Extraction method and application of rhizoma kaempferiae extract

Abstract

The invention discloses an extraction method and application of a rhizoma kaempferiae extract, and belongs to the technical field of daily chemical products. The extraction method comprises the following steps: selecting mature rhizoma kaempferiae rhizomes, cleaning, drying and crushing, adding ethanol and enzyme, extracting twice, and performing suction filtration to obtain filtrate; concentrating the filtrate under reduced pressure, performing membrane purification treatment, collecting clear liquid, performing decoloration purification on the clear liquid by using decoloration resin, and drying to obtain a rhizoma kaempferiae extract; the kaempferia galamga extract extracted by the method is relatively high in oxidation resistance, and can effectively inhibit lipid peroxidation, resist inflammation, relieve and other symptoms. The extracted rhizoma kaempferiae extract is wide in application prospect, and new innovation and breakthrough can be brought to the field of daily chemical products.

Description

Technical Field

[0001] This invention relates to the field of daily chemical product technology, and in particular to an extraction method and application of Kaempferia galanga extract. Background Technology

[0002] With economic development and consumption upgrades, the global cosmetics market is undergoing a profound transformation from basic skincare to functional skincare. Consumers, especially the younger generation, are no longer satisfied with just the moisturizing function of products, but are demanding higher standards for efficacy, safety, and natural origin. Against this backdrop, natural active ingredients derived from plants, due to their green and safe image and diverse bioactivities, have become a research hotspot for new product development, meeting the market's urgent need for "pure beauty" and "green chemistry."

[0003] In the field of functional skincare, skin aging and sensitivity are the most prominent issues, and the common key pathophysiological mechanisms behind them are oxidative stress and inflammatory response. However, current solutions for these two major problems have significant limitations:

[0004] In terms of anti-oxidation and anti-aging: Vitamin C (ascorbic acid) and Vitamin E (tocopherol) are well-known classic antioxidants. However, they have extremely poor stability in formulations and are easily deactivated or discolored by light, heat, and oxygen, thus affecting the product's shelf life and appearance. In addition, these ingredients often target only a single site, making it difficult to cope with complex oxidative stress networks, and their transdermal absorption efficiency is limited, resulting in their actual efficacy often being lower than theoretical values.

[0005] Regarding anti-inflammatory and soothing effects on sensitivity: Many soothing products on the market rely on chemically synthesized anti-inflammatory agents or even illegally added corticosteroids. While these ingredients may work quickly, long-term use can further damage the skin barrier, lead to dependence, and even cause serious side effects such as steroid dermatitis, which runs counter to consumers' pursuit of healthy and sustainable skincare.

[0006] Bottlenecks in natural raw materials: Although some natural extracts (such as licorice and centella asiatica) have been widely used, their effects are often relatively mild and difficult to address severe oxidative damage or intense inflammation. More importantly, research on many natural raw materials is not in-depth enough, with unclear active ingredients, ambiguous mechanisms of action, and crude extraction processes, resulting in unstable product quality, large batch-to-batch variations, and an inability to meet the standardization and efficiency requirements of industrial production.

[0007] While traditional cosmetics contain various anti-aging and soothing ingredients, they often suffer from limited effectiveness, insufficient safety, or complex composition. Therefore, developing a natural cosmetic ingredient that can effectively combat oxidation and aging while also reducing inflammation and soothing sensitivity is of significant practical importance and market demand.

[0008] Kaempferia parviflora, also known as black ginger, rotten ginger, or Thai ginseng, is a rare ginger family plant mainly distributed in Southeast Asia. It has been widely used in traditional medicine, and modern pharmacological studies have revealed that its rhizomes are rich in a series of polymethoxyflavonoids, such as 5,7-dimethoxyflavonoids and 3,5,7-trimethoxyflavonoids.

[0009] The key to realizing the cosmetic application value of these highly active methoxyflavonoids lies in how to efficiently and enrich them through specific extraction processes while maintaining their bioactivity. This is also a gap in existing technologies. Summary of the Invention

[0010] The purpose of this invention is to provide an extraction method and application of Kaempferia galanga extract to solve the problems existing in the prior art. The Kaempferia galanga extract extracted by this invention is non-toxic, non-irritating, has no side effects, and does not induce drug resistance; it is a green and safe pure natural extract. This extract can effectively scavenge free radicals (such as DPPH free radicals and hydroxyl free radicals) and inhibit oxidative stress damage to the skin. Simultaneously, it can inhibit the release of inflammatory factors (such as IL-6 and TNF-α), reducing skin redness, itching, and other sensitive symptoms, and can serve as a novel natural raw material with antioxidant and soothing effects.

[0011] To achieve the above objectives, the present invention provides the following solution:

[0012] In a first aspect, the present invention provides a method for extracting an extract of Kaempferia galanga, the extraction method comprising the following steps:

[0013] The small-flowered galangal was pretreated, extracted with ethanol and enzyme preparation, the filtrate was collected, concentrated and purified, the clear liquid was collected, decolorized and purified, the purified liquid was collected and dried to obtain the small-flowered galangal extract.

[0014] Preferably, ethanol is added to the pretreated *Kaempferia galanga* at a material-to-liquid ratio of 1 g:10 mL to 1 g:30 mL, followed by the addition of 0.1-1.0% by mass of an enzyme preparation. The mixture is extracted at 45-65°C for 2-3 h, and the filtrate and filter cake are collected by vacuum filtration. The filter cake is extracted again using the same method, and the filtrate is collected by vacuum filtration. The two filtrates are then combined.

[0015] Preferably, the enzyme preparation is one or more of immobilized lipase, immobilized cellulase, and immobilized β-glucosidase.

[0016] Preferably, the concentration step involves concentrating the filtrate under reduced pressure to 50-80% of its original volume.

[0017] Preferably, the purification step involves purification using a filtration membrane.

[0018] Preferably, the filtration membrane includes one or more of ceramic membranes, microfiltration membranes, and ultrafiltration membranes.

[0019] Preferably, the pore size of the filter membrane is 0.2 μm-500 nm.

[0020] Preferably, the decolorization and purification step involves purification using a decolorization resin; and / or

[0021] The purification conditions for the decolorizing resin are: 1 BV of resin adsorbs 3-4 BV of clear liquid, and the flow rate is 1-2 BV / h.

[0022] Secondly, the present invention also provides a small-flowered galangal extract prepared according to the extraction method.

[0023] Thirdly, the present invention also provides the use of the aforementioned Kaempferia galanga extract in any of the following:

[0024] (1) Preparation of antioxidant and anti-aging products;

[0025] (2) Preparation of anti-inflammatory products.

[0026] The present invention discloses the following technical effects:

[0027] (1) Kaempferia galanga is an edible Chinese medicinal herb, often used to relieve fatigue and improve blood circulation. It is also often added to food as a seasoning, and has the advantages of being green and safe. The methoxyflavonoid component extracted from Kaempferia galanga in this invention is a pure plant-based active ingredient. It is non-toxic, non-irritating, has no side effects, and will not produce drug resistance. It is stable in various types of cosmetic formulations and is suitable for a wide range of people. It is a green, safe, and environmentally friendly cosmetic raw material.

[0028] (2) This invention is the first to add one or more combinations of immobilized lipase, immobilized cellulase and immobilized β-glucosidase to the extraction of Kaempferia galanga, thereby improving the extraction rate of Kaempferia galanga.

[0029] (3) The present invention separates and purifies the methoxyflavonoids of Kaempferia galanga by combining a filter membrane and a decolorizing resin. It exhibits strong antioxidant capacity and has a significant inhibitory effect on DPPH free radicals and ABTS free radicals. It can effectively remove free radicals generated by skin oxidative stress and provide strong support for the skin to resist oxidative damage.

[0030] (4) This invention is the first to discover that Kaempferia galanga and its contained methoxyflavonoids have excellent antioxidant properties, and their ability to scavenge free radicals such as DPPH and ABTS is significantly better than that of most traditional antioxidants. Skin care products prepared with Kaempferia galanga as the core active ingredient have outstanding antioxidant effects, can effectively resist skin oxidative damage, delay the skin aging process, and show excellent effects in improving dullness and reducing wrinkles. Whether for daily protection or combating environmental aging stimuli, they can exert a powerful antioxidant effect. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a flowchart of the extraction process of the small-flowered galangal extract of the present invention. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] The immobilized lipase (derived from MedChemExpress, HY-E70310), immobilized cellulase, and immobilized β-glucosidase were all biosynthesized in-house by our company.

[0039] Both the immobilized cellulase and the immobilized β-glucosidase were synthesized using the sodium alginate encapsulation method.

[0040] Preparation method of immobilized cellulase: Weigh 2 g of sodium alginate and dissolve it in 100 mL of deionized water. Stir in a 60°C water bath until dissolved and cool to 35°C. Add 5 mL of 1 mg / mL cellulase solution (prepared with pH 5.5 acetate-sodium acetate buffer) and stir gently for 10 minutes. Then, use a syringe to inject the mixture dropwise into 5% calcium chloride solution at a rate of 1 drop per second. Let it stand for 2 hours to solidify and form gel beads with a diameter of 1-2 mm. After rinsing 3 times with deionized water, store in PBS buffer at pH 5.5 at 4°C to obtain immobilized cellulase.

[0041] Preparation method of immobilized β-glucosidase: Weigh 1.5 g sodium alginate and dissolve it in 80 mL deionized water. Stir and dissolve in a water bath at 55 °C and cool to 30 °C. Add 4 mL of 0.8 mg / mL β-glucosidase solution (prepared with pH 6.0 phosphate buffer) and stir slowly for 15 minutes. Then, drop the mixture from a height of 10 cm into 4% calcium chloride solution using a syringe. Let it stand and solidify for 1.5 hours to form gel beads. After washing twice with pH 6.0 PBS buffer, soak and store in PBS buffer at 4 °C to obtain immobilized β-glucosidase.

[0042] The extraction process flow chart of the *Kaempferia galanga* extract described in this invention can be seen in the diagram. Figure 1 The specific operating steps can be seen in the following embodiment.

[0043] Example 1: A method for extracting Kaempferia galanga extract

[0044] Mature Kaempferia galanga rhizomes were washed, dried, and pulverized. 50% (volume fraction) ethanol was added at a material-to-liquid ratio of 1:10 (g:mL), followed by 0.1% (w / w) immobilized lipase. Extraction was carried out at 45℃ for 2 h. The filtrate and filter cake were collected separately. The filter cake was extracted and filtered again using the same method, and the filtrate was combined. The filtrate was reduced to 50% of its original volume and purified using a 200 nm ceramic membrane. The supernatant was collected and purified using D941 decolorizing resin. 1 BV of resin adsorbed 3 BV of the supernatant, with a flow rate of 1.5 BV / h. The purified liquid was collected and dried to obtain Kaempferia galanga extract.

[0045] Example 2: A method for extracting Kaempferia galanga extract

[0046] The mature rhizomes of *Kaempferia galanga* were washed, dried, and pulverized. 80% (volume fraction) ethanol was added at a material-to-liquid ratio of 1:20 (g:mL), followed by 0.5% (w / w) of a complex enzyme (immobilized lipase and immobilized cellulase mixed at a 1:1 mass ratio). Extraction was carried out at 55℃ for 2 h. The filtrate and filter cake were collected separately by vacuum filtration. The filter cake was extracted and filtered again using the same method, and the filtrate was combined. The filtrate was reduced to 50% of its original volume and purified using a 0.22 μm microfiltration membrane. The supernatant was collected and purified using LX-98 decolorizing resin. 1 BV of resin adsorbed 4 BV of the supernatant, with a flow rate of 1.5 BV / h. The purified liquid was collected and dried to obtain the *Kaempferia galanga* extract.

[0047] Example 3: An extraction method for Kaempferia galanga extract

[0048] The mature rhizomes of *Kaempferia galanga* were washed, dried, and pulverized. 80% (volume fraction) ethanol was added at a material-to-liquid ratio of 1:20 (g:mL), followed by 0.5% (w / w) immobilized β-glucosidase. Extraction was carried out at 55℃ for 2 h. The filtrate and filter cake were collected separately. The filter cake was extracted and filtered again using the same method, and the filtrate was combined. The filtrate was reduced to 50% of its original volume and purified using a 0.45 μm ultrafiltration membrane. The supernatant was collected and purified using LX-98 decolorizing resin. 1 BV of resin adsorbed 4 BV of the supernatant, at a flow rate of 1.5 BV / h. The purified liquid was collected and dried to obtain the *Kaempferia galanga* extract.

[0049] Comparative Example 1

[0050] The only difference from Example 1 is that the enzymatic hydrolysis step is removed, while the other steps remain the same.

[0051] Comparative Example 2

[0052] The only difference from Example 1 is the removal of the decolorizing resin adsorption; the other steps remain the same.

[0053] Example 4: Liquid chromatography method for the determination of methoxyflavonoids in Kaempferia galanga extract

[0054] Main instruments and reagents: Shimadzu high performance liquid chromatograph (LC-2050), phosphoric acid (chromatographic grade, purity >99%, Anaiji Chemical Reagent Co., Ltd.), acetonitrile (chromatographic grade, purity >99%, Anaiji Chemical Reagent Co., Ltd.), 5,7-dimethoxyflavonoid standard (purity >99%, Aladdin Reagent Co., Ltd.).

[0055] Preparation of standards and samples: Accurately weigh 5 mg of 5,7-dimethoxyflavonoid standard using a 0.01 g / L balance and place it in a 10 mL volumetric flask. Dissolve in methanol and dilute to the mark. Shake well to obtain a 2 g / L 5,7-dimethoxyflavonoid standard stock solution. Accurately weigh 5 mg of Kaempferia galanga extract using a 0.01 g / L balance and place it in a 10 mL volumetric flask. Dissolve in methanol and dilute to the mark. Shake well. Vortex for 5 min, centrifuge at 12000 r / min for 10 min, collect an appropriate amount of the supernatant, filter through a 0.22 µm filter membrane, and reserve the filtrate.

[0056] HPLC analysis conditions: Shimadzu C18 column (4.6×150 mm, 5 μm), column temperature and cell temperature: 30℃, mobile phase: 0.2% phosphoric acid aqueous solution-acetonitrile (0.2% phosphoric acid aqueous solution and acetonitrile volume ratio of 55:45), flow rate: 1 min, detection wavelength: 270 nm, time: 30 min, injection volume: 10 μL.

[0057] Prepared standard stock solutions of 5,7-dimethoxyflavonoids were used to prepare standard solutions with concentrations of 0.01, 0.1, 0.2, and 2 g / L, respectively. The peak areas of 5,7-dimethoxyflavonoids were measured using the method described above, and linear regression was performed. The regression equation was found to be y = 2.13386 × 10⁻⁶. 7 x-176357 (x is the concentration of 5,7-dimethoxyflavonoids, y is the peak area, R...) 2 =0.9997).

[0058] The results showed that the measured results were linear in the concentration range of 0.01-2 g / L, and the content of 5,7-dimethoxyflavonoids in the test sample could be accurately determined.

[0059] The content of 5,7-dimethoxyflavonoids in the extracts obtained by the extraction methods of Examples 1-3 and Comparative Examples 1-2 was determined using the above method, and the results are shown in Table 1.

[0060] Table 1. Content of 5,7-dimethoxyflavones in the extracts obtained from the examples and comparative examples.

[0061]

[0062] Therefore, it can be seen that the content of 5,7-dimethoxyflavonoids in the extract obtained by the extraction method described in this invention is significantly higher than that in the comparative example.

[0063] Example 5 Orthogonal experimental design and results for process optimization of Kaempferia galanga extract

[0064] The combined process parameters of ethanol extraction and enzymatic hydrolysis were optimized to clarify the effect of each parameter on the content of 5,7-dimethoxyflavonoids in the extract and to verify the synergistic effect of the combination. The factor level design is shown in Table 2, and the orthogonal experimental design is shown in Table 3.

[0065] Table 2 4-Factor 3-Level Experiment

[0066]

[0067] The extraction conditions were fixed at 55℃ for 2.5 h, and the extraction was performed twice.

[0068] Table 3 Orthogonal Arrays and Experimental Combinations

[0069]

[0070] Three control groups were set up: Control 1 (single 70% ethanol group): 1 g: 20 mL material-to-liquid ratio + 70% ethanol, no enzyme added, other conditions the same as above; Control 2 (single enzymatic digestion group): 1 g: 20 mL material-to-liquid ratio + 0.5% compound enzyme, water was used instead of ethanol, other conditions the same as above; Control 3 (single 90% ethanol group): 1 g: 20 mL material-to-liquid ratio + 90% ethanol, no enzyme added, other conditions the same as above.

[0071] Table 4. Results of the orthogonal experiment

[0072]

[0073] According to the orthogonal experimental results in Table 4, the optimal extraction process for the extract of Kaempferia galanga is as follows: Take the pretreated Kaempferia galanga, add 70% ethanol at a material-to-liquid ratio of 1 g:20 mL, and then add 0.5% by mass of a compound enzyme (immobilized lipase and immobilized cellulase mixed in a 1:1 mass ratio). Extract at 55℃ for 2.5 h, filter and collect the filtrate; repeat the above extraction operation once with the filter cake, combine the two filtrates, and obtain the target extract after concentration, purification, decolorization and drying.

[0074] Under this process, the content of 5,7-dimethoxyflavonoids in the extract can reach 0.66±0.05 g / L, which is significantly higher than that of the single ethanol extraction group (0.33±0.11 g / L for 70% ethanol and 0.48±0.12 g / L for 90% ethanol) and the single enzymatic hydrolysis group (0.22±0.09 g / L), indicating that the combination of ethanol extraction and enzymatic hydrolysis has a significant synergistic effect. Specifically, 70% ethanol can efficiently dissolve 5,7-dimethoxyflavonoids, while the complex enzyme can synergistically degrade cell wall cellulose and lipid components, breaking down structural barriers and promoting the release of active ingredients. The 1 g:20 mL solid-liquid ratio ensures solvent dissolution efficiency while avoiding the increased subsequent concentration costs caused by an excessively high solid-liquid ratio, thus achieving safe and efficient extraction of active ingredients.

[0075] Example 6: Determination of antioxidant activity

[0076] Based on the optimal process parameters obtained by orthogonal experiment, the small flower galangal was extracted to obtain the small flower galangal extract, and then the antioxidant activity was determined by experiment.

[0077] 1. Determination of DPPH free radical scavenging capacity

[0078] 1,1-Diphenyl-2-picrylhydrazine is a stable nitrogen-centered free radical. The presence of multiple electron-withdrawing -NO2 groups and a large π-bond on the benzene ring in its molecule ensures the stability of this nitrogen free radical. DPPH analysis is a simple method for screening free radical scavengers and evaluating antioxidant activity. DPPH in ethanol is purple, with a maximum absorption wavelength of 517 nm. When a free radical scavenger is added to the DPPH solution, its lone pair electrons are paired, causing the absorption to disappear or weaken, resulting in a lighter color (yellow or pale yellow) and a decrease in absorbance at 517 nm. The degree of change is linearly related to the free radical scavenging intensity, thus allowing for quantitative analysis using spectrophotometry. This method can be expressed as a scavenging rate; a higher scavenging rate indicates a stronger scavenging ability.

[0079] 1.1 Reagent Preparation

[0080] DPPH (2×10) -4 Preparation of 20 mg DPPH ethanol solution: Weigh 20 mg DPPH, dissolve in anhydrous ethanol and dilute to 250 mL in a volumetric flask, and store at 0-4℃ protected from light.

[0081] 1.2 Operating Procedures

[0082] Mix 3 mL of the test solution with 3 mL of DPPH solution and measure the absorbance at 517 nm (A1). Mix 3 mL of distilled water with 3 mL of DPPH solution and measure the absorbance at 517 nm (A2). Mix 3 mL of distilled water with 3 mL of the test solution and measure the absorbance at 517 nm (A3).

[0083] The formula for calculating the clearance rate is:

[0084]

[0085] The experimental results showed that the extract of Kaempferia galanga has good antioxidant activity against DPPH, and the scavenging rate of Kaempferia galanga extract against DPPH can reach 92.65% at 1000 ppm.

[0086] 2. Determination of ABTS free radical scavenging ability

[0087] The maximum absorption wavelength of ABTS radical ions is 734 nm. 734nm It can detect the concentration of ABTS radical ions. If A 734nm A decrease in absorbance indicates that ABTS radical ions have been scavenged. The ABTS method reflects the sample's ability to scavenge ABTS radicals by measuring the degree of fading in the reaction system. ABTS is oxidized to green ABTS radicals by an oxidant. The production of ABTS radicals is inhibited in the presence of antioxidants, causing the reaction system to fade and the absorbance at 405 nm to decrease. Within a certain range, the change in absorbance is directly proportional to the degree of radical scavenging.

[0088] 2.1 Reagent Preparation

[0089] ABTS stock solution (7 mM): Weigh 0.1938 g ABTS (2,2'-azido-bis-3-ethylbenzothiazole-6-sulfonic acid) and dissolve it in 100 mL of ultrapure water. Store in the dark.

[0090] Potassium persulfate solution (2.45 mM): Weigh 0.0598 g of potassium persulfate (K2S2O8) and dissolve it in 100 mL of ultrapure water. Prepare fresh before use.

[0091] ABTS working solution: Mix ABTS stock solution and potassium persulfate solution at a 1:1 volume ratio and react at room temperature in the dark for 12 hours to form a blue-green ABTS free radical solution. Before use, dilute with phosphate-buffered saline (PBS, pH=7.4) or ethanol to an absorbance of 0.70±0.02 at 734 nm.

[0092] Sample solution: Dissolve the extract of Kaempferia galanga in ethanol.

[0093] 2.2 Operating Procedures

[0094] Blank group: 160 μL ABTS working solution + 40 μL ethanol solvent; Sample group: 160 μL ABTS working solution + 40 μL sample solution (replicas of different concentration gradients, n≥3); gently shake to mix, and react at room temperature in the dark for 6 minutes. Measure the absorbance of each well at 734 nm (A0). 样品 At the same time, the absorbance of the blank group (A0) was recorded.

[0095] The formula for calculating the clearance rate is:

[0096]

[0097] The experimental results showed that the extract of Kaempferia galanga has good antioxidant activity, and the scavenging rate of Kaempferia galanga extract against ABTS free radicals can reach 91.57% at 1000 ppm.

[0098] Example 7: Cytotoxicity and Antioxidant Activity Studies

[0099] Based on the optimal process parameters obtained by orthogonal experiments, the extract of *Kaempferia galanga* was obtained, and then its cytotoxicity and antioxidant activity were determined.

[0100] 1. Cell Culture

[0101] Human normal skin fibroblasts (HFF-1, purchased from the Cell Bank of the Chinese Academy of Sciences) were removed from the liquid nitrogen container and rapidly thawed in a 37°C water bath. They were then added to DMEM high-glucose culture medium containing 1% sodium pyruvate, 1% penicillin antibiotics, and 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO2 saturated humidity to resuscitate the cells. After digestion with 0.25% trypsin, the cells were passaged, with the medium changed every two days. Experiments were started after two passages, with each experiment using the same passaged cells. HFF-1 cells were cultured at a rate of 2.0 × 10⁶ cells / year. 5 The cells were seeded at a concentration of 100 μL / mL in 96-well plates (for CCK-8 assay) and 2 mL / well plates (for SOD and MDA assay), and cultured for 24 hours to allow the cells to adhere completely.

[0102] 2. Cytotoxicity test of Kaempferia galanga extract

[0103] After cells adhered to the 96-well plates, the original culture medium was discarded, and complete culture medium containing different concentrations of Kaempferia galanga extract (50, 100, 200 μg / mL) was added, with five replicates for each concentration. The plates were then incubated for 12 hours. After treatment, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Blank wells (containing only culture medium and CCK-8 reagent) were used as controls, and cell viability was calculated.

[0104] The formula for calculating cell viability is:

[0105]

[0106] Table 5 Effects of Kaempferia galanga extract on cell viability

[0107]

[0108] The results of the cytotoxicity test of Kaempferia galanga extract are shown in Table 5. As can be seen from Table 5, the cell survival rate was 110% at 50 μg / mL of Kaempferia galanga extract, 102% at 100 μg / mL of Kaempferia galanga extract, and 108% at 200 μg / mL of Kaempferia galanga extract. This means that Kaempferia galanga extract has no obvious toxic effect on cells.

[0109] 3. Assay of cellular antioxidant activity

[0110] The antioxidant activity assay consisted of six groups. After 24 h of pre-incubation, the normal control group received no modeling or drug intervention, maintaining normal cell conditions. The H2O2 model group was subsequently modeled using 200 μM H2O2. Low (50 μg / mL), medium (100 μg / mL), and high (200 μg / mL) doses of Kaempferia galanga extract were added, with the corresponding concentrations of extract added. The positive control group received 20 μg / mL ergothioneine. Each drug group was pretreated for 12 h with an appropriate volume covering the cells (e.g., 200 μL of drug-containing medium per well in a 96-well plate). After discarding the pretreatment solution and washing with PBS, all groups except the normal control group were treated with 200 μM H2O2 for 2 h. After treatment, cells were collected from the well plates, washed with PBS, and lysed with cell lysis buffer containing protease inhibitors. Cells were then sonicated on ice for 30 seconds, centrifuged at 12000 r / min for 10 min at 4℃, and the supernatant was collected. SOD activity (U / mg·prot) was calculated by measuring absorbance at 450 nm using the WST-1 method, and GSH content (μmol / mg·prot) was calculated by measuring absorbance at 412 nm using the DTNB colorimetric method. These results were used to assess antioxidant activity. The results are shown in Table 6.

[0111] Table 6. Effects of Kaempferia galanga extract on GSH and SOD levels due to oxidative damage.

[0112]

[0113] As shown in Table 3, Kaempferia galanga extract has a protective effect against H2O2-induced oxidative damage in HFF-1 cells. Data showed that SOD activity and GSH levels in the model group cells decreased significantly, while all doses of Kaempferia galanga extract increased SOD activity and GSH levels in a dose-dependent manner, with the high-dose group showing particularly significant effects. The GSH level in the high-dose group was similar to that of the positive control group. This indicates that Kaempferia galanga extract can protect HFF-1 cells by enhancing the cellular antioxidant defense system and reducing oxidative stress damage. However, in terms of increasing SOD activity, it still lags behind the positive control, providing experimental evidence for further investigation of its antioxidant mechanism and optimized application.

[0114] Example 8: Anti-inflammatory activity study

[0115] Based on the optimal process parameters obtained by orthogonal experiments, *Kaempferia galanga* extract was obtained and then its anti-inflammatory activity was studied.

[0116] 1. CCK-8 test

[0117] RAW264.7 macrophage cell line (Beina Biotechnology Co., Ltd.) was cultured in a special medium (Wuhan Pronosei Life Sciences Co., Ltd.). Cells were seeded into 96-well plates and cultured until they reached the logarithmic growth phase. Cells were then treated with 100 μL of sample and cultured for 24 h. After the culture medium was removed, 100 μL of 10% CCK-8 solution was added to each well. The plates were incubated for 1–4 h, and the absorbance at 450 nm was measured. Cell viability was then determined.

[0118] Cell viability is calculated using the following formula:

[0119]

[0120] In the formula, A (blank) is the absorbance of the culture medium alone; A (negative control) is the absorbance of the culture medium with only cells added and no sample added; A (sample) is the absorbance of the culture medium with both sample and cells added.

[0121] 2. Flowchart for LPS stimulation of cells and qPCR detection of anti-inflammatory genes

[0122] Cells were seeded in 24-well plates and cultured until they reached the logarithmic growth phase, after which the culture medium was discarded. Sample groups were treated with fresh culture medium containing LPS (Beijing Solarbio Science & Technology Co., Ltd.) and different concentrations of Kaempferia galanga extract (50, 100, and 200 μg / mL), model groups were treated with fresh culture medium containing LPS, positive controls were treated with fresh culture medium containing LPS and dexamethasone (1 μg / mL), and the normal control group was treated with fresh culture medium without any additives. After drug administration, the plates were incubated for 17 h. Cells were collected, and total RNA was extracted from each experimental group. The RNA was reverse transcribed into cDNA, and the expression of the internal control GADPH and the target genes IL-6 and TNF-α was detected using qPCR (RNA extraction reagent, qPCR enzyme (Nanjing Novizan Biotechnology Co., Ltd.), reverse transcription kit (Tiangen Biotech (Beijing) Co., Ltd.)).

[0123] Using GAPDH as an internal control for gene expression, the relative RNA expression level of the target gene was calculated:

[0124]

[0125]

[0126]

[0127] Table 7. Inhibitory effects of different concentrations of samples on IL-6 and TNF-α gene expression.

[0128]

[0129] As shown in Table 7, the extract of *Kaempferia galanga* inhibited the expression of IL-6 and TNF-α in an LPS-induced cellular inflammation model. Data showed that the relative expression levels of IL-6 and TNF-α genes in the model group were significantly higher than those in the control group. Furthermore, each dose of *Kaempferia galanga* extract significantly reduced the relative expression levels of IL-6 and TNF-α genes in a dose-dependent manner, with the high-dose group showing particularly significant effects. This indicates that *Kaempferia galanga* extract can alleviate cellular inflammatory damage by inhibiting the expression of inflammation-related genes, providing experimental evidence for its application in the anti-inflammatory field. Further research is needed to explore the specific mechanisms by which it regulates inflammatory pathways.

[0130] Example 9: Application of an extract of Kaempferia galanga with antioxidant properties in the preparation of emulsions.

[0131] Based on the optimal process parameters obtained by orthogonal experiments, the extract of *Kaempferia galanga* was obtained, and the emulsion was prepared based on this extract.

[0132] Adding Kaempferia galanga extract at a concentration of 0.5%-5% to cosmetics can prepare cosmetics with antioxidant effects. This example uses an emulsion with antioxidant effects as an example.

[0133] Aqueous phase components: Deionized water 71.0%, Glycerin 4.0%, Kaempferia galanga extract 3.0%, Centella asiatica extract 3.0%, Sodium hyaluronate 0.2%, Dipotassium glycyrrhizate 0.5%, Panthenol 2.0%, Acrylic acid esters / C10-30 alkanol acrylate crosspolymer (thickener) 0.3%, Phenoxyethanol + Ethylhexylglycerin (9:1 ratio) (preservative) 0.5%.

[0134] Oil phase components: Caprylic / Capric triglycerides (GTCC) 6.0%, Squalane 5.0%, Olive oil PEG-7 esters (Olivem 1000) 3.5%, Bisabolol 0.5%, Phytosphingosine 0.3%, Tocopheryl acetate 0.2%.

[0135] Aqueous phase preparation: Deionized water was heated to 75°C, and glycerol, sodium hyaluronate, and acrylate / C10-30 alkanol acrylate crosspolymer were added. The mixture was stirred until completely dissolved and free of particles. The temperature was then lowered to 60°C, and galangal extract, centella asiatica extract, and dipotassium glycyrrhizate were added. The mixture was stirred at low speed to avoid bubbles and maintained for 10 minutes to form a homogeneous aqueous phase.

[0136] Oil phase preparation: In a separate container, heat Olivem 1000 to 75°C to melt it, and then add GTCC, squalane, bisabolol, phytosphoethanolamine, and tocopheryl acetate in sequence, stirring until transparent and homogeneous.

[0137] The oil phase (75°C) was slowly injected into the aqueous phase (60°C) in a thin stream while a homogenizer was activated for high-speed shearing (5000 rpm, 3 minutes) to form a primary emulsion. The mixture was then cooled to 45°C, and panthenol and preservatives (phenoxyethanol + ethylhexylglycerin) were added. The mixture was stirred using a paddle mixer (200 rpm) for 15 minutes to form a homogeneous emulsion. When the emulsion cooled to 30°C, stirring was stopped, and the mixture was transferred to a vacuum homogenizer for degassing for 10 minutes. After filling, the mixture was aged at 25°C in the dark for 72 hours until the system properties stabilized, yielding the emulsion.

[0138] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting an extract of Kaempferia galanga, characterized in that, The extraction method includes the following steps: The small-flowered galangal was pretreated, extracted with ethanol and enzyme preparation, the filtrate was collected, concentrated and purified, the clear liquid was collected, decolorized and purified, the purified liquid was collected and dried to obtain the small-flowered galangal extract.

2. The extraction method according to claim 1, characterized in that, Ethanol was added to the pretreated Kaempferia galanga at a material-to-liquid ratio of 1 g:10 mL to 1 g:30 mL, followed by 0.1-1.0% (w / w) of enzyme preparation. The mixture was extracted at 45-65°C for 2-3 h. The filtrate and filter cake were collected by vacuum filtration. The filter cake was extracted again using the same method, and the filtrate was collected by vacuum filtration. The two filtrates were then combined.

3. The extraction method according to claim 2, characterized in that, The enzyme preparation is one or more of immobilized lipase, immobilized cellulase, and immobilized β-glucosidase.

4. The extraction method according to claim 1, characterized in that, The concentration step involves concentrating the filtrate under reduced pressure to 50-80% of its original volume.

5. The extraction method according to claim 1, characterized in that, The purification step involves purifying the material using a filtration membrane.

6. The extraction method according to claim 5, characterized in that, The filtration membrane includes one or more of ceramic membranes, microfiltration membranes, and ultrafiltration membranes.

7. The extraction method according to claim 6, characterized in that, The pore size of the filter membrane is 0.2 μm-500 nm.

8. The extraction method according to claim 1, characterized in that, The decolorization and purification step involves purification using a decolorizing resin; and / or The purification conditions for the decolorizing resin are: 1 BV of resin adsorbs 3-4 BV of clear liquid, and the flow rate is 1-2 BV / h.

9. A small-flowered galangal extract prepared by the extraction method according to any one of claims 1-8.

10. The use of the Kaempferia galanga extract as described in claim 9 in any of the following: (1) Preparation of antioxidant and anti-aging products; (2) Preparation of anti-inflammatory products.

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