Use of an alcohol extract of rubus parvifolius root in the preparation of an anti-inflammatory composition

Abstract

The application discloses application of an alcohol extract of a root of a wild angelonia in preparation of an anti-inflammatory composition. The alcohol extract of the root of the wild angelonia has obvious anti-inflammatory capacity in vitro, and can be applied to the fields of health-care food, cosmetics and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to the application of the root alcohol extract of Melastoma candidae in the preparation of anti-inflammatory compositions. Background Technology

[0002] With increasing health awareness, consumers are paying more and more attention to natural medicines and ingredients. Among them, traditional Chinese medicine (TCM), a time-tested and historically significant natural remedy, is rich in bioactive components and offers various health benefits, making it highly popular. As a country with abundant TCM resources, China has begun to apply these valuable resources to the research and development of skincare products. TCM can be extracted and separated to obtain various effective components, which can then be used in cosmetics. These components have multiple effects, such as anti-oxidation, anti-inflammation, whitening, and moisturizing, helping to improve skin texture, promote blood circulation, and provide deep repair, thus achieving long-term health and skincare benefits. Compared to chemically synthesized raw materials, TCM ingredients are safer and less harmful, with fewer side effects and less burden on the skin. Therefore, the safety and practicality of innovative TCM skincare products are greatly improved. With increasing consumer enthusiasm for TCM skincare products, more and more TCM cosmetic brands and even professional research teams specializing in TCM beauty have emerged in the market. They have incorporated the concepts of TCM research into their cosmetics to meet consumers' demand for natural and health-promoting skincare products. The development and application of traditional Chinese medicine (TCM) skincare products not only helps broaden the application areas of TCM, but also injects new vitality into the cosmetics industry. With the support of TCM resource development, the cosmetics industry will usher in a broader prospect and market space.

[0003] *Tibouchina granulosa*, an evergreen plant belonging to the genus *Tibouchina* (also known as the Glory Tree or Blue Tree) in the family Melastomataceae, adapts well to the soil conditions of Fujian province. It generally exhibits good growth, abundant and vibrant flowers, and demonstrates strong pollution resistance when planted as a street tree. However, to date, there are almost no reports on the medicinal effects of *Tibouchina granulosa*, and even fewer reports on its anti-inflammatory properties and applications. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects of the prior art and provide the application of the root alcohol extract of Melastoma candidae in the preparation of anti-inflammatory compositions.

[0005] Another object of the present invention is to provide an anti-inflammatory composition.

[0006] The technical solution of the present invention is as follows:

[0007] Application of the root alcohol extract of Melastoma hornbeam in the preparation of anti-inflammatory compositions.

[0008] In a preferred embodiment of the present invention, the preparation method of the root alcohol extract of Melastoma hornbillii includes: pulverizing the root of Melastoma hornbillii, mixing it thoroughly with 90-100% ethanol at a mass-volume ratio of 1:4-6 and dispersing it evenly, extracting it by ultrasonic vibration, then centrifuging to obtain the supernatant, vacuum rotary evaporation and freeze drying to obtain the extract.

[0009] More preferably, the ultrasonic oscillation extraction specifically involves: ultrasonic oscillation extraction, followed by a 18-25s pause, and then continuing ultrasonic oscillation extraction, repeating this cycle, with the total ultrasonic oscillation extraction time being 1-2 hours.

[0010] More preferably, the temperature of the vacuum rotary evaporation is 35-70°C.

[0011] An anti-inflammatory composition comprising an alcoholic extract of Melastoma hornbillii root.

[0012] In a preferred embodiment of the present invention, the active ingredient is the root alcohol extract of Melastoma hornbillii.

[0013] More preferably, the preparation method of the root alcohol extract of Melastoma hornbillii includes: crushing the root of Melastoma hornbillii, mixing it thoroughly with 90-100% ethanol at a mass-volume ratio of 1:4-6 and dispersing it evenly, extracting it by ultrasonic vibration, then centrifuging to obtain the supernatant, vacuum rotary evaporation and freeze drying to obtain the extract.

[0014] More preferably, the ultrasonic oscillation extraction specifically involves: ultrasonic oscillation extraction, followed by a 18-25 second pause, and then continuing ultrasonic oscillation extraction, repeating this cycle, with the total ultrasonic oscillation extraction time being 1-2 hours.

[0015] More preferably, the temperature of the vacuum rotary evaporation is 35-70°C.

[0016] The beneficial effects of this invention are: the ethanol extract of Melastoma horn stem root in this invention has significant anti-inflammatory ability in vitro and can be used in health foods and cosmetics. Attached Figure Description

[0017] Figure 1 The figure shows the experimental results of the ability of the root alcohol extract of Melastoma hornbeam in Example 1 of the present invention to inhibit lipoxygenase activity.

[0018] Figure 2 This is a graph showing the experimental results of the biochemical NO scavenging ability of the root alcohol extract of Melastoma horn in Example 1 of the present invention.

[0019] Figure 3 The figure shows the experimental results of the MTT assay of the root alcohol extract of Melastoma candidae in Example 1 of this invention.

[0020] Figure 4 This is a graph showing the experimental results of Western blot in Embodiment 1 of the present invention. Detailed Implementation

[0021] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0022] Example 1

[0023] 1. Materials and Methods

[0024] 1.1 Materials and Reagents

[0025] Tibouchina granulosa (Desr.) Cogn. (purchased from Southeast Flower City, Zhangzhou City); Vitamin C (Vc) reagent purchased from Sangon Biotech (Shanghai) Co., Ltd.; 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) purchased from Tokyo Chemical Industry Co., Ltd., Japan; Total antioxidant capacity assay kit (ABTS method) and nitric oxide assay kit purchased from Shanghai Beyotime Biotechnology Research Institute; L7395 lipoxygenase (Sigma-Aldrich); linoleic acid and Tris-HCl buffer (Tris-HCl) All buffer solutions were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; sodium nitroprusside was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; DMEM and FBS were purchased from Gibco, Inc. (USA); DMSO was purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd.; PBS buffer solution was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; methanol, sodium hydroxide, anhydrous ethanol, sodium chloride, ferrous chloride, and EDTA were purchased from Xilong Scientific Co., Ltd.; RAW264.7 cells were purchased from the Typical Culture Cell Bank of the Chinese Academy of Sciences; trypsin and MTT were purchased from Sigma-Aldrich (USA); β-actin, COX-2, and p-ERK antibodies were purchased from Cell Signaling Technology, Inc. (USA).

[0026] 2. Methods

[0027] 2.1 Preparation of Melastoma Root Alcohol Extract

[0028] After crushing the roots, a certain mass was accurately weighed and placed in a beaker. The root powder was mixed with 95% ethanol at a ratio of 1:5 (i.e., 50g of root powder to 250mL of 95% ethanol, with the ethanol level at least 1cm above the powder). The mixture was stirred to ensure complete dispersion and then subjected to ultrasonic extraction (650W, 85%) for 10 seconds, followed by a 20-second pause and another 10-second extraction. This cycle was repeated for a total ultrasonic extraction time of 60 minutes. The extracted liquid was then centrifuged for 10 minutes, and the supernatant was collected. The supernatant was then poured into a distillation flask, fitted with a rotary evaporator and secured with clamps. The rotation speed was set to approximately 80 rpm, and the vacuum pump was turned on. Once a certain vacuum was achieved, rotation began. Most of the solvent was extracted in a water bath at 45°C. The product in the distillation flask is the root extract of *Melastoma candida*. Centrifuge tubes containing Melastoma root extract were placed in a freeze dryer and freeze-dried for 48-72 hours to obtain Melastoma root alcohol extract powder.

[0029] For comparison, the settings are as follows:

[0030] A. Replace the 95% ethanol with water.

[0031] B. Replace the above roots with stems and leaves.

[0032] C. Change the ultrasonic extraction method to immersion extraction.

[0033] 2.2 Assay for the inhibitory capacity of lipoxygenase activity

[0034] The obtained Melastoma root alcohol extract powder and the control sample were diluted to different concentrations (2.0 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL), with vitamin C as the positive control. 1.0 mL of each concentration of sample and control group was taken, and 2.0 mL of lipoxygenase (2500 units) was added. Then, 1.5 mL (0.5%) of linoleic acid was used as the reaction matrix, and 95.5 mL of Tris-HCl buffer (pH 9.0) was added. The control group was based on an equal amount of DMSO. After complete and uniform mixing, the mixture was reacted in a water bath at 50℃. The OD value at 234 nm was measured after 5 min of reaction and again after 10 min. The changes in the OD values ​​were observed, and three parallel experiments were performed.

[0035] Formula for calculating enzyme activity units: A = 1000 × (OD) 10min -OD 5min ) / 2

[0036] In the formula: A is the enzyme activity unit; OD 10min The OD value is the result of the reaction after 10 minutes; OD5min The value is the OD value after 5 minutes of reaction.

[0037] The inhibition rate is calculated using the formula: (A0-A1) / A0*100%.

[0038] In the formula: A0 is the enzyme activity of the control group, and A1 is the enzyme activity when the root extract of Melastoma dodecandrum is added.

[0039] 2.3 Biochemical level NO content detection

[0040] The obtained Melastoma root alcohol extract powder and the control sample were diluted to the following concentrations (0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL), with vitamin C as the positive control. 10 mL of each concentration sample was mixed with 90 mL of sodium nitroprusside (20 mM) and reacted at 25°C for 60 min. 50 mL of Griess Reagent I and 50 mL of Griess Reagent II from a nitric oxide assay kit (Beyotime Biotechnology Co., Ltd.) were added and mixed thoroughly. The mixture was then spectrophotometrically measured at 540 nm.

[0041] The calculation formula is as follows: Relative clearance rate % = (OD0 - OD1) / OD0' × 100%

[0042] (OD0: blank control group, OD1: sample experimental group)

[0043] 2.4 MTT assay of RAW264.7 cells

[0044] Cell suspensions were prepared using culture medium containing 10% fetal bovine serum and seeded into sterile 96-well plates at 100 mL per well. The plates were incubated overnight (14 ± 2 h) at 37°C with 5% CO2. When the cell count reached approximately 85% of the wells, 20 mL of sample and 70 mL of culture medium containing 10% fetal bovine serum were added to each well. The plates were incubated for 24 h, then 10 mL of MTT solution was added to each well. The plates were incubated for another 4 h, and the culture was terminated. The supernatant was carefully aspirated from the wells, and 200 mL of DMSO was added to each well. The plates were shaken for 10 min to fully dissolve the crystals, and the absorbance was measured at 490 nm using a microplate reader.

[0045] The calculation formula is as follows: Cell viability % = OD1 / OD0 × 100%

[0046] (OD0: blank control group, OD1: sample experimental group)

[0047] 2.5 Assay of NO clearance capacity at the cellular level

[0048] A plate of RAW264.7 cells was digested with trypsin, centrifuged, and resuspended. The cells were then resuspended in culture medium containing 10% fetal bovine serum and seeded into 96-well plates (100 mL per well). The plates were incubated overnight (14 ± 2 h) at 37°C with 5% CO2. When the cell count reached approximately 85% of the wells, the culture medium was discarded, and the cells were washed with 200 μL / well of PBS. For the control group, 195 mL of culture medium and 5 mL of PBS were added to each well. For the sample group, 193 mL of cell culture medium, 2 mL of LPS working solution, and 5 mL of the sample were added to each well. The plates were incubated at 37°C with 5% CO2 for 24 h. 50 mL of supernatant was collected from each well, and 50 mL of Griess Reagent I and 50 mL of Griess Reagent II were added. The absorbance at 540 nm was measured using a microplate reader. The NO detection kit (Beyotime Biotechnology Co., Ltd.) was used to measure the NaNO2 concentration and OD value according to the kit's standard. 540 Plot a standard curve on the x and y axes, and calculate the regression equation for the standard curve. Based on the sample's detection OD... 540 The corresponding NaNO2 concentration was calculated, representing the NO concentration. The average of the three replicates was used as the final NO detection result.

[0049] 2.6 Immunoblot analysis of protein expression

[0050] Protein sample preparation: RAW264.7 cells in logarithmic growth phase were digested and centrifuged to obtain a cell suspension, which was then divided into 1×10⁶ cells per well. 6 Cells were seeded at a density of 1 / mL in 35mm cell culture dishes and cultured for 24 hours in a 5% CO2, 37℃ cell culture incubator. The experiment included a blank control group, a positive control group, and a drug intervention group. The drug intervention group received *Melastoma candida* extract at final concentrations of 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively, ensuring uniform distribution of the drug in the culture dishes. After 2 hours of culture, 15 mL of LPS (100 mg / mL) was added to the positive control group and the drug intervention group, and cultured for another 24 hours. The culture medium in the cell culture dishes was then aspirated, and 210 mL of pre-prepared, pre-chilled cell lysis buffer was added. The cells were lysed on ice for approximately 5 minutes. Cells were scraped off using a cell scraper and transferred to 1.5 mL EP tubes. After sonication to disrupt the cells, the cells were centrifuged at 13000 rpm, 4℃ for 10 minutes. Take 180 mL of the supernatant, add 60 mL of 4×SDS protein loading buffer, mix well, boil in boiling water for 10 min, cool at room temperature, and store in a -20℃ refrigerator for later use.

[0051] Preparation of SDS-PAGE adhesive: Prepare a 12% separating adhesive and pour it into the glass plate interlayer. Spray an appropriate amount of 70% ethanol onto the adhesive to achieve liquid sealing. When obvious fold lines appear in the separating adhesive in the glass plate, pour off the ethanol and drain. Prepare a 4% stacking adhesive, pour it into the glass plate interlayer, insert the adhesive comb, and let it stand until the adhesive solidifies.

[0052] Electrophoresis, transfer, and blocking: Preheat the sample for 1 min, place the gel plate into the electrophoresis tank and pull out the gel casting comb vertically upwards, add 1×Running buffer, and load 10-15 mg of protein per well; electrophoresis (U=100V, I=400mA, Time=90min), prepare 1×Transfer buffer in advance and refrigerate at -20℃ for later use; after activating the PVDF membrane with methanol, assemble the transfer sandwich structure; transfer (U=100V, I=400mA, Time=90min); after transfer, quickly place the PVDF membrane into blocking buffer (TBST containing 5% BSA) and block on a shaker at room temperature for 1 h.

[0053] Primary antibody, secondary antibody, and exposure: After cutting and marking the PVDF membrane, place it in a plastic sealant, add the corresponding primary antibody, seal, and incubate overnight at 4°C on a shaker; after incubation, recover the primary antibody, wash three times with 1×TBST for 3 min each time; pour in the secondary antibody, incubate at room temperature on a shaker for 1 h, then wash three times with 1×TBST for 3 min each time; expose using the ChemiDoc XRS+ chemiluminescence imaging system and save the results.

[0054] 2.7 Data Processing

[0055] Each sample was measured in triplicate, and the result was the average of the three parallel measurements. The results were plotted and analyzed using GraphPadPrism 8, and the images were processed using Photoshop CS6.

[0056] 3 Results

[0057] 3.1 Inhibition of lipoxygenase activity (as shown in Table 1 and...) Figure 1 (As shown)

[0058] Table 1

[0059]

[0060]

[0061] 3.2 Biochemical NO content detection (as shown in Table 2 and...) Figure 2 (As shown)

[0062] Table 2

[0063]

[0064]

[0065] 3.3 MTT assay of RAW264.7 cells (as shown in Table 3 and...) Figure 3 (As shown)

[0066] Table 3

[0067]

[0068] 3.4 Assay of NO clearance capacity at the cellular level (as shown in the table)

[0069] Table 4

[0070]

[0071] 3.5 Immunoblot analysis of protein expression (e.g., Western blot) Figure 4 (As shown)

[0072] From the above results, it can be seen that

[0073] 1. Ethanol extract is superior to water extract: At concentrations of 2.0 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, the water extract of the root showed an inhibition rate of 53% against the highest concentration of lipoxygenase activity, while the ethanol extract reached 75%. At concentrations of 0.20 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.025 mg / mL, the water extract of the root showed an inhibition rate of 54% against the highest concentration of NO content, while the ethanol extract reached 72%. Therefore, the ethanol extract has a better anti-inflammatory effect than the water extract.

[0074] 2. Ultrasonic extraction is superior to maceration: The root ethanol extract obtained by maceration at a concentration of 2.0 mg / mL showed a 70% inhibition rate of lipoxygenase activity, while the ultrasonically extracted ethanol extract at the same concentration achieved 75%. At a concentration of 0.2 mg / mL, the root ethanol maceration extract showed a 64% inhibition rate of NO content, while the ultrasonically extracted root ethanol extract achieved 72%. This indicates that ultrasonic extraction is superior to maceration in assessing inflammation inhibition.

[0075] In summary, extracts from different parts of the *Melastoma candida* plant, obtained through pure water and ethanol extraction, and different extraction processes (ultrasonic extraction and maceration), were used to conduct biochemical experiments on the inhibition of lipoxygenase activity and NO content. The results showed that the extract from the *Melastoma candida* root obtained through ultrasonic ethanol extraction had the best inhibitory effect on inflammation. Subsequently, the root ethanol extract obtained using this process was used to detect MTT activity in RAW264.7 cells and to induce an LPS (lipopolysaccharide)-induced inflammation model in RAW264.7 cells. The NO content detected in the cell supernatant of the LPS model group was 29.59 ± 1.15 (NO / mmol·L⁻¹). -1 The NO content detected in the sample group treated with an extract concentration of 0.2 mg / mL was 12.58 ± 0.64 (NO / mmol·L). -1 The NO content in the sample group was significantly lower than that in the model group. Cells treated with this drug and sample were lysed to prepare protein samples for Western blotting analysis of protein expression. Under the action of antibodies p-ERK and COX-2, the expression levels in the sample group were lower than those in the LPS model group. This further verifies that the ethanol extract of *Melastoma candida* root in this invention has excellent anti-inflammatory activity.

[0076] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. The application of the root alcohol extract of Melastoma candidae in the preparation of anti-inflammatory compositions, characterized in that: The preparation method of the root alcohol extract of Melastoma hornbillii includes: crushing the root of Melastoma hornbillii, mixing it thoroughly with 90-100% ethanol at a mass-volume ratio of 1:4-6 and dispersing it evenly, extracting it by ultrasonic vibration, then centrifuging to obtain the supernatant, vacuum rotary evaporation and freeze drying to obtain the extract.

2. The application as described in claim 1, characterized in that: The ultrasonic oscillation extraction specifically involves ultrasonic oscillation extraction, followed by a 18-25 second pause, and then continuing ultrasonic oscillation extraction, repeating this cycle. The total ultrasonic oscillation extraction time is 1-2 hours.

3. The application as described in claim 1, characterized in that: The temperature of the vacuum rotary evaporation is 35-70℃.

4. An anti-inflammatory composition, characterized in that: Its active ingredient is the root alcohol extract of Melastoma hornbillii. The preparation method of the root alcohol extract of Melastoma hornbillii includes: crushing the root of Melastoma hornbillii, mixing it thoroughly with 90-100% ethanol at a mass-volume ratio of 1:4-6 and dispersing it evenly, extracting it by ultrasonic vibration, then centrifuging to obtain the supernatant, vacuum rotary evaporation and freeze drying to obtain the extract.

5. The anti-inflammatory composition according to claim 4, characterized in that: The ultrasonic oscillation extraction specifically involves ultrasonic oscillation extraction, followed by a 18-25 second pause, and then continuing ultrasonic oscillation extraction, repeating this cycle. The total ultrasonic oscillation extraction time is 1-2 hours.

6. The anti-inflammatory composition according to claim 4, characterized in that: The temperature of the vacuum rotary evaporation is 35-70℃.

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