JP5946509B2 - Method for producing longan seed extract and its application - Google Patents

Description

The present invention relates to a method for producing longan seed extract, and the obtained longan seed extract is applied to a living body and has anti-inflammation, reduction of serum uric acid level, promotion of keratinocyte proliferation, enhancement of wound healing and bacterial There are many effects such as suppression of activation.

1. Inflammation:
Inflammation is a symptom that cannot be overlooked by drug treatment, and is a warning signal from the body before the onset of illness. When human tissue is damaged due to bacteria, injury, chemicals, or other causes, macrophages are activated in the vicinity of the damaged tissue, precipitating foreign substances, and at the same time Factors of the defense response will also be released, and if the inflammation of the tissue continues to be stimulated by foreign substances, the defense system may last for months to years, often causing inflammation. The amount of released factor is also large, but depending on the results of the study, the factor includes nitric oxide (NO), tumor necrosis factor (TNF), and interleukin (IL). , Granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), granulocyte-monocyte colony stim It has been revealed that GM-CSF is included, and scholars have found that TNF produced from mononuclear cells and macrophages is converted to TNF-α and lymphotoxin produced from T cells (Lymphotoxin, LT ) And TNF-β, respectively.

Lipopolysaccharides (LPS) are the main components of endotoxins, and according to the results of animal experiments, LPS works to make it easy for food to stay in the stomach for a long time. Proven to be involved in the increase of cytokines and nitric oxide, but when living organisms are stimulated by LPS, they act on monocyte macrophages to synthesize various cytokines such as TNF-α, IL-1β, and IL-6 And induces the body's defense response and repair, but the synthesis of three types of cytokines, TNF-α, IL-1β, and IL-6, is performed in a cascade sequence, that is, by induction of LPS The synthesis of TNF-α, the synthesis of IL-1β by the induction of TNF-α, and the synthesis of IL-6 by the induction of IL-1β, but the overproduction of cytokines has a negative effect on the living body. For example, due to excessive production of TNF-α, , Susceptible to disseminated intravascular coagulation and toxic shock syndrome, or also a risk of death for the animals treated with anti-TNF antibody is that effectively prevent the lethal caused by toxic shock syndrome.

There are many types of anti-inflammatory drugs currently used in the pharmaceutical industry, such as antibiotics (Nantibiotics), non-steroidal anti-inflammation drugs (NSAIDs), anti-histamines (Anti-histamines). drugs), which have excellent anti-inflammatory effects, but also cause side effects such as drug resistance and damage to the gastrointestinal tract.

Second, gout:
Gout is a disease caused by abnormal purine metabolism or decreased excretion of uric acid. Hyperuricemia, Recurrent acute monoarthritis, gout nodules (Tophi) deposition due to sodium urate, gout Although there are clinical symptoms such as chronic arthritis, it is often the case that Gouty nephropathy eventually results without proper treatment. The disease can be broadly divided into two types: primary and secondary, but less than 1% of patients with primary gout are said to have developed an enzyme deficiency, usually unexplained, and clinically, gouty arthritis It is often accompanied by hyperlipidemia, hypertension, diabetes, arteriosclerosis and coronary artery disease, and secondary gout is a common complication caused by kidney disease, blood disease and drugs.
Hyperuricemia, the basis for gout attacks, is not a synonym for gout, and studies show that about 5 to 18.8% of patients end up with hyperuricemia to gout, but there are patients with gout It is said that it will become hyperuricemia at any stage.

In the laboratory, serum uric acid levels can be accurately measured by the uricase differential spectrophotometric method, but hyperuricemia is largely divided into two types, absolute and relative. When the upper limit of the amount that can be dissolved in is exceeded, it is called absolute hyperuricemia, and at a temperature of 37 ° C, the saturated serum uric acid level is 7 mg / dl. It gradually becomes needle-like crystals, and in general epidemiological studies, the upper limit of two standard deviations is the upper limit of the average serum uric acid level of normal subjects, and when the male uric acid level exceeds 7 mg / dl, the female If the dl is exceeded, it is considered that the patient suffered from relative hyperuricemia, but if the serum uric acid level exceeds 7 mg / dl, the incidence of gout or kidney stones is said to be high.

There are four clinical stages of gout attacks: asymptomatic hyperuricemia, acute gouty arthritis, inter-critical gout, and chronic tophaceous gout. There is.
To diagnose gout more accurately, there is a urate salt (Monosodium urate crystal) phagocytosed by neutral leukocytes after collecting joint fluid during a gouty acute arthritis attack. Can be confirmed as gout when negative birefringent is observed. Other common clinical symptoms of gout or laboratory tests include those in which a gout attack suddenly occurs on the big toe and single joints such as the back of the foot and ankle are red and swollen or severely painful, and colchicine is effective Or hyperuricemia patients, but they are only a reference for gout diagnosis.

Although the cause of primary gout is unknown and cannot be completely cured, the objectives of clinical treatment are: 1. Immediate control of acute attacks of gouty arthritis, 2. Through long-term treatment of hyperuricemia, uric acid sodium salt It is to prevent joint damage and kidney damage due to deposition.

In the selection of uric acid drugs, if renal function is normal or mildly impaired, or if the 24-hour urinary uric acid excretion is lower than 600 mg, administer a uric acid excretion-promoting drug and moderately damage renal function (creatinine clearance If the rate is <35 ml / min), or if uric acid in the 24-hour urine rises significantly, a uric acid production inhibitor is administered, but the serum uric acid level rises significantly and gouty nodules are deposited in large quantities In order to prevent progressive gouty complications, the above two drugs may be used in combination.

The uricuric agent is mainly used to promote the uric acid excretion by suppressing the reabsorption of uric acid in the proximal tubule, but the kidney is produced by excretion of a large amount of uric acid from the kidney In order to prevent side effects such as damage and kidney stones, the administration should be started from a small amount. In 7 to 10 days, the dose should be increased little by little to allow for urinary alkalinization. This class of drugs includes probenecid and benzbromarone.
The uric acid production inhibitor (Xanthine oxidase inhibitor) is only allopurinol, and its structure is similar to hypoxanthine, and has a function to strongly inhibit xanthine oxidase, gout nodules and kidney stones. In addition to inhibiting the formation of gout, it can also promote dissolution of gouty nodules. When combined with anti-cancer drugs, such as mercaptopurine or azathioprine, blood levels of anti-cancer drugs will rise, but at that time, you should be aware of appropriate doses and clinical side effects.

3. Wound healing:
The wound healing process is a dynamic interaction in many cells, including cell migration, cell proliferation, cell differentiation, extracellular matrix synthesis and tissue remodeling, etc. (Multiple steps), the process involves the regeneration of damaged epithelium and the repair of subcutaneous connective tissue, but during the healing of skin wounds, the epidermal keratinocytes on both sides of the wound edge proliferate, While moving to the center of the wound edge to form a new epidermal layer, the healing process takes days to weeks.

Growth factors closely related to wound healing include fibroblast growth factor 2 (FGF2), platelet-derived growth factor (PDGF), epidermal growth factor (Epidermal growth factor) , EGF), keratinocyte growth factor (KGF), transforming growth factor-α (Transforming growth factor-α, TGF-α), transforming growth factor-β (TGF-β) ) And Vascular endothelial growth factor (VEGF). Among these growth factors, PDGF, EGF, TGF-β and VEGF are secreted from keratinocytes, and PDGF is a macrophage. (Macrophages) and fibroblasts (Fibroblasts) are aspirated, matrix protein (Matrix protein) is promoted, EGF promotes autocrine migration and proliferation, TGF-β is fibroblast (Fibrobl) Asts) promotes cell migration and angiogenesis, and is released in large quantities at the beginning of wound healing. VEGF promotes vascular transit, proangiogenic matrix deposition and angiogenesis, mononuclear It is said to stimulate the movement of cells (Monocytes). According to the results of the study described above, any growth factors released from these keratinocytes are closely involved in the wound healing process.

On the other hand, the National Medicinal Medicinal Edition states that longan seeds are used for the treatment of stomach pain, burning, bruise bleeding, bruise pain, trauma bleeding, scabies, and psoriasis. People use longan seeds to treat trauma and have good effects on blood-stopping, analgesia, and muscle formation. It is said that it can be cured by applying it to the affected area after it has been baked in a fire and powdered while leaving its properties. " `` Many people were healed by the warriors of 秦 and 巴 理 坤. '' "It is painted on". Although the long-standing books and drug descriptions mentioned above show that longan seeds are effective in treating cutaneous wounds and related diseases and are effective in promoting wound healing, the mechanism of action is unknown. .

Patent Publication No. 2008-512221

An object of this invention is to provide the manufacturing method of a longan seed extract, and the application of a biological body.

The present invention first includes the following procedure,
(1) Make an extract with a specific concentration,
(2) Heat the extract to a specific temperature,
(3) Put the crushed longan seed granules in the extract, extract,
(4) Filter and concentrate the extracted solution,
(5) Freeze and dry the concentrated solution.
(6) A method for producing a longan seed extract comprising the completion of the production of longan seed extract is provided.

In the present invention, the extract is an aqueous solution or an alcohol solution, the alcohol solution is an ethanol solution, and the concentration of the ethanol solution is 20% to 95%. In step (2), the extract is 70 ° C to 90 ° C. In the heating and step (3), the extraction method is characterized in that the extraction temperature is 70 ° C. to 90 ° C. and the extraction time is 1 to 3 hours.

According to the present invention, it is further provided that the components of the longan seed extract produced by the method include corilazine, ellagic acid, and gallic acid. It is characterized by doing.

Moreover, the present invention further provides the main application of the longan seed extract,
The longan seed extract is applied to the anti-inflammatory of the living body,
The longan seed extract is applied to lowering uric acid in the body,
The longan seed extract is applied to promote wound healing in the living body,
The longan seed extract is characterized in that it is applied to suppress bacterial activation.

Longan seed extract obtained by the production method of the present invention, as shown below,
Used in the body to cause an anti-inflammatory reaction, suppress uric acid production, suppress bacterial activation, promote skin keratinocyte growth, improve wound healing speed, and at the same time damage or damage organs in the body There is a merit that there is no burden.

High-performance liquid chromatographic analysis diagram (HPLC diagram) of a standard solution containing gallic acid, corrazine, and ellagic acid. The high performance liquid chromatograph analysis figure (HPLC figure) of the longan seed extract of this invention. Line graph created from Table 3. Line graph created from Table 4. Histogram created from the average values in Table 7. Histogram created from the average values in Table 7. Histogram created from the average values in Table 8. Histogram created from the average values in Table 9. Line graph created from Table 10. Line graph created from Table 11.

Hereinafter, the technical solution of the present invention will be further described with specific examples. However, the present invention includes the following embodiments, but is not limited thereto. To the extent that it does not depart from the substance of the present invention.

(1) A hydrophilic solution (for example, water) or a lipophilic solution is used as an extract. In this example, an ethanol solution having a concentration of 20 to 95% is used as an extract.
(2) Heat the temperature of the extract to 70-90 ° C,
(3) Put the crushed longan seeds in the extract, maintain the extraction temperature at 70-90 ° C, and the extraction time is 1-3 hours,
(4) Filter and concentrate the extracted solution,
(5) Further freeze and dry at low temperature and low pressure,
(6) The production of longan seed extract is completed.

Analyzing longan seed extract prepared by high performance liquid chromatography (HPLC), the main composition containing gallic acid, corilagin and ellagic acid Components can be obtained, but the structural formulas are as follows.
Corilagin:

Gallic acid:

Ellagic acid:

The analysis conditions of the high performance liquid chromatography are shown in Table 1.


A standard solution containing phagolic acid, corrazine, and ellagic acid is used as a reference solution, and analysis is performed according to the analysis conditions of the high performance liquid chromatography to obtain the results shown in FIG. 1. According to the results, Retention times of acid (42.42μg / ml), corrazine (52.72μg / ml) and ellagic acid (22.4μg / ml) were 14.409, 43.304 and 63.489 minutes, respectively. When analyzed by liquid chromatography, the results shown in FIG. 2 are obtained. According to the results, the retention times of the components of the longan seed extract peak were 14.461, 43.302 and 63.476 minutes, respectively. The main components of the extract are the same as in the case of the standard solution, that is, the longan seed extract contains gallic acid, corrazine and ellagic acid.

In order to show the therapeutic effect of the prepared longan seed extract, various in vivo tests and in vitro tests were performed.

1. In-vivo test for anti-inflammatory effect:
(1) Preparation of materials:
Longan seed extract (extract is 50% ethanol solution), longan seed extract A (extract is pure water) and longan seed extract B (extract is 20% ethanol solution).
(2) In vivo test (feeding test):
1. Use 24 male SD rats (Sprague-dawley rats), each weighing approximately 200g to 250g, the room temperature in the breeding room is 23 ° C, and the lighting is switched between light and dark every 12 hours. Using a dedicated bait, the drinking water is treated with a reverse osmosis membrane.
2. Randomly divide the rats into 9 groups, each group feeding 6 animals,
(1) The control group orally administers only reverse osmosis membrane water,
(2) Longan seed extract was orally administered (0.5 g / Kg, rat was orally administered 0.5 g longan seed extract per 1 kg body weight), and one week later, intraperitoneal injection of E. coli lipopolysaccharide (Lipopolysaccharides, LPS, 2.5mg / Kg, rats were injected with 2.5mg E. coli lipopolysaccharide per kg body weight), 24 hours later,
(3) Longan seed extract (0.5g / Kg) was orally administered, one week later, intraperitoneal injection of LPS (2.5mg / Kg) was performed, 48 hours later,
(4) LPS (2.5mg / Kg) was injected intraperitoneally, 24 hours later, longan seed extract A (0.5g / Kg) was orally administered.
(5) LPS (2.5 mg / Kg) was injected intraperitoneally, 24 hours later, longan seed extract B (0.5 g / Kg) was orally administered.
(6) LPS (2.5mg / Kg) was injected intraperitoneally, 24 hours later, longan seed extract (0.5g / Kg) was orally administered,
(7) LPS (2.5mg / Kg) was injected intraperitoneally, 48 hours later, longan seed extract (0.5g / Kg) was orally administered.
(8) A single intraperitoneal injection of LPS (2.5mg / Kg) was performed, 24 hours later,
(9) Longan seed extract (0.5 g / Kg) is orally administered alone.
After administration of the drug, fasted overnight, followed by paralysis with ethyl ether, blood is collected from the celiac artery of SD rats and used for serum immunoassay.
3. Serum immunity test:
Tumor necrosis factor (TNF-α) cell interleukin (IL-1β) is measured by enzyme-linked immunosorbent assay (ELISA).
4. Statistical methods:
All data obtained in this experiment is performed by one-way analysis of variance (ANOVA).
5. Experimental results:
As shown in Table 2, in IL-1β, oral reaction of longan seed extract and intraperitoneal injection of LPS both caused an immune reaction in SD rats, but as shown in Table 3 and FIG. In TNF-α, longan seed extract was orally administered in advance, and then intraperitoneal injection of LPS or single oral administration of longan seed extract was both anti-inflammatory and treated with LPS reverse osmosis membrane After that, when longan seed extract was orally administered, it was found that there was no anti-inflammatory effect on SD rats.

2. In-vivo test and in-vitro test for anti-gout effect:
(1) Preparation of materials: Longan seed extract (50% ethanol solution for the extract).
(2) In vivo test (feeding test):
1. Use 24 male SD rats (Sprague-dawley rats), each weighing approximately 200g to 250g, the room temperature in the breeding room is 23 ° C, and the lighting is switched between light and dark every 12 hours. Using a dedicated bait, the drinking water is treated with a reverse osmosis membrane.
2. Randomly divide the rats into three groups, each group is 8 animals, one group is a control group, the group is orally administered with reverse osmosis membrane water, the other group is an experimental group, Administer 6-hypoxanthine (Hypoxathine) with a rat weight of 300 mg / Kg, and administer oxonic acid (Oxonic acid, a urate-degrading enzyme inhibitor) with a rat weight of 250 mg / Kg. Longan seed set, which includes 6-hypoxanthine with 300 mg / Kg rat weight, Oxonic acid with 250 mg / Kg rat weight, and 0.1% (wt%) longan seed The main purpose of the combination of oxonic acid is to induce the formation of hyperuricemia in the SD rat body. The SD rat is allowed to drink water freely. During the drug administration period, animals were observed twice daily, weighed once daily, finished drug administration, and fasted overnight , Then hemp drunk with ethyl ether, bled from the tail of the SD rats, used in the serum biochemistry.
(1) Control group: Oral administration of reverse osmosis membrane water.
(2) Experimental set: 6-hypoxanthine (Hypoxathine) with a rat weight of 300 mg / Kg is orally administered, followed by administration of oxonic acid with a rat weight of 250 mg / Kg.
(3) Longan seed group Rat body weight: 300 mg / Kg of 6-hypoxanthine, 250 mg / Kg of oxonic acid and 0.1% (Wt%) longan seeds are administered.
3. Serum biochemistry:
The serum uric acid level of SD rats is measured by biochemical automatic analyzer (Ciba-cornint 550) measurement.
4. Statistical methods:
All data obtained in this experiment is performed by one-way analysis of variance (ANOVA).
5. Experimental results:
As shown in Table 4 and FIG. 4, administration of longan seed extract (the extract is an ethanol solution 50%) effectively reduces the serum uric acid level in SD rats, and the reduction effect reaches 32%.

(3) In vitro experiment (Xanthine oxidase inhibition experiment):
1. Prepare xanthine 50mmol / L with phosphate buffer solution (PBS),
2. Prepare xanthine oxidase with phosphate buffer solution (PBS) to 0.1-0.2 unit / ml,
3. Preparation of various longan seed extract samples:
(1) Extracting and preparing longan seeds with pure water
(2) Extract and prepare longan seeds with 20% ethanol solution
(3) Extract and prepare longan seeds with 50% ethanol solution
(4) Extract and prepare longan seeds with 95% ethanol solution.
4. Positive control group-Clinical drug Allopurinol is used, Xanthine oxidase is added to the positive control group, reacted for 5 minutes, and then xanthine is added.
5. Use a blank control set and add pure water only to the set.
6. Add Xanthine oxidase to each sample of longan seed extract, react for 5 minutes, and then add xanthine.
7. Using a spectrophotometer at a wavelength of 290 nm, measure the change in absorbance of each sample of the longan seed extract and the control group, measure once every 20 seconds for a total of 5 minutes, and then measure the enzyme with the measuring instrument. Calculate activity.
8. Activity inhibition rate = [1- (experimental group activity / control group activity)].
9. Experimental results:
As shown in Table 5, each longan seed extract has an inhibitory effect on xanthine oxidase activity, and the activity inhibition rate has reached 60%.

3. Toxicity test of anti-gout effect:
(1) Material preparation: Longan seed extract.
(2) Acute toxicity test: SD rats are divided into 2 groups, each group is 8-10 animals, fasted overnight before the experiment, but drinking water is consumed, but longan seed extract (450 mg / ml) is taken orally After administration, the symptoms of intoxication are observed and the change in body weight and the number of deaths within 14 days are recorded. The half-lethal dose (LD ₅₀ ) and its 95% confidence limit are calculated by the Litctchfield-Wilcoxon method.
(3) Acute toxicity test with 28 days of feeding: The rats were divided into two groups, each group consisting of 10 animals, 1 g / kg longan seed extract, 3 g / kg and deionized water (1 ml / 100 g). Oral administration is performed for 28 consecutive days. During the drug administration period, the animals are observed twice daily and weighed once a week, but after the drug administration and fasting overnight, they are paralyzed with ethyl ether, and SD rats Blood is collected from the celiac artery of the dog and used for hematology and serum biochemistry.
(4) Serum biochemistry:
Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), albumin (Albumin), globulin (Globulin), and creatinine (Greatinine) are measured by an automatic biochemical analyzer (Ciba-cornint 550).
(5) Statistical methods:
All data obtained in this experiment are performed by one-way analysis of variance (ANOVA) and Dunnett test, p <0.01 is recognized as a significant difference.
(6) Experimental results:
1. Acute toxicity:
The main purpose of the acute toxicity test is to determine the half-lethal dose of the test animal with a single drug administration. The maximum fasting dose can be administered to SD rats at a dose of 1.0 ml per 100 g body weight. The drug concentration is 450 mg / ml, therefore the dose for the acute toxic test is based on 15 g / kg, so if the SD rat does not die completely, no further action is taken.
Based on the single oral administration of 15 g / kg of longan seed extract to 10 SD rats, and observed for 14 days, there was no death, so the half-lethal dose (LD50) of SD rats was greater than 15 g / kg. Judgment, 14 days after administration of longan seed extract, it was recognized that there was no difference in the body weight of SD rats compared to the control group.
2. Toxicity test of continuous feeding for 28 days:
In principle, the maximum dose is a half-lethal dose of 1/5, and the maximum dose is 3 g / kg and 1 g / kg.
(1) Each group was 8-10 SD rats, longan seed extract 1 g / kg, 3 g / kg was orally administered for 28 consecutive days, and there was no death. We recognized that there was no significant change.
(2) Serum biochemistry:
As shown in Table 6, when longan seed extract was orally administered continuously for 28 days, it was considered that there was no difference in the serum biochemical index between the extract group and the normal control group between 1 g / kg and 3 g / kg of SD rats. It was.
(3) Major organ weights:
Longan seed extract was orally administered for 28 consecutive days, but the liver and kidney weights of SD rats 1g / kg and 3g / kg were not different from those in the control group. It was found that there was no effect on weight.
(4) Pathological examination:
Pathological examination was performed on 1g / kg and 3g / kg pairs of longan seed extract administered orally for 28 consecutive days. The heart, liver, kidney, spleen, adrenal gland, seminal vesicle, and testicles of SD rats were completely changed. None.
3. Experimental results:
The longan seed extract was determined not to cause damage to organs in the body or burden to organs.

4. Bacteriostatic test:
(1) Material:
"P bean coagulum (product name including longan seed extract)" 2.5mg / ml (2.5mg of longan seed extract per 1ml of "P bean coagulum") ), Prepare a 1 × isotonic phosphate solution with reverse osmosis membrane water (1 × PBS, isotonic solution is a solution that prevents deformation of the erythrocyte membrane), and then prepare The isotonic phosphate solution is filtered through a sterile filter (Mini pore) to prepare a sterile “P bean glue” isotonic phosphate solution.
(2) Test species:
Escherichia coli and Staphylococcus aureus:
1. Escherichia coli and Staphylococcus aureus were each LB broth liquid medium, continuously cultured at 37 ° C for 16 hours, and the cultured bacterial solution was washed three times with 1 × isotonic phosphate solution (1 × PBS), after each washing Centrifuge at 3000 rpm for 10 minutes and remove the supernatant, but measure the absorbance (OD value) of the washed bacterial solution with a photometer, and finally dilute with a 1-fold isotonic phosphate solution to obtain an absorbance of 0.3 ( Prepare and test the bacterial solution of OD = 0.3).
2. To each prepared isotonic phosphoric acid solution (1 × PBS), add 10 times serially diluted Escherichia coli and Staphylococcus aureus solution and treat at 37 ° C. for 1 hour.
3. Take 5, 10, 20, 50, 100 μl from the bacterial solution after 1 hour treatment, apply to LB medium, continue culture at 37 ° C for 18 hours, and then count the number of bacteria in each medium. Treating the solution with an isotonic phosphoric acid solution of P-bean coagulum is an experimental group, and treating the bacterial solution with an isotonic phosphate solution of reverse osmosis membrane water is a control group.
4. Repeat the above procedure three times, and finally stat the results.
5. Experimental results:
As shown in Table 7 and FIG. 5, since the number of E. coli and Staphylococcus aureus groups was significantly reduced in the experimental group compared with the control group, “P bean coagulum” mainly composed of longan seed extract was used. Certainly, it has been shown that it has an inhibitory function against Escherichia coli and Staphylococcus aureus, which is applied to the suppression of acne and pimples.


(3) Test species: Propionibacterium acne:
1. Acne bacteria are continuously cultured in anBAP broth liquid medium at 37 ° C for 48 hours, and the cultured bacteria solution is washed three times with a 1-fold isotonic phosphate solution (1 x PBS). Centrifugation for 5 minutes, remove the supernatant, but measure the absorbance (OD value) of the washed bacterial solution with a photometer, and finally dilute it with a 1-fold isotonic phosphate solution. Prepare the fungus and experiment.
2. To each prepared isotonic phosphate solution (1 × PBS), add 10-fold serially diluted acne bacteria and treat continuously at 30 ° C. for 1 hour.
3. Take 5, 10, 20, 50, 100 μl from the bacterial solution after 1 hour treatment, apply to LB medium, continuously culture at 30 ° C for 48 hours, and then count the number of bacteria in each medium. Was treated with an isotonic phosphate solution of P bean coagulum in the experimental group, and the bacterial solution was treated with an isotonic phosphate solution in reverse osmosis membrane water as a control group.
4. Repeat the above procedure three times, and finally stat the results.
5. Experimental results:
As shown in Table 8 and FIG. 6, since the number of acne bacteria in the experimental group was significantly reduced compared to the control group, the “P bean coagulum” composed mainly of longan seed extract is certainly It becomes clear that there is a suppressive function against acne bacteria, which is applied to control acne and pimples.


(Four) Test species: red white癬菌(Trichophyton rubrum):
1, the red white癬菌IMA plate (Inhibit mold Agar) medium, 96 hours continuous culture at 30 ° C., three times washing the culture already bacterial solution in one times the isotonic phosphate solution (1 × PBS), washed each Centrifuge at 3000 rpm for 10 minutes and remove the supernatant, but measure the absorbance (OD value) of the washed bacterial solution with a photometer, and finally dilute with a 1-fold isotonic phosphate solution to obtain an absorbance of 0.1 ( OD = 0.1) is prepared and experimented.
2, the ready-haploid isotonic phosphate solution (1 × PBS), a 10-fold serial diluted red white癬菌added respectively, for 1 hour continuous treatment at 30 ° C..
3. Take 5, 10, 20, 50, 100 μl from the bacterial solution after 1 hour treatment, apply to LB medium, continuously incubate at 130 ° C for 96 hours, and then count the number of bacteria in each medium. Was treated with an isotonic phosphate solution of P bean coagulum in the experimental group, and the bacterial solution was treated with an isotonic phosphate solution in reverse osmosis membrane water as a control group.
4. Repeat the above procedure three times, and finally stat the results.
5. Experimental results:
As shown in Table 9 and Figure 7, compared to the control group, experimental group from that red white癬菌decreased significantly (about 30%), as a main ingredient longan seed extract "P Mameko glue "certainly become apparent that there is suppression function against germs that red white癬菌, it is applied to produce inhibition of athlete's foot.

5. Wound healing mechanism promotion test:
(1) Preparation of materials:
1. Dissolve 5 g longan seed extract in 250 ml secondary water, filter with filter paper No. 2, and then filter with 0.45 μm and 0.22 μm filters.
2. Longan seed extract 0%, 0.25%, 2.5%, 5.0% and 10.0% (Wt%) are prepared as a keratinocyte culture solution.
3. Human epidermal keratinocytes (HEKa-C005-5C).
(2) Cell culture: human epidermal keratinocytes 1 × 104 cells / ml (HEKa-C005-5C), keratinocyte medium (Cascade biologics) supplemented with keratinocyte growth factor (KC supplements) and penicillin-streptomycin , USA) at 37 ° C. in a culture box containing 5% CO ₂ and cultured. This is a primary cell, and the medium is changed once every two days. Add trypsin-EDTA solution 0.25% (trypsin-EDTA solution, containing trypsin 0.25% and EDTA 0.02%), act at 37 ° C for 5 minutes, then suspend the keratinocytes, After washing and neutralizing with 10%, put in a centrifuge tube, centrifuge at 1500 rpm for 10 minutes, remove the supernatant, and then resuspend the cells in keratinocyte medium and dilute 1: 3 In addition, the cells are put into a culture box containing 5% CO ₂ and cultured, but the third generation cells are taken and experimented.
(3) Cell proliferative capacity measurement: Crystal violet staining is performed, but human epidermal keratinocytes (HEKa-C005-5C) are cultured for 24, 48, 72 hours, and then cell growth is first performed with an inverted microscope. Observe the actual situation, wash the cells twice with 200 μl supernatant, fix the cells with cytology fixative for 20 minutes, and then wash the cells twice with 200 μl PBST, but crystal violet Stain for 30 minutes at room temperature with 100 μl of the solution, and then wash the cells three times with 200 μl of the supernatant. Lyse the cells with SDS 1%, then shake and culture at room temperature for 1 hour, but use the crystal violet dye that stained the cell nucleus. Elution is performed, and the absorbance (OD value) at a wavelength of 595 nm is measured. At the same time, the absorbance (OD value) is corrected at a reference wavelength of 650 nm.
A group to which no longan seed extract is added is set as a control group, and the growth promotion rate is calculated.
(4) Measurement of the amount of growth factors released by human epidermal keratinocytes by ELISA:
1. After the human epidermal keratinocytes are stimulated, the supernatant is collected, and the amount of growth factors in the culture solution is measured with Commercial kits.
2. First, using a 96 well plate microtiter plate, the unbound position was inhibited with bovine serum albumin, and further washed with PBS-Tween for stimulation. Add 100 μl of the supernatant, react at 37 ° C. for 2 hours, and then wash with PBS-Tween, then add rabbit anti-growth factor Ab-HRP Chemicon, Temecula, CA After reacting at 37 ° C for 2 hours, washing and adding a chromogenic substrate (Substrate, containing o-phenylenediamine), after coloring, add 50 µl of 2NH ₂ SO ₄ and react. At the same time, the absorbance at a wavelength of 450 nm is measured, from which the vascular endothelial growth factor (VEGF) concentration of growth factors is measured.
(5) Experimental results:
1. As shown in Table 10 and FIG. 8, the proliferative ability obtained by measuring with the crystal violet staining method is consistent with the result seen under a microscope, and longan seed extract 2.5%, 5.0% and 10%. The keratinocyte growth-promoting folds in the% group are 1.25, 1.26 and 1.50 times that in the control group, respectively. Among them, only the 10% longan seed extract group has a statistically significant difference (p value <0.05). The results revealed that 10% longan seed extract was effective in promoting the growth of human epidermal keratinocytes.
2. As shown in Table 11 and FIG. 9, according to the measurement results of enzyme immunosorbent assay (ELISA), the combination of collagen I (Collagen I, CI) and fibronectin (Fibronectin, FN) was applied to longan seed extraction. After 5% and 10% of the product was added and cultured, the vascular endothelial growth factor (VEGF) in the supernatant was significantly increased by increasing the dose (p <0.05), from which the longan seed extract was In the process of promoting wound healing, it has been found that there is a function to promote the vascular growth mechanism and it can promote wound healing in the living body.

As can be seen from the in-vivo tests, in-vitro tests and toxicity tests described above, the longan seed extract of the present invention is used in the living body to cause an anti-inflammatory reaction, suppress uric acid production, suppress bacterial activation, and At the same time as promoting growth and improving wound healing speed, the longan seed extract is safe to use because it does not damage or burden the organs in the body.

Claims (10)

Acne bacteria or is in the manufacture of a medicament for inhibiting the growth of red white癬菌, use of longan seed extract. Longan seed extract has the following steps:
(1) a step of preparing an extract with a specific concentration;
(2) heating the temperature of the extract to a specific temperature;
(3) A step of putting crushed longan seeds into the extract for extraction;
(4) A step of filtering and concentrating the solution after extraction;
(5) freezing and drying the concentrated solution to produce an extract; and (6) obtaining a longan seed extract;
Use according to claim 1, produced by an extraction process comprising:   Use according to claim 2, wherein the extraction solvent is either a hydrophilic solution or a lipophilic solution.   4. Use according to claim 3, wherein the extraction solvent is an aqueous ethanol solution.   Use according to claim 4, wherein the ethanol concentration of the aqueous ethanol solution is about 20-95%.   Use according to claim 2, wherein the heating temperature in step (2) is about 70-90 ° C.   Use according to claim 2, wherein the extraction temperature in step (3) is about 70-90 ° C.   Use according to claim 2, wherein the extraction time in step (3) is about 1 to 3 hours.   The use according to claim 1, wherein the extract comprises corilazine, ellagic acid and gallic acid.   Use according to claim 1, wherein the medicament is a medicament for the treatment of acne, pimples and / or athlete's foot.