A kind of apocynum venetum leaf extract and its preparation method and application

Abstract

The present invention belongs to the field of preparation of active extracts of traditional Chinese medicine, and specifically relates to a kind of Apocynum venetum leaf extract and its preparation method and application, wherein the Apocynum venetum leaf extract is an extract of Apocynum venetum leaf Fr.BuOH part or an extract of Fr.BuOH‑40% part obtained by eluting Fr.BuOH part with 40% ethanol (v / v); its active components include hyperoside and isoquercetin. The present invention further conducts secondary development for Apocynum venetum leaf, and finds two active parts Fr.BuOH and Fr.BuOH‑40% through an activity-oriented extraction and separation method, wherein the total content of hyperoside and isoquercetin is high; based on a zebrafish evaluation model, these two active parts have a significant improvement effect on verapamil hydrochloride-induced cardiac damage (pericardial edema and venous congestion), and at the same concentration, the efficacy is comparable to that of commercially available compound Danshen dripping pills.

Description

Technical Field

[0001] The invention belongs to the field of preparation of active extracts of traditional Chinese medicines, and particularly relates to a dogbun venetum leaf extract and a preparation method and application thereof. Background Art

[0002] Apocynum venetum L. is a subshrub of the genus Apocynum in the family Apocynaceae. The 2020 edition of the Chinese Pharmacopoeia states that Apocynum venetum leaves are sweet, bitter, and cool, and belong to the liver meridian. They have the effects of "calming the liver and calming the mind, clearing away heat and promoting diuresis", and are mainly used to treat "liver yang vertigo, palpitations and insomnia, edema and oliguria", which is hypertension in modern medical terms. The main effect of the 55 approved Chinese patent medicines containing Apocynum venetum leaves is also mainly to lower blood pressure, and there are no relevant reports on the blood circulation and stasis-removing effects of Apocynum venetum leaves and extracts.

[0003] The Chinese Pharmacopoeia stipulates that the index component of Apocynum venetum leaf is hyperoside, also known as quercetin-3-O-β-D-pyranoglucoside, which has a wide range of cardiovascular-related activity reports. In addition, the isomer of hyperoside, iso-quercetin (quercetin-3-O-β-D-pyranoglucoside), also shows a wide range of cardiovascular activity. The parent nucleus of both hyperoside and isoquercetin is quercetin, while the sugar substituent of the former is pyranoglucoside and the sugar substituent of the latter is pyranoglucoside. Chinese patent CN111150741A discloses the medical use of hyperoside to reduce human aortic endothelial cell damage, and provides the use of hyperoside alone or in combination with other drugs in the preparation of drugs for preventing and treating atherosclerosis, angina pectoris, heart failure and hypertension caused by oxidized low-density lipoprotein-induced human aortic endothelial cell damage. Chinese patent CN106474139A discloses a tablet for the remission period of cor pulmonale and its preparation method, wherein the tablet is mainly composed of coral phytoncidin, cyperitoyl cyanidin C, tanshinone, coixol, hyperoside, bergenin, puerarin, glycyrrhizin, cryptoxanthin, rutin, tuckahoe polysaccharide, fucose, N-acetylglucosamine, and catechins. The treatment of the remission period of cor pulmonale can prevent infection, control arrhythmia and heart failure, resist inducing factors, enhance the patient's body's immune ability, prevent recurrence, and accelerate the recovery of the patient's cardiopulmonary function to reduce the mental burden. In existing research, there is no research on the blood circulation and blood stasis removal and heart protection of Apocynum venetum leaf extract.

[0004] A large amount of literature shows that the zebrafish model has unique advantages in screening cardioprotective active ingredients. The zebrafish heart is highly similar to the human heart in structure, function, signaling pathways and ion channels; the blood lipid composition and metabolism are basically the same as those of humans; and the embryos and their larvae are almost transparent, with visible cardiovascular systems, allowing the heartbeat, angiogenesis and vascular lesions to be observed in vivo.

[0005] Based on this, the present invention prepares a dogbane leaf extract, and uses verapamil hydrochloride to induce a zebrafish heart injury model to verify the effects of the dogbane leaf extract and its main monomer chemical components in improving drug-induced pericardial edema and venous congestion, providing a scientific basis for its clinical application. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes a kind of Apocynum venetum leaf extract and its preparation method and application, further carries out secondary development for Apocynum venetum leaf, and finds two active parts Fr.BuOH and Fr.BuOH-40% through activity-oriented extraction and separation method, wherein the total content of hyperoside and isoquercetin is high; based on the zebrafish evaluation model, these two active parts have a significant improvement effect on verapamil hydrochloride-induced cardiac damage (pericardial edema and venous congestion), and the activity is much better than that of the total extract of Apocynum venetum leaf, and at the same concentration, it is better than the monomer chemical component hyperoside or isoquercetin, and the efficacy is equivalent to that of the commercially available compound danshen dripping pills.

[0007] The technical solution of the present invention is:

[0008] The invention protects the use of apocynum venetum leaf extract in preparing medicine for preventing and / or improving and / or treating heart damage.

[0009] Furthermore, the application includes any one or more of the following:

[0010] (1) Preparing a drug for preventing and / or improving and / or treating pericardial edema;

[0011] (2) preparing drugs for preventing and / or improving and / or treating venous congestion;

[0012] (3) preparing a drug for preventing and / or improving and / or treating verapamil hydrochloride-induced pericardial edema or venous congestion;

[0013] (4) Preparing a drug for preventing and / or improving and / or treating symptoms of a zebrafish cardiac injury model induced by verapamil hydrochloride.

[0014] The present invention also protects the Apocynum venetum leaf extract, which is an extract of the Apocynum venetum leaf Fr.BuOH part; its active components include hyperoside and isoquercetin. Preferably, the Apocynum venetum leaf extract is an extract of the Fr.BuOH-40% part obtained by eluting the Fr.BuOH part with 40% ethanol (v / v).

[0015] Furthermore, the preparation method of the Apocynum venetum leaf extract comprises the following steps:

[0016] The dried Apocynum venetum leaves are crushed and sieved, 95% ethanol is added in a ratio of liquid to solid (8-10):1, ultrasonic extraction is performed for 2-4 times, each extraction lasts for 50-70 minutes, and the filtrates are combined and concentrated under reduced pressure to obtain a Fr.95%EtOH extraction part; the Fr.95%EtOH extraction part is fully dispersed in water, and extracted with ethyl acetate and n-butanol in sequence to obtain a Fr.EtOAC part, a Fr.BuOH part and a Fr.H2O part; and the Fr.BuOH part is eluted with 40% ethanol (v / v) to obtain a Fr.BuOH-40% extraction part.

[0017] The present invention also protects a Chinese medicine composition for preventing and / or improving and / or treating heart damage, the active ingredient of which is the apocynum venetum leaf extract or the apocynum venetum leaf extract prepared by the preparation method.

[0018] The present invention also protects the use of the Apocynum venetum leaf extract in preparing a heart protection drug or a blood circulation and blood stasis removing drug.

[0019] Furthermore, the application includes any one or more of the following:

[0020] (1) preparing Chinese medicinal materials for promoting blood circulation and removing blood stasis;

[0021] (2) Prepare Chinese herbal medicines with cardioprotective effects.

[0022] Beneficial effects of the present invention:

[0023] (1) The present invention discovered two active parts of Apocynum venetum leaves, Fr.BuOH and Fr.BuOH-40%, through an activity-oriented extraction and separation method, and found through a zebrafish evaluation model that the extracts of these two active parts can significantly improve verapamil hydrochloride-induced cardiac damage, thereby expanding the application scope of Apocynum venetum leaves.

[0024] (2) The total contents of active ingredients hyperoside and isoquercetin in the Fr.BuOH subfraction and Fr.BuOH-40% subfraction were 20.41% and 36.81%, respectively, which were 7.21 times and 13.01 times higher than those in the 95% EtOH subfraction, and far higher than the 0.3% content standard of hyperoside specified in the pharmacopoeia.

[0025] (3) The present invention verifies that the two active parts, Fr.BuOH and Fr.BuOH-40%, have a significant improvement effect on verapamil hydrochloride-induced cardiac damage (specifically pericardial edema and venous congestion) based on the zebrafish evaluation model, which is much better than the activity of the 95% ethanol total extract of Apocynum venetum leaves and single components hyperoside or isoquercetin; and at the same concentration, the activity of the Apocynum venetum leaf extract of the present invention is equivalent to the efficacy of the commercially available compound danshen dripping pills, and can be used to develop related new Chinese medicines for promoting blood circulation and removing blood stasis in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The HPLC chromatograms of different polarity parts of Apocynum venetum leaves (detection wavelength is 280nm), wherein A: Fr.95% EtOH part B: Fr.BuOH part; C: Fr.BuOH-40% part;

[0027] Figure 2 is the standard curve of hyperoside;

[0028] Figure 3 The experimental results of evaluating the efficacy of Fr.BuOH part of Apocynum venetum leaves on the survival rate, pericardial edema and venous congestion area of ​​zebrafish;

[0029] Figure 4 The experimental results of evaluating the efficacy of Fr.BuOH-40% of Apocynum venetum leaves on the survival rate, pericardial edema and venous congestion area of ​​zebrafish;

[0030] Figure 5 The activity of Fr.BuOH part, Fr.BuOH-40% part, hyperoside and isoquercetin in Apocynum venetum leaves was compared. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0033] Example 1

[0034] The preparation method of the apocynum venetum leaf extract comprises the following steps:

[0035] (1) Extraction and preparation of active fractions

[0036] The leaves of Apocynum venetum were dried naturally, crushed and passed through a 50-mesh sieve, 100 g of dry powder was extracted twice with 95% ethanol by ultrasonic method (frequency 40 KHz), each time for 60 min, with a liquid-to-solid ratio of 10:1 and 8:1. The filtrates were combined and concentrated under reduced pressure, and the solvent was recovered to obtain 12.02 g of ethanol extract (i.e., Fr. 95% EtOH part), with an extraction rate of 12.02%.

[0037] The above Fr.95%EtOH fraction was fully dispersed in 500mL of distilled water, extracted with ethyl acetate and n-butanol solution in turn, concentrated under reduced pressure, and the solvent was recovered to obtain 2.68g of ethyl acetate extract (i.e., Fr.EtOAC fraction), 2.51g of n-butanol extract (i.e., Fr.BuOH fraction), and the remaining 6.7g of water extract (i.e., Fr.H2O fraction).

[0038] The Fr.BuOH part was eluted on a HP-20 macroporous adsorption resin column with distilled water, 10% ethanol (v / v), 20% ethanol (v / v), 30% ethanol (v / v), 40% ethanol (v / v), 60% ethanol (v / v), 80% ethanol (v / v) and 100% ethanol (v / v) in sequence, concentrated under reduced pressure, and the solvent was recovered. It was finally verified that the Fr.BuOH part eluted with 40% ethanol (v / v) (i.e., the Fr.BuOH-40% part) had the highest activity, with a mass of 0.30 g.

[0039] (2) HPLC qualitative characterization of active sites

[0040] Accurately weigh 10 mg of each sample of Fr.95% EtOH, Fr.BuOH, and Fr.BuOH-40%, and add 1 mL of methanol-water (v / v, 60:40) to dissolve. All the above samples were filtered through a 0.22 μm microporous membrane and used as test solutions, along with commercially available reference solutions (chlorogenic acid reference, hyperoside reference, and isoquercetin reference), and analyzed by HPLC with an injection volume of 15 μL.

[0041] The HPLC chromatographic conditions were set as follows: the mobile phases were methanol (A) and 0.1% trifluoroacetic acid aqueous solution (B); 5% to 95% methanol isocratic elution for 45 min; and the column temperature was 40°C.

[0042] like Figure 1 As shown in the figure, the HPLC qualitative characterization results showed that after comparison with the commercially available reference substance, it was found that the relative contents of chlorogenic acid, hyperoside and isoquercetin in the Fr.95%EtOH part of Apocynum venetum leaf were the highest, and their HPLC retention times were 14.70min, 23.04min and 23.31min, respectively. The relative contents of the three main components in the Fr.BuOH part increased significantly; while the Fr.BuOH-40% part had basically no chlorogenic acid, and mainly contained hyperoside and isoquercetin.

[0043] (3) Quantitative detection of main components of active sites

[0044] Since the parent nucleus of hyperoside and isoquercetin is exactly the same, only the glycoside part is different, they are a pair of isomers. The two cannot be effectively separated on a conventional HPLC column. In this section, the total content of the two main chemical components, hyperoside and isoquercetin, was calculated using hyperoside as the standard.

[0045] First, draw a standard curve, accurately weigh 5.00 mg of hyperoside reference substance, place it in a 5 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well to obtain 1000 μg / mL stock solution. Dilute with methanol to obtain 7 concentration samples, namely 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL. Under the HPLC chromatographic conditions of step (2), inject 10 μL and record the chromatographic peak area. Draw a standard curve of hyperoside reference substance with concentration value (μg / mL) as the horizontal coordinate and peak area (mAU) as the vertical coordinate.

[0046] The HPLC quantitative results showed that the hyperoside injection amount had a good linear relationship in the range of 0.5 to 8.0 μg, and the regression equation was: Y = 28964X-932918, R 2 =0.9999. Standard curve see Figure 2 The total percentages of hyperoside and isoquercetin in the Fr.95%EtOH fraction, the Fr.BuOH fraction, and the Fr.BuOH-40% fraction were 2.83%, 20.41%, and 36.81%, respectively (see Table 1).

[0047] Table 1 The content of hyperoside in different extraction parts of Apocynum venetum leaves

[0048]

[0049] Test Example 1 Maximum Tolerance Concentration of Zebrafish to Fr.BuOH Part, Fr.BuOH-40% Part, Hyperoside and Isoquercitrin

[0050] Wild-type AB strain zebrafish larvae at 2 dpf and with normal development were selected and placed in 6-well plates, 30 per well, and samples dissolved in DMSO were added, of which the final concentrations of the Fr.BuOH group were 2500, 2000, 1500, 1000, 500, 250, and 125 μg / mL; the final concentrations of the Fr.BuOH-40% group were 2000, 1750, 1000, 750, 500, 250, and 125 μg / mL, the final concentrations of the hyperoside group were 2000, 1500, 1000, 500, 250, 125, and 62.5 μg / mL, and the final concentrations of the isoquercetin group were 1000, 900, 800, 700, 600, 500, and 400 μg / mL. Zebrafish experimental water was used as the control group. The 6-well plate after drug addition was placed in a 28.5°C incubator for 24 h, the status of the zebrafish was observed and recorded, and the mortality and deformity rates were calculated.

[0051] like Figure 3 A and Figure 4 As shown in A, the median lethal concentration (LC) of Fr.BuOH and Fr.BuOH-40% 50 ) were: 1621.6μg / mL, 937.2μg / mL. The zebrafish phenotypes at various concentrations were observed under a microscope. No zebrafish died at a concentration of 1000μg / mL in the Fr.BuOH site, but all zebrafish showed varying degrees of deformity in the head and yolk sac. Some zebrafish showed deformities at a concentration of 500μg / mL. The morphological indicators of zebrafish at concentrations of 250μg / mL and below were all normal. Zebrafish showed varying degrees of deformity and death at concentrations of 500μg / mL and above in the Fr.BuOH-40% site, and the morphological indicators of zebrafish at concentrations of 250μg / mL and below were all normal. Based on the above results, the final dosing concentrations of the Fr.BuOH site and the Fr.BuOH-40% site were controlled within 500μg / mL. Similarly, based on the above results, the final administration concentrations of the Fr.BuOH site and the Fr.BuOH-40% site in Experimental Examples 2 and 3 were controlled within 500 μg / mL.

[0052] When investigating the maximum tolerated concentrations of hyperoside and isoquercetin, it was found that when the dosing concentration of hyperoside reached 1500 μg / mL and the dosing concentration of isoquercetin reached 800 μg / mL, drug crystallization occurred in the 6-well plate, but at this concentration, the morphological indicators of zebrafish in the hyperoside and isoquercetin groups were normal. Therefore, in Experimental Examples 2 and 3, the final dosing concentrations of hyperoside and isoquercetin need to be controlled within 1500 μg / mL and 800 μg / mL, respectively.

[0053] Experimental Example 2: Improvement of pericardial edema and venous congestion in zebrafish by Fr.BuOH fraction and Fr.BuOH-40% fraction

[0054] Two independent experiments were set up to evaluate the protective effects of Fr.BuOH and Fr.BuOH-40% on zebrafish cardiac injury. Wild-type AB zebrafish fertilized at 2dpf and with normal development were selected in 6-well plates, with 30 tails per well, and each well was fixed to 3mL with experimental water. A normal group, a model group, a positive drug group, and different concentrations of drug administration groups were set up. The normal group was treated with experimental water only, the model group was treated with 20μg / mL verapamil hydrochloride for 20min, and the positive drug group was first treated with compound Danshen dripping pills aqueous solution (keeping the final concentration consistent with the maximum dose of the drug administration group) for 4h, and then verapamil hydrochloride was added for 20min. The drug-treated groups were treated with 100μg / mL and 50μg / mL Fr.BuOH and 125μg / mL and 62.5μg / mL Fr.BuOH-40% for 4h; except for the normal group, each group was treated with verapamil hydrochloride for 20min. Ten zebrafish were randomly selected from each group, fixed on a glass slide with methylcellulose, adjusted to a lateral position to fully expose the atria and ventricles, and recorded with white light photography under a stereomicroscope (magnification of 5 times). Image J software was used to analyze the pericardial edema area and venous congestion area of ​​zebrafish.

[0055] like Figure 3 As shown in Figures B and 3C, compared with the normal control group, the pericardial edema and venous congestion area of ​​zebrafish in the model control group were significantly reduced (P < 0.001, P < 0.001), indicating that verapamil hydrochloride successfully induced cardiac injury in zebrafish. Compared with the model group, the Fr.BuOH fraction at 100 μg / mL (P < 0.05, P < 0.05) and 50 μg / mL (P < 0.01, P < 0.05) concentrations significantly improved the pericardial edema area and venous congestion area of ​​zebrafish, with statistical differences. The effect of the 100 μg / mL concentration of Fr.BuOH fraction on improving the venous congestion area of ​​zebrafish was better than that of the positive drug compound Danshen dripping pills at the same concentration (P < 0.05), and the effect of improving the pericardial edema area of ​​zebrafish was equivalent to that of the compound Danshen dripping pills at the same concentration (P < 0.05).

[0056] like Figure 4As shown in Figures B and 4C, compared with the normal control group, the pericardial edema and venous congestion area of ​​zebrafish in the model control group were significantly reduced (P < 0.001, P < 0.001), indicating that verapamil hydrochloride successfully induced zebrafish cardiac injury. Compared with the model group, the improvement effect of Fr.BuOH-40% on the pericardial edema area and venous congestion area of ​​zebrafish at the concentrations of 125μg / mL (P < 0.05, P < 0.05) and 62.5μg / mL (P < 0.01, P < 0.01) was statistically significant. The effect of BuOH-40% at a concentration of 125μg / mL was equivalent to that of Compound Danshen Dripping Pills at the same concentration; the effect of BuOH-40% at a concentration of 62.5μg / mL on zebrafish cardiac injury was significantly better than that of the positive drug Compound Danshen Dripping Pills at a concentration of 125μg / mL (P < 0.01, P < 0.01).

[0057] Experimental Example 3 Differences in the activities of Fr.BuOH site, Fr.BuOH-40% site and monomer chemical components hyperoside and isoquercetin

[0058] Another independent experiment was set up to evaluate the differences in the effects of Fr.BuOH, Fr.BuOH-40% and monomeric chemical components hyperoside and isoquercetin in improving zebrafish heart damage at the same dosage concentration.

[0059] Wild-type AB zebrafish of 2 dpf and normal development were selected and placed in a 6-well plate, with 30 fish per well, and each well was fixed to 3 mL with experimental water. Normal group, model group, positive drug group, and drug administration groups of different concentrations were set. The normal group was treated with experimental water only; the model group was treated with 20 μg / mL verapamil hydrochloride for 20 min; the positive drug group was first treated with 100 μg / mL of compound Danshen dripping pills aqueous solution for 4 h, and then verapamil hydrochloride was added for 20 min; the drug administration group was added with 100 μg / mL concentration of Fr.BuOH part, Fr.BuOH-40% part, hyperoside and isoquercetin, respectively, for 4 h; except for the normal group, verapamil hydrochloride was added to each group for 20 min. Ten zebrafish were randomly selected from each group, fixed on a glass slide with methylcellulose, adjusted to a lateral position to fully expose the atria and ventricles, and photographed under a stereomicroscope with white light (magnification 5 times). Image J software was used to analyze the pericardial edema area and venous congestion area of ​​zebrafish.

[0060] like Figure 5As shown in A and 5B, compared with the normal control group, the pericardial edema and venous congestion area of ​​zebrafish in the model control group were significantly reduced (P < 0.01, P < 0.001), indicating that verapamil hydrochloride successfully induced heart damage in zebrafish. Compared with the model group, 100 μg / mL concentration of Fr.BuOH part (P < 0.05, P < 0.01), Fr.BuOH-40% part (P < 0.01, P < 0.01), hyperoside (P < 0.05) and isoquercetin (P < 0.01, P < 0.01) had significant effects on improving the pericardial edema area and venous congestion area of ​​zebrafish, except for the hyperoside group. Among them, the effects of Fr.BuOH part and Fr.BuOH-40% part were significantly better than hyperoside, and slightly stronger than isoquercetin. The above experimental results show that at the same concentration, the effects of Fr.BuOH and Fr.BuOH-40% on zebrafish heart damage induced by verapamil hydrochloride are better than those of single chemical components hyperoside and isoquercetin.

[0061] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a venetian leaf extract for preparing a drug for preventing or treating pericardial edema or venous congestion, characterized in that: Prepare as follows: The dried Apocynum venetum leaves were crushed and sieved, and 95% ethanol was added in a ratio of liquid to solid (8-10):1, and ultrasonic extraction was performed for 2-4 times, each time for 50-70 min, and the filtrates were combined and concentrated under reduced pressure to obtain a 95% ethanol extract; the 95% ethanol extract was fully dispersed in water, extracted with ethyl acetate and n-butanol in sequence, concentrated under reduced pressure, and the solvent was recovered to obtain an ethyl acetate extract and a n-butanol extract; the n-butanol extract was eluted on an HP-20 macroporous adsorption resin column with distilled water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and 100% ethanol in sequence, concentrated under reduced pressure, and the solvent was recovered to obtain an eluate eluted with 40% ethanol; The Apocynum venetum leaf extract is an extract obtained by eluting with 40% ethanol.

2. A Chinese medicine composition for preventing or treating pericardial edema or venous congestion, characterized in that: The active ingredient is the dogbane leaf extract prepared by the preparation method described in claim 1.

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