CN112175027A - Olive phylline derivative and preparation method and application thereof - Google Patents

Olive phylline derivative and preparation method and application thereof Download PDF

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CN112175027A
CN112175027A CN202011146085.6A CN202011146085A CN112175027A CN 112175027 A CN112175027 A CN 112175027A CN 202011146085 A CN202011146085 A CN 202011146085A CN 112175027 A CN112175027 A CN 112175027A
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oleanolic acid
methanol
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杨兆勇
金媛媛
刘玉凤
王保国
樊帅
吕广新
张茜
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Abstract

The invention provides a oleanolic acid derivative, a preparation method and an application thereof, and belongs to the technical field of antibacterial agents. The oleanolic acid derivative provided by the invention has a structure shown in a formula I, wherein R is methyl or ethyl. The oleanolic acid derivative provided by the invention has a novel structure, and has excellent antibacterial performance on staphylococcus epidermidis, staphylococcus aureus, staphylococcus hominis and enterococcus faecalis.

Description

Olive phylline derivative and preparation method and application thereof
Technical Field
The invention relates to the technical field of antibacterial agents, and particularly relates to a oleanolic acid derivative and a preparation method and application thereof.
Background
Oleanolin (English name: Elaiophylin, Azalomycin B, Gopalamicin, or Salbomycin) is a sixteen-membered ring macrocyclic dilactone antibiotic with double rotational symmetry, and has wide biological activities of resisting bacteria, resisting parasites, promoting growth of rumen animals and the like.
Streptomyces hygroscopicus (Streptomyces hygroscopicus) is a kind of actinomycetes with wide antimicrobial spectrum and stable genetic character, can produce various metabolites such as nigericins, oleanolins, geldanamycin and rapamycin, and has various biological activities such as antibiosis and immunosuppression. The method comprises the steps of separating 11,11 ' -O-dimethyl-14 ' -deethyl-14 ' methyl-oleanolic acid from the mycelium of the streptomyces hygroscopicus LP-93, wherein the oleanolic acid has strong inhibitory activity on methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus faecalis and mycobacterium tuberculosis, and the like (the liuyufeng and the like, the oleanolic acid derivatives in the mycelium of the streptomyces hygroscopicus LP-93 are separated and structurally identified, the institute of Jining medicine, 2020,43(1): 1-4). However, the current research on other oleanolic leaves in S.hygroscopicus is not deep enough.
Disclosure of Invention
In view of the above, the present invention aims to provide a folin derivative, a preparation method and an application thereof, and the folin derivative provided by the present invention has a novel structure and excellent antibacterial properties against staphylococcus epidermidis, staphylococcus aureus, staphylococcus hominis and enterococcus faecalis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a oleanolic acid derivative which has a structure shown in a formula I:
Figure BDA0002739745540000011
in the formula I, R is methyl or ethyl.
The invention provides a preparation method of the oleanolic acid derivatives in the technical scheme, which comprises the following steps:
(1) culturing seeds of the streptomyces hygroscopicus LP-93 to obtain a LP-93 seed solution;
(2) inoculating the LP-93 seed liquid into a fermentation culture medium for fermentation culture, adjusting the pH value of the obtained fermentation liquid to 3, carrying out centrifugal separation, air-drying the obtained precipitate, and crushing to obtain LP-93 fermentation dry bacterial powder;
(3) extracting the LP-93 fermented dry thallus powder with ethanol, concentrating the obtained extract, dissolving the obtained concentrate with methanol, filtering, and concentrating the obtained filtrate to obtain LP-93 mycelium extract;
(4) mixing said LP-93 mycelium extract with ethyl acetate to obtain a soluble fraction F1;
(5) subjecting the soluble part F1 to silica gel column chromatography to obtain components F1-1-F1-20 in sequence; subjecting the component F1-15 to C18Separating by HPLC to obtain a methyl-R oleanolic acid derivative;
or RP-HPLC separating component F1-18 to obtain folium Oleae Europaeae derivative with R as ethyl;
the silica gel column chromatographic separation adopts a gradient elution mode, and the gradient elution is sequentially performed by adopting eluents with volume ratios of dichloromethane to methanol of 50:1, 25:1, 12.5:1, 6.25:1 and 1: 1;
said C is18The mobile phase adopted by HPLC separation is a first methanol aqueous solution, and the volume ratio of methanol to water in the first methanol aqueous solution is 92: 8;
the mobile phase adopted by the RP-HPLC separation is a second methanol aqueous solution, and the volume ratio of methanol to water in the second methanol aqueous solution is 91: 9.
Preferably, in the step (1), the temperature of the seed culture is 25-28 ℃ and the time is 48-72 hours.
Preferably, in the step (2), the fermentation medium comprises the following components in percentage by mass: 2-4% of natural nitrogen source, 2-4% of carbon source, 0.3-0.6% of ammonium sulfate, 0.3-0.6% of potassium dihydrogen phosphate, 0.2-0.5% of calcium carbonate and the balance of water.
Preferably, in the step (2), the inoculation amount of the LP-93 seed solution accounts for 5-20% of the volume of the fermentation medium.
Preferably, in the step (2), the temperature of the fermentation culture is 25-28 ℃ and the time is 72-96 h.
Preferably, in the step (3), the ratio of the mass of the LP-93 fermentation dry bacterial powder to the volume of the ethanol is 1 g: (8-15) mL.
Preferably, in the step (3), the extraction is ultrasonic extraction, the extraction times are 2-3 times, and the time for each extraction is 1.5-2.5 hours.
The invention also provides the application of the oleanolic acid derivative in the technical scheme or the oleanolic acid derivative prepared by the preparation method in the technical scheme in antibiosis.
The invention provides a folin derivative which has a structure shown in a formula I, wherein R is methyl or ethyl. The provided olivil derivative with a brand-new structure has excellent antibacterial performance on staphylococcus epidermidis, staphylococcus aureus, staphylococcus hominis and enterococcus faecalis.
Drawings
FIG. 1 shows a sequence of oleanolic acid derivative I-11H-1HCOSY spectrum;
FIG. 2 shows the olive leaf extract I-21H-1HCOSY spectrum;
FIG. 3 is an HSQC spectrum of oleanolic acid derivative I-1;
FIG. 4 is an HSQC spectrum of oleanolic acid derivative I-2;
FIG. 5 is an HMBC spectrum of the oleanolic acid derivative I-1;
FIG. 6 is an HMBC spectrum of the oleanolic acid derivative I-2;
FIG. 7 is a ROESY spectrum of the oleanolic acid derivative I-1;
FIG. 8 is a ROESY spectrum of the oleanolic acid derivative I-2;
FIG. 9 is a DEPT spectrum of olivil derivative I-1;
FIG. 10 is a DEPT spectrum of olivil derivative I-2;
FIG. 11 is an infrared spectrum of a oleanolic acid derivative I-1;
FIG. 12 is an infrared spectrum of a oleanolic acid derivative I-2;
FIG. 13 is a UV spectrum of a folin derivative I-1;
FIG. 14 is a UV spectrum of a folin derivative I-2.
Detailed Description
The invention provides a oleanolic acid derivative which has a structure shown in a formula I:
Figure BDA0002739745540000041
in the formula I, R is methyl or ethyl.
In the invention, the carbon atom identifiers in the oleanolic acid derivatives are specifically as follows:
Figure BDA0002739745540000042
in the present invention, the oleanolic acid derivatives have the structure represented by formula I-1 or I-2:
Figure BDA0002739745540000043
the invention provides a preparation method of the oleanolic acid derivatives in the technical scheme, which comprises the following steps:
(1) culturing seeds of the streptomyces hygroscopicus LP-93 to obtain a LP-93 seed solution;
(2) inoculating the LP-93 seed liquid into a fermentation culture medium for fermentation culture, adjusting the pH value of the obtained fermentation liquid to 3, carrying out centrifugal separation, air-drying the obtained precipitate, and crushing to obtain LP-93 fermentation dry bacterial powder;
(3) extracting the LP-93 fermented dry thallus powder with ethanol, concentrating the obtained extract, dissolving the obtained concentrate with methanol, filtering, and concentrating the obtained filtrate to obtain LP-93 mycelium extract;
(4) mixing said LP-93 mycelium extract with ethyl acetate to obtain a soluble fraction F1;
(5) subjecting the soluble part F1 to silica gel column chromatography to obtain components F1-1-F1-20 in sequence; the silica gel column chromatographic separation adopts a gradient elution mode, and the gradient elution is sequentially performed by adopting eluents with volume ratios of dichloromethane to methanol of 50:1, 25:1, 12.5:1, 6.25:1 and 1: 1;
subjecting the component F1-15 to C18Separating by HPLC to obtain a methyl-R oleanolic acid derivative; said C is18The mobile phase adopted by HPLC separation is a first methanol aqueous solution, and the volume ratio of methanol to water in the first methanol aqueous solution is 92: 8;
or RP-HPLC separating component F1-18 to obtain folium Oleae Europaeae derivative with R as ethyl; the mobile phase adopted by the RP-HPLC separation is a second methanol aqueous solution, and the volume ratio of methanol to water in the second methanol aqueous solution is 91: 9.
In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.
The invention carries out seed culture on the streptomyces hygroscopicus LP-93 to obtain LP-93 seed liquid.
In the present invention, the seed culture specifically includes: placing the streptomyces hygroscopicus LP-93 on a slant culture medium for slant culture to obtain a bacterial slant; inoculating the slant to seed culture medium for seed culture.
In the invention, the source of the streptomyces hygroscopicus LP-93 is preferably China pharmaceutical microorganism culture preservation management center.
In the invention, the slant culture medium preferably comprises the following components in percentage by mass: 0.05% of Asparagus and K2HPO40.05%, glucose 1%, agar 1.2% and the balance water. In the invention, the temperature of the slant culture is preferably 25-30 ℃, and more preferably 26-28 ℃; the time is preferably 5 to 7 days, and more preferably 7 days.
In the present invention, the seed culture medium preferably comprises the following components by mass: 2-4% of natural nitrogen source, 2-4% of carbon source, 0.1-0.4% of ammonium sulfate, 0.1-0.2% of calcium carbonate and the balance of water; the content of the natural nitrogen source is more preferably 2.5-3.5%, and most preferably 3%; the content of the carbon source is more preferably 2.5-3.5%, and most preferably 3.5%; the content of the ammonium sulfate is more preferably 0.2-0.3%, and most preferably 0.3%; the content of the calcium carbonate is more preferably 0.12 to 0.18%, and most preferably 0.15%. In the invention, the natural nitrogen source preferably comprises one or more of soybean cake powder, peanut cake powder, fish meal and sesame cake powder. In the present invention, the carbon source preferably includes one or more of glucose, starch, sucrose, lactose, fructose, glycerol, dextrin, soybean oil and corn flour.
In the present invention, the inoculation is preferably performed by a block digging method, and preferably, a 1cm × 1cm slant of the bacteria is dug and inoculated in 50mL of seed culture medium. In the present invention, the container for inoculation is preferably a triangular flask.
In the invention, the temperature of the seed culture is preferably 25-28 ℃, and more preferably 26-27 ℃; the time is preferably 48 to 72 hours, and more preferably 48 to 55 hours. In the invention, the seed culture is preferably carried out in a shaking table, and the shaking speed of the shaking table is preferably 180-250 r/min, and more preferably 200-220 r/min. In the present invention, the purpose of the seed culture is to germinate, grow and multiply mycelia by spores of Streptomyces hygroscopicus LP-93, and to grow the mycelia robustly to become "seeds" with strong vitality.
After LP-93 seed liquid is obtained, the LP-93 seed liquid is inoculated into a fermentation culture medium for fermentation culture, the pH value is adjusted to 3, centrifugal separation is carried out, the obtained precipitate is dried in the air and then crushed, and LP-93 fermentation dry bacterial powder is obtained.
In the invention, the fermentation medium preferably comprises the following components in percentage by mass: 2-4% of natural nitrogen source, 2-4% of carbon source, 0.3-0.6% of ammonium sulfate, 0.3-0.6% of potassium dihydrogen phosphate, 0.2-0.5% of calcium carbonate and the balance of water; the content of the natural nitrogen source is more preferably 2.5-3.5%, and most preferably 3%; the content of the carbon source is more preferably 2.5-3.5%, and most preferably 3.5%; the content of the ammonium sulfate is more preferably 0.4-0.5%, and most preferably 0.5%; the content of the monopotassium phosphate is more preferably 0.4-0.5%, and most preferably 0.5%; the content of the calcium carbonate is more preferably 0.3 to 0.4%, and most preferably 0.3%. In the invention, the natural nitrogen source preferably comprises one or more of soybean cake powder, peanut cake powder, fish meal and sesame cake powder. In the present invention, the carbon source preferably includes one or more of glucose, starch, sucrose, lactose, fructose, glycerol, dextrin, soybean oil and corn flour.
In the invention, the inoculation amount of the LP-93 seed solution is preferably 5-20% of the volume of the fermentation medium, more preferably 10-18%, and most preferably 10-15%.
In the invention, the temperature of the fermentation culture is preferably 25-28 ℃, and more preferably 26-27 ℃; the time is preferably 72 to 96 hours, and more preferably 80 to 90 hours. In the invention, the fermentation culture is preferably carried out in a shaking table, and the shaking speed of the shaking table is preferably 180-250 r/min, and more preferably 200-220 r/min. In the present invention, the fermentation medium is intended for the growth, propagation and synthesis of the strain, enabling the LP-93 seed to grow rapidly after inoculation, and enabling the growing strain to synthesize the desired product rapidly.
The reagent used for adjusting the pH value is not particularly limited, and an acid or an alkali well known to those skilled in the art can be used, specifically, hydrochloric acid, oxalic acid, sodium bicarbonate or sodium hydroxide; the concentration of the hydrochloric acid is preferably 1-5 mol/L, and more preferably 2-4 mol/L; the sodium hydroxide is preferably used in the form of an aqueous sodium hydroxide solution, and the concentration of the aqueous sodium hydroxide solution is preferably 5 to 10mol/L, and more preferably 6 to 8 mol/L. The invention adjusts the pH value to 3 to ensure that the product can be precipitated from LP-93 seed solution, thereby achieving the purpose of primary purification.
In the invention, the rotation speed of centrifugal separation is preferably 3000-5000 r/min, and more preferably 4000 r/min; the time is preferably 10 to 45min, and more preferably 20 to 30 min.
In the invention, the air drying temperature is preferably 20-30 ℃, and more preferably 25 ℃; the time is preferably 24 to 48 hours, and more preferably 30 to 40 hours.
The crushing mode is not particularly limited, and the crushing mode known to the person skilled in the art can be adopted; in the embodiments of the present invention, the pulverization is preferably performed in a pulverizer.
After LP-93 fermentation dry bacterial powder is obtained, the invention carries out ethanol extraction on the LP-93 fermentation dry bacterial powder, concentrates the obtained extracting solution, adds methanol for dissolution, then filters, concentrates the obtained filtrate to obtain LP-93 mycelium extract.
In the present invention, the ratio of the mass of the LP-93 fermentation dry bacterial powder to the volume of ethanol is preferably 1 g: (8-15) mL, more preferably 1 g: (9-10) mL. In the present invention, the extraction is preferably ultrasonic extraction; the power of the ultrasonic extraction is not particularly limited, and the ultrasonic power well known to those skilled in the art can be adopted; the extraction frequency is preferably 2-3 times, and liquid phase materials obtained by each extraction are combined; the time of each extraction is preferably 1.5-2.5 h, and more preferably 2 h. In the present invention, the volume fraction of ethanol is preferably 95%.
The invention has no special limitation on the concentration mode of the extracting solution, and the extracting solution is concentrated to obtain thick extract by adopting the concentration mode known by the technicians in the field.
In the present invention, the filtration is preferably performed using a sand core funnel.
The invention has no special limitation on the concentration mode of the filtrate, and the filtrate can be concentrated by adopting the concentration mode known by the technicians in the field, such as rotary evaporation; the condition of the rotary steaming is not particularly limited, and the rotary steaming is carried out until the product is viscous.
After obtaining the LP-93 mycelium extract, the present invention mixes the LP-93 mycelium extract with ethyl acetate to obtain the soluble fraction F1.
In the invention, the volume ratio of the mass of the LP-93 mycelium extract to the volume of the ethyl acetate is 1 g: (10-15) mL is more preferably 1 g: (10-13) mL.
In the present invention, it is preferable that the extract-ethyl acetate mixed system obtained after mixing with ethyl acetate is filtered and dried to obtain a soluble fraction F1. In the invention, the drying temperature is preferably 2-4 ℃; in the present invention, the drying time is not particularly limited, and the drying time may be set to a constant weight.
After a soluble part F1 is obtained, the soluble part F1 is subjected to silica gel column chromatographic separation to sequentially obtain components F1-1-F1-20; the silica gel column chromatographic separation adopts a gradient elution mode, and the gradient elution is sequentially performed by adopting eluents with volume ratios of dichloromethane to methanol of 50:1, 25:1, 12.5:1, 6.25:1 and 1: 1.
In the present invention, the soluble fraction F1 is preferably applied by dry application, specifically, the soluble fraction F1 is dissolved in methylene chloride, mixed with silica gel, and applied after the solvent is evaporated. The silica gel dosage for the silica gel column chromatographic separation is not particularly limited, and the silica gel dosage well known to those skilled in the art can be adopted; in the embodiment of the present invention, the mass ratio of the soluble fraction F1 to the silica gel for loading is preferably 12.8: 15.
in the invention, the gradient elution is that the eluents with the volume ratio of dichloromethane to methanol of 50:1, 25:1, 12.5:1, 6.25:1 and 1:1 are sequentially used for elution, the eluents are respectively received, the volume of each part of the eluent is 200mL, the components in each part of the eluent are distinguished by thin-layer chromatography (TLC), the eluents containing the same components are combined, and the components F1-1-F1-20 are obtained. In the present invention, the relationship between the eluent used in the gradient elution, the number of the eluent and the components F1-1 to F1-20 is shown in Table 1:
TABLE 1 relationship between eluent, eluent number and components F1-1-F1-20
Figure BDA0002739745540000091
Figure BDA0002739745540000101
After the components F1-1-F1-20 are obtained, the component F1-15 is subjected to C18Separating by HPLC to obtain folium Oleae Europaeae derivative (marked as folium Oleae Europaeae derivative I-1) with R as methyl; said C is18The mobile phase used for the HPLC separation was a first aqueous methanol solution with a methanol to water volume ratio of 92: 8.
After components F1-1-F1-20 are obtained, the invention performs RP-HPLC separation on the components F1-18 to obtain the oleanolic acid derivatives (marked as oleanolic acid derivatives I-2) of which R is ethyl; the mobile phase adopted by the RP-HPLC separation is a second methanol aqueous solution, and the volume ratio of methanol to water in the second methanol aqueous solution is 91: 9.
The invention also provides the application of the oleanolic acid derivative in the technical scheme or the oleanolic acid derivative prepared by the preparation method in the technical scheme in antibiosis.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. 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
(1) Placing Streptomyces hygroscopicus LP-93 (China pharmaceutical culture Collection for microorganisms) on a slant culture medium, carrying out slant culture at 28 ℃ for 7 days to obtain a bacterial slant, inoculating the bacterial slant of 1cm multiplied by 1cm into a 500mL triangular flask filled with 50mL of seed culture medium by adopting a block digging method, and carrying out seed shaking culture on a shaking table at 28 ℃ and 220r/min for 48 hours to obtain LP-93 seed liquid; wherein, the composition of the slant culture medium is as follows: 0.05 wt% of Asparagus element, K2HPO40.05 wt%, glucose 1 wt%, agar 1.2 wt% and balance water; the seed culture medium comprises the following components: 3.0 wt% of soybean cake powder, 3.5 wt% of glucose, 0.3 wt% of ammonium sulfate, 0.15 wt% of calcium carbonate and the balance of water.
(2) Inoculating the LP-93 seed solution obtained in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium by an inoculation amount with the volume fraction of 10%, and performing shake fermentation culture on a shaking table at 25 ℃ and 220r/min for 96h to obtain LP-93 fermentation liquid; adding oxalic acid into the LP-93 fermentation liquor to adjust the pH value of the obtained fermentation liquor to 3.0, carrying out centrifugal separation, air-drying the obtained precipitate, and then placing the precipitate into a pulverizer to pulverize to obtain LP-93 fermentation dry bacterial powder; wherein, the fermentation medium comprises the following components: 3.0 wt% of soybean cake powder, 3.5 wt% of glucose, 0.5 wt% of ammonium sulfate, 0.5 wt% of monopotassium phosphate, 0.3 wt% of calcium carbonate and the balance of water.
(3) And (2) taking 124g of LP-93 fermented dry bacterial powder obtained in the step (1), adding 1000mL of 95% ethanol in volume fraction, performing ultrasonic extraction for 3 times, wherein the ultrasonic extraction time is 2h each time, condensing the obtained extracting solution, adding 1000mL of methanol for dissolving, filtering by a sand core funnel, and performing rotary evaporation on the obtained filtrate to form a viscous shape to obtain the LP-93 mycelium extract.
(4) 44.8g of the LP-93 mycelium extract obtained in step (3) was dissolved by adding 500mL of ethyl acetate under stirring, filtered, and dried to obtain a soluble fraction F1.
(5) Performing silica gel column chromatographic separation on the soluble part F1 by taking a dichloromethane-methanol mixed solvent as an eluent in a gradient elution mode to sequentially obtain components F1-1-F1-20;
the specific steps of silica gel column chromatographic separation are as follows: adding 30mL of dichloromethane into 12.8g of soluble part F1 for dissolution, adding 15g of 200-300-mesh silica gel for 3 times, uniformly stirring, and volatilizing the solvent to obtain a sample; and (2) completely loading the loaded sample into a silica gel column, sequentially eluting by using eluents with the volume ratios of dichloromethane to methanol of 50:1, 25:1, 12.5:1, 6.25:1 and 1:1, receiving the eluates, numbering the eluates according to the volume of 200mL of each eluent, distinguishing components in each eluent by using thin-layer chromatography (TLC), and combining the eluates containing the same components to obtain components F1-1-F1-20. Wherein the mass of the silica gel in the silica gel column is 150g of 200-300 mesh silica gel, and the height of the silica gel is about 15 cm. The relationship between the eluent used for gradient elution, the number of the eluent and the components F1-1 to F1-20 is shown in Table 1:
TABLE 1 relationship between eluent, eluent number and components F1-1-F1-20
Figure BDA0002739745540000111
Figure BDA0002739745540000121
Subjecting the component F1-15 to C18Separating by HPLC to obtain folium Oleae Europaeae derivative (folium Oleae Europaeae derivative I-1) with methyl R; said C is18The mobile phase used for the HPLC separation was a first aqueous methanol solution with a methanol to water volume ratio of 92: 8.
Subjecting component F1-18 to RP-HPLC separation to obtain folium Oleae Europaeae derivative (folium Oleae Europaeae derivative I-2) with R as ethyl; the mobile phase adopted by the RP-HPLC separation is a second methanol aqueous solution, and the volume ratio of methanol to water in the second methanol aqueous solution is 91: 9.
Process for preparing oleanolic acid derivatives I-1 and I-21H NMR data (DMSO-d6,600MHz) are shown in Table 2,13the C NMR data are shown in Table 3,1H-1the HCOSY spectrogram is shown in figure 1-2, wherein figure 1 is I-1, and figure 2 is I-2; the HSQC spectrogram is shown in fig. 3-4, wherein fig. 3 is I-1, and fig. 4 is I-2; HMBC spectrograms are shown in figures 5-6, wherein figure 5 is I-1, and figure 6 is I-2; the ROESY spectrogram is shown in figures 7-8, wherein figure 7 is I-1, and figure 8 is I-2; DEPT spectra are shown in FIGS. 9-10, wherein FIG. 9 is I-1, and FIG. 10 is I-2; the infrared spectrogram is shown in FIGS. 11-12, wherein FIG. 11 is I-1, and FIG. 12 is I-2; the ultraviolet spectrogram is shown in FIGS. 13-14, wherein FIG. 13 is I-1, and FIG. 14 is I-2.
TABLE 2 of oleanolic acid derivatives I-1 and I-21H NMR data (DMSO-d6,600MHz)
Figure BDA0002739745540000131
Figure BDA0002739745540000141
TABLE 3 preparation of oleanolic acid derivatives I-1 and I-213C NMR data (DMSO-d6,150MHz)
Figure BDA0002739745540000142
Figure BDA0002739745540000151
Figure BDA0002739745540000161
As shown in tables 2 to 3 and FIGS. 1 to 14, the high resolution electrospray mass spectrum (HR-ESIMS) of the oleanolic acid derivative I-1 gave an excimer ion peak M/z 1043.5888[ M + Na ]]+Combining nuclear magnetic resonance spectrum (NMR) data to determine the molecular formula of C55H88O17. Infrared spectrum absorption peak (1612,1638,1717 cm) of oleanolic acid derivative I-1-1) Ultraviolet spectrum (lambda)max252nm) shows the presence of a conjugated ester group system in the structure, binding1H-1Correlation peaks in HCOSY spectra, their hydrogen spectra: (H spectrum)1HNMR) showed the presence of two sets of conjugated trans double bonds in the structure [5.70(d, J ═ 15.6Hz, H-2),6.82(dd, J ═ 15.6,11.4Hz, H-3),6.10(dd, J ═ 15.0,11.4Hz, H-4),5.64(dd, J ═ 15.0,10.2Hz, H-5); 5.68(d, J ═ 15.6Hz, H-2 '), 6.79(dd, J ═ 15.6,11.4Hz, H-3'), 6.10(dd, J ═ 15.0,11.4Hz, H-4 '), 5.61(dd, J ═ 15.0,10.2Hz, H-5')]Two sugar end protons [4.91(d, J-2.4 Hz, H-22) ], 4.99(d, J-3.0 Hz, H-22')]An independent olefinic hydrogen proton [4.75(d, J ═ 2.4Hz, H-12')]One methoxy group 2.94(s, 11-OCH)3) And 12 methyl groups; in that13In the C NMR spectrum and DEPT spectrum, two carboxyl carbon signals [ C ] are shown in addition to the carbon signals corresponding to the above groupsC 167.1(C-1),166.8(C-1′)]A hemiacetal quaternary carbon signal [ alpha ] [ alpha ]C 102.8(C-11)]14 vicinal oxymethylene groups, 8 aliphatic methine groups and 5 methylene groups; using two-dimensional nuclear magnetic resonance spectroscopy (1H-1HCOSY, HSQC, HMBC, and ROESY spectra) confirmed the structure of the oleanolic acid derivative I-1; the HMBC spectrum of the oleanolic acid derivative I-1 shows 11-OCH3(H2.94) with C-11 (C)C102.8) indicating that the methoxy group is attached to C-11; correlation signals between H-12 ' and C-10 ', C-11 ', C-14 ', and between H-10 ', H-13 ', H-15 ', H-19 ' and C-11 ', indicate dehydration at the C-11 ' and C-12 ' positions, both of which form a double bond; from the above information, it was confirmed that the oleanolic acid derivative I-1 had a planar structure. Thus, the structure of the oleanolic acid derivative I-1 was determined to be 11-O-methyl-11 ', 12' -dehydrooleanolic acid.
High resolution electrospray mass spectrometry (HR-ESIMS) of the oleanolic derivative I-2 gave an excimer ion peak M/z 1057.6045[ M + Na ]]+Combining nuclear magnetic resonance spectrum (NMR) data to determine the molecular formula of C56H90O17(ii) a The ultraviolet spectrum, the infrared spectrum and the nuclear magnetic data of the oleanolic acid derivative I-2 are very similar to those of the oleanolic acid derivative I-1; by comparing the nuclear magnetic data of the two, the C-11 methoxy group in the oleanolic acid derivative I-1 is substituted by one ethoxy group [ H3.26 (1H, dq, J ═ 15.0,7.2Hz),3.19(1H, dq, J ═ 15.0,7.2Hz),1.03(3H, t, J ═ 7.2 Hz); c53.1,15.1]Namely to oleanolic acid derivative I-2; the structure of the oleanolic acid derivative I-2 also obtained two-dimensional nuclear magnetic resonance spectrum (1H-1HCOSY, HSQC, HMBC, and ROESY spectra); between the above ethyl protons1H-1The presence of ethoxy groups is evidenced by the H COSY related signal, and the HMBC related signals between the two vicinal oxymethylene protons (H3.26 and 3.19) and the methyl carbon (C15.1), the methyl proton (H1.03, 3H) and the vicinal oxymethylene carbon signal (C53.1); HMBC correlation signal between the methyl proton (H3.26) and C-11 (C102.9), indicating that the ethoxy group is attached to C-11; the ROESY and CD profiles of the oleanolic acid derivative I-2 are also very similar to those of the oleanolic acid derivative I-1, indicating that their absolute configurations are the same. Thus, the knot of the oleanolic acid derivative I-2The structure is determined to be 11-O-ethyl-11 ', 12' -dehydroolivil.
Comparative example 1
The structural formula of the oleanolic acid derivative is shown as a formula I-3 and is marked as the oleanolic acid derivative I-3:
Figure BDA0002739745540000171
comparative example 4
The structural formula of the oleanolic acid derivative is shown as a formula I-4, and is marked as the oleanolic acid derivative I-4:
Figure BDA0002739745540000172
test example
The levofloxacin, the oleanolic acid derivatives I-1 and I-2 were subjected to an antibacterial activity test, and compared with the oleanolic acid derivatives I-3 and I-4 by a two-fold gradient dilution method. The results of the Minimum Inhibitory Concentration (MIC) test of the levofloxacin and the olivil derivatives I-1 to I-4 are shown in Table 4:
TABLE 4 results of the minimum inhibitory concentrations of levofloxacin and oleanolic acid derivatives I-1 to I-4
Figure BDA0002739745540000181
As is clear from Table 4, the MIC values of the oleanolic acid derivatives I-1 and I-2 against various drug-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus faecalis (VRE) were all 32. mu.g/mL or less, and the antibacterial activity was good.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (9)

1. The oleanolic acid derivative is characterized by having a structure shown in a formula I:
Figure FDA0002739745530000011
in the formula I, R is methyl or ethyl.
2. A process for the preparation of the oleanolic acid derivatives of claim 1, comprising the steps of:
(1) culturing seeds of the streptomyces hygroscopicus LP-93 to obtain a LP-93 seed solution;
(2) inoculating the LP-93 seed liquid into a fermentation culture medium for fermentation culture, adjusting the pH value of the obtained fermentation liquid to 3, carrying out centrifugal separation, air-drying the obtained precipitate, and crushing to obtain LP-93 fermentation dry bacterial powder;
(3) extracting the LP-93 fermented dry thallus powder with ethanol, concentrating the obtained extract, dissolving the obtained concentrate with methanol, filtering, and concentrating the obtained filtrate to obtain LP-93 mycelium extract;
(4) mixing said LP-93 mycelium extract with ethyl acetate to obtain a soluble fraction F1;
(5) subjecting the soluble part F1 to silica gel column chromatography to obtain components F1-1-F1-20 in sequence; subjecting the component F1-15 to C18Separating by HPLC to obtain a methyl-R oleanolic acid derivative;
or RP-HPLC separating component F1-18 to obtain folium Oleae Europaeae derivative with R as ethyl;
the silica gel column chromatographic separation adopts a gradient elution mode, and the gradient elution is sequentially performed by adopting eluents with volume ratios of dichloromethane to methanol of 50:1, 25:1, 12.5:1, 6.25:1 and 1: 1;
said C is18The mobile phase adopted by HPLC separation is a first methanol aqueous solution, and the volume ratio of methanol to water in the first methanol aqueous solution is 92: 8;
the mobile phase adopted by the RP-HPLC separation is a second methanol aqueous solution, and the volume ratio of methanol to water in the second methanol aqueous solution is 91: 9.
3. The preparation method according to claim 2, wherein in the step (1), the seed culture temperature is 25-28 ℃ and the time is 48-72 h.
4. The preparation method according to claim 2, wherein in the step (2), the fermentation medium comprises the following components in percentage by mass: 2-4% of natural nitrogen source, 2-4% of carbon source, 0.3-0.6% of ammonium sulfate, 0.3-0.6% of potassium dihydrogen phosphate, 0.2-0.5% of calcium carbonate and the balance of water.
5. The method according to claim 2 or 4, wherein the LP-93 seed solution is inoculated in an amount of 5-20% by volume of the fermentation medium in step (2).
6. The preparation method according to claim 2 or 4, wherein in the step (2), the temperature of the fermentation culture is 25-28 ℃ and the time is 72-96 h.
7. The method according to claim 2, wherein in the step (3), the ratio of the mass of the LP-93 fermentation dry bacterial powder to the volume of the ethanol is 1 g: (8-15) mL.
8. The preparation method according to claim 2 or 7, wherein in the step (3), the extraction is ultrasonic extraction, the extraction times are 2-3 times, and the time of each extraction is 1.5-2.5 h.
9. Use of the oleanolic acid derivatives of claim 1 or the oleanolic acid derivatives obtained by the production method of any one of claims 2 to 8 for antibacterial purposes.
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