Preparation method and application of Kaempferia parviflora extract

Abstract

The present invention discloses a preparation method and application of a Kaempferia parviflora extract in the technical field of extract preparation, comprising the following steps: S1. Preparation of a Kaempferia parviflora rhizome extract, S1-1: weighing 500 g of Kaempferia parviflora rhizome, crushing it and placing it in a 1000 ml glass container; S1-2: adding 800 ml of methanol to the glass container, cold-immersing and extracting at room temperature for 7 days, filtering, repeating the extraction 3-5 times and concentrating to obtain a Kaempferia parviflora rhizome methanol extract; S1-3: adding the concentrated methanol to the flavonoids; 500 ml of water was added to the extract, and the extract and water were mixed. After mixing, the extract was extracted 3-5 times with equal volumes of petroleum ether, ethyl acetate layer, and n-butanol, respectively. The extracts were concentrated to obtain petroleum ether layer, ethyl acetate layer, and n-butanol layer extracts; S2. Determination of the antibacterial activity of the Kaempferia parviflora rhizome extract; S3. Determination of the antifungal activity of the Kaempferia parviflora rhizome extract; S4. Determination of the cytotoxic activity of the Kaempferia parviflora rhizome extract; S5. High-performance liquid chromatography analysis of the Kaempferia parviflora rhizome extract.

Description

Technical Field

[0001] The invention belongs to the technical field of extract preparation, and in particular relates to a preparation method of a Kaempferia parviflora extract and application thereof. Background Art

[0002] Kaempferia parviflora, also known as Thai black ginger, Thai ginseng, and rotten ginger, is a perennial herbaceous plant of the Zingiberaceae family and genus Kaempferia. It is native to Myanmar, Malaysia, and the Indonesian islands of Sumatra and Borneo, and is widely distributed in tropical and subtropical Southeast Asia. As one of the most species-rich groups of subtropical plants and the most distinctive group within the Zingiberales order, Kaempferia parviflora is a highly economically valuable understory plant. Kaempferia parviflora has a long history of use as a traditional medicine. The proanthocyanidins contained in its leaves are used in food and cosmetic development; its rhizomes contain a variety of flavonoids with anti-malarial and antifungal activity; and its metabolites have been shown to exhibit a variety of biological activities beneficial to humans, including anti-allergic, anti-bacterial, anti-gastric ulcer, and anti-cancer properties.

[0003] Elaeocarpus sylvestris, also known as false bayberry, plum rice, green fruit, wild olive, gall tree, olive, and margin-petaled sylvestris, is an evergreen tree in the Elaeocarpaceae family, reaching heights of up to 15 meters. Its leaves are leathery, lanceolate or oblanceolate, with small, blunt teeth on the margins. The petioles are initially slightly hairy, becoming bald as fruit sets. Its racemes are mostly borne in leaf axils, with a slender rachis. The flowers are white, with lanceolate sepals, obovate petals, and no appendages at the anther tips. The drupe is oval, with a glabrous exocarp and a bony endocarp. It blooms from June to July. This fast-growing evergreen tree offers excellent wood quality, strong adaptability, and few pests or diseases. It is an excellent choice for garden ornamental plants and for landscaping.

[0004] Among them, Eurasian pseudocryptic red shell is the main pathogen of Eurasian blight that has caused a large number of deaths in Eurasian in recent years. In the existing technology, the method of spraying pesticides is usually used to inhibit Eurasian pseudocryptic red shell, but the inhibition effect is not ideal; therefore, it is necessary to propose a preparation method and application of Kaempferia parviflora extract. Summary of the Invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a preparation method and application of Kaempferia parviflora extract which can inhibit the growth of Eurasian pseudocryptocephalides by Kaempferia parviflora extract.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows: a preparation method of Kaempferia parviflora extract and its application, comprising the following steps:

[0007] S1. Preparation of Kaempferia parviflora rhizome extract, the preparation steps are as follows:

[0008] S1-1: Weigh 500 g of washed and dried Kaempferia parviflora rhizomes, crush them, and place them in a 1000 ml glass container;

[0009] S1-2: Add 800 ml of methanol to a glass container, perform cold extraction at room temperature for 7 days, filter, and concentrate the filtrate using a rotary evaporator. Repeat the extraction 3-5 times to obtain the methanol extract of Kaempferia parviflora rhizome;

[0010] S1-3: Add 500 ml of water to the concentrated methanol extract, mix the methanol extract and water, and extract with an equal volume of petroleum ether 3-5 times. After complete extraction, extract with an equal volume of ethyl acetate and finally extract with an equal volume of n-butanol. Concentrate the extracts to obtain petroleum ether layer, ethyl acetate layer, and n-butanol layer extracts, and refrigerate at 4°C for later use.

[0011] S2. Determination of the antibacterial activity of Kaempferia parviflora rhizome extract; S3. Determination of the antifungal activity of Kaempferia parviflora rhizome extract; S4. Determination of the cytotoxicity of Kaempferia parviflora rhizome extract; S5. High-performance liquid chromatography analysis of Kaempferia parviflora rhizome extract.

[0012] Furthermore, in S2, the antibacterial activity of the Kaempferia parviflora rhizome extract is determined as follows:

[0013] S2-1. Activate the test bacteria on LB plates at 28°C in the dark for 48 hours and pick a single colony.

[0014] S2-2. Cultivate the culture medium in LB liquid medium at 28°C, 150 rpm, and darkness for 24 h. Dilute the bacterial solution to a concentration of 108 cfu·mL. -1 spare;

[0015] S2-3. Use a pipette to draw up 50 μL of bacterial solution and spread the solution onto the plate using a glass rod.

[0016] S2-4. Use a sterilized hole puncher to evenly punch three holes with a diameter of 6 mm on the bacterial culture medium plate. Add 40 μL of the test bacteria to each well. After incubation in the dark for 24 h, measure the size of the inhibition zone with a ruler. The positive control is 5 μL, and the 0.2 mg·mL -1 of streptomycin sulfate, and each treatment was repeated 3 times; the test bacteria included eucalyptus wilt pathogen, Agrobacterium tumefaciens, tomato scab pathogen, cucumber angular leaf spot pathogen, Staphylococcus aureus, Escherichia coli and Bacillus subtilis.

[0017] Furthermore, in S3, the antifungal activity of the Kaempferia parviflora rhizome extract is determined as follows:

[0018] S3-1: Weigh 240 mg of each of three different fungicides, add 0.3 mL of DMSO to each fungicide, and add 0.7 mL of sterile water after the fungicide and DMSO are completely dissolved;

[0019] S3-2: Using the half-dilution method, 30% DMSO was added to the fungicide again, and the fungicide was diluted into 8 different concentrations in sequence;

[0020] S3-3: 1 mL of each of the eight prepared fungicides of different concentrations was added to 29 mL of PDA, mixed thoroughly, and poured into three sterile culture dishes, with each dish containing 10 mL, to obtain drug-containing culture media with final fungicide concentrations of 8 mg / mL, 6 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL, respectively. Each concentration gradient was replicated three times.

[0021] S3-4: 1 mL of each of the eight prepared fungicides at different concentrations was added to 29 mL of PDA, mixed thoroughly, and poured into three sterile culture dishes, with 10 mL in each dish, to obtain drug-containing culture media with final fungicide concentrations of 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, and 0.00625 mg / mL, respectively;

[0022] S3-5: Use a hole puncher to punch the test fungus that has been cultured for 5 days and has grown well into a 7 mm diameter cake along its edge. The cake is inoculated face down on a drug-containing PDA plate; a PDA plate with sterile water added serves as a blank control, a PDA plate with 30% DMSO added serves as a negative control, and a PDA plate with 98.4% carbendazim added serves as a positive control. Each treatment is repeated three times.

[0023] S3-6: Incubate the prepared culture medium at 28°C for 5 days. When the colonies in the blank control group grow to occupy 2 / 3 of the culture dish area, measure the colony diameters under different treatments using the cross-cross method and calculate the inhibition rate. The calculation formula for the inhibition rate is as follows:

[0024]

[0025] Among them, the test fungi include Elaeocarpus pseudocryptocephalus and Colletotrichum oleifera.

[0026] Furthermore, in S4, the cytotoxicity assay of the Kaempferia parviflora rhizome extract is performed as follows:

[0027] S4-1: Prepare each test sample into sample solutions with concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL, respectively. Fibroblasts and human tongue squamous cell carcinoma cell suspensions were seeded onto 96-well plates and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0028] S4-2: Add 15 μL of CCK-8 reagent to each well, continue to culture at 37°C and 5% CO2 for 24 hours, and then use a multifunctional microplate reader to detect the absorbance of each well at 450 nm. The well plate without sample is used as a blank control.

[0029] Furthermore, in S5, the steps of HPLC analysis of Kaempferia parviflora rhizome extract are as follows:

[0030] S5-1 Weigh 10mg of different extracts of Kaempferia parviflora and dissolve the Kaempferia parviflora extract in 1mL of methanol for chromatography;

[0031] S5-2. The extract of Kaempferia parviflora was filtered through a 0.22 μm organic filter membrane, and the filtrate was transferred to a sample vial and detected using a diode array detector; wherein, the temperature of the chromatographic column was 40°C and the injection volume was 5 μL; the chromatographic analysis conditions of the extract of Kaempferia parviflora were as follows: 0-1 min, isocratic elution with 50% acetonitrile; 1-15 min, linear elution with acetonitrile from 50% to 100%; 15-20 min, isocratic elution with 100% acetonitrile; 20-21 min, linear decrease in acetonitrile concentration from 100% to 50%; 21-29 min, re-equilibration of the chromatographic column with 50% acetonitrile.

[0032] Furthermore, the extract of Kaempferia parviflora is used to prepare a biological preparation for inhibiting the fungus Pseudocryphala elaeocarpa.

[0033] The following beneficial effects are achieved by adopting the above scheme:

[0034] (1) The petroleum ether layer and ethyl acetate layer extracts of the Kaempferia parviflora rhizome of the present invention have good antibacterial and antifungal activities, while the n-butanol layer extract has a promoting effect on the growth of fibroblasts.

[0035] (2) The petroleum ether layer extract of the rhizome of Kaempferia parviflora in the present invention has good inhibitory activity against the red shell of the pseudocryptic clover of Elaeocarpus edulis, thereby providing a new idea for the biological control of Elaeocarpus blight; the ethyl acetate layer extract of the rhizome of Kaempferia parviflora has good antibacterial activity, the petroleum ether layer extract has a significant antibacterial effect on the red shell of the pseudocryptic clover of Elaeocarpus edulis, and the n-butanol layer extract has a promoting effect on the growth of fibroblasts.

[0036] (3) The results of HPLC analysis in the present invention show that the rhizomes of Kaempferia parviflora contain rich secondary metabolites. Therefore, in the future, it is necessary to further study the chemical structure and pharmacological activity of the active compounds in the rhizomes of Kaempferia parviflora, accelerate the research on germplasm preservation and breeding technology, and provide a scientific basis for clarifying the active mechanism of action and the development and utilization of Kaempferia parviflora resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the antibacterial activity of the Kaempferia parviflora rhizome extract according to an embodiment of the present invention.

[0038] Figure 2 This is a schematic diagram of the cytotoxic activity of different extracts from the rhizome of Kaempferia parviflora on fibroblasts in the examples of the present invention.

[0039] Figure 3 HPLC-UV chromatograms of different extracts from the rhizome of Kaempferia parviflora in the examples of the present invention. DETAILED DESCRIPTION

[0040] The following is further described in detail through specific implementation methods:

[0041] The embodiment is basically as shown in the attached Figure 1-3 As shown:

[0042] Preparation method and application of Kaempferia parviflora extract,

[0043] The method comprises the following steps: S1. Preparation of Kaempferia parviflora rhizome extract, the preparation steps are as follows: S1-1: weighing 500g of washed and dried Kaempferia parviflora rhizome, crushing it and placing it in a 1000ml glass container; S1-2: adding 800ml of methanol to the glass container, cold-immersing and extracting at room temperature for 7 days, filtering, and concentrating the filtrate using a rotary evaporator, repeating the extraction and concentration 3-5 times to obtain the Kaempferia parviflora rhizome methanol extract; S1-3: adding 500ml of water to the concentrated methanol extract, extracting the methanol extract; The extract was mixed with water, and then extracted with an equal volume of petroleum ether for 3-5 times. After complete extraction, it was extracted with an equal volume of ethyl acetate, and finally with an equal volume of n-butanol. The extracts were concentrated to obtain petroleum ether layer, ethyl acetate layer and n-butanol layer extracts, respectively, and refrigerated at 4°C for later use; S2. Determination of the antibacterial activity of the rhizome extract of Kaempferia parviflora; S3. Determination of the antifungal activity of the rhizome extract of Kaempferia parviflora; S4. Determination of the cytotoxicity of the rhizome extract of Kaempferia parviflora; S5. High performance liquid chromatography analysis of the rhizome extract of Kaempferia parviflora.

[0044] In S2, the antibacterial activity of the Kaempferia parviflora rhizome extract was determined as follows: S2-1. The test bacteria were activated and cultured on LB plates at 28°C in the dark for 48 h, and single colonies were picked; S2-2. The bacterial solution was diluted to a concentration of 108 cfu·mL by shaking in LB liquid culture medium at 28°C, 150 rpm, and in the dark for 24 h. -1 Set aside; S2-3. Use a pipette to draw up 50 μL of bacterial solution and spread the solution on the plate with a glass rod; S2-4. Use a sterilized hole puncher to evenly punch three holes with a diameter of 6 mm on the bacterial culture medium plate. Add 40 μL of the test bacteria to each well. After incubation in the dark for 24 hours, use a ruler to measure the size of the inhibition zone. The positive control is 5 μL, and the 0.2 mg·mL -1 of streptomycin sulfate, and each treatment was repeated 3 times; the test bacteria included eucalyptus wilt pathogen, Agrobacterium tumefaciens, tomato scab pathogen, cucumber angular leaf spot pathogen, Staphylococcus aureus, Escherichia coli and Bacillus subtilis.

[0045] In S3, the antifungal activity of the Kaempferia parviflora rhizome extract was determined as follows: S3-1: 240 mg of each of three different fungicides was weighed, 0.3 mL of DMSO was added to each fungicide, and after the fungicide and DMSO were completely dissolved, 0.7 mL of sterile water was added; S3-2: 30% DMSO was added to the fungicide again using the half-dilution method, and the fungicide was diluted to 8 different concentrations in sequence; S3-3: 1 mL of each of the 8 prepared fungicides of different concentrations was taken. L were added to 29 mL of PDA, mixed thoroughly and poured into 3 sterile culture dishes, each containing 10 mL, to obtain drug-containing culture media with final fungicide concentrations of 8 mg / mL, 6 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.125 mg / mL, respectively, and 3 parallels were run for each concentration gradient; S3-4: 1 mL of each of the 8 prepared fungicides of different concentrations was added to 29 mL of PDA After thorough mixing, pour into 3 sterile culture dishes, each dish containing 10 mL, to obtain drug-containing culture medium with final fungicide concentrations of 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL and 0.00625 mg / mL, respectively; S3-5: Use a punch to punch the test fungus that has been cultured for 5 days and has grown well along its edge into a 7 mm diameter cake, and inoculate the cake downward on the drug-containing PD A plate; a PDA plate with sterile water added was used as a blank control, a PDA plate with 30% DMSO added was used as a negative control, and a PDA plate with 98.4% carbendazim added was used as a positive control. Each treatment was repeated three times. S3-6: The prepared culture medium was cultured at 28°C for 5 days. When the colonies in the blank control group grew to occupy 2 / 3 of the culture dish area, the colony diameters under different treatments were measured using the cross-hatch method and the inhibition rate was calculated. The inhibition rate was calculated as follows:

[0046]

[0047] Among them, the test fungi include Elaeocarpus pseudocryptocephalus and Colletotrichum oleifera.

[0048] In S4, the cytotoxicity determination steps of the rhizome extract of Kaempferia parviflora are as follows: S4-1: Each test sample is prepared into a sample solution with a concentration of 50 μg / mL, 100 μg / mL, 150 μg / mL and 200 μg / mL, respectively, and the fibroblasts and human tongue squamous cell carcinoma cell suspensions are inoculated into a 96-well plate, and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours; S4-2: 15 μL of CCK-8 reagent is added to each well, and the culture is continued at 37°C and 5% CO2 for 24 hours. The absorbance of each well is detected at 450 nm using a multi-function microplate reader, and the well plate without sample is used as a blank control.

[0049] In S5, the high performance liquid chromatography analysis steps of the rhizome extract of Kaempferia parviflora are as follows: S5-1. Weigh 10 mg of different extracts of Kaempferia parviflora and dissolve the Kaempferia parviflora extract with 1 mL of chromatographic methanol; S5-2. Filter the Kaempferia parviflora extract with a 0.22 μm organic filter membrane, transfer the filtrate to a sample vial, and detect with a diode array detector; wherein, the chromatographic column temperature is 40°C and the injection volume is 5 μL; the chromatographic analysis conditions of the Kaempferia parviflora extract are: 0-1 min, 50% acetonitrile isocratic elution; 1-15 min, acetonitrile concentration linearly increases from 50% to 100%; 15-20 min, 100% acetonitrile isocratic elution; 20-21 min, acetonitrile concentration linearly decreases from 100% to 50%; 21-29 min, re-equilibrate the chromatographic column with 50% acetonitrile.

[0050] The extract of Kaempferia parviflora is used to prepare a biological preparation for inhibiting the fungus Pseudocryphala elaeocarpus.

[0051] The specific implementation process is as follows:

[0052] The first step is to prepare the Kaempferia parviflora rhizome extract

[0053] The preparation steps are as follows: weigh 500 g of washed and dried Kaempferia parviflora rhizomes, crush them and put them into a 1000 ml glass container, then add 800 ml of methanol into the glass container, cold-immerse and extract at room temperature for 7 days, filter, and concentrate the filtrate using a rotary evaporator, repeat the extraction 3-5 times to obtain the methanol extract of Kaempferia parviflora rhizomes, then add 500 ml of water to the concentrated methanol extract, mix the methanol extract and water, and extract them with an equal volume of petroleum ether for 3-5 times after mixing. After the extraction is complete, extract with an equal volume of ethyl acetate, and finally extract with an equal volume of n-butanol, concentrate the extracts separately to obtain petroleum ether layer, ethyl acetate layer and n-butanol layer extracts, and refrigerate at 4°C for later use.

[0054] Step 2: Determination and analysis of the antibacterial activity of Kaempferia parviflora rhizome extract

[0055] The measurement steps are as follows:

[0056] At 28°C, in the dark, the test bacteria were activated and cultured on LB plates for 48 hours. A single colony was picked and then shaken in LB liquid medium at 28°C, 150 rpm, in the dark for 24 hours to dilute the bacterial solution to a concentration of 108 cfu·mL. -1 Set aside; then use a pipette to draw 50 μL of bacterial solution, use a glass rod to spread the bacterial solution on the plate, and then use a sterilized puncher to evenly punch three holes with a diameter of 6 mm on the culture medium plate coated with bacteria. Add 40 μL of the test bacteria to each well, culture in the dark for 24 hours, and measure the size of the inhibition zone with a ruler. Among them, the positive control is 5 μL, 0.2 mg·mL -1 of streptomycin sulfate, and each treatment was repeated 3 times; the test bacteria included eucalyptus wilt pathogen, Agrobacterium tumefaciens, tomato scab pathogen, cucumber angular leaf spot pathogen, Staphylococcus aureus, Escherichia coli and Bacillus subtilis.

[0057] The analysis results are as follows:

[0058] The inhibitory activity of Kaempferia parviflora rhizome extract against 6 test bacteria Figure 1 As shown in the figure, extracts from the rhizomes of Kaempferia parviflora exhibited some antibacterial activity against various test bacteria, but the inhibitory effect was significantly lower than that of the positive control, streptomycin sulfate. Furthermore, the antibacterial activity of the different extracts against the different test bacteria varied significantly at different concentrations. The ethyl acetate extract exhibited the greatest antibacterial activity, followed by the petroleum ether extract, and the n-butanol extract exhibited the least. The petroleum ether and n-butanol extracts exhibited no antibacterial activity at a sample weight of 1.2 mg. As the sample weight increased, the activity gradually increased. However, at a sample weight of 3.6 mg, the petroleum ether extract exhibited no antibacterial activity against tomato scab pathogen, and the n-butanol extract exhibited no antibacterial activity against Bacillus subtilis. Regarding the inhibitory activity against different strains, the extracts from each layer exhibited relatively strong inhibitory activity against bacterial wilt of eucalyptus. At a sample weight of 3.6 mg, the ethyl acetate extract produced an inhibition zone diameter of 20.10 mm against bacterial wilt of eucalyptus, followed by angular leaf spot of cucumber. The diameter of the inhibition zone of each extract from each layer was greater than 10 mm.

[0059] Step 3: Determination and analysis of the antifungal activity of Kaempferia parviflora rhizome extract:

[0060] The measurement steps are as follows:

[0061] Weigh 240 mg of three different fungicides respectively, add 0.3 mL of DMSO to each fungicide, and add 0.7 mL of sterile water after the fungicide and DMSO are completely dissolved; use the half-dilution method to add 30% DMSO to the fungicide again, and dilute the fungicide into 8 different concentrations in turn; take 1 mL of each of the 8 prepared fungicides with different concentrations and add them to 29 mL of PDA respectively, mix them thoroughly and pour them into 3 sterile culture dishes, each dish is 10 mL, to obtain drug-containing culture media with final fungicide concentrations of 8 mg / mL, 6 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.125 mg / mL, respectively, and each concentration gradient is parallel to 3; take 1 mL of each of the 8 prepared fungicides with different concentrations and add them to 29 mL of PDA respectively, mix them thoroughly and pour them into 3 sterile culture dishes, each dish is 10 mL The final concentrations of the fungicide in the culture medium were 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, and 0.00625 mg / mL, respectively. The well-grown test fungi, cultured for 5 days, were punched along their edges into 7 mm diameter cakes using a cork punch and inoculated face-down onto drug-containing PDA plates. The PDA plates containing sterile water served as blank controls, those containing 30% DMSO served as negative controls, and those containing 98.4% carbendazim served as positive controls. Each treatment was repeated three times. The prepared culture medium was incubated at 28°C for 5 days. When the colonies in the blank control group grew to occupy 2 / 3 of the culture dish area, the colony diameters under different treatments were measured using the cross-hatch method, and the inhibition rates were calculated. The test fungi included Erythrocybe pseudocrypticum and Colletotrichum oleifera.

[0062] The analysis results are as follows:

[0063] The inhibitory activity of Kaempferia parviflora rhizome extract against Eurasian elata and Colletotrichum oleifera is shown in Table 1:

[0064]

[0065] As shown in Table 1, the inhibitory activities of the different extracts against the two fungi varied significantly. The inhibitory activity against Pseudocryptoceras euryngii was significantly superior to that against Colletotrichum oleifera. With the exception of the n-butanol extract, which exhibited some inhibitory activity against Colletotrichum oleifera, the EC50 values ​​of the other extracts against Colletotrichum oleifera were all greater than 5.00 mg / mL. The petroleum ether extract showed the strongest inhibitory activity against Pseudocryptoceras euryngii, with an EC50 value of 14.30 ± 1.30 μg / mL, followed by the ethyl acetate extract, which was significantly less active than the petroleum ether extract. The inhibitory activity of the n-butanol extract against Pseudocryptoceras euryngii was similar to that against Colletotrichum oleifera.

[0066] Step 4: Cytotoxicity assay and analysis of Kaempferia parviflora rhizome extract

[0067] The measurement steps are as follows:

[0068] Each test sample was prepared into a sample solution with a concentration of 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL, respectively. The fibroblast and human tongue squamous cell carcinoma cell suspensions were inoculated into a 96-well plate and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. S4-2: 15 μL of CCK-8 reagent was added to each well. After further incubation at 37°C and 5% CO2 for 24 hours, the absorbance of each well was measured at 450 nm using a multifunctional microplate reader. The well plate without sample was used as a blank control.

[0069] The analysis results are as follows:

[0070] like Figure 2 As shown, a is the blank group; b is the petroleum ether layer; c is the ethyl acetate layer; d is the n-butanol layer. It can be found from the figure that the extracts of the petroleum ether layer and the ethyl acetate layer have a significant inhibitory effect on the growth of fibroblasts at concentrations of 100μg / mL, 150μg / mL and 200μg / mL. The higher the concentration, the stronger the inhibitory effect, and at a concentration of about 200μg / mL, it leads to a semi-lethal dose of fibroblasts; however, the extract of the n-butanol layer has a tendency to promote the growth of fibroblasts at medium and low concentrations, especially at concentrations of 50μg / mL and 100μg / mL, there is a significant statistical difference. As the concentration increases, the promoting effect is not obvious, and there is no significant statistical difference compared with the blank group.

[0071] Step 5: HPLC analysis of Kaempferia parviflora rhizome extract

[0072] The analysis steps are as follows:

[0073] Weigh 10 mg of different extracts of Kaempferia parviflora and dissolve the Kaempferia parviflora extract in 1 mL of chromatographic methanol; filter the Kaempferia parviflora extract with a 0.22 μm organic filter membrane, transfer the filtrate to a sample vial, and detect with a diode array detector; the chromatographic column temperature is 40°C and the injection volume is 5 μL; the chromatographic analysis conditions of the Kaempferia parviflora extract are as follows: 0-1 min, 50% acetonitrile isocratic elution; 1-15 min, acetonitrile concentration linearly increases from 50% to 100%; 15-20 min, 100% acetonitrile isocratic elution; 20-21 min, acetonitrile concentration linearly decreases from 100% to 50%; 21-29 min, re-equilibrate the chromatographic column with 50% acetonitrile.

[0074] The analysis results are as follows:

[0075] like Figure 3 As shown in the figure, a is the methanol layer, b is the petroleum ether layer, c is the ethyl acetate layer, and d is the n-butanol layer extract. The figure shows distinct differences in the metabolites produced by the different solvents. The petroleum ether layer primarily contains compounds with lower polarity, with retention times concentrated after 12 minutes. The ethyl acetate layer contains compounds with intermediate polarity, primarily between 6 and 11 minutes. The n-butanol layer contains polysaccharides or glycosides with higher polarity, primarily before 4 minutes. The methanol layer extract is a mixture of all crude extracts, so the distribution ranges from 2 to 16 minutes. At 210 nm, compounds with intermediate polarity have higher UV absorbance. The petroleum ether and ethyl acetate layer extracts significantly inhibited fibroblast growth, likely due to compounds within the 6-16 minute range. However, the n-butanol layer extract exhibited a significant promoting effect at low concentrations, likely due to the higher polarity polysaccharides or glycosides.

[0076] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. The application of Kaempferia parviflora extract is characterized by: The preparation method of Kaempferia parviflora extract comprises the following steps: S1. Preparation of Kaempferia parviflora rhizome extract, the preparation steps are as follows: S1-1: Weigh 500 g of washed and dried Kaempferia parviflora rhizomes, crush them, and place them in a 1000 ml glass container; S1-2: Add 800 ml of methanol to a glass container, perform cold extraction at room temperature for 7 days, filter, and concentrate the filtrate using a rotary evaporator. Repeat the extraction 3-5 times to obtain the methanol extract of Kaempferia parviflora rhizome; S1-3: Add 500 ml of water to the concentrated methanol extract, mix the methanol extract and water, extract with an equal volume of petroleum ether 3-5 times, concentrate the extract to obtain the petroleum ether layer extract, and refrigerate at 4°C for later use; S2. Determination of antibacterial activity of Kaempferia parviflora rhizome extract; S3. Determination of antifungal activity of Kaempferia parviflora rhizome extract; S4. Cytotoxicity assay of Kaempferia parviflora rhizome extract; S5. HPLC analysis of Kaempferia parviflora rhizome extract; The extract of Kaempferia parviflora is used to prepare a biological preparation for inhibiting Pseudocryphonectria elaeocarpicola on Elaeocarpus elaeocarpicus.

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