CN113956307B - Flavonoid glycoside compound, plane tree leaf extract and preparation method and pharmaceutical application thereof - Google Patents

Abstract

The application discloses a flavonoid glycoside compound, a plane tree leaf extract and a preparation method and pharmaceutical application thereof, wherein the structural formula of the flavonoid glycoside compound is as follows:

Description

Flavonoid glycoside compound, plane tree leaf extract and preparation method and pharmaceutical application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a flavonoid glycoside compound, a plane tree leaf extract, a preparation method thereof and application thereof in preparing a medicine for treating staphylococcus aureus infection.

Background

Staphylococcus aureus (Staphylococcus aureus) is a highly pathogenic gram-positive bacterium that infects humans and animals, and is responsible for many superficial, toxin-mediated, or invasive infections. The superficial lesions caused by staphylococcus aureus range from slight papules and furuncles to severe abscesses and carbuncles. Once penetrating the skin barrier, staphylococcus aureus can cause muscle and bone infections such as osteomyelitis and suppurative arthritis; these internal infections may further lead to more severe pneumonia, bacteremia, endocarditis and sepsis. Since staphylococcus aureus can produce exfoliative toxins, superantigens, cytotoxins and the like, it is also the causative agent of many toxin-mediated infections, such as food poisoning, toxic shock syndrome and scalded skin syndrome. Over the past century, staphylococcus aureus has become the major source of infection for community-acquired infections as well as hospital-acquired infections (Hemmadi & Biswas,2021, arch. Microbiol.203, 481-498).

The widespread use of antibiotics in animal husbandry, plant agriculture and biotechnology has also rapidly reduced the efficacy of antibiotic molecules (Chevrette et al, 2019, j.ind.microbiol.biotechnol.46, 257-271). The emergence of a number of resistant strains has led to the evolution of staphylococcus aureus (s.

In particular, the emergence of methicillin-resistant strains and vancomycin-resistant strains breaks the last line of defense for antibiotics. Although new therapeutic drugs are approved for clinical treatment of infections with drug-resistant strains, such as ceftaroline, which inhibits the growth of methicillin-resistant strains, new antibiotics also face the problem of increased resistance with increasing drug use. Staphylococcus aureus is resistant to a variety of antibiotics in a short period of time, making efforts to develop new antibiotics almost futile. Therefore, there is a need for new strategies or the development of antibiotic alternatives to treat infections with staphylococcus aureus and alleviate the resistance problems thereof.

Natural products have been the major source of clinically approved antibiotics as well as anticancer, immunosuppressive agents and other therapeutically relevant molecules, and especially provide an important basis for the development of drugs against infectious diseases (Newman & Cragg,2020, j.nat. Prod.83, 770-803). Moreover, the plant secondary metabolites with antibacterial action are continuously discovered to have various advantages of pure nature, low toxicity and side effects, no drug resistance, various structures, multiple targets and the like which are incomparable with antibiotics and other chemical drugs. Therefore, the search for new antibacterial drugs from abundant plant resources is an important direction for the development of modern pharmaceutical industry.

The genus Platanus (Platanus) is a plant of the family Platanaceae (Platanaceae) that contains 7 species and 12 subspecies and is widely distributed around the world (Thai et al, 2016, phytochemistry 130, 170-181). There are only 3 species in China, namely, platanus occidentalis Linn, platanus aceifolia Willd, and Platanus orientalis Linn. One species of sycamore (p. Occidentalis) was originally produced in north america, also known as phoenix tree, and has been introduced in north and middle of our country. Sycamore (p. Orientalis) native to southeast europe and western asia, also known as phoenix tree in arborescence in china. Plane tree in this application includes two-ball sycamore (p. Acerifolia) and three-ball sycamore (p. Orientalis).

In recent years, people pay attention to chemical components and medicinal values of the two-ball sycamore. Sycamore is widely used in many countries for medical care, shade, fuel and air pollution control. As a folk traditional medicinal plant, it is mainly used for treating pain, wounds, inflammation and stomach discomfort of teeth and knees. In recent years, various pharmacological studies have shown that the extracts and isolated monomeric compounds thereof can be used directly or indirectly for the treatment of various diseases and disorders, including hoarseness and asthma, blepharitis and conjunctivitis, dysentery, toothache, skin diseases, rheumatism and kidney stones.

However, although the leaves of "street trees" plane tree (a collective name of two-ball sycamore and three-ball sycamore) are extremely abundant in yield, the research on the phytochemical components thereof is not systematic, and especially the antibacterial activity of the extracts and the monomer components thereof is lack of report.

Disclosure of Invention

The application provides a novel flavonoid glycoside compound, a plane tree leaf extract and application thereof in preparing a medicine for treating Staphylococcus aureus infection, the plane tree leaf extract and a monomer compound inhibit the growth of Staphylococcus aureus (Staphylococcus aureus), can be used for preparing a Staphylococcus aureus inhibitor or a lead compound of the antibacterial medicine, and have great potential application in the field of preparing medicines for treating pyogenic infection, mastitis, pneumonia, enteritis, endocarditis, septicemia and toxic shock caused by Staphylococcus aureus (Staphylococcus aureus) infection.

A flavonoid glycoside compound or a pharmaceutically acceptable salt thereof, wherein the structural formula of the flavonoid glycoside compound is shown as a formula (1):

the flavonoid glycoside compound is a compound 1 or a compound 2, wherein R in the compound 1 ₁ Is E-p-conumaroyl, R ₂ Is Z-p-conumaroyl; in the compound 2R ₁ Is Z-p-conumaroyl, R ₂ Is E-p-conumaroyl.

Wherein compound 1 is named quercetin-3-O-alpha-L- (2 '-E-p-coumaroyl-3' -Z-p-coumaroyl) -rhamnoside; the compound 2 was named quercetin-3-O- α -L- (2 "-Z-p-coumaroyl-3" -E-p-coumaroyl) -rhamnoside.

The application also provides application of the flavonoid glycoside compound or the pharmaceutically acceptable salt thereof in serving as or preparing a Staphylococcus aureus (Staphylococcus aureus) inhibitor.

The application also provides application of the flavonoid glycoside compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating Staphylococcus aureus (Staphylococcus aureus) infection.

The application also provides application of the flavonoid glycoside compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating pyogenic infection, mastitis, pneumonia, enteritis, endocarditis, septicemia or toxic shock caused by staphylococcus aureus infection.

The application also provides a plane tree leaf extract which contains the flavonoid glycoside compounds, wherein the flavonoid glycoside compounds comprise compounds 1 and/or compounds 2, and the structural formula is shown in the formula (1). Plane tree in this application includes two-ball sycamore (p. Acerifolia) and three-ball sycamore (p. Orientalis). Taking French phoenix tree leaves as a raw material, preparing a concentrated water extract or a concentrated alcohol extract by adopting a solvent extraction method, or further separating and purifying the concentrated water extract or alcohol extract to obtain a subfraction I and a subfraction II; the leaves of plane tree are from the leaves of plane tree (p.acerifolia) and/or plane tree (p.orientalis).

Therefore, optionally, the extract of the plane tree leaves comprises a concentrated aqueous extract or a concentrated alcoholic extract of the plane tree leaves, a subfraction I or a subfraction II separated and purified from the concentrated aqueous extract or the concentrated alcoholic extract; the leaves of the plane tree are from the two-ball sycamore (p. Acerifolia) and/or the three-ball sycamore (p. Orientalis); namely, the concentrated aqueous extract, the concentrated alcoholic extract subfraction I or the subfraction II are all the extract of the leaves of the French phoenix tree.

Optionally, the extract of the leaves of the French phoenix tree comprises a compound 1 and a compound 2; the concentration range of the total amount of the compound 1 and the compound 2 in the concentrated aqueous extract or the concentrated alcohol extract is (1-20) mg/kg; the concentration range of the total amount of the compound 1 and the compound 2 in the subcomponent I is (100-500) mg/kg; the concentration range of the total amount of the compound 1 and the compound 2 in the subcomponent II is (100-500) g/kg.

The application also provides application of the extract of the leaves of the French phoenix tree in serving as or preparing a Staphylococcus aureus (Staphylococcus aureus) inhibitor.

The application also provides an application of the extract of the leaves of the plane tree in preparing a medicament for treating Staphylococcus aureus (Staphylococcus aureus) infection.

The application also provides an application of the extract of the leaves of the plane tree in preparing a medicament for treating pyogenic infection, mastitis, pneumonia, enteritis, endocarditis, septicemia or toxic shock caused by staphylococcus aureus.

The application also provides a staphylococcus aureus inhibitor which takes the flavonoid glycoside compound or the pharmaceutically acceptable salt thereof or the extract of the leaves of the plane tree as an active component.

The application also provides a medicine for treating pyogenic infection, mastitis, pneumonia, enteritis, endocarditis, septicemia and toxic shock caused by staphylococcus aureus, the medicine takes a phoenix tree leaf extract or a monomer compound as an effective component, and comprises a medicine effective amount of the flavonoid glycoside compound or pharmaceutically acceptable salt thereof or the phoenix tree leaf extract.

The flavonoid glycoside compound or the pharmaceutically acceptable salt thereof or the extract of the leaves of the French phoenix tree can be used independently or jointly.

Optionally, a pharmaceutically acceptable carrier or excipient is also included.

Optionally, the extract of the leaves of the plane tree and an excipient are prepared into oral preparations, skin external preparations or injection preparations.

Alternatively, the strain is preferably Newman S.aureus ATCC 25904.

The application also provides a preparation method of the flavonoid glycoside compound, which comprises the following steps:

(1) Taking Platanus orientalis leaves as raw materials, and preparing into concentrated aqueous extract or concentrated alcoholic extract by adopting a solvent extraction method;

(2) Subjecting the concentrated aqueous extract or concentrated alcoholic extract to macroporous resin D101 column, and performing gradient elution with ethanol-water solvent system; wherein the initial volume fraction of ethanol is 0-30%, and the final volume fraction of ethanol is 100%; merging and concentrating 60-70% of EtOH elution fractions to obtain a subfraction I;

(3) Carrying out gradient elution on the subfraction I through a small-pore resin MCI gel column by adopting a methanol-water solvent system; wherein the initial volume fraction of the methanol is 0-30%, and the final volume fraction of the methanol is 100%; merging and concentrating 50-90% MeOH elution fractions to obtain a subfraction II;

(4) Purification of subfraction ii by semi-preparative HPLC was performed at 66 volume ratio: 34 methanol: isocratic elution with 0.05% aqueous trifluoroacetic acid collected fractions with a retention time of 22.5min and 31.7min, respectively, to give compound 1 (22.5 min) and compound 2 (31.7 min) as yellow amorphous powders, respectively.

The concentration range of the total amount of the compound 1 and the compound 2 in the French phoenix tree leaf concentrated aqueous extract or concentrated alcohol extract is (1-20) mg/kg, the concentration range of the total amount of the compound 1 and the compound 2 in the subcomponent I is (100-500) mg/kg, and the concentration range of the total amount of the compound 1 and the compound 2 in the subcomponent II is (100-500) g/kg.

The application also provides a preparation method of the plane tree leaf extract, which is characterized in that plane tree leaves are used as raw materials, a solvent extraction method is adopted to prepare a concentrated water extract or a concentrated alcohol extract, or a subfraction I and a subfraction II are obtained after the concentrated water extract or alcohol extract is further separated and purified (the method is the same as the steps (2) and (3) in the preparation method of the flavonoid glycoside compounds); the concentrated aqueous extract, concentrated alcoholic extract, subfraction I or subfraction II are all the extract of the leaves of the French phoenix tree; the plane tree leaves are from Platanus orientalis (P. Acerifolia) and/or Platanus trifoliatus (P. Orientalis).

Compared with the prior art, the preparation method and the application of the extract of the leaves of the French phoenix tree and the flavonoid glycoside monomeric compound thereof have at least one of the following beneficial effects:

on one hand, the application provides the application of the extract of the leaves of the plane tree and 2 novel flavonoid glycosides compounds in the preparation of staphylococcus aureus inhibitors.

Optionally, the extract of the leaves of the plane tree is a concentrated aqueous extract or an alcoholic extract of the leaves of the plane tree prepared by a solvent extraction method and a sub-component obtained after separation and purification. The novel flavonoid glycoside compound can be obtained by separating and purifying Sterculia Spreng extract.

On the other hand, the application provides the application of the extract of the plane tree leaves and 2 novel flavonoid glycosides compounds in preparing the medicines for treating pyogenic infection, mastitis, pneumonia, enteritis, endocarditis, septicemia and toxic shock.

The medicine takes the extract or monomer compound of the plane tree leaves as the active ingredient, can be used independently or jointly, is combined with a pharmaceutically acceptable carrier or excipient, and is prepared into an oral preparation, a skin external preparation or an injection preparation according to a conventional method.

Furthermore, the plane tree has rich raw materials only by manual collection in autumn fallen leaves and full lands. Therefore, the application changes waste into valuable, and greatly improves the added value of the fallen leaves of the plane tree.

Drawings

FIG. 1 is the main COSY and HMBC related signals of compound 1;

FIG. 2 is a HRESIMS spectrum of Compound 1 provided herein;

FIG. 3 is a drawing of Compound 1 provided herein ¹ H NMR(400MHz,CD ₃ OD) spectrum;

FIG. 4 is a drawing of Compound 1 provided herein ¹³ C NMR(150MHz,CD ₃ OD) spectrum;

FIG. 5 is a DEPT 135 (600 MHz) spectrum of Compound 1 provided herein;

FIG. 6 is a HSQC (600 MHz) spectrum of Compound 1 provided herein;

FIG. 7 is a drawing of Compound 1 provided herein ¹ H- ¹ H COSY (600 MHz) spectrum;

FIG. 8 is an HMBC (600 MHz) spectrum of compound 1 as provided herein;

FIG. 9 is the major COSY and HMBC related signals of Compound 1;

FIG. 10 is a HRESIMS spectrum of Compound 2 provided herein;

FIG. 11 is a drawing of Compound 2 provided herein ¹ H NMR(400MHz,CD ₃ OD) spectrum;

FIG. 12 is a schematic representation of Compound 2 provided herein ¹³ C NMR(150MHz,CD ₃ OD) spectrum;

FIG. 13 is an HSQC (600 MHz) spectrum of Compound 2 provided herein;

FIG. 14 is a drawing of Compound 2 provided herein ¹ H- ¹ H COSY (600 MHz) spectrum;

figure 15 is an HMBC (600 MHz) spectrum of compound 2 provided herein.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The application extracts the extract of the leaves of plane tree (Platanus acerifolia wild.) and 2 neoflavonoid glycosides (quercetin-3-O-alpha-L- (2 '-E-p-coumaroyl-3' -Z-p-coumaroyl) -rhamnoside (compound 1) and quercetin-3-O-alpha-L- (2 '-Z-p-coumaroyl-3' -E-p-coumaroyl) -rhamnoside (compound 2)) which are separated from the extracts from the leaves of plane tree (Platanus acerifolia wild.) and/or Platanus orientalis Linn.

The application discovers through screening that the extract of the plane tree leaves has certain inhibiting effect on Newman staphylococcus aureus ATCC 25904: the minimum inhibitory concentrations of subcomponent I and subcomponent II were 256. Mu.g/mL and 128. Mu.g/mL, respectively. The minimum inhibitory concentration of the new monomeric compound 1 and the compound 2 is 64 mug/mL. The extract and the monomeric compound have great potential application in the field of preparing medicaments for treating pyogenic infection, mastitis, pneumonia, enteritis, endocarditis, septicemia and toxic shock caused by staphylococcus aureus.

The extract of plane tree leaves of the present application can be prepared from plane tree leaves by methods well known to those skilled in the art. For example, the extract is prepared from leaves of Sterculia platanifolium by extracting with solvent to obtain concentrated aqueous extract or alcoholic extract, and separating and purifying to obtain subfractions I and II.

In the solvent extraction method, a single solvent or a mixed solvent of a plurality of solvents such as water, methanol, ethanol and the like can be selected. The water extract is prepared by crushing dried leaves of plane tree, adding 5-20 times of ultrapure water, heating and refluxing for extraction for 1-3 h, filtering with gauze to obtain supernatant for 3-4 times, combining filtrates, and concentrating under reduced pressure to concentration of 0.5-1.5 g/mL. The alcohol extract is prepared by selecting methanol or a mixed solvent of ethanol and water or a mixed solvent of a plurality of solvents, taking 75% ethanol as an optimal extraction solvent, performing immersion extraction for 24-48 h at room temperature or performing heating reflux extraction for 1-3 h for 3-4 times, combining filtrates, and performing reduced pressure concentration.

Subjecting the aqueous extract or alcoholic extract to macroporous resin D101 column, and gradient with ethanol-water (EtOH/H2O) solvent system; wherein the initial volume fraction of ethanol is 0-30%, and the final volume fraction of ethanol is 100%; preferably, 60-70% EtOH elution fractions are combined and concentrated to give subfraction I. Carrying out gradient elution on the subfraction I through a small-pore resin MCI gel column by adopting a methanol-water (MeOH/H2O) solvent system; wherein the initial volume fraction of the methanol is 0-30%, and the final volume fraction of the methanol is 100%; preferably, 50-90% MeOH eluted fractions are combined and concentrated to give subfraction II.

The extract of the plane tree leaves comprises new monomer compounds 1 and 2, and the compounds have the following molecular structures:

wherein, in the compound 1, R ₁ Is E-p-conumaroyl, R ₂ Is Z-p-conumaroyl; in the compound 2R ₁ Is Z-p-conumaroyl, R ₂ Is E-p-conumaroyl.

The method for separating and purifying the compounds 1 and 2 in the extract of the leaves of the French phoenix tree comprises the following steps: subfraction ii was purified by semi-preparative HPLC using methanol: 0.05% aqueous trifluoroacetic acid (66, v/v) was isocratically eluted, and the retention time 22.5min fraction and 31.7min fraction were collected, respectively, to give compound 1 (22.5 min) and compound 2 (31.7 min) as yellow amorphous powders, respectively.

The concentration range of the total amount of the compound 1 and the compound 2 in the concentrated aqueous extract or the concentrated alcohol extract of the French phoenix tree leaves is (1-20) mg/kg, the concentration range of the total amount of the compound 1 and the compound 2 in the subcomponent I is (100-500) mg/kg, and the concentration range of the total amount of the compound 1 and the compound 2 in the subcomponent II is (100-500) g/kg. Through the activity screening of the extract of the leaves of the plane tree and the flavonoid glycoside monomeric compound thereof on Newman staphylococcus aureus ATCC 25904, the extract of the leaves of the plane tree and the flavonoid glycoside monomeric compound thereof have certain growth inhibition effect on the staphylococcus aureus and can be used for preparing a staphylococcus aureus inhibitor or a lead compound of the antibacterial drug.

The following is a description of specific examples:

in the following preparations, NMR was measured using a Bruker Avance model II 400/600 apparatus; HR-MS was measured by an AB Sciex TripleTOF model 5600 instrument; MCI Gel CHP 20P (75-150 μm) is produced by Mitsubishi corporation of Japan; semi-preparative HPLC was Waters e2695 equipped with 2998PDA and 2424 evaporative light scattering double detectors and SunAire ODS (5 μm, 250X 10 mm) semi-preparative columns; the macroporous adsorption resin D101 and all reagents used are all produced by Shanghai national drug group chemical reagent limited.

In the following test examples of antibacterial activity, the test strain was Newman Staphylococcus aureus ATCC 25904, obtained from Shanghai pharmaceutical institute of Chinese academy of sciences. Brain Heart Infusion (BHI) agar and calcium ion conditioned M-H broth (CAMHB) medium were purchased from Shanghai Solebao, inc. The clean bench (SW-CJ-2F) is manufactured by Suzhou Antai air technology Co., ltd; the biochemical incubator (SPX-250B-Z) and the vertical pressure steam sterilizer (YXQ-LS-SII) are produced by Shanghai Bingmai industry Co., ltd; microplate reader (staining 5) is manufactured by beton instruments ltd.

Example 1: preparation method of extract of Sterculia Spreng

Pulverizing dried leaves of Platanus orientalis (Platanus acerifolia wild.) or Platanus orientalis Linn of Platanus orientalis of 11 months, extracting with 75% ethanol for 4 times (36 hr each time), mixing the extracts, and concentrating under reduced pressure to obtain 75% ethanol extract.

Passing 75% ethanol extract (3.5 kg) through macroporous resin D101 column, and eluting with ethanol-water (EtOH/H) ₂ O) gradient of the solvent system; wherein the initial volume fraction of ethanol is 0-30%, and the final volume fraction of ethanol is 100%; preferably, 60-70% EtOH eluate fractions are combined and concentrated to give subfraction I (70 g). Passing subcomponent I through a small pore resin MCI gel column with methanol-water (MeOH/H) ₂ O) carrying out gradient elution by using a solvent system; wherein the initial volume fraction of the methanol is 0-30%, and the final volume fraction of the methanol is 100%; preferably, 50-90% MeOH-eluting fractions are combined and concentrated to give subfraction II (70.0 mg).

Example 2: preparation method of extract of Sterculia Spreng

Pulverizing dried leaves of Platanus orientalis (Platanus acerifolia wild.) or Platanus orientalis Linn, adding 10 times of ultrapure water, heating under reflux for 2 hr, filtering with gauze to obtain supernatant, extracting for 4 times, mixing filtrates, and concentrating under reduced pressure to obtain 1.0g/mL aqueous extract.

Passing the aqueous extract through a macroporous resin D101 column, and eluting with ethanol-water (EtOH/H) ₂ O) gradient of the solvent system; wherein the initial volume fraction of ethanol is 0-30%, and the final volume fraction of ethanol is 100%; preferably, 60-70% EtOH elution fractions are combined and concentrated to give subgroupsAnd (4) dividing into I. Passing subcomponent I through a small pore resin MCI gel column with methanol-water (MeOH/H) ₂ O) carrying out gradient elution by using a solvent system; wherein the initial volume fraction of the methanol is 0-30%, and the final volume fraction of the methanol is 100%; preferably, 50-90% MeOH-eluting fractions are combined and concentrated to give subfraction II.

Example 3: preparation of flavonoid glycoside compounds

Subfraction II (70.0 mg, prepared in example 1) was purified by semi-preparative HPLC (SunAire column, 3.0 mL/min) with methanol: isocratic elution with 0.05% aqueous trifluoroacetic acid (66, 34, v/v) gave novel flavonoid glycoside compound 1 (3.0 mg, t _R =22.5 min) and compound 2 (3.2mg _R ＝31.7min)。

The compounds are elucidated by nuclear magnetic resonance spectroscopy (1D/2D nuclear magnetic resonance technique) and HRMS data:

the HR-ESI spectrum (FIG. 2) of Compound 1 showed an excimer ion peak of M/z 763.1621 ([ M + Na)] ⁺ Calcd 763.1633), and deducing the molecular formula as C by combining carbon spectrum ₃₉ H ₃₂ O ₁₅ 。

The hydrogen spectrum of compound 1 (FIG. 3, table 1) shows the 1 aromatic ring ABX spin system [ delta ] _H 7.42(1H, dd,J＝2.0,8.4Hz,H-6′),δ _H 7.38(1H,d,J＝2.0Hz,H-2′),δ _H 6.96(1H,d,J＝8.4 Hz,H-5′)]And 2 aromatic ring single peak proton signals [ delta ] _H 6.39(1H，s，H-8)，δ _H 6.21(1H，s， H-6)]This is a characteristic signal of quercetin glycosides. The hydrogen spectrum also includes 1 characteristic signal of alpha-L-rhamnose: 5 continuous oxygen methine proton signals [ delta ] _H 5.53(1H,d,J＝1.6Hz,H-1″),δ _H 5.81(1H,dd,J＝ 1.6,3.6Hz,H-2〞),δ _H 5.25(1H,dd,J＝3.6,9.6Hz,H-3″),δ _H 3.58(1H,t,J＝9.6Hz, H-4″),δ _H 3.60(1H,m,H-5〞)]And 1 bimodal methyl signal [ delta ] _H 1.03(3H,d,J＝5.2Hz, H-6″)]. In addition, the hydrogen spectrum also includes 21, 4-disubstituted aromatic rings, a pair of cis double bonds and a pair of trans double bonds, revealing that the compounds contain both trans and cis coumaroyl units attached to the α -L-rhamnose moiety. Carbon spectra (FIG. 4) and DEPT-135 (FIG. 5) show that the compounds contain 39 carbons: 15 carbons were present on the aglycone, 6 carbons were present on the sugar, and the remaining 18 were those of two coumaroyl groups. ¹ H- ¹ Spin system [ -CH-CH-CH-CH in H COSY spectrogram (figure 1, figure 7) ₃ (H-1”/H-2”/H-3”/H-4”/H-5”/H-6”)]The proton signal of the rhamnose fragment can be fully attributed. The remote correlation of H-2 '(delta 5.81), H-3' (delta 5.25) with the carbonyl carbon signal of trans coumaroyl group (delta 167.77), the carbonyl carbon signal of cis coumaroyl group (delta 167.58), respectively, is evident on HMBC spectra (FIG. 1, FIG. 8), confirming the attachment of trans coumaroyl and cis coumaroyl units at the C-2 'and C-3' positions of rhamnose, respectively. Meanwhile, the location of the sugar linkage was confirmed by correlation signals of H-1' (Δ 5.53) and C-3 (. Delta.135.56) in HMBC spectra (FIG. 1, FIG. 8).

Therefore, the structure of the compound 1 is determined to be quercetin-3-O-alpha-L- (2 '-E-p-coumaroyl-3' -Z-p-coumaroyl) -rhamnoside, the structural formula is shown as the formula (1), and the two-dimensional related signals are shown as the figure 1.

TABLE 1 NMR data Signal assignment (CD) for Compound 1 ₃ OD)

The HR-ESI spectrum (FIG. 10) of Compound 2 showed an excimer ion peak of M/z 763.1613 ([ M + Na ]] ⁺ Calcd 763.1633), and deducing the molecular formula as C by combining carbon spectrum ₃₉ H ₃₂ O ₁₅ 。

The 1D/2DNMR spectral data (fig. 11-15, table 2) for compound 2 are highly similar to compound 1, with the only difference being the opposite positions of the trans-coumaroyl and cis-coumaroyl units on the L-rhamnosyl group. The remote correlation of H-2 '(delta 5.81), H-3' (delta 5.25) with the carbonyl carbon signal of cis coumaroyl group (delta 167.58), and the carbonyl carbon signal of trans coumaroyl group (delta 167.77), respectively, is evident on HMBC spectra (FIG. 9, FIG. 15), confirming the attachment of cis coumaroyl and trans coumaroyl units at the C-2 'and C-3' positions of rhamnose, respectively.

Therefore, the compound 2 is identified as quercetin-3-O-alpha-L- (2 '-Z-p-coumaroyl-3' -E-p-coumaroyl) -rhamnoside, the structural formula is shown as the formula (1), and the two-dimensional correlation signal is shown as the figure 9.

TABLE 2 NMR data signal assignments (CD) for Compound 2 ₃ OD)

Example 3: assay for inhibitory Activity against Staphylococcus aureus

Inoculating Newman staphylococcus aureus ATCC 25904 to BHI culture plate, culturing at 35 deg.C for 18-24 hr, selecting several colonies from the plate, and directly preparing into (1-2) × 10 in CAMHB broth ⁸ CFU/mL of bacterial suspension. Diluting with CAMHB broth 100 times to give a concentration of (1-2). Times.10 ⁶ CFU/mL (concentration about 5X 10 when drug sensitive plate is added) ⁵ CFU/mL)。

Taking a sterile 96-well plate, adding saline into the A-th and H-th holes to prevent the volatilization of the internal bacterial liquid, wherein the B-G-th holes are the following operation holes, and the final volume of each hole is 200 mu L. A blank was prepared by adding 200. Mu.L of liquid medium to well 1. Adding 100 mu L of liquid culture medium into the wells of the 2 nd to 12 th columns: the 2 nd to 11 th rows are medicine holes (the number of the compound holes is 3), 100 mu L of the tested medicine with the concentration of 128 mu g/mL is added into the 2 th row of holes, and the 3 th to 11 th rows of holes are diluted in a multiple ratio, so that the final medicine concentration of each hole is respectively 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 \8230; \8230andmu g/mL.12 wells contained no drug and DMSO was added in an amount equal to 2 wells as a positive growth control. Adding (1-2) x 10 to the 2 nd-12 th row of holes ⁶ CFU/mL of bacterial suspension 100 u L.

And incubating the drug sensitive reaction plate in an incubator at 35 ℃ for 16-20 h, and taking the lowest concentration of the tested drug generated by sterile colony as the Minimum Inhibitory Concentration (MIC). Methicillin and chloramphenicol were used as positive controls. The results of the antimicrobial activity assay for the test substances are shown in table 3.

TABLE 3 antibacterial Activity data of extracts from leaves of Sterculia Sprensis and novel flavonoid glycoside Compounds

As can be seen from Table 3, the MIC values of the positive controls methicillin and chloramphenicol for Newman Staphylococcus aureus ATCC 25904 were 2 and 4. Mu.g/mL, respectively; MIC values of the subfraction I and the subfraction II in the Mallotus japonicus extract are 256 mug/mL and 128 mug/mL respectively; the minimum inhibitory concentration of the neoflavonoid glycoside compound 1 and the compound 2 is 64 mu g/mL. The application shows that the extract of the plane tree leaves and the neoflavonoid glycoside compounds 1 and 2 have certain inhibitory effect on Newman staphylococcus aureus ATCC 25904.

In conclusion, the extract of the plane tree leaves and the neoflavonoid glycoside compound can be used for preparing a staphylococcus aureus inhibitor or used as a lead compound of the antibacterial drugs, and have great potential application in the field of preparing drugs for treating pyogenic infection, mastitis, pneumonia, enteritis, endocarditis, septicemia and toxic shock caused by the staphylococcus aureus infection.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (1)

1. A method for preparing flavonoid glycoside compounds is characterized by comprising the following steps:

(1) Taking Platanus orientalis leaves as raw materials, and preparing into concentrated aqueous extract or concentrated alcoholic extract by adopting a solvent extraction method;

(2) Subjecting the concentrated aqueous extract or concentrated alcoholic extract to macroporous resin D101 column, and performing gradient elution with ethanol-water solvent system; wherein the initial volume fraction of the ethanol is 0 to 30 percent, and the final volume fraction of the ethanol is 100 percent; combining and concentrating 60 to 70 percent of EtOH elution fractions to obtain a subfraction I;

(3) Carrying out gradient elution on the subfraction I through a small-pore resin MCI gel column by adopting a methanol-water solvent system; wherein the initial volume fraction of the methanol is 0 to 30 percent, and the final volume fraction of the methanol is 100 percent; mixing and concentrating 50-90% MeOH elution fractions to obtain a subfraction II;

(4) Purification of subfraction ii by semi-preparative HPLC was performed at 66 volume ratio: 34 methanol: isocratic elution with 0.05% trifluoroacetic acid water solution, and collecting the components with retention time of 22.5min and 31.7min respectively to obtain yellow amorphous powder of compound 1 and compound 2 respectively;

the structural formulas of the compound 1 and the compound 2 are shown as the formula (1):

（1）；

wherein, in the compound 1, R ₁ Is composed ofE-p-conumaroyl、R ₂ Is composed ofZ-p-conumaroyl(ii) a In the compound 2R ₁ Is composed ofZ-p- conumaroyl、R ₂ Is composed ofE-p-conumaroyl。

Images