CN109232704B - Polygala tenuifolia willd saponin E, extract containing polygala tenuifolia willd saponin E and application of polygala tenuifolia willd saponin E - Google Patents

Polygala tenuifolia willd saponin E, extract containing polygala tenuifolia willd saponin E and application of polygala tenuifolia willd saponin E Download PDF

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CN109232704B
CN109232704B CN201810967961.8A CN201810967961A CN109232704B CN 109232704 B CN109232704 B CN 109232704B CN 201810967961 A CN201810967961 A CN 201810967961A CN 109232704 B CN109232704 B CN 109232704B
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saponin
polygalasaponin
sweet osmanthus
polygala tenuifolia
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王春华
向伟
张国栋
李正
王腾龙
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Tianjin University of Traditional Chinese Medicine
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Abstract

The embodiment of the invention provides a compound, the structure of which is shown in the following formula I, and the compound is named as polygalasaponin E. The embodiment of the invention also provides a sweet osmanthus root extract containing the compound; the embodiment of the invention also provides a pharmaceutical composition which comprises the compound or the sweet osmanthus root extract and a pharmaceutically acceptable carrier and/or excipient. The compound provided by the invention can inhibit the generation of NO and has anti-inflammatory effect, and based on the anti-inflammatory effect, the compound provided by the invention or the sweet osmanthus flower root extract or the pharmaceutical composition containing the compound can be expected to be used for preparing a medicament for treating inflammation; further, the medicine can be used for treating hepatitis, pneumonia, nephritis, gastroenteritis, urinary system infection, early mastitis, upper respiratory infection or bronchitis.
Figure DDA0001775436940000011

Description

Polygala tenuifolia willd saponin E, extract containing polygala tenuifolia willd saponin E and application of polygala tenuifolia willd saponin E
Technical Field
The invention relates to the technical field of new compounds, in particular to a new compound extracted from a root of cinnamomum japonicum and application thereof in anti-inflammation.
Background
Inflammation plays an important role in the development of various human diseases, is one of the most common pathological processes of various human diseases, can occur in any part and any tissue of the body, and is related to the inflammation process in most human diseases. The existing anti-inflammatory drugs are mostly chemically synthesized, have large toxic and side effects, and seriously limit the exertion of the curative effects of the drugs; traditional Chinese medicines and natural medicines are huge wealth in China, and have the characteristics of small toxic and side effects and the like, so that the search of anti-inflammatory medicines from the traditional Chinese medicines and the natural medicines is an effective way for solving the problems.
The sweet osmanthus fragrans (Polygala ariillata Buch. -ham. ex D.Don) is a Polygala genus (Polygala.) plant of Polygalaceae, named Polygala Polygala falcata and sparrow flower, is widely distributed in China, and is widely applied to treatment of hepatitis, pneumonia, nephritis, gastroenteritis, urinary system infection, early mastitis, upper respiratory tract infection, bronchitis, rheumatism pain, irregular menstruation, postpartum weakness and the like in folk, but the functions of the components in the sweet osmanthus fragrans are not clear.
Disclosure of Invention
The inventor carries out intensive research on the sweet osmanthus, prepares the sweet osmanthus root extract and further extracts and separates brand new compounds from the sweet osmanthus root extract; the compound has anti-inflammatory activity and can be used for preparing medicaments for treating inflammation; and have accomplished the present invention based on this.
In a first aspect, the invention provides a compound, the structural formula of which is shown in formula I, and the compound shown in formula I is named polygalasaponin E (arilatanoside E) in the text.
Figure BDA0001775436920000021
In some embodiments of the first aspect of the present invention, the compound is extracted from roots of cinnamomum japonicum.
In a second aspect, the present invention provides a process for the preparation of the aforementioned compound, comprising the steps of:
s1: crushing the roots of the sweet osmanthus, extracting by adopting an ethanol water solution, and concentrating to obtain an extract;
s2: adding water into the extract for suspension, and sequentially extracting by using petroleum ether, ethyl acetate and n-butanol;
s3: taking the n-butanol part obtained in S2, performing silica gel column chromatography, and performing gradient elution to obtain a first component containing the compound;
s4: performing silica gel column chromatography on the first component, and performing gradient elution to obtain a second component containing the compound;
s5: recrystallizing said second component to obtain said compound.
In some embodiments of the second aspect of the invention, the extraction with aqueous ethanol may be 75% aqueous ethanol cold-dip extraction 3 times, each for 7 days; or reflux-extracting with 95% ethanol for 2-4 times (each for 2-3 hr).
In some embodiments of the second aspect of the present invention, the concentration may be concentration under reduced pressure.
In some embodiments of the second aspect of the present invention, the n-butanol fraction may be concentrated under reduced pressure prior to employing silica gel column chromatography.
In some embodiments of the second aspect of the present invention, the gradient elution is performed in a volume of 100: gradient elution with chloroform-methanol system from 1 to 1: 1.
In some embodiments of the second aspect of the present invention, the fractions obtained by gradient elution are detected by thin layer chromatography to obtain a first fraction containing the compound and a second fraction.
Cold leaching extraction, reflux extraction, concentration, extraction, silica gel column chromatography, gradient elution, thin layer chromatography, recrystallization and the like used in the second aspect of the invention are all technical means commonly used in the field, and in order to achieve corresponding technical effects, other technical means can be adopted by a person skilled in the art, and the invention is not limited herein.
In a third aspect, the present invention provides an extract from roots of sweet osmanthus comprising the compound of the first aspect of the present invention.
In a fourth aspect, the present invention provides a pharmaceutical composition comprising a compound according to the first aspect of the present invention or a root extract of sweet osmanthus according to the third aspect of the present invention.
In some embodiments of the fourth aspect of the present invention, the compound is present in an amount of 1 to 99% based on the total weight of the pharmaceutical composition; preferably 20 to 80%; more preferably 40 to 60%.
In some embodiments of the fourth aspect of the present invention, the pharmaceutical composition further comprises any pharmaceutically acceptable carrier and/or excipient.
The pharmaceutical composition of the present invention can be prepared by obtaining the effective ingredients of the raw materials of the pharmaceutical composition of the present invention by the extraction method according to the present invention, mixing with one or more pharmaceutically acceptable carriers and/or excipients, and then forming a desired dosage form using conventional techniques in the field of formulation.
In a fifth aspect, the present invention provides the use of a compound of the first aspect, an extract of cinnamomum japonicum root of the third aspect, or a pharmaceutical composition of the fourth aspect, in the manufacture of a medicament for the treatment of inflammation selected from hepatitis, pneumonia, nephritis, gastroenteritis, urinary system infection, early mastitis, upper respiratory infection, or bronchitis.
In some embodiments of the fifth aspect of the invention, the medicament is administered to a subject in need thereof at a daily dose of 0.01 to 1000mg/kg body weight based on the compound; preferably 0.1-100mg/kg body weight, more preferably 1-100mg/kg body weight.
The term "treatment" as used herein has its ordinary meaning and refers herein in particular to the treatment of a mammalian subject (preferably a human) already suffering from an inflammation according to the invention with a medicament according to the invention in order to produce a therapeutic, curative, palliative etc. effect on said disease.
As used herein, "pharmaceutically acceptable" means having no substantial toxic effect when used in the usual dosage amounts, and thus being approved by the government or equivalent international organization or approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.
The "pharmaceutically acceptable carrier or excipient" useful in the pharmaceutical compositions of the invention may be any conventional carrier in the art of pharmaceutical formulation, and the selection of a particular carrier will depend on the mode of administration or the type and state of the disease used to treat a particular patient. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. For example, solvents, diluents, dispersing agents, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, antioxidants, and the like, which are conventional in the pharmaceutical field, may be included as the pharmaceutically acceptable carrier. If necessary, a flavor, a preservative, a sweetener and the like may be further added to the pharmaceutical composition.
As used herein, the term "pharmaceutical composition" has its ordinary meaning. In addition, the 'pharmaceutical composition' of the invention can also be present or provided in the form of health products, functional foods, food additives and the like. The pharmaceutical composition of the present invention can be prepared by obtaining the active ingredients of the raw materials of the pharmaceutical composition of the present invention by extraction, separation and purification means commonly used in pharmaceutical manufacturing, optionally mixing with one or more pharmaceutically acceptable carriers, and then forming a desired dosage form, using conventional techniques in the pharmaceutical field, particularly in the field of formulation. The pharmaceutical composition according to the present invention is a pharmaceutical formulation which may be suitable for oral, parenteral or topical, topical administration. The pharmaceutical composition can be prepared into various forms such as tablets, powder, granules, capsules, oral liquid and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field. Specifically, according to the pharmaceutical compositions of the present invention, the pharmaceutical dosage forms include, but are not limited to: tablet, capsule, granule, powder, injection, powder for injection, transdermal patch, ointment, gel, suppository, oral solution, oral suspension, emulsion for injection, oral emulsion, etc., sustained release tablet, and controlled release tablet. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.
Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, fine granules, pellets, elixirs and the like, without limitation. In addition to the active ingredient, these preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrating agents (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, coloring agents, flavoring agents, sweetening agents, and the like. Tablets may be prepared according to conventional mixing, granulating or coating methods.
Dosage forms for parenteral administration may include, for example, injections, drops for medical use, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches and the like, without being limited thereto.
The pharmaceutical compositions according to the present disclosure may be administered orally or parenterally, for example rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.
The pharmaceutically acceptable dose of the active ingredient, i.e., the administration dose, may vary according to the age, sex and weight of the subject to be treated, the particular disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration and the judgment of the diagnostician. Determining the dosage to be administered taking these factors into account is within the level of skill in the art. A typical dose may be from 0.01 to 1000 mg/kg/day, preferably from 0.1 to 100 mg/kg/day, more preferably from 1 to 100 mg/kg/day. However, the scope of the present disclosure is not in any way limited by the administration dosage.
The research shows that the compound shown in the formula I can inhibit the generation of NO and has anti-inflammatory effect, and based on the research, the compound provided by the invention or the sweet osmanthus flower root extract or the pharmaceutical composition containing the compound can be expected to be used for preparing the medicines for treating inflammation; further, the medicine can be used for treating hepatitis, pneumonia, nephritis, gastroenteritis, urinary system infection, early mastitis, upper respiratory infection or bronchitis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the anti-inflammatory activity results of polygalin E;
FIG. 2 shows the results of the cytotoxicity studies of polygalasaponin E.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious 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 extraction and characterization of the Compound Polygala Tenuifolia Saponin E
1. Apparatus and materials
X-5 type micro melting point apparatus (Beijing Take instruments Co.); ZF-20D dark box type ultraviolet analyzer (ZFcun electro-optic instrument factory); bruker AV-III NMR spectrometer, TMS as internal standard (Bruker, Switzerland); synapt G2Mass spectrometer (Waters, USA); agilent1260 high performance liquid chromatograph (Agilent corporation, usa); SunfireTM Prep C18 column (19X 150mm, 5 μm); waters2489 preparation of liquid chromatograph (Waters corporation, usa); silica gel for column chromatography (Qingdao ocean chemical plant); silica gel GF254 thin layer prefabricated plate (in the chemical industry Shanghai Co., Ltd.); sephadex LH-20 (Beijing Solaibao Tech Co., Ltd.); the reagents used were analytically pure and chromatographically pure.
The Hebao mountain sweet osmanthus medicinal material is collected from Yunnan province, and plant specimens are stored in a coastal modern traditional Chinese medicine laboratory of Tianjin traditional Chinese medicine university.
2. Extraction of polygalasaponin E from daylily
S1: 30kg of roots of the sweet osmanthus, crushing, cold-soaking and extracting for three times at room temperature by using 75% (v/v) ethanol for 7 days each time, filtering, and concentrating under reduced pressure to obtain extract;
s2: suspending the extract with appropriate amount of water, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol for three times;
s3: concentrating n-butanol part under reduced pressure to obtain 300g of crude product, performing silica gel column chromatography on the crude product, performing gradient elution by using chloroform-methanol systems with the volumes of 100:1, 100:3, 100:5, 100:7, 10:1, 8:2, 7:3 and 1:1, and collecting three column volumes in each gradient; detecting by thin layer chromatography, and collecting 20g of 100:7 fraction containing polygalasaponin E to obtain the first component;
s4: silica gel column chromatography was again applied to the first fraction, in a volume of 100: 1. gradient elution with chloroform-methanol systems at 100:3, 100:7, 10:1, 8:2, 1:1, collecting three column volumes per gradient; detecting by thin layer chromatography, collecting the fraction containing polygalasaponin E as the second component with chloroform-methanol (4:1) as developing agent;
s5: recrystallizing the second component to obtain 8mg of Polygala crotalarioides E pure product.
3. Characterization of Polygala fallax Diels saponin E
Compound polygalasaponin E: white powder (methanol), HR-ESI-MS (high resolution electrospray ion mass spectrometry) gave M/z 1277.5839[ M-H]-Peak (Calcd for:1277.5803), molecular weight determined to be 1278. Bonding of1H-NMR and13C-NMR to confirm the molecular formula to be C60H94O29And calculating that the unsaturation degree is 14, the purple spots are shown by 10 percent sulfuric acid-ethanol, the Liebermann-Burchard reaction is positive, and the Molish reaction is positive, which indicates that the triterpene saponin compound is obtained.1H-NMR(600MHz,CD3OD) gave 5 groups of sugar end hydrogen signals: deltaH5.46(1H, brs),5.44(1H, d, J ═ 8.4Hz),4.55(1H, d, J ═ 7.8Hz),4.51(1H, d, J ═ 7.8Hz),4.36(1H, d, J ═ 7.8 Hz). The 5-angle methyl proton signals, delta: 1.40(3H, s),1.28(3H, s),0.97(3H, s),0.94(3H, s),0.76(3H, s), and the 1-trisubstituted olefinic bond proton signals, delta: 5.67(1H, t).13C-NMR(150MHz,CD3OD) gave 60 carbon messages, 5 sugar terminal carbon signals δ:101.1,95.0,105.6,106.8,104.9,1 ester carbonyl signal δ:177.8,1 carboxyl group signal δ:182.9,1 group olefinic carbon signals δ:128.8(C-12) and 139.3(C-13), 2 vicinal oxymethylene signals δ:70.9(C-2) and 87.7(C-3), 1 vicinal oxymethylene signal δ:64.7(C-27), which are presumed to be the pentaglycoside of an oleanane triterpene, whose aglycone part is substantially identical to that of the plant-identified aglycone presenegenin of arilatanoside A (presenegenin is the predominant sapogenin in polygala plants). 20.8,172.8 is acetyl carbon signal, and HPLC comparison of acid hydrolysis derivatized with reagent and derivatized product of sugar standard shows that D-glucose, D-fucose, L-rhamnose and D-xylose are present in the compound. HMBC spectra show: deltaH1.40(H-24) and δC87.7(C-3),53.2(C-4),181.9 (C-23); deltaH1.28(H-25) and δC 44.6(C-1),53.2(C-4),50.1(C-9),37.5(C-10);δH0.76(H-26) and deltaC34.3(C-7),41.7(C-8),48.6 (C-14); deltaH0.94(H-29) and deltaC46.2(C-19),31.6(C-20),34.7(C-21),24.1 (C-30); deltaH0.97(H-30) and deltaC33.0(C-22),46.2(C-19), further verifying that the aglycone thereof is presenegenin. The order of sugar attachment was further extrapolated from HMBC spectra: deltaH5.44(Fuc-1) and δC177.8(C-28) correlation, demonstrating that fucose is attached to the 28-position of aglycone; deltaH5.48(Rha-1) and δC72.3(Fuc-2) associated, demonstrating linkage of rhamnose at the fucose 2 position; deltaH4.55(Glc-1) and δC87.7(C-3) correlation, demonstrating that glucose is attached to the aglycone 3 position; deltaH4.51(Xyl-1, inner) and δC85.0(Rha-4) demonstrates the attachment of xylose (inner) at the rhamnose 4 position; deltaH4.36(Xyl-1, outer) and δC85.9(Xyl-3, inner), demonstrating the attachment of xylose (outer) at position 3 of xylose (inner); deltaH5.10(Fuc-4) and δC172.8(CH3CO-) is present, demonstrating that the acetyl group is attached at the 4-position of fucose. The stereochemistry of the compound is not changed compared with the known arilatanolides A.1H and13the C nmr data are shown in table 1. From the above characterization, the chemical structural formula of the polygalasaponin E (arilatanoside E) is shown as formula I.
Figure BDA0001775436920000081
TABLE 1 preparation of Polygala tenuifolia saponins E (arilatanoside E)1H and13c nuclear magnetic resonance data
Figure BDA0001775436920000082
Figure BDA0001775436920000091
Figure BDA0001775436920000101
1H and13c NMR data were measured at 600MHz and 150MHz, respectively, and J is the coupling constant.
Example 2 anti-inflammatory Activity assay of Polygala fallax Diels Saponin E
1. Experimental Material
The polygalasaponin E is a pure polygalasaponin E product prepared by the extraction method in the embodiment 1; polygalasaponin E stock solution: dissolving polygalasaponin E by DMSO, and preparing a 1mg/ml solution or a uniform mixed solution by PBS buffer solution; mouse macrophage RAW 264.7 (coastal laboratory of Tianjin TCM university); culture solution: DMEM high-glucose medium (Hyclone, USA) and 10% fetal bovine serum (Hyclone, USA) and 1% double antibody (Hyclone, USA); FlexStation 3 full-automatic enzymatic target (Molecular Devices, USA); the Griess Reagent System kit (Promega, USA); bacterial lipopolysaccharides (LPS, Sigema, USA, Cat: L2880).
2. Experimental methods
NO is involved in a plurality of physiological and pathological processes, macrophages can generate a large amount of NO under the induction of LPS, and the NO up-regulation can enable the macrophages to release inflammatory cytokines such as TNF-alpha, IL-1a, gamma-IFN and the like. NO is involved in the regulation of cytokines, especially cytokines involved in inflammation, and has an anti-inflammatory protective effect in inflammatory reactions as well as certain cytotoxicity. These factors play a role in mediating inflammation and regulating cell growth and differentiation. Macrophages can produce immune protection, immune injury and immune regulation through NO when the intracellular and extracellular environments change. However, NO is extremely unstable and is rapidly metabolized into nitrite, which is relatively stable and can be detected by Griess Reagent. Therefore, in the experiment, the Griess Reagent System kit is adopted to detect the content of nitrite in mouse macrophage, reflect the level of NO generated by cells, and further evaluate the influence of polygalasaponin E on NO generation in mouse macrophage RAW 264.7 so as to reflect the anti-inflammatory activity of polygalasaponin E.
The specific experimental method comprises the following steps:
1) taking cells in logarithmic growth phase, scraping off with cell scraper, uniformly inoculating into 24-well culture plate, inoculating cells 5 × 10 per well5Adding 500 μ L of culture solution at 37 deg.C and 5% CO2Culturing for 2 hours in an incubator;
2) dividing the cells into polygalasaponin E group, positive control group (Dex group), control group (control) and LPS group; after the cells adhere to the wall, sucking out the culture solution in the holes; adding culture solution containing polygalin E with different final concentrations (polygalin E final concentrations are 50 μ g/mL, 25 μ g/mL and 12.5 μ g/mL respectively); dex group added culture medium containing dexamethasone sodium phosphate (final concentration 5. mu.g/mL); normal culture solution is adopted for a control group and an LPS group;
3) after continuing to culture for 1 hour, LPS with the final concentration of 100ng/mL is added into each hole of other groups except the control group to stimulate the cells for 16 hours; sucking the supernatant into a 1.5mL centrifuge tube, centrifuging for 10min at 1000 rpm, sucking the supernatant into a 96-well culture plate, wherein 50 mu L of the supernatant is put into each well, and 3 wells are arranged in parallel; 50 mu L of Sulfanilamide Solution reaction Solution in the Griess Reagent System kit is absorbed into each hole (operation of keeping out of the sun), and incubation is carried out for 5-10 minutes; sucking 50 μ L of NED reaction solution in the kit into each well (operation in dark place), and incubating for 5-10 min; detecting the OD value within 30 minutes, wherein the detection wavelength is 548 nm;
4) diluting sodium nitrite standard substance in turn according to the use instruction of Griess Reagent System kit, diluting with ultrapure water (resistivity is more than 18.2M omega cm) to the required concentration, setting 8 concentrations (100 MuM, 50 MuM, 25 MuM, 12.5 MuM, 6.25 MuM, 3.13 MuM, 1.56 MuM, ultrapure water) in the experiment, setting 3 auxiliary holes for each concentration, and setting 50 MuL for each hole; 50 mu L of Sulfanilamide Solution reaction Solution is absorbed into each well (operation in dark place), and incubation is carried out for 5-10 minutes; pipette 50. mu.L of NED reaction solution into each well (dark operation), incubate for 5-10 min; the OD was measured within 30 minutes at 548 nm. Obtaining a standard curve: y is 0.0072x +0.0557 (R)2=0.9995).
5) And substituting the OD value into a standard curve to calculate the content of the nitrite.
3. Results of the experiment
The amount of nitrite produced by mouse macrophage RAW 264.7 under treatment with 12.5, 25, 50 μ g/mL polygalasaponin E is shown in bar graph form in fig. 1, which in turn reflects the level of NO produced in mouse macrophage RAW 264.7. The result shows that LPS can stimulate RAW 264.7 to generate NO, polygalasaponin E can obviously inhibit the generation of NO, and the inhibition effect is enhanced along with the increase of the concentration of polygalasaponin E, which indicates that polygalasaponin E has obvious anti-inflammatory activity. P <0.05, n ═ 3; p <0.01, n-3, compared to LPS group.
Example 3 cytotoxic Activity assay of Polygala Tenuifolia Saponin E
1. Experimental Material
Cell Counting Kit-8 Kit (Dojindo, Japan).
2. Experimental methods
1) Cells in logarithmic growth phase were scraped off gently with a cell scraper, and seeded evenly in 96-well plates (no cells at the edges, filled with sterile PBS) at 100. mu.L per well, with a cell seeding count of about 1X 1045% CO at 37 ℃ per well2Culturing for 2 hours in an incubator;
2) dividing cells into polygalasaponin E group, DXM group, control group and LPS group; after the cells adhere to the wall, sucking out the culture solution in the holes; adding culture solution containing polygalin E with different final concentrations (polygalin E final concentrations are 50 μ g/mL, 25 μ g/mL and 12.5 μ g/mL respectively); DXM group was added with culture medium containing dexamethasone sodium phosphate (final concentration 5. mu.g/mL); normal culture solution is adopted for a control group and an LPS group; after 1 hour of incubation, cells were stimulated for 16 hours by addition of LPS (final concentration 100ng/mL) per well, except for the control group; add 10. mu.L of CCK-8 solution (solution in Cell Counting Kit-8 Kit, avoid light operation, avoid bubble formation) to each well, incubate for 1-4h, and measure OD at 450nm under microplate reader. The cell viability experiment was performed using CellCountying Kit-8 Kit, using the standard protocol for this Kit.
3. Results of the experiment
The results are shown in fig. 2, the survival rate of mouse macrophage RAW 264.7 is not affected by polygalasaponin E at different concentrations, which indicates that polygalasaponin E has no cytotoxic activity.
Example 4: tablets for preparing the pharmaceutical composition of the invention
Figure BDA0001775436920000131
The above ingredients are mixed according to a conventional method and made into tablets.
Example 5: capsules for preparing the pharmaceutical compositions of the invention
Figure BDA0001775436920000132
The above ingredients were mixed according to a conventional method and filled into a gelatin capsule.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims (4)

1. A method for extracting a compound of formula I, wherein the compound has a structural formula shown in formula I:
Figure FDA0002591140390000011
the compound is named as polygalasaponin E; the compound is extracted from the root of the sweet osmanthus, and the extraction method of the compound comprises the following steps:
s1: crushing the roots of the sweet osmanthus, extracting by adopting an ethanol water solution, and concentrating to obtain an extract;
s2: adding water into the extract for suspension, and sequentially extracting by using petroleum ether, ethyl acetate and n-butanol;
s3: taking the n-butanol part obtained in S2, performing silica gel column chromatography, and performing gradient elution to obtain a first component containing the compound;
s4: performing silica gel column chromatography on the first component, and performing gradient elution to obtain a second component containing the compound;
s5: recrystallizing said second component to obtain said compound.
2. An extract of roots of sweet osmanthus containing polygalasaponin E annum obtained by the extraction method of claim 1.
3. Use of a compound for the manufacture of a medicament for the treatment of inflammation, wherein the compound has the formula I:
Figure FDA0002591140390000021
the compound is named as polygalasaponin E.
4. Use according to claim 3, wherein the inflammation is selected from hepatitis, pneumonia, nephritis, gastroenteritis, urinary system infection, early mastitis, upper respiratory tract infection or bronchitis.
CN201810967961.8A 2018-08-23 2018-08-23 Polygala tenuifolia willd saponin E, extract containing polygala tenuifolia willd saponin E and application of polygala tenuifolia willd saponin E Expired - Fee Related CN109232704B (en)

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