CN104311623B - A kind of Sasanguasaponin C by name 1with Sasanguasaponin C 2pentacyclic triterpenoid and preparation method thereof and application - Google Patents
A kind of Sasanguasaponin C by name 1with Sasanguasaponin C 2pentacyclic triterpenoid and preparation method thereof and application Download PDFInfo
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- CN104311623B CN104311623B CN201410676245.6A CN201410676245A CN104311623B CN 104311623 B CN104311623 B CN 104311623B CN 201410676245 A CN201410676245 A CN 201410676245A CN 104311623 B CN104311623 B CN 104311623B
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- sasanguasaponin
- human
- oleifearsaponinc
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- XBZYWSMVVKYHQN-MYPRUECHSA-N (4as,6as,6br,8ar,9r,10s,12ar,12br,14bs)-10-hydroxy-2,2,6a,6b,9,12a-hexamethyl-9-[(sulfooxy)methyl]-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid Chemical compound C1C[C@H](O)[C@@](C)(COS(O)(=O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C XBZYWSMVVKYHQN-MYPRUECHSA-N 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000000284 extract Substances 0.000 claims abstract description 15
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 12
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 12
- 201000009030 Carcinoma Diseases 0.000 claims abstract description 12
- 241000699670 Mus sp. Species 0.000 claims abstract description 12
- 230000004069 differentiation Effects 0.000 claims abstract description 12
- 208000032839 leukemia Diseases 0.000 claims abstract description 12
- 201000007270 liver cancer Diseases 0.000 claims abstract description 12
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 12
- 210000000214 mouth Anatomy 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 10
- 239000003960 organic solvent Substances 0.000 claims abstract description 9
- 238000010298 pulverizing process Methods 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 150000002966 pentacyclic triterpenoids Chemical class 0.000 claims abstract description 3
- 230000002829 reductive effect Effects 0.000 claims abstract description 3
- 238000000638 solvent extraction Methods 0.000 claims abstract description 3
- 241001122767 Theaceae Species 0.000 claims abstract 7
- BWPGKXYWPBQBPV-ZOADXXHESA-N Theasaponin Natural products O=C(O[C@@H]1[C@@H](OC(=O)C)[C@]2(CO)[C@@H](O)C[C@@]3(C)[C@@]4(C)[C@@H]([C@]5(C)[C@H]([C@@](CO)(C)[C@@H](O[C@@H]6[C@@H](O[C@@H]7[C@H](O[C@@H]8[C@@H](O)[C@H](O)[C@H](O)CO8)[C@H](O)[C@@H](O)CO7)[C@@H](O[C@H]7[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O7)[C@H](O)[C@@H](C(=O)O)O6)CC5)CC4)CC=C3[C@@H]2CC1(C)C)/C(=C/C)/C BWPGKXYWPBQBPV-ZOADXXHESA-N 0.000 claims description 25
- BWPGKXYWPBQBPV-MWQJAWBESA-N Theasaponin Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@@H](O)CO1)O[C@H]1[C@@H]([C@@H](O)[C@H](O)CO1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(CO)C)C)(C)C[C@@H](O)[C@@]1(CO)[C@@H](OC(C)=O)[C@@H](C(C[C@H]14)(C)C)OC(=O)C(\C)=C/C)C(O)=O)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BWPGKXYWPBQBPV-MWQJAWBESA-N 0.000 claims description 25
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 14
- 238000010898 silica gel chromatography Methods 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000001641 gel filtration chromatography Methods 0.000 claims description 8
- -1 hydroxypropyl Chemical group 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000005238 degreasing Methods 0.000 claims description 2
- 230000000975 bioactive effect Effects 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 53
- 239000003921 oil Substances 0.000 description 28
- 230000001629 suppression Effects 0.000 description 26
- 244000269722 Thea sinensis Species 0.000 description 20
- 238000012360 testing method Methods 0.000 description 10
- 239000007900 aqueous suspension Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 230000000452 restraining effect Effects 0.000 description 7
- 239000013642 negative control Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 229930182490 saponin Natural products 0.000 description 3
- 235000017709 saponins Nutrition 0.000 description 3
- 238000004611 spectroscopical analysis Methods 0.000 description 3
- 229930182493 triterpene saponin Natural products 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229940090044 injection Drugs 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241001480043 Arthrodermataceae Species 0.000 description 1
- 240000001548 Camellia japonica Species 0.000 description 1
- 241000526900 Camellia oleifera Species 0.000 description 1
- 235000014143 Camellia sinensis var assamica Nutrition 0.000 description 1
- 240000008441 Camellia sinensis var. assamica Species 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 235000006468 Thea sinensis Nutrition 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000010495 camellia oil Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000037304 dermatophytes Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 238000005461 lubrication Methods 0.000 description 1
- 238000010999 medical injection Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 229940108949 paclitaxel injection Drugs 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 235000021018 plums Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- JUJWROOIHBZHMG-RALIUCGRSA-N pyridine-d5 Chemical group [2H]C1=NC([2H])=C([2H])C([2H])=C1[2H] JUJWROOIHBZHMG-RALIUCGRSA-N 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
A kind of Sasanguasaponin C by name
1with Sasanguasaponin C
2pentacyclic triterpenoid and preparation method thereof and application, at the above-mentioned Sasanguasaponin C by name of preparation
1with Sasanguasaponin C
2pentacyclic triterpenoid time, first get tea seed grouts, and pulverized; Then the tea seed grouts after pulverizing with organic solvent extraction, extracting solution obtains paste extract through concentrating under reduced pressure; Again described paste extract separation and purification is obtained Sasanguasaponin C
1with Sasanguasaponin C
2.The invention provides two kinds of above-mentioned Sasanguasaponin C by name
1with Sasanguasaponin C
2pentacyclic triterpenoid preparing the application in anti-human liver cancer cell (BEL-7402), people's low differentiation NIH mice cell (BGC-823), human breast cancer cell (MCF-7), human leukemia cell (HL60) and human oral cavity epithelial cancer cells (KB) medicine.The invention provides two and there is bioactive pentacyclic triterpenoid, agricultural and field of medicaments are had great importance, also for effective exploitation utilizes oil tea to provide wide prospect.Preparation method in the present invention is simple, and cost is lower.
Description
Technical field
The invention belongs to technical field of chemistry, be specifically related to two kinds of Sasanguasaponin C by name
1with Sasanguasaponin C
2pentacyclic triterpenoid and preparation method thereof and application.
Background technology
The root, stem, leaf, flower, seed etc. of Camellia Plants tea (Camelliasinensis) as plants such as tea, camellia, Assam tea, oil tea, tea plums all contain Triterpenoids sapogenins saponins, and theasaponin is also known as making tea saponin or TS.Theasaponin is a kind of natural nonionogenic tenside of excellent property, not only has good emulsifying, foaming, dispersion, infiltration, the effect of lubrication isoreactivity, and has restraining effect and obvious anti-inflammation-diminishing function to dermatophytes.Agriculturally can be used as sterilant, sterilant, concrete foamer, foodstuffs industry emulsifying agent, inert ingredient etc., these are all the study hotspots in agricultural and medical research field.
Oil tea (CamelliaoleiferaAbel) is the one in Camellia Plants tea, China distinctive woody edible oil material seeds, mainly be distributed in the provinces such as Anhui, Hunan, Jiangxi, Fujian, Guangdong, Guangxi, have the cultivating and growing history of more than one thousand years in China.Its tea seed is rich in tea oil and Sasanguasaponin, and the saponin(e total amount contained in the cake of camellia oleifera seeds of tea seed after extracting oil is more than 8%, and these Sasanguasaponins have adjusting blood lipid, hypotensive, the purposes such as antitumor.
About fewer from the report of the bioactive chemical fundamentals of Sasanguasaponin, if successfully this has bioactive pentacyclic triterpene saponin compound composition to research and develop resource to energy from tea seed grouts, significant contribution will be made to agricultural and medicine and other fields.
Summary of the invention
An object of the present invention is to provide and a kind ofly from tea seed grouts, is separated the Sasanguasaponin C prepared
1with Sasanguasaponin C
2pentacyclic triterpenoid, described theasaponin C
1with Sasanguasaponin C
2structural formula respectively such as formula shown in (1) and formula (2),
Two of object of the present invention is to provide a kind of above-mentioned Sasanguasaponin C by name
1with Sasanguasaponin C
2the preparation method of pentacyclic triterpenoid, it comprises the following steps:
1) extracting degreasing tea seed grouts, and pulverized;
2) the tea seed grouts after pulverizing with organic solvent extraction, extracting solution obtains paste extract through concentrating under reduced pressure;
3) described paste extract separation and purification is obtained product.
Preferably, step 2) described in organic solvent be ethanol, methyl alcohol.
Preferably, step 2) described in organic reagent be extracted as and adopt organic reagent soak extraction, soak 4 hours in organic solvent at being about to the tea seed grouts 60 DEG C after pulverizing, repeatedly extract 3 times; Or by the tea seed grouts organic solvent ultrasonic oscillation extraction after pulverizing, ultra-sonic oscillation adopt routine techniques.
Preferably, step 3) described in separation and purification be that described paste extract sherwood oil, ethyl acetate, propyl carbinol are extracted successively; The butanol extraction liquid finally obtained is utilized silica gel column chromatography, hydroxypropyl gel filtration chromatography, reversed-phase silica gel column chromatography successively separation and purification obtain product.
As further preferred version of the present invention, described silica gel column chromatography is used by butanol extraction liquid EtOAC-MeOH volume ratio 100:0 to 0:100 as gradient elution, collect the elution fraction between EtOAC-MeOH respective volume ratio respectively, again through hydroxypropyl gel filtration chromatography with methanol-eluted fractions, collect corresponding wash-out composition, finally by reversed-phase silica gel column chromatography with CH
3cN-H
2o volume ratio 45:55 wash-out, obtains product.
As further preferred version of the present invention, described butanol extraction liquid is evaporated to paste, lysate is obtained with hot water dissolving, again lysate is passed through 100-200 object silica gel column chromatography, using EtOAC-MeOH volume ratio 100:0 to 0:100 as gradient elution, collect the elution fraction wherein between 70:30 to 60:40; By the elution fraction that obtains again through hydroxypropyl gel filtration chromatography and SephadexLH-20 gel filtration chromatography, with methanol-eluted fractions, every 100 milliliters of cuts, collect 3-5 cut wherein, the 3-5 cut collected is merged, after evaporate to dryness again through ODS reversed-phase silica gel column chromatography and octadecyl silane column chromatography, with CH
3cN-H
2o volume ratio 45:55 wash-out, can obtain Sasanguasaponin C respectively
1with Sasanguasaponin C
2.
Three of object of the present invention is to provide a kind of above-mentioned Sasanguasaponin C by name
1with Sasanguasaponin C
2pentacyclic triterpenoid preparing the application in anti-human liver cancer cell (BEL-7402), people's low differentiation NIH mice cell (BGC-823), human breast cancer cell (MCF-7), human leukemia cell (HL60) and human oral cavity epithelial cancer cells (KB) medicine.
Confirmed by test, Sasanguasaponin C by name of the present invention
1with Sasanguasaponin C
2pentacyclic triterpenoid have restraining effect to cancer cells, can be applied to and prepare cancer therapy drug aspect.
Sasanguasaponin C described by name in the present invention
1with Sasanguasaponin C
2pentacyclic triterpenoid can make the formulations such as oral, external application, injection with pharmaceutically general auxiliary material, the medicinal preparation for oral administration such as such as tablet, capsule, pill; Cha agent, lotion, etc. external medicine; The medical injections such as injection liquid, mixed rotary liquid, freeze-drying powder, prepared by the ordinary method with reference to pharmacy field.
Beneficial effect of the present invention is as follows:
1) the invention provides two and there is bioactive pentacyclic triterpene saponin compound, agricultural and field of medicaments are had great importance.
2) the present invention has prepared the pentacyclic triterpene saponin compound that two have medicinal actives, for effective exploitation oil tea provides wide prospect from tea seed grouts.
3) preparation method of the present invention is simple, and cost is lower.
Accompanying drawing explanation
Fig. 1 is Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) chemical structural formula.
Fig. 2 is Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) vitro inhibition activity test schematic diagram to human liver cancer cell (BEL-7402).
Fig. 3 is Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) vitro inhibition activity test schematic diagram to people's low differentiation NIH mice cell (BGC-823).
Fig. 4 is Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) vitro inhibition activity test schematic diagram to human breast cancer cell (MCF-7).
Fig. 5 is Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) vitro inhibition activity test schematic diagram to human leukemia cell (HL60).
Fig. 6 is Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) vitro inhibition activity test schematic diagram to human oral cavity epithelial cancer cells (KB).
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is mass percentage.
Below in conjunction with specific embodiment, the present invention is described in further details.
One, Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) preparation
(1) get 4.0 kilograms of tea seed grouts, and pulverized;
(2) the tea seed grouts after at every turn pulverizing with the lixiviate at 60 DEG C of 10 liter of 70% ethanol, each immersion 4 hours, total immersion carries 3 times.The ethanol total amount of 70% is 30 liters.Gained extracting solution is condensed into medicinal extract, obtains medicinal extract 990 grams;
(3) above-mentioned gained medicinal extract is suspended in 3L water obtains aqueous suspension, the first step residue aqueous suspension that this aqueous suspension obtains after first extracting 3 times successively with the sherwood oil of 3L is the aqueous suspension that petroleum ether extraction is crossed, and now total consumption of sherwood oil is 9L;
The second step residue aqueous suspension that the aqueous suspension crossed by petroleum ether extraction obtains after extracting 3 times successively by the ethyl acetate of 3L is again the aqueous suspension that extraction into ethyl acetate is crossed, and now total consumption of ethyl acetate is 9L;
The aqueous suspension crossed by extraction into ethyl acetate extracts 3 times successively with the propyl carbinol of 3L again, gets butanol extraction liquid stand-by, and now total consumption of propyl carbinol is 9L;
Described butanol extraction liquid is concentrated into paste, with baking oven, 55 DEG C of oven dry are set, about 100 grams, then take together with the silica gel of 100 grams is stirred in the n-butyl alcohol extract of 100 grams, again through 100-200 object silica gel column chromatography, using EtOAC-MeOH volume ratio 100:0 to 0:100 as gradient elution, collect the elution fraction wherein between 70:30 to 60:40; By the elution fraction that obtains again through SephadexLH-20 gel filtration chromatography, with methanol-eluted fractions, every 100 milliliters of cuts, obtain 20 cuts altogether, collect 3-5 cut wherein, the 3-5 cut collected is merged, after evaporate to dryness again through ODS reverse phase silica gel (octadecyl silane) column chromatography, with CH
3cN-H
2o volume ratio 45:55 wash-out, can obtain Sasanguasaponin C
1(OleifearsaponinC
1, 16.7 milligrams) and Sasanguasaponin C
2(OleifearsaponinC
2, 14.0 milligrams).
Product oil theasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) characteristic as follows:
1) the two all dissolves in methyl alcohol and DMSO, white powdery solids,
2) Sasanguasaponin C
1: UV λ
max: 236nm; Sasanguasaponin C
2: UV λ
max: 210nm;
3) Sasanguasaponin C
1: IR (KBr) γ
max(cm
-1): 3416,2950,2927,1720,1644,1442,1385,1241,1159,1078,1046,916,895,852,782,639,588,533; Sasanguasaponin C
2: IR (KBr) γ
max(cm
-1): 3416,2927,1740,1701,1644,1442,1385,1241,1162,1077,1045,914,893,855,785,640,606;
4) Sasanguasaponin C
1: HR-ESI-MS:m/z1215.5770 ([M-H]
-, C
59h
92o
26, calculated value be 1216.5877); Sasanguasaponin C
2: HR-ESI-MS:m/z1231.6084 ([M-H]
-, C
60h
96o
26, calculated value be 1232.6190); Sasanguasaponin C
1with Sasanguasaponin C
2spectroscopic data of the nuclear magnetic resonance in table 1.
Table 1: Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) spectroscopic data of the nuclear magnetic resonance (
1hNMR at 400MHz,
13cNMR tests under 100MHz condition, and δ unit is ppm, coupling constant J unit is Hz, and solvent is deuterated pyridine).
Table 1.OleifearsaponinC
1and OleifearsaponinC
2spectroscopic data of the nuclear magnetic resonance
All spectral datas all pass through
1hNMR,
13cNMR, DEPT-90, DEPT-135 and
1h-
1two nuclear magnetic resonance spectrum ownership such as HCOSY, HSQC and HMBC, demonstrate the structure of gained compound.
Two, Sasanguasaponin C
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) extracorporeal suppression tumor cell activity test
Body outer suppressioning test to human liver cancer cell (BEL-7402), people's low differentiation NIH mice cell (BGC-823), human breast cancer cell (MCF-7), human leukemia cell (HL60) and human oral cavity epithelial cancer cells (KB): adopt mtt assay.
Experimental technique:
1, being digested respectively by above-mentioned five kinds of cancer cells, count, being mixed with concentration is 3 ~ 4 × 10
4the cell suspension of individual/mL, in 96 porocyte culture plates, every hole adds 100 μ L cell suspension (every holes 3 ~ 4 × 10
3individual cell);
2,96 porocyte culture plates are placed in 37 DEG C, 5%CO
2cultivate 24 hours in incubator;
3, with perfect medium dilution medicine Sasanguasaponin C respectively
1with Sasanguasaponin C
2to desired concn (100 μMs, 50 μMs, 25 μMs, 12.5 μMs, 6.25 μMs, 3.125 μMs, 1.5625 μMs, 0.78125 μM eight concentration gradients), every hole adds the corresponding pastille substratum (DMEM) of 100 μ L, set up negative control group, positive controls (paclitaxel injection 10 μ g/mL) simultaneously;
4,96 porocyte culture plates are placed in 37 DEG C, 5%CO
2cultivate 72 hours in incubator;
5,96 orifice plates are carried out MTT dyeing, λ=490nm, measure OD value;
1) every hole adds 20 μ LMTT (5mg/mL), continues cultivation 4 hours at incubator;
2) discard substratum, every hole adds 150 μ LDMSO and dissolves, and shaking table mixes for 10 minutes lightly;
3) λ=490nm, microplate reader reads the OD value in every hole, calculates inhibiting rate;
6, each group inhibiting rate and IC is calculated
50value.
Product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human liver cancer cell (BEL-7402) and IC
50value is in table 2:
Table 2. Sasanguasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human liver cancer cell (BEL-7402) and IC
50value
Product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of people's low differentiation NIH mice cell (BGC-823) and IC
50value is in table 3:
Table 3. product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of people's low differentiation NIH mice cell (BGC-823) and IC
50value
Product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human breast cancer cell (MCF-7) and IC
50value is in table 4:
Table 4. product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human breast cancer cell (MCF-7) and IC
50value
Product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human leukemia cell (HL60) and IC
50value is in table 5:
Table 5. product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human leukemia cell (HL60) and IC
50value
Product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human oral cavity epithelial cancer cells (KB) and IC
50value is in table 6:
Table 6. product oil theasaponin C
1(OleifearsaponinC
1) for the extracorporeal extracorporeal suppression of human oral cavity epithelial cancer cells (KB) and IC
50value
Product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human liver cancer cell (BEL-7402) and IC
50value is in table 7:
Table 7. product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human liver cancer cell (BEL-7402) and IC
50value
Product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of people's low differentiation NIH mice cell (BGC-823) and IC
50value is in table 8:
Table 8. product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of people's low differentiation NIH mice cell (BGC-823) and IC
50value
Product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human breast cancer cell (MCF-7) and IC
50value is in table 9:
Table 9. product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human breast cancer cell (MCF-7) and IC
50value
Product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human leukemia cell (HL60) and IC
50value is in table 10:
Table 10. product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human leukemia cell (HL60) and IC
50value
Product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human oral cavity epithelial cancer cells (KB) and IC
50value is in table 11:
Table 11. product oil theasaponin C
2(OleifearsaponinC
2) for the extracorporeal extracorporeal suppression of human oral cavity epithelial cancer cells (KB) and IC
50value
As shown in Figure 2, in Fig. 2, A, B are respectively the Sasanguasaponin C that concentration is 12.5 μMs, 6.25 μMs concentration
1(OleifearsaponinC
1), C, D are respectively the Sasanguasaponin C that concentration is 12.5 μMs, 6.25 μMs concentration
2(OleifearsaponinC
2) extracorporeal extracorporeal suppression figure to human liver cancer cell (BEL-7402), E is Vehicle controls figure, F is negative control figure.Sasanguasaponin C can be found out by the comparing result in Fig. 2
1with Sasanguasaponin C
2to human liver cancer cell (BEL-7402), there is remarkable restraining effect.
As shown in Figure 3, in Fig. 3, A, B are respectively the Sasanguasaponin C that concentration is 12.5 μMs, 6.25 μMs concentration
1(OleifearsaponinC
1), C, D are respectively the Sasanguasaponin C that concentration is 12.5 μMs, 6.25 μMs concentration
2(OleifearsaponinC
2) extracorporeal extracorporeal suppression figure to people's low differentiation NIH mice cell (BGC-823), E is Vehicle controls figure, F is negative control figure.Sasanguasaponin C can be found out by the comparing result in Fig. 3
1with Sasanguasaponin C
2to people's low differentiation NIH mice cell (BGC-823), there is remarkable restraining effect.
As shown in Figure 4, in Fig. 4, A, B are respectively the Sasanguasaponin C that concentration is 12.5 μMs, 6.25 μMs concentration
1(OleifearsaponinC
1), C, D are respectively the Sasanguasaponin C that concentration is 25 μMs, 12.5 μMs concentration
2(OleifearsaponinC
2) extracorporeal extracorporeal suppression figure to human breast cancer cell (MCF-7), E is Vehicle controls figure, F is negative control figure.Sasanguasaponin C can be found out by the comparing result in Fig. 4
1with Sasanguasaponin C
2to human breast cancer cell (MCF-7), there is remarkable restraining effect.
As shown in Figure 5, in Fig. 5, A, B are respectively the Sasanguasaponin C that concentration is 12.5 μMs, 6.25 μMs concentration
1(OleifearsaponinC
1), C, D are respectively the Sasanguasaponin C that concentration is 12.5 μMs, 6.25 μMs concentration
2(OleifearsaponinC
2) extracorporeal extracorporeal suppression figure to human leukemia cell (HL60), E is Vehicle controls figure, F is negative control figure.Sasanguasaponin C can be found out by the comparing result in Fig. 5
1with Sasanguasaponin C
2to human leukemia cell (HL60), there is remarkable restraining effect.
As shown in Figure 6, in Fig. 6, A, B are respectively the Sasanguasaponin C that concentration is 25 μMs, 12.5 μMs concentration
1(OleifearsaponinC
1), C, D are respectively the Sasanguasaponin C that concentration is 25 μMs, 12.5 μMs concentration
2(OleifearsaponinC
2) extracorporeal extracorporeal suppression figure to human oral cavity epithelial cancer cells (KB), E is Vehicle controls figure, F is negative control figure.Sasanguasaponin C can be found out by the comparing result in Fig. 6
1with Sasanguasaponin C
2to human oral cavity epithelial cancer cells (KB), there is remarkable restraining effect.
Above-mentioned test proves, the product oil theasaponin C that the present invention prepares
1(OleifearsaponinC
1) and Sasanguasaponin C
2(OleifearsaponinC
2) there is stronger cytotoxicity to human liver cancer cell (BEL-7402), people's low differentiation NIH mice cell (BGC-823), human breast cancer cell (MCF-7), human leukemia cell (HL60) and human oral cavity epithelial cancer cells (KB).These two kinds of pentacyclic triterpene Sasanguasaponin compounds will have important application prospect in the research and development of cancer therapy drug.
Claims (3)
1. a Sasanguasaponin C by name
1with Sasanguasaponin C
2pentacyclic triterpenoid, it is characterized in that, described theasaponin C
1with Sasanguasaponin C
2structural formula respectively as shown in formula I and formula II,
2. one kind is called Sasanguasaponin C as claimed in claim 1
1with Sasanguasaponin C
2the preparation method of pentacyclic triterpenoid, it is characterized in that comprising the following steps:
1) extracting degreasing tea seed grouts, and pulverized;
2) the tea seed grouts after pulverizing with organic solvent extraction, extracting solution obtains paste extract through concentrating under reduced pressure; Described organic solvent is ethanol, methyl alcohol; Described organic reagent is extracted as and adopts organic reagent soak extraction, soaks 4 hours in organic solvent, repeatedly extract 3 times at being about to the tea seed grouts 60 DEG C after pulverizing; Or by the tea seed grouts organic solvent ultrasonic oscillation extraction after pulverizing;
3) described paste extract separation and purification is obtained Sasanguasaponin C
1with Sasanguasaponin C
2; Described separation and purification is extracted successively described paste extract sherwood oil, ethyl acetate, propyl carbinol; Discard sherwood oil and acetic acid ethyl ester extract, the butanol extraction liquid finally obtained is utilized silica gel column chromatography, hydroxypropyl gel filtration chromatography, reversed-phase silica gel column chromatography successively separation and purification obtain Sasanguasaponin C
1with Sasanguasaponin C
2; Described silica gel column chromatography is that described butanol extraction liquid is evaporated to paste, lysate is obtained with hot water dissolving, again lysate is passed through 100-200 object silica gel column chromatography, using EtOAC-MeOH volume ratio 100:0 to 0:100 as gradient elution, collect the elution fraction wherein between 70:30 to 60:40; By the elution fraction that obtains through hydroxypropyl gel filtration chromatography and SephadexLH-20 gel filtration chromatography, with methanol-eluted fractions, every 100 milliliters of cuts, collect 3-5 cut wherein, the 3-5 cut collected is merged, after evaporate to dryness again through ODS reversed-phase silica gel column chromatography and octadecyl silane column chromatography, with CH
3cN-H
2o volume ratio 45:55 wash-out, can obtain Sasanguasaponin C
1with Sasanguasaponin C
2.
3. one kind is called Sasanguasaponin C as claimed in claim 1
1with Sasanguasaponin C
2pentacyclic triterpenoid preparing the application in anti-human liver cancer cell, people's low differentiation NIH mice cell, human breast cancer cell, human leukemia cell and human oral cavity epithelial cancer cells medicine.
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| CN111153956B (en) * | 2019-12-06 | 2020-12-11 | 江苏一片叶高新科技有限公司 | Tea saponin compound, preparation method and application |
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