A method for changing the composition of rosemary extract to enhance functionality

Abstract

The application provides a method for changing rosemary extract ingredients to improve its function. The method for changing rosemary extract ingredients to improve its function provided by the application comprises the following steps: collecting branches and leaves of rosemary plants after the plants are treated by UV-B, obtaining essential oil by essential oil extraction, drying solid residues after extraction, ultrasonic dissolution and extraction by an ethanol solution, concentrating by a rotary evaporator, freeze-drying after filtration, and preparing residue extracts. The anti-inflammatory and antioxidant functions of the essential oil and the extracts are detected by hyaluronidase inhibition method and DPPH method. The results show that, compared with the essential oil and the residue extracts prepared from the plants without UV-B treatment, the inhibition effect on hyaluronidase and the DPPH scavenging effect are significantly improved after the UV-B treatment, and the anti-inflammatory and antioxidant functions are enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for improving the function of rosemary extract by altering its components. Background Technology

[0002] Rosemary is an erect or creeping shrub belonging to the genus *Rosemary* in the family Lamiaceae. Native to the Mediterranean region, it is mainly produced in Morocco, Spain, the former Yugoslavia, Bulgaria, and Tunisia. Rosemary has a long history of cultivation in my country, having been introduced and cultivated as early as the Three Kingdoms period. It is an important aromatic economic plant, widely used not only in cooking to modify and enhance flavors but also extensively in traditional medicine, serving as a highly valued medicinal plant for the prevention and treatment of colds, rheumatism, and muscle and joint pain. It is one of the most popular sources of natural bioactive compounds today, possessing antioxidant, antitumor, anti-inflammatory, antibacterial, and skin-whitening properties.

[0003] The active components of rosemary mainly consist of volatile and non-volatile components. Rosemary volatile essential oil is a complex mixture containing monoterpenes, sesquiterpenes, aromatic compounds, and other derivatives. Rosemary essential oil obtained from the leaves by steam distillation (up to 2.5%) is colorless to pale yellow, insoluble in water, and has a characteristic camphor aroma. The main components of rosemary essential oil are camphor (5.0-21.0%), 1,8-cineole (15.0-55.0%), α-pinene (9.0-26.0%), borneol (1.5-5.0%), camphene (2.5-12.0%), β-pinene (2.0-9.0%), and limonene (1.5-5.0%). The non-volatile components of rosemary are mainly composed of caryopsisic acid, caryopsisol, rosmarinic acid, camphor, caffeic acid, ursolic acid, betulinic acid, etc. The specific proportions of these components vary depending on the variety, different developmental stages of the plant, and bioclimatic and cultivation conditions.

[0004] Hyaluronic acid (HA) possesses strong water-absorbing and adhesive properties, regulates cytokine secretion, and influences cell growth, proliferation, migration, and differentiation. Therefore, it plays a major role in maintaining skin moisture and elasticity, wound healing, and angiogenesis. Hyaluronic acid also participates in vasodilation and allergic reactions, thus being closely related to skin sensitivity. Hyaluronidase is a specific cleavage enzyme of hyaluronic acid; its excessive activity leads to HA degradation, resulting in the disintegration of the extracellular matrix, decreased joint viscosity, skin aging, and contributing to the development and progression of inflammation. The hyaluronidase inhibition assay is the most typical in vitro method for evaluating anti-inflammatory activity. The inhibition rate of hyaluronidase is used as an indicator to evaluate the anti-inflammatory activity of a substance; a higher inhibition rate indicates stronger anti-inflammatory activity. In recent years, numerous studies have reported the effects of plant extracts on hyaluronidase activity, but the results have been unsatisfactory due to differences in the content of active ingredients. Therefore, plant extracts with high content of active ingredients and excellent efficacy are highly valued by product developers.

[0005] Current research lacks studies on using UV-B treatment on living rosemary plants to alter the content of their active ingredients and thus enhance their functionality. This study is the first to treat living rosemary plants with UV-B and detect changes in their essential oil and extract components, which is beneficial for understanding how these changes alter their functions. Summary of the Invention

[0006] To address the above problems, this invention provides a method for improving the function of rosemary extract by altering its components.

[0007] The main steps of this invention include: ① UV-B treatment of rosemary plants; ② extraction of rosemary essential oil and preparation of rosemary residue extract; ③ determination of the main components and contents of the essential oil and residue extract; ④ detection of the anti-inflammatory and antioxidant functions of the essential oil and residue extract respectively.

[0008] This invention involves treating rosemary plants with UV-B light, collecting rosemary branches and leaves, extracting essential oil through water distillation, collecting the remaining solid residue, drying it, pulverizing it into powder, and then adding it to a 70-95% ethanol solution at a material-to-liquid ratio of 1:10-15 (g / ml). The mixture is then ultrasonically extracted, filtered, and subsequently concentrated using a rotary evaporator and freeze-dried. The components of the rosemary essential oil are determined using GC-MS, and the components of the rosemary residue extract are determined using high-performance liquid chromatography (HPLC). A comparison with untreated rosemary shows significant changes in composition. Subsequently, the anti-inflammatory function of the extracted rosemary essential oil and the residue extract are assessed using a hyaluronidase kit, and the antioxidant function is assessed using the DPPH method.

[0009] This invention utilizes UV-B treatment of living rosemary plants to collect rosemary material. Essential oil is extracted via water distillation, and the remaining solid residue is collected, dried, and used to prepare a rosemary residue extract. The effective components are analyzed, and the anti-inflammatory and antioxidant functions of the essential oil and extract are assessed using the hyaluronidase inhibition method and the DPPH method. Compared with essential oil and residue extract prepared from plants without UV-B treatment, UV-B treatment alters the content of effective components in the essential oil and extract, significantly improves the hyaluronidase inhibition and DPPH elimination effects, and enhances their anti-inflammatory and antioxidant functions.

[0010] The technical solution adopted in this invention is as follows:

[0011] 1. UV-B treatment of living rosemary plants: The treatment method is as follows: Place the rosemary plants in an environment with an intensity of 1W / m². 2 Rosemary materials were collected after 9-12 hours of UV-B irradiation followed by 6-9 hours of reaction.

[0012] The rosemary mentioned above mainly comes from varieties collected by the National Botanical Garden, and the above-ground parts (including rosemary branches and leaves) are collected.

[0013] 2. Extraction of rosemary essential oil: The extraction method is as follows: After the collected rosemary material is air-dried, the essential oil is extracted by steam distillation using a Clevenger apparatus.

[0014] 3. Preparation of rosemary residue extract: The preparation method is as follows: The solid residue after essential oil extraction is dried, pulverized into powder, and a certain amount is added to 70-95% ethanol solution at a ratio of 1:10-15 (g / mL). The mixture is ultrasonically dissolved and extracted at room temperature for 3-4 hours. After centrifugation, the supernatant is filtered through a 0.22-0.45μm polyvinylidene fluoride organic microporous membrane, concentrated by rotary evaporator, and freeze-dried to obtain rosemary residue extract powder.

[0015] 4. Component determination: Essential oil component determination: The components of the extracted essential oil were determined by GC-MS; Rosemary residue extract component determination: The components of the extracted rosemary residue extract were determined by high performance liquid chromatography.

[0016] 5. Functional verification: The essential oil and residue extract were subjected to hyaluronidase inhibition test and DPPH method to detect their anti-inflammatory and antioxidant functions, respectively. The essential oil and residue extract prepared from live rosemary plants that were not treated with UV-B were used as controls.

[0017] Anti-inflammatory function verification

[0018] Hyaluronidase inhibition test

[0019] Solution preparation: Measure 1.15 mL of glacial acetic acid into a volumetric flask, add water to bring the volume to 100 mL. Dissolve 1.64 g of sodium acetate trihydrate powder in ultrapure water and bring the volume to 100 mL. Mix 4.8 mL of the acetic acid solution and 45.2 mL of the sodium acetate solution to obtain the acetate buffer solution (pH = 5.6). Accurately weigh 33.3 mg of hyaluronidase, and bring the volume to 20 mL in the acetate buffer solution. Dissolve 100 mg of sodium hyaluronate in 200 mL of acetate buffer solution to obtain a sodium hyaluronate solution (0.5 mg / mL). Bring anhydrous calcium chloride (0.0277 g) to a final volume of 100 mL with ultrapure water to obtain a calcium chloride solution. Weigh 4.0 g of sodium hydroxide, dissolve it in water, and bring the volume to 20 mL to obtain a 5 mol / L sodium hydroxide solution. Accurately weigh 10.6 g of sodium carbonate, and bring the volume to 100 mL with ultrapure water to obtain a 1.0 mol / L sodium carbonate solution. Accurately weigh 1.6 g of P-DAB (p-dimethylbenzaldehyde), add 30 mL of concentrated hydrochloric acid and 30 mL of anhydrous ethanol, and mix well to obtain Ehrlich's reagent. Pipette 1.5 mL of acetylacetone and dissolve it in 50 mL of pre-prepared sodium carbonate solution.

[0020] Preparation of essential oil sample solution: Pipette 2 μL of essential oil into 1 ml of anhydrous ethanol to obtain a 0.2% essential oil sample solution.

[0021] Preparation of residue extract sample solution: Accurately weigh 10 mg of the prepared residue extract and dilute to 1 mL with ultrapure water to obtain a 1% extract sample solution.

[0022] The sample operation table is as follows. The experiment was repeated three times, and the hyaluronidase inhibitory activity of the essential oil and residue extracts was calculated separately. The formula for calculating hyaluronidase inhibitory activity is as follows:

[0023] Hyaluronidase inhibition rate % = ((Ac-Ad)-(Aa-Ab)) / (Ac-Ad)×100%, where Aa is the absorbance of the sample, hyaluronidase and sodium hyaluronate sample solutions; Ab is the absorbance of the blank solution (sample + acetate buffer); Ac is the absorbance of the control solution (acetate buffer + hyaluronidase + sodium hyaluronate); and Ad is the absorbance of the control blank solution (acetate buffer).

[0024]

[0025] Antioxidant function verification

[0026] DPPH determination method

[0027] Reagent Name rule Storage conditions Extract 50mL bottle of liquid Store at 2-8℃ Reagent 1 One 60mL bottle of liquid (to be provided by the customer) Store at room temperature Reagent 2 1 bottle of powder Store at 2-8℃

[0028] Solution preparation: The DPPH kit is used for detection. The kit includes the reagents listed in the table above. 1. Reagent 1: Anhydrous ethanol (prepared by the user); 2. Reagent 2: Powder placed in an EP tube inside the bottle. Prepare the working solution according to the required amount of reagent 2: reagent 1 (V:V) = 4:21. Prepare and use immediately.

[0029] I. Sample Preparation

[0030] (1) Essential oil: Weigh out rosemary essential oil and add it to methanol to prepare a sample solution of 20 mg / mL;

[0031] (2) Residue extract: Weigh out the rosemary residue extract and add it to methanol to prepare a sample solution of 20 mg / mL.

[0032] II. Measurement Procedure

[0033] 1. Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 515nm, and zero it with anhydrous ethanol.

[0034] 2. Procedure: Add the following reagents to a 1.5 mL EP tube.

[0035]

[0036] Calculation formula

[0037] Formula for calculating the free radical scavenging rate of a sample:

[0038] DPPH free radical scavenging rate D% = [[A blank - (A assay - A control)] ÷ A blank] × 100%

[0039] 3. The results showed that, compared with the control, the rosemary essential oil and residue extract prepared from the UV-B treated live rosemary plants exhibited significant hyaluronidase inhibition and DPPH scavenging effects, demonstrating significant anti-inflammatory and antioxidant functions.

[0040] After reviewing relevant literature, no reports have been found regarding the use of UV-B to directly treat rosemary plants to alter their composition and enhance their functions. The advantage of this invention lies in the discovery of a method using 1W / m²... 2 Rosemary plants were exposed to UV-B radiation at high intensity for 9-12 hours, followed by a reaction for 6-9 hours. Essential oils were extracted from the branches and leaves, and a residue extract was prepared. Compared with untreated rosemary plants, the composition showed significant changes, exhibiting significant hyaluronidase inhibition and DPPH scavenging effects, and enhanced anti-inflammatory and antioxidant functions. This invention provides a practical method for improving the application effects of rosemary extracts and essential oils. Attached Figure Description

[0041] Figure 1Comparison of hyaluronidase inhibitory activity in rosemary essential oil extracted with and without UV-B radiation.

[0042] Figure 2 Comparison of hyaluronidase inhibitory activity between rosemary residue extracts extracted with and without UV-B radiation.

[0043] Figure 3 Comparison of DPPH results between rosemary essential oil extracted with and without UV-B radiation.

[0044] Figure 4 Comparison of DPPH results between rosemary residue extracts extracted with and without UV-B irradiation. Detailed Implementation

[0045] To illustrate the technical content of this invention in detail, specific embodiments are provided below. It should be noted that the following specific embodiments are intended to further explain this invention, but the methods and scope of protection described herein are not limited thereto.

[0046] Example 1: Treatment and material collection of live rosemary plants

[0047] Rosemary plants were placed at an intensity of 1W / m 2 Rosemary materials were irradiated under UV-B for 3h, 6h, 9h and 12h respectively, and then reacted for 8h before being collected. Rosemary materials without UV-B treatment were used as a control.

[0048] The rosemary mentioned above mainly comes from varieties collected by the National Botanical Garden, and the above-ground parts (including rosemary branches and leaves) are collected.

[0049] Example 2: Preparation and component analysis of rosemary essential oil extraction, residue extract, and other components.

[0050] Extraction and component analysis of rosemary essential oil: Rosemary materials treated with UV-B radiation for 3h, 6h, 9h and 12h and untreated materials were air-dried and then the essential oil was extracted by steam distillation using a Clevenger apparatus. Five essential oils were obtained and labeled as EO-3, EO-6, EO-9, EO-12 and EO-CK. The components were determined by GC-MS and their absolute contents in the essential oils were calculated (Table 1).

[0051] Table 1. Types and contents of main components in rosemary essential oil under UV-B radiation.

[0052]

[0053]

[0054] Preparation and component analysis of rosemary residue extracts: The five types of rosemary solid residues after essential oil extraction were dried, pulverized into powder, and then a certain amount of each was added to 80% ethanol solution at a ratio of 1:10 (g / mL). The powders were ultrasonically dissolved and extracted at room temperature for 4 hours. After centrifugation, the supernatant was filtered through a 0.22 μm polyvinylidene fluoride organic microporous membrane, concentrated to liquid evaporation using a rotary evaporator, and freeze-dried to obtain five types of rosemary residue extracts, labeled as EX-3, EX-6, EX-9, EX-12, and EX-CK, respectively. The components were then determined using high performance liquid chromatography, and their absolute contents in the residue extracts were calculated (Table 2).

[0055] Table 2. Main components and content of rosemary residue extract under UV-B radiation.

[0056]

[0057]

[0058] Example 3 Functional Verification

[0059] The extracted rosemary essential oil and the prepared residue extract were subjected to hyaluronidase inhibition test and DPPH method, respectively, with the essential oil and residue extract extracted from live rosemary plants that were not treated with UV-B as controls.

[0060] Hyaluronidase inhibition test

[0061] Solution preparation: Measure 1.15 mL of glacial acetic acid into a volumetric flask, add water to bring the volume to 100 mL. Dissolve 1.64 g of sodium acetate trihydrate powder in ultrapure water and bring the volume to 100 mL. Mix 4.8 mL of the acetic acid solution and 45.2 mL of the sodium acetate solution to obtain the acetate buffer solution (pH = 5.6). Accurately weigh 33.3 mg of hyaluronidase, and bring the volume to 20 mL in the acetate buffer solution. Dissolve 100 mg of sodium hyaluronate in 200 mL of acetate buffer solution to obtain a sodium hyaluronate solution (0.5 mg / mL). Bring anhydrous calcium chloride (0.0277 g) to a final volume of 100 mL with ultrapure water to obtain a calcium chloride solution. Weigh 4.0 g of sodium hydroxide, dissolve it in water, and bring the volume to 20 mL to obtain a 5 mol / L sodium hydroxide solution. Accurately weigh 10.6 g of sodium carbonate, and bring the volume to 100 mL with ultrapure water to obtain a 1.0 mol / L sodium carbonate solution. Accurately weigh 1.6 g of P-DAB (p-dimethylbenzaldehyde), add 30 mL of concentrated hydrochloric acid and 30 mL of anhydrous ethanol, and mix well to obtain Ehrlich's reagent. Pipette 1.5 mL of acetylacetone and dissolve it in 50 mL of pre-prepared sodium carbonate solution.

[0062] Preparation of essential oil sample solutions: 2 μL of UV-B treated and untreated essential oils were pipetted into 1 mL of anhydrous ethanol to obtain 0.2% sample solutions, which were labeled as EOH-3, EOH-6, EOH-9, EOH-12, and EOH-CK, respectively.

[0063] Preparation of residue extract sample solutions: Accurately weigh 10 mg of the prepared residue extract and dilute to 1 mL with ultrapure water to obtain 1% extract sample solutions, which are labeled as EXH-3, EXH-6, EXH-9, EXH-12, and EXH-CK, respectively.

[0064] The sample preparation procedure is shown below. The experiment was repeated three times. The hyaluronidase inhibitory activity of the essential oil and residue extracts was calculated separately. The formula for calculating hyaluronidase inhibitory activity is as follows:

[0065] Hyaluronidase inhibition rate % = ((Ac-Ad)-(Aa-Ab)) / (Ac-Ad)×100%, where Aa is the absorbance of the sample, hyaluronidase and sodium hyaluronate sample solutions; Ab is the absorbance of the blank solution (sample + acetate buffer); Ac is the absorbance of the control solution (acetate buffer + hyaluronidase + sodium hyaluronate); and Ad is the absorbance of the control blank solution (acetate buffer).

[0066]

[0067] The results show that: Figure 1 As shown, rosemary essential oil exhibited varying degrees of inhibition against hyaluronidase, with inhibition rates all exceeding 65%. The inhibitory effect of UV-B treatment for 3 hours was not significantly different from that of untreated oil. However, the inhibitory effect gradually increased with prolonged treatment time. The inhibitory effects of 9 hours and 12 hours of treatment showed statistically significant differences compared to the untreated oil (P < 0.05), with 12 hours showing the best effect.

[0068] The inhibitory effect of rosemary residue extract on hyaluronidase, such as Figure 2 As shown, the residue extracts all exhibited varying degrees of inhibitory effects on hyaluronidase, with stronger effects than rosemary essential oil. After 6 hours of UV-B treatment, the inhibitory effect of the residue extracts on hyaluronidase gradually increased, reaching over 85% after 9 and 12 hours of treatment. The activity of the residue extracts was statistically significantly different from that of the residue extracts without UV-B treatment (P < 0.05).

[0069] DPPH determination method

[0070] Reagent Name rule Storage conditions Extract 50mL bottle of liquid Store at 2-8℃ Reagent 1 One 60mL bottle of liquid (to be provided by the customer) Store at room temperature Reagent 2 1 bottle of powder Store at 2-8℃

[0071] Solution preparation: The DPPH kit is used for detection. The kit includes the reagents listed in the table above. 1. Reagent 1: Anhydrous ethanol (prepared by the user); 2. Reagent 2: Powder placed in an EP tube inside the bottle. Prepare the working solution according to the required amount of reagent 2: reagent 1 (V:V) = 4:21. Prepare and use immediately.

[0072] I. Sample Preparation

[0073] (1) Essential oils: A certain amount of UV-B treated and untreated essential oils were added to methanol to prepare a sample solution of 20 mg / mL, which was labeled as EOD-3, EOD-6, EOD-9, EOD-12 and EOD-CK, respectively.

[0074] (2) Residue extract: Weigh a certain amount of UV-B treated and untreated residue extracts and add them to methanol to prepare a sample solution of 20 mg / mL, which is labeled as EXD-3, EXD-6, EXD-9, EXD-12 and EXD-CK respectively.

[0075] II. Measurement Procedure

[0076] 1. Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 515nm, and zero it with anhydrous ethanol.

[0077] 2. Procedure: Add the following reagents to a 1.5 mL EP tube.

[0078]

[0079] Calculation formula

[0080] Formula for calculating the free radical scavenging rate of a sample:

[0081] DPPH free radical scavenging rate D% = [[A blank - (A assay - A control)] ÷ A blank] × 100%

[0082] The results showed that, Figure 3 As shown, rosemary essential oil scavenged DPPH to varying degrees, with scavenging rates all above 70%. The scavenging effect of UV-B treatment for 3 hours was not significantly different from that of untreated oil. As the treatment time increased, the scavenging effect of the essential oil gradually strengthened. The inhibitory effects of treatment for 9 hours and 12 hours were statistically significantly different from those of untreated essential oil (P < 0.05), with the best effect observed after 9 hours of treatment.

[0083] The scavenging effect of rosemary residue extract on DPPH, such as Figure 4As shown, the residue extracts all exhibited varying degrees of DPPH scavenging activity. After 6 hours of UV-B treatment, the DPPH scavenging activity of the residue extracts gradually increased, showing a statistically significant difference compared to the residue extracts without UV-B treatment (P < 0.05). The effects were similar after 9 hours and 12 hours of treatment.

[0084] The results showed that, compared with the control, the rosemary essential oil and residue extracts prepared from the UV-B treated live plants, except for 3 hours of treatment, all showed significant hyaluronidase inhibition and DPPH scavenging effects, and had significant anti-inflammatory and antioxidant functions, with the best effects observed at 9 hours and 12 hours of treatment.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for modifying the components of rosemary extract to enhance its function, characterized in that, Includes the following steps: Rosemary plants were treated with UV-B radiation, and their branches and leaves were collected. After the collected rosemary branches and leaves were naturally dried, the essential oil was extracted by steam distillation. The UV-B radiation treatment conditions were: UV-B radiation time of 9-12 hours and intensity of 1 W / m². 2 The reaction time after radiation is 8 hours. The rosemary essential oil extracted and prepared under UV-B treatment can enhance the inhibition of hyaluronidase and the scavenging of DPPH.

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