Radix linderae total alkaloid extract as well as preparation method and application thereof
The total alkaloid extract of Urganyao effectively removes ROS in the organism, solving the problem of lack of safe removal of ROS in the prior art, achieving a safe and effective ROS removal effect, which is suitable for industrial production.
Patent Information
- Application Number
- CN202510689351.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-08-01
AI Technical Summary
There is a lack of effective and safe method for removing reactive oxygen species (ROS) from organisms in the prior art. ROS accumulation can lead to a variety of diseases such as diabetes and its complications, neurodegenerative diseases, etc.
The total alkaloid extract of Aconite, including norisobolding, boltine, Norjuziphine, reticulum and isobolding, were prepared by specific extraction methods to scavenge reactive oxygen species.
The total alkaloid extract of Aconite significantly eliminates ROS in the organism, is safe, non-toxic, has no obvious side effects, and has a wide range of sources. It is suitable for industrial production and can treat or alleviate diseases caused by ROS.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and particularly relates to an extract of total alkaloids from Lindera aggregata (Sims) Kosterm., a preparation method thereof, and an application thereof. Background Art
[0002] The accumulation of reactive oxygen species (ROS) can cause various harms to organisms, involving different levels such as cells, tissues, organs, and overall physiological functions. At the cellular level, it may damage cell membranes, disrupt protein structures and functions, and cause DNA damage. At the tissue level, it may lead to tissue inflammation and promote tissue fibrosis. At the organ and system levels, the accumulation of ROS can damage vascular endothelial cells, resulting in vascular endothelial dysfunction, thus affecting the normal operation of the cardiovascular system and triggering a series of cardiovascular diseases; it may also affect the normal operation of the nervous system and trigger neurodegenerative diseases, etc. Generally speaking, the accumulation of ROS may cause organisms to age rapidly and induce various diseases, such as diabetes and its complications, cancer, neurodegenerative diseases, etc.
[0003] Taking diabetes as an example, in diabetes, ROS can cause insulin resistance and pancreatic islet β-cell damage, aggravating the condition of diabetes; in recent years, the excessive production and accumulation of ROS have even been considered as one of the inducing factors of diabetes and an important factor leading to its development.
[0004] Therefore, if a substance that can safely and effectively scavenge ROS can be provided, it will have important medical value.
[0005] Lindera aggregata (Sims) Kosterm. of the genus Lindera in the Lauraceae family is a traditional Chinese medicine, and its main efficacy is promoting qi flow and dispelling cold. Modern pharmacological studies have found that alkaloids are one of the main active ingredients of Lindera aggregata. At present, there is no relevant report on the regulation of the ROS level by total alkaloids from Lindera aggregata. Summary of the Invention
[0006] The purpose of the present invention is to provide an extract of total alkaloids from Lindera aggregata, a preparation method thereof, and an application thereof.
[0007] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is: an extract of total alkaloids from Lindera aggregata, and the components in the extract of total alkaloids from Lindera aggregata include: demethylisoboldine, boldine, Norjuziphine, reticuline, and isoboldine. Preferably, the total mass ratio of demethylisoboldine, boldine, Norjuziphine, reticuline, and isoboldine in the extract of total alkaloids from Lindera aggregata is more than 80%.
[0008] Correspondingly, the preparation method of the extract of total alkaloids from Lindera aggregata includes the following steps:
[0009] (1) Take the Lindera aggregata (Sims) Kosterm. medicinal material, crush it, add it to an ethanol aqueous solution for soaking extraction (ethanol extraction), concentrate the extract to obtain an extraction paste; preferably, perform ethanol extraction 3 times, with each ethanol extraction lasting for 3 days;
[0010] (2) Add an acetic acid aqueous solution to the said extraction paste, heat and rotate to disperse the sample, and extract with dichloromethane to obtain a dichloromethane layer and an acid aqueous layer;
[0011] (3) Add ammonia water to the said acid aqueous layer, adjust the pH to 9 - 10, extract with dichloromethane, and concentrate the extract to a dry powdery solid to obtain the desired total alkaloid extract of Lindera aggregata (Sims) Kosterm..
[0012] Correspondingly, a drug, health product or food for scavenging reactive oxygen species prepared by using the said total alkaloid extract of Lindera aggregata (Sims) Kosterm.; or; a drug, health product or food for scavenging reactive oxygen species containing the said total alkaloid extract of Lindera aggregata (Sims) Kosterm..
[0013] Correspondingly, a drug, health product or food for scavenging reactive oxygen species prepared by using the total alkaloid extract of Lindera aggregata (Sims) Kosterm. prepared by the said preparation method; or; a drug, health product or food for scavenging reactive oxygen species containing the total alkaloid extract of Lindera aggregata (Sims) Kosterm. prepared by the said preparation method.
[0014] The present invention has the following beneficial effects: The total alkaloid extract of Lindera aggregata (Sims) Kosterm. obtained by the specific method of the present invention can effectively scavenge the ROS accumulated in the organism, thereby treating the related diseases caused by ROS accumulation or alleviating the related symptoms.
[0015] Compared with the existing drugs for scavenging ROS, the total alkaloid extract of Lindera aggregata (Sims) Kosterm. provided by the present invention is derived from natural traditional Chinese medicine, has multiple active components, has remarkable effects, is safe and non-toxic, and has no obvious side effects. Moreover, the preparation method of the total alkaloid extract of Lindera aggregata (Sims) Kosterm. is simple, the medicinal material source is wide, it is environmentally friendly, has low cost, is suitable for industrial production, and can promote the development and utilization of Lindera aggregata (Sims) Kosterm.. Description of the Drawings
[0016] Figure 1 UPLC - Q - TOF - MS of the total alkaloid extract of Lindera aggregata (Sims) Kosterm. 2 Component analysis spectrum;
[0017] Figure 2 Effect of the total alkaloid extract of Lindera aggregata (Sims) Kosterm. on the body weight and blood glucose of mice; among them, compared with the control group, * indicates P < 0.05, ** indicates P < 0.01; compared with the model group, # indicates P < 0.05, ## indicates P < 0.01;
[0018] Figure 3Effect of the total alkaloid extract of Lindera aggregata on the accumulation of ROS in the whole body of mice; among them, compared with the control group, * indicates P < 0.05, ** indicates P < 0.01; compared with the model group, # indicates P < 0.05, ## indicates P < 0.01;
[0019] Figure 4 Evaluation result chart of the total alkaloid extract of Lindera aggregata on the biochemical indexes of mice; among them, compared with the control group, * indicates P < 0.05, ** indicates P < 0.01; compared with the model group, # indicates P < 0.05, ## indicates P < 0.01;
[0020] Figure 5 H&E and PAS staining diagrams of mouse tissues after treatment with the total alkaloid extract of Lindera aggregata. Detailed implementation manners
[0021] The present invention provides a total alkaloid extract of Lindera aggregata, and its components include: norisoboldine, boldine, Norjuziphine, reticuline and isoboldine; the total proportion of these components in the total alkaloid extract of Lindera aggregata is more than 80%. The molecular formulas and compound structural formulas of each component are shown in Table 1.
[0022] Table 1 Comparison table of each compound in the total alkaloid extract of Lindera aggregata
[0023]
[0024]
[0025] The present invention also provides a method for extracting the total alkaloid extract of Lindera aggregata, which includes the following steps:
[0026] 1. Take the Lindera aggregata medicinal materials (the medicinal materials obtained after drying the Lindera aggregata tuberous roots), crush them, and soak and extract them in a 70% - 100% ethanol aqueous solution (100% is pure ethanol, and its extraction rate is not as ideal as that of the ethanol aqueous solution; preferably 90% ethanol aqueous solution); preferably, extract with alcohol 3 times, 3 days each time. Concentrate the obtained extract at 60 - 70 °C to obtain an extraction paste.
[0027] 2. Add an acetic acid aqueous solution (the acetic acid aqueous solution is 5% - 10%, preferably 5%) to the extraction paste, and by volume ratio, extraction paste: acetic acid aqueous solution = 1:3 - 1:5; heat and rotate to disperse the sample at 60 °C, and then extract 3 times with dichloromethane with the same volume as the acetic acid aqueous solution to obtain a dichloromethane layer and an acid water layer.
[0028] 3. Add ammonia water to the acid water layer, adjust the pH to 9 - 10, and extract 3 times with dichloromethane with the same volume as the acetic acid aqueous solution. Combine the dichloromethane extraction solutions, and concentrate them to dry powdery solids at 50 - 70 °C to obtain the required total alkaloid extract of Lindera aggregata.
[0029] The total alkaloid extract of Linderae agglomerata can effectively remove reactive oxygen species (ROS), inhibit oxidative damage, and regulate physiological metabolic disorders caused by ROS accumulation.
[0030] The following will be combined with the accompanying 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 embodiments described are only part of the embodiments of the present invention, not all of them. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The data obtained are the average values obtained after at least three repetitions, and the data obtained in each repetition are valid data.
[0031] Example 1: Extraction and identification of active ingredients of Linderae serrata total alkaloid extract
[0032] 10 kg of Linderae agglomerata medicinal material was crushed, added with 20 L of 90% (v / v) ethanol, and soaked and extracted three times at room temperature, each time for three days. The extract was concentrated to obtain an extract. 2 L of 5% (v / v) acetic acid aqueous solution was added to the obtained extract, and the sample was heated and rotated to disperse the sample at 60° C., and then extracted three times with dichloromethane equal in volume to the acetic acid aqueous solution to obtain a dichloromethane layer and an acid aqueous layer. Ammonia water was added to the acid aqueous layer to adjust the pH to 9-10, and the extract was extracted three times with dichloromethane equal in volume to the acetic acid aqueous solution. The dichloromethane extracts were combined and concentrated to dryness to obtain a powdery solid to obtain the Linderae agglomerata total alkaloid extract.
[0033] Application of UPLC-Q-TOF-MS 2 The components of the total alkaloid extract of Linderae agglomerata were analyzed, and the analysis steps and conditions were as follows: the total alkaloid extract sample of Linderae agglomerata was dissolved in pure methanol. UPLC-Q-TOF-MS 2Analysis was performed on a Waters ACQUITYTM ultra performance liquid chromatograph equipped with an ACQUITY UPLC BEH C18 column (4.6 mm × 150 mm, 1.7 μm) and connected to a SYNAPT G2 Si high-resolution mass spectrometer. The column temperature was maintained at 40 °C and the flow rate was 0.4 mL / min. Mass spectra were acquired in positive mode. The mobile phase consisted of 10 mM ammonium acetate solution (solution A) and acetonitrile (solution B), and the HPLC gradient was as follows (the remaining percentages corresponded to solution A): 0 - 2 minutes, 25% solution B; 2 - 7 minutes, 25% - 50% solution B; 7 - 10 minutes, 50% - 70% solution B; 10 - 11 minutes, 70% - 90% solution B; 11 - 13 minutes, 90% solution B; 13 - 13.1 minutes, 90% - 25% solution B; 13.1 - 16 minutes, 25% solution B. The source temperature of the mass spectrometer was 120 °C, the cone gas flow rate was 50 L / h, the desolvation gas temperature was 450 °C, the gas flow rate was 800 L / h, the sampling cone voltage was set at 40 V, and the capillary voltage was set at 2 kV. MS data were collected in sensitivity acquisition mode using the MSe method, and the scanning range was m / z 100 - 1200.
[0034] UPLC-Q-TOF-MS 2 The results of the analytical chromatogram are as Figure 1 shown in Table 2. By comparing with databases, literature, and reference standards, the main components of the total alkaloid extract of Lindera aggregata were identified as demethylisoboldine, boldine, Norjuziphine, reticuline, and isoboldine.
[0035] Table 2 Comparison table of the main components of the total alkaloid extract of Lindera aggregata
[0036]
[0037]
[0038] Example 2: Demonstration of the effect of the total alkaloid extract of Lindera aggregata
[0039] 1. Construction and grouping of diabetic mouse models
[0040] Twenty-four mature male C57BL / 6 mice (8 weeks old) with body weights between 23 and 25 g were randomly divided into three groups. The mice had free access to water and food and were housed in standard cages at room temperature (22 - 24°C) with good ventilation, a 12-h light-dark cycle, and a humidity of approximately 50 ± 5%. The mice were adaptively fed for 7 days, after which they were fasted for 8 hours. Eighteen mice were randomly selected and injected continuously with STZ (streptozotocin, 50 mg / kg, dissolved in sodium citrate-citric acid buffer) for 5 days; six normal mice were randomly selected as the control group (Control) and injected with an equal volume of sodium citrate-citric acid buffer (0.1 M, pH = 4.5). Mice with FGB (fasting blood glucose) ≥ 13.9 mmol / L and maintained for 7 days were considered to have successfully established a diabetes model and were regarded as diabetic mice.
[0041] The 18 mice with successful modeling were randomly divided into three groups, with 6 mice in each group: the model group (Model), the positive group (Positive), and the group intervened with the total alkaloid extract of Lindera aggregata (Sims) Kosterm. (RLA). All mouse groups were given a normal diet. Group RLA: The total alkaloid extract of Lindera aggregata (Sims) Kosterm. was dissolved and evenly dispersed in 0.25% sodium carboxymethylcellulose (CMC-Na) and administered by gavage at a concentration of 300 mg / kg / day; Positive group: The mice were gavaged with acarbose at 10 mg / kg / day. The model group and the control group mice were treated with an equal volume of 0.25% CMC-Na solvent. All treatments lasted for 35 days.
[0042] After the experiment was completed, mouse plasma was obtained by exsanguination through eye removal, and then the mice were sacrificed by cervical dislocation. Tissues were collected for subsequent pathological and biochemical evaluations.
[0043] 2. Effects of the total alkaloid extract of Lindera aggregata (Sims) Kosterm. on mouse body weight and blood glucose
[0044] The mice were fed corn starch (1 g / kg) every day, and the body weights of the mice were recorded daily. Fasting blood glucose and postprandial blood glucose (PPG) at 0.5, 1.5, and 2.0 hours were measured every 7 days. Blood glucose at 0, 30, 60, 90, and 120 minutes after glucose administration (2 g / kg) was recorded for an oral glucose tolerance test (OGTT), and the AUC of the OGTT within 120 minutes was calculated. The results are as Figure 2 shown.
[0045] The results showed that during the experiment, the mice in the model group showed diabetic symptoms such as polyuria and weight loss. Compared with the model group, the body weights of the control group mice increased significantly. All diabetic mouse groups showed significant weight loss within the first ten days. From around the 12th day, the treatment with the total alkaloid extract of Lindera aggregata (Sims) Kosterm. reversed this situation, while the body weights of the model group continued to decrease ( Figure 2 A). The dynamic monitoring of FBG is as Figure 2As shown in Figure B, the FBG value of the diabetic mouse model was significantly higher than that of the normal mouse model; after treatment with the total alkaloid extract of Lindera aggregata, a reverse trend of the FBG value was shown in the second week, and significant differences were observed after the third week compared with the model group. The dynamic changes of PPG were monitored by recording the PPG value every half hour for 2 hours, and the results are as Figure 2 shown in Figures E-J: compared with the model group, the total alkaloid extract of Lindera aggregata and acarbose treatment inhibited the increase in blood glucose at all stages of the experiment and remained stable at 0.5 hours after a meal. For OGTT, the blood glucose level in the model group increased significantly 30 minutes after intragastric administration of glucose ( Figure 2 Figure C). Compared with the model group, the area under the curve (AUC) of the acarbose (P < 0.05) and total alkaloid extract of Lindera aggregata (P < 0.01) treatment groups was significantly reduced ( Figure 2 Figure D), reflecting good glucose tolerance.
[0046] In summary, the total alkaloid extract of Lindera aggregata showed better or comparable hypoglycemic and diabetes symptom-relieving effects than acarbose.
[0047] 3. The ability of the total alkaloid extract of Lindera aggregata to scavenge reactive oxygen species (ROS) in mice
[0048] A ROS test kit was used to detect the ROS levels in the plasma, small intestine, large intestine, liver and kidney of mice to verify the in vivo ROS scavenging ability of RLA. The results are as Figure 3 shown.
[0049] The results showed that systemic ROS accumulation was induced under the physiological condition of high blood glucose in diabetes. After treatment with the total alkaloid extract of Lindera aggregata, it showed significantly better ability to scavenge ROS in plasma, large intestine, small intestine, liver and kidney than acarbose. It indicates that the total alkaloid extract of Lindera aggregata can effectively scavenge the accumulated ROS in visceral organs, and can not only be used to treat the complications caused by the increase in ROS due to diabetes, but also has the potential to directly scavenge ROS, and has the potential to prevent and treat diseases caused by the increase in ROS due to other factors, and also has various potential uses such as anti-aging, antioxidant and anti-inflammatory.
[0050] 4. The effects of the total alkaloid extract of Lindera aggregata on the biochemical indexes of mice
[0051] Mouse plasma was obtained by centrifuging mouse blood at 2000 rpm (8 °C) for 10 minutes. Plasma biochemical parameters, including ALB (albumin), AST (aspartate aminotransferase), ALT (alanine aminotransferase), LDL-C (low density lipoprotein cholesterol), HDL-C (high density lipoprotein cholesterol), TAG (tumor abnormal glycan protein), LACT (lactate), LIP (serum lipase) and UREA (urea), were measured with an automatic analyzer, and the results are asFigure 4 as shown
[0052] The results showed that compared with the control group, the levels of AST, ALT, LDL-C, TAG, and LACT in the model group were significantly increased (P < 0.01), indicating potential liver injury and metabolic disorders caused by hyperglycemic physiological conditions. On the contrary, the levels of ALB, HDL-C, and LIP in the model group were significantly decreased (P < 0.01), indicating potential kidney injury and abnormal lipid metabolism caused by hyperglycemic physiological conditions. After treatment with the total alkaloid extract of Lindera aggregata, the above indexes tended to be normal.
[0053] 5. Effects of the total alkaloid extract of Lindera aggregata on the physiology of mouse tissues
[0054] The large intestine, small intestine, liver, kidney, and heart tissues of all groups were removed and immersed in 10% buffered formalin for 48 hours. Subsequently, the samples were dehydrated, fixed with paraffin, and cut into 4-μm sections for hematoxylin-eosin (H&E) staining. The sections were observed under a GS3-U3-51S5M-C microscope. Fresh liver tissues were fixed overnight in 4% paraformaldehyde (PFA), dewaxed, rehydrated into 5-μm liver sections, and incubated in xylene solution at room temperature for 10 minutes. Then, the sections were stained with periodic acid Schiff (PAS) and observed using a microscope. The results are as Figure 5 shown Figure 5 In the figure, from left to right are: the control group, the model group, the positive control group, and the total alkaloid extract of Lindera aggregata group.
[0055] The results showed that the H&E staining results of the control group demonstrated the biosafety of the administration dose of the total alkaloid extract of Lindera aggregata. In these tissues, compared with the control group, pathological changes were observed in the liver tissues of the remaining groups, including vacuolar degeneration, focal necrosis, and central vein congestion. After 35 days of treatment, no liver lesions were found in the total alkaloid extract of Lindera aggregata treatment group, indicating its liver protection effect under hyperglycemic physiological conditions.
[0056] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. An extract of total alkaloids from Lindera aggregata (Sims) Kosterm., characterized in that: The components of the total alkaloid extract of Lindera aggregata include: norisoboldine, boldine, Norjuziphine, reticuline, and isoboldine.
2. The total alkaloid extract of Lindera aggregata (Sims) Kosterm. according to claim 1, wherein: The total mass ratio of norisoboldine, boldine, Norjuziphine, reticuline, and isoboldine in the total alkaloid extract of Lindera aggregata is more than 80%.
3. The preparation method of the total alkaloid extract of Lindera aggregata (Sims) Kosterm. according to claim 1 or 2, characterized in that: It includes the following steps: (1) Take Lindera aggregata herbs, crush them, add them to an ethanol aqueous solution for soaking and extraction, and concentrate the extract to obtain an extraction paste. (2) Add an acetic acid aqueous solution to the extraction paste, heat and rotate to disperse the sample, and extract with dichloromethane to obtain a dichloromethane layer and an acid aqueous layer. (3) Add ammonia water to the acid aqueous layer, adjust the pH to 9 - 10, then extract with dichloromethane, and concentrate the extract to a solid to obtain the required total alkaloid extract of Lindera aggregata.
4. The preparation method according to claim 3, wherein: In step (1), alcohol extraction is carried out 3 times, and each alcohol extraction lasts for 3 days.
5. The preparation method according to claim 3, characterized in that: In step (2), dichloromethane with the same volume as the acetic acid aqueous solution is used for extraction.
6. The preparation method according to claim 3, wherein: In step (3), dichloromethane with the same volume as the acetic acid aqueous solution is used for extraction 3 times respectively, the extraction liquids are combined, and then concentrated.
7. A drug, health product, or food for scavenging reactive oxygen species prepared from the total alkaloid extract of Lindera aggregata described in claim 1 or 2.
8. A drug, health product, or food for scavenging reactive oxygen species containing the total alkaloid extract of Lindera aggregata described in claim 1 or 2.
9. A drug, health product, or food for scavenging reactive oxygen species prepared from the total alkaloid extract of Lindera aggregata prepared by the preparation method described in any one of claims 3 - 6.
10. A drug, health product, or food for scavenging reactive oxygen species containing the total alkaloid extract of Lindera aggregata prepared by the preparation method described in any one of claims 3 - 6.