Composition of rhizoma coptidis component as well as preparation method and application of composition

Abstract

The invention discloses a rhizoma coptidis component composition as well as a preparation method and application thereof, and the rhizoma coptidis component composition comprises an alkaloid component and a phenolic acid component, the mass ratio of the alkaloid component to the phenolic acid component is (3-6): (1-3). According to the invention, the anti-inflammatory drug effects of substances with different compositions are verified through independent berberine component, coptis chinensis full-component extract, coptis chinensis alkaloid component and coptis chinensis alkaloid and non-alkaloid proportioning drugs; pharmacodynamic results prove that the inflammatory factor inhibition rate of the medicine with the alkaloid component and the phenolic acid component in the mass ratio range of (3-6): (1-3) is higher than that of the medicine prepared by directly extracting coptis chinensis and is also obviously higher than that of the medicine with the alkaloid component as the main component.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to a composition of coptis root components and a preparation method and application thereof. Background Art

[0002] Coptis chinensis is bitter in taste and cold in nature. It can ascend or descend. It enters the heart, liver, spleen, stomach, and large intestine meridians. It is yin and non-toxic. It has the effects of clearing away heat and dampness, purging fire and detoxifying. The Compendium of Materia Medica records that it can "drain liver fire, remove bad blood from the heart, and stop palpitations." Traditional Chinese medicine uses it to treat damp-heat fullness, vomiting and acid regurgitation, diarrhea, carbuncle, hyperactivity of heart fire, etc.

[0003] With the application of modern science and technology in the research of traditional Chinese medicine, the research on the chemical composition and pharmacological effects of Coptis chinensis has been continuously improved and innovated. At present, the public literature reports that 133 chemical components have been isolated from Coptis chinensis, which can be divided into two categories: alkaloids and non-alkaloids (lignin, flavonoids, phenylpropionic acid and its derivatives or others). Existing research mainly focuses on the alkaloid components of Coptis chinensis, and the main anti-inflammatory effect is also the alkaloid component. There are no reports on the research of other components in anti-inflammatory effects. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is how to optimize the chemical composition of Coptis chinensis to significantly improve the anti-inflammatory effect, and to provide a composition of Coptis chinensis components with significant anti-inflammatory effect, as well as a preparation method and use thereof.

[0005] A composition of coptis root components comprises: alkaloid components and phenolic acid components; the mass ratio of the alkaloid components to the phenolic acid components is (3-6):(1-3).

[0006] The alkaloid components include one or more of berberine, coptisine, epiberberine, jatrorrhizine, tetrandrine, palmatine and magnolamine.

[0007] The phenolic acid components include one or more of 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, 5-O-feruloylquinic acid, and 3-(3',4'-dihydroxyphenyl)-(2R)-lactic acid-4'-O-β-D-glucopyranoside. The CAS number of the 3-(3',4'-dihydroxyphenyl)-(2R)-lactic acid-4'-O-β-D-glucopyranoside is 96552-85-1.

[0008] The mass ratio of the alkaloid components to the phenolic acid components is (56-60):(20-24).

[0009] A method for preparing a composition of coptis root components, comprising:

[0010] To obtain the ethanol extract, take Coptis chinensis and extract it with ethanol to obtain the ethanol extract;

[0011] The ethanol extract is taken, water is added to prepare a sample solution, and a resin is used for dynamic adsorption; after sufficient adsorption, elution is performed with ethanol of different concentrations between 5% and 35%, respectively, to obtain different Rhizoma Coptidis preparations, at least including alkaloid components as the main components and phenolic acid components accounting for more than 20%;

[0012] Different Rhizoma Coptidis preparations can be used to prepare a composition having a mass ratio of alkaloid components to phenolic acid components of (3-6): (1-3).

[0013] The coptis root includes one or more of coptis root rhizomes, coptis root hairs, coptis leaves or the whole coptis root plant;

[0014] Preferably, after sufficient adsorption, 5 BV of 10% ethanol and 5 BV of 30% ethanol are used for elution respectively;

[0015] The 10% ethanol eluate was collected and dried to obtain the Rhizoma Coptidis preparation 1;

[0016] The 30% ethanol eluate was collected and dried to obtain the Rhizoma Coptidis Preparation 3;

[0017] Preferably, the Coptidis preparation 1 and the Coptidis preparation 3 are prepared into a composition having a mass ratio of alkaloid components to phenolic acid components of (3-6):(1-3).

[0018] After sufficient adsorption, 10 BV of the product was eluted with 20% ethanol, and the 20% ethanol eluate was collected and dried to obtain the Rhizoma Coptidis Preparation 2.

[0019] The Rhizoma Coptidis preparation 1 comprises 1% to 6% of 3-(3',4'-dihydroxyphenyl)-(2R)-lactic acid-4'-O-β-D-glucopyranoside, 1% to 6% of magnolamine, and 30% to 60% of total alkaloids.

[0020] The Rhizoma Coptidis preparation 2 contains 30-60% total alkaloids and 1-5% total phenols.

[0021] The Rhizoma Coptidis preparation 3 comprises 10% to 15% of 3-O-feruloylquinic acid, 3% to 7% of 4-O-feruloylquinic acid, 7% to 10% of 5-O-feruloylquinic acid, 20% to 40% of total alkaloids and 20% to 35% of total phenol content.

[0022] The mixing ratio of the Coptis chinensis preparation 1 and the Coptis chinensis preparation 3 is 1:(1-4).

[0023] Use of the above composition or the composition prepared by the above preparation method in the preparation of anti-inflammatory drugs.

[0024] The technical solution of the present invention has the following advantages:

[0025] The present invention provides a composition of coptis components, comprising: alkaloid components and phenolic acid components; the mass ratio of the alkaloid components to the phenolic acid components is (3-6): (1-3); by optimizing and adjusting the chemical component ratio, the purpose of significantly improving the anti-inflammatory effect can be effectively achieved. Specifically, in a chemical study based on the quality classification of traditional coptis medicinal materials, it was found that when the total alkaloid content of coptis reaches a certain amount, the non-alkaloid components in coptis also participate in the pharmacodynamic effect of coptis, and the factors leading to the quality difference of coptis are also reflected in the content difference of non-alkaloid components; in the study of the present invention, it was found that the non-alkaloid components have the effect of synergizing the alkaloid components of coptis to enhance the efficacy; therefore, the present invention optimizes the component content ratio in the coptis components so that the mass ratio of the alkaloid components to the phenolic acid components reaches (3-6): (1-3), thereby achieving the purpose of significantly improving the anti-inflammatory effect. In order to demonstrate the above effects, a method for chemically separating and preparing different components of Coptis chinensis was adopted to adjust the ratio of different components of Coptis chinensis to obtain drugs with different ratios of Coptis chinensis components. Under the condition of consistent dosage of alkaloid components, the anti-inflammatory effects of substances with different compositions were verified by comparing the berberine component alone, the whole extract of Coptis chinensis, the alkaloid components of Coptis chinensis, and the ratio of Coptis chinensis alkaloids and non-alkaloid components. The efficacy results showed that the drugs with a mass ratio of alkaloid components to phenolic acid components in the range of (3-6): (1-3) had a higher inhibition rate on inflammatory factors than the preparation of Coptis chinensis direct extract, and were also significantly higher than the drugs whose main components were alkaloid components. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 is a comparison of the swelling of the right feet of rats in each group in Experimental Example 1;

[0028] Figure 2 This is a graph of the levels of inflammatory factors in the serum of rats in each group in Experimental Example 1. DETAILED DESCRIPTION

[0029] Example 1

[0030] A composition of coptis root components, the specific preparation process of which is as follows:

[0031] Take the whole plant material of Coptis chinensis, crush it into powder, weigh it, add 70% ethanol to extract twice, add 10 times the amount each time, extract for 2 hours, concentrate the extract, and dry it under reduced pressure to obtain an ethanol extract.

[0032] Take the dry paste of the ethanol extract, add water to prepare a loading solution with a concentration of 1g / ml (raw drug amount), the loading amount (raw drug amount) and the resin volume ratio is 1g:10mL, dynamic adsorption, and the adsorption flow rate is 3BV / h.

[0033] After sufficient adsorption, wash with water for 2 BV, elute with 10% ethanol for 5 BV, elute with 20% ethanol for 10 BV, and elute with 30% ethanol for 5 BV.

[0034] The 10% ethanol eluate was collected, the solvent was recovered, and the product was dried under reduced pressure to obtain Coptis chinensis preparation 1. The mass fraction range of the main components of Coptis chinensis preparation 1 prepared from different Coptis chinensis whole plant medicinal materials was: 3-(3',4'-dihydroxyphenyl)-(2R)-lactic acid-4'-O-β-D-glucopyranoside 1% to 6%, magnolamine 1 to 6%, and total alkaloids 30 to 60%.

[0035] The 20% ethanol eluate was collected to obtain Coptis chinensis Preparation 2.

[0036] The 30% ethanol eluate was collected, the solvent was recovered, and the mixture was dried under reduced pressure to obtain Coptis chinensis Preparation 3. The mass fraction ranges of the main components of Coptis chinensis Preparation 3 prepared from different Coptis chinensis medicinal materials were as follows: 3-O-feruloylquinic acid 10%-15%, 4-O-feruloylquinic acid 3%-7%, 5-O-feruloylquinic acid 7%-10%, total alkaloids 20-40%, and total phenol content 20-35%.

[0037] The Coptidis preparation 1 and the Coptidis preparation 3 are mixed in a ratio of 1:(1-4) to obtain the composition of the Coptidis components of the present invention.

[0038] In this embodiment, the same batch of Coptis chinensis medicinal materials (batch number 20091011) is taken as an example, and the specific composition of the ethanol extract, Coptis chinensis preparation 1, Coptis chinensis preparation 2 and Coptis chinensis preparation 3 obtained by testing is shown in Table 1 below:

[0039] Table 1

[0040]

[0041]

[0042] The content of each of the above components is detected by conventional high performance liquid chromatography. The specific detection process is a conventional method for those skilled in the art and will not be described in detail here.

[0043] Among them, the mass ratio of alkaloid components to phenolic acid components in the ethanol extract of Coptis chinensis is about 30:3.5, which is 17:2 after conversion; the mass ratio of alkaloid components to phenolic acid components in Coptis chinensis preparation 2 is about 52:2. The composition of the Coptis chinensis components described in this embodiment, which is prepared by mixing Coptis chinensis preparation 1 and Coptis chinensis preparation 3 in a ratio of 1:1.2, has an alkaloid component accounting for about 40% of the composition and a phenolic acid component accounting for about 16% of the composition; after conversion, the mass ratio of alkaloid components to phenolic acid components is about 5:2.

[0044] Example 2

[0045] The Coptidis preparation 1 and the Coptidis preparation 3 in Example 1 were mixed in a ratio of 1:1 to obtain a Coptidis component composition having a mass ratio of alkaloid components to phenolic acid components of 5.4:2.

[0046] Example 3

[0047] The Coptidis preparation 1 and the Coptidis preparation 3 in Example 1 were mixed in a ratio of 1:1.5 to obtain a Coptidis component composition having a mass ratio of alkaloid components to phenolic acid components of 4.6:2.

[0048] Example 4

[0049] The Coptidis preparation 1 and the Coptidis preparation 3 in Example 1 were mixed in a ratio of 1:2 to obtain a Coptidis component composition having a mass ratio of alkaloid components to phenolic acid components of 3.8:2.

[0050] Example 5

[0051] The Coptidis preparation 1 and the Coptidis preparation 3 in Example 1 were mixed in a ratio of 1:4 to obtain a Coptidis component composition having an alkaloid component and a phenolic acid component in a mass ratio of 3:2.

[0052] Experimental Example 1

[0053] The composition of the Rhizoma Coptidis components prepared in the examples, the ethanol extract, Rhizoma Coptidis preparation 2, the aspirin group and berberine were used for animal verification, and the specific verification settings were as follows:

[0054] 1. Experimental animal modeling

[0055] The experimental animals were SD rats, SPF grade, weighing between 130-150g, and the breeding temperature was 25℃ and the relative humidity was 55%. After one week of adaptive breeding, they were divided into experimental group, model group and blank control group, and all were gavaged with the same dose of normal saline as the experimental group. 30 minutes after the administration on the 7th day, 1% carrageenan 0.1ml / rat was subcutaneously injected into the plantar part of the right hind paw of the rat to establish the model, and the blank control group was injected with the same volume of normal saline; before injection modeling, 2h, 3h, and 4h after inflammation, the toe volume meter was used to measure the swelling of the foot.

[0056] 2. Laboratory animal index detection

[0057] The basal value of the right hind paw volume of the rats was measured before modeling, and the volume of the right hind paw volume of the rats was measured 2h, 3h, and 4h after modeling. After the measurement, chloral hydrate anesthesia was used, blood was collected from the abdominal aorta, and serum was obtained by centrifugation to determine the levels of IL-1β, PGE, and TNFα in the serum.

[0058] 3 Animal Grouping

[0059] The experimental groups were administered drugs including aspirin, berberine, test drug 1 (i.e., ethanol extract of Coptis chinensis), test drug 2 (i.e., Coptis chinensis preparation 2), and test drug 3 (i.e., a combination of Coptis chinensis components consisting of Coptis chinensis preparation 1 and Coptis chinensis preparation 3 in Example 1). Animals were set in each group (n=10), and a total of 9 groups of experimental animals were set up, namely: blank control group, model group, aspirin group, berberine group, test drug 1 group, test drug 2 group, and test drug 3 group.

[0060] 4 Dosage regimen

[0061] Aspirin was used as the positive control drug in this experiment, and berberine was used as the main control drug. The dosage was calculated based on the content of berberine-represented alkaloid components in Coptis chinensis. The dosage of alkaloids in test drug group 1, test drug group 2, and test drug group 3 was the same. The specific dosage conditions are as follows:

[0062] 4.1 Aspirin Group

[0063] The adult dosage is 300 mg / 60 kg, and the rat dosage is 0.035 mg / g based on 7 times the dose.

[0064] 4.2 Berberine group

[0065] The dosage for adults is 445.5 mg / 60 kg (of which the berberine content is 100%), and the dosage for rats is 0.052 mg / g calculated based on 7 times the dosage.

[0066] 4.3 Test drug group 1

[0067] The dosage for adults is 1500 mg / 60 kg (equivalent to alkaloid dosage of 445.5 mg / 60 kg), and the dosage for rats is 0.175 mg / g, which is converted to 7 times the dosage.

[0068] 4.4 Test drug group 2

[0069] The dosage for adults is 858 mg / 60 kg (equivalent to an alkaloid dosage of approximately 445.5 mg / 60 kg). The dosage for rats is converted to 0.100 mg / g based on 7 times the dose.

[0070] 4.5 Three groups of test drugs

[0071] The dosage for adults is 1118 mg / 60 kg (equivalent to alkaloid dosage of 445.5 mg / 60 kg), and the dosage for rats is 0.130 mg / g based on 7 times the dosage.

[0072] 5 Statistical methods

[0073] SPSS 17.0 was used for statistical analysis, and data were analyzed using The data were in accordance with normal distribution, and one-way ANOVA was used to compare the means of multiple samples. LSD test was used for homogeneous variance, and Dunnett's T3 test was used for unequal variance. P<0.05 was considered statistically significant.

[0074] 6 Statistical results

[0075] 6.1 Clinical Signs Observation

[0076] The capillary amplification measurement method was used to measure the swelling of the right hind paw of rats at 2h, 3h, and 4h after inflammation. After the injection of carrageenan, the rats' feet began to become inflamed and swollen, and the clinical signs were local capillary dilation, increased vascular permeability, and edema in the rats. Swelling was obviously observed in the rats in the model group 1h after inflammation, and the foot swelling reached a peak from 2h to 3h after inflammation. Signs of foot swelling were obviously observed in each drug-treated group from 2h after inflammation, and the clinical signs gradually alleviated more obviously after 3h in each drug-treated group. The right hind paw of rats in the blank group was slightly swollen after injection of normal saline.

[0077] 6.2 Effects of different preparations of Coptis chinensis on carrageenan-induced arthritis model in rats

[0078] Among them, the results of the changes in the swelling degree of the right foot of rats with the inflammation time are shown in Table 2, Figure 1 As shown in Table 3, the results of the determination of inflammatory factors in rat serum are as follows: Figure 2 shown.

[0079] Table 2 Results of the changes in swelling of the right paw of rats with inflammation time

[0080]

[0081] Table 3 Determination results of inflammatory factors in rat serum

[0082]

[0083] Note: Compared with the blank group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0084] The experimental results in Table 2 above show that compared with the blank group, the arthritis scores of the model group rats in each time period were significantly increased, showing a significant difference (P<0.01). Compared with the model group, aspirin and test drug 3 can significantly reduce the swelling of the rats' feet 2 hours after the model replication (P<0.05, P<0.05, respectively); aspirin, test drug 1, test drug 2, and test drug 3 can significantly reduce the swelling of the rats' feet 3 hours after the model replication (P<0.01, P<0.01, P<0.01, P<0.01, respectively); aspirin, test drug 3, and test drug 2 can significantly reduce the swelling of the rats' feet 4 hours after the model replication (P<0.05, P<0.05, P<0.05, respectively); the swelling of the rats' feet in the berberine group showed a certain downward trend, but there was no statistical significance compared with the model group.

[0085] The serum inflammatory factor determination data in Table 3 above showed that the levels of inflammatory factors IL-1β, PGE2, and TNFα in the serum of rats in the model group were significantly increased compared with the blank group, showing a significant difference (P<0.01). Compared with the model group, aspirin, berberine, test drug 2, and test drug 3 can significantly reduce the levels of inflammatory factors IL-1β, PGE2, and TNFα in the serum of rats (all P<0.01 among the groups); compared with the model group, test drug 1 can significantly reduce the levels of inflammatory factors IL-1β, PGE2, and TNFα in the serum of rats (P<0.05, P<0.01, and P<0.01, respectively).

[0086] Specifically, compared with the blank group, there was no statistical difference in the levels of inflammatory factors IL-1β, PGE2, and TNFα in the serum of rats in the aspirin group; there was no statistical difference in the level of inflammatory factor IL-1β in the serum of rats in the test drug 3 group, and there were significant differences in the levels of PGE2 and TNFα (P<0.05); there were significant differences in the levels of inflammatory factors IL-1β, PGE2, and TNFα in the serum of rats in the berberine group, the test drug 1 group, and the test drug 2 group (P<0.01), indicating that the positive regulation effect of test drug 3 on inflammatory factors is better than that of test drug 1 and test drug 2.

[0087] According to the efficacy results in Table 2, the inhibitory effect of test drug 3 on the paw swelling of inflammatory rats showed significant differences from 2h to 4h, which was close to that of aspirin group and better than that of test drug 2 and test drug 1. The regulatory effect of test drug 3 on the inflammatory factor IL-1β was close to that of aspirin, and the regulatory effect on the levels of inflammatory factors PGE2 and TNF-α was better than that of other drug groups, and the significant difference was reduced compared with the blank group (P<0.05); the inhibitory effect of test drug 2 on the paw swelling of inflammatory rats showed significant differences from 3h to 4h, and the inhibitory effect on inflammatory factors The regulatory effect on IL-1β, PGE2, and TNF-α levels of the test drug group was similar to that of the berberine group (all P<0.01 among all groups); the inhibitory effect of the test drug group 1 on the paw swelling of inflammatory rats showed significant differences at 3 hours, and the regulatory effect on the inflammatory factor IL-1β level was significantly lower (all P<0.05 among all groups), and the regulatory effect on the inflammatory factor PGE2 and TNF-α levels was similar to that of the berberine group (all P<0.01 among all groups); there was no statistical difference in the inhibitory effect of the berberine group on the paw swelling of inflammatory rats.

[0088] The results of pharmacodynamic indexes showed that the drug 3 had the best efficacy, followed by drug 2, drug 1, and berberine. The contents of alkaloid components were drug 3 (39.85%), drug 2 (51.92%), drug 1 (29.71%), and berberine (100%); the contents of organic acid components were drug 3 (15.06%), drug 1 (2.94%), drug 2 (2.07%), and berberine (0%); from the changes in the content of the components, the changes in the content of organic acid components showed a positive correlation with the efficacy. The alkaloid components and non-alkaloid components in drug 3 jointly participated in the treatment of foot swelling in inflammatory rats, indicating that the multi-component synergistic anti-inflammatory effect of the Coptis chinensis extract was better than that of the berberine component. By comparing test drug 3, test drug 2 and test drug 1, it was found that when the dosage of alkaloids was the same, the anti-inflammatory effect of test drug 3 was better than that of test drug 2, and test drug 2 was better than that of test drug 1 when the dosage of organic acids increased. This shows that organic acid components significantly promote the anti-inflammatory efficacy of the drug at a specific proportion, especially enhance the regulatory effect on the inflammatory factor IL-1β.

[0089] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A composition of Coptis chinensis components, characterized in that: include: Alkaloid components and phenolic acid components; the mass ratio of the alkaloid components to the phenolic acid components is (3-6): (1-3).

2. The composition according to claim 1, characterized in that The alkaloid components include one or more of berberine, coptisine, epiberberine, jatrorrhizine, tetrandrine, palmatine and magnolamine.

3. The composition according to claim 1, characterized in that The phenolic acid components include one or more of 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, 5-O-feruloylquinic acid and 3-(3',4'-dihydroxyphenyl)-(2R)-lacticacid-4'-O-β-D-glucopyranoside.

4. The composition according to any one of claims 1 to 3, characterized in that The mass ratio of the alkaloid components to the phenolic acid components is (3-6):2, preferably (56-60):(20-24).

5. A method for preparing a composition of Coptis chinensis components, characterized in that: include: To obtain the ethanol extract, take Coptis chinensis and extract it with ethanol to obtain the ethanol extract; The ethanol extract is taken, water is added to prepare a sample solution, and a resin is used for dynamic adsorption; after sufficient adsorption, elution is performed with ethanol of different concentrations between 5% and 35%, respectively, to obtain different Rhizoma Coptidis preparations, at least including alkaloid components as the main components and phenolic acid components accounting for more than 20%; Different Rhizoma Coptidis preparations can be used to prepare a composition having an alkaloid component and a phenolic acid component in a mass ratio of (3-6): (1-3).

6. The preparation method according to claim 5, characterized in that: The coptis root includes one or more of coptis root rhizomes, coptis root hairs, coptis leaves or the whole coptis root plant; And / or, after sufficient adsorption, 10% ethanol and 30% ethanol are used to elute 5BV and 5BV respectively; the 10% ethanol eluate is collected and dried to obtain Coptis chinensis preparation 1; the 30% ethanol eluate is collected and dried to obtain Coptis chinensis preparation 3; preferably, Coptis chinensis preparation 1 and Coptis chinensis preparation 3 are configured to form a composition with a mass ratio of alkaloid components and phenolic acid components of (3-6): (1-3).

7. The preparation method according to claim 6, characterized in that: The Rhizoma Coptidis preparation 1 contains 1% to 6% of 3-(3',4'-dihydroxyphenyl)-(2R)-lactic acid-4'-O-β-D-glucopyranoside, 1% to 6% of magnolamine, and 30% to 60% of total alkaloids.

8. The preparation method according to claim 6, characterized in that: The Rhizoma Coptidis preparation 3 comprises 10% to 15% of 3-O-feruloylquinic acid, 3% to 7% of 4-O-feruloylquinic acid, 7% to 10% of 5-O-feruloylquinic acid, 20% to 40% of total alkaloids and 20% to 35% of total phenol content.

9. The preparation method according to claim 6, characterized in that: The mixing ratio of the Coptis chinensis preparation 1 and the Coptis chinensis preparation 3 is 1:(1-4).

10. Use of the composition according to any one of claims 1 to 5 or the composition prepared by the preparation method according to any one of claims 6 to 9 in the preparation of anti-inflammatory drugs.

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