New application of iso-kaurenoic acid

Abstract

The invention discloses an application of isokaurenoic acid in preparation of a medicine for treating and / or preventing pain. The invention relates to an application of isokaurenoic acid in preparation of a medicine for treating and / or preventing pain. The pain comprises but not limited to inflammatory pain, neuropathic pain, cancer pain, postoperative pain and migraine. According to the present invention, the iso-kaurenoic acid is prepared into an oral drug, a mucosal drug or a dermatological drug for preventing and / or treating pain.

Description

Technical Field

[0001] The invention belongs to the medical use of kaurenoic acid and relates to the use of kaurenoic acid in preparing a medicine for treating and / or preventing pain. Background Art

[0002] Kaurenoic acid is a diterpenoid compound found in plants such as Acanthopanax cortex and Leucoderma odoratum. The content of Kaurenoic acid in Acanthopanax cortex is about 1%. It has pharmacological activities such as antibacterial, vascular smooth muscle relaxation, anti-HIV, antispasmodic, anti-platelet aggregation, anti-tumor, antihypertensive, diuretic, osteoporosis prevention, and anti-inflammatory. There is no report on its effect on pain treatment.

[0003] In 2020, the International Association for the Study of Pain (IASP) made the latest revision to the definition of pain, calling it "an unpleasant sensory and emotional experience, usually caused by, or resembling actual or potential tissue damage." Although pain is an important part of the body's self-protection alert mechanism, it still brings both physical and mental torture to patients. Protracted pain not only seriously affects the patient's quality of life and greatly reduces their confidence in treatment, but also brings a heavy burden to the patient's family and even the entire society. Currently, commonly used pain treatment drugs in clinical practice are mainly divided into two categories: non-steroidal anti-inflammatory drugs (NSAIDs) for mild to moderate pain and opioids for moderate to severe pain. Therefore, the discovery of new analgesics with low toxicity and side effects in natural products has great research significance and social value. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide the use of kaurenoic acid in the preparation of a drug for treating and / or preventing pain. The kaurenoic acid (KA) has the following structure:

[0005]

[0006] The present invention relates to the use of kaurenoic acid in preparing a medicine for treating and / or preventing pain, wherein the pain includes but is not limited to inflammatory pain, neuropathic pain, cancer pain, postoperative pain, and migraine, and the medicine has the characteristics of being safe to use and being usable for a long time when used for the above purposes. Therefore, the use of kaurenoic acid in preparing a medicine for treating and / or preventing pain has the use of treating various pains.

[0007] Kaurenoic acid can be commercially available or prepared using existing technology.

[0008] The iso-kaurenoic acid used in the present invention can be prepared into a medicine with any pharmaceutically acceptable auxiliary material or pharmaceutical excipient, and its preparation can be any pharmaceutically acceptable dosage form, and the pharmaceutically acceptable carrier is selected from one or more of hydroxypropyl methylcellulose, sorbitol, glycerol, citric acid, yellow vaseline, liquid paraffin, stearic acid, magnesium stearate, talc, and polyethylene glycol.

[0009] The drug provided by the present invention mainly includes oral administration, mucosal administration or skin administration.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: pain is a survival mechanism and a warning signal of ongoing or impending tissue damage. In some cases, even if the wound heals, the pain will continue and become chronic. Kaurenoic acid can inhibit the expression of pro-inflammatory factors and promote the expression of anti-inflammatory factors to play a role in combating osteoarthritis, and at the same time has a significant analgesic effect; oral mucosal administration of kaurenoic acid can significantly reduce the number of mouse twists caused by acetic acid, which is equivalent to the effect of aspirin, showing good acute pain treatment activity; kaurenoic acid can significantly reduce the total time of phase I central pain and phase II inflammatory pain response in formalin-induced pain mice; topical application of kaurenoic acid ointment can significantly reduce mechanical pain and thermal pain in rat incision pain models; kaurenoic acid can significantly relieve carrageenan-induced chronic inflammatory foot pain in rats and produce a long-term low pain threshold state. It can be seen from this that kaurenoic acid can be used in the treatment and / or prevention of pain, characterized in that the pain includes but is not limited to inflammatory pain, neuropathic pain, cancer pain, postoperative pain, and migraine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention will be further described below in conjunction with specific embodiments. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental animals of the present invention were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd. [Animal Production License No.: SYXK (Su) 2023-0019], with a room temperature of 20-25°C, a relative humidity of 5%-65%, and a 12-h light / dark alternating culture. The animals were fed with free food and water for one week before the experiment.

[0012] Figure 1 Effects of kaurenoic acid on pain threshold in OA rats n=6)

[0013] Figure 2 Effects of kaurenoic acid on the levels of IL-1β, IL-6 and TNF-α in synovial fluid and serum of OA rats n=6)

[0014] Figure 3 Study on the analgesic effect of kaurenic acid, syringic acid and euphorbic acid ( n=10)

[0015] Figure 4 Effects of kaurenic acid, syringic acid and euphorbic acid on formalin-induced pain in mice n=10)

[0016] Figure 5 Study on the activity of kaurenic acid, syringic acid and eucomycin against incisional pain ( n=12)

[0017] Figure 6 Effects of kaurenoic acid, syringic acid and eugenol on carrageenan-induced chronic inflammatory foot pain in rats ( n=12) DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0020] Example 1 Effect of Kaurenoic Acid on the Activity of Osteoarthritis Model Rats

[0021] Animal model construction

[0022] Experimental rats were treated with sodium pentobarbital (40-50 mg kg -1 ) Anesthesia was given by intraperitoneal injection, the skin was prepared, and a 1-2 cm incision was made in the midline of the knee joint to expose the knee joint and cut the anterior cruciate ligament. The incision was closed and postoperative management was performed to observe the joint gradually developing osteoarthritis lesions such as cartilage degeneration and synovitis.

[0023] Animal grouping and treatment

[0024] After one week of adaptive feeding, SD rats were randomly divided into control group (Control group), model group (OA group), low-dose kaurenoic acid group (KA-L group), high-dose kaurenoic acid group (KA-H group), and positive control group (diclofenac sodium group, Dic group), with 12 rats in each group. The control group was not treated, and the other groups were surgically modeled. One week after surgery, the treatment of each group was as follows: the OA group did not receive any treatment; the KA-L group was given 10 mg kg -1 Kaurenoic acid was administered orally; KA-H group was given 30 mg kg -1Kaurenoic acid was administered orally; the Dic group was given 10 mg kg -1 Diclofenac sodium was administered orally. Treatment started one week after modeling surgery, with 7 days as one course of treatment, and a total of 2 courses of treatment.

[0025] Test indicators and results PAM stress test

[0026] The mechanical pain threshold in the experimental joint hypersensitivity model in rodents can be measured using the PAM device (Ugo Basile SRL-Italy). The PAM device directly stimulates the knee joint with a quantifiable force and automatically reads the animal's response to obtain pain threshold information. When used, only a specially designed force sensor is worn on the thumb to apply stress to the rat's knee joint. When the pain threshold is reached, the animal will respond accordingly (usually retracting the limbs), and the instrument automatically records the maximum force during the stimulation as the pain threshold (gf). The acquired data are analyzed using Ugo Basile DCA (Data Collection Application) software.

[0027] The basal pain threshold of rats in each group was measured before modeling. After successful modeling, the drug was administered for 2 consecutive weeks. The pain threshold of rats was tested before, 1 h after, and 2 h after administration in the first week; the pain threshold of rats was tested before, 1 h after, and 2 h after administration in the second week.

[0028] Before administration in the first week after modeling, the pain threshold of the model group was significantly lower than that of the control group (P < 0.05), and it has been at a lower level since then, indicating that the OA model was successfully established and the rats showed obvious osteoarthritis lesions. Compared with the model group, after administration in the first week, the pain thresholds of the low- and high-dose groups of kaurenoic acid increased, and the pain thresholds 2 hours after administration were greater than the pain thresholds 1 hour after administration; after administration in the second week, the pain thresholds of the low- and high-dose groups of kaurenoic acid continued to increase, and the difference was statistically significant compared with the model group (P < 0.05), and the pain threshold of the high-dose group of kaurenoic acid was greater than that of the positive control group, and was close to the pain threshold of the control group. This shows that kaurenoic acid has the effect of improving the pain threshold of OA rats, and the improvement effect of the high-dose group of kaurenoic acid is greater than that of the positive control group, and it has a significant analgesic effect, as shown. Figure 1 .

[0029] Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of IL-1β, IL-6 and TNF-α in joint fluid and serum

[0030] Use joint cavity puncture to collect joint fluid as much as possible, and preserve the joint fluid after low-temperature centrifugation for inflammatory factor detection; use capillary glass tubes to collect blood from the inner canthus of the orbit, collect the blood into anticoagulant tubes, separate the serum, centrifuge at 4°C, and transfer the supernatant for storage. According to the instructions of the ELISA kit, the levels of IL-1β, IL-6, and TNF-α in rat joint fluid and serum were detected.

[0031] Compared with the control group, the levels of IL-1β, IL-6 and TNF-α in the joint fluid and serum of the model group were significantly increased (P<0.01); compared with the model group, the levels of IL-1β, IL-6 and TNF-α in the joint fluid and serum of the low- and high-dose kaurenoic acid groups were significantly decreased (P<0.01), and the levels of inflammatory factors in the high-dose kaurenoic acid group were close to those in the positive control group, as shown in Table 1. Figure 2 .

[0032] In summary, the pharmacological activity of kaurenoic acid is mainly anti-inflammatory activity. This study also reflects its role in intervening in osteoarthritis model rats. It can inhibit the expression of pro-inflammatory factors, promote the expression of anti-inflammatory factors, play an anti-inflammatory role, and play a role in treating osteoarthritis. In this study, it was found that kaurenoic acid has a significant analgesic effect.

[0033] Example 2 Study on the analgesic effect of kaurenoic acid and other active compounds in Acanthopanax cortex (acetic acid writhing test)

[0034] The test drugs (1000 mg of aspirin, syringic acid, kaurenic acid and eucomycin) are micronized and set aside; take hypromellose, add appropriate amount of water to soak it to make it swell; dissolve sorbitol, glycerin and citric acid in appropriate amount of water, add the swollen hypromellose solution, and stir evenly; then add the micronized test drugs, stir evenly, add water to the full amount, and stir evenly to obtain the product.

[0035] Female mice were randomly divided into 5 groups, 10 in each group. Each group of mice was treated with normal saline (control group Control), aspirin (positive control, APC), syringic acid (Syr), kaurenoic acid (KA), and acanthoic acid (AA) by oral spray. 30 minutes after administration, 0.2 mL of 0.6% acetic acid solution was injected intraperitoneally. The number of writhing reactions of mice within 15 minutes after acetic acid injection was observed and recorded. The writhing reactions included abdominal concavity, extension of trunk and hind limbs, and high buttocks. The inhibition rate was calculated according to the following formula: inhibition rate = (number of writhing reactions in the control group - number of writhing reactions in the drug administration group) / number of writhing reactions in the control group × 100%.

[0036] The results are as follows Figure 3As shown in the results, compared with the control group, aspirin, syringic acid, kaurenoic acid, and acanthoic acid can significantly reduce the number of writhing times of mice induced by acetic acid (P<0.05). At the same dose, the three samples showed good inhibitory activity (50.3%, 65.2%, 50.3%), and the inhibition rate was greater than 50%. Only the kaurenoic acid group and the aspirin group (71.6%) had no significant difference. It can be seen that kaurenoic acid has a good analgesic activity on mice induced by acetic acid writhing, which is stronger than syringic acid and acanthoic acid.

[0037] Example 3 Study on the activity of kaurenoic acid against formalin-induced pain in mice

[0038] BALB / c mice were randomly divided into blank control group, model group, syringic acid (40 mg kg -1 , Syr), isokaurenoic acid (40 mg kg -1 , KA), Acanthopanax acid (40 mg kg -1 , Acanthoic acid, AA), 10 mice in each group, intragastrically administered, for 14 consecutive days. 30 minutes after the last administration, 5% formalin solution was injected intradermally into the plantar of the mice in each modeling group, 30 μL / mouse, and the blank control group was injected with an equal volume of pure water. Immediately after the injection, the animal licking and biting reaction was observed: ① The total time of licking and biting the foot of the mice within 0-10 minutes after the formalin injection (total time of phase I pain reaction). ② The total time of licking and biting the foot of the mice within 10-60 minutes after the formalin injection (total time of phase II pain reaction).

[0039] The results are as follows Figure 4 As shown in the figure: Compared with the blank control group, the total time of phase I pain reaction and the total time of phase II pain reaction of mice in the model group were significantly prolonged, indicating that the model was successfully established. Compared with the model group, the total time of phase I and phase II pain reaction of mice in the kaurenoic acid group was significantly shortened (P<0.001); while only the total time of phase I pain reaction of mice in the syringic acid group was significantly shortened, and only the total time of phase II pain reaction of mice in the eucomycin group was significantly shortened (P<0.001).

[0040] Example 4 Study on the activity of kaurenoic acid on incision pain

[0041] Take the test drugs (10 g of syringic acid, kaurenic acid and euphorbic acid, respectively), add them into a proper amount (about 1 / 3 amount) of a mixed matrix of 850 g of heated and melted (about 70-80°C) yellow vaseline and 140 g of liquid paraffin, stir evenly until condensed, and the mixture is obtained.

[0042] SD rats were selected, all male. Rats were randomly divided into blank control group, model group, syringic acid (Syr), kaurenoic acid (KA), and acanthopanax acid (AA) according to the basic pain threshold, with 12 rats in each group. After the rats were anesthetized, an incision of about 1 cm long was made from 0.5 cm proximal to the plantar part of the foot according to the Brennan method in each modeling group. The skin was cut open, and the plantar muscle was lifted up with ophthalmic scissors and cut longitudinally. The muscle was kept intact in its origin and attachment, and hemostasis was stopped by pressing. The skin was sutured with fine thread for a total of 2 stitches to establish a rat postoperative incision pain model. After the animals woke up, the test drug ointment prepared by sterile treatment was applied to the incision and the vicinity in groups. The blank control group and the model group were given pure water once a day for 12 consecutive days. The mean pain threshold of the rats was detected on d2, d6, d10, and d12 after administration.

[0043] The results are as follows Figure 5 As shown: Compared with the blank control group, the mean pain threshold of rats in the model group was significantly reduced (P < 0.01), indicating that the model was successfully established. Compared with the model group, the mean pain threshold of rats in the kaurenoic acid group was significantly different at d2, d6, d10, and d12 (P < 0.05, P < 0.001, P < 0.01, P < 0.01); while the mean pain threshold of rats in the syringic acid group was no different from that in the model group, and the mean pain threshold of rats in the euphorbic acid group was significantly different at d2, d10, and d12 (all P < 0.05).

[0044] Example 5 Effect of Kaurenoic Acid on Chronic Inflammatory Foot Pain Induced by Carrageenan in Rats

[0045] Male SD rats were randomly divided into blank control group, model group, syringic acid (30 mg kg -1 , Syr), isokaurenoic acid (30 mg kg -1 , KA), Acanthopanax acid (30 mg kg -1 , AA), 12 in each group. After anesthesia, rats in each modeling group were injected with 100 μL of 2% carrageenan solution into the plantar of the right foot of rats in each modeling group. On d5 after the first injection, 100 μL of carrageenan solution of the same concentration was injected again at the same site. Rats in the blank control group were injected with equal volumes of saline twice to establish a rat chronic inflammatory foot pain model. Drug administration began on d3 after the second modeling, and was intragastrically administered at 10 mL / kg for 19 consecutive days. The mean pain threshold of rats was detected on d2 (basal value) after the second modeling, and on d1, d4, d7, d10, d14, d17, and d19 after drug administration.

[0046] The results are as follows Figure 6As shown in the figure: Compared with the blank control group, the mean pain threshold of rats in the model group continued to decrease significantly (P < 0.01), indicating that the model was successfully established. Compared with the model group, the mean pain threshold of rats in the kaurenoic acid group was significantly different at d1, d4, d7, d10, d14, d17, and d19 (P < 0.05, P < 0.01, P < 0.01, P < 0.01, P < 0.01, P < 0.05); while the mean pain threshold of rats in the syringic acid group and the eucomycin group was not different from that in the model group (all P < 0.05).

Claims

1. Use of kaurenoic acid in the preparation of a medicament for treating and / or preventing pain.

2. The use according to claim 1, characterized in that The pain includes, but is not limited to, inflammatory pain, neuropathic pain, cancer pain, postoperative pain, and migraine.

3. The application according to claim 1, characterized in that The dosage form of the drug is any pharmaceutically approved dosage form.

4. The use according to claim 3, characterized in that The dosage form of the drug can be prepared into a drug with any pharmaceutically acceptable excipient or pharmaceutical excipient, and the excipient or pharmaceutical excipient is selected from one or more of the pharmaceutically acceptable carrier selected from hydroxypropyl methylcellulose, sorbitol, glycerol, citric acid, yellow vaseline, liquid paraffin, stearic acid, magnesium stearate, talc, and polyethylene glycol.

5. The use according to claim 3, wherein the administration form mainly includes oral administration, mucosal administration or skin administration.

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