A method for extracting total naphthoquinones from Lithospermum erythrorhizon in Xinjiang and its application

Abstract

This invention belongs to the field of plant extract preparation technology, specifically relating to a method for extracting total naphthoquinones from Xinjiang Lithospermum erythrorhizon and its application. The method includes pretreatment: pulverizing Xinjiang Lithospermum erythrorhizon raw material and subjecting it to high-pressure steam explosion treatment to obtain pretreated material; supercritical CO2 extraction; and molecular distillation to obtain the total naphthoquinone solution from Xinjiang Lithospermum erythrorhizon. This invention significantly disrupts cell wall structure through high-pressure steam explosion pretreatment, facilitating the release and dissolution of target components; employing supercritical CO2 extraction with a preferred entrainer achieves efficient extraction under mild conditions, increasing the yield of naphthoquinones. The extract obtained by this method has a more optimized composition of naphthoquinone components and exhibits excellent bioactivity in anti-inflammatory, antipruritic, skin repair, and improvement of atopic dermatitis effects. Furthermore, the process is simple to operate, uses green and safe solvents, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of plant extract preparation technology, specifically relating to a method for extracting total naphthoquinones from Xinjiang Lithospermum erythrorhizon and its application. Background Technology

[0002] Xinjiang Lithospermum (scientific name: *Arnebia euchroma*) is a perennial herb belonging to the genus *Arnebia* in the family Boraginaceae. Also known as soft lithospermum, its roots are harvested in spring and autumn, sun-dried, or oven-dried before being used medicinally. The *Chinese Pharmacopoeia* records that Xinjiang Lithospermum can clear heat and cool the blood, invigorate blood circulation and detoxify, and promote rash eruption and eliminate spots. It is used for blood heat and toxicity, purplish-black rashes, incomplete measles eruption, sores, eczema, and burns. The active ingredients in Xinjiang Lithospermum are mainly naphthoquinone compounds, represented by shikonin and its derivatives, primarily consisting of eight components: shikonin, isovaleryl shikonin, acetyl shikonin, deoxyshikonin, β-hydroxyisovaleryl shikonin, β,β-dimethylacryloyl shikonin, isobutyryl shikonin, and β-acetoxyisovaleryl shikonin. These components possess physiological functions such as promoting wound healing, anti-inflammation, antibacterial, anticancer, and antiviral properties. Additionally, it can be used as a natural pigment and is widely applied in the food, cosmetics, traditional Chinese medicine, and textile industries.

[0003] Currently, conventional extraction methods for total naphthoquinones from Xinjiang erythrorhizon include ethanol extraction, reflux extraction, and ultrasonic extraction. However, due to the poor stability of total naphthoquinones from Xinjiang erythrorhizon to light, heat, metal ions, and pH changes, conventional extraction processes easily lead to degradation or color changes. Furthermore, they involve the use of large amounts of toxic organic solvents such as n-hexane and petroleum ether. Existing technologies mainly focus on the extraction and purification of naphthoquinone monomers from Xinjiang erythrorhizon, which suffers from cumbersome extraction steps, low extraction rates, and difficulty in scaling up production. Summary of the Invention

[0004] As one aspect of the present invention, the present invention provides a method for extracting total naphthoquinones from Lithospermum erythrorhizon in Xinjiang, which includes the following steps:

[0005] S1. Pretreatment: The raw material of Xinjiang purple gromwell is crushed and then subjected to high-pressure steam explosion treatment to obtain the pretreated material.

[0006] S2. Supercritical CO2 extraction: The pretreated material is subjected to supercritical CO2 extraction, and an entrainer is added during the extraction process. The entrainer is caprylic / capric triglyceride, or ethanol, or vegetable oil, or a combination of ethanol and caprylic / capric triglyceride; the extract is collected to obtain crude extract.

[0007] S3. Molecular distillation: The crude extract is subjected to molecular distillation to collect the heavy components and obtain the total naphthoquinone solution of Lithospermum erythrorhizon.

[0008] As a preferred embodiment of the extraction method described in this invention, when the entrainer is a combination of ethanol and caprylic / capric triglyceride, step S2 is a stepwise extraction, including:

[0009] First extraction: Ethanol is added as an entrainer for extraction;

[0010] Second extraction: Ethanol is added as an entrainer to the material after the first extraction for further extraction;

[0011] Third extraction: Caprylic / capric triglyceride is added as an entrainer to the material after the second extraction for further extraction.

[0012] As a preferred embodiment of the extraction method described in this invention, in step S1, the pressure of the high-pressure steam explosion treatment is 0.8-1.0 MPa, and the pressure holding time is 20-30 seconds.

[0013] As a preferred embodiment of the extraction method described in this invention, in step S2, the conditions for supercritical CO2 extraction include: an extraction pressure of 20-30 MPa and an extraction temperature of 40-50 ℃.

[0014] As a preferred embodiment of the extraction method of the present invention, in step S2, the mass ratio of ethanol added in the first extraction to the pretreated material is (0.3-1):1; the mass ratio of the total mass of ethanol added in the second extraction to the mass of the material after the first extraction is (1-1.5):1; and the mass ratio of the total mass of caprylic / capric triglycerides added in the third extraction to the mass of the material after the second extraction is (1-1.5):1.

[0015] As a preferred embodiment of the extraction method described in this invention, step S2 includes three extractions. When the entrainer is caprylic / capric triglyceride, the mass ratio of the entrainer added in each extraction to the pretreated material is (0.3-1.5):1.

[0016] As a preferred embodiment of the extraction method described in this invention, in step S2, the supercritical CO2 extraction is performed with a CO2 flow rate of 45-200 L / h.

[0017] As a preferred embodiment of the extraction method described in this invention, in step S3, the conditions for molecular distillation include: an evaporator temperature of 75-85°C and a system vacuum degree of less than 50 Pa.

[0018] The present invention also provides the application of total naphthoquinones from Lithospermum erythrorhizon prepared by the extraction method described above in the preparation of cosmetic compositions for topical skin care, wherein the cosmetic compositions are used to promote skin barrier repair or relieve skin inflammation.

[0019] The present invention also provides the application of total naphthoquinones from Xinjiang Lithospermum prepared by the extraction method described above in the preparation of a drug for treating atopic dermatitis.

[0020] The beneficial effects of this invention are as follows: Pretreatment with high-pressure steam explosion significantly disrupts the cell wall structure, facilitating the release and dissolution of the target components. Supercritical CO2 extraction with a preferred entrainer achieves efficient extraction under mild conditions, increasing the yield of naphthoquinones. The resulting extract exhibits a superior composition of naphthoquinones and demonstrates excellent bioactivity in anti-inflammatory, antipruritic, skin repair, and atopic dermatitis improvement effects. Furthermore, the process is simple to operate, uses green and safe solvents, and is suitable for large-scale production.

[0021] Liquid chromatography detection method: A 5 µm Thermo Fisher C18 column (250 × 4.6 mm) was used. The mobile phase was A: acetonitrile and B: 0.05% formic acid, with isocratic elution (A:B = 70:30) for 0–30 min. Detection wavelength: 516 nm. Injection volume: 10 µL. Time: 30 min. Column temperature: 30 ℃. Retention times were as follows: shikonin: 5.33 min, β-hydroxyisovalerylshikonin: 6.616 min, acetylshikonin: 8.201 min, β-acetoxyisovalerylacanine: 11.884 min, deoxyshikonin: 13.126 min, isobutyrylshikonin: 14.724 min, β,β-dimethylacryloylacanine: 17.690 min, and isovalerylshikonin: 19.322 min. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0023] Figure 1 The relative cell survival rate is shown in Example 1.

[0024] Figure 2 This represents the relative cell survival rate in Example 2.

[0025] Figure 3 The relative cell survival rate is shown in Example 3.

[0026] Figure 4 This refers to the relative cell survival rate in Example 4.

[0027] Figure 5 This is a graph showing the immunofluorescence detection results of filaggrin. Detailed Implementation

[0028] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0029] The roots of *Arnebia euchroma* used in the following examples were harvested in Urumqi, Xinjiang. The supercritical CO2 extraction instrument, model HA220-50-06-C, was purchased from Nantong Huaan Supercritical Extraction Co., Ltd.; the molecular distillation instrument, model QYMD-80C, was purchased from Qiyu Industrial (Shanghai) Co., Ltd. The QB-200 steam explosion test bench used in this experiment was manufactured by Henan Hebi Zhengdao Heavy Machinery Factory. Caprylic / capric triglyceride (GTCC) was purchased from Hubei Chuyi New Materials Co., Ltd.

[0030] Example 1:

[0031] Step 1: Pretreatment: Crush the dried Xinjiang purple gromwell root into powder using a pulverizer, sieve (0.355mm aperture), retain the sieved portion, and load it into a high-pressure steam explosion reactor. Use high-pressure instantaneous explosion technology to maintain pressure at 0.8 MPa for 30 seconds. After the pressure holding is completed, open the explosion valve to rupture the cell walls and release the active ingredients, thus obtaining the pretreated material.

[0032] Step 2: Supercritical CO2 Extraction: 1 kg of the material obtained in Step 1 was loaded into the extraction vessel, and 0.5 kg of GTCC was added and stirred until homogeneous. The extraction vessel was heated to 45 ℃, the separation vessel to 40 ℃, and the storage tank to 35 ℃. Then, the CO2 cylinder was turned on, and the CO2 flow rate was controlled at 50 L / h. The system was pressurized by a high-pressure pump. When the pressure in the extraction vessel reached 40 MPa and the pressure in the separation vessel reached 5 MPa, the circulation extraction began, and the extraction vessel was kept at a constant temperature and pressure. After 1 h of extraction, the material was discharged from the outlet of the separation vessel, completing the first extraction and obtaining a dark red extract. After the first extraction, 1 kg of GTCC was pumped in through an entrainer pump, and the above experimental operation was repeated for 1 h to complete the second extraction. After the second extraction, 1 kg of GTCC was pumped in through an entrainer pump, and the above experimental operation was repeated for 1 h to obtain a light red third extract of Lithospermum erythrorhizon. The extracts from the above three extractions were combined to obtain a crude total naphthoquinone extract solution of Lithospermum erythrorhizon.

[0033] Step 3: Molecular distillation: Take the crude total naphthoquinone extract from *Lithospermum erythrorhizon* obtained in Step 2. Set the molecular distillation instrument parameters as follows: feed temperature 40 ℃, evaporator temperature 80 ℃, condenser -5 ℃, external condenser -20 ℃, rotor speed 200 rpm / min. Pump the feed into the molecular distillation instrument, turn on heating, condensation, vacuum (vacuum degree below 50 Pa), and stir. Discard the light components and collect the heavy component samples to obtain the total naphthoquinone extract solution from *Lithospermum erythrorhizon*. Quantitative analysis of eight components—shikonin, isovalerylshikonin, acetylshikonin, deoxyshikonin, β-hydroxyisovalerylshikonin, β,β-dimethylacryloylacarine, isobutyrylshikonin, and β-acetoxyisovalerylacarine—was performed using high-performance liquid chromatography (HPLC). The total naphthoquinone yield was 4.94%.

[0034] Total naphthoquinone yield (%) = (mass of total naphthoquinone in the extract / mass of raw material from Xinjiang Lithospermum erythrorhizon root) × 100%.

[0035] Example 2:

[0036] Step 1: Pretreatment: Crush the dried Xinjiang purple gromwell root into powder using a pulverizer, sieve (0.355mm aperture), retain the sieved portion, and load it into a high-pressure steam explosion reactor. Use high-pressure instantaneous explosion technology to maintain pressure at 0.8 MPa for 30 seconds. After the pressure holding is completed, open the explosion valve to rupture the cell walls and release the active ingredients, thus obtaining the pretreated material.

[0037] Step 2: Supercritical CO2 Extraction: 10 kg of the material obtained in Step 1 was loaded into the extraction vessel, and 10 kg of ethanol was added. The mixture was stirred and stirred until homogeneous. The extraction vessel was heated to 45 °C, the separation vessel to 35 °C, and the storage tank to 30 °C. Then, the CO2 cylinder was turned on, and the CO2 flow rate was controlled at 160 L / h. The system was pressurized using a high-pressure pump. When the pressure in the extraction vessel was 30 MPa and the pressure in the separation vessel was 6 MPa, the circulation extraction began. The extraction vessel was kept at a constant temperature and pressure. After 2 hours of extraction, the material was discharged from the outlet of the separation vessel, completing the first extraction. After the first extraction, 10 kg of ethanol was pumped in using an entrainer pump, and the above experimental operation was repeated. After 2 hours of extraction, the material was discharged from the outlet of the separation vessel, completing the second extraction. After the second extraction, 10 kg of ethanol was pumped in using an entrainer pump, and the above experimental operation was repeated. After 2 hours of extraction, the pink extract of *Lithospermum erythrorhizon* was obtained from the third extraction. The extract solutions from the three extractions were combined to obtain the crude total naphthoquinone extract solution of *Lithospermum erythrorhizon*.

[0038] Step 3: Molecular distillation: Take the crude total naphthoquinone extract solution from Xinjiang Lithospermum obtained in Step 2, and set the molecular distillation instrument parameters as follows: feed temperature 50 ℃, evaporator temperature 80 ℃, condenser -5 ℃, external condenser -20 ℃, rotor speed 300 rpm / min. Pump the feed solution into the molecular distillation instrument, turn on heating, condensation, vacuuming, and stirring. Discard the light components and collect the heavy component samples to obtain the total naphthoquinone extract solution from Xinjiang Lithospermum. High performance liquid chromatography (HPLC) was used to quantitatively detect the eight components: shikonin, isovaleryl shikonin, acetyl shikonin, deoxyshikonin, β-hydroxyisovaleryl shikonin, β,β-dimethylacryloylacarin, isobutyryl shikonin, and β-acetoxyisovalerylacarin. The total naphthoquinone yield was 4.72%.

[0039] Example 3

[0040] Step 1: Preparation of Xinjiang Lithospermum powder: The dried Xinjiang Lithospermum is pulverized into powder using a pulverizer and passed through a No. 3 sieve (0.355 mm). The portion that passes through the sieve is retained to obtain Xinjiang Lithospermum powder. The powder is loaded into an explosion reaction vessel, and high-pressure instantaneous explosion technology is used to maintain the pressure at 0.8 MPa for 30 seconds. After the pressure holding is completed, the explosion valve is quickly opened to rupture the cell walls and release the active ingredients for subsequent extraction.

[0041] Step 2: Take 10 kg of the blasted Xinjiang Lithospermum powder, add 5 kg of anhydrous ethanol, stir and mix evenly, then load into the extraction vessel. Heat the extraction vessel to 45 °C, the separation vessel to 45 °C, and the storage tank to 35 °C. Then turn on the CO2 cylinder, controlling the CO2 flow rate to 180 L / h, and pressurize the system using a high-pressure pump. When the extraction pressure in the extraction vessel is 35 MPa and the pressure in the separation vessel is 6 MPa, start the circulation extraction, maintaining constant temperature and pressure in the extraction vessel. After 2 hours of extraction, discharge from the outlet of the separation vessel to obtain a dark red Xinjiang Lithospermum extract. After the first extraction is complete, add 10 kg of anhydrous ethanol through an entrainer pump, repeat the above experimental operation, and extract for 2 hours to obtain a second Xinjiang Lithospermum extract. After the second extraction is complete, add 15 kg of GTCC through an entrainer pump, repeat the above experimental operation, and extract for 2 hours to obtain a light red third Xinjiang Lithospermum extract. Combine the above three Xinjiang Lithospermum extracts to obtain the crude total naphthoquinones from Xinjiang Lithospermum.

[0042] Step 3: Mix the crude total naphthoquinone solution from Xinjiang Lithospermum obtained in Step 2 thoroughly. Set the molecular distillation instrument parameters as follows: heating temperature of the solution at 50 °C, evaporator temperature at 85 °C, condenser at -5 °C, external condenser at -20 °C, and rotor speed at 200 rpm / min. Pump the solution into the molecular distillation instrument, and turn on heating, condensation, vacuuming, and stirring. Discard the lighter fractions, which mainly contain other components. Collect the heavier fraction samples to obtain the total naphthoquinone extract solution from Xinjiang Lithospermum. Quantitative analysis of eight components—shikonin, isovalerylshikonin, acetylshikonin, deoxyshikonin, β-hydroxyisovalerylshikonin, β,β-dimethylacryloylacanine, isobutyrylshikonin, and β-acetoxyisovalerylacanine—was performed using high-performance liquid chromatography (HPLC). The total naphthoquinone yield was calculated to be 5.88%.

[0043] Example 4

[0044] Step 1: Preparation of Xinjiang Lithospermum powder: The dried Xinjiang Lithospermum is pulverized into powder using a pulverizer and passed through a No. 3 sieve (0.355 mm). The portion that passes through the sieve is retained to obtain Xinjiang Lithospermum powder. The powder is loaded into an explosion reaction vessel, and high-pressure instantaneous explosion technology is used to maintain the pressure at 1.0 MPa for 30 seconds. After the pressure holding is completed, the explosion valve is quickly opened to rupture the cell walls and release the active ingredients for subsequent extraction.

[0045] Step 2: Take 1 kg of the blasted Xinjiang Lithospermum powder, add 1 kg of camellia oil, stir and mix evenly, then load into the extraction vessel. Heat the extraction vessel to 45 °C, the separation vessel to 45 °C, and the storage tank to 35 °C. Then turn on the CO2 cylinder, control the CO2 flow rate to 40 L / h, and pressurize the system using a high-pressure pump. When the extraction pressure in the extraction vessel is 30 MPa and the pressure in the separation vessel is 5 MPa, start the circulation extraction, and maintain constant temperature and pressure in the extraction vessel. After 1 hour of extraction, discharge from the separation vessel outlet to obtain a dark red Xinjiang Lithospermum extract. After the first extraction is completed, add 1 kg of camellia oil through an entrainer pump, repeat the above experimental operation, and after 1 hour of extraction, discharge from the separation vessel outlet to obtain a second Xinjiang Lithospermum extract. After the second extraction is completed, add another 1 kg of camellia oil through an entrainer pump, and after 1 hour of extraction, discharge from the separation vessel outlet to obtain a light red third Xinjiang Lithospermum extract. Combining the above three extracts of Lithospermum erythrorhizon yields the crude total naphthoquinones from Lithospermum erythrorhizon.

[0046] Step 3: Mix the crude total naphthoquinone solution from Xinjiang Lithospermum obtained in Step 2 thoroughly. Set the molecular distillation instrument parameters as follows: heating temperature of the solution at 50 °C, evaporator temperature at 80 °C, condenser at -5 °C, external condenser at -20 °C, and rotor speed at 200 rpm / min. Pump the solution into the molecular distillation instrument, and turn on heating, condensation, vacuuming, and stirring. Discard the lighter fractions, which mainly contain other components. Collect the heavier fraction samples to obtain the total naphthoquinone extract solution from Xinjiang Lithospermum. Quantitative analysis of eight components—shikonin, isovalerylshikonin, acetylshikonin, deoxyshikonin, β-hydroxyisovalerylshikonin, β,β-dimethylacryloylacanine, isobutyrylshikonin, and β-acetoxyisovalerylacanine—was performed using high-performance liquid chromatography (HPLC). The total naphthoquinone yield was calculated to be 4.60%.

[0047] Comparative Example 1:

[0048] Step 1: Pretreatment: Crush the dried Xinjiang purple gromwell root into powder using a pulverizer, sieve it (0.355mm aperture), and retain the sieved portion to obtain the pretreated material.

[0049] Step 2: Supercritical CO2 extraction: 1 kg of the material obtained in Step 1 was loaded into the extraction vessel. The extraction vessel was heated to 45°C, the separation vessel to 45°C, and the storage tank to 35°C. Then, the CO2 cylinder was turned on, and the CO2 flow rate was controlled at 50 L / h. The system was pressurized by a high-pressure pump. When the pressure in the extraction vessel reached 40 MPa and the pressure in the separation vessel reached 5 MPa, the circulation extraction began, and the extraction vessel was kept at a constant temperature and pressure. After 1.5 h of extraction, the material was discharged from the outlet of the separation vessel, completing the extraction and obtaining crude total naphthoquinone extract from Xinjiang Lithospermum erythrorhizon. High-performance liquid chromatography (HPLC) was used to quantitatively detect eight components: shikonin, isovaleryl shikonin, acetyl shikonin, deoxyshikonin, β-hydroxyisovaleryl shikonin, β,β-dimethylacryloyl argentin, isobutyryl shikonin, and β-acetoxyisovaleryl argentin. The total naphthoquinone yield was calculated to be 2.04%.

[0050] Comparative Example 2:

[0051] Step 1: Pretreatment: Crush the dried Xinjiang purple gromwell root into powder using a pulverizer, sieve it (0.355mm aperture), and retain the sieved portion to obtain the pretreated material.

[0052] Step 2: 1 kg of pretreated material was placed in an extraction tank, and 10 kg of 95% ethanol aqueous solution was added for soaking and extraction for 24 h. After filtration, the residue was soaked and extracted again with 8 kg of 95% ethanol aqueous solution for 24 h, and then filtered again. The three filtrates were combined and concentrated using a 50°C rotary evaporator to obtain the total naphthoquinone extract from Xinjiang Lithospermum erythrorhizon. High performance liquid chromatography (HPLC) was used to quantitatively detect eight components: shikonin, isovalerylshikonin, acetylshikonin, deoxyshikonin, β-hydroxyisovalerylshikonin, β,β-dimethylacryloylacanine, isobutyrylshikonin, and β-acetoxyisovalerylacanine. The total naphthoquinone yield was calculated to be 2.86%.

[0053] Test Example 1: Evaluation of Anti-inflammatory Effect

[0054] Human immortalized keratinocytes (HaCaT) in logarithmic growth phase were seeded at 30,000 cells / well in 96-well plates. After cell adhesion, the BC group was treated with DMEM medium, while the sample groups were treated with different DMEM-prepared samples for 24 hours. Cells were then washed with PBS, and 200 μL of 0.5 mg / mL MTT solution was added to each well. After incubation at 37°C for 4 hours, the supernatant was removed, and 100 μL of DMSO was added to each well to dissolve the crystals. 100 μL of DMSO was used as a control group. The OD value at 570 nm was measured using a microplate reader. The relative cell viability for Examples 1, 2, 3, and 4 is as follows: Figures 1-4 As shown. From Figures 1-4 It can be seen that Examples 1-4 showed no significant cytotoxicity at concentrations of 0.78 μg / mL and below.

[0055] Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were digested, resuspended, and seeded into 24-well cell culture plates. DMEM medium was added for culture, with three replicates per group. The cells were cultured at 37°C in a 5% CO2 environment. When the cells reached approximately 50% confluence, 5 μg / mL LPS was added to the NC group, and 5 μg / mL LPS and samples from each example were added to each experimental group. The naphthoquinone content in each example sample was adjusted to 0.78 μg / mL. The BC group served as a blank control group. After culturing for another 24 hours, the supernatant was collected, and the TNF-α level in each group was detected by ELISA.

[0056] Table 1

[0057]

[0058] As shown in Table 1, the naphthoquinone extracts from lithospermum erythrorhizon obtained by different extraction processes can significantly inhibit LPS-induced TNF-α secretion and all have good anti-inflammatory effects. Moreover, the anti-inflammatory effect of the naphthoquinone extract in the examples is significantly better than that of the comparative examples, indicating that the anti-inflammatory effects of naphthoquinone extracts obtained by different extraction methods are also different due to the different composition and distribution of naphthoquinone substances.

[0059] Test Example 2: Evaluation of Antipruritic Effect

[0060] Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were digested, resuspended, and seeded into 24-well cell culture plates. DMEM medium was added for culture, with three replicates per group. Cells were cultured at 37°C in a 5% CO2 environment. When the cells reached approximately 50% confluence, the NC group was treated with 20 ng / mL TNF-α and 20 ng / mL IFN-γ, while other experimental groups were treated with 20 ng / mL TNF-α, 20 ng / mL IFN-γ, and samples from each example. The naphthoquinone content in each example sample was adjusted to 0.78 μg / mL. The BC group served as a blank control group. After 24 hours of culture, the supernatant was collected, and the TSLP (thymic stromal lymphopoietin) level in each group was detected by ELISA.

[0061] Table 2

[0062]

[0063] As can be seen from Table 2, all samples in the experimental group were able to significantly inhibit the secretion of TSLP. The inhibitory effect of the naphthoquinone extract in the examples was significantly better than that of the comparative example, indicating that the naphthoquinone extracts obtained by different extraction methods have different antipruritic effects due to the different composition and distribution of naphthoquinone substances.

[0064] Test Example 3: Evaluation of Repair Effect

[0065] Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were harvested, digested, resuspended, and seeded into 24-well cell culture plates. DMEM medium was added, and each group was cultured in triplicate at 37°C with 5% CO2. When the cells reached approximately 50% confluence, samples from each example were added. The naphthoquinone content in each sample was adjusted to 0.78 μg / mL. Group BC served as a blank control. After 3 days of further culture, the supernatant was discarded, and the cells were fixed. The relative fluorescence intensity of FLG (filaggrin) in each group was detected by immunofluorescence antibody incubation and fluorescence microscopy. The immunofluorescence results are shown below. Figure 5 The statistical results of relative fluorescence intensity (%) are shown in Table 3.

[0066] Table 3

[0067]

[0068] As can be seen from Table 3, naphthoquinone extracts obtained by different extraction processes all significantly promoted the expression of FLG in keratinocytes. The naphthoquinone extract from the example was significantly better than that from the comparative example, indicating that the naphthoquinone extracts obtained by different extraction methods have different repair effects due to the different composition and distribution of naphthoquinone substances.

[0069] Test Example 4: Evaluation of efficacy against atopic dermatitis

[0070] ICR mice were randomly divided into three groups of 12 mice each, based on body weight: a normal control group (BC group), a model group (NC group), and sample groups (Examples 1-4, Comparative Examples 1-2). One day before modeling, hair was removed from a 2×2 cm area of ​​the abdomen of each mouse using depilatory cream. On the day of modeling, mice in the model group and sample groups had 100 μL of a 4:1 volume ratio solution of 1% DNFB (2,4-dinitrofluorobenzene) in acetone and olive oil evenly applied to the bare skin of the abdomen to sensitize and challenge the mice, thus establishing a mouse atopic dermatitis model. This application was repeated for two consecutive days, and on the fifth day, 20 μL of the 1% DNFB acetone and olive oil solution was evenly applied to the inner and outer ear surfaces of both ears for challenge. In the normal group, acetone-olive oil (4:1 volume ratio) solution was evenly applied to the abdomen and ears of mice. Drug administration began 12 hours after challenge. For each sample group, 20 μL of the sample solution was applied evenly to the inner and outer surfaces of both ears using a cotton swab, once for each ear, three times a day for two consecutive days. The normal and model groups received the same volume of GTCC, three times a day for two consecutive days, with each application involving repeated wiping of the inner and outer ear surfaces with a cotton swab for approximately 5 seconds. All sample groups contained a total naphthoquinone content of 500 μg / mL.

[0071] The thickness of both ears of mice was measured 48 hours after stimulation, and the average value of both ears was taken.

[0072] Two days after administration, mouse serum was collected, and the content of the inflammatory factor IL-4 in mice was detected using an ELISA kit.

[0073] Table 4

[0074]

[0075] Table 5

[0076]

[0077] As can be seen from Tables 4 and 5, the extracts of Examples 1-4 showed better improvement effects on atopic dermatitis than those of Comparative Examples 1-2. This indicates that the naphthoquinone extracts obtained by different extraction methods have different effects on improving atopic dermatitis due to the different composition and distribution of naphthoquinone substances.

[0078] Test Example 5: Skin Irritation Test

[0079] Healthy adult New Zealand rabbits, half male and half female, were divided into an intact skin group and a damaged skin group, with four rabbits in each group. Twenty-four hours before the experiment, the rabbits underwent hair removal on both sides of their backs, covering an area of ​​approximately 3cm x 4cm. In the damaged skin group, "#"-shaped incisions were made on the left and right sides of the shaved area using a sterilized needle, allowing only slight bleeding, serving as a self-control. In the sample group, 0.5mL of the sample solution (total naphthoquinone content 500 μg / mL) was applied to the left shaved area of ​​the rabbits, while GTCC was applied to the right shaved area as a control. After applying the medication, the treated area was covered with double-layered gauze and secured with breathable adhesive tape. In the intact skin group, only hair was shaved and the medication was applied, without scratching the skin. The medication was administered continuously for 7 days, 4 hours each time. After each application, the area was promptly washed with water to ensure complete removal of the medication. Simultaneously, 1 hour after each removal of the medication and before the next application, the presence and recovery time of edema, erythema, pigmentation, skin roughness, and bleeding points at the application site were observed and recorded. Within 72 hours after the last removal of the drug, observe and record the occurrence of edema and erythema at the application site in the hair removal area under natural light. According to the scoring criteria in Tables 6 and 7, score the two indicators of edema and erythema, calculate the average score of the stimulation response of each group of animals, and evaluate the degree of drug stimulation response based on this score.

[0080] Table 6 Skin Irritation Reaction Scoring Criteria

[0081]

[0082] Table 7 Skin Irritation Intensity Scoring Criteria

[0083]

[0084] Table 8 Skin irritation test in New Zealand rabbits

[0085]

[0086] As can be seen from Table 8, Examples 1-4 showed no significant skin toxicity within the range of 500 μg / mL naphthoquinone.

[0087] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for extracting total naphthoquinones from Arnebia euchroma (Jacks.) Johnst., characterized in that, Includes the following steps: S1. Pretreatment: The raw material of Xinjiang purple gromwell is crushed and then subjected to high-pressure steam explosion treatment to obtain the pretreated material. S2. Supercritical CO2 extraction: The pretreated material is subjected to supercritical CO2 extraction, and an entrainer is added during the extraction process. The entrainer is a combination of ethanol and caprylic / capric triglyceride. The extract is collected to obtain crude extract. S3. Molecular distillation: The crude extract is subjected to molecular distillation to collect the heavy components and obtain the total naphthoquinone solution of Lithospermum erythrorhizon from Xinjiang. Wherein, when the entrainer is a combination of ethanol and caprylic / capric triglycerides, step S2 is a stepwise extraction, including: First extraction: Ethanol is added as an entrainer for extraction; Second extraction: Ethanol is added as an entrainer to the material after the first extraction for further extraction; Third extraction: Caprylic / capric triglycerides are added as an entrainer to the material after the second extraction for further extraction; the mass ratio of ethanol added in the first extraction to the pretreated material is (0.3-1):1; the mass ratio of the total mass of ethanol added in the second extraction to the material after the first extraction is (1-1.5):1; the mass ratio of the total mass of caprylic / capric triglycerides added in the third extraction to the material after the second extraction is (1-1.5):

1.

2. The extraction method according to claim 1, characterized in that, In step S1, the pressure of the high-pressure steam explosion treatment is 0.8-1.0 MPa, and the pressure holding time is 20-30 seconds.

3. The extraction method according to claim 1 or 2, characterized in that, In step S2, the conditions for supercritical CO2 extraction include: an extraction pressure of 20-30 MPa and an extraction temperature of 40-50 ℃.

4. The extraction method according to claim 1 or 2, characterized in that, In step S2, the supercritical CO2 extraction is performed with a CO2 flow rate of 40-200 L / h.

5. The extraction method according to claim 1 or 2, characterized in that, In step S3, the conditions for molecular distillation include: evaporator temperature of 75-85℃ and system vacuum of less than 50 Pa.

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