CN107929395B - Preparation method and application of uncaria extract - Google Patents
Preparation method and application of uncaria extract Download PDFInfo
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- CN107929395B CN107929395B CN201711317601.5A CN201711317601A CN107929395B CN 107929395 B CN107929395 B CN 107929395B CN 201711317601 A CN201711317601 A CN 201711317601A CN 107929395 B CN107929395 B CN 107929395B
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- uncaria
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- soluble extract
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/74—Rubiaceae (Madder family)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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Abstract
The invention discloses a preparation method of an uncaria extract and application of the uncaria extract in preparation of an enkephalin degrading enzyme inhibitor and a medicine for treating moderate pain. The uncaria water-soluble extract has strong activity of inhibiting enkephalin degrading enzymes (NEP24.11, APN, ACE), shows analgesic activity both by oral administration and intraperitoneal injection, has no addiction, and has good application prospect in preparation of enkephalin degrading enzyme inhibitors and drugs with analgesic function.
Description
Technical Field
The invention relates to the field of natural medicine, in particular to a preparation method and application of an uncaria extract.
Background
Pain is an unpleasant sensation caused by actual or potential tissue damage. The pain of the patient can be relieved by effectively relieving the pain, and the life quality of the patient is improved. Particularly for critical patients, the effective analgesia can effectively relieve the stress reaction of the organism and reduce the incidence and the death rate of complications of the critical patients. The analgesic drugs commonly used in the market at present mainly comprise opioid alkaloid analgesics such as morphine; synthetic analgesics such as tramadol; non-steroidal antipyretic, analgesic and anti-inflammatory drugs, such as aspirin; other analgesics, such as rotundine, etc. As is well known, although morphine has a strong analgesic effect, morphine is extremely easy to addict and is generally only used for relieving pain at the late stage of cancer; the synthetic analgesic tramadol hydrochloride belongs to centrally acting analgesic, has dependence and addiction after long-term use, and has more and more prominent abuse phenomenon and limited application range at present; aspirin and other non-steroidal anti-inflammatory drugs are easy to induce gastrointestinal adverse reactions; rotundine is mainly used for chronic pain, but not for severe pain.
In recent years, people pay more and more attention to the development of natural medicines, and more researchers begin to screen novel analgesics from common traditional Chinese medicinal materials. Researchers find that some traditional Chinese medicine extract components have remarkable analgesic effect, such as angelica dahurica coumarin, strychnine, aescin and the like. However, at present, no analgesic drug which can be widely used and has the advantages of strong efficacy, low toxicity, no addiction and the like is available. The uncaria is used as a common traditional Chinese medicine and is clinically used for treating diseases such as dizziness, hypertension, cerebral infarction and the like, and in the prior art, the research on the analgesic activity, the action mechanism and the addiction of a water extract of the uncaria is not available, and an analgesic drug prepared from a water-soluble extract of the uncaria is also not available.
Disclosure of Invention
In view of the above, the application provides a preparation method of an uncaria extract and an application of the uncaria extract in preparation of an enkephalin degrading enzyme inhibitor and a medicine for treating moderate pain, the uncaria extract is a water-soluble extract and has very strong activity of inhibiting enkephalin degrading enzyme (NEP, 24.11), animal experiments show that the uncaria extract shows analgesic activity by oral administration and intraperitoneal injection and does not have addiction, and the uncaria extract has a good application prospect in preparation of the enkephalin degrading enzyme inhibitor and the analgesic medicine.
In order to solve the technical problems, the invention provides a preparation method of an uncaria extract, which is characterized by comprising the following steps: the method comprises the following steps:
providing uncaria;
mixing the uncaria with water to obtain a mixture, wherein the weight ratio of the uncaria to the water is 1: (8-12);
boiling the mixture for 10-30 minutes, carrying out primary filtration, collecting a first filtrate, adding water into filter residues, boiling for 18-25 minutes again, carrying out secondary filtration, collecting a second filtrate, and combining the first filtrate and the second filtrate to obtain a water-soluble extract;
concentrating the water-soluble extract to obtain an uncaria extract.
Preferably, in the mixture, the weight ratio of uncaria and water is 1: 10.
preferably, the pH of the water-soluble extract is adjusted to 4.1.
Preferably, the mixture is boiled for 30 minutes, and the filter residue is boiled again for 20 minutes.
Preferably, the method further comprises a drying and freeze-drying step to obtain uncaria extract dry powder.
Preferably, the method further comprises an activity checking step, wherein the activity checking step is carried out after obtaining the uncaria extract dry powder.
More preferably, in the activity test step, the pH is 5.0.
The invention also provides the uncaria extract obtained according to the preparation method, wherein the extract is a water-soluble extract.
The invention also provides application of the uncaria extract obtained by the preparation method in preparing an enkephalin degrading enzyme inhibitor.
Preferably, the enkephalin degrading enzyme inhibitor comprises an aminopeptidase inhibitor and a neutral endopeptidase inhibitor.
Preferably, the enkephalin degrading enzyme inhibitor further comprises an angiotensin converting enzyme inhibitor.
The invention also provides application of the uncaria extract obtained by the preparation method in preparing a medicine for treating pain.
Preferably, the pain treating drug is a non-addictive drug.
More preferably, the pain is moderate pain.
The uncaria rhynchophylla extract is a water-soluble extract and is in a dry powder state, the uncaria rhynchophylla water-soluble extract is subjected to analgesic effect research, and the inhibition effect and IC (integrated circuit) of the uncaria rhynchophylla water-soluble extract on enkephalin degrading enzyme (NEP 24.11) are verified50The value is obtained. Experiments prove that the uncaria rhynchophylla water-soluble extract has strong activity of inhibiting enkephalin degrading enzymes (NEP 24.11), and comprises three enkephalin degrading enzymes: aminopeptidase, neutral endopeptidase and angiotensin converting enzyme inhibitor. In addition, animal experiments prove that the uncaria water-soluble extract shows analgesic activity both by oral administration and intraperitoneal injection, can achieve moderate analgesic effect, and has good application prospect in preparation of drugs with moderate analgesic function.
The term "enkephalin degrading enzyme (NEP 24.11)" is a substance capable of degrading enkephalin in the brain, the enkephalin in the brain comprises methionine enkephalin and leucine enkephalin, and has an analgesic effect, but is easily degraded into inactive derivatives by the enkephalin degrading enzyme, and the uncaria rhynchophylla water-soluble extract in the application has strong inhibitory activity on the enkephalin degrading enzyme (NEP, APN, ACE), can prevent degradation reaction and prolong the analgesic effect of the enkephalin, so that the uncaria rhynchophylla water-soluble extract in the application has an analgesic effect.
Wherein the term "IC50"is the half inhibitory concentration of the active ingredient being measured, which indicates that the ingredient being measured, i.e. the half amount of the water-soluble extract of uncaria rhynchophylla in the present technical scheme, which inhibits certain biological processes, the lower the value, the stronger the inhibitory effect.
Wherein the term "moderate pain" is a grade of pain, the world health organization classifies the degree of pain as four grades, with grade 0 being no pain; level 1 is mild pain, has pain but not severe pain, can be tolerated, and the sleep is not affected; grade 2 is moderate pain, obvious pain, intolerable, disturbed sleep, requiring analgesic; grade 3 is severe pain, intolerance, sleep is severely disturbed, requiring analgesics.
In terms of action mechanism, enkephalin plays a key role in inhibiting physiological pain by improving the sensitivity to noxious stimulation caused by the ablation of the PENK gene. The duration of this effect is limited by the rapid interruption of the endogenous opioid signal, one of the reasons for which is the degradation of enkephalin, and the neutral endopeptidase (NEP, also known as CD10) and aminopeptidase N (APN, also known as CD13) are two enkephalin degrading enzymes that metabolise enkephalin to the inactive tyrosine-glycine peptide chain and tyrosine, thereby inactivating enkephalin.
Blocking the degradation pathway of enkephalin can increase the quantity level of enkephalin at basal cells. However, inhibition of the activity of neutral endopeptidase and aminopeptidase N produces an analgesic effect which is also limited by the number of opioid receptors. The intensity of the opioid receptor response therefore depends on three factors: first, the quantitative level of enkephalin released by stimulation; second, the number level of opioid receptors; third, the activity of enkephalin degrading enzymes. The roles that these three points play in the actual pathological process vary depending on the type of pathways and stimuli involved in the neurons. When administered systemically, neither humans nor mice, selective neutral endopeptidase inhibitors (NEPs) have significant analgesic effects, because the levels of protected enkephalin are too low to stimulate opioid peptide receptors. From this finding, it can be concluded that a dual inhibitor of neutral endopeptidase and aminopeptidase N is more effective.
In addition, enkephalin is not the only substrate of APN and NEP, APN and NEP can decompose several natural peptides in vitro, such as substance P, neurotensin, CCK and bradykinin, and NEP is also involved in modulating the activity of atrial natriuretic peptide and endothelin in vivo, APN is helpful for angiotensin metabolism, maintains its dynamic balance in brain and fluid in kidney, and has the function of controlling blood pressure. The uncaria water extract obtained in the invention also has an inhibiting effect on angiotensin converting enzyme, and the obtained uncaria water extract is prompted to act on a metabolic pathway of enkephalin from a plurality of targets, so that a better analgesic effect is achieved, and the uncaria water extract has no addiction due to non-singleness of the targets, and can safely obtain a satisfactory analgesic effect for a long time.
In conclusion, compared with the prior art, the application provides a preparation method of an uncaria extract and application of the uncaria extract in preparation of an enkephalin degrading enzyme inhibitor and a medicine for treating moderate pain, wherein the uncaria extract is a water-soluble extract, the preparation method of the uncaria water-soluble extract comprises the steps of crushing raw materials, soaking the raw materials, extracting, concentrating, drying and storing, the obtained uncaria water-soluble extract is in a dry powder shape by controlling parameters in the process steps, experiments prove that the uncaria water-soluble extract has very strong activity of inhibiting enkephalin degrading enzymes (NEP24.11, APN, ACE), oral administration and intraperitoneal injection show analgesic activity in animal experiments, and the inhibitory activity is not lower than that of similar medicines sold in the market, namely angiotensin converting enzyme inhibitor captopril, aminopeptidase inhibitor ubenimex and nepeptidase inhibitor thiophaninase inhibitor, and the enkephalin degrading enzyme inhibitor has no addiction, and has good application prospect in preparing enkephalin degrading enzyme inhibitors and medicines with analgesic function.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described in detail with reference to the following embodiments.
Ramulus Uncariae cum uncis is a common Chinese medicine and is a plant of Uncaria of Rubiaceae. The traditional Chinese medicine theory considers that the medicine has pharmacological activities of reducing blood pressure, resisting convulsion, tranquilizing, resisting arrhythmia and the like. The invention adopts a molecular pharmacology model to carry out high-throughput screening, and the traditional Chinese medicine uncaria rhynchophylla obtained by screening has strong effect of inhibiting the activity of rat enkephalin degrading enzymes (including aminopeptidase and neutral endopeptidase), and the inhibition effect of the uncaria rhynchophylla is not inferior to the standard chemical inhibitors (aminopeptidase inhibitor ubenix and neutral endopeptidase inhibitor Thiorphan, trade name Tiorphan). In addition, uncaria has also shown strong inhibitory effect on Angiotensin Converting Enzyme (ACE). Its inhibitory effect is also not inferior to that of captopril. It is known that a substance having a double inhibitory effect on aminopeptidase and neutral endopeptidase can be developed as a non-addictive analgesic agent internationally. And uncaria has an inhibiting effect on three enkephalin degrading enzymes (aminopeptidase, neutral endopeptidase and angiotensin converting enzyme). The uncaria is suggested to be a non-addictive analgesic traditional Chinese medicine with multiple target points.
The enzyme inhibition activity determination of the uncaria rhynchophylla water extract is mainly completed, and the action result on a morphine receptor shows that the uncaria rhynchophylla water extract has the activity effect of inhibiting three enzymes; and has antagonistic effect on morphine receptor. The research of a classical experimental animal analgesic model shows that the uncaria water extract has positive effect on mouse writhing and hot plate experiments, and the analgesic activity strength is 1/20 of morphine.
The above is a detailed description of the present invention, and the following is an example of the present invention
Examples
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example one
The preparation method of the uncaria water-soluble extract comprises the following steps:
(1) crushing raw materials: screening 124g of uncaria raw material without worm damage, mildew and impurities, and crushing to obtain uncaria powder;
(2) soaking raw materials: adding 1240mL of water into the uncaria powder in the step (1), mixing, and soaking for 10 minutes;
(3) extraction: and (3) boiling the mixture of the uncaria powder and the water in the step (2) for 30 minutes, carrying out primary filtration, collecting filtrate, adding 1000mL of water into filter residue, boiling for 20 minutes, carrying out secondary filtration, combining the two filtrates, and adjusting the pH of the filtrate to 4.1.
(4) Concentration: concentrating the filtrate obtained in the step (3) under reduced pressure or with slow fire to obtain extract;
(5) and (3) drying and storing: and (4) freeze-drying the extract in the step (4) to be dry powder, checking the activity under the condition of pH5.0, wherein the activity checking method is mouse writhing experiment detection, and sealing and refrigerating after checking to obtain 4.0g of dry powder.
(6) Water extraction and alcohol precipitation: adding 765ml absolute ethanol into the concentrated solution obtained in the step (3) to enable the ethanol concentration to reach 75%, removing substances insoluble in alcohol, filtering, concentrating, freeze-drying, checking the activity under the condition of pH5.0, sealing and refrigerating to obtain 2.6g of dry powder which is soluble in water and alcohol. The substances which are soluble in water but insoluble in alcohol were likewise concentrated and lyophilized for the activity check.
Another embodiment of the above experimental steps is that 124g of uncaria is weighed, 1480mL of water is added, the mixture is soaked and mixed, boiled for 20 minutes, first filtration is carried out, filtrate is collected, 1000mL of water is added to filter residue, then boiling is carried out for 25 minutes, second filtration is carried out, the two filtrates are combined, and the pH of the filtrate is adjusted to 4.1. The subsequent steps are the same as the steps (4) to (6), and 3.8g of water extract dry powder and 2.3g of water body alcohol sediment are obtained.
In another embodiment of the third embodiment of the above experimental steps, 124g of uncaria is weighed, 990mL of water is added, the uncaria is soaked and mixed, the mixture is boiled for 15 minutes, the first filtration is carried out, the filtrate is collected, 1000mL of water is added into the filter residue, the mixture is boiled for 18 minutes, the second filtration is carried out, the two filtrates are combined, and the pH value of the filtrate is adjusted to 4.1. The subsequent steps are the same as the steps (4) to (6), and 3.5g of water extract dry powder and 2.0g of water body alcohol sediment are obtained.
(7) And (3) activity examination:
1. administration dose: experimental groups: respectively administering B.W mg/kg of dried powder of water-soluble extract of ramulus Uncariae cum uncis, B.W mg/kg of water-extracted and alcohol-precipitated extract (soluble in water and alcohol) of ramulus Uncariae cum uncis, and 300mg/kg of B.W (H) of water-extracted and alcohol-precipitated extract (soluble in water and alcohol);
negative control group: 0.85% physiological saline;
positive control group: 10mg/kg morphine hydrochloride.
2. The administration route is as follows: abdominal injection
3. The experimental results are as follows: the number of writhing within 30min of the mice in each group was measured after the administration and the percentage of analgesia was calculated and the results are shown in table 1.
Percent (%) pain (number of animals with no writhing response in the administered group-number of animals with no writhing response in the NS group)/number of animals with writhing response in the NS group × 100%
The above results indicate that the active ingredient is a water-only and alcohol-insoluble ingredient. And after 21 hours of administration, the uncaria aqueous extract group showed 61.6% of its analgesic activity, and it was found that the analgesic component was a long-acting analgesic substance.
Example two
Endorphin degrading enzyme inhibitory activity assay
The uncaria rhynchophylla water-soluble extract is measured according to an enkephalin degrading enzyme inhibition activity measuring method, the uncaria rhynchophylla water-soluble extract is compared with the inhibition activity of a commercial product Captopril (Captopril), a commercial enkephalin inhibitor racecadotril (Thiorphan) and a commercial product ubenimex (Bestatin), and the test result and the comparison result are shown in a table 2.
Wherein MIC is minimum inhibitory concentration, IC50Is the half inhibitory concentration.
TABLE 2 measurement results of inhibitory Activity
As can be seen from the table, the water-soluble extract of Uncaria rhynchophylla has inhibitory effects on three endorphin degrading enzymes. The inhibitory concentration was 20 mg/ml. Considering that the water-soluble extract of uncaria rhynchophylla is a crude extract, it is expected that the water-soluble active ingredients of the water-soluble extract of uncaria rhynchophylla in the technical scheme of the application may not be lower than the inhibitory effect of Captopril (Captopril) and racecadotril (Thiorphan) and ubenimex (Bestatin).
EXAMPLE III
Acetic acid writhing test of mouse (I)
1. Administration dose:
experimental group (uncaria rhynchophylla water-soluble extract dry powder described in the present application): administered separately and numbered 300mg/kg B.W (A), 150mg/kg B.W (B), 75mg/kg B.W (C), 37.5mg/kg B.W (D), 18.75mg/kg B.W (E);
negative control group: 0.85% physiological saline;
positive control group: 10mg/kg morphine hydrochloride.
2. The administration route is as follows: abdominal injection
3. The experimental results are as follows: the number of writhing times within 30min was measured for each group of mice, and the percentage of analgesia was calculated, and the results are shown in Table 3.
Table 3 example three mouse writhing test results
From the data, the uncaria water-soluble extract dry powder has higher analgesic percentage on the acetic acid-induced mouse writhing test, and the ED is calculated5011.25mg/kg B.W, and in addition, it was found during observation of mice that the dried powder of the water-soluble extract of uncaria rhynchophylla described in the present application can produce significant sedative effect without hypnotic effect, unlike the existing hypnotic or psychosedative agents.
Wherein ED50Is a half effective amount, in the case of quantitative response, refers to the amount of the drug which can cause 50% of the animal response intensity, and in the case of qualitative response, refers to the amount of the drug which can cause 50% of the experimental subjects to have positive reaction.
Example four
Acetic acid writhing test of mouse (II)
1. Administration dose:
experimental group (uncaria rhynchophylla water-soluble extract dry powder described in the present application): administered separately and numbered 30mg/kg B.W (F), 110mg/kg B.W (G), 330mg/kg B.W (H), 1000mg/kg B.W (I);
negative control group: 0.85% physiological saline;
positive control group: 10mg/kg morphine hydrochloride.
2. The administration route is as follows: is administered orally
3. The experimental results are as follows: the number of writhing within 30min of the mice in each group was measured 5min after administration, and the percentage of analgesia was calculated, and the results are shown in table 4.
TABLE 4 torsion test results of four mice in example
The experimental results show that the uncaria rhynchophylla water-soluble extract can quickly take effect after being orally taken, when the dosage of the uncaria rhynchophylla water-soluble extract is 330mg/kg B.W, the analgesic percentage can reach 73.2% after being orally taken for 5m minutes.
EXAMPLE five
Acetic acid writhing test of mouse (III)
1. Administration dose:
experimental group (uncaria rhynchophylla water-soluble extract dry powder described in the present application): administered separately and numbered 110mg/kg B.W (I), 330mg/kg B.W (J), 1000mg/kg B.W (K);
negative control group: 0.85% physiological saline;
positive control group: 10mg/kg morphine hydrochloride.
2. The administration route is as follows: is administered orally
3. The experimental results are as follows: 30min after administration and 75min after administration, the number of writhing of mice in each group was measured for 30min, and the percentage of analgesia was calculated, and the results are shown in Table 5.
Table 5 example four mouse writhing test results
From the data, the test result of testing the acetic acid writhing after oral administration shows that the analgesic activity reaches 91.5% when the administration dose reaches 1000mg/kgB.W, and the preliminary long-acting analgesic activity is shown.
EXAMPLE six
Acetic acid writhing test (IV) of mouse
1. Administration dose: the uncaria water-soluble extract dry powder is 330mg/kg B.W;
2. the administration route is as follows: is administered orally
3. Experimental group setup: measuring the times of writhing of the mice within 30min after 50min, 6h, 9h, 11h and 24h after administration; morphine hydrochloride 10mg/kg B.W was used as a positive control.
4. The percentage of analgesia was calculated from the number of writhes measured and the results are shown in Table 6.
Table 6 example five mouse writhing test results
From the data, the uncaria water-soluble extract dry powder has a long-acting analgesic effect and has an application prospect in preparing long-acting analgesics. Suggesting that the uncaria water-soluble extract can be used for treating chronic pain.
EXAMPLE seven
Acetic acid writhing test (V) of mouse
In the above-mentioned mouse I-IV acetic acid writhing test, Kunming-series mice were used, and in this example, NIH-series mice were used for comparative test.
1. Administration dose:
experimental group (uncaria rhynchophylla water-soluble extract dry powder described in the present application): 300mg/kg B.W;
negative control group: 0.85% physiological saline;
positive control group: 10mg/kg morphine hydrochloride.
2. The administration route is as follows: is administered orally
3. Experimental animals: NIH mice, male and female halves
3. The experimental results are as follows: the number of writhing times within 30min was measured for each group of mice 75min after administration, and the percentage of analgesia was calculated, and the results are shown in Table 7.
TABLE 7 twist test results of seven mice in example
| Group of | Animal number (only) | Twisting for several times/30 min/body | Percentage of analgesia (%) |
| Negative control group | 12 | 36.25 | - |
| Positive control group | 12 | 0 | 100％ |
| GS | 12 | 9.8 | 77.5％ |
And (4) conclusion: the post-analgesia effect of GS on NIH mice strains is slightly lower than that on Kunming mice, but the GS still shows obvious analgesia effect, which indicates that the experimental results of different strains of animals are consistent.
Example eight
Drug addiction experiments of uncaria water-soluble extracts
1. Test drugs: water soluble extracts of quebracho, morphine and ramulus Uncariae cum uncis
2. Experimental materials: guinea pig ileum, mouse vas deferens isolated tissue, Krebs-Henseleit solution
3. The experimental steps are as follows: firstly, placing the isolated tissue into a culture solution under a certain tension to balance for 1 hour, and replacing the nutrient solution every 15 minutes; after the balance is finished, giving an electric field stimulation with certain strength to the isolated tissue, starting adding the medicine after the isolated tissue has stable contraction amplitude, and gradually adding the medicine by an accumulative concentration method; and (3) making a Logarithmic Cumulative Concentration Response Curve (LCCRC) of the test medicament in the absence of antagonists and LCCRC in the presence of naloxone with different concentrations for each in vitro tissue specimen, treating the in vitro tissue specimens by adopting a front-back sequence alternating method to prevent the reduction of contraction caused by specimen fatigue, repeating each in vitro tissue specimen only twice, and flushing 5 times by Krebs-Henseleit nutrient solution and balancing for 30 minutes at the end of a single experiment.
4. Data processing: LCCRC of the drug is converted into a straight line by a logarithm conversion method, and regression analysis is performed to obtain the concentration of the drug for inhibiting the maximum shrinkage of the isolated tissue by 50%, wherein the concentration is IC50. IC in the Presence of different concentrations of naloxone50Value and IC in the absence of naloxone50The dissociation constant K of the antagonist can be determined by substituting the ratio R into the formula B/(R-1)BB is the concentration of antagonist, and the negative logarithm is pA 2. The results are shown in Table 8.
TABLE 8 results of electrostimulation test of guinea pig ileum and mouse vas deferens
| Reagent | pD2 value | pA2 value | Slope of |
| Morphine (morphine) | 8.89 | 8.52 | 0.86 |
| Cap column wood alkali | 6.94 | 7.87 | 0.95 |
| Uncaria water soluble extract | 8.67 | 8.49 | 0.91 |
In an isolated tissue test, the water-soluble extracts of the cephalosporine and the uncaria generate obvious inhibition effect on electrical stimulation contraction of Guinea Pig Ileum (GPI) and Mouse Vas Deferens (MVD) containing different opium accepting points; the double ileum trial of the mice showed: the gambir plant water-soluble extract dry powder can inhibit the ileum contraction of mice, and the inhibition effect cannot be blocked by naloxone, which indicates that the gambir plant water-soluble extract dry powder does not contain morphine-like substances and has no drug addiction.
Example nine
Moderate pain treatment test (or treatable pain rating test)
Hot plate test of mice
1. Administration dose: in the experiment, 9 groups are set, namely a negative control group, an intraperitoneal injection group of morphine hydrochloride of 10mg/kg and groups of uncaria rhynchophylla water-soluble extract dry powder of 10mg/kg, 30mg/kg and 300mg/kg, wherein each group comprises 10 male ICR mice. Each group of mice is given 0.9% sodium chloride injection and morphine hydrochloride injection with corresponding concentration and uncaria water-soluble extract dry powder by 10mL/kg intraperitoneal injection;
2. detecting the times and time of paw-contracting and foot-lifting of the mouse 20, 40 minutes and 1, 2 and 3 hours before and after administration by using an intelligent hot plate instrument;
3. calculating the percentage of analgesia by measuring the times and time of the paw-contracting and the foot-lifting of the mouse,
the results are as follows:
20 minutes after the administration of the oral gavage 10, 30 and 300mg/kg group of mice, the times of paw-contracting and foot-lifting of the 30 and 300mg/kg group of mice are reduced, and the statistical difference (P is less than or equal to 0.05) indicates that a certain analgesic effect exists; no significant change was observed in the 10mg/kg group of mice. Has a certain analgesic effect within 20 minutes after oral gavage of 30 and 300 mg/kg.
20 minutes after the mice of 10, 30 and 300mg/kg groups are subjected to intraperitoneal injection, the times of paw-contracting and foot-lifting of the mice of 300mg/kg groups are reduced, and the statistical difference (P is less than or equal to 0.05) indicates that a certain analgesic effect exists; the number of times of paw withdrawal and foot lifting of the 10mg/kg group of mice is increased (compared with the contemporary negative control group, the statistical difference is that P is less than or equal to 0.05). The water soluble extract dry powder with the dosage of 300mg/kg is injected into the abdominal cavity, and has a certain analgesic effect within 20 minutes.
The time for contracting and lifting the paw is reduced after the administration of the morphine hydrochloride 10mg/kg group mice in intraperitoneal injection for 20 minutes, 40 minutes and 1 hour and 2 hours, which proves that the system has obvious analgesic effect and is reliable.
Example ten
Toxicity test
Acute toxicity test of mice
1. Experimental materials: 18-22g of Kunming mouse with male and female halves; water extract of ramulus Uncariae cum uncis.
2. The experimental steps are as follows:
1) pre-testing: the mice were divided into 6 groups of 4 mice, a series of doses with a large distance between groups were selected, each group was individually gavaged, the symptoms were observed and the number of deaths recorded, and the ranges of doses causing 0% mortality and 100% mortality were found.
2) And (3) formal test: within the range of 0% and 100% lethal dose obtained in preliminary experiments, 5 doses were selected, the ratio between adjacent doses being 1: 0.7. mice were randomly grouped and 10 mice per group were gavaged.
3) Observations were recorded for each reaction: latency, intoxication, time to onset of death, pre-mortem events, number of deaths in each group, etc. The observation was carried out for 7 days after the application. LD50 was calculated by modified Korbor or simplified probability unit method based on the mortality of each group of animals.
3. Results of the experiment
The dosage of the uncaria water extract selected in the embodiment is respectively 8.57, 6, 4.2, 2.94 and 2.05, the unit is g/kg.d, and the LD50 value is calculated to be 4.75g/kg.d by using the modified Korbor method (Korbor), which proves that the uncaria water extract has no toxicity under the normal dosage.
In addition, the results of a number of animal experimental studies conducted in the early stages of the present invention showed that: uncaria rhynchophylla main fat-soluble compounds such as rhynchophylline, isorhynchophylline, hirsutine dehydrogenized, isorhynchophylline and the like have no analgesic activity. Uncaria rhynchophylla C with weak water solubility has weak analgesic activity, so that the analgesic active ingredient of the Uncaria rhynchophylla is proved to be a water-soluble substance from the opposite side.
The application provides a preparation method of an uncaria rhynchophylla extract and application of the uncaria rhynchophylla extract in preparation of an enkephalin degrading enzyme inhibitor and a medicine for treating moderate pain, wherein the uncaria rhynchophylla extract is a water-soluble extract, the preparation method of the uncaria rhynchophylla water-soluble extract comprises the steps of raw material crushing, raw material soaking, extraction, concentration and drying storage, the obtained uncaria rhynchophylla water-soluble extract is in a dry powder state by controlling parameters in the process steps, experiments prove that the uncaria rhynchophylla water-soluble extract has strong activity of inhibiting enkephalin degrading enzymes (NEP24.11, APN, ACE), oral administration and intraperitoneal injection show analgesic activity in animal experiments, and the inhibitory activity of the uncaria rhynchophylla water-soluble extract is not lower than that angiotensin converting enzyme inhibitor captopril, aminopeptidase inhibitor ubenimex and nepenth, and the enkephalin degrading enzyme inhibitor has no addiction, and has good application prospect in preparing enkephalin degrading enzyme inhibitors and medicines with analgesic function.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (4)
1. The application of an uncaria extract in preparing a long-acting non-addiction medicament for treating pain is disclosed, wherein the uncaria extract is prepared by the following method: providing uncaria; mixing the uncaria with water to obtain a mixture, wherein the weight ratio of the uncaria to the water is 1: (8-12); boiling the mixture for 10-30 minutes, carrying out primary filtration, collecting a first filtrate, adding water into filter residues, boiling for 18-25 minutes again, carrying out secondary filtration, collecting a second filtrate, combining the first filtrate and the second filtrate to obtain a water-soluble extract, and adjusting the pH value of the water-soluble extract to 4.1; concentrating the water-soluble extract to obtain an uncaria extract.
2. A use as claimed in claim 1, wherein the pain is moderate pain.
3. A preparation method of uncaria extract for long-acting non-addiction pain treatment is characterized in that: the method comprises the following steps:
providing uncaria;
mixing the uncaria with water to obtain a mixture, wherein the weight ratio of the uncaria to the water is 1: 10;
boiling the mixture for 10-30 minutes, carrying out primary filtration, collecting a first filtrate, adding water into filter residues, boiling for 18-25 minutes again, carrying out secondary filtration, collecting a second filtrate, combining the first filtrate and the second filtrate to obtain a water-soluble extract, and adjusting the pH value of the water-soluble extract to 4.1; concentrating the water-soluble extract to obtain an uncaria extract.
4. The long-acting non-addictive pain-treating uncaria extract obtained by the preparation method according to claim 3, wherein the extract is a water-soluble extract.
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