CN111588717A

CN111588717A - Application of two quadruple lactone antibiotics as MRSA (methicillin resistant Staphylococcus aureus) resisting drugs and extraction and separation method thereof

Info

Publication number: CN111588717A
Application number: CN202010555152.3A
Authority: CN
Inventors: 张娟利; 张晓梅; 李慧玲; 文爱东
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2020-08-28

Abstract

The invention discloses application of two quadruple lactone antibiotics as MRSA (methicillin resistant) drugs and an extraction and separation method thereof, wherein two quadruple lactone compounds, namely dinatin and trinatin, are separated from Streptomyces puniceus S009 which is an endophyte of dracaena cochinchinensis for the first time, and the results of in-vitro MRSA activity resistance tests on the two separated compounds show that the dinatin and trinatin have stronger inhibitory action on various clinically separated MRSA strains than a positive control vancomycin and the molecular docking results show that the compounds and the MRSA target protein PBP2a have strong binding activity. Therefore, the invention not only can provide a lead compound for developing novel MRSA resistant medicines, but also can effectively protect rare and endangered medicinal plant resources, and has important significance for reducing the damage of the diversity of wild medicinal plants.

Description

Application of two quadruple lactone antibiotics as MRSA (methicillin resistant Staphylococcus aureus) resisting drugs and extraction and separation method thereof

Technical Field

The invention belongs to the technical field of natural product chemistry, and relates to application of two quadruple lactone antibiotics as anti-MRSA drugs and an extraction and separation method thereof, in particular to a preparation method of two quadruple lactone antibiotics separated from Streptomyces puniceus S009 as endophytes of dracaena cochinchinensis and application of the quadruple lactone antibiotics as anti-MRSA drugs.

Background

Methicillin-resistant staphylococcus aureus (MRSA) is a main pathogenic bacterium of hospital infection, has the characteristics of high separation rate, high drug resistance and high fatality rate, and is the first of three most difficult infectious diseases (MRSA, hepatitis B and AIDS) in the world. Glycopeptide antibiotics are currently the primary therapeutic against MRSA, with vancomycin being referred to as the "last line of defense" in the treatment of MRSA infections. However, vancomycin-resistant Staphylococcus aureus (VRSA) has emerged, and thus the search for new and effective antibacterial agents for treating MRSA infections is urgent.

The plant endophyte is used as a new microbial resource, has wide distribution and various varieties, and some endophytes can even produce natural active substances which are the same as or similar to host plants, thus being an important way for screening antibacterial drugs with high activity and novel structure. Therefore, the method for producing natural active substances such as antibacterial agents, antitumor agents and the like by utilizing the endophytes not only provides a new way for the research and development of new drugs, but also can effectively protect rare or endangered medicinal plant resources and has important significance for reducing the damage of the diversity of wild medicinal plants.

Dracaena cochinchinensis, the scientific name of latin: (Dracaena cochinchinensis (Lour.) S.C.Chen) genus Liliaceae, there has not been reported any related report on the isolation of the tetrabasic lactone compound from the endophyte of the Dracaena cochinchinensis, and there has not been reported any report on the isolated tetrabasic lactone compound against MRSA.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of two quadruple lactone antibiotics as anti-MRSA medicines and an extraction and separation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses application of two quadruple lactone antibiotics in preparation of MRSA (methicillin resistant staphylococcus aureus) resistant medicines, wherein the structural formulas of the two quadruple lactone antibiotics are shown as the following formulas 1 and 2:

preferably, the drug has strong binding activity with the MRSA target protein PBP2 a.

Preferably, the medicine is a preparation prepared by adding pharmaceutically acceptable pharmaceutical excipients into the compound shown in the formula 1 or the formula 2.

The invention also discloses application of two quadruple lactone antibiotics as lead compounds for preparing MRSA (methicillin resistant Staphylococcus aureus) resistant medicines, wherein the structural formulas of the two quadruple lactone antibiotics are shown as the following formulas 1 and 2:

the invention also discloses a method for extracting and separating the quadruple lactone antibiotics from the endophytes of dracaena cochinchinensis, which comprises the following steps:

1) taking Streptomyces puniceus S009 as a material, performing fermentation culture by adopting an oat agar culture medium, then extracting, and concentrating the extract of the fermentation product to prepare a fermentation crude extract;

2) extracting the fermented crude extract by using an ethyl acetate-water system for 3-5 times, extracting an ethyl acetate phase by using a petroleum ether-methanol system for 3-5 times, combining the extract liquor, and concentrating the extract liquor to prepare a methanol extract;

3) performing normal-phase silica gel column chromatography on the methanol extract, performing gradient elution by taking dichloromethane-methanol system as eluent according to the ratio of 100: 1-2: 1, removing pigment components, performing Sephadex LH-20 column chromatography on non-pigment components, and performing dichloromethane-methanol chromatography according to the ratio of 1:2, performing proportional isocratic elution, detecting by TLC, developing by using an improved bismuth potassium iodide reagent, and combining to obtain a bismuth potassium iodide developing component Fr.2;

4) performing medium-pressure reverse phase column chromatography on a bismuth potassium iodide chromogenic component Fr.2, collecting 90% methanol-water elution fraction to prepare a crude product of the quadruple lactone, and performing HPLC separation to obtain two quadruple lactone antibiotics shown as the following formulas 1 and 2:

preferably, in the step 1), the fermentation culture adopts large-scale plate fermentation for 20L, and is carried out for 11 days at 28 ℃ in an inverted way;

the extraction is to cut the cultured culture medium into blocks, and extract the culture medium at room temperature overnight by using extract which is ethyl acetate, methanol and glacial acetic acid according to the weight ratio of 80:15:5, extracting for 3-5 times;

the concentration is carried out by concentrating the combined fermentation product extract under reduced pressure at 45 deg.C.

Preferably, in the step 2), the ethyl acetate and the water in the ethyl acetate-water system are in equal volume ratio, and the petroleum ether and the methanol in the petroleum ether-methanol system are in equal volume ratio;

the extract was concentrated under reduced pressure at 45 ℃.

Preferably, in step 3), TLC detection is performed by using dichloromethane-methanol 10:1 for development; improving the color development of the bismuth potassium iodide reagent, and collecting the orange flow part of the main spot, namely the quadruple lactone compound.

Preferably, in step 4), the HPLC chromatographic conditions are: Inertsustainin-C₁₈5 mu m,10 × 250mm and UV193nm under the condition of eluting with acetonitrile-water according to the ratio of 95:5, and collecting fractions with the retention time of 15.6min and 19.0min respectively to obtain the compound Dinactin of the formula 1 and the compound Trinactin of the formula 2 respectively.

Compared with the prior art, the invention has the following beneficial effects:

the invention separates two quadruple lactone compounds from Streptomyces puniceus S009 which is an endophyte of dracaena cochinchinensis for the first time, and adopts spectral techniques such as high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and the like to determine that the two compounds are respectively dinatin and trinatin, and the molecular formulas are respectively C₄₂H₆₈O₁₂And C₄₃H₇₀O₁₂. The structural feature is a series of homologues consisting of four homologous quadruple lactone rings consisting of tetramino furan-hydroxycarboxylic acid and derivatives thereof. The invention carries out in-vitro MRSA (methicillin resistant Staphylococcus aureus) activity test on the two separated compoundsThe results show that the dinacotin and trinacotin have stronger inhibitory action on various clinically isolated MRSA strains than a positive control vancomycin, and the molecular docking results show that the compounds have strong binding activity with MRSA-resistant target protein PBP2 a. Therefore, the invention not only can provide a lead compound for developing novel MRSA resistant medicines, but also can effectively protect rare and endangered medicinal plant resources, and has important significance for reducing the damage of the diversity of wild medicinal plants.

The invention discloses a method for separating two quadruple lactone compounds from Streptomyces puniceus S009, which is an endophyte of dracaena cochinchinensis.

Drawings

FIG. 1 is the interaction analysis of the optimal configuration of the compounds dinatin and trinatin of the present invention with the target protein PBP2a, wherein: a is Dinactin, B is Trinacin;

FIG. 2 shows the preparation of compound Dinactin (1)¹H NMR spectrum;

FIG. 3 shows the preparation of compound Dinactin (1)¹³A C NMR spectrum;

FIG. 4 is an HSQC spectrum of compound Dinactin (1);

FIG. 5 is an HMBC profile of compound Dinactin (1);

FIG. 6 is a NOESY map of compound Dinactin (1);

FIG. 7 is an HR-EI-MS spectrum of compound Dinactin (1);

FIG. 8 shows Trinacin (2) as a compound¹H NMR spectrum;

FIG. 9 shows Trinacin (2) as a compound¹³A C NMR spectrum;

FIG. 10 is an HSQC spectrum of compound Trinacin (2);

FIG. 11 is an HMBC profile of compound Trinacin (2);

FIG. 12 is a NOESY plot of compound Trinactin (2);

FIG. 13 is an HR-EI-MS profile of compound Trinacin (2).

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

example 1: preparation of two quadruple lactone compounds

1. Strain source and identification

The compounds of formula 1 and formula 2 were isolated from the fermentation product of a. xyphoides endophyte provided by dawn Mei subject group of the basic medical college of Yunnan university of traditional Chinese medicine and identified as Streptomyces puniceus S009 by 16S rRNA sequencing analysis.

Isolation and characterization of Streptomyces puniceus S009

1.1. Strain isolation

Washing healthy plant sample with flowing water, air drying at room temperature for 48h, and ultrasonic cleaning at 160W for 5min to remove surface of plantSoil and organic residue. And the surface sterilization of the plant sample is carried out according to the following process: soaking in 70% ethanol for 5min, washing the sample with sterile water for three times, soaking in 2% sodium hypochlorite for 5min, washing the sample with sterile water for three times, soaking in 10% sodium bicarbonate for 10min, and washing the sample with sterile water for three times. Air drying the plant sample with sterilized surface in sterile environment, treating at 80 deg.C for 30min, pulverizing the sample with sterile pulverizer, smearing on the surface of the isolated culture medium, and culturing at 28 deg.C. After the actinomycetes grow out, picking the monoclone to YIM38^#Purifying and culturing on a solid culture medium, transferring the obtained monoclonal to ISP2 slant for culturing and short-term storage, named as S009, and preparing glycerin tube and milk tube of strain for long-term storage at-80 deg.C.

1.2. Identification of strains

(1) Morphology, culture characteristics: strain S009 in YIM38^#The growth vigor on the culture medium is better, the spores are produced, the spores are gray, the colony is round, the edge is neat, and the purplish red pigment is produced.

(2) Amplification and phylogenetic analysis of the 16S rRNA gene: extracting the genome of the strain, and performing gene amplification by using a bacterial 16SrRNA gene universal primer PA: 5'-CAG AGT TTG ATC CTG GCT-3', PB: 5'-AGG AGG TGA TCC AGCCGC A-3' PCR amplification was performed. The amplification conditions were: pre-denaturation at 94 ℃ for 5 min; 30 cycles: denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 1min, and extension at 72 deg.C for 3 min; total extension at 72 ℃ for 5 min. The resulting PCR product was recovered on a 0.8% agarose gel and sequenced. The resulting sequence was subjected to a similarity search by the EzTaxon-e online alignment service in ezbiocoud, i.e. S009 was identified as a Streptomyces strain (Streptomyces sp.).

1.3. The culture medium used:

ISP 2: 4g of yeast extract, 4g of glucose, 10g of malt extract, 15g of agar and 1000mL of water, and the pH value is 7.2-7.6.

YIM38 #: 5g of malt extract, 4g of yeast extract, 4g of glucose, 1mL of trace salt solution, trace vitamin B complex, 15g of agar, 1000mL of water and pH 7.2.

2. Extraction and isolation of compounds

1) Preparing an oat agar culture medium, which comprises the following specific steps: 20.0g of oatmeal (ground into powder), adding water, boiling for about 20 minutes, filtering to obtain filtrate, adding 1mL of trace inorganic salt solution, adding pure water to reach a constant volume of 1000mL, and adjusting the pH value to 7.2-7.4; the solid culture medium is added with 2% agar, and autoclaved at 121 deg.C for 30 min.

2) The endophyte Streptomyces puniceus S009 of dracaena cochinchinensis is fermented for 20L by oat agar medium plate, and is cultivated for 11 days at 28 ℃ in an inverted way. Cutting, soaking at room temperature for 3 times to extract fermented product (the extractive solution is ethyl acetate: methanol: glacial acetic acid: 80:15:5, v/v/v), mixing extractive solutions, and concentrating at 45 deg.C under reduced pressure to obtain fermented crude extract.

3) The fermented crude extract is subjected to equal-volume extraction phase separation by ethyl acetate and water, an ethyl acetate phase (anhydrous Na2SO4 for water removal) and a water phase are respectively combined, the concentrated ethyl acetate extract is subjected to equal-volume extraction phase separation by petroleum ether and 95% methanol, and the petroleum ether and methanol extracts are respectively obtained after vacuum concentration at 45 ℃ until the concentrated ethyl acetate extract is dried.

4) Performing normal-phase silica gel column chromatography on the methanol extract, performing gradient elution by taking a dichloromethane-methanol system (100: 1-2: 1) as an eluent, removing pigment components, performing Sephadex LH-20 column chromatography (160g) on non-pigment components, performing isocratic elution by using dichloromethane/methanol 1:2(v/v), receiving a sample by using an automatic collector, detecting by using silica gel thin-layer chromatography, developing by using dichloromethane-methanol 15:1, improving the color development of a bismuth potassium iodide reagent, and collecting a component with an orange main spot to obtain a component Fr.2.

5) Fr.2 by medium pressure reverse phase column chromatography (RP-18,40g), eluting with water, 30%, 50%, 70%, 90% methanol-water and 100% methanol sequentially, each eluting with 400mL gradient, collecting 90% methanol-water eluate to obtain crude quadruple lactone 2.5g, collecting 630mg of the component, and further performing HPLC (Inertsutain-C)₁₈5 μm,10 × 250mm, UV193 nm), acetonitrile-water 95:5(v/v) elution, and fractions with retention times of 15.6min and 19.0min were collected to give dinatin (93mg) and trinatin (10mg), respectively.

The chemical structural formulas of the compounds Dinactin (1) and Trinactin (2) are as follows:

example 2: structure identification of two quadruple lactone compounds

The compound Dinactin (1) prepared in example 1 is colorless oil, and is easily soluble in chloroform, acetone and DMSO. FIGS. 2 to 7 are structural analysis maps of Compound Dinactin (1),

HR-ESI-MS is: m/z 787.4590[ M + Na ]]⁺(positive ion mode), and the molecular formula is determined to be C₄₂H₆₈O₁₂. From¹H NMR and¹³c NMR data show that the nuclear magnetic data of the compound 1 is basically consistent with the data of dinactin (Diactive biotin) reported in the literature, and NOESY related signals

And optical rotation data further confirm that the stereoconfiguration of compound 1 is consistent with literature reports. Compound 1 was thus identified as dinactin, as shown in table 1:

TABLE 1 of the compound Dinactin¹H (400MHz) and¹³c (100MHz) NMR data (solvent: CDCl)₃)

The compound Trinactin (2) of example 1 is a colorless oil, readily soluble in chloroform, acetone and DMSO. FIGS. 8 to 13 are structural analysis maps of compound Dinactin (1), wherein HR-ESI-MS is as follows: m/z796.5185[ M + NH ]₃]⁺，m/z801.4741[M+Na]⁺(positive ion mode), and the molecular formula is determined to be C₄₃H₇₀O₁₂。

Of comparative compounds 1 and 2¹H NMR and¹³c NMR data show that the nuclear magnetic data of the compound 2 is basically consistent with the data of Trinacin (triple viable bacteria) reported in the literature, so that the nuclear magnetic data of the compound 2 are basically consistent with the data of Trinacin (triple viable bacteria) reported in the literatureAnd identifying the compound 2 as Trinactin.

Example 3: study of in vitro anti-MRSA effects of Compounds Dinactin (1) and Trinactin (2)

1. Indicator bacteria for experiment

9 strains of MRSA resistant bacteria used in the present invention: 1505. 1450, 1591, 1957, 2024, I-20, I-67, 28299 and 28300, which are all separated from the patients in the first civil hospital of Jingjing, are resistant to methicillin and sensitive to vancomycin.

2. Sample to be tested

Dinatin (1), trinatin (2), vancomycin (positive control), DMSO (negative control)

3. Culture medium

Liquid LB medium: 10g of peptone; 5g of yeast extract; 10g of NaCl; 15g of agar; 1000mL of water; the pH value is 7.2-7.6.

4. Experimental methods

1) Preparation of bacterial liquid

Inoculating the purified indicator bacteria into corresponding liquid culture medium respectively, inoculating MRSA into LB culture medium, culturing at 37 deg.C for 12-24 hr, diluting with corresponding liquid culture medium to obtain bacteria 0.5 × 10⁶CFU/mL。

2) Preparation of test article

Dinacin (1), Trinacin (2) and vancomycin were prepared in DMSO to be 25.6mg/mL solutions, respectively.

3) Minimum Inhibitory Concentration (MIC) test

MIC was measured by a double dilution method and 3 replicates were performed. Adding 2uL of test sample and 196uL of liquid culture medium into the test tube in the 1 st hole, uniformly mixing, putting 99uL of mixed solution into the 2 nd hole, and supplementing 99uL of culture medium into the first hole; adding 99uL culture medium into the second hole, uniformly mixing, sucking 99uL mixed solution from the second hole, adding the 99uL mixed solution into the third hole, and supplementing the 99uL culture medium into the second hole; and repeating the steps until the 11 th hole, taking 99uL and discarding, and adding 2uL of the bacterial liquid into the first 11 holes. Well number 12 for each set of experiments was: 200uL of blank liquid culture medium, 2uL of bacterial liquid and 198uL of liquid culture medium, and 2uL of test sample and 198uL of liquid culture medium. Thus, the experimental concentrations were, in order: 128, 64, 32, 16, 8,4, 2, 1, 0.5, 0.25, 0.125 ug/mL. Culturing in a constant temperature incubator for 12-24 hr, and observing and recording every 12 hr. And judging the result by visual observation, and taking the growth of no bacteria in the drug minimum concentration tube as the Minimum Inhibitory Concentration (MIC) of the test bacteria.

5. Results of the experiment

TABLE 2 minimum inhibitory concentration (ug/mL) of Compounds dinatin (1), trinatin (2) and vancomycin against MRSA strains

Experimental results show that the quadruple lactone compounds dinatin (1) and trinatin (2) have obvious inhibition effects on various MRSA strains, the inhibition activity (MIC) of the quadruple lactone compounds dinatin (1) and trinatin (2) on the MRSA strains is higher than that of a positive control vancomycin, and the MIC value of dinatin on the multiple clinically isolated MRSA strains is less than 0.125 ug/mL.

EXAMPLE 4 examination of the binding Activity of Compounds Dinactin and Trinactin to the MRSA target PBP2a

The main drug resistance mechanism of MRSA is related to mecA gene, which can encode Penicillin Binding Protein 2a (PBP2a), which differs from common PBPs in that TPasedomain has poor Binding ability to β -lactam antibiotics, i.e., very low affinity. When the activity of the beta-lactam drugs is lost due to the combination of the beta-lactam drugs and normal PBPs, the PBP2a can be used as a substitute for the original PBPs to perform functions, so that bacteria can continuously maintain the capability of synthesizing peptidoglycan, thereby ensuring that the MRSA not only can survive, but also can continuously grow. Therefore, in order to further study the target of the compound of the S009 strain against MRSA, we intend to perform molecular docking with PBP2a, which is the known target of MRSA, as a receptor and with Dinectin and Trinectin as ligands, respectively.

1. Experimental methods

Molecule docking step:

(1) the Pubchem database downloads the compound 2D/3D structure, and the 2D structure file without the 3D structure is converted into a mol2 format file by Chem 3D.

(2) Inputting the PBP2a protein information into a PDB database (https:// www.rcsb.org /), and downloading the appropriate protein-ligand complex structure;

(3) removing water molecules and ligands in the target protein by adopting PyMoL 1.7.2.1, and storing the ligands and the protein as pdb format files;

(4) converting a target protein, a ligand and a compound into a pdbqt format file by adopting AutoDock Vina 1.1.2;

(5) searching for the active site of the target protein by taking the ligand as a center, and setting Grid Box coordinates and Box size;

(6) performing molecular docking by adopting AutoDock Vina 1.1.2;

(7) the best conformation of affinity was chosen as the final docking and PyMoL was used to map the interaction.

2. Results of the experiment

The molecular docking results show that: the affinity values of the compounds 1 and 2 and the target protein PBP2a are-7.1 and-7.2 kcal/mol respectively, which indicates that the compounds 1 and 2 and the target protein PBP2a both have stronger binding activity (as shown in A, B in figure 1), and is the basis for the identification of the action mechanism of the compounds for resisting MRSA in the next step.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. The application of two quadruple lactone antibiotics in preparing MRSA (methicillin resistant Staphylococcus) resistant medicines is characterized in that the structural formulas of the two quadruple lactone antibiotics are shown as the following formulas 1 and 2:

2. the use of claim 1, wherein the medicament is a medicament having strong binding activity to the anti-MRSA target protein PBP2 a.

3. The use of claim 1, wherein the medicament is a preparation prepared by adding pharmaceutically acceptable pharmaceutical excipients into the compound shown in formula 1 or formula 2.

4. The application of two quadruple lactone antibiotics as lead compounds for preparing MRSA (methicillin resistant Staphylococcus aureus) medicines is characterized in that the structural formulas of the two quadruple lactone antibiotics are shown as the following formulas 1 and 2:

5. a method for extracting and separating quadruple lactone antibiotics from dracaena cochinchinensis endophytes is characterized by comprising the following steps:

6. the method for extracting and separating the quadruple lactone antibiotics from the endophytes of the dracaena cochinchinensis as claimed in claim 5, wherein in the step 1), the fermentation culture adopts large-scale plate fermentation for 20L, and the inversion culture is carried out at 28 ℃ for 11 days;

7. The method for extracting and separating the quadruple lactone antibiotics from the endophytes of the dracaena cochinchinensis as claimed in claim 5, wherein in the step 2), the ethyl acetate and the water in the ethyl acetate-water system are in equal volume ratio, and the petroleum ether and the methanol in the petroleum ether-methanol system are in equal volume ratio;

the extract was concentrated under reduced pressure at 45 ℃.

8. The method for extracting and separating the quadruple lactone antibiotics from the endophyte of the dracaena cochinchinensis as claimed in claim 5, wherein in the step 3), TLC detection is performed by adopting dichloromethane-methanol 10:1 for development; improving the color development of the bismuth potassium iodide reagent, and collecting the orange flow part of the main spot, namely the quadruple lactone compound.

9. The method for extracting and separating the quadruple lactone antibiotics from the endophyte of the dracaena cochinchinensis as claimed in claim 5, wherein in the step 4), HPLC chromatographic conditions are as follows: Inertsustainin-C₁₈5 mu m,10 × 250mm and UV193nm, wherein the elution condition is that acetonitrile-water is eluted according to the ratio of 95:5, and fractions with the retention time of 15.6min and 19.0min are collected to obtain the compound dinatin shown in the formula 1 and the compound trinatin shown in the formula 2 respectively.