Preparation method of amomum tsao-ko extracting solution
By frying the grass nut kernels, the problem of low utilization rate of the active ingredients of grass nuts is solved, the quality of grass nut extract and the antibacterial effect of mouthwash is improved, and the safe and reliable preparation is provided for the development of grass nut mouthwash. method.
Patent Information
- Application Number
- CN202410513364.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-26
- Publication Date
- 2025-02-07
AI Technical Summary
The existing methods of preparing grass fruits result in low leaching rate of active ingredients of grass fruits, low utilization rate, and not being used in the field of Chinese herbal mouthwash.
By frying the herb kernel, the medicinal properties of the herbs are alleviated, the leaching rate of the active ingredients of the herbs are improved, and the grass fruit extract is prepared for the development of Chinese herbal mouthwash.
It improves the utilization rate of the active ingredients of Caoguo, provides high-quality Caoguo medicinal materials, provides guarantee for the quality and efficacy of Caoguo mouthwash, and significantly improves the inhibitory effect of mouthwash on oral pathogenic bacteria.
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Abstract
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 for changing the properties of drugs, facilitating preparation and storage, removing edible and medicinal parts, and eliminating or reducing the toxicity, potency or side effects of traditional Chinese medicines. However, the existing method for processing tsaoko is to process the whole tsaoko fruit, and then remove the tsaoko kernels to obtain the processed tsaoko kernels, but the extraction rate of the active ingredients in the tsaoko is not high, and the utilization rate of the active ingredients is not high. Moreover, the processing method and processed products of tsaoko have not been applied to the field of Chinese herbal mouthwashes.
[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 the tsaoko extract.
[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 the development and utilization of Amomum villosum as a traditional Chinese medicine can be continued, and high-quality Amomum villosum medicinal raw materials are provided to ensure the quality and efficacy of later Amomum villosum mouthwashes 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 taking fried Amomum villosum kernels, crushing them, mixing them with water and then soaking them, ultrasonically extracting them, filtering them and centrifuging them;
[0010] The starting temperature of the frying is 149-151°C;
[0011] The temperature at which the frying is completed is 200-229°C;
[0012] The frying time is 7 minutes.
[0013] In some specific embodiments of the present invention, the mass ratio of the fried cardamom kernels to water is 1:10.
[0014] 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.
[0015] In some specific embodiments of the present invention, the soaking time is 7 h.
[0016] 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.
[0017] In some specific embodiments of the present invention, the filtration pore size is 200 mesh.
[0018] In some specific embodiments of the present invention, the centrifugation comprises: 4000 r / min, 15 min.
[0019] The invention also provides an extract of Amomum villosum prepared by the preparation method.
[0020] Based on the above research, the present invention also provides the application of the amomum villosum extract in antibacterial treatment.
[0021] In some specific embodiments of the present invention, the antibacterial activity includes inhibiting one or more of Staphylococcus aureus, Streptococcus mutans or Porphyromonas gingivalis.
[0022] The invention also provides application of the amomum villosum extract in preparing mouthwash.
[0023] The invention also provides a mouthwash comprising the tsaoko extract, a moisturizing agent, a freshener, a sweetener, sodium bicarbonate, citric acid and water.
[0024] In some specific embodiments of the present invention, the moisturizing agent includes glycerin and sorbitol; the refreshing agent is mint powder; and the sweetener is xylitol.
[0025] In some specific embodiments of the present invention, the composition comprises, by weight percentage, 16% to 20% of the amomum villosum extract, 0.1% to 6% of the moisturizer, 0.14% to 0.24% of the freshener, 0.1% to 0.2% of the sweetener, 0.06% of the sodium bicarbonate, 0.04% to 0.08% of the citric acid and 73.27% to 78.41% of the water.
[0026] In some specific embodiments of the present invention, the composition comprises, by weight percentage, 16% to 20% of amomum villosum extract, 5% to 6% of glycerol, 0.04% to 0.08% of citric acid, 0.1% to 0.2% of xylitol, 0.1% to 0.2% of sorbitol, 0.14% to 0.24% of mint powder, 0.06% of sodium bicarbonate and 73.27% to 78.41% of water.
[0027] In some specific embodiments of the present invention, the composition comprises, by weight percentage, 16% of tsaoko extract, 5% of glycerol, 0.04% of citric acid, 0.2% of xylitol, 0.15% of sorbitol, 0.14% of mint powder, 0.06% of sodium bicarbonate and 78.41% of water; or
[0028] 18% Amomum villosum extract, 4% glycerin, 0.06% citric acid, 0.1% xylitol, 0.1% sorbitol, 0.2% peppermint powder, 0.06% sodium bicarbonate and 77.48% water; or
[0029] 20% Amomum villosum extract, 6% glycerin, 0.08% citric acid, 0.15% xylitol, 0.2% sorbitol, 0.24% peppermint powder, 0.06% sodium bicarbonate and 73.27% water.
[0030] In some specific embodiments of the present invention, the pH of the mouthwash is 6.9-7.3.
[0031] The invention provides a method for preparing a tsaoko extract. The method comprises the steps of removing the shell of dry tsaoko and taking out the kernel, processing the tsaoko kernel, extracting the fried tsaoko kernel, raw tsaoko kernel and whole tsaoko, obtaining the extract, preparing tsaoko mouthwash, and finally evaluating the antibacterial efficacy of the tsaoko mouthwash. By comparing the antibacterial efficacy of three different tsaoko mouthwashes, it is concluded that the mouthwash prepared by the tsaoko processing method has the best antibacterial efficacy. DETAILED DESCRIPTION
[0032] 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.
[0033] The innovative point of the method for preparing the tsaoko of the present invention is that when frying the tsaoko in the present invention, the dried whole tsaoko fruit is first shelled to obtain raw tsaoko kernels, and then the raw tsaoko kernels are fried to obtain the fried tsaoko kernels, and the tsaoko mouthwash is prepared by using the fried tsaoko kernel extract. The research of the present invention shows that the fried tsaoko kernel mouthwash has a good inhibitory effect on the main pathogenic bacteria in the human oral cavity.
[0034] The invention provides a method for preparing tsaoko and tsaoko mouthwash with better antibacterial effect.
[0035] The innovation thereof lies in that in the preparation of tsaoko in the past dynasties, stir-fried tsaoko was prepared by whole tsaoko fruits with shells, and the shells were removed and the kernels were taken out when used. The present invention removes the shells and kernels of dry tsaoko fruits, prepares the tsaoko kernels, and then extracts the stir-fried tsaoko kernels, raw tsaoko kernels and whole tsaoko fruits to prepare tsaoko mouthwash after obtaining the extract, and finally evaluates the antibacterial efficacy of the tsaoko mouthwash. By comparing the antibacterial efficacy of three different tsaoko mouthwashes, it is concluded that the mouthwash prepared by the tsaoko preparation method has the best antibacterial efficacy.
[0036] 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, then breaking the shell of the dried grass fruit whole fruit to obtain kernels, and obtaining raw grass fruit kernels. The raw grass fruit kernels are placed in a pot for frying, and are fried until the surface of the grass fruit kernels is microscopically yellow fire color, which is darker than the color of the raw material, and the surface of the grass fruit kernels is bulging and the inherent fragrance is revealed, and then the kernels are cooled and sealed for storage.
[0037] The following is a further optimization description of the above invention technical solution:
[0038] When frying the grass nut kernels, take 200 g of grass nut kernels each time, the initial temperature in the pot is (150±1)°C, the frying time is 7 min, the frying temperature is strictly controlled, and the final frying temperature is 200-229°C. After frying, let it cool, sieve out impurities, and seal it for storage.
[0039] Table 1 Measurement of the initial temperature of frying grass kernels
[0040]
[0041] 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 improves the utilization rate of effective ingredients in tsaoko. The processing technology of the invention is safe and reliable, so that the development and utilization of tsaoko as a traditional Chinese medicine is continued, and high-quality tsaoko medicinal raw materials are provided to ensure the quality and efficacy of tsaoko mouthwash and tsaoko-related preparations in the later stage.
[0042] The present invention discloses a method for preparing tsaoko for improving the inhibition of main oral pathogens in mouthwash. The mouthwash prepared with the tsaoko kernels prepared in this way has the effect of improving the inhibition of main oral pathogens (Staphylococcus aureus, Streptococcus mutans, Porphyromonas gingivalis). The method for preparing tsaoko comprises shelling the tsaoko kernels, which is the main raw material of the mouthwash, and preparing the fried tsaoko kernels at (150±1)°C for 7 min. The antibacterial effect of mouthwash prepared with the fried tsaoko kernels, raw tsaoko kernels, and whole tsaoko kernels is evaluated by comparing the antibacterial effect of mouthwash prepared with the fried tsaoko kernels, raw tsaoko kernels, and whole tsaoko kernels. The efficacy of the fried tsaoko kernel mouthwash in inhibiting (Staphylococcus aureus, Streptococcus mutans, Porphyromonas gingivalis) is higher than that of the raw tsaoko kernels and whole tsaoko kernels. Therefore, the present invention provides a method for preparing tsaoko for improving the antibacterial effect of mouthwash.
[0043] 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.
[0044] The present invention will be further described below in conjunction with embodiments:
[0045] Example 1
[0046] In this embodiment, the fried grass nut kernels are obtained by the following method: raw grass nut kernels are placed in a pot and fried until the surface of the grass nut kernels is slightly yellow fire color, which is darker than the color of the raw material, and the surface of the grass nut kernels bulges and emits inherent aroma, and then cooled and sealed for storage.
[0047] 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.
[0048] In this embodiment, when frying the grass kernels, 200 g of grass kernels were taken each time, the initial temperature in the pot was 150°C for 7 minutes, the frying temperature was strictly controlled, and the final frying temperature was 223°C.
[0049] The stir-fried cardamom extract of this embodiment is obtained by the following method: take an appropriate amount of stir-fried cardamom kernels, crush them with a grinder, weigh 50 g and put them in a clean 1L beaker, add deionized water at a solid-liquid ratio (W / W) of 1:10, stir, seal with plastic wrap and soak for 7 hours, and perform low-temperature ultrasonic extraction. The extraction conditions are: 75°C constant temperature, extraction power 300W, ultrasonic on 3s, off 2s, extraction time 1h. After the extraction is completed, filter with a 200-mesh filter cloth, collect the filtrate, centrifuge the collected filtrate (4000r / min, centrifuge for 15min), and store it in a 4°C refrigerator.
[0050] The stir-fried tsaoko mouthwash is obtained by the following method:
[0051] The mouthwash of this embodiment includes the following ingredients in weight percentage: 16% of stir-fried tsaoko extract, 5% of glycerol, 0.04% of citric acid, 0.20% 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).
[0052] Example 2
[0053] In this embodiment, the fried grass nut kernels are obtained by the following method: raw grass nut kernels are placed in a pot and fried until the surface of the grass nut kernels is slightly yellow fire color, which is darker than the color of the raw material, and the surface of the grass nut kernels bulges and emits inherent aroma, and then cooled and sealed for storage.
[0054] 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.
[0055] In this embodiment, when frying the grass kernels, 200 g of grass kernels were taken each time, the initial temperature in the pot was 149°C for 7 minutes, the frying temperature was strictly controlled, and the final frying temperature was 200°C.
[0056] The stir-fried cardamom extract of this embodiment is obtained by the following method: take an appropriate amount of stir-fried cardamom kernels, crush them with a grinder, weigh 50 g and put them in a clean 1L beaker, add deionized water at a solid-liquid ratio (W / W) of 1:10, stir, seal with plastic wrap and soak for 7 hours, and perform low-temperature ultrasonic extraction. The extraction conditions are: 75°C constant temperature, extraction power 300W, ultrasonic on 3s, off 2s, extraction time 1h. After the extraction is completed, filter with a 200-mesh filter cloth, collect the filtrate, centrifuge the collected filtrate (4000r / min, centrifuge for 15min), and store it in a 4°C refrigerator.
[0057] The stir-fried tsaoko mouthwash is obtained by the following method:
[0058] The mouthwash of this embodiment includes the following ingredients in weight percentage: 18% of stir-fried tsaoko extract, 4% of glycerol, 0.06% of citric acid, 0.10% of xylitol, 0.10% of sorbitol, 0.20% of mint powder, 0.06% of sodium bicarbonate, and the balance is water. The pH of the tsaoko mouthwash is adjusted to (7.1±0.2).
[0059] Example 3
[0060] In this embodiment, the fried grass nut kernels are obtained by the following method: raw grass nut kernels are placed in a pot and fried until the surface of the grass nut kernels is slightly yellow fire color, which is darker than the color of the raw material, and the surface of the grass nut kernels bulges and emits inherent aroma, and then cooled and sealed for storage.
[0061] 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.
[0062] In this embodiment, when frying the grass kernels, 200 g of grass kernels were taken each time, the initial temperature in the pot was 151°C for 7 minutes, the frying temperature was strictly controlled, and the final frying temperature was 229°C.
[0063] The stir-fried cardamom extract of this embodiment is obtained by the following method: take an appropriate amount of stir-fried cardamom kernels, crush them with a grinder, weigh 50 g and put them in a clean 1L beaker, add deionized water at a solid-liquid ratio (W / W) of 1:10, stir, seal with plastic wrap and soak for 7 hours, and perform low-temperature ultrasonic extraction. The extraction conditions are: 75°C constant temperature, extraction power 300W, ultrasonic on 3s, off 2s, extraction time 1h. After the extraction is completed, filter with a 200-mesh filter cloth, collect the filtrate, centrifuge the collected filtrate (4000r / min, centrifuge for 15min), and store it in a 4°C refrigerator.
[0064] The stir-fried tsaoko mouthwash is obtained by the following method:
[0065] The mouthwash of this embodiment includes the following ingredients in weight percentage: 20% stir-fried tsaoko extract, 6% glycerol, 0.08% citric acid, 0.15% xylitol, 0.20% 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).
[0066] Comparative Example 1
[0067] 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.
[0068] 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 1L beaker, add deionized water at a solid-liquid ratio (W / W) of 1:10, stir it, seal it with plastic wrap and let it 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 for 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 ℃.
[0069] The raw grass nut mouthwash is obtained by the following method:
[0070] The mouthwash of this comparative example comprises the following ingredients in weight percentage: 14% of raw grass fruit kernel extract, 3% of glycerol, 0.02% of citric acid, 0.05% of xylitol, 0.05% of sorbitol, 0.08% 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).
[0071] Comparative Example 2
[0072] 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.
[0073] The whole fruit extract of Amomum villosum was obtained by the following method: take an appropriate amount of whole fruit of Amomum villosum, 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 (W / W) of 1:10, stir it, seal it with plastic wrap and 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 for 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 ℃.
[0074] The whole tsaoko fruit mouthwash is obtained by the following method:
[0075] 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).
[0076] Table 2 shows the components and fuzzy sensory evaluation of the tsaoko mouthwash prepared in Examples 1-3 and Comparative Examples 1-2.
[0077]
[0078] The fuzzy mathematical sensory scores of the tsaoko mouthwash were obtained through the tsaoko embodiments and comparative examples. As shown in Table 2, the fuzzy mathematical sensory scores of the mouthwashes prepared from the stir-fried tsaoko kernels in embodiments 1-3 were higher than the fuzzy mathematical sensory scores of the mouthwashes prepared from the whole tsaoko fruit and raw tsaoko kernels in the comparative examples. It can be seen that the sensory effects of the embodiments are better than those of the comparative examples. The fuzzy mathematical sensory scores of embodiments 1-3 were obtained, and the fuzzy mathematical sensory score of embodiment 1 was the highest. Therefore, embodiment 1 and comparative example 1, comparative example 2 were selected as the subsequent analysis of inhibiting the main oral pathogens.
[0079] Effect Example 1
[0080] Table 3 shows the detection and analysis of the main components of processed and unprocessed Amomum villosum.
[0081] Table 3 Analysis of main components of processed and unprocessed Amomum villosum
[0082]
[0083] Note: “-” means not detected
[0084] The dissolution of effective ingredients in processed Amomum villosum is higher than that in raw Amomum villosum kernels and whole Amomum villosum fruit. As shown in Table 3, β-pinene and δ-cadinene were detected in fried Amomum villosum kernels, while they were not detected in raw Amomum villosum kernels and whole Amomum villosum fruit. The relative contents of 2,4-dimethylstyrene and (-)-4-terpineol detected in fried Amomum villosum kernels were higher than those in raw Amomum villosum kernels. The relative contents of L-α-pinene, α-phellandrene and 3-phenyl-2-butenal in fried Amomum villosum kernels were higher than those in whole Amomum villosum fruit.
[0085] Example 4
[0086] Common oral pathogens Staphylococcus aureus (FSCC 223005), Streptococcus mutans (ATCC25175), and Porphyromonas gingivalis (ATCC BAA-308) were selected from Guangdong Microbiological Culture Collection Center to verify the antibacterial effect of the mouthwash.
[0087] 1. Experiment on the inhibition of Staphylococcus aureus by mouthwash prepared in Example 1 and Comparative Example 1-2
[0088] 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:
[0089] (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.
[0090] (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.
[0091] (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.
[0092] (4) Soak the blank drug-sensitive tablets in the prepared mouthwash solutions to be tested (the raw grass fruit kernel mouthwash prepared in comparative example 1, the stir-fried grass fruit mouthwash prepared in example 1, and the whole grass fruit mouthwash prepared in comparative example 2). Operate on a clean bench, aspirate 100 μL of the prepared bacterial suspension onto a plate, and spread it evenly with an L-shaped glass rod. Then, place three drug-sensitive tablets soaked with extracts on the surface of the culture medium, of which two are soaked with extracts and one is 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 stir-fried grass fruit kernel mouthwash prepared in example 1, 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. Place the plates with the drug-sensitive tablets in a 37°C incubator for 24 h, then take out the plates to observe and measure the antibacterial zones around the drug-sensitive tablets in each plate. Through the Staphylococcus aureus antibacterial experiment, it was found that the Amomum villosum mouthwash has the effect of inhibiting the growth of oral bacteria, thus achieving the experimental purpose.
[0093] 2. Experiment on inhibition of Streptococcus mutans by mouthwash prepared in Example 1 and Comparative Example 1-2.
[0094] 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:
[0095] (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.
[0096] (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°C incubator for 48 hours.
[0097] (3) Preparation of bacterial suspension: Prepare 2-3 generations of mutans streptococci, 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.
[0098] (4) Soak the prepared mouthwash solutions to be tested (the stir-fried grass kernel mouthwash prepared in Example 1, the raw grass kernel mouthwash prepared in Comparative Example 1, and the whole fruit mouthwash prepared in Comparative Example 2) in blank drug-sensitive tablets. Operate on a clean bench, aspirate 100 μL of the prepared bacterial suspension onto a plate, and spread it evenly with an L-shaped glass rod. Then, place three drug-sensitive tablets soaked with extracts on the surface of the culture medium, of which two are soaked with extracts and one is 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 stir-fried grass kernel mouthwash prepared in Example 1, the raw grass kernel mouthwash prepared in Comparative Example 1, and the whole fruit mouthwash prepared in Comparative Example 2) is paralleled 3 times, for a total of 9 groups. Place the plates with the drug-sensitive tablets in a 37°C incubator for 24 h, then take out the plates to observe and measure the antibacterial zones generated around the drug-sensitive tablets in each plate. Through the antibacterial experiment of Streptococcus mutans, it was found that the Amomum villosum mouthwash has the effect of inhibiting the growth of oral bacteria, thus achieving the experimental purpose.
[0099] 3. Experiment on the inhibition of Porphyromonas gingivalis by the mouthwash prepared in Example 1 and Comparative Example 1-2.
[0100] 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:
[0101] (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.
[0102] (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.
[0103] (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.
[0104] (4) Soak the prepared mouthwash solutions to be tested (the stir-fried grass kernel mouthwash prepared in Example 1, the raw grass kernel mouthwash prepared in Comparative Example 1, and the whole fruit mouthwash prepared in Comparative Example 2) in blank drug-sensitive tablets. Operate on a clean bench, aspirate 100 μL of the prepared bacterial suspension onto a plate, and spread it evenly with an L-shaped glass rod. Then, place three drug-sensitive tablets soaked with extracts on the surface of the culture medium, of which two are soaked with extracts and one is 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 stir-fried grass kernel mouthwash prepared in Example 1, the raw grass kernel mouthwash prepared in Comparative Example 1, and the whole fruit mouthwash prepared in Comparative Example 2) is paralleled 3 times, for a total of 9 groups. Place the plates with the drug-sensitive tablets in a 37°C incubator for 24 h, then take out the plates to observe and measure the antibacterial zones generated around the drug-sensitive tablets in each plate. Through the Porphyromonas gingivalis antibacterial experiment, it was found that the Amomum villosum mouthwash has the effect of inhibiting the growth of oral bacteria, thus achieving the experimental purpose.
[0105] Effect Example 2
[0106] The antibacterial effect of the mouthwash prepared by the process of the optimal embodiment is shown in Table 4 below. The mouthwash has an inhibitory effect on Staphylococcus aureus, Streptococcus mutans, and Porphyromonas gingivalis.
[0107] Table 4 Analysis of the antibacterial effect of the optimal mouthwash
[0108]
[0109] In summary, the above-mentioned fried tsaoko kernel mouthwash, raw tsaoko kernel mouthwash, and whole tsaoko fruit mouthwash have good inhibitory effects on Staphylococcus aureus, Streptococcus mutans, and Porphyromonas gingivalis, among which the fried 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.
[0110] 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 fried grass fruit kernels are crushed, mixed with water and soaked, ultrasonically extracted, filtered and centrifuged; The starting temperature of the frying is 149-151°C; The temperature at which the frying is completed is 200-229°C; The frying time is 7 minutes.
2. The amomum villosum extract prepared by the preparation method as claimed in claim 1.
3. Use of the tsaoko extract as claimed in claim 2 in antibacterial treatment.
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 comprises 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 16% to 20% of the tsaoko extract, 0.1% to 6% of the moisturizer, 0.14% to 0.24% of the freshener, 0.1% to 0.2% of the sweetener, 0.06% of the sodium bicarbonate, 0.04% to 0.08% of the citric acid and 73.27% to 78.41% of the water.
9. The mouthwash according to any one of claims 6 to 8, characterized in that Calculated by weight percentage, it includes 16% to 20% of amomum villosum extract, 5% to 6% of glycerin, 0.04% to 0.08% of citric acid, 0.1% to 0.2% of xylitol, 0.1% to 0.2% of sorbitol, 0.14% to 0.24% of mint powder, 0.06% of sodium bicarbonate and 73.27% to 78.41% of water.
10. The mouthwash according to any one of claims 6 to 9, characterized in that In terms of weight percentage, it includes 16% of tsaoko extract, 5% of glycerin, 0.04% of citric acid, 0.2% of xylitol, 0.15% of sorbitol, 0.14% of mint powder, 0.06% of sodium bicarbonate and 78.41% of water; or 18% Amomum villosum extract, 4% glycerin, 0.06% citric acid, 0.1% xylitol, 0.1% sorbitol, 0.2% peppermint powder, 0.06% sodium bicarbonate and 77.48% water; or 20% Amomum villosum extract, 6% glycerin, 0.08% citric acid, 0.15% xylitol, 0.2% sorbitol, 0.24% peppermint powder, 0.06% sodium bicarbonate and 73.27% water.