CN117304031A - Herba Acalyphae diterpenoid compounds, herba Acalyphae extract and application thereof in preparing antioxidant and/or antiinflammatory products - Google Patents

Herba Acalyphae diterpenoid compounds, herba Acalyphae extract and application thereof in preparing antioxidant and/or antiinflammatory products Download PDF

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CN117304031A
CN117304031A CN202311140095.2A CN202311140095A CN117304031A CN 117304031 A CN117304031 A CN 117304031A CN 202311140095 A CN202311140095 A CN 202311140095A CN 117304031 A CN117304031 A CN 117304031A
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acalypha australis
diterpene
acalypha
australis
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刘晓宇
王怀玲
洪靖怡
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Shenzhen University
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Abstract

The invention relates to the technical field of biomedicine, and particularly discloses an acalypha australis diterpenoid compound, an acalypha australis extract and application thereof in preparation of products with antioxidant and/or anti-inflammatory effects. The herba Acalyphae diterpenoid compound is one or more selected from herba Acalyphae diterpenoid-I, herba Acalyphae diterpenoid-II, herba Acalyphae diterpenoid-III, herba Acalyphae diterpenoid-IV and herba Acalyphae diterpenoid-V. Because the acalypha australis diterpenoid compounds and the acalypha australis extract have anti-inflammatory and antioxidant effects; therefore, the extract is used as an active ingredient and has important application value in preparing cosmetics, skin care products, medicines or health care products with anti-inflammatory and antioxidant effects.

Description

Herba Acalyphae diterpenoid compounds, herba Acalyphae extract and application thereof in preparing antioxidant and/or antiinflammatory products
Technical Field
The invention relates to the technical field of biomedicine, in particular to an acalypha australis diterpenoid compound, an acalypha australis extract and application thereof in preparing products with antioxidant and/or anti-inflammatory effects.
Background
Herba Acalyphae is an annual herb of Acalypha in Euphorbiaceae. Research reports that the acalypha australis is rich in nutrition and has potential anti-inflammatory activity. As reported in the study, the antioxidant substances contained in acalypha australis help to reduce the inflammatory response. In addition, the flavonoid active compound contained in the acalypha australis has antibacterial and anti-inflammatory activities, has a strong antioxidation effect, can inhibit peroxidation of cell lipid, and has a protective effect on cell DNA damage caused by UV irradiation. In addition, the polyphenol active compounds in the acalypha australis can reduce inflammatory reaction by inhibiting the generation of inflammatory mediators, thereby achieving the anti-inflammatory effect. Studies have also found that substances such as vitamin C and beta-carotene contained in acalypha australis play an important role in the anti-inflammatory process. For example, vitamin C and other antioxidant substances in Acalypha australis help to enhance the immune system, improve body resistance, and play a role in combating diseases and maintaining physical health. Several studies have also found that the active ingredients in acalypha australis also have antibacterial activity and can help to combat infections caused by inflammation.
At present, although a great deal of research and reports about the use of active ingredients or extracts thereof in acalypha australis in foods, skin care products or anti-inflammatory active foods and the like exist, the research and reports about the use of acalypha australis extracts, especially the use of active diterpenoid compounds of acalypha australis and compositions thereof in anti-inflammatory and cell anti-inflammatory injury enhancing activities are fresh.
Disclosure of Invention
In view of the above, the present invention provides a compound with a novel structure, and further researches show that the compound has antioxidant and anti-inflammatory effects.
The detailed technical scheme of the invention is as follows:
the invention firstly provides an acalypha australis diterpenoid compound, wherein the acalypha australis diterpenoid compound is selected from one or more than one of acalypha australis diterpenoid-I, acalypha australis diterpenoid-II, acalypha australis diterpenoid-III, acalypha australis diterpenoid-IV and acalypha australis diterpenoid-V;
wherein the structural formulas of the acalypha australis diterpene-I, the acalypha australis diterpene-II, the acalypha australis diterpene-III, the acalypha australis diterpene-IV and the acalypha australis diterpene-V are shown as the formula I, the formula II, the formula III, the formula IV or the formula V respectively;
the invention also provides a preparation method of the acalypha australis extract, which comprises the following steps:
(1) Preparing acalypha australis diterpene-I-acalypha australis package, acalypha australis diterpene-II-acalypha australis package, acalypha australis diterpene-III-acalypha australis package, acalypha australis diterpene-IV-acalypha australis package, acalypha australis diterpene-V-acalypha australis package;
(2) The copperleaf herb diterpene-I-copperleaf herb, copperleaf herb diterpene-II-copperleaf herb, copperleaf herb diterpene-III-copperleaf herb, copperleaf herb diterpene-IV-copperleaf herb and copperleaf herb diterpene-V-copperleaf herb are uniformly mixed to obtain the copperleaf herb extract.
Preferably, the acalypha australis diterpene-I-acalypha australis diterpene-II-acalypha australis wrapper, acalypha australis diterpene-III-acalypha australis wrapper, acalypha australis diterpene-IV-acalypha australis wrapper or acalypha australis diterpene-V-acalypha australis wrapper in the step (1) is prepared by the following method:
taking acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V, adding water, then adding the acalypha australis crude extract, uniformly mixing, and freeze-drying to obtain acalypha australis diterpene-I-acalypha australis wrapper, acalypha australis diterpene-II-acalypha australis wrapper, acalypha australis diterpene-III-acalypha australis wrapper, acalypha australis diterpene-IV-acalypha australis wrapper or acalypha australis diterpene-V-acalypha australis wrapper.
Preferably, 1 (15-25) of acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V and water and 5-10.
Most preferably, the ratio of acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V to water is 1:20:8.
Preferably, the crude copperleaf extract is prepared by the following method:
adding water into fresh acalypha australis, and crushing to obtain crushed liquid; then centrifuging the crushed liquid; the supernatant is firstly dialyzed by a dialysis membrane with the pore diameter of 30kDa, and then is dialyzed by a dialysis membrane with the pore diameter of 35 kDa; and (3) taking the dialysate which passes through a 35kDa dialysis membrane, concentrating and drying to obtain the acalypha australis crude extract.
Preferably, the mass ratio of the acalypha australis diterpene-I-acalypha australis wrapper, the acalypha australis diterpene-II-acalypha australis wrapper, the acalypha australis diterpene-III-acalypha australis wrapper, the acalypha australis diterpene-IV-acalypha australis wrapper and the acalypha australis diterpene-V-acalypha australis wrapper in the step (2) is 1 (1-100): 1-100.
Further preferably, the mass ratio of the acalypha australis diterpene-I-acalypha australis wrapper, the acalypha australis diterpene-II-acalypha australis wrapper, the acalypha australis diterpene-III-acalypha australis wrapper, the acalypha australis diterpene-IV-acalypha australis wrapper and the acalypha australis diterpene-V-acalypha australis wrapper in the step (2) is 1 (1-20): 1-20.
Most preferably, the mass ratio of the acalypha australis diterpene-I-acalypha australis wrapper, the acalypha australis diterpene-II-acalypha australis wrapper, the acalypha australis diterpene-III-acalypha australis wrapper, the acalypha australis diterpene-IV-acalypha australis wrapper and the acalypha australis diterpene-V-acalypha australis wrapper in step (2) is 1:11.8:13.9:6.95:12.1.
The invention also provides the acalypha australis extract prepared by the preparation method.
The invention also provides an application of the acalypha australis diterpenoid compound or the acalypha australis extract in preparing a product with an anti-inflammatory effect.
The invention also provides an application of the acalypha australis diterpenoid compound or the acalypha australis extract in preparing a product with an antioxidant effect.
Preferably, the product is a cosmetic, skin care product, pharmaceutical or health care product.
The beneficial effects are that: the invention provides an acalypha australis diterpenoid compound with a brand new structure and an acalypha australis extract with a brand new composition, which is prepared by a brand new method. Research shows that the acalypha australis diterpenoid compound has obvious anti-inflammatory and antioxidant effects; in particular, the anti-inflammatory and antioxidant effects of the acalypha australis extract prepared by the method are greatly higher than those of acalypha australis diterpenoid compounds, and the acalypha australis extract prepared by the conventional method is also greatly higher than those of the acalypha australis extract.
Because the acalypha australis diterpenoid compounds and the acalypha australis extract have anti-inflammatory and antioxidant effects; therefore, the extract is used as an active ingredient and has important application value in preparing cosmetics, skin care products, medicines or health care products with anti-inflammatory and antioxidant effects.
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In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only drawings of some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of acalypha australis diterpene-I ESI-MS.
FIG. 2 shows acalypha australis diterpene-I 1 H NMR spectrum.
FIG. 3 shows acalypha australis diterpene-I 13 C NMR spectrum.
FIG. 4 is a diagram of acalypha australis diterpene-IDEPT spectrum.
FIG. 5 is a diagram of acalypha australis diterpene-II ESI-MS spectrum.
FIG. 6 shows acalypha australis diterpene-II 1 H NMR spectrum.
FIG. 7 shows acalypha australis diterpene-II 13 C NMR spectrum.
FIG. 8 is a diagram of acalypha australis diterpene-IIDEPT.
FIG. 9 is a diagram of acalypha australis diterpene-III ESI-MS.
FIG. 10 shows acalypha australis diterpene-III 1 H NMR spectrum.
FIG. 11 shows acalypha australis diterpene-III 13 C NMR spectrum.
FIG. 12 is a diagram of acalypha australis diterpene-III DEPT.
FIG. 13 is a diagram of acalypha australis diterpene-IV HR-ESI-MS spectrum.
FIG. 14 shows acalypha australis diterpene-IV 1 H NMR spectrum.
FIG. 15 shows acalypha australis diterpene-IV 13 C NMR spectrum.
FIG. 16 is a diagram of acalypha australis diterpene-IV DEPT.
FIG. 17 is a diagram of acalypha australis diterpene-V HR-ESI-MS.
FIG. 18 shows acalypha australis diterpene-V 1 H NMR spectrum.
FIG. 19 shows acalypha australis diterpene-V 13 C NMR spectrum.
FIG. 20 is a diagram of acalypha australis diterpene-V DEPT.
FIG. 21 is a graph showing experimental results of the effect of acalypha australis extract on the improvement of NCM460 cell viability and on LPS-induced intracellular ROS of NCM460, MDA and GSH contents in cell supernatant and antioxidant enzyme SOD activity.
FIG. 22 is a graph showing experimental results of the effect of acalypha australis extract on the serum inflammatory factor content of LPS-induced NCM460 cells.
FIG. 23 is a graph showing experimental results of the effect of acalypha australis extract on LPS-induced expression of iNOS, COX-2 and MAPK/NF- κB/TLR4 pathway proteins in NCM460 cells.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 preparation of acalypha australis diterpenoid compounds
(1) Crushing 4000g of fresh acalypha australis with a 6L crusher to obtain crushed liquid, centrifuging the crushed liquid at 10 ℃ for 10min, collecting supernatant, dialyzing sequentially through dialysis membranes with the pore diameters of 1kDa, 1.5kDa, 3kDa, 3.5kDa, 5kDa, 10kDa, 15kDa, 30kDa, 35kDa and 50kDa, and freeze-drying the dialysate to obtain the acalypha australis extract with different molecular weights.
(2) 200g of acalypha australis extract with molecular weight less than 1kDa is dissolved in 1000mL of water, sequentially extracted with petroleum ether, ethyl acetate and chloroform, and the extracts are respectively concentrated in vacuum to obtain the extract.
(3) Separating the ethyl acetate extract obtained in the step (2) by macroporous resin (HZ-818) column chromatography, sequentially carrying out gradient elution by using 3 times of ethanol solution (15%, 30%, 45%, 60%, 75% and 95%) with the column volume, and freeze-drying the eluent to obtain different components.
(4) Concentrating and drying the 45% ethanol eluent obtained in the step (3), separating by a Sephadex column (Sephadex G15, 5.0x90 cm) chromatography, eluting by using an ethanol water solution with the volume fraction of 45%, collecting each 200mL of eluent as 1 component, collecting 15 components in total, and freeze-drying; and sequentially numbering to form active components-1 to 15.
(5) Separating and purifying the active component-10 obtained in the step (4) by different High Performance Liquid Chromatography (HPLC) to obtain acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV and acalypha australis diterpene-V;
wherein, the HPLC preparation conditions of the acalypha australis diterpene-I are as follows: using a C18 column (9.4 x 250mm,5 μm, agilent, USA); the mobile phase is: a: water (0.1% trifluoroacetic acid), B: methanol; the gradient elution procedure was: 0-15min 42% B,15-25min 55% B,25-30min 70% B; the sample injection amount is as follows: 300. Mu.L; the detection wavelength is as follows: 260nm,300nm; flow rate: 10.0mL/min; column temperature: 20 ℃;
collecting eluent corresponding to chromatographic peak with retention time of 11.00min, concentrating and drying to obtain acalypha australis diterpene-I;
the HPLC preparation conditions of the acalypha australis diterpene-II are as follows: using a C18 column (9.4 x 250mm,5 μm, agilent, USA); the mobile phase is: a: water (0.1% trifluoroacetic acid), B: methanol; the gradient elution procedure was: 0-15min 38% B,15-25min 45% B,25-30min 60% B; the sample injection amount is as follows: 300. Mu.L; the detection wavelength is as follows: 260nm,300nm; flow rate: 10.0mL/min; column temperature: 20 ℃;
collecting eluent corresponding to chromatographic peak with retention time of 13.02min, concentrating, and drying to obtain herba Acalyphae diterpene-II;
the HPLC preparation conditions of the acalypha australis diterpene-III are as follows: using a C18 column (9.4 x 250mm,5 μm, agilent, USA); the mobile phase is: a: water (0.1% trifluoroacetic acid), B: methanol; the gradient elution procedure was: 0-10min 55% B,11-25min 75% B,25-30min 80% B; the sample injection amount is as follows: 300. Mu.L; the detection wavelength is as follows: 260nm,300nm; flow rate: 10.0mL/min; column temperature: 20 ℃;
collecting eluent corresponding to chromatographic peak with retention time of 9.32min, concentrating and drying to obtain acalypha australis diterpene-III;
the HPLC preparation conditions of the acalypha australis diterpene-IV are as follows: using a C18 column (9.4 x 250mm,5 μm, agilent, USA); the mobile phase is: a: water (0.1% trifluoroacetic acid), B: methanol; the gradient elution procedure was: 75% B in 0-10min 60%B,1125min 65%B,25-30 min; the sample injection amount is as follows: 300. Mu.L; the detection wavelength is as follows: 260nm,300nm; flow rate: 10.0mL/min; column temperature: 20 ℃;
collecting eluent corresponding to chromatographic peak with retention time of 7.04min, concentrating and drying to obtain acalypha australis diterpene-IV;
the HPLC preparation conditions of the acalypha australis diterpene-V are as follows: using a C18 column (9.4 x 250mm,5 μm, agilent, USA); the mobile phase is: a: water (0.1% trifluoroacetic acid), B: methanol; the gradient elution procedure was: 0-8min 50% B,9-25min 50% B,25-30min 75% B; the sample injection amount is as follows: 300. Mu.L; the detection wavelength is as follows: 260nm,300nm; flow rate: 10.0mL/min; column temperature: 20 ℃;
collecting eluent corresponding to chromatographic peak with retention time of 10.06min, concentrating, and drying to obtain herba Acalyphae diterpene-V.
Acalypha australis diterpene-I: light yellow oil, ESI-MS (FIG. 1) m/z 623.5[ M+Na ]] + The molecular formula is estimated to be C by combining hydrogen spectrum and carbon spectrum information 35 H 52 O 8 The molecular weight was 600 and the unsaturation was 10.
1 H NMR(400MHz,CDCl 3 ) The spectrum (FIG. 2) shows that the compound contains 7 methyl proton signals [ delta ] H 1.80(3H,s),1.76(3H,s),1.72(3H,s),1.28(3H,s),1.24(3H,s),0.85(3H,d,J＝4.5Hz),0.84(3H,t,J＝4.8Hz)]3 olefin hydrogen proton signals [ delta ] H 7.56(1H,s),5.66(1H,d,J＝5.1Hz),6.79(1H,m)]Proton signal [ delta ] on 3 oxygen carbons H 5.42(1H,d,J＝10.2Hz),4.02(1H,d,J＝12.9Hz),3.93(1H,d,J＝12.9Hz)]。 13 C NMR (FIG. 3) and DEPT-135 (FIG. 4) (100 MHz, CDCl) 3 ) The spectrum shows that the compound has 35 carbon signals in total, wherein the chemical shift is delta C The 3 carbon signals of 209.5,176.7,168.0 are carbonyl carbon signals with chemical shift delta C 161.2,140.7,137.7,133.0,129.6,128.7 the 6 carbon signals are double-bond ethylenic carbon signals, chemical shift delta C 78.6,77.0,73.9,68.2,65.7 the 5 carbon signals are oxygen-carbon signals with chemical shift delta C The 7 carbon signals of 24.0,17.1,14.6,14.5,14.3,12.4,10.3 are methyl carbon signals. In combination with the degree of unsaturation that is present, 1 h NMR data speculates that the compound is a diterpene of a 4-ring parent nucleus and has 2 ester group substitutions. Wherein the chemical shift is delta C 168.0,128.7,137.7,12.4,14.6 the 5 carbon signals are tiglyl group carbon signals, chemical shift delta C The 10 carbon signals of 176.7,34.6,32.0,29.6,29.5,29.4,29.3,24.8,22.9,14.5 are decanoyl group carbon signals.
By combining the information, the structure of the final identified compound is shown as a formula I, the compound is named as acalypha australis diterpenoid-I, 1 H NMR、 13 the C NMR data are shown in Table 1.
TABLE 1 acalypha australis diterpene-I 1 H and 13 C NMR data
Acalypha australis diterpene-II: ESI-MS (FIG. 5) m/z 611.3[ M+Na ] as pale yellow oil] + The molecular formula is estimated to be C by combining hydrogen spectrum and carbon spectrum information 34 H 52 O 8 The molecular weight was 588 and the unsaturation was 9.
1 H NMR(400MHz,CDCl 3 ) The spectrum (FIG. 6) shows that the compound contains 6 methyl proton signals [ delta ] H 2.05(3H,s),1.76(3H,br s),1.23(3H,s),1.19(3H,s),0.86(3H,d,J＝6.6Hz),0.86(3H,t,J＝6.9Hz)]2 olefinic hydrogen proton signals [ delta ] H 7.57(1H,s),5.66(1H,d,J＝4.2Hz)]Proton signal [ delta ] on 3 oxygen carbons H 5.35(1H,d,J＝10.5Hz),4.03(1H,d,J＝13.2Hz),3.96(1H,d,J＝13.2Hz)]。 13 C NMR (FIG. 7) and DEPT-135 (FIG. 8) (100 MHz, CDCl) 3 ) The spectrum shows that the compound has 34 carbon signals in total, wherein, the chemical shift is delta C The 3 carbon signals of 209.2,176.0,171.1 are carbonyl carbon signals with chemical shift delta C 161.0,140.6,133.1,129.4 the 4 carbon signals are double-bond ethylenic carbon signals, chemical shift delta C 78.4,77.2,73.9,68.1,65.5 the 5 carbon signals are oxygen-carbon signals with chemical shift delta C The 6 carbon signals of 24.1,22.9,21.3,17.0,14.6,10.3 are methyl carbon signals. In combination with the degree of unsaturation that is present, 1 h NMR data speculates that the compound is a diterpene of a 4-ring parent nucleus and has 2 ester group substitutions. Wherein the chemical shift is delta C 171.1,21.3 the 2 carbon signals are acrylate group carbon signals, chemical shift delta C The 11 carbon signals of 176.0,34.6,32.1,29.8,29.7,29.6,29.5,29.3,24.7,22.9,14.3 are the dodecanoyl group carbon signals.
By combining the information, the structure of the final identified compound is shown as a formula II, the compound is named as acalypha australis diterpenoid-II, 1 H NMR、 13 the C NMR data are shown in Table 2.
TABLE 2 acalypha australis diterpene-II 1 H and 13 C NMR data
Acalypha australis diterpene-III: white powder, ESI-MS (FIG. 9) m/z 511.5[ M+Na ]] + The molecular formula is estimated to be C by combining hydrogen spectrum and carbon spectrum information 27 H 36 O 8 The molecular weight was 488 and the unsaturation was 10.
1 H NMR(400MHz,CDCl 3 ) The spectrum (FIG. 10) shows that the compound contains 7 methyl proton signals [ delta ] H 2.08(3H,s),1.81(3H,s),1.77(3H,s),1.73(3H,s),1.25(3H,s),1.18(3H,s),0.86(3H,d,J＝6.4Hz)]2 olefinic hydrogen proton signals [ delta ] H 7.57(1H,s),5.64(1H,d,J＝3.9Hz)]Proton signal [ delta ] on 3 oxygen carbons H 5.43(1H,d,J＝10.5Hz),4.03(1H,d,J＝12.6Hz),3.95(1H,d,J＝12.6Hz)]。 13 C NMR (FIG. 11) and DEPT-135 (FIG. 12) (100 MHz, CDCl) 3 ) The spectrum shows that the compound has 27 carbon signals in total, wherein the chemical shift is delta C The 3 carbon signals of 209.4,174.1,168.1 are carbonyl carbon signals with chemical shift delta C 161.1,140.7,133.1,137.9,129.5,128.7 the 6 carbon signals are double-bond ethylenic carbon signals, chemical shift delta C 78.6,76.9,73.9,68.2,65.9 the 5 carbon signals are oxygen-carbon signals with chemical shift delta C The 7 carbon signals of 24.0,21.4,17.1,14.7,14.6,12.4,10.3 are methyl carbon signals. In combination with the degree of unsaturation that is present, 1 HNMR data speculate that the compound is a diterpene of a 4-ring parent nucleus and has 2 ester group substitutions. Wherein the chemical shift is delta C 168.1,128.7,137.9,14.6,12.4 the 5 carbon signals are tiglyl group carbon signals, chemical shift delta C The 2 carbon signals of 174.1,21.4 are acetyl group carbon signals.
In combination with the information, the structure of the final identified compound is shown as a formula III, the compound is named as acalypha australis diterpenoid-III, 1 H NMR、 13 the C NMR data are shown in Table 3.
TABLE 3 acalypha australis diterpene-III 1 H and 13 C NMR data
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Acalypha australis diterpene-IV: white powder, HR-ESI-MS (FIG. 13) m/z 513.2463[ M+Na ]] + (calcd for C 27 H 38 O 8 Na, 513.2459) and its molecular formula is C 27 H 38 O 8 The molecular weight was 490 and the degree of unsaturation was 9.
1 H NMR(400MHz,CDCl 3 ) The spectrum (FIG. 14) shows that the compound contains 7 methyl proton signals [ delta ] H 2.04(3H,s),1.73(3H,s),1.22(3H,s),1.17(3H,s),1.13(3H,d,J＝7.2Hz),0.91(3H,d,J＝7.5Hz),0.86(3H,d,J＝4.8Hz)]2 olefinic hydrogen proton signals [ delta ] H 7.56(1H,s),5.58(1H,s)]Proton signal [ delta ] on 3 oxygen carbons H 5.38(1H,d,J＝10.2Hz),4.02(1H,d,J＝12.9Hz),3.95(1H,d,J＝12.9Hz)]。 13 C NMR (FIG. 15) and DEPT-135 (FIG. 16) (100 MHz, CDCl) 3 ) The spectrum shows that the compound has 27 carbon signals in total, wherein the chemical shift is delta C The 3 carbon signals of 209.3,176.5,174.1 are carbonyl carbon signals with chemical shift delta C 161.0,140.8,133.1,129.4 the 4 carbon signals are double-bond ethylenic carbon signals, chemical shift delta C 78.5,76.5,73.9,68.2,65.4 the 5 carbon signals are oxygen-carbon signals with chemical shift delta C The 7 carbon signals of 24.3,21.3,17.2,17.0,14.6,11.8,10.3 are methyl carbon signals. In combination with the degree of unsaturation that is present, 1 h NMR data speculates that the compound is a diterpene of a 4-ring parent nucleus and has 2 ester group substitutions. Wherein the chemical shift is delta C 174.1,42.0,26.9,17.2,11.8 the 5 carbon signals are the (2-methyl) butyl radical carbon signals, chemical shift delta C The 2 carbon signals of 176.5,21.3 are acetyl group carbon signals.
By combining the information, the structure of the final identified compound is shown as a formula IV, the compound is named as acalypha australis diterpene-IV, 1 H NMR、 13 the C NMR data are shown in Table 4.
TABLE 4 acalypha australis diterpene-IV 1 H and 13 C NMR data
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Acalypha australis diterpene-V: white powder, HR-ESI-MS (FIG. 17) m/z 539.2640[ M+Na ]] + (calcd for C 29 H 40 O 8 Na, 539.2615) and its molecular formula is C 29 H 40 O 8 Molecular weight 516 and degree of unsaturation 10.
1 H NMR(400MHz,CDCl 3 ) The spectrum (FIG. 18) shows that the compound contains 8 methyl proton signals [ delta ] H 1.81(3H,s),1.77(3H,s),1.74(3H,s),1.26(3H,s),1.19(3H,s),1.18(3H,d,J＝6.1Hz),1.13(3H,d,J＝7.5Hz),0.86(3H,d,J＝6.6Hz)]2 olefinic hydrogen proton signals [ delta ] H 7.57(1H,s),5.81(1H,s)]Proton signal [ delta ] on 3 oxygen carbons H 5.42(1H,d,J＝10.5Hz),4.03(1H,d,J＝12.9Hz),4.00(1H,d,J＝12.9Hz)]。 13 C NMR (FIG. 19) and DEPT-135 (FIG. 20) (100 MHz, CDCl) 3 ) The spectrum shows that the compound has 29 carbon signals in total, wherein the chemical shift is delta C The 3 carbon signals of 209.1,179.5,167.7 are carbonyl carbon signals with chemical shift delta C 160.9,140.4,137.5,132.8,129.4,128.5 the 6 carbon signals are double-bond ethylenic carbon signals, chemical shift delta C 78.3,76.7,73.7,68.1,65.4 the 5 carbon signals are oxygen-carbon signals with chemical shift delta C The 8 carbon signals of 23.9,18.6,18.7,16.9,14.4,14.4,12.3,10.1 are methyl carbon signals, combined with unsaturation, 1 h NMR data speculates that the compound is a diterpene of a 4-ring parent nucleus and has 2 ester group substitutions. Wherein, is converted intoThe chemical shift is delta C 167.7,137.5,128.5,14.4,12.3 the 5 carbon signals are tiglyl group carbon signals, chemical shift delta C The 4 carbon signal of 179.5,34.2,18.7,18.6 is the isobutyryl group carbon signal.
By combining the information, the structure of the final identified compound is shown as a formula V, and the compound is named as acalypha australis diterpenoid-V, 1 H NMR、 13 the C NMR data are shown in Table 5.
TABLE 5 acalypha australis diterpene-V 1 H and 13 C NMR data
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Example 2 preparation of Acalypha australis extract
(1) Taking acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V, adding water, then adding the acalypha australis crude extract, uniformly mixing, and freeze-drying to obtain acalypha australis diterpene-I-acalypha australis wrapper, acalypha australis diterpene-II-acalypha australis wrapper, acalypha australis diterpene-III-acalypha australis wrapper, acalypha australis diterpene-IV-acalypha australis wrapper or acalypha australis diterpene-V-acalypha australis wrapper;
1:20:8 of acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V with water;
the acalypha australis crude extract is prepared by the following method: taking 4000g of fresh acalypha australis, adding water and crushing by a 6L crusher to obtain crushing liquid, and centrifuging the crushing liquid at 10 ℃; the supernatant is firstly dialyzed by a dialysis membrane with the pore diameter of 30kDa, and then is dialyzed by a dialysis membrane with the pore diameter of 35 kDa; collecting dialysate which penetrates through a 35kDa dialysis membrane, concentrating and drying to obtain the crude extract of acalypha australis;
(2) And uniformly mixing the acalypha australis diterpene-I-acalypha australis wrapper, the acalypha australis diterpene-II-acalypha australis wrapper, the acalypha australis diterpene-III-acalypha australis wrapper, the acalypha australis diterpene-IV-acalypha australis wrapper and the acalypha australis diterpene-V-acalypha australis wrapper according to the mass ratio of 1:11.8:13.9:6.95:12.1 to obtain the acalypha australis extract.
Effect examples
Human colon epithelial cells (NCM 460 cells) were cultured with RPMI-1640 containing 10% FBS based on 5% CO at 37 ℃C 2 Culturing in a constant temperature incubator for 24 hours. At 5X 10 3 The density of each mL is inoculated on a 96-well plate, and the culture is carried out in an incubator for 12 hours. The blank group is added with complete culture medium, the other groups are added with LPS with the final mass concentration of 10 mug/L, the cells are placed into a cell culture box for continuous culture for 12 hours, then the cells are respectively collected, washed for 2 times by cold PBS, then placed into an RPMI-1640 culture medium to dissolve active substances with the final mass concentration of 50 mug/mL, and the cell culture box is used for continuous culture for 24 hours (LPS group, normal culture after cell cleaning). After the end of the experiment, cell viability was detected by CCK 8 method with reference to the instructions; ELISA kit detects ROS content in cells and MDA, GSH content, SOD activity and inflammatory factor (TNF-alpha, IL-6, IL-1 beta, PGE2 and NO) content in cell supernatant; protein immunoblotting method is used for detecting protein expression of iNOS, COX-2, TLR4, p-JNK and the like.
Experimental grouping: blank group, acalypha australis diterpene-I group, acalypha australis diterpene-II group, acalypha australis diterpene-III group, LPS exposure group, acalypha australis diterpene-IV group, acalypha australis diterpene-V group, commercial acalypha australis extract group and the acalypha australis extract group of the present invention.
As a result, as shown in fig. 21A, the LPS stimulation significantly reduced NCM460 cell viability compared to the blank (blank cell viability set to 100%). The cell viability of the LPS group was reduced by 31.1% (p < 0.01) compared to the placebo group. Compared with the LPS group, the acalypha australis diterpenoid compound and the acalypha australis extract can improve the NCM460 cell activity. Compared with LPS group, the cell viability of Acalypha australis diterpenoid-I group, acalypha australis diterpenoid-II group, acalypha australis diterpenoid-III group, acalypha australis diterpenoid-IV group and Acalypha australis diterpenoid-V group NCM460 is respectively improved by 12.4% (p < 0.05), 10.8% (p < 0.05), 9.91%, 10.4% (p < 0.05) and 10.1% (p < 0.05). Compared with the acalypha australis diterpenoid compounds, the acalypha australis extract can better improve the NCM460 cell activity. Compared with the LPS exposure group, the acalypha australis extract group NCM460 cell viability is improved by 21.3% (p < 0.01), and the NCM460 cell viability is improved most obviously. And compared with the commercial copperleaf extract, the copperleaf extract has more excellent activity of improving the NCM460 cell activity. Under the co-culture condition of the amaranth diterpenoid compounds with the same concentration, the activity of the amaranth extract for improving the NCM460 cell activity is strongest. We speculate that the active diterpene in the acalypha australis extract can exert a synergistic effect, and can better alleviate inflammatory injury caused by LPS exposure so as to improve NCM460 cell viability.
Further analysis of the experimental results showed that NCM460 cell ROS content and MDA content in cell supernatant were significantly increased in the LPS group compared to the blank group (fig. 21b, c). Compared to the blank, the ROS content in NCM460 cells and MDA content in cell supernatant of LPS group were increased by 251.3% (p < 0.01) and 167.3% (p < 0.01), respectively. It is shown that LPS exposure causes excessive accumulation of oxidative substances such as MDA, ROS and the like in NCM460 cells, thereby inducing oxidative damage and causing significant reduction of NCM460 cell viability. Compared with the LPS group, the copperleaf extract can effectively inhibit the increase of ROS in NCM460 cells and MDA content in cell supernatant induced by LPS exposure. Compared with the LPS group, the content of the ROS in the cells of the acalypha australis diterpene-I group NCM460 and the MDA in the cell supernatant is respectively reduced by 27.6 percent (p < 0.01) and 20.9 percent (p < 0.01); compared with LPS group, the content of ROS in the acalypha australis diterpene-II group NCM460 cells and MDA in the cell supernatant is respectively reduced by 23.9 percent (p < 0.01) and 12.6 percent (p < 0.05); compared with the LPS group, the content of the MDA in the intracellular ROS and the cell supernatant of the NCM460 of the commercial acalypha australis extract group is respectively reduced by 14.9 percent (p < 0.01) and 9.73 percent; compared with the commercial acalypha australis extract group, the acalypha australis diterpenoid compound has more excellent anti-inflammatory activity, and can better inhibit the increase of the ROS in NCM460 cells and MDA content in cell supernatant induced by LPS exposure. Compared with LPS group, the content of ROS in the NCM460 cells and MDA in the cell supernatant of the acalypha australis extract group is respectively reduced by 51.8% (p < 0.01) and 32.2% (p < 0.01). The acalypha australis extract provided by the invention has better activity of inhibiting the increase of the content of ROS in NCM460 cells and MDA in cell supernatant induced by LPS exposure than the acalypha australis diterpenoid compound monomer and the commercial acalypha australis extract under the same dosage condition.
Further analysis of the experimental results (fig. 21D) found that GSH content in NCM460 cell supernatants of LPS group was significantly reduced compared to the blank group. GSH content in NCM460 cell supernatants of LPS group was reduced by 28.7% (p < 0.01) compared to the blank group. Compared with the LPS group, the acalypha australis extract can effectively inhibit the reduction of GSH content in the supernatant of NCM460 cells induced by LPS exposure. Wherein GSH content in the acalypha australis diterpene-I group NCM460 cell supernatant is increased by 12.3% (p < 0.05) compared to the LPS group; GSH content in the acalypha australis diterpene-II group NCM460 cell supernatant was increased by 10.4% (p < 0.05) compared to the LPS-exposed group; compared with the LPS group, the GSH content in the NCM460 cell supernatant of the commercial copperleaf extract group is increased by 8.11%; further analysis experiments show that compared with the LPS group, the GSH content in the NCM460 cell supernatant of the acalypha australis extract group is increased by 21.8% (p < 0.01). Compared with the commercial acalypha australis extract group, the acalypha australis diterpenoid compound can better inhibit reduction of GSH content in NCM460 cell supernatant induced by LPS exposure. Compared with the commercial copperleaf extract or copperleaf compound disclosed by the invention, the copperleaf extract disclosed by the invention has the advantages that the activity of NCM460 cells is improved, the increase of ROS (reactive oxygen species) content and MDA (multiple-action) content in the NCM460 cells caused by LPS (LPS exposure) exposure can be well inhibited, and the content of antioxidant substances such as GSH (GSH) in the cells is improved. We speculate that the active diterpenoids in the copperleaf extract of the invention can exert synergistic effect and better inhibit the oxidative damage of NCM460 cells caused by LPS exposure.
Further analysis of the experimental results (fig. 21E) revealed that, after LPS exposure, the antioxidant enzyme SOD activity in NCM460 cell supernatant was significantly reduced compared to the blank, and combined with the experimental results of the effect of LPS exposure on ROS, MDA, etc. in NCM460 cells, it was demonstrated that LPS exposure induced oxidative stress and suffered severe oxidative damage to NCM460 cells. Compared with the blank group, the NCM460 cell of LPS group has 31.8 percent reduced antioxidant enzyme SOD activity (p < 0.01). Compared with the LPS group, the antioxidant enzyme SOD activity of the acalypha australis extract group is increased by 21.3 percent (p is less than 0.01). Meanwhile, compared with the acalypha australis diterpenoid compounds with the same concentration and the commercial acalypha australis extract, the NCM460 cell antioxidant enzyme SOD activity of the acalypha australis extract group is most obviously improved. We speculate that the active diterpenoid compounds in the acalypha australis extract can play a synergistic role and better improve the activity of the antioxidant enzyme SOD of the NCM460 cells, and further can better improve the antioxidant injury activity of the NCM460 cells.
Further analysis of the effect of acalypha australis extract of the present invention on the inflammatory factors of LPS-induced NCM460 cells (fig. 22) it is seen that the inflammatory factors (IL-6, IL-1 beta, NO, PGE2 and TNF-a) content in the cell supernatant of the LPS group was significantly increased compared to the blank group. Inflammatory factors IL-6, IL-1 beta, NO, PGE2 and TNF-alpha levels in the cell supernatants of LPS groups were increased by 194.2% (p < 0.01), 231.3% (p < 0.01), 97.7% (p < 0.01), 171.3% (p < 0.01) and 311.5% (p < 0.01), respectively, compared to the blank group. The experimental results in this section demonstrate that LPS exposure induces oxidative damage to NCM460 cells while also inducing inflammation to NCM460 cells, thereby increasing secretion of cytokines and causing inflammatory damage to NCM460 cells. And after the treatment of the acalypha australis diterpenoid compounds, the commercial acalypha australis extract or the acalypha australis extract, especially the acalypha australis extract group of the invention, the increase of the inflammatory factors IL-6, IL-1 beta, NO, PGE2 and TNF-alpha in the supernatant fluid of NCM460 cells is obviously inhibited compared with the LPS group. Wherein, compared with LPS stimulated group, the contents of the inflammatory factors IL-6, IL-1 beta, NO, PGE2 and TNF-alpha in the acalypha australis diterpene-I group are respectively reduced by 28.2 percent (p < 0.01), 10.2 percent (p < 0.05), 20.8 percent (p < 0.01), 32.4 percent (p < 0.01) and 28.5 percent (p < 0.01); compared with LPS stimulated group, the content of inflammatory factors IL-6, IL-1 beta, NO, PGE2 and TNF-alpha in the commercial acalypha australis extract group is reduced by 18.0% (p < 0.01), 5.74%, 13.1% (p < 0.05), 11.8% (p < 0.05) and 18.2% (p < 0.01), respectively; the inflammatory factors IL-6, IL-1 beta, NO, PGE2 and TNF-alpha content of the copperleaf extract group of the present invention were reduced by 42.5% (p < 0.01), 21.8% (p < 0.01), 38.0% (p < 0.01), 43.5% (p < 0.01) and 55.7% (p < 0.01), respectively, compared to LPS stimulated group. The acalypha australis extract can effectively inhibit excessive accumulation of NCM460 cell inflammatory factors induced by LPS exposure and relieve NCM460 cell inflammatory injury caused by LPS stimulation.
Further analysis and experiment results show that the activity of inhibiting the increase of the inflammatory factors IL-6, IL-1 beta, NO, PGE2 and TNF-alpha of NCM460 cells induced by LPS of the copperleaf extract is stronger than that of copperleaf diterpenoid compounds with the same concentration or commercial copperleaf extract. We speculate that the acalypha australis diterpenoid compounds in the acalypha australis extract can improve the antioxidant enzyme activity in NCM460 cells and improve the capacity of NCM460 cells to remove oxidative substances such as MDA and ROS through synergistic action, can improve the activity of NCM460 cells in inhibiting excessive accumulation of inflammatory factors, and further can better improve the anti-inflammatory activity of NCM460 cells and improve the anti-inflammatory capacity of NCM460 cells.
Meanwhile, the influence of the acalypha australis extract on the expression of NCM460 cell iNOS, COX-2, MAPK/NF- κB/TLR4 and other channel related proteins induced by LPS is also detected. The results of the analysis experiments showed (FIG. 23A, B) that LPS group iNOS, COX-2 protein was significantly increased (p < 0.01) compared to the blank group, and combined with LPS exposure induced increased NO and PGE2 secretion, we speculated that LPS exposure induced increased cell iNOS, COX-2 protein, and thus increased NO and PGE2 secretion. Compared with LPS, the expression of iNOS and COX-2 proteins in the copperleaf extract is obviously reduced, and the secretion increase of NO and PGE2 induced by LPS exposure is also obviously inhibited, and we speculate that the copperleaf extract can play an active role by inhibiting the over expression of iNOS and COX-2 proteins in cells induced by LPS exposure, so that the secretion increase of NO and PGE2 is inhibited.
Analytical experimental results showed (fig. 23C, D, E, F, G), compared with the blank group, the expression of TLR4, p-JNK, p-NF- κb proteins of the LPS group was significantly increased (p < 0.01), while the expression of JNK and NF- κb proteins was significantly decreased (p < 0.01), indicating that LPS exposure may induce cellular inflammation by activating cellular MAPK/NF- κb/TLR4 pathway; compared with LPS, the expression of TLR4, p-JNK and p-NF- κB proteins in the copperleaf extract group is reduced, and the expression of JNK and NF- κB proteins is increased. The experimental result in the part shows that the acalypha australis extract can play a role in inhibiting the inflammatory injury of cells induced by LPS exposure by regulating and controlling the expression of MAPK/NF- κB/TLR4 pathway related proteins so as to play a role in protecting. Further analysis and experiment results show that the acalypha australis extract has better activity than the acalypha australis diterpenoid compounds and the acalypha australis extract sold in the market thereof on inhibiting the increase of LPS-induced NCM460 cell TLR4, p-JNK and p-NF-kappa B protein expression and relieving LPS-induced NCM460 cell JNK and NF-kappa B protein inhibition. The active diterpenoid compounds in the acalypha australis extract can better regulate and control the expression of MAPK/NF- κB/TLR4 pathway proteins and iNOS and COX-2 proteins through synergistic effect so as to play a role in protecting.
The above examples illustrate that the copperleaf extract of the present invention can increase the cell viability of NCM460 cells under inflammatory injury conditions, and increase the antioxidant and anti-inflammatory injury activity of NCM460 cells. The experimental results of the embodiment show that the acalypha australis extract has better antioxidant and anti-inflammatory activities than the acalypha australis diterpenoid compounds or the commercial acalypha australis extract under the same concentration condition. We speculate that the active diterpenoid compounds in the acalypha australis extract can better exert the antioxidant and anti-inflammatory activities through the synergistic effect.
The foregoing disclosure is illustrative of the preferred embodiments of the present invention, and is not to be construed as limiting the scope of the invention, as it is understood by those skilled in the art that all or part of the above-described embodiments may be practiced with equivalents thereof, which fall within the scope of the invention as defined by the appended claims.

Claims (10)

1. The acalypha australis diterpenoid compound is characterized in that the acalypha australis diterpenoid compound is selected from one or more than one of acalypha australis diterpenoid-I, acalypha australis diterpenoid-II, acalypha australis diterpenoid-III, acalypha australis diterpenoid-IV and acalypha australis diterpenoid-V;
wherein the structural formulas of the acalypha australis diterpene-I, the acalypha australis diterpene-II, the acalypha australis diterpene-III, the acalypha australis diterpene-IV and the acalypha australis diterpene-V are shown as the formula I, the formula II, the formula III, the formula IV or the formula V respectively;
2. a preparation method of acalypha australis extract is characterized by comprising the following steps:
(1) Preparing acalypha australis diterpene-I-acalypha australis package, acalypha australis diterpene-II-acalypha australis package, acalypha australis diterpene-III-acalypha australis package, acalypha australis diterpene-IV-acalypha australis package, acalypha australis diterpene-V-acalypha australis package;
(2) The copperleaf herb diterpene-I-copperleaf herb, copperleaf herb diterpene-II-copperleaf herb, copperleaf herb diterpene-III-copperleaf herb, copperleaf herb diterpene-IV-copperleaf herb and copperleaf herb diterpene-V-copperleaf herb are uniformly mixed to obtain the copperleaf herb extract.
3. The preparation method according to claim 2, wherein the acalypha australis diterpene-I-acalypha australis wrapper, acalypha australis diterpene-II-acalypha australis wrapper, acalypha australis diterpene-III-acalypha australis wrapper, acalypha australis diterpene-IV-acalypha australis wrapper or acalypha australis diterpene-V-acalypha australis wrapper in step (1) is prepared by the following method:
taking acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V, adding water, then adding the acalypha australis crude extract, uniformly mixing, and freeze-drying to obtain acalypha australis diterpene-I-acalypha australis wrapper, acalypha australis diterpene-II-acalypha australis wrapper, acalypha australis diterpene-III-acalypha australis wrapper, acalypha australis diterpene-IV-acalypha australis wrapper or acalypha australis diterpene-V-acalypha australis wrapper.
4. The preparation method according to claim 3, wherein 1 (15-25) of acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V with water, (5-10);
most preferably, the ratio of acalypha australis diterpene-I, acalypha australis diterpene-II, acalypha australis diterpene-III, acalypha australis diterpene-IV or acalypha australis diterpene-V to water is 1:20:8.
5. The preparation method of claim 3, wherein the crude acalypha australis extract is prepared by the following method:
adding water into fresh acalypha australis, and crushing to obtain crushed liquid; then centrifuging the crushed liquid; the supernatant is firstly dialyzed by a dialysis membrane with the pore diameter of 30kDa, and then is dialyzed by a dialysis membrane with the pore diameter of 35 kDa; and (3) taking the dialysate which passes through a 35kDa dialysis membrane, concentrating and drying to obtain the acalypha australis crude extract.
6. The preparation method according to claim 3, wherein the mass ratio of the acalypha australis diterpene-I-acalypha australis package, the acalypha australis diterpene-II-acalypha australis package, the acalypha australis diterpene-III-acalypha australis package, the acalypha australis diterpene-IV-acalypha australis package and the acalypha australis diterpene-V-acalypha australis package in step (2) is 1 (1-100): 1-100;
further preferably, the mass ratio of the acalypha australis diterpene-I-acalypha australis wrapper, the acalypha australis diterpene-II-acalypha australis wrapper, the acalypha australis diterpene-III-acalypha australis wrapper, the acalypha australis diterpene-IV-acalypha australis wrapper and the acalypha australis diterpene-V-acalypha australis wrapper in the step (2) is 1 (1-20): 1-20.
7. The preparation method according to claim 6, wherein the mass ratio of the acalypha australis diterpene-I-acalypha australis package, the acalypha australis diterpene-II-acalypha australis package, the acalypha australis diterpene-III-acalypha australis package, the acalypha australis diterpene-IV-acalypha australis package and the acalypha australis diterpene-V-acalypha australis package in step (2) is 1:11.8:13.9:6.95:12.1.
8. The acalypha australis extract prepared by the preparation method of any one of claims 2 to 7.
9. Use of the acalypha australis diterpenoid compounds of claim 1 or the acalypha australis extract of claim 8 for the preparation of a product with anti-inflammatory effect.
10. Use of the acalypha australis diterpenoid compounds of claim 1 or the acalypha australis extract of claim 8 in the preparation of products with antioxidant effect.
CN202311140095.2A 2023-09-05 2023-09-05 Herba Acalyphae diterpenoid compounds, herba Acalyphae extract and application thereof in preparing antioxidant and/or antiinflammatory products Pending CN117304031A (en)

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