Preparation method of tsaoko extract

Abstract

The invention relates to the field of oral care technology, and in particular to a method for preparing an extract of tsaoko. The invention discloses a method for preparing an extract of tsaoko and a mouthwash for inhibiting major oral pathogens. The mouthwash prepared from the extract of tsaoko of the invention has the effect of enhancing the inhibition of major oral pathogens (Staphylococcus aureus, Streptococcus mutans, Porphyromonas gingivalis). The tsaoko extract is prepared by processed tsaoko kernels, and the tsaoko processing method comprises the following steps: shelling the tsaoko, which is the main raw material of the mouthwash, to obtain the kernels, preparing dough, wrapping the tsaoko kernels with the dough, adding talcum powder pre-heated to 170-175°C in a pot, stir-frying and stewing until the dough is browned, stewing for 18 minutes, taking out, sieving out the talcum powder and peeling off the dough, and obtaining stewed tsaoko kernels. By comparing the antibacterial effects of mouthwashes prepared with simmered cardamom kernels, raw cardamom kernels, and whole cardamom fruits, it was found that the simmered cardamom kernel mouthwash was more effective in inhibiting the main oral pathogens than the raw cardamom kernels and whole cardamom fruit mouthwashes.

Description

Technical Field

[0001] The invention relates to the technical field of oral care, and in particular to a method for preparing an extract of Amomum villosum. Background Art

[0002] Tsaoko is the dried mature fruit of Amomum tsao-ko Crevost et Lemaire, a plant of the ginger family. Tsaoko is pungent and warm, and enters the spleen and stomach meridians. It has the effects of drying dampness and warming the middle, stopping malaria and removing phlegm. It can be used to treat internal cold and dampness, abdominal distension and pain, fullness and vomiting, malaria, fever, and other symptoms. The effects of Tsaoko were first seen in the Yuan Dynasty's "Yinshi Zhengyao" that "treats heart and stomach pain, stops vomiting, nourishes the stomach, and relieves gas." The main component of Tsaoko is volatile oil, which has multiple pharmacological effects such as anti-inflammatory, antibacterial, antioxidant, anti-tumor, and regulating intestinal flora.

[0003] In clinical practice, the herbal material of Amomum villosum must be processed into decoction pieces before it can be used. There are many records of the processing methods of Amomum villosum in ancient herbal books and classic Chinese medicine books, such as "Compendium of Materia Medica Mengquan" "When adding materials to medicine, take the seeds and grind them into pieces" and "Compendium of Materia Medica Fengyuan" "Remove the shell and use it raw". In addition to the stir-frying method, the auxiliary material processing methods such as ginger processing and salt processing are also recorded. However, to date, except for the ginger and amomum villosum kernels, the other methods are no longer widely used.

[0004] Processing is an effective processing method to change the properties of drugs, facilitate preparation and storage, remove edible and medicinal parts, and eliminate or reduce the toxicity, potency or side effects of traditional Chinese medicines. However, one of the existing processing methods (stewed) is to stew the whole fruit of the grass fruit with shell, and then remove the shell and take out the kernel, but the extraction rate of the active ingredients in the grass fruit is not high, and the utilization rate of the active ingredients is not high. Moreover, the processing method and processed products of the grass fruit have not been applied to the field of Chinese herbal mouthwash.

[0005] Therefore, it is necessary to provide a method for preparing tsaoko with safe and reliable process and improved quality and efficacy, and to provide a Chinese herbal medicine mouthwash. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing an extract of Amomum villosum.

[0007] The present invention provides a method for preparing an extract of Amomum villosum. The present invention mitigates the medicinal properties of Amomum villosum by processing Amomum villosum kernels, improves the leaching rate of effective ingredients of Amomum villosum, and also improves the utilization rate of effective ingredients in Amomum villosum. The processing process of the present invention is safe and reliable, so that Amomum villosum can be innovatively utilized in traditional Chinese medicine, and provides a guarantee for the raw material quality of Amomum villosum mouthwash and Amomum villosum-related preparations.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a method for preparing an extract of Amomum villosum, comprising the steps of crushing the simmered Amomum villosum kernels, mixing them with water and then soaking them, performing ultrasonic extraction, filtering and centrifuging.

[0010] The simmering starting temperature is 170-175°C;

[0011] The temperature at which the simmering is completed is 215-224°C;

[0012] The simmering time is 18 minutes.

[0013] In some specific embodiments of the present invention, the simmering step includes: taking raw cardamom kernels and wrapping them with dough, and then mixing them with talcum powder and heating them.

[0014] In some specific embodiments of the present invention, each piece of the dough wraps 15 to 20 g of the grass nut kernels.

[0015] In some specific embodiments of the present invention, the mass ratio of the simmered cardamom kernels to water is 1:10.

[0016] In some specific embodiments of the present invention, the temperature of the ultrasonic extraction is 75° C., the power is 300 W, and the time is 1 hour.

[0017] In some specific embodiments of the present invention, the soaking time is 7 h.

[0018] In some specific embodiments of the present invention, the conditions for ultrasonic extraction are: constant temperature of 75°C, extraction power of 300W, ultrasound on for 3s and off for 2s, and extraction time of 1 h.

[0019] In some specific embodiments of the present invention, the filtration pore size is 200 mesh.

[0020] In some specific embodiments of the present invention, the centrifugation comprises: 4000 r / min, 15 min.

[0021] In some specific embodiments of the present invention, the preparation method prepares the amomum villosum extract.

[0022] The invention also provides application of the amomum villosum extract in antibacterial treatment.

[0023] In some specific embodiments of the present invention, the antibacterial activity includes inhibiting one or more of Staphylococcus aureus, Streptococcus mutans or Porphyromonas gingivalis.

[0024] The invention also provides application of the amomum villosum extract in preparing mouthwash.

[0025] Based on the above research, the present invention also provides a mouthwash, comprising the tsaoko extract, a moisturizer, a freshener, a sweetener, sodium bicarbonate, citric acid and water.

[0026] In some specific embodiments of the present invention, the humectant includes glycerin and sorbitol; the freshener is mint powder; and the sweetener is xylitol.

[0027] In some specific embodiments of the present invention, the composition comprises, by weight percentage, 14% to 18% of the amomum villosum extract, 0.05% to 5% of the moisturizer, 0.08% to 0.16% of the freshener, 0.05% to 0.2% of the sweetener, 0.06% of the sodium bicarbonate, 0.02% to 0.06% of the citric acid and 77.52% to 82.74% of the water.

[0028] In some specific embodiments of the present invention, the composition comprises, by weight percentage, 14% to 18% of amomum villosum extract, 3% to 5% of glycerol, 0.02% to 0.06% of citric acid, 0.05% to 0.2% of xylitol, 0.05% to 0.15% of sorbitol, 0.08% to 0.16% of mint powder, 0.06% of sodium bicarbonate and 77.52% to 82.74% of water.

[0029] In some specific embodiments of the present invention, the composition comprises, by weight percentage, 14% tsaoko extract, 3% glycerol, 0.02% citric acid, 0.05% xylitol, 0.05% sorbitol, 0.08% mint powder, 0.06% sodium bicarbonate and 82.74% water; or

[0030] 16% Amomum villosum extract, 5% glycerin, 0.04% citric acid, 0.2% xylitol, 0.15% sorbitol, 0.12% peppermint powder, 0.06% sodium bicarbonate and 78.41% water; or

[0031] 18% Amomum villosum extract, 4% glycerin, 0.06% citric acid, 0.1% xylitol, 0.1% sorbitol, 0.16% peppermint powder, 0.06% sodium bicarbonate and 77.52% water.

[0032] In some specific embodiments of the present invention, the pH of the mouthwash is 6.9-7.3.

[0033] The invention provides a method for preparing a tsaoko extract. The method comprises the steps of removing the shell of dried tsaoko and taking out the kernel, processing the tsaoko kernel, extracting the stewed tsaoko kernel, raw tsaoko kernel and whole tsaoko, preparing a tsaoko mouthwash after obtaining the extract, and finally evaluating the antibacterial efficacy of the tsaoko mouthwash. By comparing the antibacterial efficacy of three different tsaoko mouthwashes, a mouthwash with better antibacterial efficacy under the processing method is obtained. DETAILED DESCRIPTION

[0034] The present invention discloses a method for preparing an extract of Amomum villosum. A person skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0035] Studies have shown that the extract of tsaoko kernel has a good inhibitory effect on the main pathogenic bacteria in the human oral cavity. The present invention utilizes this effect of tsaoko and processes tsaoko kernel to obtain a tsaoko mouthwash with better antibacterial effect. Processing of tsaoko kernel is conducive to the dissolution of effective components, improves the utilization of effective components of tsaoko kernel, and enhances the pharmacological effect of tsaoko kernel.

[0036] The invention provides a method for preparing tsaoko fruit for inhibiting main oral pathogenic bacteria.

[0037] The innovation of the invention lies in that the dry grass fruit is shelled and kernels are taken out, the grass fruit kernels are processed, and then the stewed grass fruit kernels, raw grass fruit kernels and whole grass fruit are extracted to prepare the grass fruit mouthwash after obtaining the extract, and finally the antibacterial effect of the grass fruit mouthwash is evaluated, and a mouthwash with better antibacterial effect under the processing method is obtained by comparing the antibacterial effects of three different grass fruit mouthwashes.

[0038] The technical scheme of the present invention is achieved by the following measures: a method for preparing grass fruit kernels, firstly, taking grass fruit fruits, drying the grass fruit naturally to obtain dried grass fruit whole fruits, then breaking the shells of the dried grass fruit whole fruits to obtain kernels, and obtaining raw grass fruit kernels. Taking an appropriate amount of raw grass fruit kernels and wrapping them with wet dough, adding talcum powder into a pot and heating it, burying the wrapped grass fruit kernels in the hot talcum powder, stir-frying and stewing until the dough skin is browned, taking them out, sieving the talcum powder and peeling off the dough skin, cooling them, and sealing them for storage.

[0039] The following is a further optimization description of the above invention technical solution:

[0040] When stewing the grass nut kernels as mentioned above, each piece of dough wraps 15 to 20 g of grass nut kernels. The stewing weight is preferably based on the weight wrapped by the dough. Before stewing, talcum powder is added to the pot and heated to 170 to 175°C, and then the wrapped grass nut kernels are added. The stewing time is 18 minutes, and the stewing temperature is strictly controlled. After stewing, the talcum powder is sieved out and the dough is peeled off. After cooling, impurities are sieved out and sealed for storage.

[0041] When the grass fruit kernels are stewed in the present invention, the starting temperature of the stewing is strictly controlled to be between 170 and 175° C., the ending temperature is between 215 and 224° C., and the stewing time is 18 minutes.

[0042] Table 1 Measurement table of the starting temperature of simmered Amomum villosum

[0043]

[0044] The invention is an innovation of the traditional processing technology of tsaoko, from processing tsaoko with shell to processing tsaoko kernels without shell, most of the effective ingredients in tsaoko are concentrated in tsaoko kernels, and the processing of tsaoko kernels mitigates the medicinal properties of tsaoko, improves the leaching rate of effective ingredients in tsaoko, and the utilization rate of effective ingredients in tsaoko is also improved. The processing technology of the invention is safe and reliable, so that tsaoko is innovatively used in traditional Chinese medicine, and provides guarantee for the raw material quality of tsaoko mouthwash and tsaoko related preparations.

[0045] The invention discloses a method for preparing tsaoko for inhibiting the main oral pathogens. The mouthwash prepared with the tsaoko kernels prepared in this way has the effect of enhancing the inhibition of the main oral pathogens (Staphylococcus aureus, Streptococcus mutans, Porphyromonas gingivalis). The method for preparing tsaoko comprises the following steps: removing the shell of the tsaoko kernels, which is the main raw material of the mouthwash, and preparing dough skins; wrapping the tsaoko kernels with the dough skins; adding talcum powder pre-heated to 170-175°C in a pot, stir-frying and stewing until the dough skins are browned; stewing for 18 minutes; taking out, sieving out the talcum powder, and peeling off the dough skins to obtain stewed tsaoko kernels. By comparing the antibacterial effect evaluation of mouthwash prepared with stewed tsaoko kernels, raw tsaoko kernels, and whole tsaoko fruits, the stewed tsaoko kernel mouthwash has a higher effect of inhibiting (Staphylococcus aureus, Streptococcus mutans, Porphyromonas gingivalis) than raw tsaoko kernels and whole tsaoko fruit mouthwashes. Therefore, the present invention provides a method for preparing tsaoko fruit for inhibiting major oral pathogens.

[0046] The raw materials and reagents used in the preparation method of the tsaoko extract provided by the invention can all be purchased from the market.

[0047] The present invention will be further described below in conjunction with embodiments:

[0048] Example 1

[0049] In this embodiment, the stewed grass nut kernels are obtained by the following method: take an appropriate amount of raw grass nut kernels and wrap them with wet dough. Before stewing, add talcum powder into the pot and heat it to 170°C, add the wrapped grass nut kernels into the hot talcum powder, stir-fry and stew until the dough is browned. The stewing time is 18 min and the end temperature is 215°C. Take out, sieve out the talcum powder, peel off the dough, cool it down and seal it for storage.

[0050] In the present embodiment, the grass fruit kernel refers to the natural dried whole grass fruit, the shell of which is broken and the kernel is taken out to obtain the raw grass fruit kernel.

[0051] In the present embodiment, when stewing the grass nut kernels, each piece of dough wraps 15 to 20 g of the grass nut kernels, and the weight is preferably based on the dough wrapping.

[0052] The simmered cardamom extract of this embodiment is obtained by the following method: take an appropriate amount of simmered cardamom kernels, crush them with a grinder, weigh 50 g and put them in a clean 1 L beaker, add deionized water at a solid-liquid ratio of 1:10 (W / W), stir, seal with plastic wrap, and soak for 7 hours, extract with a low-temperature ultrasonic extractor, and the extraction conditions are: 75°C constant temperature, extraction power 300W, ultrasound on 3s, off 2s, extraction time 1 hour. After the extraction is completed, filter with a 200-mesh filter cloth, collect the filtrate, centrifuge the collected filtrate (4000 r / min, centrifuge for 15 min), and store it in a 4°C refrigerator.

[0053] The tsaoko mouthwash is obtained by the following method:

[0054] The mouthwash of this embodiment includes the following ingredients in weight percentage: 14% simmered tsaoko extract, 3% glycerol, 0.02% citric acid, 0.05% xylitol, 0.05% sorbitol, 0.08% mint powder, 0.06% sodium bicarbonate, and the balance is water. The pH of the tsaoko mouthwash is adjusted to (7.1±0.2).

[0055] Example 2

[0056] In this embodiment, the stewed grass nut kernels are obtained by the following method: take an appropriate amount of raw grass nut kernels and wrap them with wet dough. Before stewing, add talcum powder into the pot and heat it to 175°C. Add the wrapped grass nut kernels into the hot talcum powder, stir-fry and stew until the dough is browned. The stewing time is 18 min and the end temperature is 219°C. Take out, sieve out the talcum powder, peel off the dough, cool it down and seal it for storage.

[0057] In the present embodiment, the grass fruit kernel refers to the natural dried whole grass fruit, the shell of which is broken and the kernel is taken out to obtain the raw grass fruit kernel.

[0058] In the present embodiment, when stewing the grass nut kernels, each piece of dough wraps 15 to 20 g of the grass nut kernels, and the weight is preferably based on the dough wrapping.

[0059] The simmered cardamom extract of this embodiment is obtained by the following method: take an appropriate amount of simmered cardamom kernels, crush them with a grinder, weigh 50 g and put them in a clean 1 L beaker, add deionized water at a solid-liquid ratio of 1:10 (W / W), stir, seal with plastic wrap, and soak for 7 hours, extract with a low-temperature ultrasonic extractor, and the extraction conditions are: 75°C constant temperature, extraction power 300W, ultrasound on 3s, off 2s, extraction time 1 hour. After the extraction is completed, filter with a 200-mesh filter cloth, collect the filtrate, centrifuge the collected filtrate (4000 r / min, centrifuge for 15 min), and store it in a 4°C refrigerator.

[0060] The tsaoko mouthwash is obtained by the following method:

[0061] The mouthwash of this embodiment includes the following ingredients in weight percentage: 16% of simmered tsaoko extract, 5% of glycerol, 0.04% of citric acid, 0.2% of xylitol, 0.15% of sorbitol, 0.12% of mint powder, 0.06% of sodium bicarbonate, and the balance of water. The pH of the tsaoko mouthwash is adjusted to (7.1±0.2).

[0062] Example 3

[0063] In this embodiment, the stewed grass nut kernels are obtained by the following method: an appropriate amount of raw grass nut kernels are wrapped with wet dough, talcum powder is added to the pot and heated to 171°C before stewing, the wrapped grass nut kernels are added to the hot talcum powder, stir-fried and stewed until the dough is browned, the stewing time is 18 min, and the end temperature is 224°C. The kernels are taken out, the talcum powder is sieved out, the dough is peeled off, and the kernels are sealed and stored after cooling.

[0064] In the present embodiment, the grass fruit kernel refers to the natural dried whole grass fruit, the shell of which is broken and the kernel is taken out to obtain the raw grass fruit kernel.

[0065] In the present embodiment, when stewing the grass nut kernels, each piece of dough wraps 15 to 20 g of the grass nut kernels, and the weight is preferably based on the dough wrapping.

[0066] The simmered cardamom extract of this embodiment is obtained by the following method: take an appropriate amount of simmered cardamom kernels, crush them with a grinder, weigh 50 g and put them in a clean 1 L beaker, add deionized water at a solid-liquid ratio of 1:10 (W / W), stir, seal with plastic wrap, and soak for 7 hours, extract with a low-temperature ultrasonic extractor, and the extraction conditions are: 75°C constant temperature, extraction power 300W, ultrasound on 3s, off 2s, extraction time 1 hour. After the extraction is completed, filter with a 200-mesh filter cloth, collect the filtrate, centrifuge the collected filtrate (4000 r / min, centrifuge for 15 min), and store it in a 4°C refrigerator.

[0067] The tsaoko mouthwash is obtained by the following method:

[0068] The mouthwash of this embodiment includes the following ingredients in weight percentage: 18% simmered tsaoko extract, 4% glycerol, 0.06% citric acid, 0.10% xylitol, 0.10% sorbitol, 0.16% mint powder, 0.06% sodium bicarbonate, and the balance is water. The pH of the tsaoko mouthwash is adjusted to (7.1±0.2).

[0069] Comparative Example 1

[0070] In this comparative example, the raw grass nut kernels are obtained by the following method: taking grass nut fruits, drying the grass nut fruits naturally to obtain dried whole grass nut fruits, and then cracking the dried whole grass nut fruits to obtain the kernels to obtain the raw grass nut kernels.

[0071] The extract of raw grass kernel was obtained by the following method: take an appropriate amount of raw grass kernel, crush it with a grinder, weigh 50 g and put it in a clean 1 L beaker, add deionized water at a solid-liquid ratio of 1:10 (W / W), stir it, seal it with plastic wrap, soak it for 7 hours, and extract it with low-temperature ultrasonic wave. The extraction conditions are: constant temperature of 75 ℃, extraction power of 300W, ultrasonic on for 3s, off for 2s, and extraction time of 1 hour. After the extraction is completed, filter it with a 200-mesh filter cloth, collect the filtrate, centrifuge the collected filtrate (4000 r / min, centrifuge for 15 min), and store it in a refrigerator at 4 ℃.

[0072] The raw grass nut mouthwash is obtained by the following method:

[0073] The mouthwash of this comparative example comprises the following ingredients in weight percentage: 20% of raw grass fruit kernel extract, 6% of glycerol, 0.08% of citric acid, 0.15% of xylitol, 0.2% of sorbitol, 0.2% of mint powder, 0.06% of sodium bicarbonate, and the balance of water. The pH of the grass fruit mouthwash is adjusted to (7.1±0.2).

[0074] Comparative Example 2

[0075] In this comparative example, the whole Amomum villosum fruit is obtained by the following method: taking the Amomum villosum fruit, and naturally drying the Amomum villosum fruit to obtain the dried whole Amomum villosum fruit.

[0076] The whole fruit extract of Amomum villosum was obtained by the following method: take an appropriate amount of raw Amomum villosum kernels, crush them with a grinder, weigh 50 g and put them in a clean 1 L beaker, add deionized water at a solid-liquid ratio of 1:10 (W / W), stir, seal with plastic wrap and soak for 7 hours, and perform low-temperature ultrasonic extraction. The extraction conditions are: constant temperature of 75 ℃, extraction power of 300W, ultrasonic on for 3s, off for 2s, and extraction time of 1 hour. After the extraction is completed, filter with a 200-mesh three-layer filter cloth, collect the filtrate, centrifuge the collected filtrate (4000 r / min, centrifuge for 15 minutes), and store it in a 4 ℃ refrigerator.

[0077] The whole tsaoko fruit mouthwash is obtained by the following method:

[0078] The mouthwash of this comparative example comprises the following ingredients in weight percentage: 22% whole tsaoko fruit extract, 7% glycerol, 0.1% citric acid, 0.2% xylitol, 0.25% sorbitol, 0.24% mint powder, 0.06% sodium bicarbonate, and the balance is water. The pH of the tsaoko mouthwash is adjusted to (7.1±0.2).

[0079] Table 2 shows the components and fuzzy mathematical sensory evaluation of the tsaoko mouthwash prepared in Examples 1-3 and Comparative Examples 1-2.

[0080] Table 2 Tsaoko mouthwash process

[0081]

[0082] As can be seen from Table 2, the fuzzy mathematical sensory scores of Examples 1-3 and Comparative Examples 1 and 2 are obtained, and the fuzzy mathematical sensory score of the mouthwash of Example 2 is the highest. Since Examples 1-3 are all mouthwashes prepared from the extracts of simmered cardamom kernels, Example 2 with the highest sensory score is selected hereafter for antibacterial efficacy evaluation together with Comparative Examples 1 and 2.

[0083] Effect Example 1

[0084] Table 3 shows the detection and analysis of the main components of processed and unprocessed Amomum villosum.

[0085] Table 3 Analysis of main components of processed and unprocessed Amomum villosum

[0086]

[0087] Note: “-” means not detected

[0088] The results are shown in Table 3. β-pinene, trans-2-decenol and (1R)-(+)-α-pinene can be detected in the simmered grass kernels, but they cannot be detected in the raw grass kernels and whole grass fruits. The relative contents of 2-pinene and α-caryophyllene in the simmered grass kernels are higher than those in the raw grass kernels. The relative contents of terpinene, 4-terpenol and 3-phenyl-2-butenal are higher than those in the whole grass fruits.

[0089] Example 4

[0090] In order to further prove that the mouthwash has antibacterial effect, common oral pathogens Staphylococcus aureus (FSCC 223005), Streptococcus mutans (ATCC 25175), and Porphyromonas gingivalis (ATCC BAA-308) were selected from Guangdong Microbiological Culture Collection Center to verify the antibacterial effect of the mouthwash.

[0091] 1. Experiment on the inhibition of Staphylococcus aureus by mouthwash prepared in Example 2 and Comparative Example 1-2

[0092] Prepare TSA solid culture medium → activate bacteria → subculture → prepare bacterial suspension → blank drug sensitive tablet method → ​​culture at 37℃ constant temperature and aerobic conditions for 24 hours → observe and measure the diameter of the inhibition zone. The detailed steps are as follows:

[0093] (1) Prepare TSA solid culture medium: weigh 38 g TSA and add 1 L pure water. Heat and boil until completely dissolved. Pour the mixture into four 250 mL conical flasks and seal them. Autoclave at 121 °C for 20 min. When cooled to 50 °C, pour the mixture into plates to obtain TSA solid culture medium.

[0094] (2) Activation of bacterial strains: Pipette the bacterial resuscitation solution into the freeze-dried tube, shake to dissolve, and then pipette all the bacterial solution onto a TSA plate, and place it in a 37°C incubator for 24 h.

[0095] (3) Preparation of bacterial suspension: Prepare 2-3 generations of Staphylococcus aureus, pick single colonies with an inoculation loop and place them in a test tube containing sterile water, suspend them in sterile water, shake them evenly, and use an ELISA reader to measure the OD value of the bacterial suspension. 405 ≈0.4.

[0096] (4) Soak the blank drug-sensitive tablets in the prepared mouthwash solutions to be tested (the stewed tsaoko mouthwash prepared in Example 2, the raw tsaoko kernel mouthwash prepared in Comparative Example 1, and the whole tsaoko fruit mouthwash prepared in Comparative Example 2). Operate on a clean bench, pipette 100 μL of the prepared bacterial suspension onto a plate, spread evenly with an L-shaped glass rod, and then place 3 drug-sensitive tablets soaked with extracts on the surface of the culture medium, of which 2 were soaked with extracts and 1 was soaked with sterile water or saline (negative control); place a blank drug-sensitive tablet soaked with anhydrous ethanol (positive control) on the coated plate, and make 3 parallels for each group (the stewed tsaoko mouthwash prepared in Example 2, the raw tsaoko kernel mouthwash prepared in Comparative Example 1, and the whole tsaoko fruit mouthwash prepared in Comparative Example 2), for a total of 9 groups. The plates with the drug-sensitive tablets were placed in a 37°C incubator for 24 hours, and then the plates were taken out to observe and measure the antibacterial zones around the drug-sensitive tablets in each plate. Through the antibacterial experiment of Staphylococcus aureus, the tsaoko mouthwash has the effect of inhibiting the growth of oral bacteria, achieving the purpose of the experiment.

[0097] 2. Example 2. Experiment on inhibition of Streptococcus mutans by the mouthwash prepared in Comparative Example 1-2.

[0098] Prepare 5% defibrinated sheep blood TSA solid medium → activate the strain → subculture → prepare bacterial suspension → blank drug sensitive plate method → ​​culture at 37 ℃ constant temperature and aerobic conditions for 48 hours → observe and measure the diameter of the inhibition zone. The detailed steps are as follows:

[0099] (1) Prepare 5% defibrinated sheep blood TSA solid culture medium: weigh 38 g TSA and add 1 L pure water. Heat and boil until completely dissolved, then pour into four 250 mL conical flasks and seal. Autoclave at 121 °C for 20 min. When cooled to 50 °C, add 12.5 mL of sterile defibrinated sheep blood to each 250 mL culture medium. Shake evenly and pour onto a plate to obtain 5% defibrinated sheep blood TSA solid culture medium.

[0100] (2) Activation of bacterial strains: Pipette the bacterial resuscitation solution into the bacterial freeze-dried tube, shake and dissolve, then aspirate all the bacterial solution and spread it onto a 5% defibrinated sheep blood TSA plate, and place it in a 37 ℃ incubator for 48 h.

[0101] (3) Preparation of bacterial suspension: Prepare 2-3 generations of mutans Streptococcus for subculture. Use an inoculation loop to pick single colonies and place them in test tubes containing sterile water. Suspend them in sterile water and shake them evenly. Use an ELISA reader to measure the OD405≈0.4 of the bacterial suspension.

[0102] (4) Soak the blank drug-sensitive tablets in the prepared mouthwash solutions to be tested (the stewed tsaoko mouthwash prepared in Example 2, the raw tsaoko kernel mouthwash prepared in Comparative Example 1, and the whole tsaoko fruit mouthwash prepared in Comparative Example 2). Operate on a clean bench, aspirate 100 μL of the prepared bacterial suspension onto a plate, spread it evenly with an L-shaped glass rod, and then place 3 drug-sensitive tablets soaked with extracts on the surface of the culture medium, of which 2 were soaked with extracts and 1 was soaked with sterile water or saline (negative control). Place a blank drug-sensitive tablet soaked with anhydrous ethanol (positive control) on the coated plate. Each group (the stewed tsaoko mouthwash prepared in Example 2, the raw tsaoko kernel mouthwash prepared in Comparative Example 1, and the whole tsaoko fruit mouthwash prepared in Comparative Example 2) is paralleled 3 times, for a total of 9 groups. The plates with the drug sensitive tablets were placed in a 37 ℃ incubator for 24 hours, and then the plates were taken out to observe and measure the antibacterial zones around the drug sensitive tablets in each plate. Through the mutans streptococcus antibacterial experiment, the tsaoko mouthwash has the effect of inhibiting the growth of oral bacteria, achieving the experimental purpose.

[0103] 3. Experiment on the inhibition of Porphyromonas gingivalis by the mouthwash prepared in Example 2 and Comparative Example 1-2.

[0104] Prepare 5% defibrinated sheep blood TSA solid culture medium (add 4-5 mL 1% hemin solution and 4-5 mL 1% vitamin K1 solution to each liter of culture medium) → activate the strain → subculture → prepare bacterial suspension → blank drug sensitive plate method → ​​culture at 37°C constant temperature and strict anaerobic conditions for 48-72 hours → observe and measure the diameter of the inhibition zone. The detailed experimental steps are as follows:

[0105] (1) Prepare 5% defibrinated sheep blood TSA solid culture medium: weigh 38 g TSA and add 1 L pure water. Heat and boil until completely dissolved, then pour into four 250 mL conical flasks and seal. Autoclave at 121 °C for 20 min. When cooled to 50 °C, add 12.5 mL of sterile defibrinated sheep blood, 2.5 mL of 1% hemin chloride (Shanghai Yuanye Biotechnology Co., Ltd., B27376), and 2.5 mL of 1% vitamin K1 (Shanghai Yuanye Biotechnology Co., Ltd., B21297) to each 250 mL culture medium. Shake evenly and pour onto a plate to obtain 5% defibrinated sheep blood TSA solid culture medium.

[0106] (2) Activation of bacterial strains: Pipette the bacterial resuscitation solution into the bacterial freeze-dried tube, shake to dissolve, and then aspirate all the bacterial solution to spread on a 5% defibrinated sheep blood TSA plate, and place it in a 37 ℃ incubator for 72 h.

[0107] (3) Preparation of bacterial suspension: Prepare 2-3 generations of Porphyromonas gingivalis, pick single colonies with an inoculation loop and place them in a test tube containing sterile water, suspend them in sterile water, shake them evenly, and use an ELISA reader to measure the OD value of the bacterial suspension. 405 ≈0.4.

[0108] (4) Soak the blank drug-sensitive tablets in the prepared mouthwash solutions to be tested (the stewed grass fruit kernel mouthwash prepared in Example 2, the raw grass fruit kernel mouthwash prepared in Comparative Example 1, and the whole fruit mouthwash prepared in Comparative Example 2). Operate on a clean bench, aspirate 100 μL of the prepared bacterial suspension onto a plate, spread it evenly with an L-shaped glass rod, and then place 3 drug-sensitive tablets soaked with extracts on the surface of the culture medium, of which 2 were soaked with extracts and 1 was soaked with sterile water or saline (negative control). Place a blank drug-sensitive tablet soaked with anhydrous ethanol (positive control) on the coated plate. Each group (the stewed grass fruit mouthwash prepared in Example 2, the raw grass fruit kernel mouthwash prepared in Comparative Example 1, and the whole grass fruit mouthwash prepared in Comparative Example 2) is paralleled 3 times, for a total of 9 groups. The plates with the drug-sensitive sheets were placed in a 37 ℃ incubator for 24 hours, and then the plates were taken out to observe and measure the antibacterial zones around the drug-sensitive sheets in each plate. Through the antibacterial experiment of Porphyromonas gingivalis, the tsaoko mouthwash has the effect of inhibiting the growth of oral bacteria, achieving the purpose of the experiment.

[0109] Effect Example 2

[0110] The antibacterial effect of the mouthwash under the optimal process is shown in Table 4 below. The mouthwash has an inhibitory effect on Staphylococcus aureus, Streptococcus mutans, and Porphyromonas gingivalis.

[0111] Table 4 Analysis of antibacterial effect of mouthwash under optimal process

[0112]

[0113] In summary, the above-mentioned simmered tsaoko mouthwash, raw tsaoko kernel mouthwash, and whole tsaoko mouthwash have good inhibitory effects on Staphylococcus aureus, Streptococcus mutans, and Porphyromonas gingivalis, among which the simmered tsaoko mouthwash has better antibacterial effect. The mouthwash has a certain auxiliary therapeutic effect on oral diseases caused by common oral bacteria, does not contain alcohol in the formula, is mild and non-irritating, has a unique aroma of tsaoko, has a simple preparation process, is safe and reliable, and is easy to carry and use.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an extract of Amomum villosum, characterized in that: The simmered grass fruit kernels are crushed, mixed with water and soaked, ultrasonically extracted, filtered and centrifuged; The simmering starting temperature is 170-175°C; The temperature at which the simmering is completed is 215-224°C; The simmering time is 18 minutes; The simmering steps include: taking raw grass nut kernels and wrapping them with dough, mixing them with talcum powder and heating them; Each piece of the dough wraps 15 to 20 g of the grass fruit kernels; The mass ratio of the simmered grass fruit kernels to water is 1:10; The soaking time is 7 h; The conditions of the ultrasonic extraction are: constant temperature of 75°C, extraction power of 300W, ultrasound on for 3s, off for 2s, and extraction time of 1h; The pore size of the filtration is 200 mesh; The centrifugation includes: 4000 r / min, 15 min.

2. The amomum villosum extract prepared by the preparation method as claimed in claim 1.

3. Use of the amomum villosum extract as claimed in claim 2 in the preparation of an antibacterial agent.

4. The use according to claim 3, characterized in that The bacteriostasis includes inhibiting one or more of Staphylococcus aureus, Streptococcus mutans or Porphyromonas gingivalis.

5. Use of the tsaoko extract as claimed in claim 2 in preparing mouthwash.

6. A mouthwash, characterized in that The invention is composed of the tsaoko extract as claimed in claim 2, a moisturizer, a freshener, a sweetener, sodium bicarbonate, citric acid and water.

7. The mouthwash according to claim 6, characterized in that The moisturizing agent includes glycerin and sorbitol; the refreshing agent is mint powder; and the sweetener is xylitol.

8. The mouthwash according to claim 6 or 7, characterized in that Calculated by weight percentage, the composition comprises 14% to 18% of the tsaoko extract, 0.05% to 5% of the moisturizer, 0.08% to 0.16% of the freshener, 0.05% to 0.2% of the sweetener, 0.06% of the sodium bicarbonate, 0.02% to 0.06% of the citric acid and 77.52% to 82.74% of the water.

9. The mouthwash according to claim 8, characterized in that Calculated by weight percentage, it includes 14% to 18% of amomum villosum extract, 3% to 5% of glycerin, 0.02% to 0.06% of citric acid, 0.05% to 0.2% of xylitol, 0.05% to 0.15% of sorbitol, 0.08% to 0.16% of mint powder, 0.06% of sodium bicarbonate and 77.52% to 82.74% of water.

10. The mouthwash according to claim 9, characterized in that In terms of weight percentage, it comprises 14% tsaoko extract, 3% glycerol, 0.02% citric acid, 0.05% xylitol, 0.05% sorbitol, 0.08% peppermint powder, 0.06% sodium bicarbonate and 82.74% water; or 18% Amomum villosum extract, 4% glycerin, 0.06% citric acid, 0.1% xylitol, 0.1% sorbitol, 0.16% peppermint powder, 0.06% sodium bicarbonate and 77.52% water.

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