Preparation method of herba epimedii essential oil

Abstract

The invention discloses a preparation method of herba epimedii essential oil, and belongs to the technical field of plant extraction. The preparation method comprises the following steps: firstly, pre-treating epimedium leaves, namely selecting, cleaning, drying, crushing and sieving to obtain epimedium leaf powder; then performing efficient extraction under specific pressure, temperature and time by adopting a supercritical CO2 fluid extraction technology to obtain an epimedium oil crude product; and finally, dissolving the crude product with an organic solvent, drying with a dehydrating agent, filtering, and removing the solvent to obtain the pure herba epimedii essential oil. A supercritical extraction process is adopted, conditions are mild, heat-sensitive components and natural flavor can be reserved to the maximum extent, and activity loss caused by traditional high-temperature extraction is avoided; the obtained essential oil is complex and harmonious in component and rich in characteristic terpene and alcohol compounds, so that the essential oil has excellent antioxidant biological activity while keeping unique medicine fragrance and wood fragrance. The method is simple in step, good in repeatability and environment-friendly, and provides a reliable way for high-quality, standardized and industrial production of the herba epimedii essential oil.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for preparing Epimedium essential oil. Background Technology

[0002] Epimedium brevicornu Maxim. is a perennial herb belonging to the genus Epimedium in the family Berberidaceae. It is mainly distributed in Sichuan and Guizhou provinces of my country, characterized by its wide distribution and low cultivation cost. For a long time, scientific research has focused primarily on the polysaccharides of Epimedium; however, exploration of its volatile oil components has been relatively limited. This is mainly due to the extremely low content of volatile oil in its leaves and the technical bottlenecks of difficult extraction using traditional methods, resulting in low yields. These limitations restrict the in-depth understanding and application development of the value of this component.

[0003] Chinese herbal essential oils are volatile, oily liquids extracted from the leaves, roots, stems, and petals of Chinese medicinal herbs. Although derived from nature, their production and application face significant limitations. In the extraction process, traditional methods such as steam distillation require continuous heating, which can easily destroy heat-sensitive active ingredients, leading to the loss of the essential oil's natural flavor and reduced antioxidant and other biological activities. Furthermore, this method has low extraction efficiency for water-soluble and large molecular components, resulting in incomplete chemical composition and affecting the overall efficacy. In addition, process fluctuations lead to inconsistent quality between batches, impairing the product's activity, causing unstable effects, and compromising its safety.

[0004] In recent years, with advancements in extraction technology, the market has seen a surge in Epimedium essential oil products. However, the lack of unified quality evaluation standards has led to inconsistent product quality. Different extraction processes and quality standards result in significant differences in the chemical composition of the final products, directly impacting their stable function and efficacy. To address this issue, this invention delves into the effects of different extraction methods, process parameters, and storage conditions on the quality of Epimedium essential oil, solving problems associated with traditional methods such as complex operation steps, high production costs, weak reducing and antioxidant capabilities, low essential oil extraction efficiency, low extraction rates of heat-sensitive components in volatile oils, and limited flavor profiles. This invention provides an environmentally friendly and reproducible method, which is of significant importance for promoting standardized production and green extraction technology for Epimedium essential oil. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing Epimedium essential oil. This method has simple operation steps, low production cost, strong reducing and antioxidant capacity, high essential oil extraction efficiency, high extraction rate of heat-sensitive components in volatile oil, and diverse flavor components. This invention can provide an environmentally friendly and reproducible method for preparing Epimedium essential oil. This method is of great significance for promoting the standardized production and green extraction technology of Epimedium essential oil in the industry.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing epimedium essential oil includes the following steps: (1) Take the leaves of Epimedium, remove the petioles and impurities, wash with water, dry, grind into powder, pass through a No. 2 sieve, remove the debris, and finally obtain Epimedium leaf powder for use. The drying temperature is 40-60℃ and the time is 4-7h. (2) Take 120-170g of Epimedium leaf powder from step (1) and add it to a supercritical CO2 extractor for extraction. The extraction pressure of the supercritical CO2 extractor is 10-50MPa, the extraction temperature is 20-60℃, the extraction time is 1-6h, and it is left to stand for 30-70 days to obtain crude Epimedium oil for later use. (3) Take the crude Epimedium oil from step (2), dissolve it in 99% ether at 10-30 times the mass of the crude Epimedium, add 1-10% anhydrous sodium sulfate to remove water and dry, filter it with a 0.1-0.6 μm pore size filter membrane, and remove the ether with a rotary evaporator at a temperature of 40-60℃ for 0-60 min to obtain Epimedium essential oil.

[0007] In step (1) above, the drying temperature is 45-55℃ and the time is 5-6h.

[0008] Specifically, in the aforementioned step (1), the drying temperature is 50°C and the time is 6 hours.

[0009] In step (2) above, 140-160g of Epimedium leaf powder was extracted using a supercritical CO2 extractor at a pressure of 20-40MPa, a temperature of 30-40℃, and a time of 3-5h, and the product was left to stand for 40-60 days.

[0010] Specifically, in step (2) above, 150g of Epimedium leaf powder was extracted using a supercritical CO2 extractor with an extraction pressure of 30MPa, an extraction temperature of 35℃, an extraction time of 5h, and the product was left to stand for 50 days.

[0011] In step (3) above, the amount of 99% ether used is 15-25 times the mass of crude Epimedium, and the mass fraction of anhydrous sodium sulfate is 3-7%.

[0012] Specifically, in step (3) above, the amount of 99% ether used is 20 times the mass of crude Epimedium, and the mass fraction of anhydrous sodium sulfate is 5%.

[0013] In step (3) above, the pore size of the filter membrane is 0.2-0.5 μm, the temperature of the rotary evaporator is 45-55 °C, and the time is 20-50 min.

[0014] Specifically, in step (3) above, the pore size of the filter membrane is 0.45 μm, the temperature of the rotary evaporator is 50 °C, and the time is 30 min.

[0015] Beneficial effects of this invention: 1. The present invention has simple operation steps and low production cost. The supercritical fluid extraction method significantly improves the extraction efficiency of essential oils, with a yield of 1.21%, which is significantly improved compared with the traditional distillation extraction method (0.94%). Furthermore, it can be further improved to 1.31% through process optimization.

[0016] 2. This method can efficiently retain heat-sensitive components in volatile oils, avoiding component decomposition caused by prolonged heating in traditional distillation methods. This results in higher ketone and terpene content in essential oils prepared by supercritical fluid extraction, thus preserving more complete flavor components.

[0017] 3. The essential oil obtained by this invention has a variety of flavor components, among which characteristic components such as α-guaiacene, sesquiterpenes, patchouliene and patchouli alcohol have been clearly identified. These components together endow the essential oil with multiple effects such as antibacterial, anti-inflammatory, anti-allergic and immune regulation.

[0018] 4. The epimedium essential oil of the present invention has excellent reducing and antioxidant capabilities, especially in the Essential oil-C sample stored at room temperature for 50 days, exhibiting the strongest DPPH and ABTS free radical scavenging ability, and can effectively remove Fe... 3 ⁺ Reduced to Fe 2 ⁺.

[0019] 5. The Epimedium essential oil samples of the present invention all meet the microbial limit standard of GB / T26516-2011 (total bacterial count ≤1000 cfu / mL, mold and yeast ≤100 cfu / mL, pass rate 100%), and the refractive index is stable in the range of 1.509-1.520 (RSD=0.503%), providing a reliable basis for quality control. Attached Figure Description

[0020] Figure 1Results of the aroma, appearance, and color of Epimedium essential oil (Essential oil-SDE: Epimedium essential oil prepared by distillation extraction; Essential oil-SFE: Epimedium essential oil prepared by supercritical fluid extraction under normal extraction conditions; Essential oil-A: Epimedium essential oil prepared by supercritical fluid extraction with increased extraction pressure; Essential oil-B: Epimedium essential oil prepared by supercritical fluid extraction with prolonged extraction time; Essential oil-C: Epimedium essential oil prepared by supercritical fluid extraction under normal extraction conditions, after 50 days of storage). Figure 2 The results of aroma evaluation of Epimedium essential oil; Figure 3 FTIR spectra of different Epimedium essential oils; Figure 4 GC-MS spectrum of Epimedium essential oil; Figure 5 Results of gas chromatography-mass spectrometry (GC-MS) analysis of different Epimedium essential oils; Figure 6 Schematic diagram of the main components of Epimedium essential oil; Figure 7 The scavenging rates of different Epimedium essential oils against DPPH free radicals, ABTS free radicals, and their reducing abilities (A: Scavenging rate of different Epimedium essential oils against DPPH free radicals; B: Scavenging rate of different Epimedium essential oils against ABTS free radicals; C: Reducing ability of different Epimedium essential oils against ABTS free radicals). Figure 8 Epimedium essential oil quality evaluation system. Detailed Implementation

[0021] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0022] Example 1: Preparation method of Epimedium essential oil The preparation method of Epimedium essential oil includes the following steps: (1) Take the leaves of Epimedium, remove the petioles and impurities, wash with water, dry, grind into powder, pass through a No. 2 sieve, remove the debris, and finally obtain Epimedium leaf powder for use. The drying temperature is 50℃ and the time is 6h. (2) Take 150g of Epimedium leaf powder from step (1) and add it to a supercritical CO2 extractor for extraction. The supercritical CO2 extractor has an extraction pressure of 30MPa, an extraction temperature of 35℃, an extraction time of 5h, and is left to stand for 50 days to obtain crude Epimedium oil for later use. (3) Take the crude Epimedium oil from step (2), dissolve it in 99% ether at 20 times the mass of the crude Epimedium, add 5% anhydrous sodium sulfate to remove water and dry, filter it with a 0.45 μm pore size filter membrane, and remove the ether with a rotary evaporator at a temperature of 50°C for 30 min to obtain Epimedium essential oil.

[0023] Example 2: Preparation method of Epimedium essential oil The preparation method of Epimedium essential oil includes the following steps: (1) Take the leaves of Epimedium, remove the petioles and impurities, wash with water, dry, grind into powder, pass through No. 2 sieve, remove the debris, and finally obtain Epimedium leaf powder for use. The drying temperature is 40℃ and the time is 4h. (2) Take 120g of Epimedium leaf powder from step (1) and add it to a supercritical CO2 extractor for extraction. The supercritical CO2 extractor has an extraction pressure of 10MPa, an extraction temperature of 20℃, an extraction time of 1h, and is left to stand for 30 days to obtain crude Epimedium oil for later use. (3) Take the crude Epimedium oil from step (2), dissolve it in 99% ether at 10 times the mass of the crude Epimedium, add 1% anhydrous sodium sulfate to remove water and dry, filter it with a 0.1 μm pore size filter membrane, and remove the ether with a rotary evaporator at a temperature of 40°C for 0 min to obtain Epimedium essential oil.

[0024] Example 3: Preparation method of Epimedium essential oil The preparation method of Epimedium essential oil includes the following steps: (1) Take the leaves of Epimedium, remove the petioles and impurities, wash with water, dry, grind into powder, pass through No. 2 sieve, remove the debris, and finally obtain Epimedium leaf powder for use. The drying temperature is 45℃ and the time is 5h. (2) Take 140g of Epimedium leaf powder from step (1) and add it to a supercritical CO2 extractor for extraction. The supercritical CO2 extractor has an extraction pressure of 20MPa, an extraction temperature of 30℃, an extraction time of 3h, and is left to stand for 40 days to obtain crude Epimedium oil for later use. (3) Take the crude Epimedium oil from step (2), dissolve it in 99% ether at 15 times the mass of the crude Epimedium, add 3% anhydrous sodium sulfate to remove water and dry, filter it with a 0.2 μm pore size filter membrane, and remove the ether with a rotary evaporator at a temperature of 45°C for 20 min to obtain Epimedium essential oil.

[0025] Example 4: Preparation method of Epimedium essential oil The preparation method of Epimedium essential oil includes the following steps: (1) Take the leaves of Epimedium, remove the petioles and impurities, wash with water, dry, grind into powder, pass through a No. 2 sieve, remove the debris, and finally obtain Epimedium leaf powder for use. The drying temperature is 55℃ and the time is 6h. (2) Take 160g of Epimedium leaf powder from step (1) and add it to a supercritical CO2 extractor for extraction. The supercritical CO2 extractor has an extraction pressure of 40MPa, an extraction temperature of 40℃, an extraction time of 5h, and is left to stand for 60 days to obtain crude Epimedium oil for later use. (3) Take the crude Epimedium oil from step (2), dissolve it in 99% ether at 25 times the mass of the crude Epimedium, add 7% anhydrous sodium sulfate to remove water and dry, filter it with a 0.5 μm pore size filter membrane, and remove the ether with a rotary evaporator at a temperature of 55°C for 50 min to obtain Epimedium essential oil.

[0026] Example 5: Preparation method of Epimedium essential oil The preparation method of Epimedium essential oil includes the following steps: (1) Take the leaves of Epimedium, remove the petioles and impurities, wash with water, dry, grind into powder, pass through No. 2 sieve, remove the debris, and finally obtain Epimedium leaf powder for use. The drying temperature is 60℃ and the time is 7h. (2) Take 170g of Epimedium leaf powder from step (1) and add it to a supercritical CO2 extractor for extraction. The supercritical CO2 extractor has an extraction pressure of 50MPa, an extraction temperature of 60℃, an extraction time of 6h, and is left to stand for 70 days to obtain crude Epimedium oil for later use. (3) Take the crude Epimedium oil from step (2), dissolve it in 99% ether at 30 times the mass of the crude Epimedium, add 10% anhydrous sodium sulfate to remove water and dry, filter it with a 0.6 μm pore size filter membrane, and remove the ether with a rotary evaporator at a temperature of 60°C for 60 min to obtain Epimedium essential oil.

[0027] To obtain the solution of this invention and verify its technical effects, the inventors conducted extensive experimental research, some of which are recorded below: 1. Materials and Methods 1.1 Materials and Reagents Epimedium leaves (collected from the Epimedium demonstration planting base in Gaoguan Town, Chengkou County, Chongqing, 108.81°E, 31.88°N). Ethanol and acetonitrile (both chromatographic grade) were from Thermo Fisher Scientific, USA; carbon tetrachloride, anhydrous sodium sulfate, and diethyl ether (all analytical grade) were from Shanghai Maclean Biochemical Technology Co., Ltd.; polysorbate 80 (chemically pure) was from Sinopharm Chemical Reagent Co., Ltd.; sodium chloride-peptone buffer (analytical grade) was from Guangdong Wengjiang Chemical Reagent Co., Ltd.; 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and ascorbic acid (V C Anhydrous ethanol (Shanghai Yuanye Biotechnology Co., Ltd.); Deionized water (laboratory-made).

[0028] 1.2 Instruments and Equipment Electronic balance, Mettler Toledo, USA; 180E ultrasonic instrument, Shanghai Zhixin Instrument Co., Ltd., China; PG01 centrifuge, Shanghai Chenlian Biotechnology Development Co., Ltd., China; Quanta 650FEG scanning electron microscope, FEI Corporation, USA; LC2030 high performance liquid chromatograph, Shimadzu Corporation, Japan; 7890B-5977B gas chromatograph-mass spectrometer, Agilent Technologies, USA; XC-600Y small pulverizer, Zhejiang Xichu Food Machinery Co., Ltd.; Supercritical CO2 extraction device, HA121-50-01, Nantong Huaan Supercritical Extraction Co., Ltd.; SN-RE-201D rotary evaporator, Shanghai Shangyi Instrument Co., Ltd.; POMEX steam distillation apparatus (including steam generator, three-necked flask and condenser), Beijing Beibo Bomei Glass Co., Ltd.; D600 colorimeter, DataColor Corporation, USA; TU-1950 UV-Vis spectrophotometer, Beijing Purkinje General Instrument Co., Ltd.

[0029] 1.3 Methods 1.3.1 Preparation of Epimedium Leaf Powder Take the leaves of Epimedium, remove the petioles and impurities, wash with water, dry (drying temperature is 50℃, time is 6h), grind into powder, pass through No. 2 sieve, remove debris, and finally obtain Epimedium leaf powder.

[0030] 1.3.2 Preparation of Epimedium essential oil by steam distillation Each time, 100g of Epimedium leaf powder is added to 500g of distilled water and extracted by steam distillation for 4 hours. The sample is collected at the condenser of the steam distillation apparatus using a separatory funnel to obtain Epimedium essential oil.

[0031] 1.3.3 Preparation of Epimedium essential oil by distillation extraction method (Code: Essential oil-SDE) Each time, 100g of Epimedium leaf powder is added to 400mL of water and soaked for about 16 hours. The mixture is heated and cooled with cooling water. After distillation for 3 hours, the resulting extract is extracted with carbon tetrachloride. After the aqueous phase and organic phase separate into layers, the organic phase is collected and concentrated by rotation to obtain Epimedium essential oil.

[0032] 1.3.4 Preparation of Epimedium essential oil by supercritical fluid extraction Take 150g of Epimedium leaf powder and add it to a supercritical CO2 extractor for extraction (the extraction pressure of the supercritical CO2 extractor is 30MPa, the extraction temperature is 35℃, and the extraction time is 5h). After standing for 50 days, crude Epimedium oil is obtained. Crude Epimedium oil was dissolved in 20 times its weight of 99% diethyl ether, and then dried with 5% anhydrous sodium sulfate. The solution was filtered through a 0.45 μm pore membrane, and the diethyl ether was removed using a rotary evaporator at 50°C for 30 minutes to obtain Epimedium essential oil. (See Table 1) Table 1: Specific process optimization schemes are shown in Table 1. 1.3.5 Color Analysis Take 1 mL of Epimedium essential oil into a cuvette, place the cuvette in the test port of a colorimeter, use a D65 light source, and test the color parameters (L*, a*, and b*) of the essential oils obtained from each treatment using the specular light mode. Among them, L*, a*, and b* are parameters of the CIEL*a*b* color space, which are mainly used for quantitative description of color. The L* parameter represents the brightness of the sample being measured, the a* parameter represents the color characteristics of the sample on the red-green axis (the larger the positive value, the redder it is, and the larger the negative value, the greener it is), and the b* parameter represents the color characteristics of the sample on the yellow-blue axis (the larger the positive value, the yellower it is, and the larger the negative value, the bluer it is).

[0033] 1.3.6 Aroma Assessment A quantitative rating and descriptive analysis method was used to evaluate the aroma components of Epimedium essential oil. The evaluation panel consisted of 5 men and 5 women, aged between 25 and 40. The descriptive vocabulary for Epimedium essential oil included: medicinal, woody, spicy, fresh, and bitter. The evaluation panel members uniformly used a ten-point scale to evaluate the essential oil; the specific meanings are shown in Table 2.

[0034] Table 2: Meaning of the Ten-Point Scale Levels 1.3.7 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis Gas chromatography conditions: Column: DB-5MS (30m×0.25mm, 0.25μm); Injector temperature: 260℃; Injection mode: splitless; Flow rate: 1mL / min; Column pressure: 9.08Psi; Column temperature: Initial temperature: 30℃, heating rate 5℃ / min, to 330℃.

[0035] Mass spectrometry conditions: ion source temperature: 250℃, transfer temperature: 280℃, solvent switching time: 2 min, mass spectrometry ion source: electron impact source (EI), scan mode: Scan scan. Chemical components in the essential oils were identified by comparing the spectra with those in the standard spectral library NIST14.

[0036] Quantitative analysis was performed using the internal standard method. 2.0 mL of the essential oil sample was accurately measured into 20 mL of dichloromethane, and 50 μL of n-tetradecane was added as an internal standard. The density of the n-tetradecane internal standard was calculated as 0.767 g / mL, therefore the concentration of the internal standard was 19.175 ug / mL, or 19,175 ppm. The content of each substance was calculated by integrating the area of ​​the target compound (see formula (1), and qualitative analysis was performed using the matching results from the NIST14 database. The matching threshold of the NIST database was set to above 80% to ensure the accuracy and reliability of the identification results.

[0037] (1) Where Atarget is the peak area of ​​the target compound, AIS is the peak area of ​​the internal standard, and CIS is the concentration of n-tetradecane.

[0038] 1.3.8 Other characterization conditions FTIR spectroscopy testing conditions: The ATR method was used. First, a background scan was performed. Then, a small amount of essential oil sample was placed on the ATR crystal and the test began, with a range of 400-4000 cm⁻¹. -1 The scanning speed is 400 nm·mm. -1 ; Relative density test conditions: The relative density of the essential oil was determined according to the Webster's specific gravity balance method as specified in the General Chapter of the 2020 edition of the Chinese Pharmacopoeia.

[0039] Refractive index testing conditions: The refractive index of Epimedium essential oil was measured according to the refractive index testing method specified in the General Chapter of the 2020 edition of the Chinese Pharmacopoeia.

[0040] 1.3.9 Microbial Indicator Testing According to the microbial limit test method specified in the General Chapter of the 2020 edition of the Chinese Pharmacopoeia, the total bacterial count, mold count, and yeast count in Epimedium essential oil were tested. The specific procedure is as follows: Take 1 mL of the essential oil, add sterile sodium chloride-peptone buffer (pH 7.0) to 10 mL, mix well to prepare a 1:10 test solution, and then add 5 mg of sterile polysorbate 80 to disperse the test sample evenly. Referring to GB / T26516-2011 "Massage Essential Oils," which stipulates that the total bacterial count should be ≤1000 CFU / mL and the mold and yeast count should be ≤100 CFU / mL, the microbial limit evaluation of Epimedium essential oil was performed.

[0041] 1.3.10 Evaluation of antioxidant capacity 1.3.10.1 Determination of DPPH free radical scavenging ability of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) Five batches of Epimedium essential oil (Essential oil-SDE, Essential oil-SFE, Essential oil-A, Essential oil-B, and Essential oil-C) were diluted with anhydrous ethanol to concentrations of 0.5, 1, 1.5, 2, and 2.5 mg / mL, respectively. 4 mL of each sample solution was added to 0.5 mL of DPPH solution (0.15 mmol / L), and the mixture was reacted in the dark for 20 min. The absorbance was measured at 517 nm. Ascorbic acid of the same concentration was used as a positive control. The DPPH free radical scavenging rate (K1) was calculated according to formula (2), and the IC50 was also calculated. 50 value.

[0042] ×100%(2) In the formula, A0 is the absorbance measured after mixing DPPH and anhydrous ethanol, A1 is the absorbance measured after mixing anhydrous ethanol and epimedium essential oil sample, and A2 is the absorbance measured after mixing epimedium essential oil sample and DPPH solution.

[0043] 1.3.10.2 2,2'-Diammonium bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) + Free radical scavenging capacity determination Five batches of Epimedium essential oil sample solutions were prepared according to method 1.3.9.1. 3.5 mL of the sample solution was mixed with 2.8 mL of ABTS working solution, and the mixture was reacted in the dark for 6 min. The absorbance was measured at 734 nm. Ascorbic acid of the same concentration was used as a positive control. The ABTS scavenging rate (K2) was calculated according to formula (3), and the IC50 was calculated. 50 value.

[0044] ×100%(3) In the formula, A0 is the absorbance measured after mixing ABTS with anhydrous ethanol, A1 is the absorbance measured after mixing anhydrous ethanol with epimedium essential oil sample, and A2 is the absorbance measured after mixing epimedium essential oil sample with ABTS solution.

[0045] 1.3.10.3 Reducing power determination Five batches of Epimedium essential oil sample solutions were prepared according to method 1.3.9.1. 0.5 mL of the sample solution, 2 mL of phosphate buffer (pH=6.6, 0.2 mol / L), and 2 mL of potassium ferricyanide solution (1%) were mixed thoroughly and incubated at 50 °C for 20 min. After cooling, 2.0 mL of trichloroacetic acid (10%) was added, followed by centrifugation at 3000 rpm for 10 min. 2 mL of the supernatant was mixed with 0.1 mL of ferric chloride solution (0.05%) and 2 mL of distilled water and incubated for 10 min. The absorbance was then measured at 700 nm. Ascorbic acid of the same concentration was used as a positive control.

[0046] 2. Results and Analysis 2.1 Analysis of the extraction effect of Epimedium essential oil In this study, three methods were used to prepare Epimedium essential oil: steam distillation, distillation extraction, and supercritical fluid extraction. Steam distillation is the main method for extracting essential oils from traditional Chinese medicines, as recorded in the *Pharmacopoeia of Chinese Materia Medica*, and it has advantages such as simple operation and low cost. However, the experimental results showed that steam distillation failed to effectively extract the oily components from Epimedium leaves. This may be related to the low content of oily substances, resulting in the inability of steam distillation to effectively enrich the oily components in the leaves. Therefore, only distillation extraction and supercritical fluid extraction successfully prepared Epimedium essential oil, with yields of 0.94% and 1.21%, respectively. Appropriate pressure and extended extraction time could improve the yield of Epimedium essential oil, to 1.27% and 1.31%, respectively.

[0047] 2.2 Sensory index analysis of Epimedium essential oil 2.2.1 Color This study further investigated the effects of different process parameters and storage time during supercritical fluid extraction on the appearance characteristics of the essential oils. The results are shown in Table 3. The results showed that the essential oil of Epimedium extracted by laboratory distillation was light yellow, while the essential oils of Epimedium prepared by supercritical fluid extraction (Essential oil-SFE, Essential oil-A, Essential oil-B) were clear liquids ranging from deep yellow to deep green. After prolonged storage at room temperature, the color further darkened, a phenomenon consistent with similar literature reports. Figure 1 .

[0048] Table 3: Appearance Analysis Results of Epimedium Essential Oil Note: L* parameter represents the brightness of the measured sample, a* parameter represents the color characteristic of the sample on the red-green axis, and b* parameter represents the color characteristic of the sample on the yellow-blue axis. 2.2.2 Aroma The aroma evaluation results of Epimedium essential oil under different extraction methods, processes, and storage conditions were analyzed by mean value analysis. The results are as follows: Figure 2 As shown. The main volatile flavor characteristics of epimedium essential oil include medicinal, woody, spicy, refreshing, and bitter. Among them, the medicinal and woody flavors are the most prominent, while there are also some spicy, refreshing, and bitter flavors, which together constitute the overall flavor characteristics of epimedium essential oil.

[0049] Experimental results showed that the essential oil of Epimedium extracted by distillation (Essential oil-SDE) had a weaker medicinal and fresh aroma, which may be closely related to the prolonged heating during the extraction process. High temperatures easily lead to the decomposition of heat-sensitive volatile components, affecting the aroma characteristics of the essential oil. In contrast, the essential oils of Epimedium prepared by supercritical fluid extraction (Essential oil-SFE, Essential oil-A, Essential oil-B, Essential oil-C) were more effective at extracting heat-sensitive components from the volatile oil and retained more flavor components.

[0050] 2.3 Physicochemical analysis of Epimedium essential oil 2.3.1 Physical Properties 2.3.1.1 Relative density The relative density of Epimedium essential oil was tested according to the methods specified in the Chinese Pharmacopoeia, and the results are shown in Table 4. The results show that the coefficient of variation (RSD) of the relative density of Epimedium essential oil is 0.843%, indicating that the relative density of the essential oil is relatively stable. No significant differences were found between essential oils prepared by different processing methods, and the relative density range was 0.965-0.986.

[0051] Table 4: Density test results of different Epimedium essential oils 2.3.1.2 Refractive index The refractive index of Epimedium essential oil was tested according to the methods specified in the Chinese Pharmacopoeia, with an accuracy of ±0.0001. The specific results are shown in Table 5. The results show that the coefficient of variation (RSD) of the refractive index of Epimedium essential oil is 0.503%, and there is no significant difference between essential oils prepared by different processes and from different sources. Its refractive index range is 1.509-1.520.

[0052] Table 5: Refractive index test results of different Epimedium essential oils 2.3.1.3 Microbial Limit Determination Microbial limits were evaluated for Epimedium essential oil according to the methods in the Chinese Pharmacopoeia, and the results are shown in Table 6. The results indicate that all five Epimedium essential oils prepared in this study (Essential oil-SDE, Essential oil-SFE, Essential oil-A, Essential oil-B, and Essential oil-C) meet the requirements of GB / T26516-2011, with a pass rate of 100% (total bacterial count ≤1000 cfu / mL, molds and yeasts ≤100 cfu / mL).

[0053] Table 6: Detection results of microorganisms in Epimedium essential oil 2.3.2 Composition Analysis 2.3.2.1 FTIR Spectroscopy Study The functional group signals of the overall components of Epimedium essential oil were tested using Fourier Transform Infrared Spectroscopy (FTIR), and the results are as follows: Figure 3 As shown. All essential oils are at 3300cm. -1 The stretching vibration signals of the OH bonds on both sides are not visible, indicating that Epimedium essential oil basically does not contain water (H2O), alkaloids, or sugars and sugar derivatives containing hydroxyl groups. This is significantly different from the processed and liquid-extracted components of Epimedium reported in the literature, which is also a characteristic difference between herbal essential oils and traditional Chinese medicine preparations. At 2910 cm⁻¹ -1 The asymmetric stretching vibrations of the CH groups appearing in the absorption bands on the left and right, and at 1650 cm⁻¹ -1 1060cm -1 The peak at 1250 cm⁻¹ corresponds to the stretching vibrations of C=C and COC bonds, suggesting an exceptionally rich content of aromatic rings, ketones, and amino compounds in Epimedium essential oil. -1 The CO bond is generated in ketones and terpenes.

[0054] Notably, infrared spectroscopy analysis revealed relatively low levels of ketones and terpenes in the essential oil (SDE) extracted by distillation. Ketones and terpenes are typically highly volatile, rapidly released into the air and perceived by the olfactory system; therefore, their concentration directly determines the aroma characteristics of the essential oil. This finding aligns with sensory evaluation results, further confirming that supercritical fluid extraction is more effective in extracting key components such as ketones and terpenes from Epimedium.

[0055] 2.3.2.2 Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS Analysis This study employed the method described in section "1.3.7" for GC-MS analysis of essential oil components, and qualitative analysis of the main volatile signal peaks was performed using a search of the NIST14 standard mass spectrometry library (see [link to study]). Figure 4 The results are shown in Table 7. Taking the essential oil (SDE) extracted by distillation extraction of Epimedium as an example, a total of 117 volatile organic compounds were detected. The main components were alkenes, alcohols, esters, and ketones, with the proportions of each type being 57.56%, 14.45%, 15.42%, and 1.77%, respectively. Among them, 19 compounds had a concentration greater than 10,000 ppm. The three compounds with the highest concentrations were α-pinene (94,098.78 ppm), Δ-juniperene (62,156.76 ppm), and 2-(decanoyloxy)propane-1,3-dioctanoic acid diester (60,222.32 ppm).

[0056] Table 7: GC-MS Test and Matching Results of Epimedium Essential Oil The epimedium essential oils prepared by the other four extraction methods also mainly consist of four types of compounds: alkenes, alcohols, esters, and ketones, with specific content ratios as follows: Figure 5 As shown in the figure. Consistent with the infrared spectroscopy results, GC-MS results showed that the relative contents of alcohols and esters in Epimedium essential oil (SDE) extracted by distillation were low. In all Epimedium essential oils, the contents of α-guaiacene, sesquiterpenes, patchouliene, and patchouli alcohol all exceeded 1%, indicating that these are characteristic substances of Epimedium essential oil. Terpenes such as α-guaiacene and sesquiterpenes have antibacterial, anti-inflammatory, and anti-allergic effects, while patchouliene and patchouli alcohol have immunomodulatory and antioxidant effects. Furthermore, patchouliene is a metabolite of patchouli alcohol; the two components can work synergistically to form better pharmacological activity, providing a molecular basis for the functional verification of the essential oil. See details... Figure 6 .

[0057] 2.4 Evaluation of antioxidant capacity 2.4.1 Determination of DPPH and ABTS free radical scavenging capabilities like Figure 7As shown in Figure A, five Epimedium essential oil samples exhibited DPPH free radical scavenging ability within a concentration range of 0.5-2.5 mg / mL. The results showed that the DPPH free radical scavenging rate increased with increasing essential oil concentration, indicating that the DPPH free radical scavenging effect of Epimedium essential oil is concentration-dependent. At a concentration of 2.5 mg / mL, the scavenging rates of each group were: 51.65% (Essential oil-A), 19.84% (Essential oil-B), 89.56% (Essential oil-C), 25.53% (Essential oil-SDE), 86.81% (Essential oil-SFE), and 86.46% (Vc), with Essential oil-C showing a slightly higher scavenging rate than Vc. Figure 7 Five Epimedium essential oil samples (shown in B) exhibited ABTS free radical scavenging ability within a concentration range of 0.5-2.5 mg / mL. The results showed that the scavenging rate of ABTS free radicals increased with increasing essential oil concentration, indicating that the scavenging effect of Epimedium essential oil on ABTS free radicals is concentration-dependent. At a concentration of 2.5 mg / mL, the scavenging rates of each group were: 97.39% (Essential oil-A), 46.22% (Essential oil-B), 98.41% (Essential oil-C), 97.61% (Essential oil-SDE), 94.42% (Essential oil-SFE), and 99.59% (Vc), with the scavenging rate of Essential oil-C being close to that of Vc.

[0058] Table 8 lists the IC50 values ​​of each group of samples against DPPH and ABTS radicals. 50 Value. IC 50 The IC50 value is an important indicator of a sample's antioxidant capacity, representing the sample concentration required to inhibit 50% of free radical activity. 50 The smaller the value, the stronger its ability to scavenge free radicals. The IC50 value of DPPH... 50 The values, in order of magnitude, are: V C (0.04mg / mL)<Essential oil-C (0.05mg / mL)<Essential oil-SFE (1.02mg / mL)<Essential oil-A (2.19mg / mL)<Essential oil-B (8.60mg / mL)<Essential oil-SDE (12.95mg / mL). ABTS IC 50 The values ​​are V in order of magnitude. CThe concentrations of essential oils were: (0.00001 mg / mL) < Essential oil-C (0.0003 mg / mL) < Essential oil-SDE (0.46 mg / mL) < Essential oil-SFE (0.63 mg / mL) < Essential oil-A (1.57 mg / mL) < Essential oil-B (3.16 mg / mL). These results indicate that Essential oil-C exhibited the best DPPH and ABTS radical scavenging abilities among all Epimedium essential oil samples. This may be because Essential oil-C was stored sealed at room temperature for 50 days; the storage process may have promoted the transformation of unstable components or the formation of new antioxidant complexes, thereby enhancing the antioxidant activity of the essential oil.

[0059] Table 8. Antioxidant IC50 of Epimedium essential oil 50 value 2.4.2 Reducing power determination like Figure 7 C shows the effects of five types of Epimedium essential oil on Fe in the concentration range of 0.5-2.5 mg / mL. 3+ The reducing power of the essential oils was measured. As the concentration increased, the absorbance of the reaction system gradually increased, indicating that the reducing power of various essential oils is concentration-dependent. At a test concentration of 2.5 mg / mL, all samples showed good reducing power, with Essential oil-C exhibiting the highest absorbance (0.653). Under the same conditions, the absorbance of Vitamin C was 0.781, indicating that the reducing power of Essential oil-C was similar to that of Vitamin C. The results show that Essential oil-C can effectively reduce ferric ions (Fe3+) to Fe2+. 3+ ) is reduced to ferrous ions (Fe) 2+ The presence of this compound indicates a strong reducing ability, suggesting that Essential oil-C may contain a large number of compounds that can donate electrons or react with other reactive oxygen species, thereby exerting an antioxidant effect.

[0060] 2.5 Overview of the Quality Evaluation System This study, through the integration of colorimeter-assisted color parameter measurement, multi-dimensional sensory evaluation methods, and detection of key physical parameters such as density and refractive index, and systematically integrating chemical fingerprint analysis based on gas chromatography-mass spectrometry (GC-MS), has for the first time constructed a complete quality control system for Epimedium essential oil that integrates visual characteristics, olfactory properties, physical constants, and microbial limits. Figure 8Epimedium essential oil is a clear, yellow to yellowish-green liquid with a unique aroma, primarily medicinal and woody, mixed with complex notes of spiciness and freshness, making it highly recognizable. Its main physical properties include a relative density between 0.965 and 0.986 and a refractive index between 1.509 and 1.520, providing important criteria for quality control. Chemically, epimedium essential oil is mainly composed of four major classes of compounds: alkenes, alcohols, esters, and ketones. Among these, α-guaiacol, sesquiterpenes, patchouliene, and patchouli alcohol are present in high amounts and can be considered characteristic substances of epimedium essential oil. These components not only give the oil its unique aroma but also possess significant pharmacological activities, such as anti-inflammatory, antibacterial, immunomodulatory, and antioxidant effects. Regarding hygiene indicators, the total bacterial count of Epimedium essential oil should be controlled at ≤10 CFU / mL, and the content of mold and yeast should be controlled at ≤10 CFU / mL. The establishment of this system not only achieves standardized characterization of product appearance, aroma characteristics, physicochemical properties, and safety indicators, but also provides a scientific basis for the standardized development of the industry by innovatively introducing chemical component monitoring.

[0061] 3. Conclusion This study compared the effects of different extraction methods, processes, and storage conditions on the quality of Epimedium essential oil, proposing supercritical fluid extraction (SFE) as the optimal extraction method. SFE offers high extraction efficiency and effectively preserves key active components such as esters and alcohols from Epimedium leaves. Addressing the issue of insufficient extraction efficiency in traditional steam distillation due to the low volatile oil content of Epimedium, this study compared the advantages and disadvantages of steam distillation, steam distillation, and SFE for the first time. The results showed that SFE significantly increased the essential oil yield (up to 1.31%) and effectively preserved heat-sensitive components (such as ketones and terpenes), providing a new approach for the efficient industrial production of Epimedium essential oil.

[0062] This study further clarifies the quality standards for qualified Epimedium essential oil, integrating sensory evaluation (colorimeter quantification of color, quantitative rating method for aroma analysis), physicochemical indicators (density, refractive index), and chemical composition analysis. It establishes for the first time a quality control framework for Epimedium essential oil covering appearance, odor, physical properties, and hygiene standards, filling a gap in standardization research in this field. Compositional analysis shows that Epimedium essential oil is mainly composed of four major categories: alkenes, alcohols, esters, and ketones, significantly different from processed and liquid extracts of Epimedium containing flavonoids and alkaloids. This provides a scientific basis for distinguishing different forms of Epimedium products. Furthermore, α-guaiacol, sesquiterpenes, patchouliene, and patchouli alcohol were identified as characteristic substances of Epimedium essential oil. Regarding antioxidant capacity, this study found that, due to the transformation of unstable components, oil prepared by supercritical fluid extraction and stored sealed at room temperature for 50 days exhibited stronger antioxidant capacity. The findings in this paper provide a reference for the development of Epimedium essential oil products and the construction of a quality control system.

Claims

1. A method of preparing an Epimedium essential oil, characterized by: It comprises the following steps: (1) Take the Epimedium leaf, remove the petiole and impurities, wash with water, dry, whip into powder, pass through No. 2 sieve, sieve off the debris, and finally obtain Epimedium leaf powder for standby, the drying temperature is 40-60℃, and the time is 4-7h; (2) Take 120-170g of Epimedium leaf powder in step (1), add it to the supercritical CO2 extraction instrument for extraction, the extraction pressure of the supercritical CO2 extraction instrument is 10-50MPa, the extraction temperature is 20-60℃, the extraction time is 1-6h, and it is placed for 30-70 days, to obtain crude Epimedium oil for standby; (3) Take the crude Epimedium oil in step (2), dissolve it with 99% ether in an amount of 10-30 times the mass of the crude Epimedium, add anhydrous sodium sulfate with a mass fraction of 1-10% to dry, filter with a filter membrane with a pore size of 0.1-0.6um, remove the ether with a rotary evaporator, and the temperature of the rotary evaporator is 40-60℃, and the time is 0-60min, to obtain Epimedium essential oil.

2. The method of claim 1, wherein the method is characterized by: In step (1), the drying temperature is 45-55℃, and the time is 5-6h.

3. The method of claim 2, wherein the method is characterized by: In step (1), the drying temperature is 50℃, and the time is 6h.

4. The method of claim 1, wherein the method is characterized by: In step (2), the Epimedium leaf powder is 140-160g, the extraction pressure of the supercritical CO2 extraction instrument is 20-40MPa, the extraction temperature is 30-40℃, the extraction time is 3-5h, and it is placed for 40-60 days.

5. The method of claim 4, wherein the method is characterized by: In step (2), the Epimedium leaf powder is 150g, the extraction pressure of the supercritical CO2 extraction instrument is 30MPa, the extraction temperature is 35℃, the extraction time is 5h, and it is placed for 50 days.

6. The method of claim 1, wherein the method is characterized by: In step (3), the amount of 99% ether used is 15-25 times the mass of the crude Epimedium, and the mass fraction of anhydrous sodium sulfate is 3-7%.

7. The method of claim 6, wherein the method is characterized by: In step (3), the amount of 99% ether used is 20 times the mass of the crude Epimedium, and the mass fraction of anhydrous sodium sulfate is 5%.

8. The method of claim 1, wherein the method is characterized by: In step (3), the pore size of the filter membrane is 0.2-0.5um, and the temperature of the rotary evaporator is 45-55℃, and the time is 20-50min.

9. The method for preparing Epimedium essential oil according to claim 8, characterized in that: In step (3), the pore size of the filter membrane is 0.45um, and the temperature of the rotary evaporator is 50℃, and the time is 30min.

Images