CN117603178A - Two anti-inflammatory compounds extracted from herba Alternantherae Alternifolia, and extraction method and application thereof - Google Patents
Two anti-inflammatory compounds extracted from herba Alternantherae Alternifolia, and extraction method and application thereof Download PDFInfo
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- 230000003110 anti-inflammatory effect Effects 0.000 title claims abstract description 24
- 238000000605 extraction Methods 0.000 title claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000000126 substance Substances 0.000 claims description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 20
- -1 2,7, 9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one Chemical compound 0.000 claims description 15
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
- C07D241/06—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members
- C07D241/08—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the technical field of medicines, in particular to two anti-inflammatory compounds extracted from a common lantana leaf, and an extraction method and application thereof. The application firstly separates and identifies 2 compounds from the plum of the leaves of the largeflower, and the compounds are respectively: 2,7, 9-trihydroxy-3-method-1-methyl-6H-benzyl-6-one (1), 3, 6-diisoopyyl-2, 5-diisoketopiperazine (2), wherein 1 new compound (compound 1), compound 1 and compound 2 are separated from the genus plant for the first time. The compounds 1 and 2 obtained by separation and identification in the application have excellent anti-inflammatory properties, and can be used for preparing anti-inflammatory medicaments.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to two anti-inflammatory compounds extracted from a common lantana leaf, and an extraction method and application thereof.
Background
All plants of the genus primula (Lysimachia L.) belonging to the family primula of the family primula (primula) can be used as medicines, and the whole plants are mainly distributed in southwest areas of China and are common medicines for minority nationalities such as Guizhou Miao nationality. Has effects of dispelling pathogenic wind, activating collaterals, promoting blood circulation and relieving pain, and can be used for treating rheumatalgia, hemiplegia, limb spasm, infantile convulsion, traumatic injury, etc.
At present, the researches on chemical components of the rheum officinale are less, and the chemical components are the material basis for the traditional Chinese medicine to exert the traditional effects. Because the traditional Chinese medicine has the characteristics of 'multi-component, multi-target and multi-path synergistic effect', the basic research still has a plurality of difficulties. Therefore, in order to further explore the pharmacodynamic substances in the rheum palmatum, the chemical composition research of the medicinal materials needs to be continued in the later period, and meanwhile, the in-vivo and in-vitro pharmacodynamic research needs to be carried out in combination with the subjects of metabonomics, network pharmacology and the like, so as to strive for further separating various chemical compositions from the rheum palmatum, confirming the efficacy of the chemical compositions and realizing the application direction of the chemical compositions.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides two anti-inflammatory compounds extracted from the common lantana leaf, and an extraction method and application thereof.
An anti-inflammatory compound (compound 1) extracted from herba Lespedezae Cuneatae has the following specific structural formula:
the chemical name of the compound 1 is 2,7, 9-trihydroxy-3-methoxy-1-methyl-6H-benzol [ c ]]chromen-6-one of formula C 15 H 12 O 6 The molecular weight is 288.25.
An anti-inflammatory compound (compound 2) extracted from herba Lespedezae Cuneatae has the following specific structural formula:
the compound is2, the chemical name of which is 3, 6-dioropyl-2, 5-diotopiperazine, and the molecular formula of which is C 10 H 18 N 2 O 2 The molecular weight is 198.26.
The obtained compound 1 and compound 2 have obvious inhibition effect on the generation of nitric oxide in RAW264.7 cells of inflammatory response induced by LPS, have excellent anti-inflammatory properties, and can be used for preparing anti-inflammatory drugs.
The extraction method of the anti-inflammatory compounds (compound 1 and compound 2) specifically comprises the following steps (corresponding steps can be omitted correspondingly when a certain compound is not required to be obtained):
(1) Soaking and extracting coarse powder of herba Alternantherae Alternifolia in methanol water with different gradients at normal temperature, mixing extractive solutions, concentrating under reduced pressure until no alcohol smell exists to obtain total extract of herba Aristolochiae Mollissimae; dispersing the total extract with appropriate amount of hot water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and recovering under reduced pressure to obtain petroleum ether part, ethyl acetate part and n-butanol part;
(2) Decolorizing the ethyl acetate part by MCI column chromatography, sequentially eluting with gradient of 50:50, 60:40, 70:30, 80:20, 90:10, 100:0 by adopting a methanol-water system, detecting the obtained fractions by TLC, and combining to obtain 7 parts, wherein the parts are Fr.A-Fr.F in sequence;
(3) Separating and purifying the Fr.A part by silica gel column chromatography, eluting with dichloromethane-methanol system with gradient of 100:1, 50:1, 30:1, 10:1, 5:1, 1:1, and separating into 6 components by TLC, wherein Fr.A 1-Fr.A 6 are sequentially carried out;
(4) Subjecting Fr.A3 to silica gel column chromatography, eluting with dichloromethane-methanol system sequentially with gradient of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1, and identifying by TLC and separating into 3 components, fr.A3-1-Fr.A3-3; fr.A3-1 is subjected to silica gel column chromatography, and is eluted with a petroleum ether-ethyl acetate system by gradient of 30:1, 20:1, 10:1, 5:1 and 1:1 in sequence to obtain a compound 2.
(4) The Fr.C fraction is separated and purified by silica gel column chromatography, and is eluted with a gradient of 30:1, 20:1, 10:1, 5:1, 3:1, 1:1 in sequence by using a petroleum ether-ethyl acetate system to obtain the compound 1.
Further, the coarse powder is 60-80 meshes.
Further, the normal temperature soaking extraction is carried out by adopting methanol water with different gradients, specifically, 100 percent methanol, 90 percent methanol and 70 percent methanol are sequentially added for extraction twice for 5 days each time.
Compared with the prior art, the invention has the technical effects that:
(1) The application separates and identifies 2 compounds from the plum of the leaves of the garden balsam for the first time, and the compounds are respectively: 2,7, 9-trihydroxy-3-method-1-methyl-6H-benzyl-6-one (1), 3, 6-diisoopyyl-2, 5-diisoketopiperazine (2), wherein 1 new compound (compound 1), compound 1 and compound 2 are separated from the genus plant for the first time.
(2) The compounds 1 and 2 obtained by separating and identifying the plum from the leaves of the plum for the first time have excellent anti-inflammatory properties, and can be used for preparing anti-inflammatory medicaments.
(3) In the application, the compound 1 (2, 7, 9-trihydroxy-3-methyl-1-methyl-6H-benzol [ c ] chromen-6-one (1)) separated from the plum is a new compound obtained by first extraction, separation and purification. And has excellent anti-inflammatory properties, and can be used for preparing anti-inflammatory drugs. There has not been found a publication of the related research literature reporting the compound 1, and the application of the compound 1 to the research aspect of preparing anti-inflammatory drugs. The primary classification of the compound 1 belongs to phenylpropanoids, the secondary classification belongs to diphenyl-alpha-pyrones, the compound is less common in vegetable medicines, only 4 kinds of compounds are found in the plant of the genus Phyllostachys at present, and the compounds are separated from the medicinal materials of the pholiota nameko. The compound 1 obtained by separation and identification of the eupatorium is enriched in the variety of the chemical components of the eupatorium plant, and scientific basis is provided for the subsequent research on the chemical components and other aspects of the eupatorium.
(4) Compound 2 (3, 6-diotopyl-2, 5-diketopirazine (2)) isolated for the first time from Prunus angustifolia in this application. The compound 2 is separated from the herba schizophragmatis integrifolii radicis for the first time, a novel preparation method and a source are provided for the compound 2, the preparation method of the compound 2 is enriched, and scientific basis is provided for the subsequent research on chemical components and other aspects of the rheum officinale.
(5) The application researches the chemical components and the quality control of the rheum palmatum, not only lays a foundation for the development and the utilization of the medicinal material resources and the research and the development of new products in the later period, but also lays a certain foundation for the improvement of the quality control level of the rheum palmatum and the filling of the ground standard blank.
Drawings
FIG. 1 is a diagram of Compound 1 1 H-NMR(600MHz，DMSO-d 6 ) And (5) a map.
FIG. 2 is a diagram of Compound 1 13 C-NMR(150MHz，DMSO-d 6 ) And (5) a map.
FIG. 3 is HMBC (600 MHz, DMSO-d) of Compound 1 6 ) And (5) a map.
FIG. 4 is the HMQC of Compound 1 (600 MHz, DMSO-d 6 ) And (5) a map.
FIG. 5 is a diagram of Compound 1 1 H- 1 H COSY(600MHz，DMSO-d 6 ) And (5) a map.
FIG. 6 is DEPT of Compound 1 (150 MHz, DMSO-d 6 ) And (5) a map.
FIG. 7 is NOESY (600 MHz, DMSO-d) of Compound 1 6 ) And (5) a map.
Fig. 8 is a high resolution mass spectrum of compound 1.
FIG. 9 is an infrared spectrum of Compound 1.
FIG. 10 is a diagram of Compound 2 1 H-NMR(600MHz，CD 3 OD) profile.
FIG. 11 is a diagram of Compound 2 13 C-NMR(150MHz，CD 3 OD) profile.
Figure 12 is a graph showing a significant analysis of NO release by compounds 1, 2.
Detailed Description
The technical scheme of the present invention is further defined below in conjunction with the specific embodiments, but the scope of the claimed invention is not limited to the description.
Example 1
Research on chemical components of rheumatalgia-relieving umbrella
1 instrument and materials
1.1 sources of medicinal materials
The medicinal material was purchased from Fuqing county Guan Xingzhen, guizhou province, and identified by the professor Wu Zhikun of the university of Guizhou traditional Chinese medicine as whole grass of Meissima angustifolia (Lysimachia paridiformis var stenophylla Franch.) belonging to the genus Primulaceae (Primalaceae) and the genus Lysimachia. And (3) drying the sample in the shade naturally, and crushing the sample for later use. The original plant is reserved in the national medicine key laboratory of Guizhou traditional Chinese medicine university.
1.2 instruments
The details of the apparatus used in this experiment are shown in Table 1-1.
Table 1-1 laboratory apparatus
1.3 reagents and fillers
The reagents and fillers used in this experiment are shown in tables 1-2.
TABLE 1-2 Experimental reagents and fillers
Note that: the color developer used in this experiment: 10% sulfuric acid ethanol solution and iodine simple substance.
2 experimental methods and results
2.1 extraction and separation of medicinal materials of Cornus trabeculatus
35.58kg of the crude powder (60-80 meshes) of the whole herb of the rheum officinale is taken, normal-temperature soaking extraction is carried out by adopting methanol water with different gradients (100, 90 and 70 percent methanol are sequentially added for two times each for 5 days), the extracting solutions are combined for multiple times, and the decompression concentration is carried out until no alcohol smell exists, thus obtaining the total extract (6.96 kg, and the extract yield is 19.56%) of the rheum officinale. Dispersing the total extract with appropriate amount of hot water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and recovering under reduced pressure to obtain 993g of petroleum ether part, 328g of ethyl acetate part and 4.04kg of n-butanol part.
The ethyl acetate fraction was decolorized by MCI column chromatography using a methanol-water system (50:50, 60:40, 70:30, 80:20, 90:10, 100:0) gradient elution, and the resulting fractions were combined to give 7 fractions (Fr. A-Fr. F) after TLC detection. The Fr.A fraction (71 g) was isolated and purified by column chromatography on silica gel, eluted with a gradient of methylene chloride-methanol system (100:1, 50:1, 30:1, 10:1, 5:1, 1:1), and identified by TLC and separated into 6 fractions (Fr.A 1 to Fr.A 6).
Fr.A3 was eluted by silica gel column chromatography with a gradient of methylene chloride-methanol system (50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1) and was identified by TLC and separated into 3 components (Fr.A3-1 to Fr.A3-3). Fr.A3-1 was subjected to silica gel column chromatography, and gradient elution was performed with a petroleum ether-ethyl acetate system (30:1, 20:1, 10:1, 5:1, 1:1) to give Compound 2.
The Fr.C fraction (11.3 g) was isolated and purified by column chromatography on silica gel, eluting with a gradient of petroleum ether-ethyl acetate system (30:1, 20:1, 10:1, 5:1, 3:1, 1:1) to give compound 1.
2.2 structural identification of Compounds
After the compound is separated and purified, the structure identification is carried out by physicochemical property, nuclear Magnetic Resonance (NMR), infrared (IR), high resolution mass spectrum (HR-ESI-MS) and other analysis methods by means of a micro-spectrum database, sciFinder Scholar and the like.
1. Structural identification of Compound 1
Compound 1: white powder. Optical rotation:(c 0.207, pyridine). Is easily dissolved in DMSO, pyridine, indissolvable methanol, insoluble ethyl acetate, dichloromethane and petroleum ether. TLC detection: dichloromethane-methanol (10:1) is used as a developing agent, and fluorescence is generated at ultraviolet 254 and 365nm wavelengths after developing; spraying 10% ethanol solution of sulfuric acid, heating at 105deg.C, and making single orange yellow spot (R f A value of 0.56). According to HR-ESI-MS, the test value m/z287.0573[ M-H ]] - (calculated 287.0561, C) 15 H 11 O 6 ) Thereby determining the molecular formula as C 15 H 12 O 6 The degree of unsaturation was 10. According to IR display, at 1654.2, 3419.6cm -1 The absorption at this point shows that the compound may have carbonyl and hydroxyl groups, respectively.
According to 1 H-NMR(600MHz，DMSO-d 6 ) Delta is known H 11.78 (1H, s, 7-OH), 10.82 (1H, s), 8.80 (1H, s) may be 3 active hydrogens, delta H 7.21 (1 h, d, j=1.5 hz, h-10), 6.30 (1 h, d, j=1.6 hz, h-8) suggests that an aromatic signal with a set of meta-couplings is possible by 1 H- 1 The H COSY spectrum has been shown to indicate that a tetra-substituted aromatic ring system is possible in the compounds. Delta H 6.86 (1H, s, H-4) suggests 1 aromatic hydrogen, indicating that a pentasubstituted aromatic ring system may also be present in the compound. Delta H 3.83(3H，s，-OCH 3 ) May be 1 methoxy group, delta H 2.51(3H，s，-CH 3 ) Possibly 1 benzhydryl group. According to 13 C-NMR(150MHz，DMSO-d 6 ) DEPT, HMQC spectra show that the compound contains 15 carbon atoms, presumably 10 quaternary carbons (including a carbonyl carbon delta C 165.3 3 methine carbons [ delta ] C 104.7(C-10)、101.1(C-8)、98.3(C-4)]1 methoxy carbon [ delta ] C 56.1(-OCH 3 )]1 methyl carbon [ delta ] C 15.8(-CH 3 )]. From the fact that the unsaturation degree of the compound is 10, two benzene rings share 8 unsaturation degrees, 1 carbonyl group shares 1 unsaturation degree, and the rest 1 unsaturation degree speculates that the compound possibly has a tricyclic ring system. In summary, the reference shows that compound 1 has similar data to the reported 2-hydroxy-alternariol (2-OH-AOH), and can be primarily presumed to be a phenylpropanoid compound which has 1 more methoxy group and one less hydroxy hydrogen than 2-OH-AOH.
By observing HMBC spectra and 1 H- 1 the H COSY spectrum shows that: 1 H-NMR Spectroscopy delta H 11.78 (1H, s, 7-OH) and 13 C-NMRδ C 97.9 (C-6 a), 101.1 (C-8), 164.1 (C-7) have remote correlation signals; delta H 6.30 (1 h, d, j=1.6 hz, h-8) and δ C 97.9(C-6a)、104.7(C-10)、164.1(C-7) remote correlation signal, delta H 7.21 (1 h, d, j=1.5 hz, h-10) and δ H 6.30 (1 h, d, j=1.6 hz, h-8) indicating a hydroxyl group attached at the C-7 position, a delta value of 164.1ppm for the 7-OH attached carbon, and a continuous 5 carbon signal on the tetra-substituted aromatic ring system (ring a) for C-6a, C-7, C-8, C-9, C-10. Delta H 7.21 (1 h, d, j=1.5 hz, h-10) and δ C 97.9 (C-6 a), 101.1 (C-8), 110.1 (C-10 b), 164.9 (C-9) had remote related signals, indicating that a hydroxyl group was attached at the C-9 position, the delta value of the 7-OH attached carbon was 164.9ppm, and C-6a, C-7, C-8, C-9, C-10 were the consecutive 5 carbon signals in the A ring. Delta H 2.51(3H，s，-CH 3 ) And delta C 104.7 (C-10), 110.1 (C-10B), 121.3 (C-1), 141.9 (C-2) have remote related signals, indicating that the two benzene rings are connected by C-10B, C-2, C-1, C-10B are 3 carbon signals in succession on the pentasubstituted aromatic ring system (B ring). Delta H 3.83(3H，s，-OCH 3 ) And delta C 149.2 (C-3) having a remote correlation signal; delta H 6.86 (1H, s, H-4) and delta C 110.1 (C-10B), 141.9 (C-2), 144.9 (C-4 a), 149.2 (C-3) had a remote correlation signal, which, in combination with the HMQC spectrum, indicated a carbon delta value associated with methoxy of 149.2ppm, C-2, C-3, C-4a, C-10B being the 5 carbon signal consecutive on the B ring. In the NOESY spectrum (see figure 1), 1 H-NMR Spectroscopy delta H 2.51(3H，s，-CH 3 ) And delta H 7.21 (1 h, d, j=1.5 hz, h-10) indicating that the a-and B-rings are spatially one whole with a correlation.
In summary, compound 1 was identified as a phenylpropanoid compound, designated: 2,7, 9-trihydroxy-3-methoxy-1-methyl-6H-benzol [ c ]]chrome-6-one. Compound 1 was identified as a new compound by searching SciFinder Scholar and looking up the relevant literature. The compound is 1 H-NMR 13 The C-NMR data are shown in the following Table.
Datawere recorded inDMSO on aBrukerAV-600MHzspectrometer.
The structural formula of compound 1 is shown below:
compound 1 1 H- 1 H COSY(-)、HMBC(→)、NOESYThe spectrum related signal is as follows:
2. structural identification of Compound 2
Compound 2: white powder. Is easy to dissolve in methanol, slightly dissolve in ethyl acetate, and insoluble in dichloromethane and petroleum ether. TLC detection: dichloromethane-ethyl acetate-formic acid (20:2:1) is used as a developing agent, and fluorescence is generated at ultraviolet 254 and 365nm wavelengths after developing; spraying 10% sulfuric acid ethanol solution, heating at 105deg.C, and making single dark orange spot (R f A value of 0.43); molecular formula C 10 H 18 N 2 O 2 . According to 1 H-NMR(600MHz，CD 3 OD) of delta H 3.84 (2 h, d, j=3.4 hz, h-3, 6) is shown as a hydrogen signal on the azodicarbon, δ H 1.07 (6 h, d, j=7.0 hz, h-8,8 '), 0.97 (6 h, d, j=6.7 hz, h-9, 9') showed 4 methyl groups. According to 13 C-NMR(150MHz，CD 3 OD) data shows that the compound contains 10 carbon atoms. Delta C 168.8 (C-2, 5) shows 2 carbonyl groups, delta C 16.4 (C-8, 8 '), 17.9 (C-9, 9') show 4 methyl groups, and on the whole, it can be primarily presumed that the compound is a alkaloid compound. According to the above data, the compound 2 was identified as 3, 6-dioropyl-2, 5-diotopiperazine and was reported to be substantially identical to the literature 1 H-NMR 13 The C-NMR data are shown in the following Table.
Datawere recorded in CD 3 OD on aBrukerAV-600MHzspectrometer.
The structural formula of compound 2 is shown below:
2.3 partial physicochemical constant and Spectrum data for Compounds
2,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one (1): white powder. Development of dichloromethane-methanol (10:1), 10% sulfuric acid in ethanol revealed a single orange-yellow spot (R) f A value of 0.58). Optical rotation:(c 0.207, pyridine); IR (KBr) V max 1654.2、3419.6cm -1 The method comprises the steps of carrying out a first treatment on the surface of the HR-ESI-MS test value M/z287.0573[ M-H ]] - (calculated 287.0561, C) 15 H 11 O 6 )； 1 H-NMR(600MHz，DMSO-d 6 )δ11.78(1H，s，7-OH)，7.21(1H，d，J＝1.5Hz，H-10)，6.86(1H，s，H-4)，6.30(1H，d，J＝1.6Hz，H-8)，3.83(3H，s，-OCH 3 )，2.51(3H，s，-CH 3 )； 13 C-NMR(150MHz，DMSO-d 6 )δ121.3(C-1)，141.9(C-2)，149.2(C-3)，98.3(C-4)，144.9(C-4a)，165.3(C-6)，97.9(C-6a)，164.1(C-7)，101.1(C-8)，164.9(C-9)，104.7(C-10)，138.3(C-10a)，110.1(C-10b)，56.1(-OCH 3 )，15.8(-CH 3 )。
3, 6-Diisopropyyl-2, 5-Diketopiperazine (2): white powder. Development of dichloromethane-ethyl acetate-formic acid (20:2:1), 10% ethanol solution of sulfuric acid revealed a single dark orange spot (R) f A value of 0.43). 1 H-NMR(600MHz，CD 3 OD)δ3.84(2H，d，J＝3.4Hz，H-3，6)，2.30(2H，dq，J＝12.4，6.1，5.7Hz，H-7，7')，1.07(6H，d，J＝7.0Hz，H-8，8')，0.97(6H，d，J＝6.7Hz，H-9，9')； 13 C-NMR(150MHz，CD 3 OD)δ168.8(C-2，5)，59.8(C-3，6)，31.8(C-7，7')，16.4(C-8，8')，17.9(C-9，9')。
Example 2
Anti-inflammatory Activity assay of two Compounds
In vitro anti-inflammatory Activity study of Compounds 2,7, 9-trihydroxy-3-method-1-methyl-6H-benzol [ c ] chrome-6-one (1) and 3, 6-diisoopropyl-2, 5-diketopiperazine (2) on LPS-induced RAW264.7 cells
1. Principle of experiment
(1) The English language of CCK-8 is called Cell Counting Kit-8. The chemical which plays a main role is WST-8, and the chemical name is 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt. Its core group is the same as MTT, which is an upgraded version of MTT. In the presence of an electron-coupling reagent (i.e., the cell is living, respiring, has energy metabolism), it is oxidized and reduced by NAD+ to a water-soluble yellow Formazan product (Formazan). The more living cells, the more formalzan is produced and the darker the color.
(2) NO is easily oxidized into NO in the environment such as in vivo or aqueous solution 2- Under acidic conditions, NO reacts in series to form a pink specific substance having a maximum absorption peak at 540nm, and the concentration of NO can be estimated from the OD value.
2. Experimental procedure
(1) Cell Activity assay of Compounds 1,2
RAW264.7 cells in logarithmic growth phase were digested with trypsin containing 0.25% edta (1×) until the cells were in suspension, and then the whole medium was added to dilute the pancreatin concentration, and the digestion was terminated. Centrifuging, and preparing a density of 1×10 with complete medium after resuspension 5 The single cell suspension of cells/mL is inoculated into a 96-well plate at a concentration of 100 mu L/well, and 100 mu LPBS buffer solution is added into the outer ring cell culture well, so that the influence of the edge effect on experimental results is reduced. At 37 ℃,5% CO 2 And (5) standing and culturing for 20h in an incubator. After completion of the culture, 100. Mu.L of culture solutions containing the above-mentioned compounds at different concentrations were added. The concentration range of the compound test solution is set as follows: 6.25, 12.5, 25, 50. Mu.M, 3 multiplex wells per concentration. Three wells were left without drug as normal group, without drug and without cells as blank group. After culturing for 20 hours, discarding cell supernatant, adding 10 mu L of CCK-8 working solution, continuously placing into an incubator for culturing for 2 hours, and measuring absorbance value at 450nm wavelength by using an enzyme-labeling instrument. Cell relative viability= [ OD experiment-OD blank],/[ OD Normal-OD blank]×100％。
(2) Detection of anti-inflammatory Activity of Compounds 1,2
Preparation of RAW264.7 cells in logarithmic growth phase with complete Medium at a density of 1×10 5 The single cell suspension is inoculated into a 96-well plate at 100 mu L/well, and 100 mu L of PBS buffer solution is added into the outer ring cell culture well, so that the influence of edge effect on experimental results is reduced. At 37 ℃,5% CO 2 And (5) standing and culturing for 20h in an incubator. After the completion of the culture, 100. Mu.L of a culture medium containing the above compound at a concentration of 50. Mu.M was added thereto, and 3 wells were formed. Three wells were left without drug as normal group, without drug and without cells as blank group. Adding medicine at 37deg.C and 5% CO 2 After static culture for 1h in an incubator, adding LPS with the concentration of 1 mug/mL to stimulate the inflammation, and finally adding 37 ℃ and 5% CO 2 Culturing in an incubator for 20h.
Taking out Griess Reagent I and II, allowing to return to room temperature, preparing standard curve according to Reagent specification, adding standard substance and sample into new 96-well plate at 50 μl/well, adding Griess Reagent I into each well, adding Griess Reagent II, and adding 5% CO at 37deg.C 2 After 15min of stationary culture in an incubator, the absorbance was measured at 540nm using a microplate reader, and the concentration of Nitric Oxide (NO) in the sample was calculated from the standard curve.
3. Experimental results
The relative viability of RAW264.7 cells after 20h at 6.25, 12.5, 25, 50 μm compound 1,2, respectively, is shown in table 1, indicating that compound 1,2 has no toxic effect on the cells at the above concentrations.
Table 1 relative Activity of Compounds 1,2 on RAW264.7 cells
As shown in fig. 12, after LPS stimulation of RAW264.7 cells, the amount of cellular NO release was extremely significantly increased compared to the control group without LPS stimulation. The amount of NO released by LPS groups to which compounds 1 and 2 were not added after LPS stimulation reached 14.10. Mu.M. The NO production was significantly reduced in the experimental group with continued culture with the addition of compounds 1,2 compared to the LPS group. Specifically, after compound 1 (50. Mu.M) was added, the amount of NO released from the cells was reduced to 7.75. Mu.M; after addition of compound 2 (50. Mu.M), the amount of NO released by the cells was reduced to 10.76. Mu.M.
The above results demonstrate that compounds 1 and 2 extracted from the lantana camara have a remarkable inhibitory effect on the production of nitric oxide by RAW264.7 cells of LPS-induced inflammatory response, reflecting excellent anti-inflammatory properties.
Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the invention is not limited to the above-described embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (7)
1. An anti-inflammatory compound extracted from a largeflower holly leaf is characterized by having the following specific structural formula:
2. an anti-inflammatory compound as claimed in claim 1, wherein the chemical name is 2,7, 9-trihydroxy-3-methoxy-1-methyl-6H-benzol [ c ]]chromen-6-one of formula C 15 H 12 O 6 The molecular weight is 288.25.
3. Use of an anti-inflammatory compound as defined in claim 1 for the manufacture of an anti-inflammatory medicament.
4. The application of an anti-inflammatory compound extracted from the herba schizophragmatis integrifolii radicis in the aspect of preparing anti-inflammatory drugs is characterized in that the specific structural formula of the compound is as follows:
the chemical name of the compound is 3, 6-dioopyyl-2, 5-diotopiperazine, and the molecular formula is C 10 H 18 N 2 O 2 The molecular weight is 198.26.
5. The extraction method of the anti-inflammatory compound in the largeflower lantana is characterized by comprising the following steps of:
(1) Soaking and extracting coarse powder of herba Alternantherae Alternifolia in methanol water with different gradients at normal temperature, mixing extractive solutions, concentrating under reduced pressure until no alcohol smell exists to obtain total extract of herba Aristolochiae Mollissimae; dispersing the total extract with appropriate amount of hot water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and recovering under reduced pressure to obtain petroleum ether part, ethyl acetate part and n-butanol part;
(2) Decolorizing the ethyl acetate part by MCI column chromatography, sequentially eluting with gradient of 50:50, 60:40, 70:30, 80:20, 90:10, 100:0 by adopting a methanol-water system, detecting the obtained fractions by TLC, and combining to obtain 7 parts, wherein the parts are Fr.A-Fr.F in sequence;
(3) Separating and purifying the Fr.A part by silica gel column chromatography, eluting with dichloromethane-methanol system with gradient of 100:1, 50:1, 30:1, 10:1, 5:1, 1:1, and separating into 6 components by TLC, wherein Fr.A 1-Fr.A 6 are sequentially carried out;
(4) Subjecting Fr.A3 to silica gel column chromatography, eluting with dichloromethane-methanol system sequentially with gradient of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1, and identifying by TLC and separating into 3 components, fr.A3-1-Fr.A3-3; fr.A3-1 was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system, and eluted sequentially with a 30:1, 20:1, 10:1, 5:1, 1:1 gradient to give 3, 6-diotopyl-2, 5-diotopiperazine.
(4) Separating and purifying the Fr.C part by silica gel column chromatography, eluting with petroleum ether-ethyl acetate system sequentially with gradient of 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1 to obtain 2,7,
9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one。
6. the extraction method according to claim 5, wherein the coarse powder is 60 to 80 mesh.
7. The extraction method according to claim 5, wherein the extraction is performed by using methanol water with different gradients at normal temperature, specifically by sequentially adding 100%, 90% and 70% methanol for extraction twice for 5 days each.
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