AU2020100748A4 - Preparation method and application of alkaloids from cynanchum komarovii - Google Patents

Preparation method and application of alkaloids from cynanchum komarovii Download PDF

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AU2020100748A4
AU2020100748A4 AU2020100748A AU2020100748A AU2020100748A4 AU 2020100748 A4 AU2020100748 A4 AU 2020100748A4 AU 2020100748 A AU2020100748 A AU 2020100748A AU 2020100748 A AU2020100748 A AU 2020100748A AU 2020100748 A4 AU2020100748 A4 AU 2020100748A4
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alkaloids
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cynanchum komarovii
ethanol
filtrate
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Changcai Bai
Chengqian Cui
Lu Han
Bingge Li
Fengying Ren
Li Tao
Ruizhou WANG
Yi Wang
Shipeng Zhao
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Ningxia Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
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    • A61K36/27—Asclepiadaceae (Milkweed family), e.g. hoya
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
    • 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
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • 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
    • A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
    • 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
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction

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Abstract

Abstract The present invention discloses a preparation method and application of total alkaloids from Cynanchum Komarovii. In the present invention, a method for extracting alkaloids stepwise is used. The used solvents are dichloromethane, petroleum ether and ethanol, and have low boiling points. The resultant product has a small amount of solvent residues, and is simple in process and advantageous for large-scale production. Moreover, the prepared alkaloids from Cynanchum Komarovii have good analgesic effects.

Description

Description
Preparation Method and Application of Alkaloids from Cynanchum Komarovii
Technical Field
The present invention belongs to the technical field of preparation of alkaloids, and in particular relates to a preparation method and application of alkaloids from Cynanchum Komarovii ( Latin name: Cynanchum Komarovii Al. Iljinski).
Background Art
Alkaloids are a kind of nitrogen-containing alkaline organic compounds in the natural world. Most of the alkaloids have a complex ring structure, and nitrogen is typically contained in the ring. The alkaloids have remarkable biological activity, and are one of the important effective ingredients in Chinese herbal medicines. The alkaloids have anti-pathogenic microorganisms, and 15 anti-inflammatory, anti-tumor, anti-allergic, immunoregulatory and anti-arrhythmic effects, etc.
Cynanchum Komarovii is a medicinal material distributed in the northwest desert areas of China, and has rich resources and large reserves. Herb of Cynanchum Komarovii can be for external use in treatment of various types of arthritis, traumatic injuries and so on. Researches show that alkaloids from Cynanchum Komarovii have obvious analgesic and anti-inflammatory effects and 20 also significant inhibitory effects on various acute and chronic inflammation models.
Phenanthroindolizidine alkaloids such as tylophorine in Cynanchum Komarovii have significant activity against rheumatoid arthritis (RA), and the same type of alkaloids separated from the plants of this genus also have outstanding anti-RA activity. At present, most natives consider Cynanchum Komarovii as weed, so the medicinal value of Cynanchum Komarovii has not been concerned 25 seriously. Therefore, it is of great significance to extract alkaloids from Cynanchum Komarovii.
However, there are no reports on the extraction of alkaloids from Cynanchum Komarovii in the prior art.
Summary of the Invention
A first object of the present invention is to provide a preparation method of alkaloids from Cynanchum Komarovii, which leads to high extraction rate and a small amount of impurities.
A second object of the present invention is to provide a botanical drug containing alkaloids from Cynanchum Komarovii, which has a significant analgesic effect.
To achieve the objects given above, the present invention employs the following technical 35 solution.
2020100748 13 May 2020
Provided a preparation method of alkaloids from Cynanchum Komarovii, characterized in that the method comprises the steps of:
(1) drying the collected herb Cynanchum Komarovii in the air, and crushing into powder by a crusher;
(2) putting the powder into a glass container, and soaking for 10 h to 12 h with 75% ethanol;
(3) ultrasonically extracting the soaked powder 3 times in an ultrasonic extraction tank by using 75% ethanol as a solvent to obtain filtrate;
(4) merging the filtrate, filtering, concentrating under reduced pressure at 50 U to recover ethanol, so as to obtain a crude extract of Cynanchum Komarovii;
(5) dissolving the crude extract with 2% hydrochloric acid, filtering to remove insoluble substances, and extracting the filtrate 3 times with petroleum ether to remove liposoluble components; and (6) merging the acidic water layer, adjusting the acidic water layer to be alkaline, extracting with dichloromethane, and merging the extract layer; concentrating under reduced pressure at 25 U to 15 recover the solvent, so as to obtain the alkaloids from Cynanchum Komarovii.
Preferably, the ratio of the mass of the Cynanchum Komarovii to the volume of the 75% ethanol is 1:10.
Preferably, in the step (3), each ultrasonic extraction is carried out for 2 h.
Preferably, in the step (5), the volume ratio of the petroleum ether to the filtrate is 1:2.
Preferably, in the step (6), the reagent used for adjusting the acidic water layer to be alkaline is strong ammonia water, and the pH value is adjusted to 9 to 11.
The present invention further discloses a drug with an analgesic effect, which contains the alkaloids from Cynanchum Komarovii prepared by the preparation method described above. Tests prove that the drug has a good analgesic effect. The drug may be tablets, capsules, granules and 25 the like. The dosage form of the drug is not limited to a large extent.
The present invention has the following advantages.
In the present invention, Cynanchum Komarovii is used as a raw material, and it is advantageous for the sustainable utilization of resources since there are abundant resources and the environmental compatibility is high.
In the present invention, a method for extracting alkaloids stepwise is used. The used solvents are dichloromethane, petroleum ether and ethanol, and have low boiling points. The resultant product has a small amount of solvent residues, and is simple in process and advantageous for large-scale production.
Detailed Description of the Invention
2020100748 13 May 2020
The present invention will be further explained through specific examples below, but it should be understood that the present invention can be implemented in various forms and should not be limited by the examples set forth herein. Rather, these examples are provided to enable a more thorough understanding of the invention, and to fully convey the scope of the invention to those 5 skilled in the art.
As mentioned throughout the description and claims, include or comprise is an open-ended term and should be construed as including but not limited to. Preferred examples for implementing the present invention are described below, but the following are for the general principles of the description, and not intended to limit the scope of the present invention. The 10 protection scope of the present invention shall be determined by the scope defined by the appended claims.
Unless otherwise specified, various methods adopted in the present invention are conventional methods, and various materials and reagents can be commercially available.
Embodiment 1: Preparation of alkaloids from Cynanchum Komarovii 5 kg of herb Cynanchum Komarovii was crushed into crude powder, and the crude powder was soaked for 10 h to 12 with 75% ethanol and then filtered to obtain herb residues for further use. Then, the herb residues were ultrasonically extracted 3 times by using 75% ethanol as a solvent, and each ultrasonic extraction was carried out for 2 h. The extract liquid and the filtrate were merged and then filtered. The 20 merged liquid was concentrated under reduced pressure at 50 U to recover ethanol, so as to obtain an ethanol-free suspension liquid. The suspension liquid was left to cool (about 6500 ml), then dissolved with 2% hydrochloric acid and filtrated to remove insoluble substances, so as to obtain a crude extract of Cynanchum Komarovii. The crude extract was dissolved with 2% hydrochloric acid and filtered to remove insoluble substances, and the filtrate was extracted 3 times with petroleum ether to remove liposoluble components. The acidic water layer was merged, adjusted to be alkaline (the pH value is adjusted to 9 to 11) and extracted with dichloromethane, and the extract layer was merged. The merged extract layer was concentrated under reduced pressure at 25 U to recover the solvent, so as to obtain alkaloids from Cynanchum Komarovii.
Embodiment 2: Application of alkaloids from Cynanchum Komarovii in analgesic drugs
The inventor has conducted the following experiments on the application of alkaloids from Cynanchum Komarovii(TACKIs) in analgesic drugs.
Experiment 1: Analgesic effect of alkaloids from Cynanchum Komarovii (TACKIs) on mice with heat pricking
In this experiment, there were a positive control group using 40 mg/kg of tramadol hydrochloride 35 (THSRT), TACKI (400, 200 and 100 mg/kg) groups and a blank control Model group. The results
2020100748 13 May 2020 were shown in Table 1.
Table 1 Changes in PWL of mice in each group after administration (* ± S, n = 8)
Group Dosage (mg/kg) PWL(s)
1 h 2 h 3 h 4h
Model group — 6.10 ±0.24 5.991 ±0.81 5.99 ± 1.20 5.94 ±0.36
THSRT group 40 mg/kg 6.50 ± 1.03 _ _ *** 7.70 ±0.70 6.70 ±0.77 6.25 ±0.58
TACKI group 400 mg/kg 6.73 ±0.61 6.23 ± 0.60 6.65 ±0.80 6.74 ± 0.46*
200 mg/kg 7.51 ±0.92* 7.14 ±0.89* 6.64 ± 1.13 6.17 ±0.73
100 mg/kg 6.27 ± 1.35 5.99 ±0.60 5.86 ±0.95 5.20 ±0.57
Notes: Compared with the Model group, * indicated that p<0.05, and *** indicated that p<0.001.
It could be seen from Table 1 that, the PWL of mice in the TACKI 200 mg/kg group was 5 obviously reduced after administration for 1 h and 2 h (p<0.05), whereas the PWL of mice in the TACKI 400 mg/kg group was obviously reduced after administration for 4 h (p<0.05), and the PWL of mice in the positive drug THSRT group was significantly reduced after administration for 2h(p<0.001).
Experiment 2: Observation of the analgesic effect of TACKIs by formalin-induced pain test 10 There were a positive drug group (20 mg/kg of Dolantin, Dolantin group), TACKI (400, 200 and 100 mg/kg) groups and a black control group. The licking time in phase I (0-5 min) and phase II (15-30 min) was observed and recorded. The results were shown in Table 2.
Table 2 Changes in the licking time of mice in each group after administration (x ± S, n = 10)
Group Dosage (mg/kg) Licking time
Phase I Phase II
Model group — 78.33 ± 19.15 133.80 ±44.40
Dolantin group 20 mg/kg *** 13.83 ±9.01 *** 35.70 ±25.29
TACKI group 400 mg/kg *** 36.42 ± 17.50 *** 42.70 ±25.48
200 mg/kg 53.58 ± 15.19** 92.00 ±28.91*
100 mg/kg 61.17 ±18.72* 104.20 ±24.46
Notes: Compared with the Model group, * indicated that p<0.05, ** indicated that p<0.01, and 15 *** indicated that p<0.001.
It could be seen from Table 2 that, the TACKIs significantly reduced the licking time of the phase
I mice in a dose-dependent manner, and in the TACKI 400 mg/kg group and the TACKI 200
2020100748 13 May 2020 mg/kg group, the licking time of the phase II mice was reduced significantly (p<0.01, p<0.05).
Experiment 3: Observation of the analgesic effect of TACKIs by acetic acid writhing test
There were a positive drug group (20 mg/kg of diclofenac sodium, Die group), TACKI (400, 200 and 100 mg/kg) groups and a black control Model group. Mice in each group were 5 intraperitoneally injected with 1% acetic acid after the last administration for 2 h, and the duration of first writhing and the number of times of writhing within 15 min after injection were observed. The results were shown in Table 3.
Table 3 Changes in the duration of first writhing and the number of times of writhing of mice in each group after administration (^ ± S, n = 8)
Group Dosage (mg/kg) Duration of first writhing The number of times of of writhing
Model group — 243.40 ±47.09 26.25 ±5.45
Die group 20 mg/kg 272.50 ± 87.43 *** 3.88 ±5.89
TACKI group 400 mg/kg 346.20 ± 103.00* __ *** 5.63 ± 6.65
200 mg/kg 252.00 ±40.61 24.13 ±4.70
100 mg/kg 269.40 ± 45.80 19.88 ± 4.45
Notes: Compared with the Model group, * indicated that p<0.05, and *** indicated that p<0.001.
It could be seen from Table 3 that, in the TACKI 200 mg/kg group and the TACKI 100 mg/kg group, the duration of first writhing and the number of times of writhing were not influenced, but in the TACKI 400 mg/kg group, the duration of first writhing (p<0.05) and the number of times of writhing (p<0.001) of the mice could be significantly inhibited.
The tests above revealed that the TACKIs had good analgesic effects. Therefore, the TACKIs may be used as an analgesic drug, which may be in the form of common tablet, capsule, granule or the like.
Although the present invention has been described in detail with general descriptions and specific examples, it is obvious for those skilled in the art that some modifications or improvements can be 20 made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims (7)

    Claims
  1. (1) drying the collected herb Cynanchum Komarovii in the air, and crushing into powder by a crusher;
    1. A preparation method of alkaloids from Cynanchum Komarovii, characterized in that the method comprises the steps of:
  2. 2. The preparation method according to claim 1, characterized in that the ratio of the mass of the Cynanchum Komarovii to the volume of the 75% ethanol is 1:10.
    (2) putting the powder into a glass container, and soaking for 10 h to 12 h with 75% ethanol;
  3. 3. The preparation method according to claim 1, characterized in that, in the step (3), each ultrasonic extraction is carried out for 2 h.
    (3) ultrasonically extracting the soaked powder 3 times in an ultrasonic extraction tank by using 75% ethanol as a solvent, to obtain filtrate;
  4. 4. The preparation method according to claim 1, characterized in that, in the step (5), the volume ratio of the petroleum ether to the filtrate is 1:2.
    (4) merging the filtrate, filtering, concentrating under reduced pressure at 50 U to recover ethanol, so as to obtain a crude extract of Cynanchum Komarovii;
  5. 5. The preparation method according to claim 1, characterized in that, in the step (6), the reagent used for adjusting the acidic water layer to be alkaline is strong ammonia water, and the pH value is adjusted to 9 to 11.
    (5) dissolving the crude extract with 2% hydrochloric acid, filtering to remove insoluble substances, and extracting the filtrate 3 times with petroleum ether to remove liposoluble components; and (6) merging the acidic water layer, adjusting the acidic water layer to be alkaline, extracting with dichloromethane, and merging the extract layer; concentrating under reduced pressure at 25 U to recover the solvent, so as to obtain the alkaloids from Cynanchum Komarovii.
  6. 6. A drug with an analgesic effect, characterized in that the drug contains the alkaloids from Cynanchum Komarovii prepared by the preparation method according to any of claims 1 to 5.
  7. 7. The drug according to claim 6, characterized in that the drug comprises tablets, capsules and granules.
AU2020100748A 2020-05-13 2020-05-13 Preparation method and application of alkaloids from cynanchum komarovii Ceased AU2020100748A4 (en)

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