A low-toxicity water extract of rhododendron dauricum, a preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIAMUSI UNIVERSITY
- Filing Date
- 2026-03-25
- Publication Date
- 2026-06-09
AI Technical Summary
Existing technologies struggle to effectively reduce the toxicity risks of Rhododendron dauricum water extracts, particularly cardiotoxicity and developmental toxicity, while preserving the main active ingredients. There is a lack of precise methods for removing toxic components.
Membrane separation technology was used to fractionate the water extract of Rhododendron dauricum to remove the 5-10 kDa enriched fraction. Through systematic molecular weight fractionation and toxicity tracking experiments, the targeted removal of components with significant toxicity was achieved.
It significantly reduced the developmental and cardiotoxicity of Rhododendron dauricum water extract, increased the LC50 value, reduced the pericardial edema area, and maintained the stability and safety of the main active ingredients.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of natural plant extract preparation and detoxification technology, and more specifically to a low-toxicity water extract of Rhododendron dauricum, its preparation method and application. Background Technology
[0002] Rhododendron dauricum ( Rhododendron dauricum L. Rhododendron dauricum, a traditional herbal medicine, is widely used in East Asia to treat respiratory diseases, exhibiting significant antitussive, expectorant, and anti-inflammatory effects. However, despite its widely recognized efficacy, certain components in the aqueous extract of Rhododendron dauricum may have toxic effects on humans. Studies have found that the toxicity of Rhododendron dauricum aqueous extract is closely related to certain specific components, especially diterpenoids and their derivatives. These compounds may lead to pathophysiological changes such as cardiac dysfunction, arrhythmia, and abnormal embryonic development through their effects on calcium ion channels, oxidative damage to cardiac muscle cells, and toxicity to the nervous system.
[0003] Existing methods for extracting and reducing the toxicity of traditional Chinese medicine, such as solvent partitioning extraction and adsorption material treatment, can remove impurities or some hazardous components to a certain extent. However, they often suffer from problems such as insufficient selectivity, simultaneous loss of effective components, affected efficacy, and unstable process repeatability, making it difficult to achieve precise removal of toxic components. Meanwhile, the applicant also found prior art US6238675B1, which discloses a method for removing cat's claw herb (…). Uncaria The proposed method for reducing the toxicity of water extracts involves dialysis to remove large molecular weight toxic components (such as polysaccharides and tannins) while retaining low molecular weight active fractions, thereby achieving detoxification while preserving antitumor activity. This literature clearly indicates that the toxic components of *Rhododendron simsii* are large molecules, therefore the focus should be on removing these large molecular weight components. However, this existing technology targets *Rhododendron simsii*, and the toxic substances and specific distribution patterns of *Rhododendron dauricum* differ from those of *Rhododendron dauricum*, making it lack relevance for comparison. Therefore, how to reduce the overall toxicity risk of water extracts while preserving the main effective components as much as possible has become a key technical problem that urgently needs to be solved in improving the safety of *Rhododendron dauricum* preparations.
[0004] The toxicological basis of water extracts from Rhododendron dauricum still needs systematic elucidation, especially lacking stable process routes for identifying, classifying, and removing key toxic components or toxic accumulation segments. Without achieving gentle classification and controllable removal of toxic components, it will be difficult to significantly reduce developmental and cardiotoxic risks while preserving the main effective components.
[0005] Therefore, how to systematically elucidate the toxic substances in the water extract of Rhododendron dauricum and construct a stable removal method, while minimizing the overall toxicity risk of the water extract while preserving the main effective components, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the present invention provides a low-toxicity water extract of Rhododendron dauricum, its preparation method, and its application, to solve the problems of insufficient selectivity for toxicity-related components and poor stability of toxicity reduction effects in existing herbal medicine detoxification processes. The present invention achieves targeted removal of the 5-10 kDa enrichment segment, which is significantly related to toxicity, by performing molecular weight range fractionation on the water extract of Rhododendron dauricum, thereby effectively reducing the toxicity of the water extract, and features a mild process and good reproducibility.
[0007] To solve the above-mentioned technical problems, this application adopts the following technical solution:
[0008] The primary objective of this application is to provide a method for preparing a low-toxicity water extract of Rhododendron dauricum, comprising the following steps: (1) Obtaining total water extract of Rhododendron dauricum; (2) The total water extract is fractionated by membrane separation technology to remove the fractions with molecular weights between 5 and 10 kDa; (3) The components in the remaining molecular weight range were combined and post-processed to obtain low-toxicity water extract of Rhododendron dauricum.
[0009] As a preferred technical solution, the step (1) of obtaining the total water extract of Rhododendron dauricum includes: taking Rhododendron dauricum leaves, crushing them, adding water for soaking pretreatment, heating and extracting multiple times, combining the extracts, centrifuging to obtain the supernatant, and obtaining the total water extract of Rhododendron dauricum.
[0010] As a preferred technical solution, the soaking pretreatment time is 12 hours; the water addition ratio is 1:10 w / v; the heating extraction operation is as follows: heating to boiling and maintaining a gentle boil; the heating extraction time is 2 hours; the centrifugation speed is 4000 rpm and the time is 10 minutes.
[0011] As a preferred technical solution, the membrane separation technology in step (2) is to use membrane modules with different molecular weight cutoffs to separate the components in sequence. The separated components include segments with molecular weights >50 kDa, 10-50 kDa, 5-10 kDa and 100 Da-5 kDa, and the 5-10 kDa segment is discarded.
[0012] As a preferred technical solution, the post-processing operation in step (3) is as follows: the components in the remaining molecular weight range are concentrated by rotary evaporation and dried into powder.
[0013] One objective of this application is to provide a low-toxicity water extract of Rhododendron dauricum prepared by the above method.
[0014] As a preferred technical solution, the low-toxicity water extract of Rhododendron dauricum has an LC50 value.50 ≥300 mg / L.
[0015] One object of this application is to provide the use of the low-toxicity aqueous extract of Rhododendron dauricum in the preparation of a drug for treating respiratory diseases.
[0016] As a preferred technical solution, the respiratory diseases include cough, sputum production, or inflammation.
[0017] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (I) The molecular weight distribution pattern of toxic substances in Rhododendron dauricum is revealed for the first time. This invention, through systematic molecular weight classification and toxicity tracking experiments, reveals for the first time that toxic substances in the aqueous extract of Rhododendron dauricum are not dispersed as conventionally expected, but are highly enriched in the molecular weight range of 5-10 kDa. This discovery overturns the conventional understanding based on the known molecular weight of diterpenoid monomers (300-600 Da)—indicating that toxic substances may exist in the form of complexes or aggregates, with apparent molecular weights far exceeding those of monomers. This is a scientific principle never before reported in existing literature.
[0018] (ii) Achieve quantifiable and significant toxicity reduction effects This invention utilizes membrane separation technology to precisely remove the 5-10 kDa fraction, successfully reducing the developmental and cardiotoxic effects of the water extract. Zebrafish experiments showed that after removing this fraction, the LC... 50 The value increased from the original 229 mg / L to 321 mg / L, and the pericardial edema area was significantly reduced and the developmental malformation was significantly alleviated.
[0019] (III) The process is simple, efficient and green. This invention employs a mild, solvent-free membrane separation process, avoiding the component loss problems that may occur in traditional attenuation methods, and boasts high processing efficiency. Furthermore, the method is simple, low-cost, highly operable, and has broad application prospects, contributing to improved safety of natural plant extracts. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0021] Figure 1The results are as follows: A) Representative toxicity phenotypes of zebrafish larvae in different treatment groups; B) Comparison of median lethal concentration (LC50) of each group of samples on zebrafish embryos; C) Quantitative analysis of body length of zebrafish larvae in each group; D) Quantitative analysis of pericardial edema area of zebrafish larvae in each group. Detailed Implementation
[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0023] Materials and equipment used in the embodiments: Rhododendron leaves: Fresh Rhododendron leaves collected from Jiamusi City, Heilongjiang Province.
[0024] Membrane separator and membrane module: purchased from Chengdu Hecheng Filtration Technology Co., Ltd.
[0025] Experimental animals: wild-type AB strain zebrafish.
[0026] Example 1 Preparation of >50 kDa fractions from Rhododendron dauricum water extract (1) Obtaining the total water extract of Rhododendron dauricum: Fresh Rhododendron dauricum leaves collected in Jiamusi City, Heilongjiang Province in 2025 were collected. After removing impurities and washing thoroughly, the leaves were cut into small pieces and weighed out to 3.5 kg. The leaves were added to distilled water and pretreated for 12 hours at a ratio of 1:10 (w / v). Then, the leaves were placed in an electric thermostatic heating pot and heated to boiling and maintained at a gentle boil for 2 hours. After extraction, the extract was centrifuged at 4000 rpm for 10 minutes and the supernatant was collected. The above extraction and centrifugation steps were repeated three times. All supernatants were combined to obtain the total water extract of Rhododendron dauricum. (2) Take the total water extract of Rhododendron dauricum obtained in step (1) and perform molecular weight fractionation using a small membrane separator. First, collect the retentate through a membrane module with a molecular weight cutoff of 50 kDa to obtain the component with a molecular weight >50 kDa. The obtained retentate is concentrated using a rotary evaporator at 60°C and dried in a vacuum drying oven to obtain powder. It is then sealed and stored in a -20°C refrigerator for later use.
[0027] Example 2 Preparation of 10-50 kDa fractions from Rhododendron dauricum water extract The filtrate from the 50 kDa membrane module in Example 1 was collected and then passed through a membrane module with a molecular weight cutoff of 10 kDa. The retentate was collected to obtain components with molecular weights between 10 and 50 kDa. The obtained retentate was concentrated using a rotary evaporator at 60°C and dried in a vacuum drying oven to obtain powder, which was then sealed and stored in a -20°C refrigerator for later use.
[0028] Example 3 Preparation of 5-10 kDa fractions from Rhododendron dauricum water extract The filtrate from the 10 kDa membrane module in Example 2 was collected and then passed through a membrane module with a molecular weight cutoff of 5 kDa. The retentate was collected to obtain components with molecular weights between 5 and 10 kDa. The obtained retentate was concentrated using a rotary evaporator at 60°C and dried in a vacuum drying oven to obtain powder, which was then sealed and stored in a -20°C refrigerator for later use.
[0029] Example 4 Preparation of 100 Da-5 kDa fraction from Rhododendron dauricum water extract The filtrate from the 5 kDa membrane module in Example 3 was collected and then passed through a membrane module with a molecular weight cutoff of 100 Da. The retentate was collected to obtain components with molecular weights between 100 Da and 5 kDa. The obtained retentate was concentrated using a rotary evaporator at 60°C and dried in a vacuum drying oven to obtain powder, which was then sealed and stored in a -20°C refrigerator for later use.
[0030] Example 5 Preparation of attenuated water extract (recombinant product) of Rhododendron dauricum Based on the total aqueous extract of Rhododendron dauricum prepared according to step (1) of Example 1, membrane separation was performed according to the methods of Examples 1-4 to sequentially obtain Rhododendron dauricum aqueous extracts with concentrations >50 kDa, 10-50 kDa, 5-10 kDa, and 100 Da-5 kDa. The 5-10 kDa aqueous extract was then discarded, while the other three fractions were retained to obtain a recombinant extract. This recombinant extract was concentrated using a rotary evaporator at 60°C and dried in a vacuum drying oven to obtain the final detoxified aqueous extract powder, which was then sealed and stored in a -20°C refrigerator for later use.
[0031] Comparative Example 1 Preparation of total water extract of Rhododendron dauricum Fresh leaves of *Rhododendron dauricum* collected in Jiamusi City, Heilongjiang Province in 2025 were harvested. After removing impurities and thoroughly washing, the leaves were cut into small pieces, and 3.5 kg were weighed. These leaves were added to distilled water at a ratio of 1:10 (w / v) for a 12-hour pretreatment. Subsequently, the leaves were placed in an electric thermostatic heating pot and heated to boiling, maintaining a gentle boil for 2 hours. After extraction, the extract was centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected. The extraction and centrifugation steps were repeated three times, and all supernatants were combined to obtain the total aqueous extract of *Rhododendron dauricum*. The total aqueous extract was concentrated by rotary evaporation at 60℃ and vacuum dried into powder, serving as the sample for Comparative Example 1.
[0032] Toxicity assessment (zebrafish embryo model) The toxicity of Rhododendron dauricum extract was evaluated using a wild-type AB strain zebrafish embryo model. Adult fish were reared in a recirculating aquaculture system under controlled environmental conditions: water temperature 28.5±0.5℃, pH 7.0–7.5, and a 14-hour light / 10-hour dark photoperiod. They were fed freshly hatched artichokes three times daily. For breeding, adult males and females were placed in a 1:1 ratio in a breeding tank at 17:00 for natural mating. Fertilized eggs were collected within one hour of spawning the following morning and incubated at 28.0℃. Embryos that reached 50% coverage at 6 hours post-fertilization (hpf) were selected for subsequent experiments.
[0033] The exposure experiment began at 6 hpf: screened embryos were randomly assigned to 24-well plates, 30 embryos per well, and 1 mL of embryo culture medium containing different concentrations of Rhododendron dauricum extract (Comparative Example 1, Examples 1, 2, 3, 4, 5) was added (the blank control group received an equal volume of culture medium). The exposure medium was changed every 24 h. Phenotypic observation and recording were performed using a Leica stereomicroscope at 24, 48, 72, and 96 hpf. The survival rate of embryos / larvae in each group was calculated, and typical toxic phenotypes such as pericardial edema were assessed. Representative images were acquired at 96 hpf, and ImageJ software was used to quantitatively analyze indicators such as pericardial edema area and body length to comprehensively evaluate the developmental and cardiotoxic effects of the extract.
[0034] Experimental results ( Figure 1 ): Toxicity phenotype observation: Compared with the blank control group, zebrafish larvae treated with Comparative Example 1 (total water extract) showed obvious developmental toxicity phenotypes, mainly pericardial edema (PE), yolk sac edema (YSE) and accompanied by abnormal eye development (ED), suggesting that the total water extract has certain developmental and cardiotoxic risks.
[0035] Toxicity differences across molecular weight fractions: After fractionating the total water extract according to molecular weight, significant differences in toxicity were observed among the different components. Zebrafish from Examples 1 (>50 kDa), 2 (10-50 kDa), 4 (100 Da-5 kDa), and 5 (recombinant) showed only mild or atypical edema changes, indicating relatively weak toxicity. However, Example 3 (5-10 kDa) showed the most obvious abnormalities in PE, YSE, and ED, suggesting that toxic substances are mainly enriched in this molecular weight range.
[0036] Acute toxicity quantitative results: based on the median lethal concentration (LC50) of zebrafish embryos. 50 To measure the toxicity, the LC50 of Comparative Example 1 (total water extract) 50 The concentration of toxicity was 229 mg / L in Example 1 (>50 kDa); 147 mg / L in Example 2 (10–50 kDa); 121 mg / L in Example 3 (5–10 kDa); and 80 mg / L in Example 4 (100 Da–5 kDa). These results indicate that the 5–10 kDa range is the primary toxicity enrichment range.
[0037] Verification of toxicity reduction effect: In terms of toxicity reduction verification, after removing the toxic fraction of 5-10 kDa, the total water extract obtained by recombination of the remaining components (Example 5) showed a significant reduction in toxicity: its LC... 50 The concentration increased to 321 mg / L, which was 92 mg / L (approximately 40%) higher than the total water extract without attenuation (229 mg / L), indicating a significant increase in the concentration required to achieve the same lethal effect. Meanwhile, in terms of developmental indicators, the trend of decreasing larval body length in Example 5 was alleviated, and the pericardial edema area decreased significantly, consistent with phenotypic observations, demonstrating that removing the 5-10 kDa fraction can effectively reduce the developmental and cardiotoxic effects of the Rhododendron dauricum water extract.
[0038] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0039] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for preparing a low-toxicity water extract of Rhododendron dauricum, characterized in that, Includes the following steps: (1) Obtaining total water extract of Rhododendron dauricum; (2) The total water extract is fractionated by membrane separation technology to remove the fractions with molecular weights between 5 and 10 kDa; (3) The components in the remaining molecular weight range were combined and post-processed to obtain low-toxicity water extract of Rhododendron dauricum.
2. The preparation method according to claim 1, characterized in that, The steps for obtaining the total water extract of Rhododendron dauricum in step (1) include: taking Rhododendron dauricum leaves, crushing them, adding water for soaking pretreatment, heating and extracting multiple times, combining the extracts, centrifuging to obtain the supernatant, and obtaining the total water extract of Rhododendron dauricum.
3. The preparation method according to claim 3, characterized in that, The soaking pretreatment time is 12 hours; the water addition ratio is 1:10 w / v; the heating extraction operation is as follows: heat to boiling and maintain a gentle boil; the heating extraction time is 2 hours; the centrifugation speed is 4000 rpm and the time is 10 minutes.
4. The preparation method according to claim 1, characterized in that, The membrane separation technology described in step (2) involves sequentially separating components using membrane modules with different molecular weight cutoffs. The separated components include segments with molecular weights >50 kDa, 10-50 kDa, 5-10 kDa, and 100 Da-5 kDa, and the 5-10 kDa segment is discarded.
5. The preparation method according to claim 1, characterized in that, The post-processing operation in step (3) is as follows: the components in the remaining molecular weight range are concentrated by rotary evaporation and dried into powder.
6. A low-toxicity water extract of Rhododendron dauricum, characterized in that, It is prepared by the preparation method according to any one of claims 1-5.
7. The low-toxicity water extract of Rhododendron dauricum according to claim 6, characterized in that, Its LC 50 ≥300 mg / L.
8. The use of the low-toxicity aqueous extract of Rhododendron dauricum according to any one of claims 6-7 in the preparation of a drug for treating respiratory diseases.
9. The application according to claim 8, characterized in that, The respiratory illnesses include cough, sputum production, or inflammation.