CN109879926B - Triterpene glycoside compounds in Glechomae herba and extraction and separation method thereof - Google Patents
Triterpene glycoside compounds in Glechomae herba and extraction and separation method thereof Download PDFInfo
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Abstract
The invention discloses a triterpenoid glycoside compound in longtube ground ivy herb and an extraction method thereof, relates to the technical field of medicines, and particularly relates to two novel triterpenoid glycoside compounds which are obtained from longtube ground ivy herb through certain extraction and separation and are respectively named longtube ground ivy herb saponin A and longtube ground ivy herb saponin B. Subjecting Glechomae herba Saponin A and Glechomae herba Saponin B to multiple detection such as superconducting nuclear magnetic resonance spectrum and mass spectrum to determine that their molecular formulas are C48H78O20The molecular weight is 974, and the chemical structural formulas are respectively shown as a formula (I) and a formula (II). The invention discloses physicochemical properties and optical activity of Glechomae herba Saponin A and Glechomae herba Saponin B, and in vitro activity screening is performed by MTT method, and the results show that the Glechomae herba Saponin A and Glechomae herba Saponin B have inhibitory effect on human gastric cancer cell, human liver cancer cell, human colon cancer cell, human ovarian cancer cell and human lung cancer cell, can be used as lead compound for developing novel antitumor drugs, and can also be used as drug for developing and treating various common clinical multiple cancers.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a triterpene glycoside compound which is firstly separated from longhairy antenoron herb serving as a raw material, an extraction method and application thereof. The compound has an inhibiting effect on tumor cell strains, can be used as a lead compound for developing new anti-tumor medicaments, and can also be used for developing medicaments for treating various clinically common multiple cancers.
Background
Glechomae herba is aerial part of Glechoma longituba (Nakai) Kupr) belonging to Labiatae. The quality standard is recorded in the section of pharmacopoeia of the people's republic of China 2015 edition. Pungent, slightly bitter and slightly cold in nature, entering liver, kidney and bladder meridians; has the effects of clearing away heat and toxic materials, inducing diuresis, removing urinary calculus, removing blood stasis and relieving swelling, and can be used for treating heat stranguria, stranguria with stone, jaundice due to damp-heat pathogen, sore and carbuncle, swelling and pain, and traumatic injury. Modern pharmacology indicates that the longtube ground ivy herb has the effects of promoting urination and benefiting gallbladder, reducing blood fat, dissolving stones, reducing blood sugar, resisting inflammation, resisting bacteria and the like, but the report of the longtube ground ivy herb on the inhibition effect of cancer cells is not found.
The chemical components contained in the glechoma longituba are complex and have various structures, and the triterpenoids separated from the glechoma longituba at present comprise oleanolic acid, ursolic acid, betulin and betulinic acid, but no triterpene glycoside component is reported.
Disclosure of Invention
One aspect of the present invention relates to triterpene glycosides of formula (I) and formula (II) which can be extracted from Glechomae herba. Herein, the compound of formula (I) may be named Glechomasaponin a, and the compound of formula (II) may be named Glechomasaponin B. The molecular formulas of Glechomae herba saponin A and Glechomae herba saponin B are both C48H78O20The chemical structural formula is as follows:
another aspect of the invention relates to a process for the preparation of compounds of formula (I) and/or formula (II). The method comprises the following steps: (1) ethanol reflux extraction; (2) concentrating the ethanol extract; (3) sequentially extracting chloroform, ethyl acetate and water saturated n-butanol; (4) concentrating the water-saturated n-butanol extract; (5) performing column chromatography separation; and optionally (6) purifying. Specifically, the method comprises the following steps:
(1) heating and refluxing Glechomae herba with ethanol, and filtering to obtain ethanol extractive solution;
(2) concentrating the ethanol extract to obtain an ethanol extract;
(3) dissolving the ethanol extract with water, sequentially extracting with chloroform, ethyl acetate and water-saturated n-butanol, and retaining water-saturated n-butanol extract phase to obtain water-saturated n-butanol extract;
(4) concentrating the water saturated n-butanol extract to obtain n-butanol extract;
(5) performing column chromatography separation on the n-butanol extract to obtain crude products of the compounds of the formula (I) and the formula (II);
(6) optionally, the crude product is subjected to purification of the monomeric compound.
In one embodiment of the method, in step (1), it is preferable that the glechoma longituba medicinal material dried in the shade is used as a raw material, the raw material is pulverized, heated and refluxed with ethanol for extraction, and filtered to obtain an ethanol extract. Wherein the ethanol is preferably high concentration ethanol aqueous solution, such as more than 70%, more than 80%, more than 90%, and 100% pure ethanol, more preferably 70% -80% ethanol aqueous solution, most preferably 75%.
In one embodiment, in step (2), the concentration is preferably concentration under reduced pressure.
In one embodiment, in step (3), the water is preferably distilled water.
In one embodiment, in step (4), the concentration is preferably concentration under reduced pressure.
In one embodiment, in step (5), the column chromatography separation preferably comprises the steps of: (a) dissolving n-butanol extract with water, transferring to macroporous resin column, gradient eluting with ethanol-water solution, performing thin layer detection on the eluate, concentrating, mixing similar (spots with same color at same position of thin layer plate) eluate fractions to obtain 6 fractions; (b) mixing the 3 rd fraction with silica gel, performing column chromatography, performing gradient elution with chloroform-methanol mixture, performing thin layer inspection on the eluate, concentrating, and mixing similar (spots with same color at the same position of thin layer plate) eluate fractions to obtain 10 fractions; (c) mixing the 8 th fraction with C18Mixing the sample, and transferring into C18Performing reverse phase column chromatography with methanolGradient eluting with alcohol-water solution, subjecting the eluate to thin layer chromatography, concentrating, and mixing similar eluates (spots with same color at the same position of thin layer plate) to obtain crude compounds of formula (I) and formula (II).
Wherein the term "macroporous resin" is used in the art, and is a resin having a macroporous structure, also known as a fully porous resin.
Wherein, in the ethanol-water used as eluent in the step (5) (a), the volume concentration of the ethanol can be 0-100%; in the chloroform-methanol mixture used as eluent in step (5) (b), the chloroform-methanol volume ratio may be from 20:1 to 1:1, such as 20:1 or 10:1 or 5:1 or 3:1 or 1: 1; the granularity of silica gel used in the column chromatography of the step (5) and (b) is preferably 100-200 meshes; in the methanol-water solution used as the gradient eluent in the step (5) (c), the volume concentration of the methanol can be 20-80%.
In one embodiment, step (6) of the method comprises: and (4) purifying the crude product obtained in the step (5) by using acetonitrile-water as an eluent through preparative liquid chromatography to obtain a monomer compound. The volume concentration of the acetonitrile aqueous solution may be 21%. Step (6) may additionally comprise further purification steps, such as recrystallization and the like.
After purification in step (6), the purity of the resulting compound may be, for example, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99%, or higher.
The above process steps (1) - (6) are relatively independent, so that the above various embodiments can be combined arbitrarily, and the technical scheme and process conditions obtained by combining the above embodiments are all within the scope of the present disclosure.
An exemplary embodiment of the process for the preparation of the compounds of formula (I) and formula (II) of the present application is shown in FIG. 1.
According to the structures of formula (I) and formula (II), the organic synthesis skilled person can also appropriately design a synthetic route to prepare the compounds of formula (I) and formula (II) by a method of organic synthesis.
The inventor has proved through experiments (such as MTT method for measuring the proliferation of tumor cells and the like) that the compound of the invention has obvious inhibition effect on human cancer cells, such as human gastric cancer cells, human liver cancer cells, human colon cancer cells, human ovarian cancer cells and human lung cancer cells.
Thus, another aspect of the present invention relates to a pharmaceutical composition comprising a compound of formula (I) and/or formula (II) as an active ingredient together with a pharmaceutically acceptable carrier and/or excipient. There is no particular requirement for pharmaceutically acceptable carriers and excipients, so long as they meet the relevant pharmaceutical regulatory requirements and are compatible with the compounds of the present invention. One skilled in the art can select a suitable pharmaceutically acceptable carrier or excipient depending on the route of administration, dosage form, and the like.
As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like, compatible with pharmaceutical administration. The pharmaceutically acceptable carrier may be solid or liquid. Exemplary solid carriers are lactose, sucrose, mica, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers are syrup, peanut oil, olive oil, water, pharmaceutical buffers, and the like.
Exemplary pharmaceutically acceptable excipients include the following: fillers such as starches (e.g., corn starch, wheat starch, rice starch, potato starch, etc.), sugars (including lactose, sucrose, mannitol, sorbitol, etc.); binders such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; humectants, such as glycerol; disintegrants, for example povidone, sodium starch glycolate, sodium carboxymethylcellulose, agar-agar, sodium carbonate and sodium bicarbonate; agents for delaying decomposition, such as paraffin; resorption accelerators, such as quaternary ammonium compounds; surfactants such as cetyl alcohol, glycerol monostearate; adsorptive carriers, for example kaolin and bentonite, and lubricating agents, for example talc, magnesium and calcium stearate, and solid polyethylene glycols.
The pharmaceutical composition can be formulated into any suitable dosage form, such as a liquid dosage form (e.g., solutions, suspensions, syrups, emulsions, and the like) or a semisolid dosage form (e.g., ointments, gels, and the like) or a solid dosage form (e.g., tablets, pills, granules, capsules, and the like). The pharmaceutical composition may be administered, for example, orally, parenterally, intradermally, subcutaneously, intramuscularly, intraperitoneally, etc., as required.
The compounds of the invention may be used in combination with other pharmaceutically active substances. The other pharmaceutically active substances may be administered simultaneously with the compounds of the invention or separately in any suitable order. Alternatively, the pharmaceutical compositions of the invention may contain more than one active ingredient. Other active substances may be, for example, antimitotic drugs (e.g., paclitaxel or vincristine, etc.), antimetabolites (e.g., gemcitabine, etc.), drugs directed against DNA (e.g., doxorubicin, etc.), drugs directed against topoisomerase (e.g., etoposide, etc.), drugs directed against biological targets in tumor cells (e.g., heusurdite, etc.).
The pharmaceutical compositions may be prepared by combining at least one compound of the invention (as the active ingredient) with one or more pharmaceutically suitable carriers or excipients that facilitate processing of the active compound into the final pharmaceutical formulation.
The invention further relates to a method for treating cancer in a mammal, in particular a human, comprising the steps of: administering to the mammal a therapeutically effective amount of a compound of formula (I) and/or formula (II).
The invention further relates to a method for treating cancer in a mammal, in particular a human, comprising the steps of: administering to said mammal a therapeutically effective amount of a pharmaceutical composition of the invention comprising as active ingredient a compound of formula (I) and/or formula (II) and a pharmaceutically acceptable carrier and/or excipient.
The invention also relates to the use of a compound of formula (I) and/or formula (II) for the treatment of a disease in a mammal, particularly a human, preferably the disease is cancer.
The invention also relates to the use of a compound of formula (I) and/or formula (II) in the manufacture of a medicament for the treatment of cancer in a mammal, particularly a human.
The "cancer" as mentioned above is, for example, breast cancer, prostate cancer, bladder cancer, pancreatic cancer, lung cancer, esophageal cancer, laryngeal cancer, liver cancer, colon cancer, thyroid cancer, melanoma, kidney cancer, testicular cancer, leukemia, ovarian cancer, stomach cancer, hepatocellular cancer, and the like. Preferably, the cancer is gastric cancer, liver cancer, colon cancer, ovarian cancer or lung cancer.
The term "therapeutically effective amount" as used herein means: an amount of a compound of the present invention that is sufficient to have an effective therapeutic effect when administered to a mammal (particularly a human) in need of such treatment. Thus, a therapeutically effective amount of a compound of the invention may be an amount sufficient to inhibit, reduce or eliminate cancer cells. The inventors have found that: the compound of the invention has obvious inhibition effect on human gastric cancer cells, human liver cancer cells, human colon cancer cells, human ovarian cancer cells and human lung cancer cells. In the case of a specific treatment, the physician may adjust and specifically determine the specific dose to be administered to the patient, depending on the subject, severity of the condition, presence or absence of complications, drug combination, etc.
The term "treating" as used herein means: preventing, alleviating, eliminating or curing the corresponding diseases.
Unless otherwise stated, reference herein to a compound includes amorphous forms thereof, various crystalline forms thereof, stereoisomers thereof, tautomers thereof and isotopically labeled forms thereof. All such compounds, in different forms, fall within the scope of the present invention and the structural formulae given.
The term "stereoisomers" refers to compounds having the same chemical composition but differing in the spatial arrangement of their atoms or groups. Unless otherwise indicated, all stereoisomers (e.g., enantiomers and diastereomers and racemic mixtures thereof) of the compounds disclosed herein are included within the scope of the compounds of the invention. In addition, all tautomers or cis-trans isomers of the compounds disclosed herein are also included within the scope of the compounds of the present invention. The various isomers of the compounds of the present invention may be separated by conventional techniques well known to those skilled in the art, such as fractional crystallization, chiral chromatographic resolution, and the like.
The invention also encompasses isotopically-labeled compounds of the present invention, wherein one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion into compounds of the invention include isotopes of hydrogen, for example2H and3isotopes of H, carbon, e.g.11C、13C and14c, isotopes of oxygen, e.g.15O、17O and18and O. For example, compounds of the present invention, incorporating a radioisotope, may be used in tissue distribution studies of drugs. Radioisotope tritium (i.e. tritium3H) And carbon-14 (i.e.14C) Are particularly suitable for this purpose because they are easy to incorporate and easy to detect. Isotopically-labelled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art.
Drawings
FIG. 1 is a flow chart of an exemplary embodiment of the extraction and separation of Glechomae herba Saponin A and Glechomae herba Saponin B, which are triterpene glycoside compounds of the present invention.
FIG. 2 is an ultraviolet spectrum of the triterpene glycoside compound Glechomae herba saponin A of the present invention.
FIG. 3 is a high resolution mass spectrum of the triterpene glycoside compound Glechomae herba saponin A of the present invention.
FIG. 4 shows the triterpene glycoside compound Glechomae herba saponin A of the present invention1H-NMR spectrum chart.
FIG. 5 shows the triterpene glycoside compound Glechomae herba saponin A of the present invention13C-NMR spectrum chart.
FIG. 6 is a nuclear magnetic resonance HSQC spectrum of triterpenoid glycoside compound glechoma longituba saponin A of the invention.
FIG. 7 is a nuclear magnetic resonance HMBC spectrum of the triterpenoid glycoside compound glechoma longituba saponin A of the invention.
FIG. 8 shows NMR of triterpenoid glycoside compound Glechomae herba saponin A1H-1HCOSY spectrum.
FIG. 9 is a nuclear magnetic resonance NOESY spectrum of a triterpene glycoside compound Glechomae herba saponin A of the invention.
FIG. 10 is a UV spectrum of the triterpene glycoside compound Glechomae herba saponin B of the present invention.
FIG. 11 is a high resolution mass spectrum of the triterpene glycoside compound Glechomae herba saponin B of the present invention.
FIG. 12 shows the triterpene glycoside compound Glechomae herba saponin B of the present invention1H-NMR spectrum chart.
FIG. 13 shows the triterpene glycoside compound Glechomae herba saponin B of the present invention13C-NMR spectrum chart.
FIG. 14 is a nuclear magnetic resonance HSQC spectrum of triterpenoid glycoside compound glechoma longituba saponin B of the invention.
FIG. 15 is a nuclear magnetic resonance HMBC spectrum of the triterpene glycoside compound Glechomae herba saponin B of the invention.
FIG. 16 is a nuclear magnetic resonance NOESY spectrum of a triterpene glycoside compound Glechomae herba saponin B of the present invention.
Detailed Description
The invention is further illustrated by the following examples. The following examples must be interpreted to illustrate the invention without limiting it. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.
Instruments and reagents
Shimadzu 2010 series high performance liquid chromatograph (Shimadzu corporation, Japan) and active Technologies 1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA), Agilent 1200 type preparative high performance liquid chromatograph, BUCHI Medium pressure preparative liquid chromatograph, UV-260 ultraviolet spectrophotometer (Shimadzu corporation, Japan), Perkin-Elmer 341 polarimeter (PERKIN ELMER, Inc., USA), Waters ACQUITY UPLC/Xevo G2 QTOF Mass spectrometer (Waters, Inc., USA), Varian UNITY INOVA 600 type superconducting nuclear magnetic resonance spectrometer (Varian, Inc., USA), EYESB-1000 rotary evaporator (EYELA, Japan), RY-IG type melting point tester (Tianjin optical instruments, Inc., China), C18The reverse phase packing is produced by YMC, and column chromatography silica gel and thin layer chromatography silica gel are produced by Qingdao ocean chemical factories.
Acetonitrile is chromatographically pure, water is ouaha purified water, and other reagents are analytically pure.
Example 1: extraction and separation of triterpenoid glycoside compound glechoma longituba saponin A, B in glechoma hedracea
Glechomae herba is collected from Huoshan county of Liu Anhui city of Anhui province in 2016 (6 months and 12 days), and identified as whole herb of Glechoma longitaba (Nakai) Kupr of Labiatae by pharmacist in the drug inspection and detection research institute of Jiangxi province. The sample is kept in the sample room for drug examination and detection research (sample No. JXSYJY2016012)
The extraction and separation steps of the triterpenoid glycoside compound glechoma longituba saponin A, B are as follows in sequence:
(1) heating ethanol and refluxing for extraction: drying Glechomae herba in the shade, pulverizing, extracting with 75% ethanol under reflux for 3 times (50.0 Kg), and filtering to obtain 75% ethanol extract;
(2) concentrating 75% ethanol extract: concentrating 75% ethanol extractive solution (EYELA SB-1000 rotary evaporator (product of EYELA, Japan)) under reduced pressure to obtain 10.0Kg ethanol extract, and recovering ethanol during the concentration under reduced pressure;
(3) and (3) extraction: dissolving 75% ethanol extract with distilled water, sequentially extracting with chloroform, ethyl acetate, and water saturated n-butanol for 3 times to obtain n-butanol extractive solution;
(4) concentrating the n-butanol extract: concentrating n-butanol extract (EYELA SB-1000 rotary evaporator (EYELA corporation) under reduced pressure to obtain 520g of n-butanol extract, and recovering n-butanol during the concentration under reduced pressure;
(5) and (3) column chromatography separation: dissolving n-butanol extract with water, transferring to macroporous resin column, and gradient eluting with ethanol-water eluent at volume ratio of 0:100, 30:70, 50:50, 70:30, 100: 0; collecting ethanol-water (30:70) part (EYELA SB-1000 rotary evaporator (product of EYELA corporation) and concentrating eluate, mixing with silica gel, subjecting to column chromatography with silica gel particle size of 100-200 meshes, and gradient eluting with chloroform-methanol eluent at chloroform-methanol volume ratio of 20:1, 10:1, 5:1, 3:1, and 1: 1; subjecting the eluate to thin layer assay, (EYELA SB-1000 rotary evaporator (product of EYELA Co., Japan)) concentration and combination of phases at the same position on the thin layer plateEluting fractions from the same color spot to obtain 10 fractions, 8 th fraction and C18Mixing the sample, and transferring into C18Performing reverse phase column chromatography (BUCHI medium pressure preparative liquid chromatograph), performing gradient elution with methanol-water eluent with volume concentration of 20%, 30%, 40%, 50%, 80%, and performing thin layer chromatography (EYELA SB-1000 rotary evaporator (product of EYELA, Japan)) to concentrate and combine the eluate fractions with spots of the same color at the same position on the thin layer plate to obtain crude product of Glechomae herba saponin A, B.
(6) Purification of monomeric compound: the crude Glechomae herba Saponin A, B is prepared into Glechomae herba Saponin A and Glechomae herba Saponin B by (Shimadzu 2010 series high performance liquid chromatograph (Shimadzu corporation) or Aglient Technologies 1260 high performance liquid chromatograph (Agilent Technologies, Inc. of America)) determining that the ratio of the prepared mobile phase is acetonitrile-water (21:79, v/v), and preparing liquid phase by (Agilent 1200 type high performance liquid chromatograph) with acetonitrile-water (21:79, v/v, 7mL/min) as eluent.
Example 2: structural identification of triterpenoid glycoside compound glechoma longituba saponin A, B
Identifying to obtain two new triterpene glycoside compounds with molecular formulas of C48H78O20The chemical structural formulas of the Glechomae herba saponin A and the Glechomae herba saponin B are respectively as follows:
table 1 shows the nuclear magnetic data (Varian UNITY INOVA 600 superconducting nuclear magnetic resonance apparatus (Varian Corp.) for the two novel triterpene glycoside compounds:1H-NMR of13C-NMR in pyridine-d5In (1).
Table 1: the invention relates to nuclear magnetic data of triterpenoid glycoside compounds, namely glechoma longituba saponin A and glechoma longituba saponin B.
Recorded in pyridine-d5.
Please refer to FIGS. 2-9 for structural identification and derivation of Glechomae herba Saponin A.
Glechomae herba Saponin A: white powder, dissolved in methanol. The melting point of the alloy is 219-221 ℃ measured by an RY-IG type melting point tester (Tianjin optical instruments Co., Ltd., China), and the melting point is measured by a Perkin-Elmer 341 polarimeter (PERKIN ELMER Co., Ltd., U.S.)]20 D26.7(c 0.045, MeOH); UV-260 ultraviolet Spectrophotometer (Shimadzu, Japan) for measuring UV (MeOH) lambdamax(log ε): 205.0(0.67) nm. HRESIMS M/z 973.5031[ M-H ] was determined by a Waters ACQUITY UPLC/Xevo G2 QTOF Mass spectrometer (Waters Co., Ltd., USA)]–，(calcd for C48H77O20,973.5008). Determining the molecular formula as C48H78O20。
1The high field region of the H NMR spectrum shows a single-peak signal delta of 6 triterpenoid characteristic methyl groupsH1.19, 1.06, 0.96, 0.84, 0.83 and 0.77(each 3H, s), a broad singlet vinyl proton signal δ is visible in the high field regionH5.44(1H, br s), 2 vicinal oxymethylene signals deltaH4.28(1H, m) and 4.15(1H, m), one hydroxymethyl signal 3.83(1H, d, J ═ 10.8Hz), 3.67(1H, d, J ═ 10.8 Hz); and 3 sugar-terminal matrix signals deltaH 6.21(1H，d，J＝8.4Hz)，5.70(1H，d，J＝7.8Hz)，5.19(1H，d，J＝7.2Hz)。
13C NMR spectrum showed 48 carbon signals, 18 carbon signals of sugar were removed, and 30 carbons remained while13The C NMR spectrum also gives 2 alkene carbon signals delta 122.6(C-12) and delta 145.2(C-13), which indicate that the compound is oleanane-12-alkene pentacyclic triterpene.
811.4482[ M-H-162 ] on TOF-MS spectrum showing characteristic fragment ion peak M/z]–The peak is the fragment anion peak of 1 six carbon sugar (162) removed; m/z 649.3965[ M-H-162-]–To remove the debris of 2 six-carbon sugars (162)An ion peak; m/z 487.3434[ M-H-162)]–The peak is the fragment anion peak of 3 six carbon sugars (162) removed. Presumably, the compound contains 3 six carbon sugar groups, and the compound is consumed with 5% H2SO4Acid hydrolysis and monosaccharide separation, TLC comparison and specific optical rotation comparison with a standard product prove that the obtained 3 sugars are all D-glucose, and the relative configuration is judged to be beta configuration by the terminal proton coupling constants of the D-glucose respectively being (J ═ 8.4Hz), (J ═ 7.8Hz) and (J ═ 7.2 Hz).
HMBC spectra show, Glc1-1 (delta)H6.21) related to C-28 (delta 177.2), Glc2-1 (delta)H5.70) related to Glc1-2 (delta 78.8), Glc3-1 (delta)H5.19) was associated with Glc2-6 (. delta.71.8), indicating that glucose 1 is linked to the C-28 position of the triterpene core, glucose 2 is linked to the 2 position of glucose 1, and glucose 3 is linked to the 6 position of glucose 2. Mother nucleus deltaH 1.94、δH 1.81(δC43.3) and δC 17.7(C-25)、δC 38.8、δC 79.7、δC66.8 and deltaC43.8 correlation, δH 4.15(δC79.7) and δC18.2、δC 42.2、δC66.8 correlation, δH 0.77(δC18.2) and δC 79.7、δC71.8、δC42.2、δC43.8 correlation, knowing δC43.3 is C-1, deltaC66.8 is C-2, deltaC79.7 is C-3, deltaC42.2 is C-4, deltaC43.8 is C-5, deltaC71.8 is C-23.
NOESY spectra show that H-2/H-24, H-2/H-25 and H-3/H-24 are respectively related, which indicates that hydroxyl groups at positions 2 and 3 are alpha configuration and methyl group at position 24 is beta configuration. Based on the above information, it can be determined that the novel triterpene glycoside has the structure described above.
Please refer to FIGS. 10-16 for structural identification and derivation of Glechomae herba Saponin B.
Glechomae herba Saponin B: white powder, dissolved in methanol. The melting point of the alloy is 216-217 ℃ by an RY-IG type melting point tester (Tianjin optical instruments Co., Ltd., China), and the melting point is determined by a Perkin-Elmer 341 polarimeter (PERKIN ELMER Co., Ltd., U.S.)]20 D54.5(c 0.077, MeOH); UV-260 ultraviolet spectrophotometer(Shimadzu, Japan) measurement of UV (MeOH). lambda.max(log ε): 205.5(0.90) nm. HRESIMS M/z 973.4966[ M-H ] was determined by a Waters ACQUITY UPLC/Xevo G2 QTOF Mass spectrometer (Waters Co., Ltd., USA)]–，(calcd for C48H77O20,973.5008). Determining the molecular formula as C48H78O20。
1The high field region of the H NMR spectrum shows 6 triterpenoid characteristic methyl signals, of which 2 are split methyl signals deltaH0.94(3H, d, J ═ 6.0Hz), 0.83(3H, d, J ═ 6.0Hz) and 4 methyl unimodal signals δH1.11, 1.10, 0.96 and 0.77(each 3H, s), a broad singlet vinyl proton signal δ is visible in the high field regionH5.44(1H, br s), 2 vicinal oxymethylene signals deltaH4.28(1H, m) and 4.15(1H, m), one hydroxymethyl signal 3.84(1H, d, J ═ 10.8Hz), 3.68(1H, d, J ═ 10.8 Hz); and 3 sugar-terminal matrix signals deltaH 6.16(1H，d，J＝8.4Hz)，5.64(1H，d，J＝7.8Hz)，5.16(1H，d，J＝7.8Hz)。
13C NMR spectrum showed 48 carbon signals, 18 carbon signals of sugar were removed, and 30 carbons remained while13The C NMR spectrum also gives 2 alkene carbon signals delta 126.1(C-12), delta 139.4(C-13), which indicates that the compound is ursane-12-alkene pentacyclic triterpene.
811.4482[ M-H-162 ] on TOF-MS spectrum showing characteristic fragment ion peak M/z]–The peak is the fragment anion peak of 1 six carbon sugar (162) removed; m/z 649.3963[ M-H-162-]–The peak is the fragment anion peak of 2 six-carbon sugars (162) removed; m/z 487.3434[ M-H-162)]–The peak is the fragment anion peak of 3 six carbon sugars (162) removed. Presumably, the compound contains 3 six carbon sugar groups, and the compound is consumed with 5% H2SO4Acid hydrolysis and monosaccharide separation, TLC comparison and specific optical rotation comparison with a standard product prove that the obtained 3 sugars are all D-glucose, and the relative configuration is judged to be beta configuration by the terminal proton coupling constants of the D-glucose respectively being (J ═ 8.4Hz), (J ═ 7.8Hz) and (J ═ 7.8 Hz).
HMBC spectra show, Glc1-1 (delta)H6.16) withC-28 (delta 176.9) -related, Glc2-1 (delta)H5.64) associated with Glc1-2(Δ 79.3), Glc3-1(Δ 79.3)H5.16) was associated with Glc2-6 (. delta.71.8), indicating that glucose 1 is linked to the C-28 position of the triterpene core, glucose 2 is linked to the 2 position of glucose 1, and glucose 3 is linked to the 6 position of glucose 2. Mother nucleus deltaH 1.93、δH 1.80(δC43.3) and δC 17.7(C-25)、δC 38.8、δC 79.7、δC66.8 and deltaC43.8 correlation, δH4.15 and deltaC 18.2、δC42.2、δC71.8、δC66.8 correlation, δH 0.77(δC18.2) and δC 79.7、δC71.8、δC42.2、δC43.8 correlation, knowing δC43.3 is C-1, deltaC66.8 is C-2, deltaC79.7 is C-3, deltaC42.2 is C-4, deltaC43.8 is C-5, deltaC71.8 is C-23.
NOESY spectra show that H-2/H-24, H-2/H-25 and H-3/H-24 are respectively related, which indicates that hydroxyl groups at positions 2 and 3 are alpha configuration and methyl group at position 24 is beta configuration. Based on the above information, it can be determined that the novel triterpene glycoside has the structure described above.
Example 3: in vitro anti-tumor activity test of triterpenoid glycoside compound glechoma longituba saponin A, B
Tumor cell growth inhibition (%) was (test well assay/control well assay) × 100%
The test principle is as follows: MTT method: in mitochondria of living cells, dehydrogenase related to NAPP (nicotinamide adenine dinucleotide phosphate, coenzyme II) exists, and succinate dehydrogenase can reduce exogenous yellow thiazole blue MTT (3- (4,5) -dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit in cells, and the enzyme disappears in dead cells, and MTT is not reduced. After formazan was dissolved in dimethyl sulfoxide (DMSO), absorbance was measured at 570nm and 630nm using a microplate reader, and the optical density was proportional to the number of living cells.
The cell lines used were: BGC-823 (human gastric cancer cells), Bel (human hepatoma cells), HCT-8 (human colon cancer cells), A2780 (human ovarian cancer cells), and A549 (human lung cancer cells) (all purchased from ATCC).
The test method comprises the following steps: MTT method: taking logarithmic growth cells, digesting, fully blowing and beating into single cell suspension, counting and diluting into 1 × 104cells/mL, seeded in 96-well plates, 100. mu.L of cell suspension per well, placed at 37 ℃/5% CO2After culturing in a saturated humidity incubator for 24 hours, the stock culture solution was discarded. Designing 6 concentration gradients for each sample, and then adding 100 μ L of the culture medium containing the above-prepared compound and paclitaxel (positive control) for each concentration gradient to the test wells, each concentration being in parallel with 6 wells; the control group was added with an equal volume of solvent. Placing 96-well culture plate at 37 deg.C/5% CO2After culturing for 72 hours in a saturated humidity incubator, 20. mu.L of a freshly prepared serum-free medium containing 5mg/mL MTT was added to each well, and after further culturing for 4 hours at 37 ℃, the supernatant was removed. Add 150. mu.L of LDMSO to each well to dissolve the Formazan precipitate, and shake the mixture on a micro-shaker for 5 minutes to fully dissolve the Formazan precipitate. The absorbance at 570nm and 630nm is measured on a microplate reader, and the number of living cells can be reflected. Inhibitory Concentration (IC) of drug was calculated by SPSS software50) The value is obtained.
TABLE 2 inhibition of BGC-823 (human gastric carcinoma cells) by two novel compounds
TABLE 3 inhibition of Bel (human hepatoma cells) by two novel compounds
| Sample (I) | Concentration (μ g/ml) | Inhibition ratio (%) | IC50(μg/ml) |
| Taxol (positive) | 1 | 78 | 0.05 |
| 0.5 | 80 | ||
| 0.1 | 76 | ||
| 0.05 | 42 | ||
| 0.01 | 23 | ||
| 0.001 | 12 | ||
| Glechomae |
100 | 92 | 8.171 |
| 50 | 90 | ||
| 25 | 83 | ||
| 12.5 | 60 | ||
| 6.25 | 42 | ||
| 3.12 | 31 | ||
| 1.56 | 12 | ||
| 0.78 | 1 | ||
| 0.39 | 2 | ||
| Glechomae |
100 | 82 | 10.93 |
| 50 | 80 | ||
| 25 | 70 | ||
| 12.5 | 62 | ||
| 6.25 | 50 | ||
| 3.12 | 20 | ||
| 1.56 | 10 | ||
| 0.78 | 1 | ||
| 0.39 | 1 |
TABLE 4 inhibitory Effect of two novel Compounds on HCT-8 (human colon cancer cells)
TABLE 5 inhibitory Effect of two novel Compounds on A2780 (human ovarian carcinoma cells)
TABLE 6 inhibitory Effect of two novel Compounds on A549 (human Lung cancer cell)
And (4) conclusion: glechomae herba Saponin A, B has obvious inhibitory effect on human BGC-823 (human gastric cancer cell), Bel (human liver cancer cell), HCT-8 (human colon cancer cell), A2780 (human ovarian cancer cell) and A549 (human lung cancer cell), and can be used as medicines for treating or researching cancer or tumor.
Claims (6)
1. A compound of formula (I) or formula (II):
2. a pharmaceutical composition comprising as an active ingredient a compound of formula (I) and/or formula (II) according to claim 1 and a pharmaceutically acceptable carrier or excipient.
3. A process for the preparation of a compound of formula (I) and/or formula (II) as defined in claim 1, comprising the steps of:
(1) heating reflux extraction of Glechomae herba with 70-95 vol% ethanol water solution, and filtering to obtain ethanol extractive solution;
(2) concentrating the ethanol extract to obtain an ethanol extract;
(3) dissolving the ethanol extract with water, sequentially extracting with chloroform, ethyl acetate and water-saturated n-butanol, and retaining water-saturated n-butanol extract phase to obtain water-saturated n-butanol extract;
(4) concentrating the water saturated n-butanol extract to obtain n-butanol extract;
(5) performing column chromatography separation on the n-butanol extract to obtain crude products of the compounds of the formula (I) and the formula (II), wherein the column chromatography separation comprises the following steps:
dissolving n-butanol extract with water, transferring to macroporous resin column, and gradient eluting with ethanol-water eluent at volume ratio of 0:100, 30:70, 50:50, 70:30, 100: 0; collecting ethanol-water (30:70), concentrating the eluate, mixing with silica gel, performing column chromatography with silica gel particle size of 100-200 meshes, performing gradient elution with chloroform-methanol eluate at a volume ratio of chloroform-methanol of 20:1, 10:1, 5:1, 3:1, 1: 1; performing thin layer inspection on the eluate, concentrating and mixing eluate fractions with spots having the same color at the same position on the thin layer plate to obtain 10 fractions, and mixing 8 th fraction with fraction C18Mixing the sample, and transferring into C18Performing reverse phase column chromatography, gradient eluting with methanol-water eluent with methanol volume concentration of 20%, 30%, 40%, 50%, 80%, performing thin layer inspection on the eluate, concentrating and mixing eluate fractions with spots having the same color at the same position on the thin layer plate to obtain fractions of formula (I) and formula (II)Crude product of the compound;
(6) optionally subjecting the crude product to purification of monomeric compounds, said purification comprising: the monomer compound is purified by preparative liquid chromatography using acetonitrile-water as an eluent.
4. The method according to claim 3, wherein the concentration in steps (2), (4) is concentration under reduced pressure.
5. Use of a compound of formula (I) and/or formula (II) as defined in claim 1 in the manufacture of a medicament for the treatment of cancer.
6. The use of claim 5, wherein the cancer is gastric or liver or colon or ovarian or lung cancer.
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