family Saxifragaceae, the name of Tibetan medicine: the traditional Chinese medicine named as 'Luda' is mainly distributed under conifer bush with elevation of 2900-4900 m in Qinghai, Gansu, Tibet, Sichuan and Dan and Kaishi regions. Thanggu saxifrage is a common Tibetan medicine, and can be used as a whole herb, which is recorded in the Chinese medicine dictionary: it is slightly bitter, pungent and cold in nature. The main effects are as follows: clearing liver-fire, promoting bile flow, invigorating spleen and stomach. The main treatment is as follows: hepatitis, cholecystitis, influenza. Modern pharmacological studies have demonstrated that phenols are the major active ingredient. The phenolic compounds have good free radical scavenging activity reported in the literature. However, only 8 antioxidant phenolic compounds published in this subject have been isolated and identified from Saxifraga tangutica (Jun Dang, Yandau Tao, Yun Shao, et al. antioxidant extracts and phenols isolated from Jianhai-Tibet Plateau media plant Saxifraga tandutifolia Industrial Crops and Products 2015,78: 13-18). In order to further accelerate the quality evaluation, production and sale and research and development steps of related new drugs of the saxifrage tangutica, more active ingredients with novel structures need to be excavated from the saxifrage tangutica.
The research related to the subject group has applied for a national invention patent (application number: 202010041021.3) describing the separation and preparation process and application of six galloyl natural radical scavengers with known structures in saxifrage tangutica, and no literature or patent report of a novel diarylnonane radical inhibitor is related to the present. Therefore, a method for separating and preparing the diarylnonane free radical inhibitor from the saxifrage tangutica with simple process and large scale is needed to be established.
Disclosure of Invention
Based on the technical problems, the invention aims to provide novel diarylnonane IV and III free radical inhibitors in saxifraga tangutica, and a separation preparation process and application thereof.
The invention protects new diaryl nonane IV and III free radical inhibitors in saxifraga tangutica, wherein the diaryl nonane IV and III free radical inhibitors are brown yellow oil, are named as diaryl nonane Saxitansin A free radical inhibitor and Saxitansin B free radical inhibitor respectively, and have molecular formulas C21H26O6And C27H36O10The chemical structural formulas are respectively as follows:
the invention provides a separation and preparation process of a new diaryl nonane IV and III free radical inhibitor in saxifraga tangutica, which specifically comprises the following steps:
and 3, screening the components of the online free radical inhibitor: adding 70-100% methanol into the Thalictrum aquilegifolium target component Fr5 for dissolving, preparing a sample with the concentration of 70.0-120.0 mg/mL, filtering with a 0.45-micrometer microporous filter membrane to obtain a Thalictrum aquilegifolium target component Fr5 sample solution, namely a filtrate B, taking 1mL of the filtrate B, and screening a free radical inhibitor in the Thalictrum aquilegifolium target component Fr5 by using an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a 7-X10(250 multiplied by 4.6mm, 7 mu m) reversed phase chromatographic column resistant to pure water, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm;
and 4, separating by using a high-pressure reverse phase chromatographic column: sample solution B of the objective component Fr5 of thalictrum tanguticum in terms of the amount of polyamide: mixing the components of the thalictrum alutahensis Fr5 in an amount of 2:1, and drying under reduced pressure to obtain a sample mixture of the thalictrum alutahensis extract, separating the sample by a preparation column filled with reverse 7-X10 filler, detecting by an ultraviolet detector with the detection wavelength of 254nm, collecting a second main chromatographic peak fraction Fr5-2 and a third main chromatographic peak fraction Fr5-3 in a preparation chromatogram, and drying under reduced pressure to obtain a new diaryl nonane radical inhibitor Saxitansin A with the purity of more than 95%; drying the chromatographic peak fraction Fr5-3 under reduced pressure to obtain a component containing a new diaryl nonane radical inhibitor Saxitanide B; wherein, the working parameters for preparing the high-pressure reversed-phase preparation column are as follows: preparing a column with the length of 250mm and the diameter of 20mm, wherein a stationary phase of a reversed phase chromatographic column is 7 mu m 7-X10 filler, a mobile phase A is an aqueous solution, a mobile phase B is an acetonitrile solution, a target component Fr5 is subjected to gradient elution according to 0-60 min and 10-45% of B, the sample injection amount is 1.0g, and the flow rate is 19 mL/min;
Further, in step 1, step 2, step 4 and step 6, the conditions of reduced pressure drying are as follows: the vacuum degree is 50-250 mbar, and the temperature is 40-60 ℃.
Further, in the step 3, the mobile phase a adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the ratio of 0-60 min and 10-45% B; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the reaction ring length was 15 m.
Further, in the step 5, the mobile phase a adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the ratio of 0-40 min to 27% B; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the reaction ring length was 15 m.
Further, the pure water resistant C18 reverse phase chromatographic column in the steps 5 and 6 is a pure water resistant Reprosil C18 reverse phase chromatographic column or a pure water resistant Megres C18 reverse phase chromatographic column.
The invention also protects the application of the new diaryl nonane IV and III free radical inhibitors in the saxitansine, wherein the diaryl nonane Saxitansin A and Saxitansin B free radical inhibitors can be applied to the preparation of free radical inhibition drugs or health-care foods, and concretely, the diaryl nonane Saxitansin A and Saxitanside B free radical inhibitors can be used as active ingredients to prepare various medicinal preparations according to any pharmaceutically acceptable carrier, or can be used as the active ingredients to prepare various health-care foods according to any food scientifically acceptable carrier.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention has low cost and high product purity
The solvent used for extraction, the solvent used for separation of the microporous resin column and the high-pressure reversed-phase chromatographic column can be recycled; materials used for separation can be recycled, the recycled solvent and the recycled separation materials ensure lower average separation cost, and the high-pressure preparative chromatographic separation can ensure that the purity of the product is more than 95%.
(2) The preparation method can meet the requirement of large-scale production
The raw material requirement is not high, the cost is low, the wild or commercial thalictrum sibiricum can be used, and the batch preparation is easy; the methanol is extracted by cold immersion at room temperature, and the operation is easy; the separation material can be arranged in a medium-pressure column chromatography system by adopting the coarse separation of a microporous resin column, and the large scale is easy to realize; the high-pressure reversed-phase preparative liquid chromatography used in the separation and purification is also very suitable for mass production.
Drawings
FIG. 1 is a chromatogram of a microporous resin separation of methanol extract of saxifraga tangutica according to the present invention;
FIG. 2 is an on-line HPLC-DPPH screening chromatogram of a target component Fr5 of saxifraga tangutica according to the present invention;
FIG. 3 is a diagram of a high pressure reverse phase chromatographic column for a target component Fr5 of Saxifraga tangutica according to the present invention;
FIG. 4 is an on-line HPLC-DPPH screening chromatogram of a target component Fr5-3 of saxifraga tangutica according to the present invention;
FIG. 5 is a reverse phase chromatographic analysis and preparative chromatogram of a target component Fr5-3 of Saxifraga tangutica according to the present invention;
FIG. 6 is a chromatogram for verifying the purity and activity of the new diarylnonane Saxitansin A (Fr5-2) free radical inhibitor of saxitan grass in accordance with the present invention;
FIG. 7 is a chromatogram for verifying the purity and activity of the new diarylnonane Saxitanside B (Fr5-3-1) free radical inhibitor of saxitansine of the present invention;
FIG. 8 is a mass spectrum of a novel diarylnonane Saxitansin A radical inhibitor of Saxitansin according to the present invention;
FIG. 9 shows the preparation of the novel diarylnonane Saxitansin A free radical inhibitor of Saxitansin A of the present invention1H NMR nuclear magnetic map;
FIG. 10 shows the preparation of the novel diarylnonane Saxitansin A free radical inhibitor of Saxitansin A of the present invention13C NMR nuclear magnetic map;
FIG. 11 is a DEPT nuclear magnetic map of the novel diarylnonane Saxitansin A free radical inhibitor of Saxitansin A of the present invention;
FIG. 12 is a two-dimensional nuclear magnetic diagram of HSQC of the new diarylnonane Saxitansin A free radical inhibitor in saxifraga tangutica of the present invention;
FIG. 13 is a two-dimensional nuclear magnetic map of HMBC of the novel diarylnonane Saxitansin A radical inhibitor of Saxitansin A in saxifraga tangutica according to the present invention;
FIG. 14 is a two-dimensional nuclear magnetic diagram of H-HCOSY of the novel diarylnonane Saxitan sin A radical inhibitor of saxitan in saxifrage tangutica of the present invention;
FIG. 15 is a two-dimensional nuclear magnetic diagram of NOESY of the novel diarylnonane Saxitansin A radical inhibitor of Saxitansin A in saxifrage tangutica of the present invention;
FIG. 16 is an infrared spectrum of a novel diarylnonane Saxitansin A radical inhibitor of Saxitansin A in saxifraga tangutica according to the present invention;
FIG. 17 is a UV spectrum of a novel diarylnonane Saxitan sin A radical inhibitor from saxifraga tangutica according to the present invention;
FIG. 18 is a graph showing the optical rotation test of the novel diarylnonane-type Saxitan sin A radical inhibitor in saxifraga tangutica according to the present invention;
FIG. 19 is a CD test chart of the new diarylnonane Saxitansin A free radical inhibitor in saxifraga tangutica of the present invention;
FIG. 20 is a mass spectrum of a novel diarylnonane Saxitanside B radical inhibitor of Saxitanside of the invention;
FIG. 21 shows the preparation of the novel diarylnonane Saxitanside B free radical inhibitors of Saxitanside B of the present invention1H NMR nuclear magnetic map;
FIG. 22 shows the preparation of a novel diarylnonane Saxitanside B radical inhibitor of Saxitanside of the invention13C NMR nuclear magnetic map;
FIG. 23 is a DEPT nuclear magnetic map of the novel diarylnonane Saxitanside B radical inhibitor of Saxitanside of the invention;
FIG. 24 is a two-dimensional nuclear magnetic diagram of HSQC of the novel diarylnonane Saxitanside B radical inhibitor of saxitansine in saxifrage tangutica of the present invention;
FIG. 25 is a two-dimensional nuclear magnetic map of HMBC of the novel diarylnonane Saxitanside B radical inhibitor of saxitansine in saxifrage tangutica of the present invention;
FIG. 26 is a two-dimensional nuclear magnetic diagram of the H-HCOSY of the novel diarylnonane Saxitanside B radical inhibitor of saxitansine in saxifrage tangutica of the present invention;
FIG. 27 is a two-dimensional nuclear magnetic diagram of the NOESY of the novel diarylnonane Saxitanside B radical inhibitor of saxitansine in saxifrage tangutica of the present invention;
FIG. 28 is an infrared spectrum of a novel diarylnonane Saxitanside B radical inhibitor from saxitansine of the present invention;
FIG. 29 is a UV spectrum of a novel diarylnonane Saxitanside B radical inhibitor from saxitansine of the present invention;
FIG. 30 is a graph showing the optical rotation of a novel diarylnonane-type Saxitanside B radical inhibitor in saxifrage tangutica of the present invention;
FIG. 31 is a CD test chart of the novel diarylnonane Saxitanside B radical inhibitor of Saxitanside B in saxifrage tangutica of the present invention;
FIG. 32 is a structural diagram of the novel diarylnonane Saxitansin A and Saxitansin B radical inhibitors of Saxitansin according to the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The separation and preparation process of the new diaryl nonane IV and III free radical inhibitor in the saxifraga tangutica concretely comprises the following steps:
and 3, screening the components of the online free radical inhibitor: adding 70% methanol in volume concentration into the Thalictrum aquilegifolium target component Fr5 for dissolving, preparing a sample with concentration of 70.0mg/mL, filtering with a 0.45 μm microporous membrane to obtain a sample solution of the Thalictrum aquilegifolium target component Fr5, namely a filtrate B, taking 1mL of the filtrate B, and screening a free radical inhibitor in the Thalictrum aquilegifolium target component Fr5 by using an online HPLC-DPPH chromatography combined system (detailed in figure 2); in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a 7-X10(250 multiplied by 4.6mm, 7 mu m) reversed phase chromatographic column resistant to pure water, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm; the mobile phase A adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the conditions of 0-60 min and 10-45% of B; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the length of the reaction ring is 15 m;
and 4, separating by using a high-pressure reverse phase chromatographic column: sample solution B of the objective component Fr5 of thalictrum tanguticum in terms of the amount of polyamide: mixing the components of the thalictrum alutahensis Fr5 at a ratio of 2:1, and drying under reduced pressure to obtain a mixed sample of the thalictrum alutahensis extract, separating the sample by a preparation column filled with reversed-phase 7-X10 filler, detecting the sample by an ultraviolet detector with a detection wavelength of 254nm, collecting a second main chromatographic peak fraction Fr5-2 and a third main chromatographic peak fraction Fr5-3 (shown in detail in figure 3) in a preparation chromatogram, and drying the chromatographic peak fraction Fr5-2 under reduced pressure to obtain a new diarylnonane radical inhibitor Saxitansin A with the purity of more than 95%; drying the chromatographic peak fraction Fr5-3 under reduced pressure to obtain a component containing a new diaryl nonane radical inhibitor Saxitanide B; wherein the reduced pressure drying condition is as follows: vacuum degree of 50mbar, temperature of 40 deg.C to obtain 637.4mg of herba Saxifragae Fr5-2 sample and 120.3mg of Fr5-3 target component sample, respectively; wherein, the working parameters for preparing the high-pressure reversed-phase preparation column are as follows: preparing a column with the length of 250mm and the diameter of 20mm, wherein a stationary phase of a reversed phase chromatographic column is 7 mu m 7-X10 filler, a mobile phase A is an aqueous solution, a mobile phase B is an acetonitrile solution, a target component Fr5 is subjected to gradient elution according to 0-60 min and 10-45% of B, the sample injection amount is 1.0g, and the flow rate is 19 mL/min;
step 5, on-line free radical inhibitor screening: adding 70% methanol into the Thalictrum aquilegifolium target component Fr5-3 for dissolving, preparing a sample with a concentration of 50.0mg/mL, filtering with a 0.45 μm microporous membrane to obtain a solution of the Thalictrum aquilegifolium target component Fr5-3, namely a filtrate C, taking 1mL of the filtrate C, and screening a free radical inhibitor in the Thalictrum aquilegifolium target component Fr5-3 by using an on-line HPLC-DPPH chromatography combined system (detailed in figure 4); in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a pure water resistant RepurSil C18(250 multiplied by 4.6mm, 5 mu m) reversed phase chromatographic column, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm; the mobile phase A adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the proportion of 27% B in 0-40 min; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the length of the reaction ring is 15 m;
The novel diaryl nonane IV and III free radical inhibitor in the saxitansine can be applied to the preparation of free radical inhibition medicines or health-care foods, and specifically can be used as an effective component to prepare various medicinal preparations according to any pharmaceutically acceptable carrier, or can be used as an effective component to prepare various health-care foods according to any food scientifically acceptable carrier.
Example 2
The separation and preparation process of the new diaryl nonane IV and III free radical inhibitor in the saxifraga tangutica concretely comprises the following steps:
and 3, screening the components of the online free radical inhibitor: adding methanol with the volume concentration of 100% into the thalictrum aizoon target component Fr5 for dissolving, preparing a sample with the concentration of 120.0mg/mL, filtering by a 0.45-micrometer microporous filter membrane to obtain a thalictrum aizoon target component Fr5 sample solution, namely a filtrate B, taking 1mL of the filtrate B, and screening a free radical inhibitor in the thalictrum aizoon target component Fr5 by utilizing an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a 7-X10(250 multiplied by 4.6mm, 7 mu m) reversed phase chromatographic column resistant to pure water, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm; the mobile phase A adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the conditions of 0-60 min and 10-45% of B; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the length of the reaction ring is 15 m;
and 4, separating by using a high-pressure reverse phase chromatographic column: sample solution B of the objective component Fr5 of thalictrum tanguticum in terms of the amount of polyamide: mixing the components of the thalictrum alutahensis Fr5 in an amount of 2:1, and drying under reduced pressure to obtain a sample mixture of the thalictrum alutahensis extract, separating the sample by a preparation column filled with reverse 7-X10 filler, detecting by an ultraviolet detector with the detection wavelength of 254nm, collecting a second main chromatographic peak fraction Fr5-2 and a third main chromatographic peak fraction Fr5-3 in a preparation chromatogram, and drying under reduced pressure to obtain a new diaryl nonane radical inhibitor Saxitansin A with the purity of more than 95%; drying the chromatographic peak fraction Fr5-3 under reduced pressure to obtain a component containing a new diaryl nonane radical inhibitor Saxitanide B; wherein the reduced pressure drying condition is as follows: vacuum degree of 250mbar, temperature of 60 deg.C to obtain 1158.6mg of herba Saxifragae Fr5-2 sample and 231.4mg of Fr5-3 target component sample, respectively; wherein, the working parameters for preparing the high-pressure reversed-phase preparation column are as follows: preparing a column with the length of 250mm and the diameter of 20mm, wherein a stationary phase of a reversed phase chromatographic column is 7 mu m 7-X10 filler, a mobile phase A is an aqueous solution, a mobile phase B is an acetonitrile solution, a target component Fr5 is subjected to gradient elution according to 0-60 min and 10-45% of B, the sample injection amount is 1.0g, and the flow rate is 19 mL/min;
step 5, on-line free radical inhibitor screening: adding methanol with the volume concentration of 100% into the Thalictrum aquilegifolium target component Fr5-3 for dissolving, preparing a sample with the concentration of 100.0mg/mL, filtering by a 0.45-micrometer microporous filter membrane to obtain a solution of the Thalictrum aquilegifolium target component Fr5-3, namely a filtrate C, taking 1mL of the filtrate C, and screening a free radical inhibitor in the Thalictrum aquilegifolium target component Fr5-3 by utilizing an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a pure water resistant Megres C18(250 multiplied by 4.6mm, 5 mu m) reversed phase chromatographic column, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm; the mobile phase A adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the proportion of 27% B in 0-40 min; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the length of the reaction ring is 15 m;
The novel diaryl nonane IV and III free radical inhibitor in the saxitansine can be applied to the preparation of free radical inhibition medicines or health-care foods, and specifically can be used as an effective component to prepare various medicinal preparations according to any pharmaceutically acceptable carrier, or can be used as an effective component to prepare various health-care foods according to any food scientifically acceptable carrier.
Example 3
The separation and preparation process of the new diaryl nonane IV and III free radical inhibitor in the saxifraga tangutica concretely comprises the following steps:
and 3, screening the components of the online free radical inhibitor: adding 80% methanol in volume concentration into the thalictrum aizoon target component Fr5 for dissolving, preparing a sample with concentration of 90.0mg/mL, filtering by a 0.45-micrometer microporous filter membrane to obtain a thalictrum aizoon target component Fr5 sample solution, namely a filtrate B, taking 1mL of the filtrate B, and screening a free radical inhibitor in the thalictrum aizoon target component Fr5 by utilizing an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a 7-X10(250 multiplied by 4.6mm, 7 mu m) reversed phase chromatographic column resistant to pure water, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm; the mobile phase A adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the conditions of 0-60 min and 10-45% of B; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the length of the reaction ring is 15 m;
and 4, separating by using a high-pressure reverse phase chromatographic column: sample solution B of the objective component Fr5 of thalictrum tanguticum in terms of the amount of polyamide: mixing the components of the thalictrum alutahensis Fr5 in an amount of 2:1, and drying under reduced pressure to obtain a sample mixture of the thalictrum alutahensis extract, separating the sample by a preparation column filled with reverse 7-X10 filler, detecting by an ultraviolet detector with the detection wavelength of 254nm, collecting a second main chromatographic peak fraction Fr5-2 and a third main chromatographic peak fraction Fr5-3 in a preparation chromatogram, and drying under reduced pressure to obtain a new diaryl nonane radical inhibitor Saxitansin A with the purity of more than 95%; drying the chromatographic peak fraction Fr5-3 under reduced pressure to obtain a component containing a new diaryl nonane radical inhibitor Saxitanide B; wherein the reduced pressure drying condition is as follows: vacuum degree of 150mbar, temperature of 50 deg.C to obtain 1308.2mg of herba Saxifragae Fr5-2 sample and 265.1mg of Fr5-3 target component sample, respectively; wherein, the working parameters for preparing the high-pressure reversed-phase preparation column are as follows: preparing a column with the length of 250mm and the diameter of 20mm, wherein a stationary phase of a reversed phase chromatographic column is 7 mu m 7-X10 filler, a mobile phase A is an aqueous solution, a mobile phase B is an acetonitrile solution, a target component Fr5 is subjected to gradient elution according to 0-60 min and 10-45% of B, the sample injection amount is 1.0g, and the flow rate is 19 mL/min;
step 5, on-line free radical inhibitor screening: adding 80% methanol in volume concentration into the Thalictrum aquilegifolium target component Fr5-3 for dissolving, preparing a sample with concentration of 90.0mg/mL, filtering with a 0.45-micrometer microporous filter membrane to obtain a solution of the Thalictrum aquilegifolium target component Fr5-3, namely a filtrate C, taking 1mL of the filtrate C, and screening a free radical inhibitor in the Thalictrum aquilegifolium target component Fr5-3 by using an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a pure water resistant RepurSil C18(250 multiplied by 4.6mm, 5 mu m) reversed phase chromatographic column, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm; the mobile phase A adopted by the first high performance liquid chromatograph is an aqueous solution, the mobile phase B is an acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to the proportion of 27% B in 0-40 min; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the length of the reaction ring is 15 m;
The novel diaryl nonane IV and III free radical inhibitor in the saxitansine can be applied to the preparation of free radical inhibition medicines or health-care foods, and specifically can be used as an effective component to prepare various medicinal preparations according to any pharmaceutically acceptable carrier, or can be used as an effective component to prepare various health-care foods according to any food scientifically acceptable carrier.
Example 4
The activity of the novel diaryl nonane IV and III free radical inhibitor in the saxifraga tangutica is verified:
adding chromatographic methanol into new diaryl nonane Saxitansin A and Saxitanside B free radical inhibitors in the separated saxitans respectively for dissolving, preparing sample solutions with the concentrations of 0.2mg/mL and 0.1mg/mL respectively, filtering by a 0.45-micrometer microporous filter membrane to obtain new diaryl nonane Saxitansin A and Saxitanside B sample solutions in the saxistor, taking 1mL of sample, and verifying the activity of the new diaryl nonane Saxitansin A and Saxitanside B samples in the saxistor by utilizing an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a pure water resistant reversed phase chromatographic column 7-X10(250 multiplied by 4.6mm, 7 mu m), a mobile phase A adopted by the first high performance liquid chromatograph is an aqueous solution, a mobile phase B is an acetonitrile solution, the flow rate of the mobile phase is 1.0mL/min according to 0-60 min and 10-45% B, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, the concentration of the DPPH solution is 50 mu g/mL, and the flow rate of a mobile phase is 0.5 mL/min; the reaction ring length is 15m, the detection wavelength is 517nm, and the activity verification chromatogram map (detailed in figure 6 and figure 7) is shown. The mass spectrum, nuclear magnetic diagram, infrared spectrum diagram, ultraviolet spectrum diagram, optical rotation test diagram, CD test diagram and structural diagram of new diaryl nonane Saxitansin A and Saxitanside B free radical inhibitor (see the attached figures 8-32 in detail).
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The new diaryl nonane IV and III free radical inhibitor in the saxifrage tangutica is characterized in that the diaryl nonane IV and III free radical inhibitor is brown yellow oil, the names of which are respectively diaryl nonane Saxitansin A free radical inhibitor and Saxitansin B free radical inhibitor, the molecular formulas of which are respectively C21H26O6And C27H36O10The chemical structural formulas are respectively as follows:
2. the process for the isolation and preparation of the novel diarylnonanes IV and III radical inhibitors of saxifraga tangutica as claimed in claim 1, comprising the steps of:
step 1, extraction: drying the whole herb of the saxifrage tangut in the shade, coarsely crushing the herb, and mixing the crushed herb according to the ratio of 1 g: extracting with 5-100 mL of methanol at room temperature for 2-4 times, each time for 2-4 hours, filtering, and combining the filtrates to obtain a filtrate A, wherein the filtrate A is obtained by mixing the following raw materials in percentage by weight: mixing the saxifrage with the amount of 1:5, and drying under reduced pressure to obtain mixed sample of the saxifrage extract;
step 2, roughly dividing the microporous resin column: mixing a sample with the thalictrum aizoon extract, carrying out medium-pressure chromatographic separation on the sample by using microporous resin, detecting by using an ultraviolet detector with the detection wavelength of 254nm, collecting the fifth main chromatographic peak fraction in a preparative chromatogram, and drying the fraction under reduced pressure to obtain a target component Fr5, wherein the crude separation working parameters of the microporous resin column are as follows: the length of a chromatographic column is 460mm, the diameter of the chromatographic column is 49mm, the stationary phase of a microporous resin column is CHP20P, the mobile phase A is water, the mobile phase B is ethanol, the chromatographic conditions are 0-120 min, 0-100% of B, 120-150 min and 100% of B, the sample injection amount is 40g, and the flow rate is 30 mL/min;
and 3, screening the components of the online free radical inhibitor: adding 70-100% methanol into the Thalictrum aquilegifolium target component Fr5 for dissolving, preparing a sample with the concentration of 70.0-120.0 mg/mL, filtering with a 0.45-micrometer microporous filter membrane to obtain a Thalictrum aquilegifolium target component Fr5 sample solution, namely a filtrate B, taking 1mL of the filtrate B, and screening a free radical inhibitor in the Thalictrum aquilegifolium target component Fr5 by using an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a 7-X10(250 multiplied by 4.6mm, 7 mu m) reversed phase chromatographic column resistant to pure water, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm;
and 4, separating by using a high-pressure reverse phase chromatographic column: sample solution B of the objective component Fr5 of thalictrum tanguticum in terms of the amount of polyamide: mixing the components of the thalictrum alutahensis Fr5 in an amount of 2:1, and drying under reduced pressure to obtain a sample mixture of the thalictrum alutahensis extract, separating the sample by a preparation column filled with 7-X10 reverse phase filler, detecting by an ultraviolet detector with the detection wavelength of 254nm, collecting a second main chromatographic peak fraction Fr5-2 and a third main chromatographic peak fraction Fr5-3 in a preparation chromatogram, and drying under reduced pressure to obtain a new diaryl nonane radical inhibitor Saxitansin A with the purity of more than 95%; drying the chromatographic peak fraction Fr5-3 under reduced pressure to obtain a component containing a new diaryl nonane radical inhibitor Saxitanide B; wherein, the working parameters for preparing the high-pressure reversed-phase preparation column are as follows: preparing a column with the length of 250mm and the diameter of 20mm, wherein a stationary phase of a reversed phase chromatographic column is 7 mu m 7-X10 filler, a mobile phase A is an aqueous solution, a mobile phase B is an acetonitrile solution, a target component Fr5 is subjected to gradient elution according to 0-60 min and 10-45% of B, the sample injection amount is 1.0g, and the flow rate is 19 mL/min;
step 5, on-line free radical inhibitor screening: adding 70-100% methanol into the Thalictrum aquilegifolium target component Fr5-3 for dissolving, preparing a sample with the concentration of 50.0-100.0 mg/mL, filtering with a 0.45-micrometer microporous filter membrane to obtain a solution of the Thalictrum aquilegifolium target component Fr5-3, namely a filtrate C, taking 1mL of the filtrate C, and screening a free radical inhibitor in the Thalictrum aquilegifolium target component Fr5-3 by using an online HPLC-DPPH chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a pure water resistant C18(250 multiplied by 4.6mm, 5 mu m) reversed phase chromatographic column, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm;
step 6, preparing a high-pressure reverse phase column: separating the filtrate C by a reversed phase C18 preparation column, detecting by an ultraviolet detector with the detection wavelength of 254nm, collecting a corresponding chromatographic peak fraction Fr5-3-1 in a preparative chromatogram, and drying the chromatographic peak fraction Fr5-3-1 under reduced pressure to obtain a new diaryl nonane radical inhibitor Saxitanside B with the purity of more than 95%; wherein, the working parameters of the preparation of the high-pressure reversed-phase C18 preparation column are as follows: preparing pure water-resistant C18 filler with the column length of 250mm and the diameter of 20mm, the stationary phase of a reversed phase chromatographic column of 5 microns, the mobile phase A being an aqueous solution, the mobile phase B being an acetonitrile solution, the target component Fr5-3 being eluted according to the isocratic ratio of 0-40 min and 27% B, the sample injection volume being 1.0mL, and the flow rate being 19 mL/min.
3. The process for the isolation and preparation of the novel diarylnonanes IV and III radical inhibitors of saxifraga tangutica as claimed in claim 2, wherein drying under reduced pressure in step 1, step 2, step 4 and step 6 is carried out under the following conditions: the vacuum degree is 50-250 mbar, and the temperature is 40-60 ℃.
4. The process for separating and preparing the novel diarylnonane IV and III free radical inhibitors in saxifraga tangutica as claimed in claim 2, wherein in step 3, the mobile phase A adopted by the first HPLC is aqueous solution, the mobile phase B is acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min according to 0-60 min and 10-45% of B; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the reaction ring length was 15 m.
5. The process for separating and preparing the novel diarylnonane IV and III free radical inhibitors in saxifraga tangutica as claimed in claim 2, wherein in step 5, the mobile phase A adopted by the first HPLC is aqueous solution, the mobile phase B is acetonitrile solution, and the flow rate of the mobile phase is 1.0mL/min at 0-40 min and 27% B; the DPPH solution used by the second high performance liquid chromatograph has the concentration of 50 mug/mL and the flow rate of the mobile phase is 0.5 mL/min; the reaction ring length was 15 m.
6. The process for the isolation and preparation of the novel diarylnonanes IV and III radical inhibitors from saxifraga tangutica as claimed in claim 2, wherein said pure water resistant C18 reverse phase chromatography column in steps 5 and 6 is a pure water resistant Reprosil C18 reverse phase chromatography column or a pure water resistant Megres C18 reverse phase chromatography column.
7. The novel diarylnonane IV and III free radical inhibitors in Saxitansin as claimed in claim 1, wherein the diarylnonane Saxitansin A and Saxitansin B free radical inhibitors can be used in the preparation of free radical inhibitors or health foods, and specifically can be used as active ingredients to prepare various pharmaceutical preparations according to any pharmaceutically acceptable carriers, or used as active ingredients to prepare various health foods according to any pharmaceutically acceptable carriers.
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