A kind of oleanane type triterpenoid compound and its preparation method and application

Abstract

The invention discloses an oleanane-type triterpenoid compound and a preparation method and application thereof, belonging to the technical field of triterpenoid compound preparation. The oleanane-type triterpenoid compound of the invention has the following structural formula: The oleanane-type triterpenoid compound of the invention is extracted and prepared from northeastern tripterygium wilfordii, has inhibitory activity on the release of NO in macrophage RAW264.7, and can be used to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of triterpenoid compounds, and in particular to an oleanane-type triterpenoid compound and a preparation method and application thereof. Background Art

[0002] Northeast Tripterygium wilfordii is a plant of the genus Tripterygium in the family Celastraceae. The dried roots and rhizomes of Northeast Tripterygium wilfordii are mainly distributed in Northeast my country. Northeast Tripterygium wilfordii has the effects of dispelling wind and dampness, promoting diuresis and reducing swelling, killing insects and detoxifying. It is commonly used by the people to treat rheumatoid arthritis, swelling and edema, jaundice, accumulation of mass, traumatic injury, sore swelling and pain, tinea capitis, skin itching and snake bites. However, the prior art has little research on the anti-inflammatory active ingredients of Northeast Tripterygium wilfordii. Summary of the invention

[0003] The purpose of the present invention is to provide an oleanane-type triterpenoid compound and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is: an oleanane-type triterpenoid compound, the structural formula of which is shown in formula (1):

[0006]

[0007] The second technical solution of the present invention is a method for preparing the above-mentioned oleanane-type triterpenoid compound, comprising the following steps:

[0008] The northeastern tripterygium wilfordii and ethanol solution are mixed and then heated under reflux for extraction, the extract is extracted with ethyl acetate, and the extract is purified to obtain the oleanane-type triterpenoid compound.

[0009] Furthermore, the concentration of the ethanol solution is 70 vol. %; the number of times of heating reflux extraction is 3 times, the extraction temperature is 70-80° C., and the time of each extraction is 1.5 h.

[0010] Furthermore, the purification method comprises the following steps:

[0011] The extract was subjected to alumina column chromatography, and gradient elution was performed using a mixed solution of ethyl acetate and methanol, and the eluate with a volume ratio of ethyl acetate to methanol of 40:1 was collected to obtain an elution fraction A;

[0012] The eluted fraction A was subjected to silica gel column chromatography, and gradient elution was performed using a mixed solution of dichloromethane and methanol, and an eluate having a volume ratio of dichloromethane to methanol of 50:1 was collected to obtain an eluted fraction B;

[0013] The eluted fraction B was subjected to silica gel column chromatography, and a mixed solution of petroleum ether and ethyl acetate was used for gradient elution, and the eluate with a volume ratio of petroleum ether to ethyl acetate of 1:100 was collected to obtain an eluted fraction C;

[0014] Eluted fraction C was subjected to reverse phase semi-preparative C 18 HPLC column separation to obtain the oleanane-type triterpenoid compound; the reverse phase semi-preparative C 18 The eluent used for HPLC separation is a mixed solution of methanol and water in a volume ratio of 9:1, and the mixed solution contains 0.03 vol.% trifluoroacetic acid.

[0015] When the methanol content in the mixed solution of methanol and water is greater than 9:1 (the volume ratio of methanol to water is 9:1), the purity of the extracted target compound will decrease. When the methanol content in the mixed solution of methanol and water is less than 9:1 (the volume ratio of methanol to water is 9:1), the target compound will not be eluted.

[0016] Furthermore, when the extract was subjected to alumina column chromatography, a mixed solution of ethyl acetate and methanol in a volume ratio of 100:1 and 40:1 was used for gradient elution.

[0017] Furthermore, when the eluted fraction A was subjected to silica gel column chromatography, a mixed solution of dichloromethane and methanol in a volume ratio of 100:1 and 50:1 was used for gradient elution.

[0018] Furthermore, when the eluted fraction B was subjected to silica gel column chromatography, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 100:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 1:100 was used for gradient elution.

[0019] Further, the eluted fraction C was subjected to reverse phase semi-preparative C 18 The eluent flow rate during HPLC separation was 3.0 mL / min.

[0020] The third technical solution of the present invention: an application of the above-mentioned oleanane-type triterpenoid compound in the preparation of anti-inflammatory drugs.

[0021] Furthermore, the anti-inflammatory drug includes an active drug that inhibits the release of NO from macrophages.

[0022] The present invention discloses the following technical effects:

[0023] (1) The oleanane-type triterpenoid compounds of the present invention have a significant inhibitory effect on the production of NO by macrophages RAW264.7.

[0024] (2) The oleanane-type triterpenoid compound of the present invention is extracted and prepared from Tripterygium wilfordii, has inhibitory activity on NO release from macrophage RAW264.7 cells, and can be used to prepare anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The HRESIMS spectrum of compound 1 prepared in Example 1 of the present invention;

[0027] Figure 2 The key to compound 1 prepared in Example 1 of the present invention is 1 H- 1 Schematic diagram of H COSY and HMBC correlation;

[0028] Figure 3 Compound 1 prepared in Example 1 of the present invention 1 H NMR spectrum;

[0029] Figure 4 Compound 1 prepared in Example 1 of the present invention 13 C NMR spectrum;

[0030] Figure 5 Compound 1 prepared in Example 1 of the present invention 1 H- 1 H COSY spectrum;

[0031] Figure 6 HSQC spectrum of compound 1 prepared in Example 1 of the present invention;

[0032] Figure 7 The HMBC spectrum of compound 1 prepared in Example 1 of the present invention;

[0033] Figure 8 The NOESY spectrum of compound 1 prepared in Example 1 of the present invention;

[0034] Fig. 9 NO2 - Standard curve. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] A method for preparing oleanane-type triterpenoid compounds:

[0042] (1) The fresh root tuber of Tripterygium wilfordii was sliced, and extracted three times with 70% ethanol by heating and refluxing (the mass / volume ratio of Tripterygium wilfordii to ethanol was 1:5, the extraction temperature was 70-80°C, and the extraction time for each time was 1.5 h). The extracts were combined, concentrated under reduced pressure, and the ethanol was recovered to obtain a Tripterygium wilfordii extract; the Tripterygium wilfordii extract was dispersed with water, and extracted with petroleum ether, ethyl acetate, and n-butanol for multiple times with equal volumes (each time, the substances in the extract were extracted), and concentrated under reduced pressure to obtain a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract.

[0043] (2) The ethyl acetate extract was subjected to alumina column chromatography, and gradient elution was performed using a mixed solution of ethyl acetate and methanol in volume ratios of 100:1, 40:1, 1:1, and 1:100, respectively. Four elution fractions were collected for each gradient, and the four elution fractions obtained in sequence were respectively recorded as HM2-1 to HM2-4.

[0044] HM2-1 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 100:1;

[0045] HM2-2 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 40:1;

[0046] HM2-3 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 1:1;

[0047] HM2-4 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 1:100.

[0048] (3) The eluted fraction HM2-2 was subjected to silica gel column chromatography, and gradient elution was performed using a mixed solution of dichloromethane and methanol in volume ratios of 100:1, 50:1, 15:1, 10:1, and 5:1, respectively. The 26 eluted fractions obtained were recorded as E1 to E26.

[0049] (4) The eluted fractions E5 to E6 (the eluted fractions obtained by eluting with a mixed solution of dichloromethane and methanol in a volume ratio of 50:1) were combined and then subjected to silica gel column chromatography. Gradient elution was performed using a mixed solution of petroleum ether and ethyl acetate in volume ratios of 100:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 1:100, respectively. The 24 eluted fractions obtained were recorded as ED1 to ED24.

[0050] (5) The eluted fractions ED23 to ED24 were combined (the eluted fractions were obtained by eluting with a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:100), and then subjected to reverse phase semi-preparative C 18 The product was separated by HPLC column, with the eluent being a mixed solution of methanol and water in a volume ratio of 9:1, the mixed solution containing trifluoroacetic acid at a concentration of 0.03 vol.%, and a flow rate of 3.0 mL / min, to obtain a white amorphous powder, a monomer compound (compound 1, purity of 98%).

[0051] The HRESIMS spectrum of compound 1 prepared in this example is shown in Figure 1 ;key 1 H- 1 H COSY and HMBC correlation diagrams are shown in Figure 2 ; 1 H NMR spectrum is shown in Figure 3 ; 13C NMR spectrum is shown in Figure 4 ; 1 H- 1 H COSY spectrum see Figure 5 HSQC spectra are shown in Figure 6 ; HMBC spectrum see Figure 7 ; NOESY spectrum see Figure 8 .

[0052] The H NMR and C NMR data of compound 1 prepared in this example are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] Note: 1 H-NMR (600MHz, CDCl3), 13 C-NMR (150 MHz, CDCl3).

[0057] According to (+)-HR-ESIMS m / z 541.3486[M+H] + (calcd for C 33 H 49 O6,541.3524) combined with NMR data and 10 degrees of unsaturation to determine its molecular formula is C 33 H 48 O6( Figure 1 ). Compound 1 1 H NMR spectrum showed the presence of eight methyl signals [δ H 1.11 (s, H3-23), 1.07 (s, H3-24), 1.26 (s, H3-25), 1.17 (s, H3-26), 1.42 (s, H3-27), 0.93 (s, H3-28), 1.33 (s, H3-30), 2.05 (s, COCH3-22)], one methoxy signal [δ H 3.68 (s, OCH3-29)], a methine signal connected to oxygen [δ H 4.87 (dd, J = 12.0, 4.8 Hz, H-22)] and an olefinic hydrogen signal [δ H 5.66(s,H-12)](Table 1). 13 The CNMR spectrum showed 33 resonance carbon signals, including two keto carbonyl carbon signals [δ C 217.2 (C-3) and 199.3 (C-11)], two ester carbonyl carbon signals [δ C177.4 (C-29) and 170.6 (OAc-22)], two olefinic carbon signals [δ C 129.1 (C-12), 167.2 (C-13)], an oxygen-linked carbon signal [δ C 76.5(C-22)] (Table 1). The above spectral data combined with the purple-red color of the compound with 10% sulfuric acid ethanol indicate that the compound is a lanolin-type triterpenoid compound, and the HSQC, 1 H- 1 The structural formula of the compound was determined by H COSY, HMBC and NOESY analytical techniques as follows:

[0058] And named methyl3,11-dioxo-22β-acetoxy-olean-12-en-29-oate.

[0059] Effect Example 1

[0060] Evaluation of the anti-inflammatory activity of compound 1 prepared in Example 1 of the present invention:

[0061] Evaluation of anti-inflammatory activity: The Griess reduction method was used to evaluate the effect of compound 1 on LPS-induced NO production in RAW264.7 macrophages.

[0062] The specific method is as follows:

[0063] (1) Cell culture: RAW264.7 macrophages in good growth condition were placed in a constant temperature incubator and cultured at 37°C and 5% CO2. The cell confluence was 40% for each passage during the culture process. When the cell confluence reached 70%, the cell suspension was collected by blowing the cells gently, the supernatant was discarded after centrifugation, the cells were resuspended in DMEM complete medium containing 10% FBS and plated, and the plated concentration of 96-well plates was 10,000 cells per well.

[0064] (2) Reagent preparation: Under sterile conditions, indomethacin and compound 1 were dissolved in DMSO to prepare a 100 mM positive control drug solution and a 100 mM test drug stock solution, respectively. After filtering through a 0.22 μm sterile filter membrane, the solution was stored at -20°C for later use. LPS was prepared in the same manner as above to prepare a 1 mg / mL stock solution for later use. PBS and Griess reaction reagents were purchased from Bio-Tech.

[0065] (3) Experimental methods: The experiment was divided into a blank control group, an LPS model group, a positive drug control group and a drug administration group, and each group of experiments had 3 replicate wells.

[0066] Blank control group: 0.4 μL DMSO was added to 200 μL cell culture medium.

[0067] LPS group: 0.2 μL of 1 mg / mL LPS solution and 0.2 μL of blank DMSO were added to 200 μL of cell culture medium (the volume change was ignored) to make the final concentration of LPS 1 μg / mL.

[0068] Positive drug control group (indomethacin): add 0.2 μL of 1 mg / mL lipopolysaccharide LPS solution and 0.2 μL of 100 mM indomethacin control drug solution to 200 μL cell culture medium (ignore the volume change) to make the final concentration of lipopolysaccharide LPS 1 μg / mL and the final concentration of indomethacin 100 μM.

[0069] Drug administration group: 0.2 μL of 1 mg / mL LPS solution and 0.2 μL of 100 mM drug solution were added to 200 μL cell culture medium (ignore the volume change), so that the final concentration of LPS was 1 μg / mL and the final concentration of the drug (Compound 1) was 100 μM.

[0070] Then continue to culture for 24 hours at 37°C and 5% CO2. Take 50 μL of cell culture supernatant, add 50 μL of Griess reagent A solution and 50 μL of Griess reagent B solution, and measure the OD value at 540 nm with a microplate reader within 10 minutes.

[0071] According to NO2 - The standard curve is used to calculate the NO content. Standard curve drawing method: The concentration of the standard substance (NaNO2) can be 3.906, 7.813, 15.625, 31.25, 62.5, 125 μM. The standard curve is drawn according to the absorbance value of the standard substance at 540 nm using the Griess reagent method (see the results). Fig. 9 ).

[0072] The inhibition rate of compound 1 on NO production in macrophage RAW264.7 cells was calculated.

[0073]

[0074] The compound 1 prepared in Example 1 and indomethacin were used as inhibitors to inhibit the production of NO by macrophages RAW264.7. The results are shown in Table 2.

[0075] Table 2

[0076] Compound NO inhibition rate (%) Compound 1 92.9 Indomethacin 91.3

[0077] As can be seen from Table 2, compound 1 has a significant inhibitory effect on the production of NO by macrophages RAW264.7.

[0078] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing an oleanane-type triterpenoid compound, characterized in that: The following steps are involved: (1) Slice the fresh root tuber of Tripterygium wilfordii, extract it three times with 70% ethanol by volume concentration by heating and refluxing, the mass / volume ratio of Tripterygium wilfordii to ethanol is 1:5, the extraction temperature is 70-80°C, the extraction time for each time is 1.5 hours, combine the extracts, concentrate under reduced pressure and recover ethanol to obtain Tripterygium wilfordii extract; disperse the Tripterygium wilfordii extract with water, extract it with petroleum ether, ethyl acetate and n-butanol in equal volumes in sequence, and concentrate under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract; (2) The ethyl acetate extract was subjected to alumina column chromatography, and gradient elution was performed using a mixed solution of ethyl acetate and methanol with a volume ratio of 100:1, 40:1, 1:1, and 1:100, respectively. Four elution fractions were collected for each gradient, and the four elution fractions obtained in sequence were respectively recorded as HM2-1 to HM2-4; HM2-1 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 100:1; HM2-2 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 40:1; HM2-3 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 1:1; HM2-4 is the elution fraction of a mixed solution of ethyl acetate and methanol in a volume ratio of 1:100; (3) The eluted fraction HM2-2 was subjected to silica gel column chromatography, and gradient elution was performed using a mixed solution of dichloromethane and methanol with a volume ratio of 100:1, 50:1, 15:1, 10:1, and 5:1, respectively, to obtain 26 eluted fractions, which were respectively recorded as E1 to E26; (4) The eluted fractions E5 to E6 were combined and then subjected to silica gel column chromatography. Gradient elution was performed using a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 100:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 1:100, respectively. The 24 eluted fractions obtained were respectively recorded as ED1 to ED24; The elution fractions E5 to E6 are eluted with a mixed solution of dichloromethane and methanol in a product ratio of 50:1; (5) The eluted fractions ED23 to ED24 were combined and then separated by reverse phase semi-preparative C18 column HPLC. The eluent was a mixed solution of methanol and water in a volume ratio of 9:1, and the mixed solution contained trifluoroacetic acid at a concentration of 0.03 vol.%, and the flow rate was 3.0 mL / min to obtain a white amorphous powder, monomer compound 1; The elution fractions ED23 to ED24 are eluted from a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:100; The monomer compound 1 is the oleanane-type triterpenoid compound, and its structure is shown in formula (1): Formula (1).

Images