KR101932856B1 - Korean medicine material composition for treating Chemotherapy-induced peripheral neuropathy - Google Patents

Abstract

The present invention relates to a medicinal herb composition for treatment of anticancer drug-induced peripheral neuropathy, and more specifically, to a medicinal herb composition for treatment of anticancer drug-induced peripheral neuropathy without side effects. The present invention provides the medicinal herb composition for the treatment of the peripheral neuropathy caused by the anticancer drugs extracted by mixing ginseng, Paeonia lactiflora, Ostrea gigas and Angelica gigas. In addition, the present invention also provides the medicinal herb composition for treatment of anticancer drug-induced peripheral neuropathy, which is obtained by mixing and extracting 150 to 230 parts by weight of the Paeonia lactiflora, 180 to 250 parts by weight of the Ostrea gigas and 150 to 230 parts by weight of the Angelica gigas with 100 parts by weight of the ginseng. The present invention also provides a pharmaceutical composition comprising the medicinal composition for treatment of the anticancer drug-induced peripheral neuropathy.

Description

The present invention relates to a medicinal composition for treating peripheral neuropathy which is chemotherapy-induced peripheral neuropathy

The present invention relates to a medicinal herb composition for the treatment of cancer-induced peripheral neuropathy, and more specifically, to a medicinal herb composition capable of treating cancer-induced peripheral neuropathy without side effects.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and serious clinical problem affecting many patients receiving chemotherapy.

Peripheral neuropathy (PN) can be defined as injury, inflammation, or degeneration of peripheral nerve fibers. Symptoms of peripheral neuropathy can be divided into sensory, motor, and autonomic symptoms. Typical symptoms of sensory neuropathy are pain in the hands or feet and numbness. Pain is typically expressed as low or burning, and the numbness or loss of sensation is often compared to a sensation of wearing a thin stocking or gloves. If the motor nerve is damaged, muscle weakness and muscle atrophy can occur, and with autonomic nerves, symptoms such as constipation, orthostatic hypotension, and urinary incontinence may appear (Constance Visovsky et al., 2007).

Peripheral neuropathy can be caused by a variety of causes, including genetic, chronic diseases, environmental toxins, alcoholism, malnutrition and drugs (F. H. Hausheera et al., 2006). Cancer patients experiencing peripheral neuropathy are known to be the cause of their chemotherapy.

Among the symptoms of peripheral neuropathy due to chemotherapy, symptoms related to the senses and motor nerves include finger, toe tingling, numbness, burning, stabbing, There are shooting, pricking, and generally bilateral, and more severe. Symptoms can worsen to the point of feeling pain. These symptoms may be acute, mild or transient, and may be recovered after termination of chemotherapy, but in some cases it may be worse.

The anticancer drugs known to cause peripheral neuropathy include platinum series, taxane series, vinca alkaloids, bortezomib or thalidomide, and the type, The degree of peripheral neuropathy that follows is different (FH Hausheera et al., 2006). In particular, platinum-based anticancer drugs include cisplatin, carboplatin and oxaliplatin, which are widely used in various cancer treatments, and peripheral neuropathy is known to be one of the serious toxicities.

Cisplatin is an anticancer drug widely used for lung cancer, stomach cancer, esophageal cancer, cervical cancer, ovarian cancer, prostate cancer and bladder cancer. It is associated with serious toxicity due to its strong anticancer effect. You can do harm to the patient you are receiving. Peripheral neuropathy caused by cisplatin is usually sensory neuropathy, which occurs symmetrically with sensory disturbances and numbness spreading to the feet and hands. This symptom is progressively reduced or lost in affected limbs.

Carboplatin is clinically similar to cisplatin in peripheral neuropathy. Carboplatin is usually reported to cause lower neurotoxicity than cisplatin, but in some studies it appears that both drugs similarly cause peripheral neuropathy (F. H. Hausheera et al., 2006).

Oxaliplatin is a third-generation platinum complex, which is less renal toxic than the first-generation platinum complex, cisplatin, and has little hematological and gastrointestinal toxicity, but characteristic and chronic neurological toxic symptoms. Acute is characterized by sensory paralysis caused by cold, muscle spasms, and excitability of the peripheral nerves, affecting the face, upper limb and lower limb. Chronic is characterized by terminal sensory abnormalities that occur during the treatment cycle, which can lead to impairment in gait or fine motor ability, and can become more severe as the drug accumulates in the body.

To date, there is no method for the prevention, symptom relief, and treatment of peripheral neuropathy induced by anticancer drugs. In clinical practice, antiepileptic drugs (Gabapentin) and antidepressants (Amitriptyline) are used to alleviate symptoms when peripheral neuropathy occurs, but they are often not effectively controlled. In addition, current therapeutic agents have side effects such as dizziness and drowsiness, and there is a problem that a high dose can not be administered due to a low safety margin.

Phytolacca esculenta Van Houtte is a perennial plant belonging to the genus Phytolaccaceae. Its height is about 1 meter. Leaves are alternate, lanceolate or broad lanceolate, and petiole is short. In May ~ June, white flowers bloom between the tip ends of the branches and the fruit is purple and arranged in such a way that eight corollaes are adjacent to each other and are toxic. The leaves are edible and the roots are diuretics. It is originated from China and distributed in various parts of Korea. The root portion of the tail is used as a medicinal product for the landing (Phytolaccae Radix). Compounds isolated from landings include phenolic compounds such as gallic acid, protocatechuic acid, clorogenic acid, caffeic acid, m-hydroxybenzoic acid, coumaric acid and cinnamic acid. Saponin esculentosides A, E, F and H are known. In addition, triterpene components are also known. have.

As a related art, Patent No. 10-1695207 (a pharmaceutical composition for preventing or treating an anticancer drug-induced peripheral neuropathy containing the landing extract as an active ingredient) discloses " Phytolaccae Radix extract " a pharmaceutical composition for the prevention and treatment of chemotherapy-induced peripheral neuropathy (CIPN) ".

The present invention provides a medicinal herb composition for the treatment of cancer-induced peripheral neuropathy using natural materials with little side effects.

In order to solve the above problems and needs,

The present invention provides a medicinal herb composition for treating cancer-induced peripheral neuropathy, which is obtained by mixing ginseng, peony root, moxa, and Angelica gigas.

The present invention also provides a medicinal herb composition for treating cancer-induced peripheral neuropathy, which comprises 150 to 230 parts by weight of peanuts, 180 to 250 parts by weight of peaches, and 150 to 230 parts by weight of Angelica gigas L. in 100 parts by weight of ginseng.

The present invention also provides a pharmaceutical composition comprising a medicinal herb composition for the treatment of cancer-induced peripheral neuropathy.

The medicinal herb composition for the treatment of cancer-induced peripheral neuropathy according to the present invention has little side effects and is very environmentally friendly and has excellent effects in the treatment of cancer-induced peripheral neuropathy using natural materials.

1 is a diagram showing the morphological changes of sciatic nerve axons and DRG neurons after treatment with taxol and BGJTD.
FIG. 2 is a view showing the effect of treatment of a visceral fluid (BGJTD) on neurite growth of DRG neurons. FIG.
Fig. 3 is a view showing the regulation of survival of Schwann cells by treatment with Bengjutong (BGJTD). Fig.
FIG. 4 is a graph showing the effect of treatment of TESOL and VEGJTD on Erk1 / 2 and Cdc2 activity in the sciatic nerve.
FIG. 5 is a graph showing the effect of treatment of a visceral fluid (BGJTD) on axon regeneration after sciatic nerve injury. FIG.
FIG. 6 is a graph showing the effect of the treatment of the herbicide growth of Neurite outgrowth of the DRG sensory neurons treated with TESOL in a small-scale trough subgroup (Be, herbal composition composition for treating cancer-induced peripheral neuropathy).
FIG. 7 is a graph showing the effect of treatment of Texels, Vogtjest (BGJTD) and Be medicaments on the thermal sensitivity of experimental animals.
8 is a diagram showing a comparison of neurite outgrowth of DRG neurons after discrete pharmacological treatment of Example BGJTD.
FIG. 9 is a graph showing blood glucose level, TNF-.alpha. Production, and treatment effect of small-dose hydrogenation (Ba Be) on neurite outgrowth.
Figure 10 shows a comparison of TNF-a levels in sciatic nerves of STZ-diabetic animals after multiple treatments.
11 is a diagram showing the formation of phospho-Erk1 / 2 and p38 proteins in the sciatic nerve of an STZ-induced diabetic animal. (CTL: normal control group, SAL: physiological saline treated group).
FIG. 12 is a graph showing the effect of treating the visceral fluid (BGJTD) and the beverage with respect to the thermal sensitivity of an experimental animal.
FIG. 13 is a diagram showing HPLC analysis data for a viscous liquid (BGJTD) and a small group drug extract (Ba Be).

BRIEF DESCRIPTION OF THE DRAWINGS FIG.

The present invention provides a medicinal herb composition for the treatment of cancer-induced peripheral neuropathy, which is obtained by mixing ginseng, peony root, moxa, and Angelica gigas.

The present invention provides a herbal composition composition for treating cancer-induced peripheral neuropathy by mixing and extracting 150 to 230 parts by weight of peanut, 180 to 250 parts by weight and 150 to 230 parts by weight of ginseng in 100 parts by weight of ginseng.

The present invention also provides a pharmaceutical composition comprising a medicinal composition for the treatment of the above-mentioned anticancer drug induced peripheral neuropathy.

The ginseng of the present invention means the ginseng which is normally cultivated.

In Korea and Japan, it refers to the root of Panax ginseng C. A. Meyer and the removal of thin roots and cork layer. In Korea, white ginseng (raw), red ginseng (red ginseng: steamed) and ginseng (蔘 蔘: thin roots) are recorded separately. In the private sector, wild ginseng camphor and wild ginseng are distinguished. In China, it refers to the roots and rootstocks of ginseng and distinguishes it from raw ginseng (red ginseng), red ginseng and wild ginseng (wild ginseng).

The above-mentioned peonies of the present invention are grown in mountain areas. Several stems come out of a stand and stand straight, about 60cm high, with no hairs on leaves and stems. There are several roots, but they are thin and thick with a pointed cylindrical shape with both ends.

Leaves are alternate and lower leaves are double leaves with three small leaves. Small leaves are basal or elliptical, sometimes split into 2-3, and the veins and petiole are red. The leaf on the upper part is a leaf or a leaf which is simple in shape and has three small leaves. The surface of the leaf is glossy, the back is light green and the edge is flat.

Flowers bloom in May to June at the end of the stem. It is large and beautiful. It grows about 10cm in diameter. There are many kinds of horticultural varieties such as red and white. The calyx is green with 5, the edge is flat and attached to the end, the outermost one is leaf-shaped. The number of petals is about 10, but the number of basic species is 8 ~ 13, and it looks like inverted egg and is about 5cm long.

The stamen is very large and yellow, with 3 to 5 pistils, the stigma is backward, and the egg-shaped ovary has no or little hair. The fruit is ovate, the end is bent like a hook, it is split along the inner line, and the seed is spherical.

The flowers are beautiful and they are used for gardening. Root is used as medicine for pain, abdominal pain, dysmenorrhea, amenorrhea, hematemesis, anemia, bruise. In China, it has been cultivated for ornamental purposes in the Jin and Ming periods, and its cultivation history is older than Moran. There are dozens of varieties recorded in Song during the Qing period. They are distributed in Korea, Mongolia, and East Siberia.

The other name of the above-mentioned motif of the present invention is 蛤 蛤 모 모 (牡蛤). Oyster shells and animal ostriches Ostrea gigas Thunb. · Cave oyster Ostrea rivuralis Gould. And shells of oyster shells belonging to the same genus. Oyster shellfish are located in the east, west, and south sea of our country. I take the shellfish and grab the shellfish at any time and wash it and dry it. The taste is salty and the quality is fair.

It acts on the nerve (肾 经) and liver (肝 经). It strengthens and nourishes the pitch, removes the phlegm, softens the firmness, and stops sweating. It also heals the oil, stops the diarrhea and heals the boil. It has been found that the acidity of the stomach is lowered. My heart is aching and pounding with hunger. My head is dizzy, aching and sweating. I sweat in oil, diarrhea, diarrhea, scrape, schizophrenia, gastric hyperplasia, and ulcer. I make 10 ~ 30g a day by making a tablet, powder or pill form. When you use it as an external medicine, it is put into the base (medicine) agent in powder.

The Angelica gigas Nakai of the present invention refers to the dried root of Angelica gigas Nakai in Korea and the mountain type and Angelica sinensis (Oliv.) Diels in China. In Japan, the roots of Angelica acutiloba (Siebold. & Zucc.) Kitag. Or Angelica acutiloba (Siebold. & Zucc.) Kitag. Var. Sugiyamae Hikino) are dried and used.

This medicinal herb is warm and tastes sweet and spicy [溫 甘辛], Generally, Angelica angelica is more sweet than Chinese angelica angelica and Angelica giganta, and has a strong spicy taste.

The efficacy of angiosperms is due to the blood-producing (blood-clotting) action of producing blood when blood is scarce. Chinese Angelica, why Angelica, made from the root of Angelica gigas, is excellent in blood circulation. However, Angelica root, made from the root of Angelica gigas, has better anticancer effect and blood pressure lowering effect than antihypertensive action.

<Examples>

1. Composition extraction

(1) Extracting Bottled Water (BGJTD) Extract

 I purchased the ingredients (dry ones) from Daejeon Oriental Medicine Hospital,

30g of ginseng, 4g of ginseng, 7.5g of ginseng, 10g of ginseng, 7.5g of ginseng, 7.5g of ginseng, 12g of ginseng, 7.5g of safflower, 7.5g of safflower, 12g of ginseng, 10g of ginseng leaves, 5g of earthworms, 8g of ginseng, 8g of ferns, , 12 g of mackerel, 8 g of mackerel, 12 g of mackerel were immersed in water for 2 hours, boiled for 3 hours, filtered with Whatman filter paper, and frozen at 70 ^° C for 4 hours and then freeze-dried for 24 hours.

The yield of the product obtained from freeze drying was 13%, and 24g of the purified drug was obtained from 178g of the initial product. The obtained drug was dissolved in water and stored at 20 ^° C before use in the experiment.

The Example of the present invention includes all the constituents of the herbal medicine composition (Be) for treating cancer-induced peripheral neuropathy, which is an embodiment of the present invention. The herbal medicine composition for treating cancer-induced peripheral neuropathy of the present invention (Be (Be) of the present invention for treating cancer-induced peripheral neuropathy exhibits the same or higher effect in the treatment of peripheral nerve pathway induced by anti-cancer drugs and anticancer drugs .

(2) Anticancer drug-induced herbal composition (hereinafter referred to as " Be ") for the treatment of peripheral neuropathy

Ingredients (dried ones) were purchased from Daejeon University Oriental Medicine Hospital in accordance with the present invention, and 4 g of ginseng, 7.5 g of peony pomace, 8 g of moxa and 8 g of Angelica gigas were soaked in water for 2 hours, boiled for 3 hours and filtered through Whatman filter paper After freezing at 70 ^° C for 4 hours, freeze-drying was performed for 24 hours.

The yield of the product obtained from freeze drying was 13%, and 24g of the purified drug was obtained from 178g of the initial product. The obtained drug was dissolved in water and stored at 20 ^° C before use in the experiment.

2. Experiment and analysis

(1) HPLC analysis

BGJTD and Be were purified by filtration through a 0.45 μm PVDF membrane and standard solutions such as amygdalin, chlorogenic acid, ginsenosides Rg1 and Rb1, nodakenin, albiflorin, paeoniflorin, puerarin, and 5-HMF were separated from ultrapure water Lt; / RTI &gt;

The drug extracts and standard solutions were analyzed by LC20A Series HPLC system (Shimadzu, Kyoto, Japan). HPLC analysis was carried out with a constant agilent eclipse plus C18 (250 x 4.6 mm) under a gradient of acetonitrile at a rate of 1.0 ml / min. In the column set at 40 ^° C, a sample of 10 μl was injected. Amygdalin was 215 nm, Rg1 and Rb1 were 203 nm, puerarin, albiflorin and paeniflorin were 230 nm, albiflorin, paeniflorin and 5-HMF. 280 nm, and nodakenin was detected at 330 nm.

(2) Experimental animals and surgery

Sprague-Dawley rats (male, 200-250g) and Balb / c mice (22-25g) were purchased from companies in the fountain. The animals were kept in a 22 ^o C animal room and maintained a 12 hour night / day cycle.

All animals were maintained and used in accordance with the provisions of the Animal Management Committee of Daejeon University.

In taxol-treated BGJTD efficacy assays for sciatic nerve, DMSO solvent treatment group, TESOL treatment group, TESOL and BGJTD treatment group were used.

For analysis of neurons, the posterior ganglia (DRG) and sciatic nerve of lumbar 4 and 5 were separated and used for tissue analysis and biochemical experiments.

The animals were anesthetized with intraperitoneal injection of a mixture of ketamine (80 mg / kg) and xylazine (5 mg / kg). Texols (3.6 mg / ml of 5% DMSO; 1.25 mg / kg of body weight) were microinjected using a HemilTon syringe.

Experiments were conducted to evaluate the integration of microtubules in the axon of peripheral nerves. BGJTD (400 mg / kg) was orally injected 24 hours after the drug injection, and the same amount was injected for 4 days every day. Animals were treated for 7 days and analyzed.

In order to evaluate the effect of BGJTD on regenerative reactivity of damaged nerves, we divided into three groups: sciatic nerve injury (SNI), SNI, saline infusion group, and normal control group. For nerve damage, the middle part of the hind leg was opened, the nerve was exposed, and the fine tweezers were subjected to pressure damage twice for 30 seconds. After 24 hours of injury, BGJTD or saline was injected into the mouth, followed by the same dose for 4 days, and the animals were treated on the 7th day for analysis.

(3) Reverse tracing of the posterior ganglion neurons

In order to test the inverse tracing of the sensory neurons in the posterior ganglia, the rat was anesthetized and the sciatic nerve was injured. The fluorescent dye DiI (l, l'-dioctodecyl-3,3,3 ', 3' tetramethylindocarbocyanine perchlorate (5 μl of 0.5% in DMSO). BGJTD was orally injected 24 hours after suturing and the same amount was replenished for 4 days. Seven days after the initial surgery, the posterior ganglia were separated and 20 μm tissue sections were prepared and fluorescently labeled cells were observed under a fluorescence microscope.

(4) Primary Schwann cells and cultured root ganglion cells

For Schwann cell culture, 1-cm sciatic nerve was isolated from the hindlimb region of the rat and treated with 0.5 mg / ml type XI collagenase (Sigma, USA) at 37 ^° C for 90 minutes.

Posterior ganglion tissues were separated from lumbar spine 4 and 5 at 3 days after pretreatment with sciatic nerve injury and used for cell culture. After washing with BME medium, the cells were treated with type SII trypsin (5 μg / ml) for 15 min and inhibited with 50 μg / ml soybean trypsin inhibitor, 0.5 mM EDTA, and 20 μg / ml DNase I for 5 min. The cells were then plated on a 12-mm coverslip placed on a 24-well plate at a concentration of 1 × 10 ⁴ and cultured for 12 hours.

Then, the medium to be tested was treated with the medium (BME containing 5% fetal bovine serum, 5% horse serum, 2 mM glutamine, 1% penicillin-streptomycin) and cultured for 48 hours. The cultured cells were then recovered and immunofluorescent staining was performed.

For mixed Schwann cells and neurons, Schwann cells were first cultured at a concentration of 1 × 10 ⁴ for 24 hours, and 1.5 × 10 ² posterior ganglion neurons were added. Mixed cultures were maintained in BME culture medium supplemented with 10% serum. Cells were harvested 48 hours after treatment with Texol (0.01 mg / ml) and BGJTD (0.5 mg / ml) and used for immunofluorescence analysis. (1: 400 dilution, Sigma), βIII-tubulin antibody (TUJ1, 1: 400 dilution, Covance), S100β antibody (H-56 diluted 1: 400, Santa Cruz Biotech.) For immunofluorescence analysis. , And caspase 3 antibody (1: 500 dilution, Cell Signaling).

For the analysis of neurite outgrowth of cultured neurons, images were acquired with a digital camera attached to a fluorescence microscope and analyzed with a computer Photoshop program. The length of the neurite was measured using the i-Solution software program and the mean value of at least 30 cell analyzes in each experiment was used for statistical analysis with other experimental groups.

(5) Western blot analysis

The sciatic nerve of 1 cm in length was immersed in 100-200 μl volume of triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM β-glycerophosphate, pH 7.14, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na ₃ VO ₄ , 10 μg / ml leupeptin, 5 μg / ml aprotinin, 3 μM benzamidine, 0.5 mM DTT, 1 mM PMSF) and sonicated. Then, the sample was centrifuged at 12,000 rpm at 4 ^o C for 10 minutes, and the supernatant was recovered to obtain a protein extract. The protein concentration was then quenched using a spectrophotometer.

Protein (15 μl) was subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting. For immunoassay, anti-phospho-Erk1 / 2 antibody (1: 4000, Cell Signaling), anti-Cdc2 antibody, anti-phospho-vimentin antibody (1: 2,000, MBL) and horseradish peroxidase (1: 1000; goat anti-rabbit; Santa Cruz, or sheep anti-mouse; Amersham Biosciences, Buckinghamshire, UK). Protein bands on the X-ray film were quantitatively analyzed using i-Solution software (Image & Microscope Technology).

(6) Immunofluorescent staining

Biological tissue and cultured cell samples were fixed in 4% paraformaldehyde, 4% sucrose in PBS for 40 min at room temperature and blocked for 16 h in 2.5% horse serum and 2.5% bovine serum albumin. (1: 100, Santa Cruz Biotech.), Vimentin (1: 1,000, Chemicon, Temecula, USA), phospho-vimentin antibody (1: 400, MBL), NF-200 antibody ), βIII-tubulin antibody (TUJ1, 1: 400, Cavance), S100β antibody (1: 400, Santa Cruz Biotech.), phospho-Erk1 / 2 antibody (1: 400, Molecular probes, Eugene, OR, USA) or rhodamine-goat anti-rabbit secondary antibody (1: 400, Santa Cruz Biotech.) For 4 hours at room temperature. (1: 400, Invitrogen, Carlsbad, CA, USA) for 90 minutes at room temperature in the dark.

When cell nuclear staining was required, the cells were washed twice with PBS solution and 0.1% Triton X-100, followed by 10 minutes of Hoechst dye 33258 (bis-benzimide; Sigma) at a concentration of 2.5 μg / ml. The samples were observed using a Nikon fluorescence microscope and images were analyzed using a digital camera for depth analysis. And analyzed by quantitative qualitative analysis.

(7) Hot plate test

The sciatic nerve of Balb / C mice was intraperitoneally injected with medicinal material (400 mg / kg) 24 hours after the local injection of Texol (1.25 mg / kg) in the middle of the hind leg. Behavioral tests were performed on animals 7 days after the same dose for 4 days. The animals were maintained on a hot plate at 30 ^° C for 10 minutes and immediately moved to a 50 ^° C hot plate. The number of times the animal was hung on the hind leg for 30 seconds on the hot plate and the lag time was measured until the initial hind limb was lifted. For quantitative analysis of behavior, behavior was recorded in real - time video and subsequently analyzed by blind analysis.

(8) Statistical analysis

The obtained numerical data are shown by the standard deviation (SEM), and the mean values among the experimental groups were analyzed by Student's t-test and one-way ANOVA and Tukey test post test method. Statistical significance was reported as * P <0.05, ** P <0.01, *** P <0.001.

3. Experimental Results

A. In order to investigate the modulating effect of neuropathic responsiveness of the visceral fluid, taxol or nerve injury was applied to the sciatic nerve, and after treatment of the visceral fluid and the small-scale fluidized bed subgroup (Ba Be) Changes were investigated.

As shown in the main research contents below, VEGETABLE and Be sub-group medicinal herb extracts have an effect of contributing to improvement of neuronal regeneration reactivity and pathology.

(1) Treatment of Taxol with nerve fibers of peripheral sciatic nerve It observed the dorsal root Sikkim inhibit the nerve cell and connective of the axons of the ganglion, also observe the recovery of the nervous system result of processing the first view tongtang to the Taxol treated animals.

FIG. 1 shows the morphological changes of sciatic nerve axons and DRG neurons after treatment with taxol and VEGF.

(A, C) and lumbar vertebra DRG (D) tissue sections (D) were treated with DMSO solution, Texol, Texol and Vogtjut (BGJTD) 200 and βII-tubuline proteins for immunofluorescent staining.

The number of axons longer than 100 μm identified in the dotted line of the dotted line in (A) was measured, and the mean value from three discontinuous tissue sections was obtained and compared among the three experimental groups. Quantitative data on this is shown graphically in (B).

(D), arrows indicate the cell bodies of DRG neurons.

 * p <0.05, *** p <0.001 (ANOVA, animal population used by experimental group = 4)

 Size bars of figures A, C and D = 100 μm.

Immunofluorescent staining for NF-200 (neurofilament) proteins in the sciatic nerve was investigated to investigate the effect of Texol and BGJTD on the structural integrity of peripheral nerves. The longitudinal axonal continuity of the longitudinal axillary nerve tissue treated with Texol was significantly reduced compared to the control group, but it was observed to be improved by the treatment of BGJTD.

Taking into account the fact that tecseol acts on the structural stability of microtubule, immunological analysis of nerve cell specific protein, beta 3 tubulin, was performed on neural tissue. As shown in FIG. 1C, the axonal staining pattern of the experimental group was significantly degraded, but the BGJTD treatment significantly improved the staining pattern. Neurons in the posterior ganglion tissue of lumbar vertebra 5 were structurally disrupted as a result of treatment with Texol, but were improved by treatment with BGJTD (Fig. 1D).

(2) It was confirmed that the growth of the neurite was improved by treating the posterior ganglion neuron with the growth inhibition of neurite by Taxol treatment .

FIG. 2 shows the effect of treatment of BGJTD on neurite outgrowth of DRG neurons.

(A) Quantitative comparison results of neurite outgrowth

(B) Representative photographs of mixed cultures of DRG neurons and Schwann cells. Neurites were identified by immunostaining with anti-β tubulin antibody (green), and the distribution of mixed cultured Schwann cells was confirmed by Hoechst nuclear staining. In (A), the length of the neurite was determined by analysis of more than 20 neurons, with 7 to 10 random selection in the microscope field. The experimental results were the average of 4 independent experimental results, which were compared among experimental groups. Error bars indicate the standard error of the mean. *** p <0.001 (one-way ANOVA, number of independent experiments = 4), size bar = 50 μm

Treatment of primary neurons isolated from the posterior ganglia with Texol significantly reduced neurite outgrowth but significantly improved neurite outgrowth by treatment with BGJTD (FIG. 2A). FIG. 2B shows a representative image of the morphology of the cultured cells. In particular, the non-neuronal cells present on the cultured cells by Hoechst staining show that they are closely associated with the neurites in the case of BGJTD treatment in addition to the treatment with TEX. have.

3 view, but is processed with self-destruction of cells of Taxol observed at a Schwann cell culture isolated from the sciatic nerve of claim tongtang serves apoptosis levels of these cells was lower.

FIG. 3 shows the regulation of survival of schwann cells by treatment with Bogjutong (BGJTD).

The Schwann cells isolated from the sciatic nerves of normal rats were treated with DMSO solution, TEXSOL, TEXSOL and VEGJTD. Formalin-fixed cells were used for immunofluorescence staining (A) with anti-S100β antibody and (B) anti-caspase 3 antibody (confirmed as red). And stained with Hoechst 33258 (observed in blue) to observe the nuclei of the cells.

(A) Morphological comparison of S100β-stained Schwann cells after different treatments.

(B) Immunofluorescent staining of caspase 3 in cultured Schwann cells.

(C) Quantitative comparison of caspase 3-positive cells in cultured cells after different treatments

Was determined as a percentage of the total number of cells identified by nuclear staining of caspase 3-positive cells identified in randomly selected 5 or more microscopic fields. * p <0.05, *** p <0.001 (one-way ANOVA, number of independent experiments = 4), size bar of (A-C) = 100 μm

Since Texols are known to be involved in the structural stability of microtubules of neurons, cultured Schwann cells examined how Tetsol affects Schwann cells present in the sciatic nerve. Some cultured Schwann cells were found to be thin and long, and the treatment of Texol resulted in a more rounded and smaller shape of the cells. However, treatment with BGJTD resulted in a number of long-form cells similar to the control group (Fig. 3A). In order to investigate the effect of treatment with TEXAS and BGJTD on apoptosis, we investigated the level of caspase 3 protein activation during apoptosis. As shown in Fig. 4B, caspase-3 positive cells were observed after treatment with TESOL, but they were significantly decreased by BGJTD treatment (Fig. 3B, C).

(4) Taxol induces axon responsiveness after scarring in sciatic nerve , that is, phospho-Erk1 / 2 production , induction of Cdc2 and phosphorylation of vimentin protein.

View my hot water  Of these proteins Increase the level of production Axonal  Resulting in tissue reactivity similar to that of regeneration.

FIG. 4 shows the effect of treatment of Texol and Vesicle (BGJTD) on Erk1 / 2 and Cdc2 activity in sciatic nerve.

Results of western blot analysis of phospho-Erk1 / 2 production levels in sciatic nerve (A) and DRG (B) after treatment with TESOL and BJJTD (T + B) (C) Results of western blot analysis of Cdc2 and phospho-vimentin production levels in sciatic nerves treated with DMSO solution, Texol, and Texol + Vogtutong (BGJTD). (D) Representative photographs showing Cdc2 and phospho-vimentin-producing signals in S100β-labeled Schwann cells. In this experiment, endothelial sections of distal nerves treated with TESOL and VEGJTD were used.

(A - C) western blot images are representative results of three independent experiments. A quantitative comparison of the average band intensity of the relative target proteins against the actin control in western blot is presented at the bottom of (A-C). (D) = 100 [mu] m.

Changes in the level of Erk1 / 2 protein phosphorylated in the sciatic nerve and posterior ganglia were investigated in order to investigate whether the treatment of Texols and BGJTD changes the activation of neurons. The production level of the phosphorylated Erk1 / 2 protein did not change as compared to the control group at the time of treatment with TESOL but increased by BGJTD treatment (Fig. 4A). In the posterior ganglia, the level of phosphorylated Erk1 / 2 decreased by Texol treatment, which increased again to the control level by BGJTD treatment (Fig. 4B). (Han et al., 2007; Chang et al., 2012), which is known to be strongly induced in injured peripheral nerves as a different indicator of activation of neuronal tissue, has been analyzed for Cdc2 kinase and vimentin protein as a phosphorylation substrate.

As shown in FIG. 4C, the production of Cdc2 and Cdc2 mediated vimenitin phosphorylated proteins was induced by treatment with Texol, and the level of production was further increased by BGJTD treatment. Immunofluorescent staining of the sciatic nerve tissue revealed that the formation of phosphorylated vimentin by Cdc2 was induced in Schwann cells (FIG. 4D).

(5) View my hot water To the damaged sciatic nerve  about Axonal  Thereby promoting regeneration reactivity.

FIG. 5 shows the effect of the treatment of the visceral fluid (BGJTD) on axon regeneration after sciatic nerve injury.

(A) Results of western blot analysis of phospho-Erk1 / 2 production after sciatic nerve injury (SNI) and vesicle treatment (BGJTD).

(B) Western blot analysis of the production of Cdc2 and phospho-vimentin after treatment of sciatic nerve injury (SNI).

In (A) and (B), the western blot image is representative of 2-3 independent experiments.

The quantification results of the relative protein band intensities for the actin control are presented at the bottom.

(C) The distribution of axons in the sciatic nerve after impaction of the crest. The axon was subjected to immunofluorescence staining for NF-200 in the proximal section, the damaged section, and the 6-mm-long nerve tissue piece in the distal section. The lower end graph counts the number of axons touching the dotted line across the center and determines the mean value from the results of several noncontinuous tissue segments. After that, statistical comparison analysis was carried out among experimental groups. (One-place ANOVA, ** p <0.01, animal population in group = 4). (D) Backward tracing of DiI-labeled sensory neurons in DRG (left). Quantitative comparison of DiI-labeled DRG neurons in individuals of the population treated with saline and BGJTD (right).

 * p < 0.05 (Student's t-test, number of independent experiments = 4). (C, D): 100 m.

The effect of BGJTD on axon regeneration after sciatic nerve injury was investigated. The production of the phosphorylated Erk1 / 2 protein in injured sciatic nerve increased and was further elevated by treatment with BGJTD (Fig. 5A). Cdc2 protein was induced at the proximal and distal parts of the damaged muscle at 7 days after neuronal damage, and it was found that the treatment of BGJTD increased intensively at the damaged distal part. Phosphorylated vimentin was observed weakly after injury but increased by BGJTD treatment (Fig. 5B). Observation of axons by immunofluorescence staining of nerve tissue with NF-200 showed that axon regeneration was constant at 3 days after injury, and this change was significantly improved by BGJTD treatment (Fig. 5C). In order to quantitatively measure axon regeneration level, posterior ganglion neurons were retrospectively stained with DiI fluorescent dye. The fluorescence-labeled cells that were stained with the regenerated axons were found to show a significant increase in the BGJTD-treated group compared to the control group (Fig. 5D).

(6) It is suggested that small group liquids of tea juice are treated on cultured proximal ganglion cells to induce characteristic neurite growth. Especially, Be group is most effective in inducing neurite outgrowth .

FIG. 6 shows the treatment effect of the treatment for the treatment of neurite outgrowth of the TESOL-treated DRG sensory neurons in the sub-tidal group (Be, herbal composition composition for the treatment of cancer-induced peripheral neuropathy).

After 6 days of in vivo injection of Texol or DMSO solution into the sciatic nerve, Ba · Be (0.5 mg / ml) was separately treated with DRG neurons isolated and cultured for 48 hours and used for immunofluorescence staining.

 (A) Comparison of the length of neurite growth between experimental groups.

 (B) Representative photograph of cells cultured after immunofluorescence staining with NF-200 (green) and GAP-43 (red).

 Cell nuclei were identified by nuclear staining of Hoechst 33258 (blue).

In (A), neurite lengths were analyzed in more than 20 neurons in 7-10 microscopic fields and averaged over four independent experiments, and then compared between the experimental loads.

 * P <0.05, ** P <0.01, *** p <0.001 (one place ANOVA, number = 4).

(B) = 100 μm

BGJTD is a combination prescription consisting of 18 drugs. In order to classify the effective ingredients showing the improvement effect on the nervous tissues treated with Texols among these many constituent drugs, first, they were classified into four subgroups (Ba-Bd) based on the oriental medicine theory.

Then, all of the respective medicines were treated with cultured proximal ganglion cells to analyze changes in neurite growth. Each subgroup was selected one of the drugs that most effectively induces neurite outgrowth, and a new group of drugs Be was formulated.

Treatment of the late ganglion cells with Texol and treatment with each subgroup of Ba-Be revealed that the Be group was most effective in inducing neurite outgrowth (Fig. 6A). We also observed that the treatment of Be showed the most pronounced production of GAP-43 protein, an indicator protein of axon regeneration. In particular, GAP-43 protein signal was well observed in the neurite branch, which is presumed to reflect the shift to axonal processing for axon regeneration (Figure 6B)

(7) View Tongue and Be subgroup extracts show similar levels of efficacy in behavioral responses to sensory functioning .

FIG. 7 shows the effect of treatment of Texels, Vogtjest (BGJTD) and Be medicaments on the thermal sensitivity of experimental animals.

 (A) and the incubation period (B) until the reflex reaction time after animals were placed on a hot plate. The animals were placed on a hot plate, Respectively.

The error bars in (A) represent the standard error of the mean.

 * P <0.05, ** P <0.01, *** p <0.001 (one place allocation analysis, number of animals in group = 4).

We investigated whether BGJTD and Be medicinal treatment were involved in the behavioral changes of heat stimuli. In the animals treated with Texol, the frequency of shrinking of the hind legs was significantly lower in the 50 ^o C hot plate than in the control group, and the reaction time was significantly increased until the initial reaction. As a result of treatment with BGJTD or Be in the treated animals, the frequency of the withdrawal increased to a level similar to that of the normal control, and the initial reaction time showed a significant decrease (FIG. 7A, B).

B. Diabetic animal models Peripheral neuropathy  For reactivity BJT's  Selective treatment effect

The improvement of diabetic response was observed in diabetic animal model induced by treatment with STZ.

(1) A small group of medicinal herb extracts were treated with Ba ~ Bd , and a small group of medicines with high efficacy for inducing growth of the nerve processes in each subgroup was selected and a new group of medicines Be was established

Figure 8 shows a comparison of neurite outgrowth of DRG neurons after treatment of the individual constituent medicaments of Example BGJTD.

The drug (0.5 mg / ml) was treated with DRG neurons for 48 hours and immunofluorescent staining for NF-200 (green) and GAP-43 (red) proteins.

(A) Comparison of neurite outgrowth by individual medication treatment in each subgroup (Ba Bd). The red dotted lines at the top and bottom indicate the length of neurite length of DRG neurons isolated from preconditioning sciatic nerve injury (SNI) and normal control (CTL).

(B) Representative immunofluorescent staining for DRG neurons treated with a single medicinal substance showing the highest neurite outgrowth in each subgroup, with normal control (CTL) and SNI population. Size bar = 100 μm

(2) After the administration of the extracts of the passbook subgroups into the STZ-induced diabetic rats, the changes in glucose levels, TNF- alpha production and neurite outgrowth on the posterior ganglion cells were analyzed .

Be drug overall Efficacy  Respectively.

FIG. 9 shows the effects of treatment of small-dose hydrogenation (Ba Be) on blood glucose level, TNF-a production and neurite outgrowth.

 (A) Changes in blood glucose levels in other animal groups for 7 weeks after STZ injection. Error bars indicate standard error (SEM) (number of animals in each group = 5).

 In (B) and (C), western blot analysis of protein extracted from sciatic nerve was performed on TNF-α after oral administration of 2 weeks of Ba be bath. Western blot for actin protein is performed by control analysis of sample loading.

(C) The sciatic nerves isolated from STZ-induced diabetic animals are subjected to immunofluorescent staining with NF-200 and TNF-α. The arrow in the figure indicates that the TNF-α signal is located in the axonal region stained with NF-200.

(D, E) 7 days after STZ injection, DRGs of lumbar spine 4 and 5 were collected from animals having a blood glucose level of 3 mg / ml or more, and neuronal cells were cultured. The cultured cells were treated with the drug (0.5 mg / ml) for 48 hours and then subjected to immunofluorescence staining with NF-200.

 (D) Comparison of neurite outgrowth of DRG neurons prepared from STZ-induced diabetic animals.

 (E) Representative image of DRG neurons immunofluorescently stained with NF-200.

 * P <0.05, ** P <0.01 (error bars: standard error of the mean, number of individual experiments = 4).

 Size bars of (C) and (E) = 100 μm.

(3) Treatment of Diet and Be drugs decreased the level of TNF-α mRNA and protein production in the sciatic nerve model of the STZ-induced diabetic model

Figure 10 shows a comparison of TNF-a levels in sciatic nerves of STZ-diabetic animals after multiple treatments.

 (CTL: untreated control, SAL: saline injection).

 One week after the STZ injection, the animals were treated with herbal medicine for 2 weeks.

 (A) Western blot analysis of TNF-α levels in protein extracts from sciatic nerve.

 The western blot for actin was used as an internal loading control.

 (B) Real-time PCR for comparison of TNF-α mRNA expression in DRG neurons in treated animals as shown in the figure.

 * P <0.05, ** P <0.01 (error bars: standard error of the mean, number of independent experiments = 4).

(4) View Jae-tang and Be drugs as cell survival factors phospho - generating levels of Erk1 / 2, while increasing, the p38 protein produced is the effect of reducing apoptosis path protein appears.

Fig. 11 shows the formation of phospho-Erk1 / 2 and p38 proteins in the sciatic nerve of STZ-induced diabetic animals. (CTL: normal control group, SAL: physiological saline treated group).

 (A) Western blot analysis of phospho-Erk1 / 2 production in sciatic nerve.

 (B) Immunofluorescence of phospho-Erk1 / 2 protein signal in sciatic nerve section stained with NF200

 image. The phospho-Erk1 / 2 signal on the epinurial sheath is indicated by an arrow.

 (C) Western blot analysis of p38 in sciatic nerve. The western blot for actin was used as a sample loading control from (A) to (C). (B) = 100 μm.

(5) View my earthenware  Be drugs were administered in diabetic model rats Sensory nerve  And it was found that it acted positively in the improvement of function.

FIG. 12 shows the effect of treatment of the viscous fluid (BGJTD) and the bean liquid on the thermal sensitivity of the experimental animals.

 (BGJTD) and Be were orally administered to STZ - induced diabetic rats for 2 weeks and incubation period (B) was measured.

 The error bars in (A) represent the standard error of the mean.

 * P <0.05, ** P <0.01, *** p <0.001 (one-way ANOVA, number of animals used in individual group = 4).

(6) BGJTD , Ba Of ~ Be HPLC  analysis; Each drug was identified to contain known indicator chemicals.

FIG. 13 shows HPLC analysis data for Bogjutong (BGJTD) and small group drug extract (Ba Be).

 The peak corresponding to the standard chemical is indicated by the vertical arrow, and the UV value applied to detect the peak is shown in the figure.

The present invention provides a medicinal herb composition for the treatment of cancer-induced peripheral neuropathy comprising the above-described composition and function.

The present invention is very useful in an industry that produces, manufactures, distributes and researches compositions for treating cancer-induced peripheral neuropathy.

In particular, the present invention is very useful in an industry for producing, manufacturing, distributing and researching a medicinal composition for treating cancer-induced peripheral neuropathy using natural medicinal materials and a pharmaceutical composition containing the same.

Claims (3)

Ginseng, peony, mulberry, and angelica are mixed and extracted,
Wherein the composition comprises 150 to 230 parts by weight of peanut, 180 to 250 parts by weight of peony, and 150 to 230 parts by weight of Angelica gigas L. in 100 parts by weight of ginseng.
delete A pharmaceutical composition for treating cancer-induced peripheral neuropathy comprising the herbal composition composition for treating cancer-induced peripheral neuropathy according to claim 1.

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