TWI779941B - Extraction method of clinacanthus nutans, and use of it's extract for treating osteoporosis, neurodegenerative diseases, skeletal muscle atrophy and improving wound healing - Google Patents

Abstract

The present invention provides a method for extracting Clinacanthus nutans, its extracts and its use for treating osteoporosis, neurodegenerative diseases, skeletal muscle atrophy and improving wound healing. Clinacanthus nutans was firstly extracted with ethanol and water, and partitioned between ethyl acetate and water to obtain a first aqueous layer and an ethyl acetate layer. The first aqueous layer was partitioned with n-butanol to obtain an n-butanol layerand a second aqueous layer. The ethyl acetate layer was portioned between ethanol aqueous solution and n-hexane to obtain a ethanol aqueous layer and an n-hexane layer.

Description

The extraction method of Sabah Snake Grass and its extracts are used to treat osteoporosis, neurodegeneration degenerative diseases, skeletal muscle atrophy and promoting wound healing

The present invention relates to an extract of Sabah Snake Grass and its pharmacological activity. Specifically, the present invention relates to an extract of Sabah Snake Grass and its use in the treatment of osteoporosis, neurodegenerative diseases, skeletal muscle atrophy and wound healing the purpose of.

Sabah snake grass ( Clinacanthus nutans ) is a traditional herbal medicine in Southeast Asia. It is used by folks to treat various diseases and its biological activity has been confirmed by considerable research.

Osteoporosis refers to an imbalance phenomenon in which the bone resorption led by osteoclasts is greater than the bone formation led by osteoblasts, which eventually leads to the destruction of bone microstructure and the decrease of bone density. Taiwan has entered an aging society. Osteoporosis is a common disease in the elderly population, and fractures caused by osteoporosis are more often accompanied by other diseases. Therefore, effective prevention and improvement of osteoporosis in the elderly will not only contribute to the health care of the elderly, but also reduce the expenditure on health care and long-term care.

Neurodegenerative disease refers to the degeneration of nerve cells due to genetic, endocrine or age and other reasons, which makes the patient's behavioral ability, cognition and intelligence decline, such as Alzheimer's disease (also known as Alzheimer's disease) Alzheimer's disease; Alzheimer's disease) and Parkinson's disease; Parkinson's disease) are one of the common neurodegenerative diseases. Alzheimer's disease and Parkinson's disease have the largest population and bring the greatest pressure on families and society. If Alzheimer's disease and Parkinson's disease can be prevented or treated, it will help reduce the burden on the elderly burden of care.

Now that the average life expectancy of human beings continues to increase, the number of cancer patients known as modern civilization diseases is also increasing. Depending on the type of cancer, surgery or chemotherapy can be used for treatment, and chemotherapy usually causes discomfort during the treatment process. side effect. Among them, skeletal muscle atrophy is one of the key issues, as it debilitates patients, ultimately leading to decreased quality of life, and increased patient mortality and morbidity. If there is a way to slow down or treat the skeletal muscle atrophy caused by chemotherapy, it is expected to improve the quality of life of patients after surgery and reduce the chance of recurrence.

In addition, wounds will be left on the patient's body after various operations. If wound healing can be promoted, the incidence of infection complications can be reduced, and the burden of postoperative care can be reduced.

To sum up, in order to reduce the burden on medical care and the cost of care in Taiwan, products that can treat osteoporosis, neurodegenerative diseases, skeletal muscle atrophy, and promote wound healing are needed.

In view of the above purpose, the present invention provides a method for preparing the extract of Snakeweed Sabah, comprising: extracting Snakeweed Sabah with aqueous ethanol, concentrating the extract to obtain a crude extract; adding the crude extract to ethyl acetate and Water is subjected to liquid-liquid distribution extraction to obtain a first water layer and an ethyl acetate layer; further adding n-butanol to the first water layer for liquid-liquid distribution extraction, and the two extraction separation layers After concentration, obtain a n-butanol layer extract (or claim the first extract) and a second water layer extract (or claim the second extract); after the ethyl acetate layer extract is concentrated, add ethanol aqueous solution and The n-hexane is subjected to liquid-liquid partition extraction, and after the two extraction separation layers are further concentrated, an ethanol aqueous solution layer extract (or called the third extract) and a n-hexane layer extract (or called the fourth extract) are obtained.

The present invention further provides the first extract, the second extract, the third extract and the fourth extract respectively obtained according to the aforementioned preparation method, which are used to prepare and treat osteoporosis, neurodegenerative diseases, bone Use of drugs for muscle atrophy and promoting wound healing.

The Sabah Snake Grass extract prepared by the above method can be used to prepare medicines for treating osteoporosis, neurodegenerative diseases, skeletal muscle atrophy and promoting wound healing, thereby reducing the burden on medical care and the cost of care in Taiwan .

The accompanying drawings are included below to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the inventive concept.

Fig. 1 is a preparation process of Snake Grass Sabah extract according to an embodiment of the present invention.

FIG. 2 shows the results of the cell viability and ALP activity tests of the MC3T3-E1 cell line with different concentrations of the third extract of the Sabah Snake Grass extract according to the embodiment of the present invention.

Fig. 3 is the results of the cell survival rate and ALP activity test of the MC3T3-E1 cell line with different concentrations of the fourth extract of the Sabah Snake Grass extract according to the embodiment of the present invention.

FIG. 4 shows the results of the cell survival rate and TRAP activity test of the RAW 264.7 cell line with different concentrations of the third extract of the Snakeweed extract according to the embodiment of the present invention.

Fig. 5 shows the results of the cell viability and TRAP activity test of the RAW 264.7 cell line with different concentrations of the fourth extract of the Sabah Snake Grass extract according to the embodiment of the present invention.

Fig. 6 is a photo of the staining of TRAP (+) multinucleated osteoclasts on RAW 264.7 cell line with the third and fourth extracts of Snakewort sabah extract according to the embodiment of the present invention.

FIG. 7 is a test result of the anti-neurinflammation activity of the first to fourth extracts of Snake grass extract according to the embodiment of the present invention.

Fig. 8 is the experimental procedure for verifying the anti-Alzheimer's disease of the second extract of Sabah Snake Grass extract in APPsw/PSEN1dE9 (APP/PS1) transgenic mice.

FIG. 9 shows the results of the anti-Alzheimer's disease test of the second extract of Snakewort sabah extract according to the embodiment of the present invention.

FIG. 10 is an experimental flow chart of the aging experiment of the second extract of Snakewort sargali extract according to the embodiment of the present invention.

Fig. 11 shows the results of the excavation ability experiment of mice in the aging experiment of the second extract of the Snakegrass sargali extract according to the embodiment of the present invention.

Fig. 12 is a DCX ⁺ quantification diagram of the region inside the dentate gyrus in the hippocampus of mice in the aging experiment of the second extract of Snake Grass Sabah extract according to an embodiment of the present invention.

FIG. 13 is a BrdU ⁺ quantification diagram of the region inside the dentate gyrus in the hippocampus of mice in the aging experiment of the second extract of Snakegrass sabaica extract according to an embodiment of the present invention.

Fig. 14 is a flow chart of the skeletal muscle cytotoxicity test of the fourth extract of Snakeweed extract according to the embodiment of the present invention.

FIG. 15 is a flow chart of the animal test on skeletal muscle atrophy performed by the fourth extract of Snakewort sargali extract according to the embodiment of the present invention.

FIG. 16 is the results of animal experiments on skeletal muscle atrophy with the fourth extract of Sabah Snake Grass extract according to an embodiment of the present invention.

Fig. 17 is the result of the migration experiment of the isolated endothelial cells of the third extract of Snakewort sabah extract according to the embodiment of the present invention.

Fig. 18 is the result of animal wound experiment of the third extract of Snake grass extract according to the embodiment of the present invention.

In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various exemplary embodiments or implementations of the invention. It is evident, however, that the various illustrative embodiments may be practiced without these specific details, or with one or more equivalent arrangements. Furthermore, the various illustrative embodiments may be different, but are not necessarily exhaustive. For example, the specific shapes, configurations, and characteristics of an exemplary embodiment may be used or implemented in another exemplary embodiment without departing from the inventive concept.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined, for example, in commonly used dictionaries should be construed as having meanings consistent with their meanings in related fields, and should not be construed in an idealized or overly formal sense unless explicitly defined herein.

A numerical range stated herein may include all subranges within that range with equal numerical precision. For example, the range "1.0 to 10.0" may include all subranges between a minimum value of 1.0 and a maximum value of 10.0, that is, having a minimum value equal to or greater than 1.0 and a maximum value equal to or less than 10.0, for example, 2.4 to 7.6 . Any maximum numerical limit stated herein may be inclusive of all lower Numerical limitations, and all minimum numerical limitations stated in this specification may include all higher numerical limitations. Accordingly, Applicants reserve the right to amend this specification (including claimed claims) to expressly enumerate any sub-ranges contained within the expressly recited ranges herein. All of these ranges are inherent descriptions in this specification.

Preparation of Sabah Snake Grass Extract

Please refer to FIG. 1 . FIG. 1 is a process for preparing an extract of Snake Grass Sabah according to an embodiment of the present invention.

The extract of Sabah Snake Grass according to the embodiment of the present invention can be obtained through the following preparation method, comprising: extracting Snake Grass Sabah with ethanol aqueous solution, taking the extract and concentrating to obtain a crude extract; adding the crude extract to ethyl acetate and Water is subjected to liquid-liquid partition extraction to obtain a first water layer and an ethyl acetate layer; further adding n-butanol to the first water layer for liquid-liquid partition extraction, and after the two extraction separation layers are concentrated, a n-butanol layer is obtained. layer extract (or claim the first extract) and a second aqueous layer extract (or claim the second extract); after the ethyl acetate layer extract is concentrated, add ethanol aqueous solution and normal hexane to carry out liquid-liquid partition extraction, After the two extraction and separation layers were further concentrated, an ethanol aqueous layer extract (or called the third extract) and a n-hexane layer extract (or called the fourth extract) were obtained.

Next, the properties of the first to fourth extracts prepared according to the above method are tested through the examples to prove their effects.

1. Osteoblast differentiation analysis

In this example, osteoblasts (mouse pre-bone blast cell line MC3T3-E1 cell line) were used for osteoblast differentiation analysis.

The experimental steps include: MC3T3-E1 cell line (1x10 ⁴ cells/well in 96-well plate) was cultured in a 96-well plate, cultured for 1 day, and replaced in a medium containing 5 mg/ml ascorbic acid (ascorbic acid), 1 mM sodium glycerophosphate (sodium glycerophosphate) MEM medium, and then add different concentrations (5μg/mL, 10μg/mL, 20μg/mL, 40μg/mL) of Sabah snake grass extract (the third extract (90% ethanol divided layer), The 4th extract (normal hexane divides layer), and set the control group that does not add, and β-glycerol phosphate (B-GP) as control group.After carrying out 5 days of cultivating, culture medium is removed, after washing with ice PBS , add 0.01% SDS to treat for 30 minutes, then add phosphoric acid p-nitrophenol solution (pNPP) solution to react for 30 minutes, add 3N NaOH to stop the reaction. Determine the release amount of nitrophenol (nitrophenol) by measuring the absorbance at a wavelength of 405nm Alkaline phosphatase (ALP) activity is used as an indicator of osteoblast differentiation. At the same time, the cell viability test of the same extract sample is also carried out by MTT assay.

The results are shown in Figure 2 and Figure 3. FIG. 2 shows the results of the cell viability and ALP activity tests of the MC3T3-E1 cell line with different concentrations of the third extract of the Sabah Snake Grass extract according to the embodiment of the present invention. Fig. 3 is the results of the cell survival rate and ALP activity test of the MC3T3-E1 cell line with different concentrations of the fourth extract of the Sabah Snake Grass extract according to the embodiment of the present invention.

From the results in Fig. 2 and Fig. 3, it can be seen that the survival rate of the third and fourth extracts on osteoblasts is higher than that of the control group, and it can be known that the third and fourth extracts can promote the growth of osteoblasts. In the ALP test, it can be seen that the measured ALP values of the third and fourth extracts with different concentrations are higher than those of the control group and the B-GP group, and there are statistically significant differences. That is to say, both the third and fourth extracts of Snakewort Sabah according to the script embodiment can significantly stimulate the differentiation of osteoblasts.

2. Analysis of osteoclast formation

In this example macrophages (RAW 264.7 cells) were used for the osteocytogenesis assay.

The experimental steps include: first culture the RAW 264.7 cell line for 1 day, add RANKL to induce the RAW 264.7 cell line to differentiate into osteoclasts, and add nuclear factor kappa-B ligand receptor activator (RANKL) at the same time , add different concentrations (5μg/mL, 10μg/mL, 20μg/mL, 40μg/mL) Sabah snake grass extract (the third extract (90% methanol layering), the fourth extract (n-hexane layering)) For treatment, remove the culture medium after culturing for 3 days, treat with 3.7% formaldehyde (formaldehyde) for 10 minutes to fix the cells on the bottom of the plate, then treat with 75% ethanol (alcohol) for 1 minute to strengthen the fixed cells, and put them in an oven to dry After drying, add TRAP solution (10mM Na ₂ C ₄ H ₄ O ₆ , 3mM p-nitrophenyl phosphate) and shake under the condition of avoiding light to carry out the reaction. When the reaction time is up, absorb the TRAP solution to 0.1 In the NaOH of N, the reaction was stopped, and the absorbance value at a wavelength of 410nm was measured, which was the total TRAP activity. In addition, after the cells were fixed and dried, TRAP dye (5mg Naphthol AS-MX phosphate, 30mg Fast Red Violet LB Salt, 0.5mg Dimethylformamide, 10mM Na ₂ C ₄ H ₄ O ₆ , 50ml H ₂ O) was added, and the After shaking for 20 minutes, the dye was removed, and the stained TRAP protein was photographed with an optical microscope, and the number of multinucleated (number ≥ 5) cells was counted to indicate the degree of cell fusion. According to the total TRAP activity test and the observed results of TRAP staining, it was used as an index of osteoclast differentiation. At the same time, the cell viability test of the same extract sample was also carried out by MTT assay.

The results are shown in Figures 4 to 6. FIG. 4 shows the results of the cell survival rate and TRAP activity test of the RAW 264.7 cell line with different concentrations of the third extract of the Snakeweed extract according to the embodiment of the present invention. Fig. 5 shows the results of the cell viability and TRAP activity test of the RAW 264.7 cell line with different concentrations of the fourth extract of the Sabah Snake Grass extract according to the embodiment of the present invention. Figure 6 is a photograph of staining of TRAP (+) multinucleated osteoclasts on the RAW 264.7 cell line with the third and fourth extracts of different concentrations of the Sabah Snake Grass extract according to the embodiment of the present invention, the top is before adding, and the bottom is For photos added 3 days later.

The results showed that both the third extract and the fourth extract of Sabah Snake Grass could significantly inhibit the formation of osteoclasts in a dose-dependent manner.

3. Anti-neuritis

In this example, primary cultured mouse cerebral cortex mixed glial cells were used to test the anti-neurinflammation activity.

Experimental steps include:

1. Microglia culture: Primary cultures of neocortical microglia will be prepared from the cerebral cortex of Sprague Dawley rat pups at postnatal day 5. Briefly, 5-day-old pups were anesthetized with ether and sacrificed by decapitation. Primary mixed glial cells were prepared from the cerebral cortex and stored in DMEM/F12 medium containing 10% FBS for 5 days. Neurobasal medium supplemented with B27 was used as an alternative medium and incubated for 11 days or until microglial cells branched. The mixed gel cell culture was treated with extract (100μg/ml) for 2 hours, and then treated with 100ng/ml LPS for 22 hours, and the culture medium was taken for the determination of nitrite.

2. Determination of nitrite: by incubating the medium with an equal volume of Griess reagent (0.05% N-(1-naphthyl)-ethylenediamine dihydrochloride, 0.5% sulfonamide and 1.25% phosphoric acid) Measurement of nitrite levels (nitric oxide NO release). After incubation, the optical density was detected at a wavelength of 540 nm using a microplate reader using sodium nitrite (NaNO ₂ ) as a standard.

The experimental results are shown in Figure 7. Fig. 7 is the first to fourth extracts (n-butanol division layer, second water division layer, ethanol division layer and n-hexane division layer) of Sabah Snake Grass extract (100g/ml) according to the embodiment of the present invention Test results for anti-neuroinflammatory activity. The amount of nitric oxide (NO) produced can estimate the intensity of inflammation.

It can be seen from Figure 7 that the first extract (butanol stratification), the second extract (second water stratification) and the third extract (ethanol layer) have anti-neurinflammation activity, which inhibits LPS-stimulated One nitric oxide generation reached 77%, 56% and 55%.

4. Anti-Alzheimer's Disease Activity

In this example, APPsw/PSEN1dE9 (APP/PS1) gene transgenic mice were used to verify the anti-Alzheimer's activity of the extract of Sabah snake grass. The animals used in the experiment were APP/PS1 gene transgenic mice (8 weeks), with a body weight of 25-30 grams. Breeding environment: The cycle of artificial lighting is 12 hours a day (07:00~19:00), the temperature is controlled at 24±1°C, the humidity is 55-65%, and the food and drinking water are adequately supplied. The animals were divided into two groups: the control group and the second extract group, with 6 animals in each group, and the dose was 100 mg/kg. After 28 days of treatment, the nesting test was completed. One week after administration of BrdU, the mice were sacrificed, and the brain, liver and blood were collected to analyze the pathological changes.

Fig. 8 is the experimental procedure for verifying the anti-Alzheimer's disease of the second extract of Sabah Snake Grass extract in APPsw/PSEN1dE9 (APP/PS1) transgenic mice. Referring next to FIG. 9 , FIG. 9 is the results of the anti-Alzheimer's disease test of the second extract of Snakeweed Sabah extract according to an embodiment of the present invention. Significant differences between treatment and control groups are indicated by *, p<0.05; **, p<0.01. As shown in FIG. 9 , the results of the nesting test found that the second extract could improve the nesting score and unshredded nest pieces of mice (A in FIG. 9 ). The second extract also reduced the number of amyloid scars and glial cell clusters (Figure 9B). However, the second extract could not promote neurogenesis in the hippocampus (Fig. 9C). From the above results, the activity of the second extract in the APP/PS1 transgenic mice was to reduce the number of amyloid scars and glial cell clusters, but not to promote the regeneration of hippocampal neurons.

5. Anti-aging activity

Please refer to Figure 10 and Figure 11. FIG. 10 is an experimental flow chart of the aging experiment of the second extract of Snakewort sargali extract according to the embodiment of the present invention. Fig. 11 shows the results of the excavation ability experiment of mice in the aging experiment of the second extract of the Snakegrass sargali extract according to the embodiment of the present invention.

In this example, the C57BL/6J mice were divided into 4 groups (1. control group (4 males), 1. induction group (administered D-galactose, 4 males) and 2. drug treatment group (one group administered D-galactose + second extract 300 mg/kg, 4 kg, another group was given D-galactose + second extract 100 mg/kg, 4 kg), n=8). Induction group and drug treatment group at 6 D-galactose was administered at the age of one week (subcutaneous injection on the back of the neck once a day for 8 consecutive weeks). The drug-treated group was given the vehicle or the second extract (300 mg/kg and 100 mg/kg) at the age of 7 weeks (feeding, 5 times a week, for 7 consecutive weeks). Nest building behavior test was carried out at 12 weeks of age, and mice were finally sacrificed after 7 days of continuous administration of BrdU.

The results are shown in Figure 11, and the data are mean ± standard deviation (n=4). Analyze parameter data through unpaired two-tailed Student's t test to analyze the results between drug and D-galactose groups: ##, p<0.01; analyze parameter data through unpaired two-tailed Student's t test to analyze D-galactose group Compared with the blank control group: **, p<0.01. Administration of the second extract (300mg/kg) significantly improved the excavation ability of D-galactose-induced aging mice, while administration of the second extract (100mg/kg) had a significant effect on D-galactose-induced aging mice The digging ability has been slightly improved. It can be seen that the second extract has anti-aging ability.

6. D-galactose-induced neuronal regeneration in the hippocampus of aging mice

In this example, D-galactose-induced aging mice were fed with vehicle and the second extract (100 mg/kg; 300 mg/kg). BrdU (50mg/Kg/day) was injected for seven consecutive days before sacrifice, and then sacrificed, and brain sections were taken for tissue immunofluorescence staining. BrdU antibody was used to detect Type 2 neural progenitor cells, and neuron migration protein (doublecortin, DCX) antibody was used to detect newborn neurons.

Fig. 12 is a DCX ⁺ quantification diagram of the region inside the dentate gyrus in the hippocampus of mice in the aging experiment of the second extract of Snake Grass Sabah extract according to an embodiment of the present invention. FIG. 13 is a BrdU ⁺ quantification diagram of the region inside the dentate gyrus in the hippocampus of mice in the aging experiment of the second extract of Snakegrass sabaica extract according to an embodiment of the present invention. The data were analyzed by unpaired two-tailed Student's t-test to analyze parameter data statistics between drug and D-galactose groups: #, p<0.05; parameter data were analyzed by unpaired two-tailed Student's t-test to D-galactose group Compared with the blank control group: *, p<0.05.

It can be seen from Figure 12 and Figure 13 that feeding the second extract can significantly increase the number of newborn neurons in D-galactose aged mice, and can also increase the number of type II neural progenitor cells.

Combining the above experiments 3 to 6, it can be found that the second extract of Sabah Snake Grass extract has anti-inflammation, anti-Alzheimer's disease and anti-aging effects by promoting the regeneration of hippocampal neurons in aging model rats.

7. Anti-skeletal muscle atrophy

In this example, a myotube toxicity detection platform was established by using mouse myoblasts C2C12, which are easily differentiated into myotubes, for drug screening. The flow chart is shown in FIG. 14 .

Using this platform, we found that the fourth extract of Sabah Snakeweed (n-hexane fractionated layer) was nontoxic to myotubes at a concentration of 100 μg/ml. However, the survival rate of myotubes treated with 40 μM cisplatin for 2 days was only about 60%. After co-treatment with cisplatin (40 μM) and the fourth extract of Sabah Snake Grass (100 μg/ml), the survival rate was greatly increased to about 90%, indicating that the fourth extract of Sabah Snake Grass had an effect on cisplatin-induced muscular Tube toxicity is protective.

The protective effects of the fourth extract of Sabah Snakeweed were also evaluated in vivo. The design of animal experiments followed the previously described procedures (Chiou et al., 2018). The scheme of animal experiments in vivo is shown in Figure 15. Figure 15 is the fourth extract of Sabah snake grass extract according to the embodiment of the present invention. Flow chart of animal experiments for muscular atrophy. Briefly, 8-week-old Balb/c mice were obtained from the National Center for Laboratory Animals. Mice were randomly housed and habituated for two weeks. Cisplatin, purchased from Sigma, was dissolved in normal saline and prepared before each use. Mice were intraperitoneally injected with cisplatin (2 mg/kg) or vehicle (normal saline) for 5 days, followed by rest for 9 days, and repeated administration for 5 days. From week 1 to the end of the experiment, a fourth extract of Snakeweed was mixed into the feed. Body weight and food intake of the mice were monitored once a week to assess the physical performance of the mice.

The results of the animal experiment are shown in Figure 16, which is the result of the animal experiment of the fourth extract of Sabah snake grass extract according to the embodiment of the present invention for skeletal muscle atrophy, including the effect on cisplatin-induced malignant mice (A) Body weight change, (B) food intake, (C) forced swim test, and (D) grip strength test. a and b represent P values less than 0.05 compared with sham and cisplatin, respectively. Cisplatin induced symptoms of chemotherapy-induced cachexia, including weight loss, decreased appetite, and reduced physical activity in mice. The fourth extract of Sabah snake grass did not improve body weight loss (Figure 16A) and food intake (Figure 16B). However, in the forced swimming test ( FIG. 16C ) and grip strength test ( FIG. 16D ), it was found that the fourth extract of Sabah Snakeweed could improve physical activity tolerance and maintain forelimb muscle strength in cisplatin-treated mice. These results suggest that the fourth extract of Sabah Snake Grass can potentially attenuate muscle atrophy induced by cancer chemotherapy.

8. Promote wound healing

This embodiment includes two parts, including the migration test of isolated endothelial cells and the animal wound test. The isolated endothelial cell migration test steps include: culture the endothelial cell line in a 24-well plate, and put ibidi Culture-Insert 2 to create a wound of equidistant width, wait until the cells are fully covered, remove the Insert and add the drug, Continue to culture for 8 hours to observe the change of cell moving area at different time points. At different times, an optical microscope was used to take pictures under a 10x objective lens, and the area of cell movement was calculated using image J software, and the multiple ratio of the moving rate per hour (moving area/hour) to the control group was calculated.

The animal wound test steps include: after six-week-old C57BL/6 mice were depilated on their backs, a 5mm wound was made on the back with a surgical sampler, and the third extract of Sabah Snake Grass extract (divided by ethanol) layer) mixed with carbopol (carbopol) to form an ointment, which was applied to the wound every day, and the size of the wound was confirmed by taking pictures, and then the change of the wound area was calculated by image J software, and the ratio was calculated.

The results are shown in Figure 17 and Figure 18. Fig. 17 is the result of the migration experiment of the isolated endothelial cells of the third extract of Snakewort sabah extract according to the embodiment of the present invention. Fig. 18 is the result of animal wound experiment of the third extract of Snake grass extract according to the embodiment of the present invention.

It can be seen from Fig. 17 that the third extract (ethanol divided layer) of Sabah Snake Grass extract at 1 μg/ml can increase the cell mobility. Using the calculation of the moving area, increase the moving speed by about 1.5 times. In the results of the animal wound experiment in Figure 18, it was found that 1% Sabah Snake Herb ointment dressing can promote wound healing, and at the same time, section staining (Masson stain) showed that it can increase muscle fiber cell proliferation (increased red staining) and promote wound healing.

To sum up, the extract of Sabah Snake Grass can be used to prepare medicines for treating osteoporosis, neurodegenerative diseases, skeletal muscle atrophy and promoting wound healing, thereby reducing the burden on medical care and the cost of care in Taiwan.

Claims (1)

The use of a Sabah Snake Grass extract is for the preparation of drugs for the treatment of Alzheimer's disease and anti-aging, wherein the Sabah Snake Grass extract is the second water layer extract extracted by the following preparation method ( or called the second extract): extracting Snakegrass sabba with ethanol aqueous solution, taking the extract and concentrating to obtain a crude extract; adding the crude extract to ethyl acetate and water for liquid-liquid partition extraction to obtain a first water layer and an ethyl acetate layer; further adding n-butanol to the first water layer for liquid-liquid partition extraction, after the two extraction and separation layers were concentrated, a second water layer extract (or second extract) was obtained.

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