Method for combining polygala tenuifolia with semen strychni and application thereof
By decocting Yuanzhi and Ma Qianzi with a dosage ratio of 1:5, we prepared a water decoction solution, which solved the problem of Ma Qianzi's toxicity, significantly reduced the toxicity and enhanced the anti-inflammatory and analgesic effects, and achieved improved safety and effect.
Patent Information
- Application Number
- CN202411822024.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-11
AI Technical Summary
The toxic components of Macako are overlapping, resulting in a narrow range of drug safety, and the existing compatibility methods cannot effectively reduce toxicity and enhance anti-inflammatory and analgesic effects.
The decoction of Yuanzhi and Matsuzi with a dosage ratio of 1:5 was used to decoct or use in combination. The decoction was decocted twice and filtered and concentrated to obtain a water decoction containing 1g/mL of sand-boiled Matsuzi and 5g/mL of Yuanzhi.
It significantly reduces the toxicity of marzizi and increases its anti-inflammatory and analgesic effects. The LD50 value has been increased by 2.0 times to 5.9 times, and the analgesic rate has reached 80%, and its anti-inflammatory effect is comparable to that of aspirin.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of medicine, and in particular to a method for combining polygala tenuifolia with strychnos nux vomica and application thereof. Background Art
[0002] Strychnos nurvomica L. is a dried mature seed of the plant Strychnos nurvomica L. of the Logarithmaceae family. It is a commonly used medicine in traumatology and has the effects of promoting blood circulation and removing blood stasis, reducing swelling and relieving pain. Strychnos nurvomica has a long history of use and has been used for cancer pain, traumatic injuries, rheumatic pain and other symptoms since ancient times. It has significant anti-inflammatory and analgesic effects. However, the toxic and effective components of Strychnos nurvomica overlap, and strychnine and brucine are both its main toxic components and its effective components, which makes the safety range of Strychnos nurvomica relatively narrow and its application is greatly limited.
[0003] The 2020 edition of the Chinese Pharmacopoeia stipulates that the strychnine content of raw and processed Strychnine should be 1.2% to 2.2%, and the content of strychnine should not be less than 0.8%, and it must be processed before being used in pills and powders; while the strychnine content of Strychnine powder should be 0.78% to 0.82%, and the content of strychnine should not be less than 0.5%. The Chinese Pharmacopoeia stipulates that the processing method of Strychnine is the sand scalding method, and it is processed into pills and powders, but its processed products are still highly toxic. How to reduce the toxicity of Strychnine while retaining its efficacy has become a key issue facing the application of Strychnine.
[0004] Common methods for reducing toxicity of toxic Chinese herbal medicines include compatibility and processing, and Chinese medicine often uses these methods to expand its application range. At present, the common single herbs that are combined with Strychnos nux vomica to reduce toxicity include: licorice, raw rehmannia root, cooked rehmannia root, white peony root, cinnamon bark, cassia twig, coptis root, etc. However, there are certain defects, such as: licorice combined with Strychnos nux vomica can only reduce the toxicity of processed Strychnos nux vomica; although raw rehmannia root and cooked rehmannia root can reduce the toxicity of Strychnos nux vomica, raw rehmannia root and cooked rehmannia root cannot enhance the anti-inflammatory and analgesic effects of Strychnos nux vomica; although cassia twig combined with Strychnos nux vomica can reduce the toxicity of Strychnos nux vomica, it cannot enhance the analgesic effect of Strychnos nux vomica. Therefore, it is urgent to find a more effective method of processing or combining Strychnos nux vomica that can reduce toxicity and increase efficacy, so that the safety of Strychnos nux vomica can be further improved.
[0005] The team of the present invention found in the previous study that the combination of Polygala and Strychnos nux vomica can significantly reduce the mortality of mice with a lethal dose of Strychnos nux vomica and significantly prolong the time of toxicity onset. Therefore, based on the previous study, the present invention designed a new method of combining Polygala with Strychnos nux vomica to further verify the detoxification effect of Polygala on Strychnos nux vomica and observe the effect of Polygala and Strychnos nux vomica on its toxicity and effective ingredients. At the same time, a preliminary evaluation was conducted on the method of combining Polygala with Strychnos nux vomica. It provides new ideas for the use, compatibility and processing of Strychnos nux vomica and contributes to the improvement of the safety of Strychnos nux vomica medication. Summary of the invention
[0006] In order to solve the problems existing in the prior art, the present invention provides a new method of using Polygala tenuifolia in combination with Strychnos nux vomica, which can significantly reduce the toxicity of Strychnos nux vomica and increase the anti-inflammatory and analgesic effects of Strychnos nux vomica in a dosage ratio of 1:5 by decocting or using the two together.
[0007] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions and steps:
[0008] The method for combining Polygala tenuifolia with Strychnos nux vomica of the present invention comprises the following steps:
[0009] S1, taking sand-scalded Strychnos nux vomica, crushing it through a 20-mesh sieve to obtain sand-scalded Strychnos nux vomica particles, and setting aside;
[0010] S2, weigh appropriate amounts of cored Polygala and blanched Strychnos nux vomica granules respectively, mix and decoct twice, add double distilled water in an amount of 6-10 times the total mass of the medicinal materials for the first time, decoct for 1-3 hours, and filter; add double distilled water in an amount of 4-6 times the total mass of the medicinal materials for the second time to the residue, decoct for 0.5-1.5 hours, and filter; combine the filtrates and concentrate them to a decoction containing 1 g / mL blanched Strychnos nux vomica and 5 g / mL Polygala tenuifolia.
[0011] Preferably, step S2 of the present invention is specifically as follows: weigh appropriate amounts of cored Polygala and blanched Strychnos nux vomica granules respectively, mix and decoct twice, add double distilled water in an amount of 8 times the total mass of the medicinal materials for the first time, decoct for 2 hours, and filter; add double distilled water in an amount of 5 times the total mass of the medicinal materials to the residue for the second time, decoct for 1 hour, and filter; combine the filtrate and concentrate it to a decoction containing 1 g / mL blanched Strychnos nux vomica and 5 g / mL Polygala.
[0012] Step S2 of the present invention can also be: weighing an appropriate amount of cored Polygala, decocting twice, adding double distilled water in an amount of 6-10 times the mass of the medicinal material for the first time, decocting for 1-3 hours, and filtering; adding double distilled water in an amount of 4-6 times the mass of the medicinal material to the residue for the second time, decocting for 0.5-1.5 hours, and filtering; combining the filtrate and concentrating to a water decoction containing 1 g / mL of scalded Strychnos nux vomica; taking scalded Strychnos nux vomica particles, using the same decoction conditions as mentioned above, decocting and filtering, combining the filtrate and concentrating to a water decoction containing 5 g / mL of Polygala; then mixing the water decoction containing 1 g / mL of scalded Strychnos nux vomica and the water decoction containing 5 g / mL of Polygala tenuifolia in equal volumes.
[0013] Preferably, step S2 of the present invention can also be: weigh an appropriate amount of cored Polygala, decoct twice, add double distilled water in an amount of 8 times the mass of the medicinal material for the first time, decoct for 2 hours, and filter; add double distilled water in an amount of 5 times the mass of the medicinal material to the residue for the second time, decoct for 1 hour, and filter; combine the filtrate and concentrate to a water decoction containing 1 g / mL of scalded Strychnos nux vomica; take scalded Strychnos nux vomica particles, use the same decoction conditions as above, decoct and filter, combine the filtrate and concentrate to a water decoction containing 5 g / mL of Polygala; then mix the water decoction containing 1 g / mL of scalded Strychnos nux vomica and the water decoction containing 5 g / mL of Polygala tenuifolia in equal volumes.
[0014] The mass ratio of the cored Polygala tenuifolia to the blanched Strychnos nux vomica is 1:2.5-5.
[0015] Preferably, the mass ratio of the cored Polygala tenuifolia to the blanched Strychnos nux vomica in the present invention is cored Polygala tenuifolia: blanched Strychnos nux vomica = 1:5.
[0016] The water decoction prepared by the method of combining Polygala tenuifolia with Strychnos nux vomica is used in medicine for reducing the toxicity of Strychnos nux vomica and enhancing the anti-inflammatory and analgesic effects of Strychnos nux vomica.
[0017] The invention discloses an application of a combination of Polygala tenuifolia and Strychnos nux vomica in a medicine for reducing the toxicity of Strychnos nux vomica and enhancing the anti-inflammatory and analgesic effects of Strychnos nux vomica.
[0018] The dosage ratio of Polygala tenuifolia and Strychnos nux vomica in the medicine of the present invention is 1:2.5-5.
[0019] Preferably, the dosage ratio of Polygala tenuifolia to Strychnos nux vomica in the medicine of the present invention is 1:5.
[0020] The Polygala tenuifolia and Strychnos nux vomica in the present invention are both cored Polygala tenuifolia and sand-scalded Strychnos nux vomica, and their processing methods are all the processing methods specified in the 2020 edition of the Chinese Pharmacopoeia.
[0021] Beneficial effects of the present invention:
[0022] 1. The present invention is the first to discover a method of using Polygala tenuifolia in combination with Strychnos nux vomica. By decocting or using Polygala tenuifolia and Strychnos nux vomica in a dosage ratio of 1:2.5-5, not only can the toxicity of Strychnos nux vomica be significantly reduced, but also the anti-inflammatory and analgesic effects of Strychnos nux vomica can be increased.
[0023] 2. The present invention has passed the acute toxicity test and anti-inflammatory and analgesic test on mice, and the results are as follows:
[0024] (1) In the acute toxicity test of Strychnos nux vomica treated with different Polygala tenuifolia, all the different treatments of Polygala tenuifolia significantly increased the LD50 of Strychnos nux vomica 50 The LD value of the decoction obtained by decocting 2.5 times and 5 times the amount of Polygala tenuifolia and sand-scalded Strychnos nux vomica was 50The values increased by 2.0 times and 4.8 times, respectively, indicating that Polygala tenuifolia has a significant effect on reducing the acute toxicity of scalded Strychnos nux vomica, and this detoxification effect increases with the increase in the dosage of Polygala tenuifolia. The LD 50 The value was 5.9 times higher than that of the single decoction of scalded Strychnos nux vomica.
[0025] (2) In the experiment, the effects of different methods of treating Polygala on the analgesic effect of Strychnos nux vomica were investigated. The single decoction of scalded Strychnos nux vomica and different methods of treating Strychnos nux vomica with Polygala could significantly reduce the number of writhing times in mice with pain (P<0.01). The reduction in the number of writhing times was more significant when Polygala and Strychnos nux vomica were decocted together and mixed by oral gavage (P<0.01). The analgesic rate of scalded Strychnos nux vomica was about 40%, while the analgesic rate of 5-fold amount of Polygala co-decocted reached 80%, and the analgesic effect was dose-dependent, that is, as the amount of Polygala used increased, its analgesic effect was further enhanced. The analgesic rate of 5-fold amount of co-decocted and mixed by oral gavage was equivalent to that of the positive drug aspirin.
[0026] (3) In the experiment of the effect of different methods of Polygala on the anti-inflammatory effect of Strychnos nux vomica, the method of using sand-scalded Strychnos nux vomica alone decoction and different methods of Polygala treatment on sand-scalded Strychnos nux vomica had a significant effect on inhibiting mouse ear swelling (P<0.05), among which the difference between the low and high doses of the co-decoction group and the control group was extremely significant (P<0.01); the anti-inflammatory effect of sand-scalded Strychnos nux vomica alone decoction was slightly worse than that of the positive drug aspirin group, while the anti-inflammatory effect of the mixed high-dose group was equivalent to that of aspirin, and the anti-inflammatory effect of the low and high doses of sand-scalded Polygala co-decoction was better than that of aspirin, and was significantly enhanced with the increase of the dosage of Polygala. The above experiments verified and clarified that Polygala can reduce the toxicity of Strychnos nux vomica and enhance the anti-inflammatory and analgesic effects of Strychnos nux vomica, and the effect is significant.
[0027] 3. The method of the present invention provides a new method for enhancing the efficacy and reducing the toxicity of Nux Vomica, and provides a new idea for the use, compatibility and processing of Nux Vomica. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 :Effects of different methods of treating Nux vomica with Polygala tenuifolia on hot plate test in mice DETAILED DESCRIPTION
[0029] The present invention is described in detail below with reference to specific embodiments.
[0030] Example 1 Preparation of a decoction of scalded Strychnos nux vomica and cored Polygala tenuifolia (1:5)
[0031] S1, taking sand-scalded Strychnos nux vomica, crushing it through a 20-mesh sieve to obtain sand-scalded Strychnos nux vomica particles, and setting aside;
[0032] S2, weigh 50g of blanched Strychnos nux vomica granules and 250g of cored Polygala tenuifolia respectively, add double distilled water to mix and decoct twice, the first time adding 2400mL of double distilled water, decoct for 2h, and filter; the second time adding 1500mL of double distilled water to the residue, decoct for 1h, and filter; combine the filtrate and concentrate to 50mL to obtain a decoction containing 1g / mL blanched Strychnos nux vomica and 5g / mL cored Polygala tenuifolia.
[0033] Example 2 Preparation of a decoction of scalded Strychnos nux vomica and cored Polygala tenuifolia (1:5)
[0034] S1, taking sand-scalded Strychnos nux vomica, crushing it through a 20-mesh sieve to obtain sand-scalded Strychnos nux vomica particles, and setting aside;
[0035] S2, weigh 50g of blanched Strychnos nux vomica granules and 250g of cored Polygala tenuifolia respectively, add double distilled water to mix and decoct twice, the first time adding 2400mL of double distilled water, decoct for 2h, and filter; the second time adding 1500mL of double distilled water to the residue, decoct for 1h, and filter; combine the filtrate and concentrate to 50mL to obtain a decoction containing 1g / mL blanched Strychnos nux vomica and 5g / mL cored Polygala tenuifolia.
[0036] Example 3 Preparation of a decoction of scalded Strychnos nux vomica and cored Polygala tenuifolia (1:5)
[0037] S1, taking sand-scalded Strychnos nux vomica, crushing it through a 20-mesh sieve to obtain sand-scalded Strychnos nux vomica particles, and setting aside;
[0038] S2, weigh 50g of blanched Strychnos nux vomica granules and 250g of cored Polygala tenuifolia respectively, add double distilled water to mix and decoct twice, the first time adding 1800mL of double distilled water, decoct for 2h, and filter; the second time adding 1200mL of double distilled water to the residue, decoct for 1h, and filter; combine the filtrate and concentrate to 50mL to obtain a decoction containing 1g / mL blanched Strychnos nux vomica and 5g / mL cored Polygala tenuifolia.
[0039] Example 4 Preparation of a decoction of scalded Strychnos nux vomica and Polygala tenuifolia (1:5)
[0040] S1, taking sand-scalded Strychnos nux vomica, crushing it through a 20-mesh sieve to obtain sand-scalded Strychnos nux vomica particles, and setting aside;
[0041] S2, weigh 250g of cored Polygala, add double distilled water and decoct twice, add 2000mL of double distilled water for the first time, decoct for 2h, and filter; add 1250mL of double distilled water to the residue for the second time, decoct for 1h, and filter; combine the filtrate and concentrate to 50mL to obtain a decoction containing 5g / mL of cored Polygala; take 50g of scalded Strychnos nux vomica granules, use the same decoction conditions as mentioned above, decoct and filter, combine the filtrate and concentrate to a decoction containing 1g / mL of scalded Strychnos nux vomica; then mix the scalded Strychnos nux vomica decoction and the Polygala tenuifolia decoction in equal volumes.
[0042] Example 5 Preparation of a decoction of scalded Strychnos nux vomica and cored Polygala tenuifolia (1:2.5)
[0043] S1, taking sand-scalded Strychnos nux vomica, crushing it through a 20-mesh sieve to obtain sand-scalded Strychnos nux vomica particles, and setting aside;
[0044] S2, weigh 50g of blanched Strychnos nux vomica granules and 125g of cored Polygala tenuifolia respectively, add double distilled water to mix and decoct twice, the first time adding 1400mL of double distilled water, decoct for 2h, and filter; the second time adding 875mL of double distilled water to the residue, decoct for 1h, and filter; combine the filtrate and concentrate to 50mL to obtain a decoction containing 1g / mL blanched Strychnos nux vomica and 2.5g / mL cored Polygala tenuifolia.
[0045] In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:
[0046] 1 Experimental Materials
[0047] 1.1 Experimental animals and materials
[0048] 400 male and 50 female SPF Kunming mice, weighing 18-22 g, were purchased from Chongqing Tengxin Biotechnology Co., Ltd., animal license number: SCXK (Guizhou) 2021-0005. The animal breeding environment was well ventilated, with a temperature of 22°C to 25°C and a relative humidity of 50% to 60%. The mice in each group were free to eat and drink water and were adaptively fed for 3 days before the experiment was carried out. The experimental process followed the animal experiment ethics of Guizhou University of Traditional Chinese Medicine.
[0049] The Chinese medicinal materials of scalded Strychnos nux vomica and cored Polygala tenuifolia were purchased from Hongyi Agricultural Products Store in Nanming District, Guiyang City; the experimental mouse maintenance feed was purchased from Chongqing Tengxin Co., Ltd.
[0050] 1.2 Experimental instruments
[0051] SPD-20A high performance liquid chromatograph (Shimadzu Co., Ltd., Japan); TG16B low temperature high speed centrifuge (Kate Experimental Instrument Co., Ltd., Yancheng City); FA2004 electronic analytical balance (Shanghai Hengping Instrument Factory); HH-S4 water bath (Beijing Kewei Yongxing Instrument Co., Ltd.); KQ-3200DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).
[0052] 2 Experimental methods
[0053] 2.1 Preparation of different drug decoctions
[0054] Take the sand-scalded Strychnos nux vomica, grind it into smaller particles with a powder grinder, and pass it through a 20-mesh sieve for later use; take the cored Polygala tenuifolia, weigh it and set aside.
[0055] ① Preparation of sand-scalded Strychnos nux vomica decoction: Take 50g of sand-scalded Strychnos nux vomica crushed particles, add 400mL of double distilled water, reflux and decoct for 2h, filter with gauze, add 250mL of double distilled water to the residue and reflux and decoct for 1h, filter with gauze, and combine the filtrates. Evaporate and concentrate all the combined filtrates to 50mL. Obtain 50mL of 1g / mL sand-scalded Strychnos nux vomica decoction.
[0056] ② Preparation of decoctions of co-cooking of Strychnos nux vomica and Polygala tenuifolia (1:2.5) and Strychnos nux vomica and Polygala tenuifolia (1:5): Take 50g of Strychnos nux vomica crushed granules and 125g and 250g of Polygala tenuifolia, respectively, decoct according to the above method, and concentrate the decoctions to 50mL, respectively. 50mL of decoctions containing 1g / mL Strychnos nux vomica, 2.5g / mL and 5.0g / mL of Polygala tenuifolia are obtained.
[0057] ③ Preparation of Polygala tenuifolia decoction: Take 250 g of cored Polygala tenuifolia, decoct according to the above method, concentrate the decoction to 50 mL, and obtain 50 mL of decoction containing 5 g / mL of Polygala tenuifolia.
[0058] ④ Preparation of the mixed decoction of Strychnos nux vomica and Polygala tenuifolia (1:5): Mix 25 mL of Strychnos nux vomica and 5 times the amount of Polygala tenuifolia decoction in a volume ratio of 1:1 to obtain 50 mL of a decoction containing 0.5 g / mL Strychnos nux vomica and 2.5 g / mL Polygala tenuifolia.
[0059] Finally, 5 kinds of decoctions were obtained, cooled, divided and stored in a -20℃ refrigerator for later use. When needed, they were thawed in a 4℃ refrigerator overnight and then diluted with double distilled water to the required concentration for various experiments.
[0060] 2.2 Acute toxicity test of Strychnos nux vomica before and after different treatments of Polygala tenuifolia and determination of LD50
[0061] 2.2.1 Preliminary experiment
[0062] The mice were divided into four groups: the sand-scalded Strychnos nux vomica (abbreviated as Strychnos nux vomica) decoction group, the Strychnos nux vomica and Polygala tenuifolia decoction group (1:2.5), the Strychnos nux vomica and Polygala tenuifolia decoction group (1:5), and the Strychnos nux vomica mixed group (1:5). Through the acute toxicity test pre-experiment, the maximum total mortality dose (Dn) and the minimum total mortality dose (Dm) of each group were obtained as the upper and lower limits of each group dose in the acute toxicity formal experiment, and then according to the formula: Determine the dose ratio between groups and the dosage of each group.
[0063] 2.2.2 Formal Experiment
[0064] Male Kunming mice were divided into groups according to the method under "2.2.1". The drug solution was prepared in advance and diluted to the corresponding concentration. The formula: drug concentration = drug dose / dose volume was used for calculation. The drug volume of the mixed gavage group was 20mL / kg, and that of the other groups was 10mL / kg. After a single gavage of each group of animals, the symptoms of poisoning of the mice were observed, the number of deaths of each group of animals within 7 days was recorded, and the mortality rate was calculated. The LD of each group was calculated using the modified Koch method. 50 Value. The formula is as follows:
[0065] lg(LD 50 )=Xm-i(∑p-0.5)
[0066] Note: Xm: the logarithm of the maximum dose; i: the difference between the logarithms of adjacent doses; p: the mortality rate of each dose group (expressed as 0 to 1).
[0067] 2.3 Effects of different treatments of Polygala tenuifolia on the analgesic effect of Nux vomica using acetic acid writhing method
[0068] 42 male Kunming mice were randomly divided into a control group (administered with normal saline), a sand-scalded Strychnos nux vomica (50 mg / kg), a sand-boiled Polygala tenuifolia (1:2.5, 50 mg / kg), a sand-boiled Polygala tenuifolia (1:5, 50 mg / kg), a sand-boiled Polygala tenuifolia mixed (1:5, 50 mg / kg), and an aspirin group (20 mg / kg), with 7 mice in each group. The mice were given oral administration at 10 mL / kg for 3 consecutive days. After the last administration on the 3rd day, the acetic acid writhing test was performed to observe the analgesic effect of different groups. The room temperature should be kept at about 20°C for the experiment. 0.5% acetic acid aqueous solution was injected intraperitoneally at 0.2 mL / mouse. Generally, the writhing reaction began to appear at 2 minutes, and the frequency was the highest at 15 minutes. Therefore, the number of mouse writhing within 15 minutes after administration was observed and recorded, and the analgesic rate of each group was calculated.
[0069] Analgesia rate = (number of writhings of mice in the control group - number of writhings of mice in the drug-treated group) / number of writhings of mice in the control group × 100%.
[0070] 2.4 Effects of different treatments of Polygala tenuifolia on the pain latency of Nux vomica by hot plate method
[0071] Fifty female Kunming mice were screened for basal pain thresholds. Mice with a latency of less than 5s or greater than 30s were eliminated, and the remaining mice were grouped and dosed according to the method under "2.3" for 3 consecutive days. After the last dose, the mice were placed in a beaker on a 55±0.5℃ constant temperature water bath, and the time (s) when the mice first licked their hind paws was observed and recorded as the pain latency. The cut-off observation time was set to 60s, and those greater than 60s were counted as 60s. Before drug administration, the basal pain threshold of each group was observed and recorded, and the average value was taken for 2 observations, with an interval of 5min between the 2 observations. After the last dose, the pain latency (s) of each group of mice was observed and recorded at 30, 60, 90, and 120min after drug administration, retaining two decimal places.
[0072] 2.5 Xylene experiment to determine the effect of different treatments of Polygala on the anti-inflammatory effect of Nux vomica
[0073] 42 male Kunming mice were grouped and dosed according to the method under "2.3" for 3 consecutive days. After the last dose, the mice were lightly anesthetized with ether, and xylene was evenly applied to the surface of the right ear of the mice, 20μL / mouse, and the left ear was not treated. After 3 hours, the mice in each group were killed, and the left and right ear tissues of the mice in fixed positions were punched with a 6mm puncher, and the weight of the left and right ears (mg) was measured, retaining two decimal places, and the swelling of the mouse ears was calculated.
[0074] Mouse ear swelling (mg) = inflamed ear mass (mg) - non-inflamed ear mass (mg)
[0075] Inflammation inhibition rate = (ear swelling degree of control group - ear swelling degree of drug administration group) / ear swelling degree of control group × 100%
[0076] 2.6 HPLC determination of the effects of different treatments of Polygala tenuifolia on the contents of strychnine and brucine in Strychnos nux vomica
[0077] 2.6.1 Chromatographic conditions
[0078] Chromatographic column: 99603 Diamonsil C18 reverse phase column (250 mm × 4.6 mm, 5 μm); mobile phase: 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B); gradient elution: 0-5 min, 5%-10% B; 5-25 min, 10%-15% B; 25-35 min, 15%-35% B; 35-45 min, 35%-55% B; 45-55 min, 55%-75% B; 55-57 min, 75% B. Analysis time: 57 min; volume flow rate: 1 mL / min; column temperature: 30°C; injection volume: 5 μL; detection wavelength: 260 nm.
[0079] 2.6.2 Preparation of reference substances, test substances and negative samples
[0080] Preparation of reference substance: Take 6 mg of strychnine and 5 mg of strychnine reference substance, weigh them accurately, place them in 10 ml volumetric flasks, add appropriate amount of methanol to dissolve them and dilute to the mark, shake well. Accurately measure 1 mL of each, place them in the same volumetric flask, dilute to the mark with methanol, shake well. (Each 1 mL contains 0.06 mg of strychnine and 0.05 mg of strychnine). Filter with a 0.22 μm microporous filter membrane and set aside.
[0081] Preparation of test samples: Take 1 mL of each drug suspension and place them in 1.5 mL EP tubes. Centrifuge at 4°C and 12000 r / min for 30 min to separate the supernatant and precipitate. The supernatant is directly filtered through a 0.22 μm microporous membrane for later use. Add methanol to each group of precipitates to 1 mL, sonicate for 40 min, make up the lost volume of methanol, filter through a 0.22 μm microporous membrane for later use.
[0082] Preparation of negative samples: Do not add the test sample, only add 1 mL of methanol and filter through a 0.22 μm microporous membrane to prepare a negative sample for use.
[0083] 2.6.3 Drawing of standard curve
[0084] Dilute the reference substance by the doubling dilution method, draw a standard curve with concentration as the x-axis and peak area as the y-axis, and then substitute the peak area of the test substance into the formula to calculate its concentration.
[0085] 2.6.4 Sample determination
[0086] Take the reference substance, test substance and negative sample under "2.6.2" and determine the content of strychnine and brucine in the sample according to the method under "2.6.1". Component percentage = amount of component in 100mg original medicinal material / 100mg×100%.
[0087] 2.7 Data Processing
[0088] The experimental results all obey the normal distribution. It indicates that SPSS20.0 statistical software was used for one-way analysis of variance, and P<0.05 was considered to be statistically significant.
[0089] 3 Experimental results
[0090] 3.1 Acute toxicity of different treatments of Polygala tenuifolia on Nux vomica and LD 50 Determination of
[0091] The experimental results are shown in Table 1. In the acute toxicity test of Strychnos nux vomica treated with different Polygala tenuifolia, the LD 50The value was 302 mg / kg, the lowest value, showing strong toxicity. Different treatment methods of Polygala tenuifolia can significantly increase the LD of sand-scalded Strychnos nux vomica 50 Among them, the LD value of the decoction obtained by decocting 2.5 times and 5 times the amount of Polygala tenuifolia and sand-scalded Strychnos nux vomica was 50 The values increased by 2.0 times and 4.8 times, respectively, indicating that Polygala tenuifolia has a significant effect on reducing the acute toxicity of scalded Strychnos nux vomica, and this detoxification effect increases with the increase in the dosage of Polygala tenuifolia, which also confirms the important role of Polygala tenuifolia in the detoxification process. 50 The value was 5.9 times higher than that of the single decoction of scalded Strychnos nux vomica.
[0092] Table 1 The results of acute toxicity test on Strychnos nux vomica with different treatments of Polygala tenuifolia and the corresponding LD 50 Value (n=10)
[0093]
[0094]
[0095] 3.2 Effects of different treatments of Polygala tenuifolia on the analgesic effect of Nux vomica using acetic acid writhing method
[0096] To further investigate the effects of different methods of treating Strychnos nux vomica with Polygala tenuifolia on the analgesic effect of Strychnos nux vomica, we conducted an acetic acid writhing experiment in mice.
[0097] The experimental results are shown in Table 3. After intraperitoneal injection of acetic acid aqueous solution, the average number of body twisting of mice in the control group was as high as 40 times within 15 minutes, which was manifested as: the mouse abdomen was sunken, the trunk and hind limbs were stretched, and the buttocks were raised. Compared with the control group, the sand-scalded Strychnos nux vomica decoction alone and the Strychnos nux vomica treated with different methods of Polygala tenuifolia could significantly reduce the number of body twisting of mice in pain (P<0.01); the number of body twisting of Polygala tenuifolia and Strychnos nux vomica decoction and mixed gavage was more significantly reduced (P<0.01). The analgesic rate of sand-scalded Strychnos nux vomica was about 40%, while the analgesic rate of 5 times the amount of Polygala tenuifolia decoction reached 80%, and it showed a dose-dependence on the amount of Polygala tenuifolia, that is, with the increase of the amount of Polygala tenuifolia, its analgesic effect was further enhanced, and the analgesic rate of 5 times the amount of decoction and mixed gavage was equivalent to that of the positive drug aspirin.
[0098] Table 2 Effects of different methods of polygala treatment and sand-scalding of Strychnos nux vomica on the number of acetic acid writhing in mice (n=7, )
[0099]
[0100] Note: Compared with the control group, *P<0.05, **P<0.01. Same as the following table.
[0101] 3.3 Effects of different treatments of Polygala tenuifolia on the pain latency of Nux vomica by hot plate method
[0102] This study further applied the mouse hot plate method to investigate the effects of different methods of polygala treatment on the pain latency of strychnine.
[0103] The experimental results are shown in Table 4. Figure 1 There was no significant difference in the basic pain threshold of mice in each group, and the data of each group were comparable. Compared with the control group at the same time, sand-scalded Strychnos nux vomica significantly increased the pain latency of the hot plate method at 90 minutes after administration (P<0.05), and the increase was more significant at 120 minutes (P<0.01); while co-cooked low and high doses and mixed oral administration could significantly increase the pain latency of mice from 60 minutes after administration (P<0.05 or P<0.01). Figure 1 It can be seen that the improvement of co-cooking with high dose is more significant. Positive drug aspirin can significantly increase the pain latency of mice from 30 minutes after administration (P<0.05).
[0104] Table 3 Effects of different methods of polygala treatment on the hot plate test in mice (n=7, )
[0105]
[0106] 3.4 Xylene experiment to determine the effect of different treatments of Polygala on the anti-inflammatory effect of Nux vomica
[0107] The xylene-induced ear swelling method was used to investigate the effects of different methods of polygala treatment and sand-scalded strychnos nux vomica on its anti-inflammatory effect.
[0108] The results showed that compared with the control group, the methods of using sand-scalded Strychnos nux vomica decoction alone and treating the sand-scalded Strychnos nux vomica with different methods had a significant effect on inhibiting mouse ear swelling (P<0.05), among which the low and high doses of the co-decoction group had extremely significant differences compared with the control group (P<0.01); the anti-inflammatory effect of the sand-scalded Strychnos nux vomica decoction alone was slightly worse than that of the positive drug aspirin group, while the anti-inflammatory effect of the mixed high-dose group was equivalent to that of aspirin, and the anti-inflammatory effect of the low and high doses of sand-scalded Strychnos nux vomica co-decoction was better than that of aspirin, and was significantly enhanced with the increase of the dosage of Polygala tenuifolia. The results are shown in Table 5.
[0109] Table 5 Effects of different methods of polygala treatment and sand-scalding of Strychnos nux vomica on mouse ear swelling experiment (n=7, )
[0110]
[0111]
[0112] 3.5 HPLC determination of the effects of different methods of polygala treatment on the contents of strychnine and brucine in strychnine. The contents of strychnine and brucine in the supernatant and precipitate of decoctions of different groups were determined by HPLC. The experimental results are shown in Table 5.
[0113] As shown in Table 5, the contents of strychnine and brucine in the single decoction of scalded Strychnos nux vomica were 0.71% and 0.88% respectively, and they were mainly concentrated in the supernatant, with less content in the precipitate; while low and high doses of Polygala tenuifolia and scalded Strychnos nux vomica decoction could reduce the contents of strychnine and brucine in the decoction, and with the increase of the dosage of Polygala tenuifolia, strychnine and brucine tended to accumulate in the precipitate. However, the amount of the two components in the precipitate of the mixed high dose did not increase, and the total amount of the two components was basically the same as that of the single decoction of scalded Strychnos nux vomica.
[0114] Table 5 Contents of strychnine and brucine in the decoction of Strychnos nux vomica and the decoction of Strychnos nux vomica treated with different methods (%)
[0115]
[0116] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to 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 claimed by the present invention.
Claims
1. A method for combining Polygala tenuifolia with Strychnos nux vomica, characterized in that: The steps include: S1, taking sand-scalded Strychnos nux vomica, crushing it through a 20-mesh sieve to obtain sand-scalded Strychnos nux vomica particles, and setting aside; S2, weigh appropriate amounts of cored Polygala and blanched Strychnos nux vomica granules respectively, mix and decoct twice, add double distilled water in an amount of 6-10 times the total mass of the medicinal materials for the first time, decoct for 1-3 hours, and filter; add double distilled water in an amount of 4-6 times the total mass of the medicinal materials for the second time to the residue, decoct for 0.5-1.5 hours, and filter; combine the filtrates and concentrate them to a decoction containing 1 g / mL blanched Strychnos nux vomica and 5 g / mL Polygala tenuifolia.
2. The method of combining Polygala tenuifolia with Strychnos nux vomica according to claim 1, characterized in that: The step S2 specifically comprises: weighing appropriate amounts of cored Polygala and blanched Strychnos nux vomica granules respectively, mixing and decocting twice, adding double distilled water in an amount of 8 times the total mass of the medicinal materials for the first time, decocting for 2 hours, and filtering; adding double distilled water in an amount of 5 times the total mass of the medicinal materials to the residue for the second time, decocting for 1 hour, and filtering; combining the filtrates and concentrating to a water decoction containing 1 g / mL blanched Strychnos nux vomica and 5 g / mL Polygala.
3. The method of combining Polygala tenuifolia with Strychnos nux vomica according to claim 1, characterized in that: The step S2 can also be: weigh an appropriate amount of cored Polygala, decoct twice, add double distilled water in an amount of 6-10 times the weight of the medicinal material for the first time, decoct for 1-3 hours, and filter; add double distilled water in an amount of 4-6 times the weight of the medicinal material to the residue for the second time, decoct for 0.5-1.5 hours, and filter; combine the filtrates and concentrate to a decoction containing 5 g / mL of Polygala; take the scalded Strychnos nux vomica particles, use the same decoction method as mentioned above, decoct twice, filter, combine the filtrates and concentrate to a decoction containing 1 g / mL of Strychnos nux vomica; then mix the scalded Strychnos nux vomica decoction and the Polygala tenuifolia decoction in equal volumes.
4. The method of combining Polygala tenuifolia with Strychnos nux vomica according to claim 3, characterized in that: The step S2 can also be: weigh an appropriate amount of cored Polygala, decoct twice, add double distilled water in an amount of 8 times the weight of the medicinal material for the first time, decoct for 2 hours, and filter; add double distilled water in an amount of 5 times the weight of the medicinal material to the residue for the second time, decoct for 1 hour, and filter; combine the filtrates and concentrate to a decoction containing 5 g / mL of Polygala; take the scalded Strychnos nux vomica particles, use the same decoction conditions as above, decoct and filter, combine the filtrates and concentrate to a decoction containing 1 g / mL of Strychnos nux vomica; then mix the Strychnos nux vomica decoction and the Polygala tenuifolia decoction in equal volumes.
5. The method for combining Polygala tenuifolia with Strychnos nux vomica according to any one of claims 1 to 4, characterized in that: The mass ratio of the cored Polygala tenuifolia and the blanched Strychnos nux vomica particles is: cored Polygala tenuifolia: blanched Strychnos nux vomica = 1:2.5-5.
6. The method of combining Polygala tenuifolia with Strychnos nux vomica according to claim 5, characterized in that: The mass ratio of the cored Polygala tenuifolia to the blanched Strychnos nux vomica is: cored Polygala tenuifolia: blanched Strychnos nux vomica = 1:
5.
7. Use of the decoction obtained by the method of combining Polygala tenuifolia with Strychnos nux vomica as claimed in any one of claims 1 to 4 in medicines for reducing the toxicity of Strychnos nux vomica and enhancing the anti-inflammatory and analgesic effects of Strychnos nux vomica.
8. Use of a combination of Polygala tenuifolia and Strychnos nux vomica in a medicine for reducing the toxicity of Strychnos nux vomica and enhancing the anti-inflammatory and analgesic effects of Strychnos nux vomica.
9. The use according to claim 8, characterized in that: The dosage ratio of Polygala tenuifolia and Strychnos nux vomica is 1:2.5-5.
10. The use according to claim 9, characterized in that: The dosage ratio of Polygala tenuifolia and Strychnos nux vomica is 1:5.