Use of gastrodia and uncaria granules

Abstract

The use of Gastrodia and Uncaria granules, which is used for preparing a medicine for treating peripheral nerve injury, such as sciatic nerve injury. The present application confirms that Gastrodia and Uncaria granules can accelerate the nerve regeneration and functional recovery after peripheral nerve injury, and the effective dose of the effect is 1.5 g / kg of Gastrodia and Uncaria granules. The present application expands the medication range of Gastrodia and Uncaria granules, and provides a theoretical basis for the clinical application of Gastrodia and Uncaria granules in treating peripheral nerve injury.

Description

Technical Field

[0001] This invention relates to new uses of traditional Chinese medicine products, and particularly to the use of Gastrodia elata and Uncaria rhynchophylla granules. Background Technology

[0002] Peripheral nerve injury (PNI) is a clinical condition caused by accidental direct or indirect trauma to the trunk or branches of peripheral nerves, resulting in dysfunction of sensory neurons, motor neurons, and autonomic nerves in the trunk and limbs. Due to incomplete and slow recovery of nerve function and target organs after peripheral nerve injury, patients experience severe disruptions to their daily lives. Therefore, finding suitable drug treatments to accelerate recovery after peripheral nerve injury remains of great importance.

[0003] Gastrodia and Uncaria granules are derived from the Gastrodia and Uncaria decoction in "New Interpretations of the Treatment of Miscellaneous Internal Diseases in Traditional Chinese Medicine" compiled by the renowned Chinese medicine expert Hu Guangci. It is used to treat hypertension, Parkinson's disease, Alzheimer's disease, Meniere's syndrome, epilepsy, and other neurological diseases, and has shown significant efficacy in treating dizziness. Studies have found that gastrodin in Gastrodia can promote peripheral nerve regeneration in rats by regulating the P13K / AKT / mTOR pathway and modulating the miR-497 / BDNF axis, demonstrating a good therapeutic effect on peripheral nerve regeneration. Peripheral nerve injury leads to atrophy of the muscles it innervates; Uncaria has the effect of alleviating muscle atrophy and can be used for cardiovascular and central nervous system diseases, exhibiting neuroprotective effects. Uncaria alkaloids in Uncaria can regulate the P13K-AKT pathway, which has been found to be related to peripheral nerve growth. In this compound preparation, Gastrodia and Uncaria are the principal herbs, while the other nine herbs act as adjuvant, assistant, and guiding herbs, enhancing the effect of the principal herb. In summary, we hypothesize that Gastrodia and Uncaria granules may also promote peripheral nerve growth, and their effect may be stronger than that of Gastrodia or Uncaria alone. Furthermore, Gastrodia and Uncaria granules are already marketed drugs with a good safety profile. Therefore, we used a rat sciatic nerve transection and anastomosis model to observe the regenerative and repair effects of Gastrodia and Uncaria granules on peripheral nerve injury, providing experimental evidence for their use in the treatment of peripheral nerve injury. Currently, there are no reports of Gastrodia and Uncaria granules being used for peripheral nerve injury. Summary of the Invention

[0004] The present invention aims to solve the technical problem of regeneration and repair of peripheral nerves after injury by Gastrodia elata and Uncaria rhynchophylla granules.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] The purpose of Gastrodia elata and Uncaria rhynchophylla granules is to treat peripheral nerve injury, specifically sciatic nerve injury.

[0007] The effective dose of the Gastrodia elata and Uncaria rhynchophylla granules is 1.5 g / kg.

[0008] The beneficial effects of adopting the above technical solution are:

[0009] 1. This invention clarifies that Gastrodia elata and Uncaria rhynchophylla granules can accelerate nerve regeneration and functional recovery after peripheral nerve injury, and the effective dose for this effect is 1.5 g / kg of Gastrodia elata and Uncaria rhynchophylla granules.

[0010] 2. This invention expands the scope of application of Gastrodia elata and Uncaria rhynchophylla granules, providing a theoretical basis for their clinical application in the treatment of peripheral nerve injury. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the paw reflex in rats with sciatic nerve anastomosis treated with Gastrodia elata and Uncaria rhynchophylla granules on the 4th week after surgery, as shown in Example 1.

[0012] Figure 2 This is a schematic diagram of the paw reflex in rats undergoing sciatic nerve anastomosis after surgery using Gastrodia elata and Uncaria rhynchophylla granules at week 6 post-surgery.

[0013] Figure 3 This is a schematic diagram of the paw reflex in rats undergoing sciatic nerve anastomosis after surgery using Gastrodia elata and Uncaria rhynchophylla granules at week 8.

[0014] Figure 4 This is a statistical chart showing the claw reflex scores of rats with sciatic nerve anastomosis treated with Gastrodia elata and Uncaria rhynchophylla granules from week 4 to week 8 after administration, as described in Example 1.

[0015] Figure 5 The image shows the results of the analysis of the effect of Gastrodia elata and Uncaria rhynchophylla granules on the toes of rats with sciatic nerve anastomosis and the sciatic nerve function index after eight weeks of administration in Example 1.

[0016] Figure 6 This is a morphological image of the pathological changes in the gastrocnemius muscle after sciatic nerve transection and anastomosis observed with HE staining in Example 1.

[0017] Figure 7 This is a morphological diagram showing the pathological changes of rat spinal cord anterior horn motor neurons observed by HE staining in Example 2.

[0018] Figure 8 This is a morphological diagram showing the pathological changes of rat spinal cord anterior horn motor neurons observed by Nissl staining in Example 2.

[0019] Figure 9 This is a morphological image showing the pathological changes of the distal sciatic nerve after injury, as observed by Masson staining in Example 2.

[0020] Figure 10 This is a diagram showing the results of Masson staining in Example 2 to count the myelinated nerve fibers distal to the sciatic nerve after injury. Detailed Implementation

[0021] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0022] Example 1: Observation of the effect of Gastrodia elata and Uncaria rhynchophylla granules on sciatic nerve function in rats with sciatic nerve transection and anastomosis model.

[0023] 1.1. Establishing a rat model of sciatic nerve transection and anastomosis

[0024] SPF-grade male SD rats of appropriate age and weight (200-220g) were selected, ensuring they were in good health. Forty rats were randomly divided into five groups: a control group, a model group, a low-dose Gastrodia and Uncaria granules group, a high-dose Gastrodia and Uncaria granules group, and a mecobalamin group. Anesthetics and other necessary medications, as well as surgical instruments, were prepared and disinfected with 75% alcohol. Rats were anesthetized by intraperitoneal injection of 2% sodium amobarbital at 0.25ml / 100g, and the anesthesia was allowed to fully develop. Anesthetized rats were fixed in a supine position on a wooden board. The surgical area was disinfected by wiping the skin on the medial side of the rat's left hind limb with povidone-iodine. A longitudinal skin incision of about 3 cm was made. The sciatic nerve was gradually exposed by sharp dissection of the vastus medialis intermuscular space. The field of vision was opened with a retractor. The sciatic nerve was completely severed about 5 mm from the lower edge of the piriformis muscle. The severed ends of the nerve epineurium were immediately sutured with 10-0 microsutures. After ensuring that the nerve was sutured, the skin incision was closed with appropriate sutures and the surgical area was disinfected again with povidone-iodine. A sciatic nerve transection and anastomosis model was established. This model is suitable for studying muscle atrophy and functional recovery after absence trauma.

[0025] 1.2. Observation after gavage

[0026] Gastrodia and Uncaria granules were purchased from Chengdu Jiuzhitang Jinding Pharmaceutical Co., Ltd. (National Drug Approval Number 251021084). Gavage administration began on the second day after surgery. The following groups were administered the medication once daily at a dose of 1 ml / 100 g: blank group (distilled water), model group (distilled water), high-dose TGg group (3.0 g / kg), low-dose TGg group (1.5 g / kg), and mecobalamin group (0.3 mg / kg). The physiological status of the rats was observed and recorded. The paw extension reflex was photographed weekly, and the modified Tarlov score was recorded.

[0027] 1.3. Calculation of the sciatic nerve functional index

[0028] During weeks 4, 6, and 8 of gavage, a sheet of white paper was laid flat at the bottom of a cardboard box measuring 60cm long, 15cm wide, and 10cm high. The rats' hind limbs were dipped in sufficient blue ink and placed at the entrance of the box, allowing them to walk freely from the entrance to the exit. The white paper was removed, and clear, complete footprints were selected from the paper for measurement. The results were then calculated using the Bain formula.

[0029] SFI=-38.3(EPL-NPL) / NPL+109.5(ETS-NTS) / NTS+13.3(EIT- NIT) / NIT- 8.8.

[0030] E: Injured side, N: Normal side, PL: Distance between the heel and the third toe (footprint length), TS: Distance between the first and fifth toes, IT: Distance between the second and fourth toes.

[0031] Footprint measurements were taken on both the normal and injured sides to measure toe spread (NTS; experimental toe spread (ETS), print length (NPL; experimental print length (NPL), and intermediary toe spread (NIT; experimental intermediary toe spread (EIT)). A SFI of 0 indicates normal sciatic nerve function, while an SFI of -100 indicates complete loss of sciatic nerve function. This method is accurate, reliable, and non-invasive, making it a commonly used behavioral method for assessing sciatic nerve function in experiments.

[0032] 1.4. HE staining of gastrocnemius muscle

[0033] Three rats were taken from each group. After the rats were completely anesthetized with 2% sodium amobarbital, they were perfused with PBS until the rat liver turned completely white. Then, they were perfused with 4% paraformaldehyde until the rats were rigid and the gastrocnemius muscle was taken. The gastrocnemius muscle was fixed with 4% paraformaldehyde, embedded in paraffin, and then stained with HE. The specific steps of HE staining are as follows: (1) Xylene dewaxing: Xylene I (5-10 min) → Xylene II (5-10 min). (2) Ethanol rehydration: Anhydrous ethanol I (5 min) → Anhydrous ethanol II (5 min) → 95% ethanol (3-5 min) → 80% ethanol (3 min). (3) Hematoxylin staining: Distilled water (1 min) → Hematoxylin staining solution (5-10 min) → Rinse slightly with running water to remove hematoxylin (2-3 s) → Hydrochloric acid ethanol differentiation (3-4 s). (4) Eosin staining: Eosin (eosin) staining solution (2-3 min) → Rinse slightly with distilled water (1-2 s). (5) Ethanol dehydration: 80% ethanol (2s) → 95% ethanol (1-2s) → 95% ethanol (2-5s) → anhydrous ethanol (5-10s) → anhydrous ethanol (10-30s) (6) Xylene clearing → neutral resin sealing. (7) Observation.

[0034] During the gavage, the skin wounds of rats in all groups healed well and there was no infection. However, except for the control group, all rats in all groups gnawed on their toes, resulting in bleeding and even toe loss. The rats with toe injuries were wiped with iodine to prevent the infection from worsening.

[0035] Compared with the model group, the rats in the low-dose TGg group (1.5 g / kg) and the methylcobalamin group showed significant recovery in their paw digits, such as... Figure 1 , 2 As shown in Figure 3, the modified Tarlov score is significantly improved, as... Figure 4 As shown, SFI increases significantly, as Figure 5 As shown, in HE staining, the gastrocnemius muscle fibers in the low-dose TGg group (1.5 g / kg) and the mecobalamin group appeared softer, fuller, and more orderly arranged than those in the model group, with increased area and neat arrangement. Figure 6 As shown, this indicates that a low dose (1.5 g / kg) of Gastrodia elata and Uncaria rhynchophylla granules can promote functional recovery after sciatic nerve injury. * P<0.01, # P<0.05).

[0036] Example 2: Observation of the effect of Gastrodia elata and Uncaria rhynchophylla granules on nerve regeneration in rats with sciatic nerve anastomosis model.

[0037] 2.1. Observe the pathological changes of motor neurons in the anterior horn of the spinal cord.

[0038] The L4-L6 segment of the spine was taken, fixed in formalin, and the spinal cord was dissected and embedded in paraffin. The paraffin sections of the spinal cord were stained with Nissl and HE to observe the pathological changes of the anterior horn motor neurons. The specific steps of Nissl staining are as follows: (1) Paraffin sections were 3 μm thick, dewaxed with xylene, and rehydrated with ethanol. (2) Nissl staining solution (toluidine blue) was placed in a 60℃ incubator for 20-40 min. Rinse briefly with distilled water. (3) Rapid differentiation was achieved with 95% ethanol. (4) Dehydration was achieved with anhydrous ethanol, clearing with xylene, and mounting with neutral resin. The HE staining steps are as described above.

[0039] 2.2. Count of myelinated nerve fibers of the sciatic nerve

[0040] The distal nerve of the sciatic nerve was routinely fixed and embedded, and paraffin sections were prepared. Masson staining was performed to observe the pathological changes of the distal sciatic nerve. At the same time, myelinated nerves of the distal nerve were counted by microscopy to evaluate the speed of nerve regeneration. The specific steps of Masson staining are as follows: (1) The sections were 3 μm thick, dewaxed with xylene, and rehydrated with ethanol. (2) The sections were immersed in mordant solution (covered) and mordanted overnight at room temperature or in a 60°C incubator for 1 hour. Then, the sections were rinsed with running water until the yellow color disappeared. (3) Azurite blue staining solution was applied for 2-3 minutes, and excess staining solution was washed away with water. (4) Mayer hematoxylin staining solution was applied for 2-3 minutes, and excess staining solution was washed away with water. (5) The sections were differentiated with acidic ethanol differentiation solution for a few seconds to remove the over-stained parts and make the staining clearer. Then, the sections were rinsed with running water for 10 minutes to ensure that the differentiation solution and excess dye were thoroughly washed away. (6) Stain with Ponceau S and Fuchsia solution for 10 min, then wash twice with distilled water for 10-15 s each time. (7) Treat with phosphomolybdic acid solution for about 10 min. (8) Pour off the supernatant, do not wash the section with water, and directly add aniline blue staining solution for 5 min. (9) After washing away the aniline blue solution with a weak acid solution, continue to add weak acid working solution to cover the section for 2 min. (10) Dehydrate with 95% ethanol for 30 s → dehydrate with anhydrous ethanol for 30 s → dehydrate with anhydrous ethanol for 1 min. (11) Clear with xylene. Mount with neutral resin.

[0041] like Figure 7 , 8As shown, compared with the blank group, neurons in the model group exhibited significant cytoplasmic shrinkage and nucleolar dissolution, along with a significant reduction in the number of Nissl bodies, an increase in intraneuronal vacuoles, and irregular and disordered morphological arrangement of cell repair. These changes indicate that the structure and function of neurons in the model group may have been impaired. However, in the low-dose group of Gastrodia elata and Uncaria rhynchophylla granules, the high-dose group of Gastrodia elata and Uncaria rhynchophylla granules, and the mecobalamin group, we observed significant improvement in these pathological changes. In these treatment groups, intact neurons could be clearly seen, with uniform staining, centered nucleoli, an increased number of Nissl bodies, and a significant reduction in intraneuronal vacuoles compared to the model group. * P<0.01, # P<0.05).

[0042] like Figure 9 , 10 As shown, in the blank group, nerve fibers were evenly and densely arranged with clear morphology. However, in the model group, the situation changed significantly, with obvious collagen fiber proliferation and fewer intact nerve fibers. In contrast, in the Gastrodia elata and Uncaria rhynchophylla granule dosage groups and the mecobalamin group, multiple dense and intact nerve fibers were observed. We performed a statistical analysis on the number of myelinated nerve fibers in each group. Compared with the blank group, the number of myelinated nerve fibers in the model group was significantly reduced, and the difference was statistically significant. In the Gastrodia elata and Uncaria rhynchophylla granule dosage groups and the mecobalamin group, the number of myelinated nerve fibers increased significantly, and this change was also statistically significant. * P<0.01, # P<0.05). This indicates that Gastrodia elata and Uncaria rhynchophylla granules can accelerate the regeneration of nerves after sciatic nerve injury, thereby promoting the recovery of nerve function.

Claims

1. The uses of Gastrodia elata and Uncaria rhynchophylla granules, characterized in that: The intended use is for preparing a drug for treating peripheral nerve injury, specifically sciatic nerve injury.

2. The use of the Gastrodia elata and Uncaria rhynchophylla granules according to claim 1, characterized in that: The effective dose of the Gastrodia elata and Uncaria rhynchophylla granules is 1.5 g / kg.

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