CN113149938A - Labdane diterpenoid compound, preparation method and application - Google Patents
Labdane diterpenoid compound, preparation method and application Download PDFInfo
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- CN113149938A CN113149938A CN202110415715.3A CN202110415715A CN113149938A CN 113149938 A CN113149938 A CN 113149938A CN 202110415715 A CN202110415715 A CN 202110415715A CN 113149938 A CN113149938 A CN 113149938A
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/58—One oxygen atom, e.g. butenolide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Neurosurgery (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Psychology (AREA)
- Hospice & Palliative Care (AREA)
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- Physical Education & Sports Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a labdane diterpenoid compound, a preparation method and application, and relates to the technical field of natural medicines. The labdane diterpenoid compound is separated from callicarpa nudiflora leaves, has the effect of neuroprotection, and can be used for preparing a neuroprotection composition for treating or preventing neurodegenerative diseases. The preparation method is simple and easy to implement, and has good economic benefit and application prospect.
Description
Technical Field
The invention relates to the technical field of natural medicines, and particularly relates to a labdane diterpenoid compound, a preparation method and application thereof.
Background
Callicarpa nudiflora (Callicarpa nudiflora hook. et Arm) is a Callicarpa L plant of Verbenaceae (Verbenaceae), the application part is dry leaf, and the Callicarpa nudiflora mainly grows in the domestic areas of Hainan, Jiangxi, Guangdong, Guangxi and the like, and the foreign areas of Singapore, India, Vietnam, Malaysia and the like. The beautyberry is recorded in the first Chinese redbud in Tang dynasty collection (materia medica, Shi Yi); the beautyberry wine is widely used by native medicine of Li nationality in Hainan province, and is named independently in the 50 s of the last century; in 2020, a part of 'Chinese pharmacopoeia' (2020 edition) is newly added and recorded. Callicarpa nudiflora is bitter and slightly pungent in taste and mild in nature, has the effects of stopping bleeding, resisting inflammation, reducing swelling, clearing away heat and toxic materials, eliminating dampness and removing turbidity and the like, and is mainly used for clinically treating various diseases such as inflammatory pyogenic infections caused by internal and external injury bleeding and bacterial infection, acute infectious hepatitis and the like.
At present, the chemical components of Callicarpa nudiflora and the active components thereof are not clear.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a labdane diterpenoid compound, a preparation method and application to solve the technical problems.
The invention is realized by the following steps:
the invention provides a labdane diterpenoid compound which has the following structural formula:
a preparation method of labdane diterpenoid compounds comprises the following steps:
extracting folium Callicarpae Formosanae with ethanol under reflux, and concentrating to obtain total extract;
subjecting the obtained total extract to adsorption resin, gradient eluting with water and 25% -100% ethanol respectively, collecting ethanol eluate, and removing ethanol and water to obtain crude extract;
separating the crude extract by silica gel column chromatography, carrying out gradient elution by eluent to obtain 6 components Fr.1-Fr.6, carrying out silica gel column chromatography on the Fr.3 component, carrying out gradient elution to obtain 8 components Fr.3-1-Fr.3-8, carrying out gradient elution on the Fr.3-2 component by silica gel column to obtain 7 components Fr.3-2-1-Fr.3-2-7, carrying out ODS column chromatography on the Fr.3-2-4 component, carrying out gradient elution by using 20%, 40% and 60% acetonitrile aqueous solution in sequence, and carrying out liquid phase column chromatography on the eluent after gradient elution of acetonitrile aqueous solution to obtain the labdane diterpenoid compound.
In a preferred embodiment of the present invention, the adsorption resin is DA-201 macroporous adsorption resin, and gradient elution is performed with water, 25%, 50%, 75% and 95% ethanol, respectively.
In a preferred embodiment of the present invention, the elution part of the collected 50% ethanol solution is subjected to a rotary evaporation method to obtain a crude extract.
In a preferred embodiment of the present invention, the crude extract is purified by silica gel column chromatography using chloroform in a volume ratio of 200-1: gradient eluting with methanol, combining and collecting to obtain 6 components Fr.1-Fr.6; and the Fr.3 component is eluted by silica gel column chromatography in the volume ratio of 50-1:1 of chloroform: gradient elution with methanol, merging and collecting to obtain 8 components Fr.3-1-Fr.3-8, passing the Fr.3-2 component through silica gel column, sequentially mixing chloroform: gradient eluting with methanol, combining and collecting 7 Fr.3-2-1-Fr.3-2-7 components, subjecting the Fr.3-2-4 component to ODS column chromatography, and gradient eluting with 20%, 40%, and 60% acetonitrile water solution sequentially.
In a preferred embodiment of the invention, the Fr.3-2-4 component is subjected to ODS column chromatography and gradient elution by acetonitrile in water, acetonitrile and water are removed by rotary evaporation, then the product obtained after removing the acetonitrile and the water is subjected to liquid phase column chromatography, gradient elution is carried out by taking acetonitrile and water with the volume ratio of 20-40:1 as a mobile phase, the detection wavelength is 265nm and/or 300nm, the flow rate is 8-12ml/min, and the retention time is 65.72 +/-10 min, so as to obtain the labdane diterpenoid compound.
In the preferred embodiment of the invention, in the ethanol reflux extraction step, 60-70% ethanol is used for reflux extraction for at least 2 times, each time for 1.5-4h, the extracting solutions are combined and concentrated to prepare the total extract;
the reflux extraction is a continuous reflux extraction method.
The invention also provides an application of the labdane diterpenoid compound or the labdane diterpenoid compound prepared by the preparation method in preparing the neuroprotective composition.
The invention also provides a neuroprotective composition comprising the labdane diterpenoid compound or the labdane diterpenoid compound prepared by the preparation method.
In a preferred embodiment of the present invention, the neuroprotective composition further comprises a pharmaceutically acceptable additive or adjuvant; preferably, the neuroprotective composition is in a dosage form selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays, or ampoules.
In a preferred embodiment of the present invention, the above neuroprotective composition is used for treating or preventing neurodegenerative diseases, preferably, neurodegenerative diseases include at least one of the following diseases: alzheimer's disease, mild cognitive impairment, Huntington's disease, prion-induced diseases, frontotemporal dementia, Lewy body dementia, vascular dementia, amyotrophic lateral sclerosis, chronic traumatic encephalopathy, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, pick's disease and olivopontocerebellar atrophy.
The invention has the following beneficial effects:
the labdane diterpenoid compound is separated from callicarpa nudiflora leaves, has the effect of neuroprotection, and can be used for preparing a neuroprotection composition for treating or preventing neurodegenerative diseases. The preparation method is simple and easy to implement, and has good economic benefit and application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the structure of labdane diterpenoid compound isolated in example 1 and key HMBC, B,1H-1An H COSY signal;
FIG. 2 is a liquid chromatogram of separated labdane diterpenoid;
FIG. 3 is a drawing of Compound 11H-NMR spectrum (deuterated methanol);
FIG. 4 is a drawing of Compound 113C-NMR spectrum (deuterated methanol);
figure 5 is an HMQC spectrum (deuterated methanol) of compound 1;
figure 6 is an HMBC map (deuterated methanol) of compound 1;
FIG. 7 is a drawing of Compound 11H-1H COSY spectrum (deuterated methanol);
FIG. 8 is a NOESY spectrum (deuterated methanol) of Compound 1;
FIG. 9 is a HR-ESI-MS spectrum (deuterated methanol) of Compound 1.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
The existing research shows that the chemical components in the callicarpa nudiflora mainly comprise flavonoids, phenethyl alcohol glycosides, diterpenes, triterpenes, iridoids, glycosides and the like, and the inventor continuously performs chemical component research on the callicarpa nudiflora on the basis of previous research, finds and separates to obtain a new compound, namely a labdane diterpenoid compound. The compound has neuroprotective effect, and is suggested to be used for new pharmaceutical application.
Specifically, the invention provides a labdane diterpenoid compound.
The name of labdane diterpenoid is:
(5E) -4-methyl-5- [ ((1R,2S,3S,4R,4aS,8aS) -decahydro-2,3,4-trihydroxy-2,5,5,8a-tetramethyl-1-naphthalenyl) methyl ] -2(5H) -furanone, (5E) -4-methyl-5- [ ((1R,2S,3S,4R,4aS,8aS) -decahydro-2,3,4-trihydroxy-2,5,5,8 a-tetramethyl-1-naphthyl) methylene ] -2(5H) -furanone, hemieicosane-type diterpenoids having the following structural formula:
the inventors named the above labdane-type diterpenoid compound callicapen M6.
HR-ESI-MS showed M/z 373.1981 [ M + Na ]]+(calculated 373.1985), 723.4072 [2M + Na]+(calculated 723.4078), and recombination of the compounds1H-NMR of13C-NMR spectral data of the compound of the formula C20H30O5The unsaturation degree was 6.
The invention also provides a preparation method of the labdane diterpenoid compound, which comprises the following steps:
extracting folium Callicarpae Formosanae with ethanol under reflux, and concentrating to obtain total extract;
subjecting the obtained total extract to adsorption resin, gradient eluting with water and 25% -100% ethanol respectively, collecting ethanol eluate, and removing ethanol and water to obtain crude extract;
separating the crude extract by silica gel column chromatography, carrying out gradient elution by eluent to obtain 6 components Fr.1-Fr.6, carrying out silica gel column chromatography on the Fr.3 component, carrying out gradient elution to obtain 8 components Fr.3-1-Fr.3-8, carrying out gradient elution on the Fr.3-2 component by silica gel column to obtain 7 components Fr.3-2-1-Fr.3-2-7, carrying out ODS column chromatography on the Fr.3-2-4 component, carrying out gradient elution by using 20%, 40% and 60% acetonitrile aqueous solution in sequence, and carrying out liquid phase column chromatography on the eluent after gradient elution of acetonitrile aqueous solution to obtain the labdane diterpenoid compound.
In other embodiments, additional columns may be selected to separate the crude extract as desired.
It should be noted that the labdane diterpenoid compounds and sterebin A (stevia rebaudiana Bertoni A) can be obtained simultaneously after the eluent is separated by liquid column chromatography. The separation of the two can be realized according to the sequence of the products eluted by the liquid phase column chromatography.
Separating the acetonitrile water solution eluent by liquid phase column chromatography, taking acetonitrile and water with the volume ratio of 40:1 as mobile phases, detecting the wavelength of 265nm and/or 300nm, the flow rate of 10ml/min and the retention time of 51.56min to prepare sterebin A.
It should be noted that, sterebin a may be collected alone at the detection wavelength of 265nm or 300nm, or may be collected at the detection wavelengths of 265nm and 300nm at the same time.
In a preferred embodiment of the present invention, the adsorption resin is DA-201 macroporous adsorption resin, and gradient elution is performed with water, 25%, 50%, 75% and 95% ethanol, respectively. The DA-201 macroporous adsorption resin can adsorb various organic compounds with polarity which are difficult to dissolve in water and highly dissolved in an ethanol organic solvent, and the aim of enriching the phenylethanoid glycosides compounds can be achieved.
In other embodiments, the gradient elution may also be performed with ethanol selected from water, 25%, 50%, 60%, 70%, 80%, 95%. It should be noted that the above-mentioned gradient elution with water and 25% -100% ethanol is not limited to the several gradient elution embodiments of the present invention.
In a preferred embodiment of the present invention, the elution part of the collected 50% ethanol solution is subjected to a rotary evaporation method to obtain a crude extract. And dealcoholizing and dehydrating the elution part of the 50 percent ethanol solution by a rotary evaporator. In other embodiments, the elution part of 25% and 50% ethanol solution can be collected for dealcoholization and dehydration, and the products after dealcoholization and dehydration are mixed to obtain the corresponding crude extract. The above-mentioned collection of the elution portion of the 50% ethanol solution to carry out the dealcoholization and dehydration treatment is only one preferred embodiment.
In another embodiment, 25%, 50% and 75% ethanol solution elution parts can be collected for dealcoholization and dehydration treatment, and products after dealcoholization and dehydration treatment are mixed to obtain corresponding crude extracts.
Subjecting the crude extract to silica gel column chromatography with chloroform at volume ratio of 200-1: gradient eluting with methanol, merging and collecting to obtain 6 components Fr.1-Fr.6. For example, 200:1, 180:1, 150:1, 120:1, 100:1, 70:1, 40:1, 10:1, 1:1 chloroform: methanol gradient elution.
And the Fr.3 component is eluted by silica gel column chromatography in the volume ratio of 50-1:1 of chloroform: gradient eluting with methanol, merging and collecting to obtain 8 components Fr.3-1-Fr.3-8. For example, 50:1, 40:1, 30:1, 20:1, 10:1, 1:1 chloroform: methanol gradient elution.
The Fr.3-2 components are treated by a silica gel column, and chloroform with the volume ratio of 200-8:1 is sequentially used: gradient eluting with methanol, combining and collecting 7 Fr.3-2-1-Fr.3-2-7 components, subjecting the Fr.3-2-4 component to ODS column chromatography, and gradient eluting with 20%, 40%, and 60% acetonitrile water solution sequentially.
The crude extract was subjected to silica gel column chromatography in order to subject the crude extract to a preliminary separation treatment to remove a part of impurities. The mixed liquid of chloroform and methanol is selected as the eluent used for the silica gel column chromatography, because the polarity of the mixed liquid is consistent with that of the target product, the spots are favorably separated to a better degree, and the inventor finds that if the mixed eluent of ethyl acetate and methanol is used, the separation of each point cannot be realized, and the separation effect is poor.
In a preferred embodiment of the invention, the Fr.3-2-4 component is subjected to ODS column chromatography and gradient elution by acetonitrile in water, acetonitrile and water are removed by rotary evaporation, then the product obtained after removing the acetonitrile and the water is subjected to liquid phase column chromatography, gradient elution is carried out by taking acetonitrile and water with the volume ratio of 20-40:1 as a mobile phase, the detection wavelength is 265nm and/or 300nm, the flow rate is 8-12ml/min, and the retention time is 65.72 +/-10 min, so as to obtain the labdane diterpenoid compound.
After removing acetonitrile and water by rotary evaporation, a small amount of solvent is added for dissolution, so that the subsequent separation on a chromatographic column is convenient. The inventors have found that the eluate can be collected by detection at a wavelength of 265nm, at a wavelength of 300nm, or at both 265nm and 300 nm.
In one embodiment, the collection is selective as long as peaks are present, and the retention time is related to the type of column and chromatographic conditions of the chromatographic separation.
In one embodiment, the flow rate may be set at 10 ml/min.
The acetonitrile water solution is used as the eluent of ODS column chromatography, so that the separation of the target product can be better realized.
In a preferred embodiment of the present invention, in the step of ethanol reflux extraction, 60-70% ethanol is used for reflux extraction at least 2 times, each time for 1.5-4h, the extract solutions are combined, and concentrated to obtain a total extract;
the reflux extraction is a continuous reflux extraction method. The reflux extraction is a continuous reflux extraction method. In another embodiment, the extraction step of the raw material may be performed by other methods such as ultrasonic extraction, as needed.
The number of reflux extractions may be 2,3,4 or more.
The invention also provides an application of the labdane diterpenoid compound or the labdane diterpenoid compound prepared by the preparation method in preparing the neuroprotective composition.
The neuroprotective composition can be a medicine, a preparation or a freeze-dried powder. Can be used for preparing the medicine for protecting the nerves of the brain, the optic nerve and the like.
The invention also provides a neuroprotective composition comprising the labdane diterpenoid compound or the labdane diterpenoid compound prepared by the preparation method.
In a preferred embodiment of the present invention, the neuroprotective composition further comprises a pharmaceutically acceptable additive or adjuvant; preferably, the dosage form of the medicament is selected from tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays or injections.
In a preferred embodiment of the present invention, the above neuroprotective composition is used for treating or preventing neurodegenerative diseases, preferably, neurodegenerative diseases include at least one of the following diseases:
alzheimer's Disease (AD), Parkinson's Disease (PD), Mild Cognitive Impairment (MCI), Huntington's Disease (HD), prion-induced diseases, frontotemporal dementia (FTD), lewy body dementia, vascular dementia, Amyotrophic Lateral Sclerosis (ALS), Chronic Traumatic Encephalopathy (CTE), Progressive Supranuclear Palsy (PSP), Multiple System Atrophy (MSA), corticobasal degeneration (CBGD), pick's disease, and olivopontocerebellar atrophy (OPCA).
In other embodiments, the neurodegenerative diseases (NDD) described above are clinically manifested in patients with symptoms such as decreased memory, cognitive impairment, dementia, motor balance disorders, and loss of motor ability.
It should be noted that the above list is only a few possible diseases listed by the inventor, and in other embodiments, the invention is not limited to the types of the above listed diseases as long as the effects of alleviating and curing neurodegenerative diseases can be achieved within the scope of the present invention.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
The equipment used in the examples:
bruker AVANCE III HD 600MHz type nuclear magnetic resonance spectrometer (Bruker, Switzerland), AB SCIEX Triple ESI 5600+ type high resolution time-of-flight mass spectrometer (AB SCIEX, USA), Agilent 1260-II type high performance liquid phase (Agilent, USA), Elite P3500 semi-preparative liquid chromatograph (Elite analytical instruments, Inc., Dalianite), YMC-Triart C18 semi-preparative chromatographic column (50 mm. times.250 mm,10 μm, Japan YMC, Inc.), DA-201 macroporous adsorbent resin (Zheng and New technology, Inc.), 100-200 mesh, 200-300 mesh column chromatographic silica gel (Qingdao ocean), thin-layer silica gel plate 254 (Qingdao ocean), MS204S/01 type analytical balance (Mettler Toledo, Switzerland), Milli Q ultra pure water machine (Millipore), REPROSTAR type 3 thin layer chromatographic scanning imaging system (CAMAG, Switzerland CMA), TLC Plate Heater (CAMAG, Switzerland) the reagents used were either chromatographically pure or analytically pure.
Example 1
The example provides a preparation method of labdane diterpenoid compounds, the Callicarpa nudiflora medicinal material in the example is purchased from Henan Baisha Hensheng source plant professional cooperative, is identified as Callicarpa nudiflora hook.et Arm by professor Van tresson of Jiangxi traditional Chinese medicine university, and a voucher specimen (NO. L201905Z) is stored in a scientific research center of Jiangxi traditional Chinese medicine valley.
The preparation method comprises the following steps:
extracting dry folium Callicarpae Formosanae 5.0kg with 15 times of 65% ethanol under reflux for 2 times each for 2 hr, filtering while hot, mixing extractive solutions, and concentrating under reduced pressure until no alcohol smell exists (to obtain total extract).
Centrifuging, separating the supernatant with DA-201 macroporous adsorbent resin column chromatography, gradient eluting with water, 25%, 50%, 75% and 95% ethanol, and recovering solvent under reduced pressure to obtain 25% ethanol, 50% ethanol (250.93g), 75% ethanol and 95% ethanol. The 50% ethanol fraction was subjected to rotary evaporator to remove alcohol and water to obtain 50% ethanol extract (250.93g crude extract).
Separating the extract (250.93g) by silica gel column chromatography (8.0cm is multiplied by 100.0cm), sequentially carrying out gradient elution by chloroform and methanol in a volume ratio of 200: 1-1: 1 (in the embodiment, the gradient elution is sequentially carried out by 200:1, 50:1, 10:1, 5:1 and 1:1, so as to achieve the purpose of coarse separation and ensure that the polar span of the eluent is large), and combining more consistent main spots by using a spot plate to obtain 6 components Fr.1-Fr.6.
Passing the Fr.3 component (21.52 g of Fr.3 component in the present example collected from the 10:1 eluate) through a silica gel column, sequentially eluting with chloroform at a volume ratio of 50:1 to 1: methanol is subjected to gradient elution (the volume ratio of gradient elution in the embodiment is 50:1, 25:1, 15:1, 10:1, 8:1, 4:1, 1:1), and the spot plates are combined with a large and consistent amount of main spot parts to obtain 8 components from Fr.3-1 to Fr.3-8. 6.12g of the thus-collected Fr.3-2 (in this example, the Fr.3-2 fraction was collected from the 50:1 eluate for the Fr.3 fraction) was passed through a silica gel column, and gradient-eluted with chloroform and methanol (gradient-eluted at a volume ratio of 200:1, 100:1, 50:1, 20:1, 10:1, 8:1, respectively) to obtain 7 fractions Fr.3-2-1 to Fr.3-2-7. Subjecting Fr.3-2-4(Fr.3-2-4 fraction collected from the eluate of 50:1 from Fr.3-2 fraction, 0.82g) to ODS column chromatography, gradient eluting with 20%, 40%, and 60% acetonitrile water solution, combining clean and consistent main spots on the spot plate, and separating by semi-preparative liquid phase column chromatography to obtain labdane diterpenoid (2.8mg) and steviosin A (18.2 mg).
In this example, the Fr.3-2-4 components were purified by ODS column chromatography using, in order, 20 volume percent: 80. 40:60, 50: gradient elution is carried out on 50 acetonitrile aqueous solution (the elution adjustment is shown in the following table), clean and consistent main spots are combined on a spot plate, acetonitrile and water are removed by evaporation of the acetonitrile aqueous solution eluent through a rotary evaporator, a solvent (methanol is used as the solvent) capable of dissolving part of a rotary evaporation product is added into the rotary evaporation product, liquid phase column chromatographic separation is carried out, the acetonitrile and the water with the volume ratio of 20:1, 30:1 and 40:1 are used as a mobile phase, the detection wavelength is 265nm and 300nm, the flow rate is 10ml/min, the retention time is 65.72min, the labdane diterpenoid compound is prepared, and sterebin A is obtained at the retention time of 51.56min, and the liquid chromatogram is shown in figure 2.
| Time (min) | Flow rate (ml/min) | Acetonitrile (%) | Water (%) |
| 0 | 10 | 20 | 80 |
| 60 | 10 | 40 | 60 |
| 70 | 10 | 40 | 60 |
| 85 | 10 | 50 | 50 |
And (3) structural identification: mainly using nuclear magnetic resonance (1H-NMR of13C-NMR spectrum data), further analyzing the HMBC spectrum data of the compound while combining the HMQC spectrum data, thereby constructing a planar structure of the compound (shown in fig. 1), wherein the labdane diterpenoid compound is a pale yellow solid,
molecular formula C20H30O5The unsaturation degree was 6. HR-ESI-MS showed M/z 373.1981 [ M + Na ]]+(calculated 373.1985), 723.4072 [2M + Na]+(calculated 723.4078).
The spectral data are shown in Table 1.
TABLE 1 preparation of Compound 11H and13C-NMR data (600/125MHz, CD)3OD)
aRepresenting overlapping hydrogens.
FIG. 3 is a drawing of Compound 11H-NMR spectrum (deuterated methanol); FIG. 4 is a drawing of Compound 113C-NMR spectrum (deuterated methanol); figure 5 is an HMQC spectrum (deuterated methanol) of compound 1; figure 6 is an HMBC map (deuterated methanol) of compound 1; FIG. 7 is a drawing of Compound 11H-1H COSY spectrum (deuterated methanol); FIG. 8 is a NOESY spectrum (deuterated methanol) of Compound 1; FIG. 9 is a HR-ESI-MS spectrum (deuterated methanol) of Compound 1.
The data in Table 1 show 5 unimodal methyl signals [ Delta ]H：2.20(3H,s,H-16)、1.23(3H,s,H-17)、1.16(3H,s,H-18)、1.01(3H,s,H-19)、1.02(s,3H,H-20)]Two alkene hydrogen proton signals [ delta ]H：5.55(1H,d,J＝11.2Hz,H-11)、5.99(1H,s,H-14)]2 continuous oxygen methine proton signals [ delta ]H：3.61(1H,dd,J＝8.6,2.4Hz,H-6)、3.35(1H,d,J＝9.5Hz,H-7)]。13The C-NMR spectrum (Table 1) showed a shift signal of 20 carbon atoms, of which there were 5 methyl carbon signals [ delta ]C：11.8(C-16)、19.1(C-17)、37.0(C-18)、22.5(C-19)、17.5(C-20)]3 pieces of continuous oxygen carbon lettersNumber [ delta ]C：73.0(C-6)、85.5(C-7)、76.9(C-8)]4 alkene carbon signals [ delta ]C：111.1(C-11)、154.8(C-12)、157.1(C-13)、117.0(C-14)]1 carbonyl carbon signal [ delta ]C：171.8(C-15)]. Shows unimodal methyl delta in HMBC spectraH: 2.20 remote correlation of (3H, s, H-16) with C-12/13/14/15, olefin proton δH: 5.55(1H, d, J. 11.2Hz, H-11) remote correlation with C-8/10/12/13, olefin proton δH: 5.99(1H, s, H-14) is remotely related to C-12/13/15 and is known to have two double bonds and to be conjugated, with the methyl group at the C-16 position attached to the C-13 position, incorporating the 1 ester carbonyl carbon signal [ delta. ]C：171.8(C-15)]It is presumed that the structure has a-4-methyl-5-methylene group]-2(5H) -furanone structural fragment. In that1H-1H-9/H-11 correlation is shown in the H COSY map, and the correlation of C-11 and C-9 can be known, and the correlation of H-1/H2/H3 and the correlation of H-6 and H-5/H-7 can also be observed. The planar structure of the compound was constructed by further analyzing HMBC (heteronuclear multiple carbon correlation spectrum of 1H) profile data of the compound while combining HMQC profile data (see fig. 1).
The labdane diterpenoid compounds are 1 new compound through sci-finder search. The NOESY spectrum shows that H-6 and H-9/11/16/17/18/20 are remotely coupled to each other. The relative configuration of the compound is constructed by combining literature (Oshima Y, stepbins A, B, C and D, bisnortripterylides of Stevia rebaudiana leaves, Tetrahedron,1986) and knowing that the optical rotation of the compound and the optical rotation of the sterebin A-D are positive, C-6 is positioned in R configuration, C-7 is positioned in S configuration, so C-9 is in R configuration and C-5/8/10 is in S configuration. This compound was therefore named:
(5E) -4-methyl-5- [ ((1R,2S,3S,4R,4aS,8aS) -decahydro-2,3,4-trihydroxy-2,5,5,8a-tetramethyl-1-naphthalenyl) methyl ] -2(5H) -furan one, (5E) -4-methyl-5- [ ((1R,2S,3S,4R,4aS,8aS) -decahydro-2,3,4-trihydroxy-2,5,5,8 a-tetramethyl-1-naphthyl) methylene ] -2(5H) -furanone.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A labdane diterpenoid compound is characterized by having a structural formula as follows:
2. a process for the preparation of the labdane diterpenoid compound according to claim 1, characterized in that it comprises the following steps:
extracting folium Callicarpae Formosanae with ethanol under reflux, and concentrating to obtain total extract;
subjecting the obtained total extract to adsorption resin, gradient eluting with water and 25% -100% ethanol respectively, collecting ethanol eluate, and removing ethanol and water to obtain crude extract;
separating the crude extract by silica gel column chromatography, carrying out gradient elution by eluent to obtain 6 components Fr.1-Fr.6, carrying out silica gel column chromatography on the Fr.3 component, carrying out gradient elution to obtain 8 components Fr.3-1-Fr.3-8, carrying out gradient elution on the Fr.3-2 component by silica gel column to obtain 7 components Fr.3-2-1-Fr.3-2-7, carrying out ODS column chromatography on the Fr.3-2-4 component, carrying out gradient elution by using 20%, 40% and 60% acetonitrile water solution in sequence, and carrying out liquid phase column chromatography on the eluent after the acetonitrile water solution gradient elution to obtain the labdane diterpenoid compound.
3. The method for preparing labdane diterpenoid compounds according to claim 2, wherein the adsorption resin is DA-201 macroporous adsorption resin, and gradient elution is performed with water, 25%, 50%, 75% and 95% ethanol, respectively;
preferably, the elution part of the 50% ethanol solution is collected and subjected to a rotary evaporation method to obtain a crude extract.
4. The method for preparing labdane diterpenoid compounds according to claim 2, wherein the crude extract is purified by silica gel column chromatography at a volume ratio of 200-1: 1 with chloroform: gradient eluting with methanol, combining and collecting to obtain 6 components Fr.1-Fr.6; and the Fr.3 component is eluted by silica gel column chromatography in turn by the volume ratio of 50-1:1 of chloroform: gradient elution with methanol, merging and collecting to obtain 8 components Fr.3-1-Fr.3-8, passing the Fr.3-2 component through silica gel column, sequentially mixing chloroform: gradient eluting with methanol, combining and collecting 7 Fr.3-2-1-Fr.3-2-7 components, subjecting the Fr.3-2-4 component to ODS column chromatography, and gradient eluting with 20%, 40%, and 60% acetonitrile water solution sequentially.
5. The method for preparing labdane diterpenoid compounds according to claim 2, wherein the Fr.3-2-4 component is subjected to ODS column chromatography and gradient elution with acetonitrile in water, acetonitrile and water are removed by rotary evaporation, the product from which acetonitrile and water are removed is subjected to liquid phase column chromatography, gradient elution is performed with acetonitrile and water as mobile phases at a volume ratio of 20-40:1, the detection wavelength is 265nm and/or 300nm, the flow rate is 8-12ml/min, and the retention time is 65.72 ± 10min, thereby obtaining the labdane diterpenoid compounds.
6. The method for preparing labdane diterpenoid compounds according to claim 2, wherein in the ethanol reflux extraction step, 60-70% ethanol is used for reflux extraction at least 2 times, each time for 1.5-4h, the extract is combined, and concentrated to obtain the total extract;
the reflux extraction is a continuous reflux extraction method.
7. Use of a labdane diterpenoid according to claim 1 or prepared by a method of preparation of a labdane diterpenoid according to any one of claims 2-6 for the preparation of a neuroprotective composition.
8. A neuroprotective composition comprising the labdane diterpenoid of claim 1 or prepared by the method of preparation of the labdane diterpenoid of any one of claims 2-6.
9. The neuroprotective composition according to claim 8, further comprising a pharmaceutically acceptable additive or adjuvant; preferably, the neuroprotective composition is in a dosage form selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays, or injections.
10. The neuroprotective composition according to claim 8 or 9, wherein the neuroprotective composition is used for the treatment or prevention of a neurodegenerative disease, preferably wherein the neurodegenerative disease comprises at least one of the following diseases: alzheimer's disease, parkinson's disease, mild cognitive impairment, huntington's disease, prion-caused diseases, frontotemporal dementia, dementia with lewy bodies, vascular dementia, amyotrophic lateral sclerosis, chronic traumatic encephalopathy, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, pick's disease and olivopontocerebellar atrophy.
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| CN113754533A (en) * | 2021-08-26 | 2021-12-07 | 海南师范大学 | Oxidized labdane diterpenoid compounds and separation method and application thereof |
| CN116354811A (en) * | 2023-02-22 | 2023-06-30 | 江中药业股份有限公司 | Labdane diterpenoid compound, preparation method and application |
| CN116813633A (en) * | 2023-06-29 | 2023-09-29 | 黑龙江中医药大学 | Chromone compound and preparation method and application thereof |
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| CN113754533A (en) * | 2021-08-26 | 2021-12-07 | 海南师范大学 | Oxidized labdane diterpenoid compounds and separation method and application thereof |
| CN113754533B (en) * | 2021-08-26 | 2023-12-01 | 海南师范大学 | Oxidized labdane diterpenoid compound, and separation method and application thereof |
| CN116354811A (en) * | 2023-02-22 | 2023-06-30 | 江中药业股份有限公司 | Labdane diterpenoid compound, preparation method and application |
| CN116354811B (en) * | 2023-02-22 | 2024-02-27 | 江中药业股份有限公司 | Labdane diterpenoid compound, preparation method and application |
| CN116813633A (en) * | 2023-06-29 | 2023-09-29 | 黑龙江中医药大学 | Chromone compound and preparation method and application thereof |
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