CN115177639A - Application of leech extract in preparation of analgesic - Google Patents
Application of leech extract in preparation of analgesic Download PDFInfo
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- CN115177639A CN115177639A CN202210913245.8A CN202210913245A CN115177639A CN 115177639 A CN115177639 A CN 115177639A CN 202210913245 A CN202210913245 A CN 202210913245A CN 115177639 A CN115177639 A CN 115177639A
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Abstract
The invention discloses application of a leech extract in preparing an analgesic. The invention discovers for the first time that the extract obtained by extracting the Hainan leech with water at the temperature of-2 to 32 ℃ has good analgesic effect on physical pain of animals, and obviously improves the pain threshold value. The pain threshold increasing rate of the mice after 105min of drug administration reaches about 150%. The extraction method of the leech extract is extremely simple and convenient, simple to operate and easy to popularize and use. The extract has low administration dosage, and can achieve good analgesic effect at the mass concentration of 0.25-1.0%.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of a hirudo extract in preparation of an analgesic.
Background
The hirudo nipponica belongs to 1 genus of annelid (Hirudinea) in the class of hirudo, and common species of the hirudo nipponica include Hirudinaria japonica (Haemadipsa. Japonica), hirudinaria japonica (H.tianmushana), hirudinaria hainanensis (H.hainana), hirudinaria salina (H.yanyuanansis), and the like. Hainan leech, commonly called leech, is a characteristic organism in Hainan and belongs to the genus Hirudinaria in the family Hirudinidae. Mainly inhabit spring streams in the eastern mountainous area of the south island of the China sea, at lower levels of bamboo groves and terrains, and are extremely likely to be hidden in wet muddy places, such as wet gravel, rotten wood and drier cattle droppings, grass joints, wet ponding places. Hainan has abundant Hainan leech resources due to unique geographical environment and climate conditions, and has huge potential medicinal value. Currently, no research is carried out on the medicinal activity of Hainan leech, and the Hainan leech is in a state of stasis. There is no report about the extraction of substance with analgesic effect from Hirudinaria manillensis.
Disclosure of Invention
In view of the defects of the prior art, the research is dedicated to the separation and extraction of the polypeptide from the Hainan hirudo and the analgesic activity is further verified by utilizing a mouse hotplate experiment, so that the application of the Hainan hirudo in the aspect of pharmacology is expected to be further expanded, and a foundation is laid for the next research.
The scheme of the invention is as follows:
the leech extract is used for preparing analgesic drugs, and is water extract, and the water extraction temperature is-2-32 ℃.
Preferably, the water extraction temperature is-2 to 22 ℃.
Preferably, the water extraction temperature is 0. + -. 2 ℃.
Preferably, the leech extract is an aqueous extract of live leech slurry.
Preferably, the method for extracting the water extract comprises the following steps: crushing living leech into homogenate, adding distilled water with the volume of 3-10 times, standing overnight at the temperature of-2-32 ℃, and centrifuging to obtain supernatant fluid which is the required leech extract.
Preferably, the method for extracting the water extract comprises the following steps: crushing living leech into homogenate, adding distilled water with the volume of 3-10 times, standing at the temperature of-2-32 ℃ for at least 8h, and centrifuging to obtain supernatant fluid which is the required leech extract.
Preferably, the pain is physical pain.
Preferably, the leech is Hainan leech.
In another aspect, the invention also relates to a leech extract with analgesic effect, and the extraction method of the leech extract comprises the following steps: crushing living leech into homogenate, adding distilled water with the volume of 3-10 times, standing overnight at the temperature of-2-32 ℃, and centrifuging to obtain supernatant fluid which is the required leech extract.
Compared with the prior art, the invention has the beneficial effects that:
the invention discovers for the first time that the extract obtained by extracting the Hainan leech with water at the temperature of-2 to 32 ℃ has good analgesic effect on physical pain of animals, and obviously improves the pain threshold value. The pain threshold increase rate of the mice after 105min of drug administration reaches about 150%.
The extraction method of the leech extract is extremely simple and convenient, simple to operate and easy to popularize and use.
The extract has low administration dosage, and can achieve good analgesic effect at the mass concentration of 0.25-1.0%.
Drawings
FIG. 1: experimental example 2 results chart. In the figure, P is < 0.05, and the pain threshold increase rate of the extract is statistically different from that of the negative control group; * P is less than 0.01, and the pain threshold increase rate of the extract is significantly different from that of a negative control group in statistics; * P < 0.001, the pain threshold increase rate of the extract was statistically significantly different from that of the negative control group.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
Fresh Hainan hirudo is collected from Haikou city of Hainan province, and is identified as a species of Hirudinaria in Hirudinidae.
The reagents and products used in the following examples are all commercially available products unless otherwise specified.
The experimental mice were adult female Kunming mice (3 weeks old).
Example 1 extraction-extraction of Hainan Hirudinaria analgesic active substance (1)
In the experiment, 20 live Hainan leeches are taken, about 40g of the live Hainan leeches are placed in a high-speed stirring and crushing machine to be prepared into homogenate, distilled water with the volume being 10 times that of the homogenate is added, the mixture is placed in a constant-temperature magnetic stirrer in an ice bath (0 +/-2 ℃) overnight (8 h), the mixture is centrifuged for 15min at 4 ℃ under the power of 9000r/min, and the supernatant is the required leeches extract and is frozen and dried in vacuum to be powder for later use.
Example 2 extraction of Hainan Hirudinaria manillensis analgesic active substance-ultrasonic extraction method
The sediment obtained after the centrifugation in the example 1 is taken, about 35g is added with a proper amount of distilled water, the mixture is smashed for 10min by ultrasonic waves (SCIENTZ-IID ultrasonic wave cell crusher) under the power of 70 percent, the solution after the ultrasonic waves is centrifugated for 15min under 9000r/min, and the supernatant fluid is frozen and dried into powder for standby application in vacuum.
Example 3 extraction-Water extraction of Hainan Hirudinaria analgesic active substance (2)
In the experiment, 20 live Hainan leeches are taken, about 40g of the live Hainan leeches are placed in a high-speed stirring and crushing machine to be prepared into homogenate, 10 times of distilled water is added, the homogenate is placed in a constant-temperature magnetic stirrer at 10 +/-2 ℃ overnight (8 h), the mixture is centrifuged for 15min at 4 ℃ under the power of 9000r/min, and the supernatant is the required leeches extract and is frozen and dried in vacuum to be powder for later use.
Example 4 extraction-Water extraction of Hainan Hirudinaria analgesic active substance (3)
In the experiment, 20 live Hainan hirudo (about 40 g) are taken and put into a high-speed stirring and crushing machine to be made into homogenate, distilled water with the volume 10 times that of the homogenate is added, the mixture is placed in a constant-temperature magnetic stirrer at the temperature of 20 +/-2 ℃ overnight (8 h), the mixture is centrifuged for 15min at the temperature of 4 ℃ under the power of 9000r/min, and the supernatant is the required hirudo extract which is frozen and dried into powder for later use.
Example 5 extraction of Hainan Hirudinaria manillensis analgesic active substance-Water extraction method (4)
In the experiment, 20 live Hainan leeches are taken, about 40g of the live Hainan leeches are placed in a high-speed stirring and crushing machine to be prepared into homogenate, 10 times of distilled water is added, the homogenate is placed in a constant-temperature magnetic stirrer at the temperature of 30 +/-2 ℃ overnight (8 h), the mixture is centrifuged for 15min at the temperature of 4 ℃ under the power of 9000r/min, and the supernatant is the required leeches extract, and the leeches extract is frozen and dried in vacuum to be powder for later use.
Experimental example 1
The molecular weights of the Hirudinaria manillensis extract and Poecilobdella manillensis extract of the above examples were determined by SDS-PAGE. The results show that different hirudo extracts have different electrophoretograms in terms of the number of protein bands, moving distance, protein concentration and the like. Wherein, each extract has protein band distribution within the range of 14.4-97.4 kDa, but the distribution positions of the bands are obviously different. The number of protein components of the poecilobdella manillensis extract is the largest, and the poecilobdella manillensis extract has 12 strips, is obviously different from the poecilobdella manillensis extract, and has different distribution positions. Analysis results show that the protein species expressed by two different leech animals of Hainan hirudo and Poecilobdella manillensis are obviously different, and the protein species and the protein concentration of the Hainan hirudo extract obtained by each extraction method are also different.
Experimental example 2: research on analgesic activity of Hainan hirudo
1.1 preliminary screening of injection mode
The pain threshold value of the mouse is measured on a hot plate instrument, the temperature of the hot plate instrument is set to be 55 +/-0.5 ℃, the feet of the mouse licked are taken as observation indexes, the measurement is repeated for 2 times, and the time interval is more than five minutes, so that the normal pain threshold value of the mouse is obtained. 12 mice with positive reactions within 30 seconds were pre-selected and randomly divided into four groups of 3 mice each. The first group was injected intraperitoneally with 0.1ml/10g of 0.25% hirudo extract (i.e. the extract obtained in example 1, with deionized water to a constant volume); the second group was injected intramuscularly with 0.1ml/10g of 0.25% leech extract (i.e. the extract obtained in example 1, volume fixed with deionized water); the pain threshold is measured 15min, 30min, 45min, 60min, 75min, 90min, 105min and 120min after the intraperitoneal injection, 5min is separated for the first 15min and 15min is separated for the next 75min after the intramuscular injection, if the mouse reaction is too slow and no pain reaction occurs in nearly one minute, the hot plate instrument should be removed in time to avoid scalding the skin of the rear foot of the mouse, and the pain threshold is calculated according to 60 s.
The data can observe that the pain threshold value rises slowly after the intraperitoneal injection administration, and the possible reason is that substances can play a role after the intraperitoneal injection administration is absorbed, the pain threshold increasing rate rises quickly in a period of 60-105 min, and the increase amplitude is larger than 100%; the intramuscular injection has quick-acting effect, once the injection is carried out on a mouse, the pain threshold value is improved in a short time, but the improvement range is smaller, and the maximum pain threshold improvement rate is less than 30%; in conclusion, the best injection mode for the experiment was selected to be intraperitoneal injection.
1.2 comparison of analgesic Activity of Hirudinaria Manillensis extract
1.2.1 the pain threshold of the mouse is determined on a hot plate instrument, the temperature of the hot plate instrument is set to be 55 +/-0.5 ℃, the normal pain threshold of the mouse is obtained by taking the foot of the mouse licked as an observation index and repeating the measurement for 2 times with the time interval of more than five minutes. Mice with positive reactions within 30 seconds were pre-selected and randomly grouped, 3 per group. The first group was administered 0.1ml/10g of 0.25% aqueous extract of hirudo (i.e. the extract obtained in example 1, to volume of deionized water); the second group was administered 0.1ml/10g of 0.25% ultrasonic extract of Hirudinaria manillensis (i.e., the extract obtained in example 2, diluted with deionized water); third group: equal amounts of saline were administered; the injection method is intraperitoneal injection, the pain threshold is measured 15min, 30min, 45min, 60min, 75min, 90min, 105min and 120min after administration, if the mouse reaction is too slow and no pain reaction occurs within nearly one minute, the hot plate instrument should be removed in time so as to avoid scalding the skin of the hind feet of the mouse, and the pain threshold is calculated according to 60 s.
1.2.2 the pain threshold of the mouse is determined on a hot plate instrument, the temperature of the hot plate instrument is set to be 55 +/-0.5 ℃, the normal pain threshold of the mouse is obtained by taking the foot of the mouse licked as an observation index and repeating the measurement for 2 times with the time interval of more than five minutes. Mice with positive reactions within 30 seconds were pre-selected and randomly grouped, 3 per group. The first group was administered 0.1ml/10g of 1% of the extract of hirudo of example 1; the second group was administered 0.1ml/10g of 1% of the extract of hirudo of example 3; the third group was administered 0.1ml/10g of 1% of the extract of Hirudinaria manillensis from example 4; the fourth group was administered 0.1ml/10g of 1% of the extract of hirudo of example 5; a fifth group: equal amounts of saline were administered; the injection method is intraperitoneal injection, the pain threshold is measured 105min after administration, if the mouse reaction is too slow and no pain reaction occurs within nearly one minute, the hot plate instrument should be removed in time so as to avoid scalding the skin of the hind feet of the mouse, and the pain threshold is calculated according to 60 s.
1.2.3 the pain threshold value of the mouse is determined on a hot plate instrument, the temperature of the hot plate instrument is set to be 55 +/-0.5 ℃, the normal pain threshold value of the mouse is obtained by taking the foot of the mouse licked as an observation index and repeating the measurement for 2 times with the time interval of more than five minutes. Mice with positive reactions within 30 seconds were pre-selected and randomly grouped into 6 mice per group. The first group was administered 0.1ml/10g of 0.25% hirudo extract (i.e. the extract obtained in example 1, with deionized water to volume); the second group was administered 0.1ml/10g of 0.50% hirudo extract (i.e. the extract obtained in example 1, with deionized water to volume); the third group was administered 0.1ml/10g of 1.00% hirudo extract (i.e., the extract obtained in example 1, to a volume of deionized water); the fourth group was administered 0.1ml/10g of a 1.00% solution of Poecilobdella manillensis extract (i.e., the extract obtained in example 1, in deionized water); the fifth group was administered 0.1ml/10g of 1.00% tramadol solution as a positive control; the sixth group was given an equal amount of physiological saline as a negative control. The injection method is intraperitoneal injection, the pain threshold is measured 15min, 30min, 45min, 60min, 75min, 90min, 105min and 120min after administration, if the mouse reaction is too slow and no pain reaction occurs within nearly one minute, the hot plate instrument should be removed in time so as to avoid scalding the skin of the hind feet of the mouse, and the pain threshold is calculated according to 60 s. The pain threshold increase rate of each group of mice was calculated according to the following formula.
Pain threshold increase rate = [ (mean pain threshold after administration-mean pain threshold before administration)/mean pain threshold before administration ] × 100%
1.3 analysis of results
1.3.1 it can be observed from the data that the pain threshold increase rate of the leech extract obtained by the water extraction method reaches the maximum value in 90min after the administration by intraperitoneal injection, and the increase amplitude is more than 100%. Therefore, the hirudo deliciosa extraction method is judged to be the best extraction method of the hirudo deliciosa analgesic polypeptide, and the pain threshold increasing rate of the mice can be improved to the greatest extent.
1.3.2 it can be observed from the data that after the administration by intraperitoneal injection, the increase rate of the pain threshold of the leech extract obtained by the extraction under the ice bath condition in the example 1 reaches 150%, the increase rate of the pain threshold of the leech extract obtained by the extraction under the condition of 10-20 ℃ reaches about 120%, and the increase rate of the pain threshold of the leech extract obtained by the extraction under the condition of 30 ℃ reaches about 100%. The leech extract obtained by the extraction of the leech by the water extraction method under the ice bath condition has the highest analgesic activity.
1.3.3 the results are shown in FIG. 1. The pain threshold increasing rate of low-dose and medium-dose administration of the hirudo nipponica extract can be used for observing that the pain threshold values of different administration doses have a rising trend after the administration of intraperitoneal injection, but the pain threshold increasing values are slightly different. The high-dose administration has the largest influence on the pain threshold increasing rate, the pain threshold increasing rate is larger than 100% on average and reaches about 150% at the maximum, and the pain threshold increasing rate is close to the positive control after 105min of administration; the change curves of the pain threshold increase rates of the low-dose group and the medium-dose group have little difference, and the change curves have the influence on the pain threshold increase rate within 75min: medium dose group > low dose group, 75min later: the low dose group > the medium dose group, so it is presumed that the medium dose may be used if a quick analgesic effect is to be achieved, and the low dose may be used if a long-acting analgesic effect is desired; in conclusion, the pain threshold increasing rate can be greatly improved by selecting high dose administration (1.0%), and the increase amplitude is more than 100%. Therefore, the analgesic activity of the leech extract is judged to be increased along with the increase of the dosage, and a better leech analgesic effect is achieved.
According to the data, the pain threshold values of the positive control, the Hirudinaria manillensis extract and the Poecilobdella manillensis extract after intraperitoneal injection administration tend to rise, but the pain threshold increase values are different. The positive control (tramadol) has the greatest influence on the pain threshold increase rate, and the effect is long in action time, and the pain threshold increase rate is generally more than 100%. The change curves of the pain threshold increase rate of the poecilobdella manillensis extract and the negative control (normal saline) are not very different, so that the poecilobdella manillensis extract is judged to have no analgesic activity. Compared with a negative control (normal saline), the pain threshold increasing rate of the hirudo nipponica extract is obviously increased, so that the hirudo nipponica extract is proved to have analgesic activity; compared with the poecilobdella manillensis extract, the change curve of the pain threshold increase rate of the poecilobdella manillensis extract is obviously increased, which shows that the analgesic activity of the poecilobdella manillensis extract is superior to that of the poecilobdella manillensis extract.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The application of the leech extract is characterized in that the leech extract is applied to preparing analgesic drugs, the leech extract is a water extract, and the water extraction temperature is-2-32 ℃.
2. The use of the leech extract according to claim 1, wherein the water extraction temperature is-2 to 22 ℃.
3. The use of a hirudo extract according to claim 1, characterized in that the water extraction temperature is 0 ± 2 ℃.
4. The use of the hirudo extract according to claim 1, characterized in that the hirudo extract is an aqueous extract of live slurry of hirudo.
5. The use of the leech extract according to claim 1, wherein the water extract is extracted by a method comprising the steps of: crushing living leech into homogenate, adding distilled water with the volume of 3-10 times, standing overnight at the temperature of-2-32 ℃, and centrifuging to obtain supernatant fluid which is the required leech extract.
6. The use of the hirudo extract according to claim 1, characterized in that the water extract is extracted by a process comprising the following steps: crushing a living body of the leech into homogenate, adding distilled water with the volume of 3-10 times, standing for at least 8 hours at the temperature of-2-32 ℃, and centrifuging to obtain supernatant fluid which is the required leech extract.
7. The use of a hirudo extract according to claim 1, characterized in that the pain is a physical pain.
8. The use of the leech extract according to claim 1, wherein the leech is leech hainanensis.
9. The hirudo extract with the analgesic effect is characterized in that the extraction method of the hirudo extract comprises the following steps: crushing living leech into homogenate, adding distilled water with the volume of 3-10 times, standing overnight at the temperature of-2-32 ℃, and centrifuging to obtain supernatant fluid which is the required leech extract.
10. The hirudo extract having an analgesic effect according to claim 9, wherein the hirudo is hirudo hainanensis.
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| CN105687274A (en) * | 2016-02-18 | 2016-06-22 | 成都中医药大学 | Novel application of duramen of Acanthopanax giraldii Harms or extract of duramen of Acanthopanax giraldii Harms |
| CN105732789A (en) * | 2016-03-03 | 2016-07-06 | 中国科学院昆明动物研究所 | Haemadipsa sylvestris analgesic peptide mh2620 and gene and application thereof |
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2022
- 2022-08-01 CN CN202210913245.8A patent/CN115177639A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1732897A (en) * | 2005-08-16 | 2006-02-15 | 周维海 | Face-beautifying skin-caring lotion with leech extract and its preparation process |
| CN101579360A (en) * | 2008-12-24 | 2009-11-18 | 贵州信邦制药股份有限公司 | Application of high-activity leeches animal extractive in preparing medicament and preparation method of high-activity leeches animal extractive |
| CN105687274A (en) * | 2016-02-18 | 2016-06-22 | 成都中医药大学 | Novel application of duramen of Acanthopanax giraldii Harms or extract of duramen of Acanthopanax giraldii Harms |
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