Preparation method and application of Huangshan pine needle essential oil

Abstract

The invention discloses a preparation method and application of Huangshan pine needle essential oil, according to the method, release of bioactive substances in Huangshan pine needles is promoted through ultrahigh pressure processing combined with a microbial liquid fermentation technology, and the Huangshan pine needle essential oil is prepared through negative pressure distillation by adopting green solvent glycerol combined with a high-speed shearing extraction technology. According to the obtained Huangshan pine needle essential oil, the content of specific active substances of Huangshan pine needles is increased, secondary metabolites of beneficial microorganisms are newly formed, and the biological efficacy of fermentation products is enhanced. The anti-inflammatory soothing cream containing the Huangshan pine needle essential oil has the advantages of excellent anti-inflammatory effect and stable performance, and can be widely applied to the field of cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing and applying Huangshan pine needle essential oil. Background Technology

[0002] In recent years, due to environmental factors, endocrine disorders, insect bites, and nutritional deficiencies, skin inflammation has become increasingly common, leading to symptoms such as itching, peeling, and redness, which has become a major factor affecting human health. With the improvement of people's living standards, the demand for anti-inflammatory skin products is vast. In the current pharmaceutical and cosmetic market, there are numerous topical medications for common skin problems such as insect bites, itching, and redness, but most products suffer from limited efficacy, significant side effects, or poor comfort. Traditional topical medications often focus on simply relieving itching or swelling, neglecting the importance of anti-inflammatory effects, resulting in limited therapeutic efficacy and a higher risk of secondary infections. Furthermore, with increasing health awareness, people have higher demands for the safety, comfort, and overall efficacy of topical medications. Consumers not only want products to quickly relieve symptoms but also expect them to have anti-inflammatory functions to reduce the risk of infection, while providing a comfortable cooling sensation during use.

[0003] Huangshan Pine ( Pinus taiwanensis *Hayata* is a species endemic to China, formed under unique landforms and climates in places like Huangshan, Anhui province. Its needles are thicker and shorter than those of *Pinus massoniana* and *Pinus massoniana*, and its resin duct characteristics differ from those of *Pinus tabuliformis*. It grows on mountain peaks, steep slopes, ridges, and bare rocks at altitudes above 800 meters. While my country has abundant *Pinus massoniana* needle resources, research on the preparation of essential oils from these needles and their application in cosmetics is limited. This is primarily due to the differences between *Pinus massoniana* and *Pinus massoniana*, and the low extraction rate and long extraction cycle of *Pinus massoniana* needle essential oil, as well as the significant damage to its active ingredients caused by current processes. Furthermore, traditional pine needle essential oils often limit their compatibility with conventional formulas in skincare applications due to their high irritancy, easy oxidation, and difficulty in stable release of active ingredients, potentially leading to skin irritation or reduced efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying Huangshan pine needle essential oil, thereby solving the problems of low levels of active substances in existing Huangshan pine needle essential oil and poor anti-inflammatory effects when applied to cosmetics.

[0005] The technical solution adopted by this invention to solve its technical problem is: a method for preparing Huangshan pine needle essential oil, comprising the following steps: (1) Raw material pretreatment: The fresh Huangshan pine needles are dried until the moisture content is ≤7%; low temperature hot air at 38-55℃ is used for drying to ensure that the active substances and essential oils in the pine needles are not damaged.

[0006] (2) Pulverization: The dried Huangshan pine needles are transferred to a wall-breaking machine for air-jet pulverization. The temperature inside the machine is adjusted to -10℃~15℃, and a high-speed airflow is introduced. The air-jet pulverization pressure is 0.5~2.5 MPa, and the pulverization time is 5~10 min to obtain Huangshan pine needle powder. (3) Soaking: Add 0.05-0.5 mol / L phosphate buffer (pH range 5.8-6.8) to Huangshan pine needle powder at a mass ratio of 1:1-10, at room temperature (i.e. 20℃-25℃), and soak for 0.5-2 h; (4) Ultra-high pressure treatment: The Huangshan pine needle powder soaked in step (3) is put into a high-pressure resistant packaging bag, the air in the packaging bag is removed, and the bag is sealed tightly; then it is put into an ultra-high pressure chamber, the pressure value is set to 100-400 MPa, the pressure holding time is 5-20 min, the pressure increase rate is 10-50 MPa / s, the temperature is room temperature, and the pressure transmission medium is water; after the set pressure holding time is reached, the pressure is quickly released within 0.5-3 s, and the pressure release rate is 50-150 Mpa / s; the method of pressurization and rapid pressure release helps to quickly break the cell wall and release the contents.

[0007] (5) Microbial liquid fermentation: The Huangshan pine needle powder after ultra-high pressure treatment in step (4) is placed into a fermentation bottle along with phosphate buffer. 2%–15% microbial suspension is inoculated into each bottle, and the bottle is then placed in a shaking incubator for fermentation. The fermentation temperature is set at 25℃–35℃, the fermentation time is 5–12 days, and the shaking speed is 100–200 rpm. The microbial suspension is composed of *Dictyophora indica* (red-topped bamboo fungus). Dictyophora rubrovalvata ) or Fomitopsis pinnatifida ( Fomes fomentarius The concentration of the microbial suspension was 1×10⁻⁶. 7 ~1×10 9 CFU / mL; (6) Filtration: After coarse filtration through a quartz sand filter, the fermentation broth is then filtered through a ceramic membrane filter with an average pore size of 20-40 μm to remove bacteria and obtain fermentation supernatant A. The pressure during the ceramic membrane filtration process is set to 0.15-0.45 MPa. (7) Cell disruption and extraction: Add glycerol at a mass ratio of 1:1 to 30 and a mass concentration of 10% to 80% to the cells in step (6). Shear at a speed of 5000 to 15000 rpm, at a shearing temperature of 25℃ to 45℃, and at a shearing time of 60 to 180 s. Then centrifuge at a speed of 3000 to 5000 rpm for 10 to 20 min. Collect the supernatant and rotary evaporate to recover the glycerol. The remainder is the cell activity solution B. (8) Negative pressure distillation: The fermentation supernatant A and the bacterial cell active liquid B prepared in steps (6) and (7) are mixed and distilled at a mass ratio of 1 to 10:1. Since the contents of fermentation supernatant A and bacterial cell active liquid B are different, this ratio is used to better prepare the anti-inflammatory and soothing ointment. The negative pressure is set to 0.04 MPa to 0.08 MPa, and the distillation is carried out at a temperature of 65℃ to 85℃ for 60 to 150 min. The mixture of essential oil and steam produced by distillation is condensed to obtain the distillate. The distillate is then subjected to oil-water separation treatment, and the essential oil floating on the upper layer is collected, which is Huangshan pine needle essential oil.

[0008] The present invention also discloses an anti-inflammatory and soothing ointment containing Huangshan pine needle essential oil, comprising the following components by weight percentage: 0.1%–0.6% Huangshan pine needle essential oil, 2%–7% wintergreen oil, 0.2%–1% Litsea cubeba oil, 0.5%–1% camellia seed oil, 1%–3% Lithospermum erythrorhizon extract, 8%–13% menthol, 1%–2% borneol, 1%–2% camphor, 1%–3% natural white beeswax, 1%–3% natural candelilla wax, 5%–7% microcrystalline wax, 60%–72% petrolatum, and the remainder being distilled water.

[0009] The preparation method of the above-mentioned anti-inflammatory and soothing ointment containing Huangshan pine needle essential oil includes the following steps: (1) Put petroleum jelly, microcrystalline wax, natural candelilla wax, camphor and natural white beeswax into the reaction vessel, heat to 75℃~95℃, stir and mix well; cool to 45℃~65℃, then add borneol and menthol, mix well, and obtain solution A. (2) Add Huangshan pine needle essential oil, wintergreen oil, Litsea cubeba oil and camellia seed oil to the aqueous phase and shear at high speed of 8000-15000 rpm to form a crude emulsion; then emulsify with 100-500 W ultrasound at a temperature of 35℃-55℃ for 3-7 min, and then cool with an ice water bath to obtain mixed essential oil nano-emulsion, liquid B; (3) Slowly add liquid B to liquid A in the reactor of step (1) and homogenize it at a speed of 5000 to 18000 rpm; add it intermittently three times, each time for 10 to 30 s; (4) Heat the mixture in step (3) to 55℃~75℃ for sterilization for 20~35 min, and then cool it to room temperature to finally form a light yellow, uniform soothing cream with a characteristic odor.

[0010] The beneficial effects of this invention are: 1. This invention employs a microbial liquid fermentation process to treat pine needles, eliminating the need for added enzymes or other exogenous substances and significantly promoting the release of bioactive substances from Huangshan pine needles. After fermentation, the content of active compounds such as α-pinene, β-pinene, and p-cymene in Huangshan pine needle essential oil increases significantly. For example, the content of p-cymene, a unique bioactive substance in Huangshan pine needles, reaches as high as 9.51% after microbial fermentation, an increase of 1.23 times, and new chemical components, such as (E)-acariene and nonanal, are also produced. This method not only saves production costs but also simplifies the production process to the greatest extent, enabling this fermentation technology to achieve mass production and industrial-scale production.

[0011] 2. This invention employs a green solvent glycerol combined with high-speed shear extraction technology, and obtains Huangshan pine needle essential oil through negative pressure distillation. It has advantages such as short extraction time, low extraction temperature, high extraction rate, and environmental friendliness. For example, the yield of Huangshan pine needle essential oil prepared by this invention is as high as 0.89%, which is 78% higher than the yield of 0.51% by the traditional steam distillation method. It also retains the heat-sensitive active ingredients of Huangshan pine needle essential oil to the greatest extent, and improves the bioactivity of the essential oil. For example, in the RAW264.7 cell experiment, the inhibitory effect of IL-6 inflammatory factor was significantly increased by 5.53 times.

[0012] 3. Another objective of this invention is to provide an anti-inflammatory and soothing ointment containing Huangshan pine needle essential oil, which exhibits excellent anti-inflammatory effects and stable performance. This invention utilizes an innovative Huangshan pine needle essential oil extraction process, combined with a specific ratio of plant-based soothing components and a stabilizer system, significantly reducing irritation while enhancing synergistic anti-inflammatory effects and long-lasting sustained-release capacity. This ensures stable release of anti-inflammatory activity, solving the key problem of balancing stability and gentleness in traditional formulations. Clinical use has shown that the Huangshan pine needle essential oil soothing ointment of this invention is gentle, safe, and has significant anti-inflammatory effects with an efficacy rate as high as 92.5%, maintaining healthy skin and meeting people's needs.

[0013] The present invention will now be described in more detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 Changes in mycelial weight during microbial fermentation of Huangshan pine needles Figure 2 Changes in enzyme activity during microbial fermentation of Huangshan pine needles Detailed Implementation

[0015] Example 1: A method for preparing Huangshan pine needle essential oil, comprising the following steps: (1) Raw material pretreatment: The freshly picked Huangshan pine needles are dried until the moisture content is reduced to a certain level. The drying temperature was 6%; specifically: first, dry at 38℃ for 1 hour; then dry at 45℃ for 3 hours; and finally dry at 55℃ for 0.5 hours.

[0016] (2) Pulverization: The dried Huangshan pine needles are transferred to a wall-breaking machine for air-jet pulverization. The temperature inside the machine is adjusted to 10℃, and a high-speed airflow is introduced. The air-jet pulverization pressure is 2 MPa, and the pulverization time is 5 min to obtain Huangshan pine needle powder.

[0017] (3) Soaking: Add Huangshan pine needle powder to 0.1 mol / L phosphate buffer (pH range 6.8) at a mass ratio of 1:10 and soak for 1.5 h at room temperature (i.e. 20℃~25℃).

[0018] (4) Ultra-high pressure treatment: The Huangshan pine needle powder soaked in step (3) is placed together in a high-pressure resistant packaging bag, the air in the packaging bag is removed, and the bag is sealed tightly. Then it is placed in an ultra-high pressure chamber, the pressure is set to 300 MPa, the pressure holding time is 10 min, the pressure increase rate is 30 MPa / s, the temperature used in the ultra-high pressure equipment is room temperature (i.e., 20℃~25℃), and the pressure transmission medium is water. After the set pressure holding time is reached, the pressure is quickly released within 3s (i.e., the pressure release rate is 100 MPa / s).

[0019] (5) Microbial liquid fermentation: The Huangshan pine needle powder after ultra-high pressure treatment in step (4) is placed into a fermentation bottle along with phosphate buffer, and 5% of red-topped bamboo fungus is inoculated. Dictyophora rubrovalvata Bacterial suspension, with a concentration of 1×10⁻⁶. 8 The mycelium was collected at CFU / mL and placed in a shaking incubator. The fermentation temperature was set to 25℃, the fermentation time to 9 days, and the shaking speed to 120 rpm. Fermentation was considered complete when the weight of the mycelium in the fermentation flask no longer increased and remained constant for a certain period of time.

[0020] (6) Filtration: After the fermentation broth is coarsely filtered through a quartz sand filter, it is then sent to a ceramic membrane filter with an average pore size of 40 μm to remove the bacteria and obtain fermentation supernatant A. The pressure during the ceramic membrane filtration process is set to 0.25 MPa.

[0021] (7) Cell disruption and extraction: Add glycerol (45% mass concentration) at a mass ratio of 1:15 to the cells obtained by the ceramic membrane filter in step (6), shear at 12000 rpm, shear temperature of 42℃, shear time of 150 s, and then centrifuge at 5000 rpm for 15 min. Collect the supernatant and then perform rotary evaporation to recover the glycerol, thus obtaining the cell activity solution B.

[0022] (8) Negative pressure distillation: The fermentation supernatant A and the cell activity liquid B prepared in steps (6) and (7) are mixed at a mass ratio of 3:1. The negative pressure is set to 0.08 MPa, and the mixture is distilled at 75°C for 120 min. The mixture of essential oil and steam produced by distillation is condensed to obtain the distillate. The distillate is subjected to oil-water separation treatment, and the essential oil floating on the upper layer is collected, which is the Huangshan pine needle essential oil of the present invention, with a yield of 0.82%.

[0023] Example 2: A method for preparing Huangshan pine needle essential oil, comprising the following steps: (1) Raw material pretreatment: The fresh Huangshan pine needles were dried in sections until the moisture content was 6%; specifically as follows: first, they were dried at 38℃ for 1 h; then at 45℃ for 3 h; and finally at 55℃ for 0.5 h.

[0024] (2) Pulverization: The dried Huangshan pine needles are transferred to a wall-breaking machine for air-jet pulverization. The temperature inside the machine is adjusted to 10℃, and a high-speed airflow is introduced. The air-jet pulverization pressure is 2 MPa, and the pulverization time is 5 min to obtain Huangshan pine needle powder.

[0025] (3) Soaking: Add Huangshan pine needle powder to 0.1 mol / L phosphate buffer (pH range 6.8) at a mass ratio of 1:10 and soak for 1.5 h at room temperature (i.e. 20℃~25℃).

[0026] (4) Ultra-high pressure treatment: The Huangshan pine needle powder soaked in step (3) is placed together in a high-pressure resistant packaging bag, the air in the packaging bag is removed, and the bag is sealed tightly. Then it is placed in an ultra-high pressure chamber, the pressure is set to 300 MPa, the pressure holding time is 10 min, the pressure increase rate is 30 MPa / s, the temperature used in the ultra-high pressure equipment is room temperature (i.e., 20℃~25℃), and the pressure transmission medium is water. After the set pressure holding time is reached, the pressure is quickly released within 3s (i.e., the pressure release rate is 100 MPa / s).

[0027] (5) Microbial liquid fermentation: The Huangshan pine needle powder after ultra-high pressure treatment in step (4) was placed into a fermentation bottle along with phosphate buffer, and 5% of Fomitopsis pinicola was inoculated into each bottle. Fomes fomentarius Bacterial suspension, with a concentration of 1×10⁻⁶. 8 The mycelium was collected at CFU / mL and placed in a shaking incubator. The fermentation temperature was set to 25℃, the fermentation time to 9 days, and the shaking speed to 120 rpm. Fermentation was considered complete when the weight of the mycelium in the fermentation flask no longer increased and remained constant for a certain period of time.

[0028] (6) Filtration: After the fermentation broth is coarsely filtered through a quartz sand filter, it is then sent to a ceramic membrane filter with an average pore size of 40 μm to remove the bacteria and obtain fermentation supernatant A. The pressure during the ceramic membrane filtration process is set to 0.25 MPa.

[0029] (7) Cell disruption and extraction: Add glycerol (45% mass concentration) at a mass ratio of 1:15 to the cells obtained by the ceramic membrane filter in step (6), shear at 12000 rpm, shear temperature of 42℃, shear time of 150 s, and then centrifuge at 5000 rpm for 15 min. Collect the supernatant and then perform rotary evaporation to recover the glycerol, thus obtaining the cell activity solution B.

[0030] (8) Negative pressure distillation: The fermentation supernatant A and the cell activity liquid B prepared in steps (6) and (7) are mixed at a mass ratio of 3:1. The negative pressure is set to 0.08 MPa, and the mixture is distilled at 75°C for 120 min. The mixture of essential oil and steam produced by distillation is condensed to obtain the distillate. The distillate is subjected to oil-water separation treatment, and the essential oil floating on the upper layer is collected, which is the Huangshan pine needle essential oil of the present invention, with a yield of 0.89%.

[0031] Comparative Example 1: A method for preparing Huangshan pine needle essential oil, comprising the following steps: (1) Raw material pretreatment: The fresh Huangshan pine needles were dried in sections until the moisture content was 6%; specifically as follows: first, they were dried at 38℃ for 1 h; then at 45℃ for 3 h; and finally at 55℃ for 0.5 h.

[0032] (2) Pulverization: The dried Huangshan pine needles are transferred to a wall-breaking machine for air-jet pulverization. The temperature inside the machine is adjusted to 10℃, and a high-speed airflow is introduced. The air-jet pulverization pressure is 2 MPa, and the pulverization time is 5 min to obtain Huangshan pine needle powder.

[0033] (3) Soaking: Add Huangshan pine needle powder to 0.1 mol / L phosphate buffer (pH range 6.8) at a mass ratio of 1:10 and soak for 1.5 h at room temperature (i.e. 20℃~25℃).

[0034] (4) Ultra-high pressure treatment: The Huangshan pine needle powder soaked in step (3) is placed together in a high-pressure resistant packaging bag, the air in the packaging bag is removed, and the bag is sealed tightly. Then it is placed in an ultra-high pressure chamber, the pressure is set to 300 MPa, the pressure holding time is 10 min, the pressure increase rate is 30 MPa / s, the temperature used in the ultra-high pressure equipment is room temperature (i.e., 20℃~25℃), and the pressure transmission medium is water. After the set pressure holding time is reached, the pressure is quickly released within 3s (i.e., the pressure release rate is 100 MPa / s).

[0035] (5) Filtration: After the ultra-high pressure treatment, the liquid is coarsely filtered through a quartz sand filter and then filtered through a ceramic membrane filter with an average pore size of 40 μm to obtain the supernatant. The pressure during the ceramic membrane filtration process is set to 0.25 MPa.

[0036] (6) Negative pressure distillation: The supernatant prepared in step (5) is subjected to negative pressure distillation. The negative pressure is set to 0.08 MPa, and the distillation is carried out at 75°C for 120 min. The mixture of essential oil and vapor produced by distillation is condensed to obtain the distillate. The distillate is subjected to oil-water separation treatment, and the essential oil floating on the upper layer is collected, which is Huangshan pine needle essential oil, with a yield of 0.65%.

[0037] Comparative Example 2: Extraction of Huangshan pine needle essential oil using traditional steam distillation. 100.0g of fresh Huangshan pine needles were weighed and placed in a flask, 350 mL of water was added, and the mixture was extracted for 6 hours using a volatile oil extractor and anhydrous diethyl ether via steam distillation. The anhydrous diethyl ether layer was collected, and the solvent was evaporated. Then, anhydrous sodium sulfate was added for drying, yielding a pale yellow, transparent oily substance with a pine needle aroma, which was Huangshan pine needle essential oil, with a yield of 0.51%.

[0038] Figure and Table Description (1) Determination of liquid fermentation of Huangshan pine needles and changes in mycelial weight The weight of mycelium during the liquid fermentation of Huangshan pine needles was measured at 1, 3, 5, 7, and 9 days. The mycelium was collected by filtration, freeze-dried, and then weighed. Unfermented Huangshan pine needles were designated as Comparative Example 1, inoculated with an equal volume of sterile distilled water.

[0039] The weight changes of various mycelia after Huangshan pine needles were fermented with red-topped bamboo fungus and *Fomitopsis pinnatifida* are as follows: Figure 1 As shown. In the early growth stage (0-7 days), the mycelium of Examples 1 and 2 grew rapidly, that is, as fermentation proceeded, the weight of the mycelium continuously increased, gradually forming mycelial bundles; by day 9, the weight of the mycelium of *Dictyophora indicum* and *Fodium argentea* reached 16.47±0.25 g and 18.93±0.67 g, respectively, indicating that both *Dictyophora indicum* and *Fodium argentea*, two medicinal (edible) fungi, can fully utilize Huangshan pine needles for liquid fermentation.

[0040] (2) Determination of enzyme activity before and after liquid fermentation of Huangshan pine needles Take 2.5 g of samples fermented for 9 days using *Dictyophora indicum* and *Fomitopsis pinnatifida*, respectively, add 50 mL of 0.1 mol / L pH 6.0 citric acid buffer solution, and stir thoroughly for 30 min. Centrifuge at 4 ℃ and 10,000 r / min for 20 min, and collect the supernatant as the crude enzyme solution. Amylase activity was determined according to the DNS reagent method, where amylase activity (unit) is defined as the amount of enzyme required to hydrolyze glucose to 1 µg per minute at 37 ℃. Protease activity was determined according to the Folin-Ciocalteu reagent method, where protease activity (unit) is defined as the amount of enzyme required to hydrolyze tyrosine to 1 µg per minute at 40 ℃. Cellulase activity was determined according to the CMC saccharification power method, where cellulase activity (unit) is defined as the amount of enzyme required to hydrolyze glucose to 1 µg per minute at 37 ℃.

[0041] Liquid fermentation of Huangshan pine needles was carried out using *Dictyophora indicum* and *Fomitopsis pinnatifida*. The activities of amylase, protease, and cellulase in the Huangshan pine needles before and after fermentation were compared. The results are shown in [Table missing]. Figure 2 Compared to Example 1, which showed low cellulase activity (only 2.33 units / g), the cellulase activity in Examples 1 and 2 significantly increased after liquid fermentation with *Dictyophora indica* and *Fotomyces cerevisiae*. P <0.05), 462.67±12.22 and 583.00±14.53 units / g, respectively. Compared with the cellulase activity of 338.40±8.25 units / g in solid-state fermentation of Huangshan pine needles by *Trametes versicolor* and *Fomitopsis pinnatifida*, the cellulase activity of Huangshan pine needles fermented by liquid fermentation by *Trametes versicolor* and *Fomitopsis pinnatifida* increased by 36.72% and 71.28%, respectively. Compared with Comparative Example 1, the protease activity and amylase activity in Huangshan pine needles after fermentation in Examples 1 and 2 were significantly increased ( P <0.05). Among them, the cellulase and amylase activities in Huangshan pine needles after fermentation in Example 2 were higher than those in Example 1. The results show that, compared with solid-state fermentation, the two medicinal (edible) fungi, *Dictyophora indica* and *Fomitopsis pinnatifida*, can produce highly active amylase, protease, and cellulase during the liquid fermentation of Huangshan pine needles, which is more conducive to promoting the release of bioactive substances in Huangshan pine needles after fermentation.

[0042] (3) Determination of chemical composition of Huangshan pine needle essential oil The chemical composition of Huangshan pine needle essential oil prepared in Examples 1, 2, Comparative Example 1, and Comparative Example 2 was determined by GC-MS. Chromatographic conditions: HP-5 MS flexible quartz capillary column (0.25 μm, 30 m × 0.25 mm); high-purity helium as carrier gas at a flow rate of 1.0 mL / min; split ratio of 40:1; injection volume of 1.0 μL; injection port temperature of 280℃; initial column temperature of 60℃ for 3 min, increased to 280℃ at 5℃ / min, and held for 10 min. Mass spectrometry conditions: ion source temperature of 230℃, ionization mode of EI, electron energy of 70 eV, quadrupole temperature of 150℃; mass scan range of 35-450 u; solvent delay time of 3 min; NIST08 standard spectral library was used for searching.

[0043] After searching the NIST08 spectral library and verifying the mass spectra, 12 major compounds were identified in Huangshan pine needle essential oil. Table 1 shows that, compared to Comparative Examples 1 and 2, the content of compounds such as α-pinene, β-pinene, and p-cymene was significantly increased after liquid fermentation of Huangshan pine needles with *Dictyophora indica* and *Fomitopsis pinnatifida*. p-cymene is a unique active substance of Huangshan pine needles; its content in Comparative Example 2 was only 4.26%, but after liquid fermentation with *Dictyophora indica* and *Fomitopsis pinnatifida*, its relative content increased to 7.56% and 9.51%, respectively, representing increases of 0.77 times and 1.23 times. Furthermore, new chemical substances were generated through microbial fermentation. For example, fermentation with *Dictyophora indicum* produced a unique active substance, (E)-acorine, in Huangshan pine needle oil, with a content of 4.05%; fermentation with *Fodium argentea* produced a unique active substance, nonanal, in Huangshan pine needle oil, with a content of 2.12%. The results confirm that the microbial liquid fermentation method of this invention can significantly promote the release of active substances from Huangshan pine needles. This is related to the fact that *Dictyophora indicum* and *Fodium argentea*, two medicinal (edible) fungi, can produce highly active amylase, protease, and cellulase during the fermentation of Huangshan pine needles.

[0044] Table 1. Main chemical components and their relative contents of Huangshan pine needle essential oil (4) Determination of the anti-inflammatory effect of Huangshan pine needle essential oil RAW264.7 cell culture and grouping: Mouse RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C, 5% CO2, and relative humidity. Experimental setups included a blank control group, an LPS model group, Example 1 group, Example 2 group, and a comparative Example 1 group.

[0045] Cytotoxicity assay: RAW264.7 cells in logarithmic growth phase were analyzed at a concentration of 1 x 10⁻⁶ cells / mL.5 Samples were seeded at 100 μL / mL in 96-well plates and incubated for 24 h. After sample treatment, the plates were incubated for another 24 h, and 10 μL of MTT was added to each well. After incubation for another 3 h, the supernatant was discarded, and 200 μL of DMSO solution was added to each well. The plates were shaken in the dark to dissolve the purple crystals completely, and the absorbance of each well was measured at 570 nm.

[0046] NO content detection: RAW264.7 cells were prepared at a concentration of 1 x 10⁻⁶ cells / mL. 6 Cells / mL were seeded in 96-well plates at 200 μL per well and cultured for 24 h. Samples were added to the experimental groups, and after 1 h of incubation, 20 μL of LPS (1 μg / mL) was added, followed by incubation for 18 h. The supernatant was collected, and the NO level in the cell supernatant was detected using the Griess method.

[0047] IL-6 level detection: The IL-6 level in the cell culture medium was determined using ELISA. The procedure was followed according to the kit instructions. A standard curve of absorbance versus mass concentration was plotted using IL-6 standards: Y = 0.003X + 0.031 ( R 2 =0.995), the concentration of IL-6 in the cell supernatant was calculated using a standard curve.

[0048] Table 2 shows that Huangshan pine needle essential oil had no toxic effect on RAW264.7 cells at a concentration of 0.02 mg / mL. The NO content in the cell sap of the blank control group RAW264.7 cells was (16.89±1.35)%. After LPS stimulation, the NO content of the cells increased significantly, indicating that the in vitro RAW264.7 cell inflammation model was successfully established. Compared with Comparative Examples 1 and 2, the Huangshan pine needle essential oil prepared by liquid fermentation of *Dictyophora indicum* and *Pterocarpus styracifolium* showed a more significant effect in inhibiting the production of NO and IL-6. For example, the IL-6 content in Comparative Example 1 was (1.96±0.41) ng / mL, with an inhibition rate of only 20%, while the IL-6 content in Examples 1 and 2 was reduced to (1.49±0.32) and (0.85±0.13) ng / mL, respectively, with inhibition rates as high as 39.18% and 65.31%, respectively. These inhibition rates were 0.96 times and 2.27 times higher than those in Comparative Example 1. Similarly, the IL-6 content in Comparative Example 2 was (2.21±0.13) ng / mL, with an inhibition rate of only 10%, while the IL-6 inhibition rates in Examples 1 and 2 were 2.91 times and 5.53 times higher than those in Comparative Example 1, respectively. The results showed that the anti-inflammatory effect of Huangshan pine needle essential oil prepared by microbial liquid fermentation was more significant, which improved the anti-inflammatory efficacy and laid a theoretical foundation for the development of functional cosmetics based on Huangshan pine needle essential oil.

[0049] Table 2. Effects of Huangshan pine needle essential oil on RAW264.7 cell viability and LPS-induced NO and IL-6 levels. Example 3: An anti-inflammatory and soothing ointment containing Huangshan pine needle essential oil. The ointment comprises the following components by weight percentage: 0.5% Huangshan pine needle essential oil, 5% wintergreen oil, 0.2% Litsea cubeba oil, 1.0% camellia seed oil, 0.5% Lithospermum erythrorhizon extract, 10% menthol, 1% borneol, 1% camphor, 1% natural white beeswax, 1.5% natural candelilla wax, 5.5% extra-refined food-grade microcrystalline wax, 68% petrolatum, and the remainder being distilled water.

[0050] A method for preparing an anti-inflammatory and soothing ointment containing Huangshan pine needle essential oil includes the following steps: (1) Put petroleum jelly, extra-grade refined food-grade microcrystalline wax, natural candelilla wax, natural white beeswax and camphor into a reaction vessel, heat to 85°C and stir to mix well; cool to 50°C, add ice skin and menthol, mix well to obtain solution A.

[0051] (2) Wintergreen oil, Litsea cubeba oil and Camellia seed oil were prepared by negative pressure distillation.

[0052] (3) Add Huangshan pine needle essential oil, wintergreen oil, Litsea cubeba oil and camellia seed oil to the aqueous phase and form a crude emulsion by high-speed shearing at 12000 rpm. Emulsify using 300 W ultrasound at 45℃ for 5 min, and then cool using an ice water bath to obtain the mixed essential oil nanoemulsion, solution B.

[0053] (4) Slowly add liquid B to liquid A in the reactor of step (1) and homogenize it at a speed of 10,000 rpm. Repeat this process three times intermittently, with each time lasting 20 s.

[0054] (5) After dispersing and mixing, heat the external circulating water to 65°C and sterilize for 30 min; cool to room temperature to finally form a light yellow, uniform soothing cream with a characteristic odor.

[0055] (5) Evaluation of the anti-inflammatory effect of a soothing ointment containing Huangshan pine needle essential oil Sixty volunteers aged 20 and above were selected who exhibited symptoms such as mosquito bites, itching, and redness. Volunteers aged 18 to 60 years old, with an equal number of men and women, were randomly divided into three groups. Volunteers in the first and second groups used the soothing cream containing Huangshan pine needle essential oil prepared in Example 3, while volunteers in the third group used a commercially available soothing cream as a control.

[0056] Before the trial, all volunteers underwent routine skin disinfection of the affected areas. Then, an appropriate amount of the soothing cream of this invention was applied to the affected areas, several times daily. During the trial, volunteers were asked to record their feelings and changes in symptoms after application, and were followed up regularly to assess the effectiveness of the treatment. The treatment period was one week.

[0057] The efficacy criteria refer to the efficacy criteria for pruritus in "Practical Integrated Traditional Chinese and Western Medicine Diagnosis and Treatment", which are specifically divided into the following three levels: Cured: Itching and secondary skin damage (such as scratch marks, blood crusts, rough skin) completely disappear; Significantly effective: Itching symptoms are significantly reduced, and secondary skin lesions disappear by more than 80%; Ineffective: Itching symptoms are not reduced or aggravated, and secondary skin lesions disappear by less than 20%.

[0058] As shown in Table 3, over 92.5% of the participants believed that the soothing cream, made with Huangshan pine needle essential oil, wintergreen oil, Litsea cubeba oil, camellia seed oil, and Lithospermum erythrorhizon extract, effectively inhibited skin inflammation, promoted the repair of inflamed skin, and maintained the healthy state of the skin. No adverse reactions or allergic reactions occurred during the experiment, proving that the cooling and soothing cream of this invention has good safety and effectiveness.

[0059] Table 3 Evaluation of the anti-inflammatory effect of Huangshan pine needle essential oil soothing cream (experimental period of 1 week) The present invention has been described above by way of example with reference to the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvements made using the inventive concept and technical solution; or the direct application of the inventive concept and technical solution to other situations without modification, are all within the protection scope of the present invention.

Claims

1. A method for preparing Huangshan pine needle essential oil, characterized in that, Includes the following steps: (1) Raw material pretreatment: The freshly picked Huangshan pine needles are dried; (2) Crushing: The dried Huangshan pine needles are crushed to obtain Huangshan pine needle powder; (3) Soaking: Soak Huangshan pine needle powder in phosphate buffer for 0.5-2 h; (4) Ultra-high pressure treatment: The Huangshan pine needle powder soaked in step (3) is put into a packaging bag and vacuum sealed; then it is placed in an ultra-high pressure chamber, the pressure is set to 100-400 MPa, the pressure holding time is 5-20 min, and the pressure increase rate is 10-50 MPa / s; after the set pressure holding time is reached, the pressure is quickly released within 0.5-3 s, and the pressure release rate is 50-150 MPa / s; (5) Microbial liquid fermentation: The Huangshan pine needle powder soaked in step (4) after ultra-high pressure treatment is put into a fermentation bottle, inoculated with 2% to 15% microbial suspension, and then placed in a shaking incubator for fermentation; (6) Filtration: The fermentation broth is filtered to remove the bacterial cells, yielding fermentation supernatant A. (7) Cell disruption and extraction: Glycerol was added to the cells in step (6) for rotary cutting and centrifugation. The supernatant was collected and the glycerol was recovered by rotary evaporation to obtain cell activity solution B. (8) Negative pressure distillation: The fermentation supernatant A and the cell activity liquid B prepared in steps (6) and (7) are mixed and distilled under negative pressure. The mixed gas produced by distillation is condensed to obtain distillate. The distillate is then separated into oil and water, and the essential oil floating on the upper layer is collected, which is Huangshan pine needle essential oil.

2. The method for preparing Huangshan pine needle essential oil according to claim 1, characterized in that: The step (1) involves drying in stages: first drying at 38℃~45℃ for 1~2 hours, then drying at 40℃~55℃ for 3~5 hours, and finally drying at 50℃~65℃ for 0.5~2 hours until the moisture content is ≤7%.

3. The method for preparing Huangshan pine needle essential oil according to claim 1, characterized in that: The microbial suspension in step (5) contains *Dictyophora indica* (red-topped bamboo fungus). Dictyophora rubrovalvata ) or Fomitopsis pinnatifida ( Fomes fomentarius The concentration of the microbial suspension was 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

4. The method for preparing Huangshan pine needle essential oil according to claim 3, characterized in that: In step (5), the fermentation temperature is set to 25℃~35℃, the fermentation time is 5~12 days, and the oscillation speed is 100~200 rpm.

5. The method for preparing Huangshan pine needle essential oil according to claim 1, characterized in that: In step (6), the fermentation broth is first coarsely filtered through a quartz sand filter, and then sent to a ceramic membrane filter with an average pore size of 20-40 μm to remove bacteria. The pressure during the ceramic membrane filtration process is set to 0.15-0.45 MPa.

6. An anti-inflammatory and soothing ointment containing Huangshan pine needle essential oil, characterized in that, The product comprises the following components by weight percentage: 0.1%–0.6% Huangshan pine needle oil, 2%–7% wintergreen oil, 0.2%–1% Litsea cubeba oil, 0.5%–1% camellia seed oil, 1%–3% Lithospermum erythrorhizon extract, 8%–13% menthol, 1%–2% borneol, 1%–2% camphor, 1%–3% natural white beeswax, 1%–3% natural candelilla wax, 5%–7% microcrystalline wax, 60%–72% petrolatum, and the remainder being distilled water.

7. The preparation method of the anti-inflammatory and soothing ointment containing Huangshan pine needle essential oil as described in claim 6, comprising the following steps: (1) Put petroleum jelly, microcrystalline wax, natural candelilla wax, camphor and natural white beeswax into the reaction vessel, heat to 75℃~95℃, stir and mix well; cool to 45℃~65℃, then add borneol and menthol, mix well, and obtain solution A. (2) Add Huangshan pine needle essential oil, wintergreen oil, Litsea cubeba oil and camellia seed oil to the aqueous phase and shear at high speed of 8000-15000 rpm to form a crude emulsion; then emulsify with 100-500 W ultrasound at a temperature of 35℃-55℃ for 3-7 min, and then cool with an ice water bath to obtain mixed essential oil nano-emulsion, liquid B; (3) Slowly add liquid B to liquid A in the reactor of step (1) and homogenize it at a speed of 5000 to 18000 rpm; add it intermittently three times, each time for 10 to 30 s; (4) Heat the mixture in step (3) to 55℃~75℃ for sterilization for 20~35 min, and then cool it to room temperature to finally form a light yellow, uniform soothing cream with a characteristic odor.

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