CN109705188B - Triterpenoid compound in exocarpium Juglandis Immaturum, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a triterpene compound, a preparation method and application thereof, belonging to a new compound and medicinal application thereof. Characterized in that the organic solvent is used for extracting the combined riceA new triterpene compound is prepared from fresh pericarp of exocarpium Juglandis Immaturus by treating with rad reagent, and separating and purifying by column chromatographyα-cyclopentanone-21, 22-combined 3, 10-epoxy-5-hydroxy-oleanane-19, 22-bicyclo [2, 2, 0 ]]Hexane, named cyclonortriterpenone a. Experiments prove that the compound has better effect of inhibiting the growth of tumor cells and has an application prospect in the aspect of tumor resistance.

Description

Triterpenoid compound in exocarpium Juglandis Immaturum, and preparation method and application thereof

Technical Field

The invention particularly relates to a novel triterpenoid with a cancer cell inhibiting effect.

Background

Malignant tumor is one of three diseases harmful to human health, and has high morbidity and mortality. Modern western medicine treatment mainly adopts chemotherapy, has large toxic and side effects, and obviously reduces the life cycle quality of patients. The traditional Chinese medicine has unique advantages in the aspects of resisting and preventing cancers, reducing adverse reactions and the like, and is a treasure house for researching and developing novel anti-tumor medicines. At present, the anticancer preparation derived from Chinese herbal medicines accounts for more than 30 percent of the total anticancer medicines, such as taxus, camptothecin, vincristine and the like, which are taken as the first choice medicines for resisting tumors.

The pericarpium Juglandis Immaturus is Juglans mandshurica Maxim of Juglandaceae family, genus JuglansJuglands mandshuricaMaxim, and walnutJ.ragiaThe epicarp of immature fruit has wide application base in China folk, and is often used for acute and chronic stomachache, borborborygmus and diarrhea, mange and the like. Modern pharmacological research shows that it has the functions of resisting inflammation, relieving pain, resisting bacteria and resisting tumor, etc., and its anti-tumor effect is obvious. The pericarpium juglandis has been reported to have good curative effects on various malignant tumors such as gastric cancer, lung cancer, liver cancer, cervical cancer and the like, but the naphthoquinone which is the main component type discovered at present has significant effect on inhibiting tumor growth, but has high toxicity and poor stability, and has great potential safety hazard when being used as a clinical medicament. In order to fully utilize the medicinal resources, the research team continuously carries out deep and systematic chemical component research, and aims to screen out a high-efficiency, low-toxicity and stable medicinal effect substance basis. In the process of separating the component of the clothes of the Juglans regia, a novel triterpenoid ketone compound is obtained, and the chemical structure, the pharmacological activity and the application of the compound are clarified.

Disclosure of Invention

The invention aims to provide a novel triterpenoid compound, a preparation method thereof and application of the novel triterpenoid compound in preparing an anti-tumor medicament, so that the defects of high toxicity and poor stability of main anti-tumor active ingredients in the existing pericarpium juglandis are overcome, and the source of the medicament for treating tumors is expanded.

The new triterpenoid is extracted, separated and purified from pericarpium juglandis and has a chemical name ofα-cyclopentanone-21, 22-combined 3, 10-epoxy-5-hydroxy-oleanane-19, 22-bicyclo [2, 2, 0 ]]Hexane, trivial name cyclonorubine A.

The chemical structural formula is as follows:

the invention also provides a preparation method of the Qinglongyi triterpenoid ketone A, which comprises the following steps: the walnut green seedcase is used as a raw material and is prepared by sequentially carrying out alcohol extraction, macroporous resin enrichment, Girard reagent purification treatment, column chromatography separation and the like.

The column chromatography comprises a macroporous resin column, a normal phase silica gel column and a sephadex column in sequence.

The preparation method of the exocarpium Juglandis Immaturum triterpenoid ketone A comprises the following steps:

(1) alcohol extraction: cortex Juglandis MandshuricaeJuglans mandshuricaDrying fresh Maxim Chinese olive peel at low temperature of 40 deg.C to obtain 5kg of dried product as raw material, extracting with 95% ethanol 30L under reflux for 3 times (2 hr each time), filtering, mixing with ethanol extractive solution, recovering solvent under reduced pressure, and drying to obtain ethanol extract;

(2) enrichment and purification: dispersing the extract obtained in the step (1) with water to a solution with the relative density of 1.18 +/-0.05 g/mL, carrying out enrichment and purification by an AB-8 or D101 type macroporous resin column chromatography, sequentially eluting with water, 50% ethanol and 80% ethanol respectively, collecting 80% ethanol eluent, recovering ethanol under reduced pressure, and adjusting to an aqueous solution with the density of 1.05 g/mL. Repeatedly passing the water solution through an active carbon layer (layer height is 10cm, diameter is 30 cm) for 5 times, carrying out vacuum filtration to remove impurities, concentrating the water solution, evaporating to dryness, adding absolute ethanol, Girard reagent P and 12% (V/V) acetic acid respectively for kneading and dissolving, heating in a water bath at 50 ℃ for 10h, adding water for dilution after the reaction is finished, and adding the same volume of diethyl ether for extraction for three times to obtain diethyl ether extract;

(3) normal phase silica gel column chromatography: carrying out normal phase silica gel column chromatographic separation on the ether extract obtained in the step (2), sequentially carrying out gradient elution by using a dichloromethane-methanol mixed solvent with volume ratios of 50:1, 25:1 and 15:1, eluting 3 column volumes in each ratio, and carrying out reduced pressure recovery on an eluted part of the dichloromethane-methanol mixed solvent with the volume ratio of 15:1 to obtain a separation product;

(4) and (3) separating the glucan gel column: and (3) taking the product separated in the step (3), passing through a sephadex column, and mixing the product with methanol: water =1:2 (V/V) elution 3 column volumes were discarded, followed by methanol: eluting 4 column volumes with water =1:1 (V/V), discarding the eluent of the first column volume, collecting the eluent of the next three column volumes, and recovering the solvent to obtain a crude product;

(5) and (3) recrystallization treatment: and (4) recrystallizing the crude product obtained in the step (4) twice by adopting methanol to obtain a pure product of the compound.

The invention also provides the application of the cyclocarya paliurus triterpenoid ketone A in the preparation of antitumor drugs. Can inhibit the proliferation of human gastric cancer cell BGC-823, human hepatoma cell HepG-2 and human lung cancer cell A549 in vitro cytotoxicity experiments, has cytotoxic effect, and can be used for preparing medicines for preventing and treating human gastric cancer, hepatocarcinoma and lung cancer.

The invention has the beneficial effects and meanings that: by combining with the Gilard reagent pretreatment technology, the carbonyl substance in the pericarpium juglandis medicinal material can be efficiently purified and enriched, and the application limit of low yield caused by large interference of other impurities on the components in the traditional solvent extraction process is overcome; in addition, the invention finds the triterpenoid with special structural skeleton characteristics and also provides an important medicinal material source for further antitumor clinical research.

Drawings

FIG. 1 is a chemical structural formula of a compound of the present invention;

FIG. 2 is a positive HR-ESI-MS spectrum of a compound of the present invention;

FIG. 3 is a drawing of a compound of the present invention¹H-NMR spectrum;

FIG. 4 is a drawing of a compound of the present invention¹³A C-NMR spectrum;

FIG. 5 is a DEPT spectrum of a compound of the present invention;

FIG. 6 is an HSQC spectrum of a compound of the present invention;

FIG. 7 is an HMBC spectrum of a compound of the present invention;

FIG. 8 is a drawing of a compound of the present invention¹H-¹H COSY spectrogram;

FIG. 9 shows HMBC spectra and¹H-¹h COSY spectrum main correlation diagram;

FIG. 10 is an IC of compounds of the invention with cisplatin on three different types of cancer cells₅₀And (6) comparing the values.

Detailed Description

Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the present invention, and the following embodiments are provided by way of example only. Various modifications and improvements can be made to the present invention without departing from the spirit and scope of the invention. Such variations are intended to be within the scope of the present invention. The present invention will be described in detail with reference to specific examples.

Example a process for the preparation of the compounds of the invention:

(1) alcohol extraction: cortex Juglandis MandshuricaeJuglans mandshuricaDrying fresh Maxim Chinese olive peel at low temperature of 40 deg.C to obtain 5kg of dried product as raw material, extracting with 95% ethanol 30L under reflux for 3 times (2 hr each time), filtering, mixing with ethanol extractive solution, recovering solvent under reduced pressure, and drying to obtain 328g of ethanol extract;

(2) enrichment and purification: dispersing the extract obtained in the step (1) with water to a solution with a relative density of 1.18 +/-0.05 g/mL, carrying out enrichment and purification by an AB-8 or D101 type macroporous resin column chromatography (the specification of a chromatographic column is that the inner diameter is multiplied by the length =6.5cm multiplied by 1.5m, and the effective height of the resin is 1.0 m), sequentially eluting with water, 50% ethanol and 80% ethanol respectively, collecting 80% ethanol eluent, recovering ethanol under reduced pressure, adjusting to a water solution with a density of 1.05g/mL, repeatedly passing the water solution through an active carbon layer (the layer height is 10cm, and the diameter is 30 cm) for 5 times to carry out vacuum filtration and impurity removal, concentrating and evaporating the water solution to dryness, respectively adding absolute ethanol, Girard reagent P and 12% (V/V) acetic acid to carry out kneading and dissolving, then heating in a water bath at 50 ℃ for 10 hours, adding water for dilution after the reaction is finished, adding ether with the same volume, and extracting for three times to;

(3) normal phase silica gel column chromatography: carrying out normal phase silica gel column chromatographic separation on the ether extract obtained in the step (2) (the specification of a chromatographic column is that the inner diameter is multiplied by the length =3.0cm multiplied by 1.5m, and the effective height of silica gel is 1.0 m), sequentially carrying out system gradient elution by adopting dichloromethane-methanol (50: 1, V/V, 3 column volume) → dichloromethane-methanol (25: 1, V/V, 3 column volume) → dichloromethane-methanol (15: 1, V/V, 3 column volume), decompressing and recovering a solvent from an elution part of a mixed solvent with a dichloromethane-methanol volume ratio of 15:1 until the mixed solvent is dry, weighing, and obtaining 1.26g of a separation product;

(4) and (3) separating the glucan gel column: taking the product separated by the silica gel in the step (3), passing through a sephadex column (the specification of the chromatographic column is that the inner diameter is multiplied by the length =1.5cm multiplied by 1.8m, and the effective height of the gel is 1.2 m), and mixing the product with methanol: water =1:2 (V/V) elution 3 column volumes were discarded, followed by methanol: eluting 4 column volumes with water =1:1 (V/V), discarding the eluent of the first column volume, collecting the eluents of the latter three column volumes, recovering solvent to dryness to obtain crude product 0.06 g;

(5) and (3) recrystallization treatment: recrystallizing the crude product obtained in the step (4) twice by using 30mL of methanol to obtain 35.5mg of the pure product of the compound.

Example two structural characterization of the compounds of the invention:

the compound of the invention is a white amorphous powder (MeOH). Positive HR-ESI-MS spectrum, as shown in FIG. 2, inm/z494.3498 see [ M]⁺Ion peak, indicating that the molecular weight of the compound is 494. Bonding of¹H-NMR、¹³C-NMR and DEPT spectra and the like presume that the molecular formula is C₃₃H₅₀O₃The unsaturation was calculated to be 9.

In the presence of the compound¹H-NMR (Methanol-d ₄400MHz) spectrum, as shown in FIG. 1, showing 8 methyl protons eachδ _H0.92 (H-26), 0.96 (H-26), 1.04 (H-30), 1.05 (H-27), 1.07 (H-29), 1.12 (H-24), 1.15 (H-28) and 1.15 (H-23), and a methine proton signal linked to oxygen is shown inδ _H3.67 (H-3)。¹³C-NMR (Methanol-d ₄100MHz) spectrum combined with DEPT spectrum, as shown in fig. 4 and 5, shows 33 carbon signals including 8 methyl groups, 10 methylene groups, 5 methine groups, 10 quaternary carbons (including 2 oxygen-containing quaternary carbons and 1 carbonyl group).

In the HMBC spectra of this compound, as shown in FIG. 7, it is clearly observed that the 3-, 24-and 25-positions constitute³ JThe long-range correlation of (A) shows that the hydroxyl group is at the 5-position and that C-5 and C-4, C-6, C-10 are in substantial agreement with the spectroscopic data of the compound klodorol A isolated from Kleinia odora (Tori M, Matsuda R, Sono M, Asakawa Y. 1988).¹³CNMR assignment of dammarane triterpenes and dendropanoxide:application of 2Dlong-range¹³C–^lH correlation spectrum, Magn Reson chem.26: 581- & 590, Estrada S, Acevedo L, Rodriguez M, Toscano R, Mata R.2002, New terpenoids from the organisms Scahyglottis livida and Nidema bothii. Nat Prod Res.16: 81-86), indicating that the hydroxyl group isαConfiguration. The compound of the invention has only partial chemical structure similarity with the known compound klodorol A, and the E and F rings of the compound and the known compound have obvious difference. In HMBC spectra, it was observed that C-19, which is related to H-18, H-29, H-30, and C-22, which is related to H-21, H-28, H-32, H-33, are changed in type from the original secondary carbon to the tertiary carbon and the quaternary carbon, respectively, and thus it is presumed that a C-C single bond exists between C-19 and C-22. At the same time¹H-¹In the H COSY spectrum, 33 bits are observed as shown in FIG. 8δ _H1.94 and 32 bitsδ _H2.48 correlation, indicating the presence of an ethyl moiety at positions 32, 33 and a carbonyl groupδ _C220.1 is remotely related to the presence of the above fragment, presumably the presence of-CH for this compound₂-CH₂-C = O structure and forms a five-membered F ring with positions 21, 22, see fig. 9 in particular. In conclusion, the chemical structure of the compounds of the present invention was determined asα-cyclopentanone-21, 22-combined 3, 10-epoxy-5-hydroxy-oleanane-19, 22-bicyclo [2, 2, 0 ]]Hexane, i.e. the triterpene ketone A of the exocarpium Juglandis Immaturum, the chemical structural formula of which is shown in figure 1.

TABLE 1 NMR Signal assignment of Compounds of the invention

Effect example-inhibition of tumor cell proliferation in vitro

(1) Materials and methods

1) Cell line and reagent human gastric cancer cell line BGC-823, human hepatoma cell line HepG-2, and human lung cancer cell line A549, which were purchased from Seal reagent company. The tumor cells were cultured in RPMI1640 medium containing 10% L-glutamine fetal bovine serum (100. mu.g/mL penicillin, 100. mu.g/mL streptomycin), and then incubated at 37 ℃ with 5% saturated humidity CO₂Culturing in an incubator. The compounds of the invention are obtained from the preparation of example one.

2) Cell proliferation analysis BGC-823, HepG-2 and A549 cells in a logarithmic growth phase are taken and inoculated into a 96-well plate according to different cell strains and a certain density, each well is 100 mu L of cell suspension, drugs with different concentrations (a sample is dissolved in DMSO, the DMSO is gradually diluted by a culture medium, the final concentration of the drug solution added into the cells is lower than 1%) are immediately added after the cells are inoculated, so that the final concentration of the cell solution reaches 5, 10, 20, 40, 80 and 160 mu M, a blank control group is 100 mu L of RPMI1640, 10 mu L of a drug-free solvent is added as a control group, and each group is provided with 3 multiple wells; after 48 h incubation, 20. mu.L of MTT (methanol/methanol) was added at 5mg/mL per well, incubation was continued for 4 h at 37 ℃ and then centrifuged (2000 rpm, 10 min), the supernatant was carefully aspirated, 150. mu.L of DMSO was added per well and mixed well, OD value per well was measured at 570nm for each group, and the cell growth inhibition rate was calculated using wells containing only DMSO as zeroing wells. Cell growth inhibition% = [1- (drug treatment well mean OD value-zero adjustment well OD value)/(cell control well mean OD value-zero adjustment well OD value)]X 100%. Calculation of IC of drugs using the Logit method₅₀The value is obtained.

(2) Results

Calculation of IC by Linear regression₅₀The value shows that the compound has the effect IC on human gastric cancer BGC-823 cells, human hepatoma cells HepG-2 and human lung cancer cells A549 cells₅₀The values were 52.96. + -. 4.22. mu.M, 32.15. + -. 3.19. mu.M and 22.95. + -. 2.19. mu.M, respectively, with cisplatin as the positive pairIC effect of medicine on human gastric cancer BGC-823 cell, human hepatoma cell HepG-2 and human lung cancer cell A549 cell₅₀The values were 6.23. + -. 1.08. mu.M, 4.88. + -. 0.59. mu.M and 5.12. + -. 2.02. mu.M, respectively, as shown in FIG. 10.

The results show that the compound has stronger inhibition effect on the growth of human gastric cancer BGC-823 cells, human hepatoma cells HepG-2 and human lung cancer cells A549 cells, and has the prospect of preparing clinical tumor prevention and treatment medicines.

Claims (3)

1. A triterpene compound is characterized by the chemical name:α-cyclopentanone-21, 22-combined 3, 10-epoxy-5-hydroxy-oleanolic acid-19, 22-bicyclo [2, 2, 0 ]]-hexane, named cycloartane A, having the chemical formula:

。

2. a process for the preparation of the compound of the extract of claim 1, characterized in that: the compound is obtained by taking walnut green seedcase as a raw material, performing alcohol extraction → AB-8 or D101 macroporous resin enrichment → Gilard reagent purification treatment → ether extraction, and performing normal phase silica gel column chromatography, Sephadex LH-20 column chromatography separation and recrystallization on the extract, wherein the specific preparation steps are as follows:

(1) alcohol extraction: cortex Juglandis MandshuricaeJuglansmandshuricaDrying fresh Maxim Chinese olive peel at low temperature of 40 deg.C to obtain 5kg of dried product as raw material, extracting with 95% ethanol 30L under reflux for 3 times (2 hr each time), filtering, mixing with ethanol extractive solution, recovering solvent under reduced pressure, and drying to obtain ethanol extract;

(2) enrichment and purification: dispersing the extract obtained in the step (1) with water to a solution with the relative density of 1.18 +/-0.05 g/mL, carrying out enrichment and purification by an AB-8 or D101 type macroporous resin column chromatography, sequentially eluting with water, 50% ethanol and 80% ethanol respectively, collecting 80% ethanol eluent, recovering ethanol under reduced pressure, and adjusting to an aqueous solution with the density of 1.05 g/mL; repeatedly passing the water solution through the activated carbon layer for 5 times, wherein the activated carbon layer has a height of 10cm and a diameter of 30cm, performing vacuum filtration to remove impurities, concentrating the water solution, evaporating to dryness, adding absolute ethanol, Girard reagent P and acetic acid with a volume ratio concentration of 12% respectively, kneading and dissolving, heating in a water bath at 50 ℃ for 10h, adding water for dilution after the reaction is finished, and adding diethyl ether with the same volume for extraction for three times to obtain diethyl ether extract;

(3) normal phase silica gel column chromatography: carrying out normal phase silica gel column chromatographic separation on the ether extract obtained in the step (2), sequentially carrying out gradient elution by using dichloromethane-methanol mixed solvent with volume ratios of 50:1, 25:1 and 15:1, eluting 3 column volumes in each ratio, and recovering the solvent from an elution part of the dichloromethane-methanol mixed solvent with the volume ratio of 15:1 under reduced pressure to obtain a separation product;

(4) and (3) separating the glucan gel column: taking the product separated in the step (3), passing through a sephadex column, eluting 3 column volumes with a methanol-water mixed solvent with a volume ratio of 1:2, discarding, eluting 4 column volumes with a methanol-water mixed solvent with a volume ratio of 1:1, discarding the eluent with the 1 st column volume, collecting the eluent with the following 3 column volumes, and recovering the solvent to obtain a crude product;

(5) and (3) recrystallization treatment: and (4) recrystallizing the crude product obtained in the step (4) twice by adopting methanol to obtain a pure product of the compound.

3. Use of the compound of the present claim 1, namely the cycloartane A, in the preparation of antitumor drugs.

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