Aconitine A and its extraction method and application

Abstract

The present invention relates to the technical field of extraction and application of traditional Chinese medicine, and in particular to aconitine A and its extraction method and application. 18 The new compound of diterpenoid alkaloids has a good ability to scavenge free radicals. 50 The value is 87.2±2.4μg / mL, which can be used as an alkaline antioxidant and can be used in sunscreen cosmetics and free radical scavenging related drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine extraction and application, and in particular to aconitine A and an extraction method and application thereof. Background Art

[0002] Free radicals in organisms are primarily reactive oxygen species (ROS). As normal metabolic products, they play an important role in transmitting cellular information, attacking invading foreign bodies, and participating in energy production. However, when the body experiences oxidative stress or free radical imbalance (OS), its ability to scavenge free radicals decreases (due to reduced antioxidant enzyme activity or decreased antioxidant concentrations), leading to an excess of free radicals in the body, which in turn damages cellular components and biomolecules.

[0003] The body maintains an oxidative defense system, including superoxide dismutase (SOD), reduced glutathione (GSH), and antioxidant nutrients, to combat oxidative stress. Once this dynamic balance between oxidation and antioxidant activity is disrupted, as in the case of disease or exogenous drugs and toxins, free radicals can exert powerful damaging effects, causing lipid peroxidation damage to biomembranes, oxidative damage to enzymes, amino acids, and proteins, and impacting the morphology and function of internal organs and the immune system, leading to illness and even death. Consequently, a large number of anti-free radical compounds and drugs are being developed, and natural antioxidants, particularly those that work under alkaline conditions, are gaining increasing attention.

[0004] Aconitum sinomontanum Nakai, also known as "Seven Sackcloth," is a plant of the genus Aconitum in the Ranunculaceae family. It has the effects of dispelling wind and dampness, regulating qi and relieving pain, and promoting blood circulation and reducing swelling. The main chemical components of Aconitum sinomontanum Nakai are diterpenoid alkaloids, which have analgesic, antiarrhythmic, anti-inflammatory, anti-rheumatic, anticancer, and antibacterial biological activities. The lappaconitine component in Aconitum sinomontanum Nakai has significant anti-inflammatory and analgesic effects and is widely used in traditional medicine in China, Pakistan, and other Asian countries.

[0005] At present, there are few reports on the antioxidant activity of alkaloids in Aconitum root. Therefore, in the field of traditional Chinese medicine extraction and application technology, it would be beneficial to provide a C 18 -type diterpene alkaloids, and provides C 18 It is necessary to find new compounds of type-diterpenoid alkaloids that have strong ability to scavenge free radicals. Summary of the Invention

[0006] The purpose of the present invention is to provide a C 18-type diterpene alkaloids, the C 18 -Diterpenoid alkaloid compounds have a good ability to scavenge free radicals, and their IC 50 The value is 87.2±2.4μg / mL. As an alkaline antioxidant, it can be used in sunscreen cosmetics and free radical scavenging related drugs.

[0007] In a first aspect, aconitine A is provided, wherein the aconitine A has a structure as shown in formula (I):

[0008]

[0009] Preferably, the molecular formula of the homoaconitine A is C 24 H 39 NO6, the molecular weight of the aconitine A is 438.2839.

[0010] In a second aspect, a method for extracting aconitine A is provided, the extraction method comprising the following steps:

[0011] Step S1: crushing the root of Aconitum carmichaelii, extracting it with methanol solution under reflux, distilling the extract under reduced pressure to remove methanol, letting it stand and filtering to remove sediment, to obtain an aqueous suspension;

[0012] Preferably, the mixture is extracted with 90% methanol solution under reflux for 3 times.

[0013] Step S2: extracting the aqueous suspension with petroleum ether and ethyl acetate in sequence, and concentrating the extract under reduced pressure to obtain an ethyl acetate extract;

[0014] Step S3: subjecting the ethyl acetate extract to silica gel column chromatography and combining the obtained fractions under TLC monitoring to obtain twelve fractions, namely, a first fraction, a second fraction, a third fraction, a fourth fraction, a fifth fraction, a sixth fraction, a seventh fraction, an eighth fraction, a ninth fraction, a tenth fraction, an eleventh fraction, and a twelfth fraction;

[0015] Preferably, in step S3, the eluents for silica gel column chromatography on the ethyl acetate extract are dichloromethane and methanol, and the volume ratios of the dichloromethane to the methanol are 100:0, 98:2, 95:5, 90:10, 85:15, 80:20, 70:30 and 60:40, respectively.

[0016] Step S4: subjecting the seventh fraction to silica gel column chromatography to obtain a thirteenth fraction, a fourteenth fraction, a fifteenth fraction, a sixteenth fraction, and a seventeenth fraction;

[0017] Preferably, in step S4, the seventh component is subjected to silica gel column chromatography gradient elution using a dichloromethane, methanol and ammonia system to obtain five components, namely the thirteenth component, the fourteenth component, the fifteenth component, the sixteenth component and the seventeenth component.

[0018] More preferably, the volume ratios of dichloromethane, methanol and ammonia water in the dichloromethane, methanol and ammonia water system are 100:5:1, 100:10:1, 100:20:1 and 100:30:1 respectively.

[0019] Step S5: The sixteenth component is sequentially subjected to decontamination by a dextran gel column, elution separation, and purification to obtain the target compound, namely aconitine A.

[0020] Preferably, in step S5, the sixteenth component is removed from the mixture using a dichloromethane and methanol system and a Sephadex LH-20 dextran gel column, separated by a gradient elution of ethyl acetate, ethanol and aqueous ammonia on a forward silica gel column, and further purified by a Sephadex LH-20 column using methanol as an eluent to prepare the target compound, i.e., aconitine A.

[0021] In the present invention, the homoaconitine A is C 18 -type diterpene alkaloids new compounds.

[0022] More preferably, the volume ratio of dichloromethane to methanol in the dichloromethane and methanol system is 1:1; the volume ratios of ethyl acetate, ethanol and aqueous ammonia are 20:1:0.1, 10:1:0.1 and 10:2:0.1, respectively.

[0023] In a third aspect, an alkaloid compound is provided, wherein the alkaloid compound includes the aconitine A described in the present invention.

[0024] In a fourth aspect, an antioxidant is provided, wherein the antioxidant comprises the aconitine A or alkaloid compound described in the present invention.

[0025] In a fifth aspect, a sunscreen cosmetic is provided, comprising a safe and effective amount of aconitine A or an alkaloid compound as described in the present invention.

[0026] In a sixth aspect, a free radical scavenger is provided, wherein the free radical scavenger comprises a safe and effective amount of aconitine A or an alkaloid compound as described in the present invention.

[0027] In a seventh aspect, an anti-aging drug is provided, wherein the anti-aging drug comprises a safe and effective amount of aconitine A or an alkaloid compound as described in the present invention.

[0028] In an eighth aspect, aconitine A or an alkaloid compound of the present invention is provided for use in the preparation of antioxidants or sunscreen cosmetics.

[0029] In a ninth aspect, a use of the aconitine A or alkaloid compound of the present invention in the preparation of free radical scavengers or anti-aging drugs is provided.

[0030] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0031] The present invention successfully extracted and separated C from the root of Aconitum carmichaelii for the first time by using the steps of reflux extraction, organic solvent extraction, positive and reverse phase silica gel column chromatography separation and Sephadex LH-20 dextran gel purification. 18 -Diterpene alkaloid compound aconitine A.

[0032] C 18 -Diterpene alkaloid compound aconitine A has good antioxidant capacity, and the half inhibitory concentration (IC 50 ) value was 87.2±2.4μmol / L, which was comparable to ascorbic acid (IC 50 The scavenging ability of the two groups was on the same order of magnitude.

[0033] C 18 -The diterpene alkaloid compound aconitine A is easy to extract and separate, and has strong operability. It has good application prospects as an antioxidant used in alkaline environments and can also be used in sunscreen cosmetics, free radical scavengers or anti-aging drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings involved in the description of the embodiments. Obviously, the drawings in the description are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is a flow chart for the preparation of the target compound of Example 1;

[0036] Figure 2 HR-ESI-MS spectrum of the target compound in Example 2;

[0037] Figure 3 is the IR spectrum of the target compound in Example 2;

[0038] Figure 4 For the target compound in Example 2 1 H-NMR spectrum;

[0039] Figure 5 For the target compound in Example 2 13 C-NMR spectrum;

[0040] Figure 6 DEPT135° spectrum of the target compound in Example 2;

[0041] Figure 7 is the HSQC spectrum of the target compound in Example 2;

[0042] Figure 8 For the target compound in Example 2 1 H- 1 H COSY spectrum;

[0043] Figure 9 is the HMBC spectrum of the target compound in Example 2;

[0044] Figure 10 This is the NOESY spectrum of the target compound in Example 2. DETAILED DESCRIPTION

[0045] The following examples illustrate the technical solutions of the present invention, but the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0046] Example 1

[0047] Separation and preparation of C 18 -type diterpene alkaloids new compounds (see process Figure 1 )

[0048] (1) 2.5 kg of dried Aconitum root was crushed into 30 mesh pieces and extracted three times with a 90% methanol / water solution (20 L x 3 times, 2 hours each time). The extracts were combined and distilled under reduced pressure to remove the methanol. The extracts were allowed to stand overnight and the sediment was filtered to obtain 0.80 kg of extract. The extract was suspended in 2 L of water to obtain an aqueous suspension. The aqueous suspension was extracted three times with petroleum ether and ethyl acetate, respectively. The combined extracts were concentrated under reduced pressure at 50°C to obtain 0.49 kg of ethyl acetate extract.

[0049] (2) 0.45 kg of ethyl acetate extract was mixed with 0.50 kg of 100-200 mesh silica gel, and 2.5 kg of silica gel (300-400 mesh) was used for silica gel column chromatography. The mixture was eluted with a gradient of dichloromethane and methanol (100:0, 98:2, 95:5, 90:10, 85:15, 80:20, 70:30, and 60:40, v / v). Twelve fractions were obtained by TLC monitoring and merging, which were recorded as Fr.A-Fr.L.

[0050] (3) 19.1 g of the seventh fraction was subjected to silica gel column chromatography using a gradient elution of dichloromethane, methanol, and aqueous ammonia (100:5:1, 100:10:1, 100:20:1, and 100:30:1, v / v). The fractions were combined and analyzed by TLC to obtain five fractions, designated as Fr.G1 to Fr.G5.

[0051] (4) The fourth component Fr.G4 (3.5 g) was passed through a Sephadex LH-20 polyacrylamide gel column (dichloromethane and methanol, 1:1, v / v) to remove impurities, and further separated using a normal silica gel column (ethyl acetate, ethanol and ammonia water: 20:1:0.1, 10:1:0.1 and 10:2:0.1, v / v), and purified through a Sephadex LH-20 column (methanol) to prepare the target compound, i.e., aconitine A.

[0052] Example 2

[0053] C 18 Structural analysis of new compounds of -type diterpenoid alkaloids

[0054] (1) Comprehensive application of high-resolution mass spectrometry (HR-ESI-MS; see Figure 2 ), infrared (IR; see Figure 3 ) and NMR spectroscopy ( 1 H-NMR, 13 C-NMR, DEPT-135°, HSQC, 1 H- 1 H COSY, HMBC, and NOESY; see Figures 4-10 ; and Table 1 below) identifies the target compound structure;

[0055] (2) The target compound is a white amorphous powder. According to the HR-ESI-MS spectrum ( Figure 2 ) ion peak at m / z 438.2839 [M+H] + (C 24 H 40 The calculated value of NO6 is 438.2856), and the molecular formula of the compound is determined to be C 24 H 39 NO6, has 6 degrees of unsaturation. Infrared spectrum ( Figure 3 ) showed that there were hydroxyl groups in the compound structure (3506, 3394 cm -1 ) and methoxy (2856, 2824 cm -1 ).

[0056] 1 H-NMR spectrum ( Figure 4 ) shows that the compound has an N-ethyl unit [δ H 1.09 (3H, t, J = 7.3 Hz), 2.57-2.52 (2H, m)] and four methoxy proton signals [δ H 3.13, 3.28, 3.31 and 3.40 (all (3H, s)]. 13 C-NMR, DEPT and HSQC spectra ( Figure 5-Figure 7) shows that the compound has 24 carbon atoms, including one N-ethyl unit (δ C 13.39 and 49.19), four methoxy groups (δc 48.52, 56.13, 56.51 and 57.96), and seven methylene groups (δ C 36.32, 47.49, 49.84, 62.10, 83.00, 84.92 and 90.24), six methylene groups (δ C 22.84, 26.52, 29.70, 30.84, 44.86 and 49.19) and four quaternary carbons (δ C 45.52, 74.98, 75.75 and 78.56). The compound has only one non-oxygenated carbon signal (δ C 50.63, CH), and the weak field signals at δc 30.84 and 45.52 are attributed to C-3 and C-5, respectively, due to the β effect of 4-OCH3. From the biosynthesis and NMR data, the compound has C 18 -Diterpene alkaloid skeleton.

[0057] 1 H- 1 H COSY spectrum ( Figure 8 ), we can see δ H 1.81 (H-2) and 3.10 (H-1), 1.99 (H-3), δ H There are related signals between 2.26 (H-6), 2.08 (H-5) and 2.16 (H-7). H 2.44 (H-12) and 2.04 (H-10), 2.36 (H-13); δ H 2.36 (H-13) and 3.56 (H-14) and δ H There are related signals between 3.33 (H-16), 2.36 (H-13), and 2.42 (H-15). H There are also related signals at 2.55 (H-21) and 1.09 (H3-22). Combined with the HSQC spectrum ( Figure 7 ), it can be deduced that the structure contains the following fragments: C1—C2—C3, C5—C6—C7, C 10 —C 12 —C 13 —C 14 、C 13 —C 16 —C 15 and C 21 —CH3.

[0058] In the HMBC spectrum ( Figure 9 ), the hydrogen proton signal HC(17)[δ H2.95]、HC(19)[δ H 2.27] and HC(22)[δ H 1.09] and C-21[δ C 49.19], indicating that the N-ethyl C atom is located at the C(21) position. H 3.31, s) and C-1(δ C 84.92, CH), 4-OCH3(δ H 3.13, s) and C-4 (δ C 74.98, C), 14-OCH3(δ H 3.40, s) and C-14 (δ C 90.24, CH) and 16-OCH3 (δ H 3.28, s) and C-16 (δ C 83.00, CH) exists 1 H- 13 The C-terminals are remotely related, so the four methoxy groups are located at the C-1, C-4, C-14 and C-16 positions, respectively.

[0059] NOESY spectrum ( Figure 10 The key correlations between H-1 and H-10, H-10 and H-7, H-10 and H-13, and H-10 and H-14 in the molecule reveal that the proton orientations at C-7, C-10, C-13, and C-14 are β-type. 4-OCH3 / H-5 and 4-OCH3 / H-7 are both β-oriented. H-17 and H α -15, H α The cross peaks between H-15 and H-16 and between H-17 and H-16 prove that H-16 is in the α position. β -2, H α -2 and H α -3, H-7 and H β -6, H-19 and H α -6), H-17 and H α -12, H-13 and H β -12 and H-16 with H α The NOESY correlation between C-15 and C-2 shows that there are two H atoms at each of the C-2, 3, 6, 12, and 15 positions. In addition, the spectral characteristics of aconitine A are similar to those of the known compound Lappaconine. The signal δ C 70.9 moves down to δ due to the electron attraction of the methyl group C 74.98.

[0060] (3) In summary, it was determined that the compound of Example 1 was a new aconite C with 8-, 9-dihydroxy and 4-methoxy groups. 18 -Diterpene alkaloid, named aconitine A.

[0061] Table 1 Target compounds 1 H-, 13 C-NMR and HMBC NMR data (500 / 125 MHz, CDCl3, δ, ppm, J / Hz)

[0062]

[0063]

[0064] Example 3

[0065] In vitro antioxidant activity test of compounds

[0066] DPPH powder was prepared into a 20mM DPPH stock solution with ethanol solvent. The DPPH stock solution was diluted 50 times before testing. The OD value was between 0.8 and 1 when tested with a microplate reader at 517nm. Ascorbic acid was positive. The sample concentration was 80μg / mL. Compound aconitine A and reagent were added to the well plate. After mixing evenly in the well plate, the mixture was reacted at room temperature in the dark for 30 minutes. The OD value was measured with a microplate reader at 517nm. The clearance rate was calculated as shown in formula (II). Then the half inhibitory concentration (IC50) was calculated. 50 )value.

[0067]

[0068] Where:

[0069] An——negative control group;

[0070] As——test sample / positive drug;

[0071] Ab—blank control group.

[0072] The results of antioxidant activity test showed that the compound aconitine A had good antioxidant capacity, and the half inhibition concentration (IC 50 ) value was 87.2±2.4μmol / L, which was comparable to ascorbic acid (IC 50 The scavenging ability of the two groups was on the same order of magnitude.

[0073] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should fully understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

[0074] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0075] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. Aconitine A, characterized in that The homoaconitine A has a structure as shown in formula (I):

2. The extraction method of aconitine A is characterized in that: The extraction method comprises the following steps: Step S1: crushing the root of Aconitum carmichaelii, extracting it with methanol solution under reflux, distilling the extract under reduced pressure to remove methanol, letting it stand and filtering to remove sediment, to obtain an aqueous suspension; Step S2: extracting the aqueous suspension with petroleum ether and ethyl acetate in sequence, and concentrating the extract under reduced pressure to obtain an ethyl acetate extract; Step S3: subjecting the ethyl acetate extract to silica gel column chromatography, and combining the obtained components to obtain a first component, a second component, a third component, a fourth component, a fifth component, a sixth component, a seventh component, an eighth component, a ninth component, a tenth component, an eleventh component, and a twelfth component; Step S4: subjecting the seventh fraction to silica gel column chromatography to obtain a thirteenth fraction, a fourteenth fraction, a fifteenth fraction, a sixteenth fraction, and a seventeenth fraction; Step S5: The sixteenth component is sequentially subjected to decontamination by a dextran gel column, elution separation, and purification to obtain the target compound, namely aconitine A.

3. The extraction method according to claim 2, characterized in that In the step S3, the eluents for silica gel column chromatography of the ethyl acetate extract are dichloromethane and methanol, and the volume ratios of the dichloromethane to the methanol are 100:0, 98:2, 95:5, 90:10, 85:15, 80:20, 70:30 and 60:40, respectively.

4. An alkaloid compound, characterized in that The alkaloid compound includes the aconitine A according to claim 1.

5. An antioxidant, characterized in that The antioxidant comprises the aconitine A according to claim 1 or the alkaloid compound according to claim 4.

6. Sunscreen cosmetics, characterized in that The sunscreen cosmetic comprises a safe and effective amount of the homoaconitine A according to claim 1 or the alkaloid compound according to claim 4.

7. A free radical scavenger, characterized in that The free radical scavenger comprises a safe and effective amount of aconitine A according to claim 1 or the alkaloid compound according to claim 4.

8. An anti-aging drug, characterized in that The anti-aging drug comprises a safe and effective amount of the aconitine A according to claim 1 or the alkaloid compound according to claim 4.

9. Use of the aconitine A according to claim 1 or the alkaloid compound according to claim 4 in the preparation of antioxidants or sunscreen cosmetics.

10. Use of the aconitine A according to claim 1 or the alkaloid compound according to claim 4 in the preparation of free radical scavengers or anti-aging drugs.

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