TWI436771B - Flos lonicerae extract, manufacturing method and use thereof - Google Patents

Description

Honeysuckle extract and preparation method and application thereof

The present invention relates to a honeysuckle extract, and more particularly to a honeysuckle extract having the efficacy against Propionibacterium acnes , and a preparation method and application thereof.

Due to geographical and environmental factors, and the abundant natural resources are available in the eastern part of Taiwan, the annual output value of natural products is about 1.2 billion yuan. In particular, there are many Taiwanese native plants in the east, including the honeysuckle in Taitung and the Tanjung in Hualien. The area is the most widely spread and unique. How to derive the use of Eastern special products for their products helps to drive the economic value of the East. One of the special uses - honeysuckle, in the "Compendium of Materia Medica" records that it is good at poisoning, so cure phlegm, swollen poison, sore sputum, etc.. Since ancient times, honeysuckle is often used for detoxification, treatment of fever, fever Toxic blood stasis, phlegm and ulcers are also used in wind-heat flu, bronchitis and other diseases. Modern pharmacological research shows that honeysuckle has antibacterial, antiviral, anti-inflammatory, antipyretic, immune regulation and other effects. It is pointed out that honeysuckle contains cyclohexanol, flavonoids, saponins, etc., which has a wide range of antibacterial effects. The literature indicates that it has strong inhibitory effects on Staphylococcus aureus, dysentery bacilli, diplococcus, and Bacillus cereus, and It also inhibits various pathogenic microorganisms such as Leptospira and influenza virus.

Acne is a chronic skin follicle inflammation caused by Propionibacterium acnes . Most of the current treatments for acne use Benzoyl Peroxide, Sulfur, Triclosan, Oral Antibiotics - Tetrahymena Tetracycline, topical antibiotics - erythromycin (Erythromycin), clindamycin (Clindamycin) and the like. The pathogen causing acne is Propionibacterium acnes , which also contains other commensal bacteria such as Staphylococcus aureus . Therefore, if a Chinese herbal medicine can be found and has the ability to resist such species, it will help. Used in the development of acne care related products.

The People's Republic of China patent application number CN200710003451.0, "Medicine containing gentamycin" patent, although the previous patent mentioned the use of silver flowers of the same family as honeysuckle, accounting for 8.6% of Chinese herbal medicines, however This extraction method is based on water extraction. The main purpose is to apply the Chinese medicine extract for the conditioning of blood and blood. The effect of the honeysuckle extract on the anti-acne bacillus is not discussed.

U.S. Patent No. 6,083,921, "Pharmaceutical Composition and method of using same", discloses a composition of a drug which is obtained by extracting active substances from Radix scutellariae, Flos Lonicera, and Fructus forsythiae. The antibacterial effect is different, but there is no anti-acne bacillus effect, and the previous honeysuckle extract is water-soluble chlorogenic acid, rather than extracting oil-soluble active substances.

The Republic of China Patent Publication No. 200932205 "External Agent for Skin for Acne Skin", the skin external preparation for acne skin of the previous case is characterized by at least one of rhododendrol and its derivatives, and a plant extract which combines an inhibitory effect on inflammation. For example, an extract from Gardenia Florida, Paeonia Albiflora root, and Lonicera japonica. The case did not provide an extraction method for honeysuckle extract, and did not investigate the efficacy of honeysuckle against acne bacteria.

U.S. Patent No. 5,834,000 "Antiviral and antimicrobial herbal complex" discloses the extraction of honeysuckle with 95% ethanol and 5% isopropyl alcohol at 82 ° C. The extract is mainly composed of flavonoids such as wood rhinoceros. Luteolin, Lonicerin, Luteolin glucoside, inositol, etc., have antibacterial and slowing sore throat effects, but the previous case did not reveal that honeysuckle extract has anti-acne Bacillus or the efficacy of treating acne.

U.S. Patent No. 6,063,383, "Pharmaceutical suppository composites for fever and influenza and method of producing the composites", discloses a suppository composition for the treatment of fever and epidemic cold, which contains honeysuckle (flos lonicerae japonicae). Previously, the case was extracted by high-temperature decoction, which has antibacterial effect. In addition to inhibiting fever and detoxification, it also mentioned that it can treat acne.

Since natural skin care is currently being sought to avoid the burden of skin caused by excess chemical products, it is necessary to seek other natural Chinese herbal medicines against acne bacteria for the development of acne care related materials.

The present invention provides a honeysuckle extract comprising: 20-50 parts by weight of chlorogenic acid; 1-5 parts by weight of oleanolic acid; and 0.001-5 parts by weight of bear Ursolic acid.

The invention also provides a method for preparing a honeysuckle extract, comprising: performing ultrasonic extraction on a honeysuckle by using a polar solvent containing ethanol to obtain a crude extract of honeysuckle, wherein the ultrasonic extraction temperature is about 15-45 ° C; and purification of the above crude honeysuckle extract by a molecularly imprinted polymer to obtain a honeysuckle extract.

The present invention also provides a dermatological drug comprising the above-described honeysuckle extract.

The invention provides a process for extracting and purifying honeysuckle, and the obtained honeysuckle brittle material has the active ingredient inhibiting the acne bacillus. The above-mentioned extraction and purification process of honeysuckle is to extract the active ingredient in the honeysuckle by the Chinese herbal medicine-honeysuckle in a low temperature ultrasonic process suitable for the solvent, which has the anti-acne bacteria effect. The invention further utilizes Molecular Imprinted Polymer (MIP) technology, which can be applied to the purification of the extract by short tube column chromatography or immersion filtration, and simultaneously improves the multiple active ingredients of the honeysuckle extract, Used in acne care related skin medications. In addition, the invention can also adopt a high-speed cell disruption-assisted extraction process to accelerate the extraction effect of the active ingredients.

In one embodiment, the composition of the above honeysuckle extract may include: 20-50 parts by weight, preferably 20-30 parts by weight of chlorogenic acid; 1-5 parts by weight, preferably 3-5 weight A portion of oleanolic acid; and 0.001 to 5 parts by weight, preferably 0.002 to 5 parts by weight, of ursolic acid. In another embodiment, the honeysuckle extract of the present invention consists essentially of the above-described chlorogenic acid, oleanolic acid, and ursolic acid (consisting essentially of) . In still another embodiment, the honeysuckle extract of the present invention consists solely of the above-described chlorogenic acid, oleanolic acid, and ursolic acid.

The method for preparing the above-mentioned crude extract of honeysuckle may include: selectively performing a cell disruption process on the honeysuckle by using a homogenizer to miniaturize the honeysuckle; and then using a polar solvent containing ethanol to treat gold and silver The flower is subjected to an ultrasonic extraction, followed by filtration to remove the solid residue to obtain a crude extract of honeysuckle. The honeysuckle here can use the commercially available dry goods of honeysuckle. The cell disruption process described above is an optional step to increase extraction efficiency, and the skilled artisan may also use other tools or means than conventional homogenizers for disruption. The above polar solvent containing ethanol may have an ethanol concentration of about 50-95 vol%, preferably about 70-95 vol%, and if the ethanol concentration is less than 50 vol%, it has an efficacy component for inhibiting acne bacillus, and is extracted. The amount coming out is insufficient. The ultrasonic extraction time is about 1-5 hours, preferably 2-3 hours, and the ultrasonic extraction temperature is about 15-45 ° C, preferably 15-35 ° C.

Next, the obtained crude extract of honeysuckle is purified by a molecularly imprinted polymer to obtain a honeysuckle extract. The invention adopts Molecular Imprinted Polymer (MIP) technology to extract and purify the active ingredients in honeysuckle. Molecularly-printed polymer is a synthetic polymer commonly used to identify molecules. It mainly uses the interaction force between a functional molecule (Function Monomer) and a template to achieve separation. It is a specific selectivity. Identification method, usually when the column column filling can enhance the purification effect.

In one embodiment, the method for purifying the obtained crude extract of honeysuckle by using the above molecular printing polymer comprises: preparing an active ingredient to be extracted as a template molecule, and then using a functional monomer (Functional monomer) The template molecule is captured, and the functional monomer is polymerized with a matrix monomer, and then the active ingredient is removed to obtain a molecularly imprinted material. In one embodiment, the above active ingredient may include: chlorogenic acid, oleanolic acid, ursolic acid, or a combination thereof. The functional monomer has a carboxylic acid group than the substrate monomer, and can form a hydrogen bond with the active ingredient to facilitate capturing the active ingredient. The above functional monomer may have a carboxylic acid, a sulfonic acid, or a heterocyclic group (for example, nitrogen and oxygen). Rings), or may include, but are not limited to, Methacrylic Acid (MAA), Acrylic Acid (AA), Itaconic Acid, 4-vinylbenzoic acid, Propylene 2-Acrylamido-2-methyl-1-propanesulfonic acid, 2-Vinylpyridine, 4-Vinylpyridine, or a combination thereof. The above substrate monomers may include, but are not limited to, methylmethacrylate (MMA), 4-divinylbenzene, ethylene glycol dimethacrylate (EGDMA), Or Pentaerythritol triacrylate (PETA).

Then, the above molecular rubbing material is further loaded into a column or a filter bag; and the above-mentioned crude honeysuckle extract is selectively subjected to a concentration and/or freeze-drying step, and then dissolved in ethanol/ Or methanol to obtain a honeysuckle crude extract ethanol / or methanol solution, and pass it through the above column or the above filter bag, to capture the honeysuckle rough extract by using the molecular column material in the above-mentioned column or the above filter bag The active ingredient in the product. The above ethanol or methanol concentration is about 30-95 vol%, preferably about 60-95 vol%, and if the ethanol concentration is less than 30 vol% or more than 95 vol%, the solubility of the crude extract of the honeysuckle is lowered, and further Reduce the recovery of the purification step. In another embodiment, the step of purifying the molecularly imprinted polymer may also directly use the unconcentrated honeysuckle crude extract to pass through the column or the filter bag to utilize the column. Or the molecular rubbing material in the above filter bag captures the active ingredient in the crude extract of honeysuckle. Finally, the above active ingredient is washed off with warm water of, for example, 40-60 ° C and collected to obtain a honeysuckle extract.

The Molecular Imprinted Polymer (MIP) technique used in the present invention uses a short column chromatography (<10 cm) or a immersion filtration method to make the separation effect relatively fuzzy, so that the size is equivalent and the same Several active ingredients of a functional group, such as chlorogenic acid, oleanolic acid, and ursolic acid, etc., for the acne care related products, the honeysuckle of the present invention The extract can also have the effect of resisting the pathogenic bacteria Acne and other commensal bacteria such as Staphylococcus aureus, and is more suitable for the application of acne care related materials.

In one embodiment, the method for preparing the silver flower extract of the present invention may further comprise: after obtaining a honeysuckle extract by polymer blotting, performing a concentration and freeze drying step on the honeysuckle extract to obtain a solid state. Honeysuckle extract.

In one embodiment, the honeysuckle extract obtained by the method for preparing the honeysuckle extract described above can be used as a dermatological drug, wherein the dermatological drug is directly filtered, concentrated and freeze-dried without a polymer printing and purification process. The concentration of the crude extract of the honeysuckle in the step is greater than about 10 mg/mL. If the solid honeysuckle extract is subjected to the polymer purification process, concentration and freeze-drying steps, the concentration is greater than about 0.1 mg/mL, preferably It is about 0.5-10 mg/mL. The types of the above drugs may include, but are not limited to, an emulsion type, a patch type, or a colloid type, and provide a new Chinese herbal medicine selection target for acne care.

In summary, the honeysuckle extract provided by the present invention, in addition to the efficacy of honeysuckle against Staphy1ococcus aureus , is found to have anti-Acne bacillus ability, so the honeysuckle extract of the present invention Propionibacterium acnes , which is simultaneously resistant to the pathogen of acne (acne), and the commensal Staphylococcus aureus , can be applied to dermatological conditions and have conditions for acne care.

The above and other objects, features, and advantages of the present invention will become more apparent and understood.

Example 1. Preparation of crude extract of honeysuckle

200g of dried honeysuckle is applied to the homogenizer, and the raw material is miniaturized and the cell wall is broken at a high speed of more than 10,000 rpm. It is expected that the high-speed cell disruption assists the extraction process to increase the extraction rate of the active material.

Next, the miniaturized honeysuckle powder was added with 1.8 liters of 95% high-concentration alcohol as a solvent, stirred and uniformly mixed, and then extracted by ultrasonic equipment at room temperature (25 ° C) for 2 hours.

Further, a filtration step is carried out: the extract after ultrasonic extraction is filtered with TOYO qualitative filter paper NO1 (pore size 6 μm/thickness 0.2 mm/diameter 110 mm/filter paper material: cotton fiber) to remove solid residue, and each filter paper is filtered. Replace the filter paper in about 45 seconds to avoid clogging the filter paper.

Then, a concentration step is carried out: the filtrate after filtration is removed by a rotary vacuum concentrator, the temperature of the water bath is 40 ° C, and the degree of vacuum is 650-720 mmHg; the initial volume is about 2000 ml, and the concentration is about 300 ml, and then added twice. Double deionized water, concentrated to 200 mL, divided into two bags, about 100 ml per bag, placed in a refrigerator at -20 ° C for one day.

Finally, a freeze-drying step is carried out: using a vacuum freeze dryer (temperature: -86.9 ° C, vacuum: 4.9 Pa), the drying time is about two days, more than water and solvent are removed, and the dried crude extract is obtained to facilitate subsequent components. use. This freeze-drying step is omitted for samples to be MIP purified.

Example 2. Analysis of the components of the crude extract of honeysuckle and the effect of anti-acne bacillus and Staphylococcus aureus

The test strain was cultured in an anaerobic state at 37 ° C for 24 to 48 hours, and after 2 days, the bacterial concentration was adjusted by serial 10-fold dilution with the culture solution. The test bacteria concentration was 10 ⁶ -10 ⁷ CFU/mL. The test substance and the test bacteria are mixed in an appropriate volume ratio, and the respective final concentrations are 50, 40, 30, 20, 10, 0.5, 0.3 mg/mL and the like, and (1~5)×10 ⁶ CFU/mL. (The test bacteria at the above concentration experiments were 2.7 × 10 ⁶ , 2.5 × 10 ⁶ , 2.5 × 10 ⁶ , 2.5 × 10 ⁶ , 3.5 × 10 ⁶ , 6.5 × 10 ⁶ , 9.2 × 10 ⁶ CFU / mL, respectively). At the time of design, 0.1 mL of the mixed culture solution was taken and applied to the vegetable medium, and placed in an incubator at 37 ° C for 24 to 48 hours under an anaerobic state, and counted after the colony appeared.

Inhibition rate = (A-B) / A × 100%

Due to the long acne, acne bacteria such as Staphylococcus aureus are often accompanied by acne bacteria, and the crude extract also contains chlorogenic acid. Table 1 shows that the crude extract of 95% alcohol of honeysuckle 20 mg/mL and the control group are treated with golden yellow. The antibacterial effect of Staphylococcus aureus (ATCC 6538P) after 18 hours, the data shows that the crude extract of honeysuckle alcohol of the present invention has the effect of inhibiting Staphylococcus aureus, thereby enhancing the effect of treating acne.

See Table 2 for the antibacterial effect of the crude extract of honeysuckle extracted at different alcohol concentrations. Among them, the anti-acne bacillus with 95% alcohol extract is better, and it is inferred from the component analysis (HPLC) of the crude extract that it should be the contribution of oleanolic acid and ursolic acid, so that 95% of the alcohol extracts are The ability to fight acne bacteria.

In order to verify the anti-acne bacillus active substance type, the standard was used to evaluate the anti-acne bacillus effect. Table 3 and Table 4 show the results of different standard quantitative strain culture counting methods, among which Oleanolic acid and bear fruit Ursolic acid has an anti-acne bacillus effect, while chlorogenic acid (Chlorogenic acid) will cause acne bacteria to germinate.

Table 5 shows the results of the culture count method for quantitative strains of different concentrations of crude extracts according to Example 2 of the present invention. Table 5 shows that the crude extract of honeysuckle obtained by extracting honeysuckle with 95% alcohol can achieve an effect of more than 98% against acne bacteria (ATCC 6919) at a concentration greater than 20 mg/ml.

1a-1d shows the results obtained by HPLC analysis of the crude extract of the honeysuckle by subtracting the background value of the chromatographic solution according to Example 1 of the present invention, wherein the first graph shows the crude honeysuckle obtained by extracting the honeysuckle with 95% alcohol. The composition of the extract is clearly found to have chlorogenic acid, oleanolic acid, and ursolic acid active ingredients (latitude of oleanolic acid and ursolic acid) The peak of time almost overlaps).

Example 3. The crude extract of honeysuckle was purified by molecular rubbing to obtain honeysuckle extract

Molecular printing polymer preparation method: 60mg of template molecule (Oleanolic acid), and 1.5 ml of methacrylic acid (MAA), after mixing, add 10.5ml methyl methacrylate (MMA) solution and then mix Stir well and deaerate by blowing nitrogen gas. Next, the solution was heated to 70 ° C under an inert gas, and then 10 mg of azobisisobutyronitrile (AIBN, Initiator) was added for about 2 to 3 hours to form a high molecular polymer. The above polymer was then pulverized by mechanical force, ground into a fine powder, and sieved through a 100 mesh metal mesh. Then, the sieved white powdery MIP material is added to warm water of 60 ° C to wash away the template molecules, washed several times, and then dried to obtain a dried MIP material (molecular blotting polymer).

Molecular rubbing purification method: The dried honeysuckle crude extract is first dissolved in 30% or 70% alcohol. Taking the dried MIP material, loading into a short pipe column, or loading into a filter bag, and then passing the above-mentioned honeysuckle crude extract ethanol solution through the above-mentioned pipe string or the above-mentioned filter bag to utilize the above-mentioned pipe string or the above filter bag The above-mentioned molecular rubbing material captures active ingredients such as Ursolic acid and Chlorogenic acid, which are similar to Oleanolic acid in the above-mentioned crude extract of honeysuckle, and finally The MIP of the active ingredient has been taken, added to warm water of 60 ° C to extract the active ingredient and collected, and dried and purified by freeze-drying to obtain a solid honeysuckle extract.

2a-2b is a diagram showing the composition of the purified honeysuckle extract by HPLC analysis according to an embodiment of the present invention. Figure 2a shows the HPLC component analysis of the crude extract of honeysuckle without molecular blotting (the background value of the chromatographic solution has been subtracted). Figure 2b shows the results of HPLC analysis of the honeysuckle extract purified by molecular blotting. In the molecularly purified and purified honeysuckle extract of Fig. 2b, the retardation time 3.49 peak (chlorogenic acid) and 63.15 peak (oleanolic acid) are taken as examples, and the ratio is increased by >10% compared with the second graph. And the purity of 2 times, the composition ratio calculated by the HPLC analysis result of the 2a-2b figure is compiled in Table 6.

Example 4. Comparison of the anti-acne bacillus effect of the crude extract of honeysuckle and the purified solid honeysuckle extract

The anti-acne bacillus effect containing 20 mg/ml of dried crude honeysuckle extract was analyzed to be 98%. Table 7 shows the results of quantitative counting of cultures of honeysuckle extract after molecular rubbing purification according to Example 3 of the present invention. The purified extract has a 2-fold increase in Oleanolic acid, which requires only 10 mg/ml of dried solid honeysuckle extract to achieve >99.99% antibacterial effect.

Example 5. Antioxidant analysis of DPPH (α,α-diphenyl-β-picrylhydrazyl) of crude extract of honeysuckle

Prepare 5 ml of 1000 ppm crude extract, then take 0.0197 g of DPPH (MW=394.32), prepare 50 ml of methanol solution (concentration of 1 mM), then take 5 ml of extract and add 1 ml of freshly prepared DPPH. (1 mM), mix and let stand for 30 minutes, and measure the absorbance at 517 nm. The sample was replaced with a solvent of the extract as a control group.

DPPH free radical scavenging percentage = [1 - (sample absorbance / unabsorbed control group absorbance)] × 100

Figure 3 is a diagram showing the DPPH free radical scavenging ability of the crude extract of honeysuckle obtained by crude extraction of honeysuckle with different concentrations of ethanol according to Example 5 of the present invention, wherein vitamin C and vitamin E are used as control groups. Today's silver flower extract has a DPPH removal capacity of more than 80% at a concentration of 250 ppm, and is close to about 95% of the DPPH removal capacity of vitamin C and vitamin E. Table 8 shows the antioxidant capacity obtained according to the DPPH test results of Fig. 3.

The anti-acne bacillus effect was verified by standard data. The data showed that Oleanolic acid and ursolic acid have anti-acne bacillus effect, and the acne care related products have the best anti-pathogenic bacteria acne And other commensal bacteria such as Staphylococcus aureus, so the best extraction and purification process can contain several active substances, such as chlorogenic acid, oleanolic acid, and ursolic acid Etc. Therefore, the molecularly-printed polymer is applied to the purification of short column (<10cm) chromatography or immersion filtration, which has the opportunity to simultaneously improve the active substances of honeysuckle extract and more opportunities for acne care related materials. .

The present invention has been disclosed in several embodiments, and is not intended to limit the invention, and any one of ordinary skill in the art can make any changes and modifications without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims.

1a-1d shows the results obtained by HPLC component analysis of the crude extract of honeysuckle (the background value of the chromatographic solution has been subtracted) according to an embodiment of the present invention, wherein the 1a-1d chart shows the standard, and 30% respectively. 60%, and 95% alcohol extracts of honeysuckle obtained from the honeysuckle crude extract.

2a-2b is a diagram showing the composition of the purified honeysuckle extract by HPLC analysis (after subtracting the background value of the chromatographic solution) according to an embodiment of the present invention, wherein the 2a image shows that the honeysuckle is not subjected to molecular rubbing and purification. The HPLC component analysis results of the extract, and the 2b chart shows the HPLC component analysis results of the molecularly purified honeysuckle extract.

Figure 3 is a graph showing the DPPH free radical scavenging ability of the crude extract of honeysuckle obtained by crude extraction of honeysuckle with different concentrations of ethanol according to an embodiment of the present invention.

Claims (9)

A honeysuckle extract comprising: 20-50 parts by weight of chlorogenic acid; 1-5 parts by weight of oleanolic acid; and 0.001-5 parts by weight of ursolic acid ( Ursolic acid). The honeysuckle extract according to claim 1, wherein the composition comprises: 20-30 parts by weight of chlorogenic acid; 3-5 parts by weight of oleanolic acid; and 0.002 - 5 parts by weight of ursolic acid. A method for preparing a honeysuckle extract comprises: performing ultrasonic extraction on a honeysuckle by using a polar solvent containing ethanol to obtain a crude extract of honeysuckle, and then performing a molecular imprinting on the crude extract of the honeysuckle The purification process is carried out to obtain a honeysuckle extract, wherein the ultrasonic extraction temperature is between about 15 and 45 °C. The method for preparing a honeysuckle extract according to claim 3, wherein the ultrasonic extraction time is between about 1 and 5 hours. The method for producing a honeysuckle extract according to claim 3, wherein the ethanol-containing polar solvent has an ethanol concentration of about 50 to 95 vol%. The method for producing a honeysuckle extract according to claim 3, wherein the ethanol-containing polar solvent has an ethanol concentration of about 70 to 95 vol%. A dermatological drug for treating a skin disease associated with acne bacteria and Staphylococcus aureus, including as described in claim 1 Honeysuckle extract. The dermatological application according to claim 7, wherein the concentration of the honeysuckle extract is >0.1 mg/mL. The dermatological drug according to claim 7, wherein the drug comprises: an emulsion type, a patch type, or a gel type.