A kind of component-reconstructed perilla leaf volatile oil and its preparation method and application

Abstract

The present invention relates to a component-reconstructed perilla leaf volatile oil, a preparation method, and an application thereof, and relates to the field of pharmaceutical technology. The component-reconstructed perilla leaf volatile oil comprises a perilla ketone reduction product, wherein the perilla ketone reduction product is 1-(3-furyl)-4-methyl-1-pentanol, and the perilla ketone reduction product is represented by Formula I. The component-reconstructed perilla leaf volatile oil has significantly reduced acute toxicity, increased safety, significantly reduced skin irritation, and has a good penetration-promoting effect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a component-reconstructed perilla leaf volatile oil, a preparation method and an application thereof. Background Art

[0002] Perilla (Perilla frutescens L.) is a traditional crop in China, South Korea, Japan, India, Nepal, and Thailand. It belongs to the species Perilla in the Lamiaceae family and is an annual herb. As a dual-purpose medicinal and edible plant, Perilla has been cultivated in my country for over 2,000 years. Currently, numerous researchers both domestically and internationally are conducting in-depth research on its components and pharmacological activities. Perilla has broad development value and application prospects in the food, pharmaceutical, and health product sectors. Japanese researchers have classified Perilla plants into PA, PK, EK, PL, PP, and C6 types based on the chemical composition of their volatile oils. These types, whose main components are perilla aldehyde, perilla ketone, odoriferone, perilla ene, phenylpropanoids, and trans-citral, respectively, are the main components. Modern pharmaceutical research on Perilla plants in my country began relatively late, but my country's vast territory provides a wide variety of Perilla varieties for research, giving it a resource advantage over other countries.

[0003] Volatile oil is the primary component of perilla leaves. This oil, which includes aromatic, terpenoid, and aliphatic compounds, possesses significant medicinal value. Currently, extraction techniques for perilla volatile oil are undergoing continuous optimization and improvement. In addition to traditional steam distillation and organic solvent extraction, newer and more efficient methods, such as optimized ultrasonic-assisted organic solvent extraction, liquid-liquid extraction, and supercritical CO2 extraction, are also being employed.

[0004] As research progressed, it was discovered that perilla contains active substances such as perilla ketone and rosmarinic acid. Through continuous extraction and experimentation, it was determined that the main active ingredient in perilla is its volatile oil, which is also the source of its aroma. This volatile oil is primarily found in perilla leaves and includes aromatic, terpenoid, and aliphatic compounds, possessing significant medicinal value. Compared to perilla leaves, perilla volatile oil has more concentrated and purified active ingredients, making it even more valuable when formulated into pharmaceuticals and health supplements. Currently, the pharmacological effects of perilla leaf volatile oil include antibacterial, antioxidant, anti-inflammatory, anti-allergic, depression relief, gastrointestinal motility promotion, diuresis, and blood pressure reduction.

[0005] However, reports of toxicity in essential oils from the leaves of Lamiaceae plants are increasing. For example, studies have shown that excessive doses of peppermint oil can cause acute liver damage in rats. Researchers have also shown toxicity in mice using essential oil from Hubei perilla leaves. PK-type perilla contains large amounts of perilla ketones, which are toxic, irritating, and allergenic. Perilla leaves also contain large amounts of oxalic acid, which, when consumed frequently, can accumulate in the body. Over time, this can damage the nervous and digestive systems, even affecting hematopoiesis and posing a serious threat to cardiovascular and cerebrovascular health, and even lead to death. Previous research on perilla essential oil has primarily focused on its pharmacological effects, overlooking the potential harm of ketone compounds in excessive amounts. However, practical applications should not blindly focus on the pharmacological effects of perilla essential oil while ignoring its toxicity. Therefore, perilla, a vitally important medicinal material, has been underutilized. A thorough and systematic investigation of the toxic effects of perilla essential oil is crucial for its practical application.

[0006] Transdermal drug delivery systems involve delivering drugs through the skin at a constant rate into the bloodstream, offering an alternative to oral and intravenous administration. The Shengji Zonglu contains a simple description of the therapeutic mechanism of Chinese herbal patch application. This has led to the rapid development of acupoint application, highlighting its advantages in treating various internal medicine conditions. In 1979, the United States approved a three-day scopolamine patch, the first transdermal system for systemic drug delivery, for the treatment of motion sickness. Currently, drugs such as estradiol, fentanyl, and lidocaine have transdermal drug delivery systems. Unlike traditional drug delivery methods, these systems deliver drugs or macromolecules painlessly from the skin into the bloodstream at a constant rate. In contrast, they prevent the first-pass effect in the liver, improve therapeutic efficacy, maintain stable plasma levels, overcome the drawbacks of traditional drug delivery, and offer broad development prospects for transdermal drug delivery systems.

[0007] There are three main ways for drugs to enter the systemic circulation through the skin: (1) The transcellular pathway, in which drugs pass through keratinocytes to the active epidermis and are then absorbed by the capillaries in the dermis and enter the systemic circulation. This process requires multiple water / lipid distribution processes, so the transcellular pathway of drugs only accounts for a very small part of the drug's transdermal absorption. (2) The transcellular interstitial pathway, in which drugs pass through the lipid bilayer between keratinocytes. This pathway is the main route for drug transdermal absorption. (3) The transdermal appendage (hair follicles, sweat glands, and sebaceous glands) pathway, in which when the drug begins to penetrate, it is first absorbed through the skin appendage pathway until it reaches a steady state. This pathway is the main route for the transdermal absorption of ionic and polar macromolecular drugs.

[0008] Drugs can only pass through the stratum corneum through these three pathways, and only certain drugs that meet certain conditions can enter the systemic circulation through these pathways, which limits the penetration of hydrophilic and biomacromolecule drugs. In order to overcome the barrier effect of the stratum corneum, a variety of methods have been developed in recent years to promote the transdermal absorption of drugs, including physical methods such as iontophoresis, electroporation, microneedles, etc.; pharmaceutical methods such as microemulsions, liposomes, nanoemulsions, etc.; and the combined application of biochemical methods and permeation enhancement methods. Among them, permeation enhancers (PE) are the best choice for increasing drug transdermal absorption due to their low cost, flexible formulation design, and convenient process. The mechanism of action of conventional chemical permeation enhancers (CPEs) is to change the properties of the skin, disrupt the arrangement of lipids, form gaps in tight connections, and thus increase the water content of the stratum corneum. Its permeation enhancement process is non-specific, and high concentrations or long-term use can cause side effects such as skin irritation and allergic reactions. The ideal PE should have the following characteristics: (1) low toxicity and no irritation. (2) fast onset and easy skin absorption. (3) It has a unidirectional effect, only promoting the permeation of therapeutic drugs through the skin without affecting the in vivo processes of endogenous substances. (4) It has no interaction with excipients and drugs.

[0009] In recent years, with the continuous deepening of research, researchers have discovered that Chinese herbal volatile oils possess excellent transdermal penetration-enhancing activity and can be used in topical preparations, acting as a "drug-adjuvant-in-one" approach. To date, approximately 34 pungent Chinese herbal volatile oils have been verified to exhibit transdermal penetration-enhancing activity. Domestic researchers have classified and analyzed the toxicity and permeation-enhancing properties of volatile oils based on the nature, flavor, and meridians of Chinese herbs, finding that most herbs with permeation-enhancing activity are attributed to the spleen, liver, and lung meridians. Furthermore, while volatile oils from heat-releasing Chinese herbs have superior permeation-enhancing abilities compared to warm-releasing Chinese herbs, they are also more toxic. Compared to traditional PE, Chinese herbal volatile oils not only enhance transdermal absorption and cause less skin irritation, but also possess certain therapeutic benefits and can synergize with transdermally absorbed drugs. Currently available Chinese herbal volatile oil perdermal absorption enhancers are primarily derived from pungent Chinese herbs. Research has shown that menthol, the most abundant compound in peppermint oil, enhances the permeation of ligustrazine, ferulic acid, ephedrine, and zidovudine. Borneol, commonly used in topical skin preparations, has been shown in numerous studies to enhance the transdermal absorption of drugs such as gastrodin, osthole, ursolic acid, and tetrahydropalmatine. The classic theory that "pungent properties enter the lungs, and the lungs are involved in the body and skin" suggests that pungent volatile oils from traditional Chinese medicines may increase skin permeability.

[0010] While current research on the main components of perilla leaves has mature methods for extracting volatile oils, flavonoids, and phenolic compounds, research on the composition of the volatile oil is still lacking, its safety remains unclear, and further research is needed on its skin irritation and acute toxicity. Therefore, there is a need for a perilla leaf volatile oil that combines low skin irritation and acute toxicity with a high safety profile. Summary of the Invention

[0011] In response to the above technical problems, the present invention provides a component-reconstructed perilla leaf volatile oil, which has significantly reduced acute toxicity, increased safety, significantly reduced skin irritation, and good penetration-promoting effect.

[0012] The present invention provides a reconstructed perilla leaf volatile oil, comprising a perilla ketone reduction product, wherein the perilla ketone reduction product is 1-(3-furyl)-4-methyl-1-pentanol, and the perilla ketone reduction product is shown in the following formula I:

[0013]

[0014] Transdermal drug delivery systems, due to their unique advantages, have become a key focus and research area for formulation development. Their effectiveness depends on sufficient drug penetration through the skin to achieve therapeutic levels. However, the stratum corneum of the skin affects drug absorption, so percutaneous absorption enhancers (i.e., penetration enhancers) are often used to increase drug absorption. Perilla leaf, a pungent and warming agent, enters the lung and spleen meridians, showing potential as a percutaneous penetration enhancer for transdermal drug delivery systems. However, its application is limited by toxicity and other issues. Therefore, the inventors proposed to reconstruct the components of perilla volatile oil to prepare a perilla leaf volatile oil with significantly reduced acute toxicity, increased safety, and significantly reduced skin irritation, and evaluated the safety of the reconstructed perilla leaf volatile oil through skin irritation experiments and mouse acute toxicity experiments; then, using isolated rat skin as a transdermal barrier, the inventors explored the promoting effect of the reconstructed perilla leaf volatile oil on the in vitro transdermal permeation behavior of model drugs (such as rutin, luteolin, ferulic acid, and ligustrazine), and verified that the perilla leaf volatile oil reconstructed with the above components has a good penetration-promoting effect and can be used as a penetration enhancer, and provided a theoretical basis and data support for the application of the reconstructed perilla leaf volatile oil in transdermal drug delivery systems.

[0015] In one embodiment, the perilla leaf volatile oil further comprises at least one of 1-octen-3-ol, linalool, caryophyllene, or (Z,E)-α-farnesene.

[0016] In one embodiment, the component-reconstructed perilla leaf volatile oil is obtained by reducing perilla leaf volatile oil with sodium borohydride.

[0017] The present invention also provides a preparation method of the reconstructed perilla leaf volatile oil, comprising the following steps: mixing the perilla leaf volatile oil with an organic solvent, adding sodium borohydride, stirring until no bubbles are generated, stirring, adding a terminator, standing, taking the filtrate, extracting, evaporating, dehydrating, extracting, evaporating, and obtaining the reconstructed perilla leaf volatile oil.

[0018] In one embodiment, the molar ratio of the sodium borohydride to the perilla leaf volatile oil is (0.5-2):2.

[0019] In one embodiment, the organic solvent includes methanol, and the terminator includes a saturated aqueous solution of ammonium chloride;

[0020] In the preparation method, after stirring until no bubbles are generated, stirring is continued for 1.8-2.2 hours, and a terminator is added.

[0021] The present invention also provides the use of the reconstructed perilla leaf volatile oil in the preparation of a penetration enhancer and / or a medicine.

[0022] The present invention also provides a penetration enhancer, comprising the reconstructed perilla leaf volatile oil.

[0023] The present invention also provides a medicine comprising the perilla leaf volatile oil reconstructed with the components.

[0024] In one embodiment, the drug is in the form of a transdermal preparation.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a component-reconstructed perilla leaf volatile oil, a preparation method and an application thereof. The component-reconstructed perilla leaf volatile oil has significantly reduced acute toxicity, increased safety, significantly reduced skin irritation, and has a good penetration-promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the reconstructed perillone in Example 1 1 H NMR spectrum;

[0028] Figure 2 is the reconstructed perillone in Example 1 13 C NMR spectrum;

[0029] Figure 3 This is the HMBC spectrum of perillone after reconstruction in Example 1;

[0030] Figure 4 This is a schematic diagram of HMBC of perillone after reconstruction in Example 1;

[0031] Figure 5 This is the rabbit skin red pigment test result diagram in Example 2;

[0032] Figure 6 This is a graph showing the results of the rabbit skin water loss test in Example 2;

[0033] Figure 7This is a diagram showing the results of rabbit skin tissue pathology observation in Example 2;

[0034] Figure 8 The results of ALT content in mouse serum in Example 3 are shown, where ##: p < 0.01, vs. blank; **: p < 0.01, vs. before reconstitution;

[0035] Figure 9 The results of AST content in mouse serum in Example 3 are shown, where ##: p < 0.01, vs. blank; #: p < 0.05, vs. blank; **: p < 0.01, vs. before reconstruction;

[0036] Figure 10 This is the result graph of AKP content in mouse serum in Example 3;

[0037] Figure 11 The results of BUN content in mouse serum in Example 3 are shown, where ##: p < 0.01, vs. blank; **: p < 0.01, vs. before reconstitution;

[0038] Figure 12 The results of CRE content in mouse serum in Example 3 are shown, where ##: p < 0.01, vs. blank; **: p < 0.01, vs. before reconstruction;

[0039] Figure 13 The results of the SOD content in mouse serum in Example 3 are shown, where ##: p < 0.01, vs. blank, **: p < 0.01, vs. before reconstitution;

[0040] Figure 14 The results of MDA content in mouse serum in Example 3 are shown, where ##: p < 0.01, vs. blank; **: p < 0.01, vs. before reconstitution;

[0041] Figure 15 This is a graph showing the in vitro percutaneous enhancement of rutin by the permeation enhancer in Example 4;

[0042] Figure 16 This is the result of the in vitro percutaneous enhancement of luteolin by the permeation enhancer in Example 4;

[0043] Figure 17 This is a graph showing the in vitro percutaneous enhancement of ferulic acid by the permeation enhancer in Example 4;

[0044] Figure 18 This is the result of the in vitro percutaneous enhancement of ligustrazine by the permeation enhancer in Example 4. DETAILED DESCRIPTION

[0045] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0047] source:

[0048] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; and the experimental methods, unless otherwise specified, are all conventional experimental methods in the art.

[0049] Example 1

[0050] Reconstruction and identification of the components of the volatile oil from Perilla frutescens leaves.

[0051] 1. Experimental medicinal materials and reagents.

[0052] Reagents: Perilla leaves (collected in Jilin City, Jilin Province, identified as Perilla frutescens of the family Labiatae by the Pharmacognosy Teaching and Research Office of the School of Pharmacy of Beihua University), anhydrous ether (Chongqing Chuandong Chemical Co., Ltd.), anhydrous sodium sulfate (Chemical Plant No. 4, Chaoyang District, Beijing), methanol (analytical grade, Liaoning Quanrui Reagent Co., Ltd.), sodium borohydride (Dingguo Changsheng Biotechnology Co., Ltd.).

[0053] Experimental instruments: electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), gas chromatography-mass spectrometry (Ang-ilent Technology Co., Ltd., USA), electronic temperature-controlled heating mantle (Tianjin Test Instrument Co., Ltd.), volatile oil extraction device (Tianjin Test Instrument Co., Ltd.).

[0054] 2. Experimental methods

[0055] 1. Extraction of volatile oil from perilla leaves.

[0056] (1) Soak: Select fresh perilla leaves and cut into pieces about 1cm 2 , placed in a 5000mL round-bottom flask, add distilled water at a material-liquid ratio of 1:10, seal with plastic wrap and place in a cool place to soak for 3h.

[0057] (2) Extraction: Soak thoroughly for 3 hours, place in a volatile oil extraction device, and use about 1 mL of ether as the extractant. Heat to a slight boil, maintaining a condensation reflux equilibrium rate of 80 drops per minute. Continue heating for 3 hours and cool to room temperature to complete the extraction of volatile oil. Repeat this step until all fresh perilla leaves of the same batch are fully extracted. Then accurately weigh and calculate the extraction rate of volatile oil from the perilla leaves.

[0058] Perilla volatile oil extraction rate (%) = mass of extracted perilla volatile oil (g) / mass of perilla leaves (g) × 100%.

[0059] 2. Extraction of volatile oil from perilla leaves.

[0060] Collect ether as the extraction agent, and collect the oil in the separatory funnel. Rinse the separatory funnel three times with the solvent so that the perilla volatile oil is completely collected. Let it stand for stratification, separate the organic layer and dehydrate it with anhydrous sodium sulfate. Volatilize the ether at room temperature, retain the upper solution (perilla volatile oil), combine the perilla leaf volatile oil, weigh it, seal it, and store it in a refrigerator at 4°C for later use.

[0061] 3. Reconstruction of Perilla leaf volatile oil.

[0062] Get 3g Perilla leaf volatile oil, add 15mL methanol (analytical pure) and stir evenly, make volatile oil be distributed in methanol completely.For ensuring experimental safety, beaker is placed in a large amount of ice water and guarantees temperature during reaction. Take 342mg sodium borohydride, repeatedly add in volatile oil very slightly and stir rapidly, each reaction adds again after no longer producing bubbling, no longer produces bubble after adding to the last time, continue to stir 2h and make reaction fully complete, add saturated ammonium chloride aqueous solution to quench reaction. Leave standstill, produce stratification, centrifugation insolubles, add a small amount of ether in solution and extract, evaporate solvent, add anhydrous sodium sulfate and dehydrate.By the product after dehydration with normal hexane extraction, evaporate solvent, seal and keep in dark.

[0063] In this reconstruction step, since perillone accounts for over 95% of the essential oil, the approximate molar amount of the essential oil was calculated based on the molecular weight of perillone. The essential oil and sodium borohydride were added in a molar ratio of 2:1. Therefore, the molar ratio of 3 g of perilla leaf essential oil to 342 mg of sodium borohydride was approximately 2:1.

[0064] 4. GC-MS conditions.

[0065] GC conditions: TG-5M (30m×0.25mm×0.25μm) capillary column. Temperature program: start at 60℃, then increase the temperature at 10℃·min -1The temperature was raised to 280°C and maintained for 5 min. The injection port temperature was 280°C. The column flow rate was 1.61 mL / min. The injection mode was split injection. The split ratio was 10:1. The split time was 0.80 min. The pressure was 100.00 kPa.

[0066] MS conditions: ion source: EI source; voltage: 70 eV; ion source temperature: 250°C; solution delay: 3 min; scanning range: 345-550 m / z.

[0067] 3. Experimental results.

[0068] 1. Extraction and reconstruction of volatile oil from Perilla leaves.

[0069] As shown in the table below, the experiment was divided into three steps to extract perilla volatile oil. A total of 14.5285 kg of fresh perilla leaves were used, and a total mass of 11.71 g of perilla leaf volatile oil was extracted. The extraction rate of perilla leaf volatile oil was 0.08%.

[0070] Table 1 Extraction of volatile oil from perilla leaves

[0071]

[0072] 2. GC-MS analysis results of Perilla leaf volatile oil before reconstruction.

[0073] The main component of the volatile oil from perilla leaves is perilla ketone, which accounts for 95.9% calculated by peak area normalization method.

[0074] The GC-MS identification results of the chemical components of Perilla leaf volatile oil before reconstruction are shown in the table below.

[0075] Table 2 GC-MS identification of chemical components of Perilla frutescens leaf volatile oil before reconstitution

[0076]

[0077]

[0078] 3. GC-MS analysis results of the reconstructed components of perilla leaf volatile oil.

[0079] After reconstitution, peak area normalization was used to calculate the proportion of perillone reduction products at 96.71%. GC-MS analysis revealed that a large number of components in the original volatile oil had been removed during the reaction and subsequent processing. Only four relatively abundant components in the original volatile oil, besides perillone, remained, but their concentrations were lower in the reconstituted volatile oil.

[0080] The NIST database failed to identify the structure of the perillone reduction product. Therefore, further structural analysis and identification of the perillone reduction product was performed using nuclear magnetic resonance (NMR). NMR conditions were: 500 MHz for H-spectroscopy and 298 K for C-spectroscopy; 125 MHz for C-spectroscopy and 298 K for C-spectroscopy.

[0081] Table 3 GC-MS identification of chemical components of perilla leaf volatile oil after reconstitution

[0082]

[0083] The structure of the perilla ketone reduction product in the above table is shown in Formula I below after identification by nuclear magnetic resonance analysis.

[0084]

[0085] Reconstructed perilla ketone (Formula I) 1 H and 13 C NMR spectrum (such as Figure 1 、 Figure 2 The results are shown in the table below (the solvent is CDCl3).

[0086] [δ H 7.34 (s, 2H)] is the chemical shift of hydrogen on C-2a and C-5a of the furan ring, and the chemical shift of C-5a [δ C 143.12], the chemical shift of C-2a [δ C 138.93].

[0087] [δ H 6.37 (s, 1H)] is the chemical shift of hydrogen on C-4a of the furan ring. The chemical shift of C-4a [δ C 108.41].

[0088] [δ H 4.56 (t, J = 6.7 Hz, 1H)] is the chemical shift of hydrogen attached to the hydroxyl carbon, and the chemical shift of C-1 [δ C 66.96].

[0089] [δ H 1.70(m,2H)] is the chemical shift of the two hydrogen atoms connected to C-3. C 34.82].

[0090] [δ H 1.52 (m, 1H)] is the chemical shift of hydrogen connected to the methine, and the chemical shift of C-4 [δ C 27.86].

[0091] [δ H 1.27 (m, 1H) and δ H1.13(m,1H)] is the chemical shift of the two hydrogen atoms connected to the methylene C-2. Since C-2 is connected to the chiral carbon C-1, the two hydrogen atoms on it have different chemical shifts. C 35.55].

[0092] [δ H 0.86 (d, J = 6.7 Hz, 6H)] is the chemical shift of the six hydrogen atoms on the two methyl groups, and the chemical shifts of C-5 and C-6 [δ C 22.44].

[0093] C-3a on the furan ring is a quaternary carbon, and the chemical shift [δ C 129.17].

[0094] Table 4 1 H(500MHz), 13 C (125 MHz) NMR data (J in Hz)

[0095]

[0096] In the HMBC spectrum (such as Figure 3 、 Figure 4 As shown), the hydrogen on C-5a in the furan ring shows the same C 108.41(C-4a), δ C 138.93(C-2a), δ C 129.17 (C-3a). The hydrogen on the hydroxyl carbon C-1 shows a correlation with δ C 138.93(C-2a), δ C 129.179(C-3a), δ C 108.41 (C-4a) and δ C 35.55(C-2), δ C 34.82 (C-3) correlation. The hydrogen on the C-2 methylene group shows a correlation with δ C 129.179(C-3a), δ C 66.96(C-1), δ C 34.82(C-3), δ C 27.86 (C-4). The hydrogen on the C-3 methylene group shows a correlation with δ C 66.96(C-1), δ C 35.55(C-2), δ C 27.86(C-4)δ C 22.44 (C-6, C-5). The hydrogen on the C-4 methyl group shows a correlation with δ C 35.55(C-2), δC 34.82(C-3), δ C The correlation of 22.44 (C-6, C-5).

[0097] 4. Result analysis.

[0098] Studies have shown that overdose of peppermint oil, a pungent and warming herb, causes acute liver damage in rats. Studies have also shown that perilla leaf essential oil, also a pungent and warm herb, contains ketone compounds, making it somewhat toxic. Based on the results of a gastrointestinal odor analysis, the oil was found to contain the following ketone compounds: perilla ketone, perilla ketone, and isoperilla ketone. Ketone compounds are active and easily react with other substances, so the content of ketone compounds in perilla ketone essential oil should be carefully considered when using it to prevent toxicity. A comparison of the chemical composition of the perilla leaf essential oil before and after reconstitution indicates that the reconstitution successfully reduced the ketone carbonyl group in the perilla ketone structure to an alcoholic hydroxyl group, while retaining some of the terpenes and unsaturated fatty alcohols present in the original oil. The combination of these components can enhance drug transdermal absorption, further enhancing the perilla leaf essential oil's permeation-enhancing properties.

[0099] In this example, sodium borohydride was used as a reducing agent to reduce ketone compounds in perilla frutescens volatile oil. GC-MS confirmed the disappearance of the ketone compounds perilla frutescens, perilla frutescens, and isoperilla frutescens in the reconstituted perilla frutescens volatile oil. This demonstrates that sodium borohydride effectively reconstitutes perilla frutescens volatile oil, achieving the intended experimental objectives. Furthermore, the reconstituted perilla frutescens volatile oil contains an additional hydroxyl compound, 1-(3-furyl)-4-methyl-1-pentanol, the aforementioned perilla frutescens volatile oil reduction product.

[0100] Example 2

[0101] Skin irritation test of Perilla frutescens leaf essential oil after component reconstitution.

[0102] 1. Experimental medicinal materials and reagents.

[0103] Reagents: sodium dodecyl sulfate (SDS, GENVIEW), azone (Tianjin Guangfu Fine Chemical Research Institute), liquid paraffin (Shanghai Hualing Rehabilitation Equipment Factory), urethane (Sinopharm Chemical Reagent Co., Ltd.), and 0.9% normal saline (Kunming Nanjiang Pharmaceutical Co., Ltd.).

[0104] Experimental instruments: electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), MPA580 skin elasticity tester (Shanghai Kaikai Technology Trading Co., Ltd.), Acradia H&C paraffin embedding machine and cold stage (Leica), RM2245 semi-automatic rotary microtome (Leica), BA400 microscope (McOdy Industrial Group Co., Ltd.).

[0105] 2. Experimental animals

[0106] SPF-grade male rabbits weighing 2.3-2.5 kg were provided by Changchun Yisi Laboratory Animal Technology Co., Ltd. Rabbits were housed in separate cages in an air-conditioned laboratory at a room temperature of 22-25°C and a relative humidity of 40%-60%, fed with standard rabbit chow, and had free access to water.

[0107] 3. Experimental methods.

[0108] 1. Skin irritation evaluation.

[0109] The experiment used the self-control method to determine the irritation degree of the reconstructed perilla leaf volatile oil on rabbit skin.

[0110] Five components were tested before and after reconstitution: perilla leaf essential oil, azone, 10% SDS, and a blank. A solvent group (normal saline) served as a blank control, and the active drug SDS served as a positive control. Rabbits were weighed and anesthetized with 5 mL / kg urethane solution. Twenty-four hours before the experiment, the rabbits' abdomens were shaved, and five 1 cm × 1 cm areas of intact skin were carved out: four for treatment and one for the blank. The skin was gently wiped with normal saline, and basal water loss and red pigmentation were measured using a skin tester. Perilla leaf essential oil was diluted 1:9 with liquid paraffin before and after reconstitution, and then 20 μL of azone and 10% SDS were applied to each area. The area was covered with double gauze and secured with medical anti-allergic tape. Erythema and edema at the application site were visually observed and recorded at 2, 4, 6, 8, and 24 hours. Skin irritation was assessed using the Dresser method. Water loss and red pigmentation were measured at each observation time point using a skin tester. The rabbits were killed, and the reaction areas of the rabbit skin were taken and immersed in tissue fixative for preservation, followed by routine dehydration, paraffin embedding, and tissue sectioning. The tissue sections were stained with HE and observed under a 400x microscope.

[0111] The skin reaction score was calculated according to Table 5, and the average score of the test animals was used for comprehensive evaluation. The skin irritation intensity was determined according to Table 6.

[0112] Table 5 Skin irritation evaluation standards

[0113]

[0114] Table 6 Skin irritation intensity classification standard

[0115]

[0116] 4. Experimental results.

[0117] 1. Rabbit skin red pigment test.

[0118] The red pigment data of rabbit skin measured using a skin tester are shown in the following table.

[0119] Table 7 Red pigment value of rabbit skin at the drug administration site

[0120]

[0121]

[0122] like Figure 5 As shown in the figure, the red pigment value of the volatile oil of Perilla leaves after component reconstruction is lower than that before component reconstruction.

[0123] 2. Rabbit skin water loss test.

[0124] The water loss data of rabbit skin measured by skin tester are shown in the following table. Figure 6 As shown in the figure, the water loss value of the volatile oil of Perilla leaves after component reconstruction is lower than that of the volatile oil of Perilla leaves before component reconstruction.

[0125] Table 8 Skin water loss index at the drug administration site in rabbits

[0126]

[0127] 3. Delay method to evaluate skin irritation.

[0128] In accordance with the "Technical Specifications for Chemical Toxicity Identification," visual observation of rabbit skin erythema and edema was used to evaluate the reconstituted perilla leaf essential oil. As shown in the table below, the reconstituted perilla leaf essential oil exhibited mild skin irritation, significantly less than the pre-reconstitution level.

[0129] Table 9 Evaluation of rabbit skin irritation

[0130]

[0131]

[0132] 4. Pathological observation of rabbit skin tissue.

[0133] Depend on Figure 7 As can be seen, the epidermis and dermis of the rabbit skin in the blank control group were well-structured, with clear demarcations and an intact basement membrane. No edema, necrosis, or shedding of epidermal cells was observed. After reconstitution, the skin in the perilla leaf essential oil area showed slight thickening. Before reconstitution, the skin in the perilla leaf essential oil area showed significant thickening and inflammatory cells. The dermis was disrupted in the azone group. In the SDS group, the epidermis was significantly thickened, and the dermis was structurally disrupted, with fractures appearing.

[0134] 5. Result analysis.

[0135] The skin tester uses optical principles to measure transepidermal water loss and red pigmentation on the skin surface. Higher values ​​indicate a higher level of red pigmentation on the skin surface and greater transepidermal water loss. Rabbits are generally believed to be more sensitive to irritants or corrosive substances than humans. Fluctuations in the blank control group values ​​may be due to physical irritation during the wiping process. Subsequently, the test sites remained generally stable at all time points, indicating the safety of the reconstituted perilla leaf essential oil.

[0136] Through observation of skin tissue sections, it was found that the changes in skin tissue caused by the reconstructed perilla leaf volatile oil were significantly smaller than those caused by the reconstructed perilla leaf volatile oil. It was judged that the irritation to the skin was reduced after the components of the perilla leaf volatile oil were reconstructed.

[0137] Example 3

[0138] Acute toxicity study of reconstituted Perilla frutescens leaf essential oil.

[0139] 1. Experimental medicinal materials and reagents.

[0140] Reagents: physiological saline (Jilin Cornell Pharmaceutical Co., Ltd.), 4% paraformaldehyde (Dingguo Changsheng Biotechnology Co., Ltd.), soybean oil (Jiusan Group Harbin Wellcome Food Co., Ltd.), anhydrous ethanol (Shenyang Huadong Reagent Factory), anhydrous ether (Shenyang Huadong Reagent Factory), anhydrous sodium sulfate (Yantai Shuangshuang Chemical Co., Ltd.), BCA protein concentration assay kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), aspartate aminotransferase (AST / GOT) test kit, alanine aminotransferase (ALT / GPT) test kit, creatinine (CRE) test kit, urea nitrogen (BUN) test kit, alkaline phosphatase (AKP) test kit, superoxide dismutase (SOD) test kit, malondialdehyde (MDA) test kit (all purchased from Nanjing Jiancheng Bioengineering Institute).

[0141] Experimental instruments: UV-2550 ultraviolet-visible spectrophotometer (Shimadzu International Trading Co., Ltd.), 5424 desktop low-temperature high-speed centrifuge (Eppendorf), Ifinite M200 microplate reader (TECAN), GZX-9140ME digital display blast drying oven (Shanghai Boxun Industrial Co., Ltd.), AL204 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.).

[0142] 2. Experimental animals

[0143] One hundred SPF-grade ICR male mice, weighing (20 ± 2) g, were provided by Changchun Yisi Laboratory Animal Technology Co., Ltd. (License Number: SCXK(Ji)-2020-0002). Five mice per group were housed in separate cages at room temperature of 22–25°C and relative humidity of 55%–65%.

[0144] 3. Experimental methods.

[0145] 1. Determination of LD 50 .

[0146] Eighty ICR mice weighing 20 ± 2 g were acclimated to their new environment for one week. Forty mice were divided into eight groups of five mice each, treated with perilla leaf essential oil before reconstitution. The reconstitution grouping was the same as the previous grouping. The 0% and 100% values ​​in the pilot study were adjusted in geometric progression. To achieve a mortality rate of at least 50% in half of the groups, the dose was increased or decreased in a geometric progression, with a ratio of 1:0.6 to 0.9 between adjacent doses. Four dose groups were established. After grouping and dose calculation, the mice were administered orally. The four groups receiving the reconstituted perilla leaf essential oil were given crude drug doses of 98.60 g / kg, 212.00 g / kg, 455.8 g / kg, and 979.97 g / kg, respectively. The four groups receiving the reconstituted perilla leaf essential oil were given crude drug doses of 456.88 g / kg, 982.29 g / kg, 2121.75 g / kg, and 4561.76 g / kg, respectively. Mice were fasted for 24 hours prior to dosing and continued to fast for 3-4 hours after dosing, with no water restriction. After fasting, they were given a normal amount of mouse chow.

[0147] Observe and record the poisoning reaction and death of mice. LD was calculated and recorded using Horn's method. 50 If a mouse dies, it is immediately dissected and its organs are observed with the naked eye to see if there are any lesions.

[0148] Calculated according to Horn's formula: LD 50 =log-1[Xm-i(∑P-0.5)]

[0149] Where i is the group interval, that is, the difference between the logarithmic doses of two adjacent groups, Xm is the logarithm of the maximum dose, and P is the mortality rate at each dose.

[0150] 2. Determination of the content of related factors in mouse serum.

[0151] Perilla leaf volatile oil was administered orally at crude drug doses of 98.60 g / kg, 212.00 g / kg, 455.8 g / kg, and 979.97 g / kg, respectively, before and after reconstitution. Blood was collected from the eyeballs of mice at the onset of acute toxicity and near-death. The mice were incubated at room temperature for 1 hour and then centrifuged at 3000 rpm at 4°C for 20 minutes. The supernatant serum was collected and stored at -80°C. Serum levels of ALT, AST, CRE, AKP, and BUN were measured using biochemical assays.

[0152] 3. Determination of the content of related factors in mouse liver tissue.

[0153] Take mouse liver, dilute to 10% saline, and centrifuge at 3000 rpm at 4°C for 10 minutes. Homogenize and aliquot for storage. Optimal sampling concentration was determined. SOD and MDA levels were determined.

[0154] 4. Statistical methods.

[0155] Statistical analysis was performed using SPSS 16.0 software, with p < 0.05 considered statistically significant. Statistical graphs were generated using GraphPad Prism 7.0.

[0156] 4. Experimental results.

[0157] 1. Physiological reaction.

[0158] After oral administration of the component-constructed perilla leaf volatile oil and soybean oil (as a blank control) to mice, the four dosage groups of 456.88g / kg, 982.29g / kg, 2121.75g / kg, and 4561.76g / kg showed that as time went on, the mice in the drug-treated groups showed varying degrees of slowed and weakened breathing, quiet inactivity, and disappearance of the righting reflex. The larger the dose, the more obvious the toxic reaction. In the dying mice, body fluid exudation could be observed at the corners of the mouth, eyes, and genitals, and severe opisthotonos could be observed before death, which lasted for more than 3-5 seconds. It was observed that compared with the blank group, the liver of the mice in the drug-treated group was darker and dark red in color.

[0159] 2. LD 50 Calculation results.

[0160] Median lethal dose (LD50) 50 ) is an important indicator commonly used in acute toxicity experiments. It refers to the dose of a toxic substance that causes half of the experimental animals of a certain weight or age to die. It is the basis for the safety evaluation of toxic substances. 50 The LD and the corresponding confidence interval are the most commonly used statistics derived from the dose-response model. 50 The larger the value, the safer the drug, that is, the lower the toxicity. The mortality of mice at different doses is shown in the table below.

[0161] Table 10 Number of mouse deaths corresponding to different concentrations before component reconstitution

[0162]

[0163] Table 11 Number of mouse deaths corresponding to different concentrations after component reconstitution

[0164]

[0165] LD before reconstruction 50: 311.576g / kg Confidence limit: 183.396-530.468g / kg;

[0166] Reconstructed LD 50 :1964.584g / kg Confidence limit:1210.732-3189.0224g / kg.

[0167] 3. The content of related factors in mouse serum.

[0168] ALT: Compared with the blank group, the ALT levels in the serum of mice in all drug-treated groups were significantly increased (p<0.01); compared with the perilla volatile oil group before reconstitution, the ALT levels in the serum of mice in the perilla volatile oil group after reconstitution were extremely significantly decreased (p<0.01). Figure 8 shown.

[0169] AST: Compared with the blank group, the serum AST content of the mice in the perilla volatile oil group before reconstitution was extremely significantly increased (p < 0.01), and the serum AST content of the mice in the perilla volatile oil group after reconstitution was significantly increased (p < 0.05); compared with the perilla volatile oil group before reconstitution, the serum AST content of the mice in the perilla volatile oil group after reconstitution was extremely significantly decreased (p < 0.01). Figure 9 shown.

[0170] AKP: Compared with the blank group, there was no significant difference in the AKP content in the serum of mice in all drug-treated groups (p>0.05). Figure 10 shown.

[0171] BUN: Compared with the blank group, the serum BUN content of the mice in the perilla volatile oil group before reconstitution was extremely significantly increased (p < 0.01); there was no significant difference in the serum BUN content of the mice in the perilla volatile oil group after reconstitution (p > 0.05). Compared with the perilla volatile oil before reconstitution, the serum BUN content of the mice in the perilla volatile oil group after reconstitution was extremely significantly decreased (p < 0.01). Figure 11 shown.

[0172] CRE: Compared with the blank group, the serum creatinine level of mice in the perilla volatile oil group before reconstitution was extremely significantly increased (p < 0.01); the serum creatinine level of mice in the perilla volatile oil group after reconstitution did not change significantly (p > 0.05). Compared with the perilla volatile oil group before reconstitution, the serum creatinine level of mice in the perilla volatile oil group after reconstitution was extremely significantly decreased (p < 0.01). Figure 12 shown.

[0173] 4. The content of related factors in mouse liver tissue.

[0174] SOD: Compared with the blank group, the SOD content in the liver tissue of mice in all drug-treated groups was extremely significantly increased (p < 0.01); compared with the perilla volatile oil group before reconstitution, the SOD content in the liver tissue of mice in the perilla volatile oil group after reconstitution was significantly decreased (p < 0.05). Figure 13 shown.

[0175] MDA: Compared with the blank group, the MDA content in the liver tissue of mice in the perilla volatile oil administration group before reconstruction was extremely significantly increased (p < 0.01); there was no significant difference in the MDA content in the liver tissue of mice in the perilla volatile oil administration group after reconstruction (p > 0.05); compared with the perilla volatile oil group before reconstruction, the MDA content in the liver tissue of mice in the perilla volatile oil administration group after reconstruction was extremely significantly decreased (p < 0.01). Figure 14 shown.

[0176] 5. Result analysis.

[0177] The median lethal dose (LD50) 50 ), the median lethal dose of perilla leaf volatile oil after component reconstruction was significantly increased, indicating that the safety of perilla leaf volatile oil after component reconstruction was improved, providing data support for the application of perilla leaf volatile oil as a safe and effective permeation enhancer in transdermal drug delivery system.

[0178] The ALT and AST serum assays obtained for reconstructed perilla leaf essential oil were lower than those for unreconstructed perilla leaf essential oil, indicating that the reconstructed perilla leaf essential oil significantly reduced liver damage in mice (p < 0.01). The BUN and CRE serum assays obtained for reconstructed perilla leaf essential oil were lower than those for unreconstructed perilla leaf essential oil, indicating that the reconstructed perilla leaf essential oil significantly reduced kidney damage in mice (p < 0.01). The AKP assay showed this result because AKP is not sensitive enough to hepatocyte damage and fibrosis, or because the degree of liver damage is not severe enough to cause excessive AKP release. The SOD and MDA liver assays obtained for reconstructed perilla leaf essential oil were lower than those for unreconstructed perilla leaf essential oil, indicating that the reconstructed perilla leaf essential oil significantly reduced oxidative damage in mouse livers (p < 0.01).

[0179] Skin irritation evaluation and acute toxicity studies in mice showed that the reconstituted perilla leaf essential oil had lower skin irritation levels and water loss values ​​compared to the unreconstituted perilla leaf essential oil; the median lethal dose of the crude drug was increased; and liver oxidative damage and renal impairment were reduced. The reconstituted perilla leaf essential oil is safer than the unreconstituted perilla leaf essential oil.

[0180] Example 4

[0181] Experiment on the transdermal penetration enhancing effect of volatile oil from Perilla leaves in vitro.

[0182] 1. Experimental reagents.

[0183] Azone (Tianjin Guangfu Fine Chemical Research Institute), Span 80 (Tianjin Ruijinte Chemical Co., Ltd.), Tween 80 (Tianjin Ruijinte Chemical Co., Ltd.), isopropyl palmitate (Guangzhou Zhonghong Biotechnology Co., Ltd.), isopropyl myristate (IPM) (Shandong Yousuo Chemical Technology Co., Ltd.), menthol (Aladdin Industrial Company), sodium chloride (Liaoning Quanrui Reagent Co., Ltd.), potassium chloride (Tianjin Damao Chemical Reagent Factory), disodium hydrogen phosphate (Liaoning Quanrui Reagent Co., Ltd.), potassium dihydrogen phosphate (Liaoning Quanrui Reagent Co., Ltd.), methanol (HPLC) (Liaoning Quanrui Reagent Co., Ltd.), normal saline (Jilin Dubang Pharmaceutical Co., Ltd.), propylene glycol (Tianjin Chemical Reagent Wholesale Company), oleic acid (Tianjin Yongda Chemical Reagent Co., Ltd.), acetonitrile (HPLC) (Liaoning Quanrui Reagent Co., Ltd.), rutin (Sinopharm Chemical Reagent Co., Ltd.), luteolin (Bibo Pharmaceuticals), ferulic acid (Liaoning Quanrui Reagent Co., Ltd.), ligustrazine (Baoji Rundekang Biotechnology Co., Ltd.).

[0184] Experimental instruments: TM-1 transdermal diffusion instrument (Shenyang Tianmeida Scientific Instrument Co., Ltd.), high performance liquid chromatograph (Agela Technologies), HH-501 super constant temperature water bath (Jintan Huacheng Kaiyuan Experimental Instrument Factory), AS series ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.), 5424 desktop low-temperature high-speed centrifuge (Eppendorf Company), AL204 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), WD-A drug stability tester (Tianjin Pharmacopoeia Standard Instrument Factory), XW-80A vortex mixer (Hangzhou Youning Instrument).

[0185] 2. Experimental animals

[0186] Twenty male SPF SD rats (Changchun Yisi Laboratory Animal Technology Co., Ltd.), weighing 180-220 g, were used with free access to food and water. The license number is SCXK (Ji)-2020-0002.

[0187] 3. Experimental methods.

[0188] 1. Determination of rutin content.

[0189] (1) Chromatographic conditions: Chromatographic column: Venusil XBP C18 (L) (4.6 mm*250 mm); mobile phase: methanol-0.4% phosphoric acid aqueous solution (55:45), flow rate 1.0 mL / min, column temperature 30°C, detection wavelength 340 nm, injection volume 10 μL.

[0190] (2) Preparation of reference substance: Accurately weigh 4.0 mg of rutin standard and add 50% methanol to make a solution of 80.0 μg / mL.

[0191] (3) Investigation of linear relationship: Accurately pipette 0.25, 0.5, 1.0, 1.5, and 2.5 mL of the reference solution into a 10 mL volumetric flask. Add 50% methanol to the mark, shake well, and inject the sample under the chromatographic conditions described for the determination of rutin content (1). Record the chromatogram. Construct a linear regression equation using the peak area as the ordinate (Y) and the injection volume as the abscissa (X).

[0192] 2. Determination of luteolin content.

[0193] (1) Chromatographic conditions: Chromatographic column: Venusil XBP C18 (L) (4.6 mm*250 mm), mobile phase: acetonitrile-water-phosphoric acid (30:70:0.1), flow rate 1.0 mL / min, column temperature 30°C, detection wavelength 350 nm, injection volume 10 μL.

[0194] (2) Preparation of reference substance: Accurately weigh 6.2 mg of luteolin standard and add methanol to make a 0.62 mg / mL solution.

[0195] (3) Investigation of linear relationship: Accurately pipette 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 mL of the reference solution into a 10 mL volumetric flask, dilute to the mark with methanol, and shake well. Inject the sample and analyze under the chromatographic conditions described in the luteolin content determination (1), and record the chromatogram. Develop a linear regression equation using the peak area as the ordinate (Y) and the injection volume as the abscissa (X).

[0196] 3. Determination of ferulic acid content.

[0197] (1) Chromatographic conditions: Chromatographic column: Venusil XBP C18 (L) (4.6 mm*250 mm), mobile phase: acetonitrile-water-phosphoric acid (25:75:0.1), flow rate 1.0 mL / min, column temperature 30 °C, detection wavelength 321 nm, injection volume 10 μL.

[0198] (2) Preparation of reference substance: Accurately weigh 2.0 mg of ferulic acid standard and add 70% methanol to make a 0.08 mg / mL solution.

[0199] (3) Investigation of linear relationship: Accurately pipette 0.25, 0.5, 1.25, 2.5, and 5 mL of the reference solution into a 10 mL volumetric flask, add 70% methanol to the mark, shake well, and inject the sample under the chromatographic conditions described in the ferulic acid determination (1). Record the chromatogram. Construct a linear regression equation using the peak area as the ordinate (Y) and the injection volume as the abscissa (X).

[0200] 4. Determination of the content of ligustrazine.

[0201] (1) Chromatographic conditions: Chromatographic column: Venusil XBP C18 (L) (4.6 mm*250 mm), mobile phase: methanol-water-phosphoric acid (60:40:0.1), flow rate 0.8 mL / min, column temperature 30 °C, detection wavelength 298 nm, injection volume 10 μL.

[0202] (2) Preparation of reference substance: Accurately weigh 6.2 mg of ligustrazine standard and add methanol to make a 0.62 mg / mL solution.

[0203] (3) Investigation of linear relationship: Accurately pipette 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 mL of the reference solution into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and inject the sample under the chromatographic conditions described in the determination of ligustrazine content (1). Record the chromatogram. Develop a linear regression equation using the peak area as the ordinate (Y) and the injection volume as the abscissa (X).

[0204] 5. Transdermal absorption experiment.

[0205] (1) Preparation of ex vivo rat skin: Rats were anesthetized by intraperitoneal injection of 2% urethane, the abdomen was shaved, and the rats were killed by cervical dislocation. The hairless skin below the xiphoid process of the rat abdomen was excised, and the subcutaneous fat and mucosa were removed. The skin was repeatedly rinsed with saline until the residual blood was drained, and stored at -80°C. Thawed at room temperature before use, rinsed with saline, and the skin integrity was checked before the experiment. Surface moisture was adsorbed with filter paper.

[0206] (2) Preparation of solution: PBS receiving solution: 4 g sodium chloride, 0.1 g potassium chloride, 0.12 g potassium dihydrogen phosphate and 0.72 g disodium hydrogen phosphate are placed in a 500 mL volumetric flask, distilled water is added to 500 mL, and the pH is adjusted to 7.4; test solution: accurately measure 0.35 g of the drug and add it to 20 mL isopropyl palmitate, mix it with a vortex mixer, and add 1 mL of perilla volatile oil before reconstitution, perilla volatile oil after reconstitution, isopropyl myristate, Tween 80, menthol, Span 80, oleic acid, propylene glycol, and azone respectively, mix well and set aside.

[0207] (3) Transdermal absorption experiment: The receiving cell volume of the horizontal double-chamber diffusion cell is 3 mL, and the effective penetration area is 0.95 cm 2 , set the system temperature to 32°C, the stirrer length to 1.0cm, and the speed to 300r / min. Fix the system, add the stirrer, exhaust the air, and fix the SD rat skin between the drug supply tank and the receiving tank, keeping the skin stratum corneum facing the drug supply tank. Add 3mL of PBS solution to one side of the receiving tank and 3mL of the test solution to the other side of the drug supply tank, seal it, wait until the temperature is constant, turn on the instrument, and keep the stirrer stirring at a constant speed. Aspirate 2mL of the receiving solution at 1, 2, 3, 4, 5, 6, 7, and 8 hours respectively, and add 2mL of PBS receiving solution. The receiving solution sample was centrifuged at a speed of 10,000r / min and a temperature of 20°C for 10 minutes. The receiving solution sample was filtered through a microporous filter membrane with a pore size of 0.45um and measured by HPLC.

[0208] 6. Data processing.

[0209] The cumulative permeation (Q n ).

[0210]

[0211] Among them, C n : drug concentration measured at the nth sampling point (ug / mL); V: volume of receiving solution (3mL); C i : drug concentration measured at the i-th sampling point (ug / mL); V i : sampling volume (2.0 mL); A: penetration area (0.95 cm 2 ).

[0212] Cumulative transmittance Q n The slope of linear regression for time t is the steady-state permeation rate J of the drug. ss [ug / (cm 2 / h)], the enhancing rate (ER) is the ratio of the cumulative permeation, that is, ER = Q 促透剂 / Q 空白 .

[0213] 4. Experimental results.

[0214] 1. The penetration-promoting effect of perilla leaf volatile oil on rutin.

[0215] (1) Rutin linear regression equation: y = 26031x - 9028.8, R 2 =0.9991; the results showed that rutin had a good linear relationship in the range of 8-80 μg / mL.

[0216] (2) Transdermal absorption test results: With time t as the horizontal axis (x), the cumulative amount of permeation Q n The linear regression equation is drawn for the ordinate (y). Reconstructed perilla leaf volatile oil: y = 2.7804x-3.269, R 2 =0.9996; Perilla leaf volatile oil before reconstruction: y=1.2429x+0.3283, R 2 =1; Tween 80: y = 1.9735x - 0.7345, R 2 =1; IPM: y=0.7777x+0.3889, R 2 =1; menthol: y = 1.7874x - 0.8641, R 2 =1; Siban 80: y = 1.0283x + 0.3153, R 2 =1; its cumulative transmittance Q n It has a good linear relationship with time t.

[0217] As shown in the following table and Figure 15 As shown in the figure, the reconstituted perilla leaf essential oil has the most significant effect on the permeation enhancement of rutin. The permeation enhancement effect of the permeation enhancers is as follows: reconstituted essential oil > Tween 80 > menthol > unreconstituted essential oil > Span 80 > isopropyl myristate (IPM).

[0218] Compared with the volatile oil before reconstitution, the drug permeation ratio of the reconstituted perilla leaf volatile oil was 1.860 (p < 0.01), and the steady-state permeation rate increased from 1.37 μg·cm -2 ·h -1 increased to 2.78 μg·cm -2 ·h -1 , the cumulative penetration amount increased by 1.86 times.

[0219] Table 12 In vitro permeation parameters of rutin by permeation enhancers

[0220] Permeation enhancer (PE) <![CDATA[J ss (μg·cm -2 ·h -1 )]]> <![CDATA[Q 8h (μg·cm -2 )]]> ER Perilla frutescens essential oil before reconstruction 1.37±0.1 10.27±0.27 1.002 Reconstructed Perilla frutescens volatile oil 2.78±0.39 19.06±2.71 1.860 Isopropyl myristate (IPM) 0.78±0.05 6.61±0.40 0.645 Menthol 1.79±0.27 13.50±2.47 1.317 Twain 80 1.97±0.57 15.50±3.89 1.512 Span 80 1.03±0.06 8.54±0.51 0.833 blank 0.85±0.11 10.25±0.72 1

[0221] 2. The penetration-enhancing effect of perilla leaf volatile oil on luteolin.

[0222] (1) Luteolin linear regression equation: y = 41440x-78602.R 2 =0.9994; the results showed that luteolin showed a good linear relationship in the range of 3.1-124 μg / mL.

[0223] (2) Transdermal absorption test results: With time t as the horizontal axis (x), the cumulative amount of permeation Q n Draw a linear regression equation for the ordinate (y). Perilla leaf volatile oil before reconstruction: y = 4.8505x + 0.3908, R 2=0.9997; reconstructed perilla leaf volatile oil: y=5.3327x+0.0884, R 2 =0.9997; IPM: y=4.0167x+2.0083, R 2 =1; menthol: y = 4.919x + 0.7861, R 2 =1; Siban 80: y = 4.1528x + 1.8867, R 2 =1; Tween 80: y = 4.7773x + 0.7891, R 2 =1 its cumulative transmission amount Q n It has a good linear relationship with time t.

[0224] As shown in the following table and Figure 16 As shown in the figure, reconstituted perilla leaf essential oil had the most significant permeation-enhancing effect on luteolin. The permeation-enhancing effect of the permeation enhancers was as follows: reconstituted essential oil > menthol > unreconstituted essential oil > Span 80 > IPM > Tween 80.

[0225] Compared with the volatile oil before reconstitution, the drug permeation ratio of the reconstituted perilla leaf volatile oil was 4.185 (p < 0.01), and the steady-state permeation rate increased from 4.85 μg·cm -2 ·h -1 increased to 5.33 μg·cm -2 ·h -1 , the cumulative penetration amount increased by 1.091 times.

[0226] Table 13 Permeation parameters of various permeation enhancers on luteolin

[0227] Permeation enhancer (PE) <![CDATA[J ss (μg·cm -2 ·h -1 )]]> <![CDATA[Q 8h (μg·cm -2 )]]> ER Perilla frutescens essential oil before reconstruction 4.85±0.35 39.32±2.37 3.836 Reconstructed Perilla frutescens volatile oil 5.33±0.13 42.90±0.91 4.185 Isopropyl myristate (IPM) 4.02±0.20 34.14±0.20 3.331 Menthol 4.92±0.27 40.15±1.75 3.917 Twain 80 11.44±4.19 15.05±3.89 1.468 Span 80 4.15±0.05 35.11±0.37 3.425 blank 0.85±0.11 10.25±0.72 1

[0228] 3. The penetration-promoting effect of perilla leaf volatile oil on ferulic acid.

[0229] (1) Ferulic acid linear regression equation: y = 128231x - 23007, R 2 =0.9996; the results showed that ferulic acid had a good linear relationship in the range of 2-40 μg / mL.

[0230] (2) Transdermal absorption test results: With time t as the horizontal axis (x), the cumulative amount of permeation Q n Draw a linear regression equation for the ordinate (y). Perilla leaf essential oil before reconstruction: y = 1.4873x + 18.12, R 2 =0.996; reconstructed perilla leaf volatile oil: y=2.6542x+22.786, R 2 =0.964; oleic acid: y = 0.8661x + 10.134, R 2=0.9717; Propylene glycol: y = 1.0987x + 6.9594, R 2 =0.9866; Siban 80: y=1.6763x+4.5783, R 2 =0.9904; Tween 80: y = 1.055x + 6.8512, R 2 =0.9948; without penetration enhancer: y=0.8641x+3.8222, R 2 =0.9942; its cumulative transmittance Q n It has a good linear relationship with time t.

[0231] As shown in the following table and Figure 17 As shown in the figure, reconstituted perilla leaf essential oil has the most significant permeation-enhancing effect on ferulic acid. The permeation-enhancing effect of the permeation enhancers is as follows: reconstituted essential oil > unreconstituted essential oil > Span 80 > oleic acid > propylene glycol > Tween 80.

[0232] Compared with the volatile oil before reconstitution, the drug permeation ratio of the reconstituted perilla leaf volatile oil was 4.799 (p < 0.01), and the steady-state permeation rate increased from 1.49 μg·cm -2 ·h -1 Increased to 4.05 μg·cm -2 ·h -1 , the cumulative penetration amount increased by 1.649 times.

[0233] Table 14 Effects of various penetration enhancers on transdermal absorption of ferulic acid

[0234] Permeation enhancer (PE) <![CDATA[J ss (μg·cm -2 ·h -1 )]]> <![CDATA[Q 8h (μg·cm -2 )]]> ER Perilla frutescens essential oil before reconstruction 1.49±0.26 29.85±5.34 2.912 Reconstructed Perilla frutescens volatile oil 4.05±0.38 49.19±4.61 4.799 Oleic acid 0.87±0.08 17.09±0.57 1.667 Propylene glycol 3.41±2.30 15.90±0.78 1.551 Twain 80 0.85±0.06 10.48±3.07 1.022 Span 80 1.68±0.16 17.74±2.18 1.731 blank 0.85±0.11 10.25±0.72 1

[0235] 4. The penetration-enhancing effect of perilla leaf volatile oil on ligustrazine.

[0236] (1) Ligustrazine linear regression equation: y = 67005x + 116029, R 2 =0.9999; the results showed that ligustrazine had a good linear relationship in the range of 5.0-200 μg / mL.

[0237] (2) Transdermal absorption test results: With time t as the horizontal axis (x), the cumulative amount of permeation Q n Draw a linear regression equation for the vertical axis (y). Perilla leaf volatile oil before reconstruction: y=0.5452x-0.0934, R 2 =0.9997; reconstructed perilla leaf volatile oil: y=1.8838x-0.9799, R 2 =0.9998; oleic acid: y = 2.193x - 2.5839, R 2 =0.9997; Propylene glycol: y = 0.4416x - 0.2871, R 2=0.9992; Siban 80: y = 1.4372x - 1.2594, R 2 =0.9987; Tween 80: y = 1.3735x - 0.5821, R 2 =0.9998. Its cumulative transmission amount Q n It has a good linear relationship with time t.

[0238] As shown in the following table and Figure 18 As shown in the figure, reconstituted perilla leaf essential oil significantly enhances the permeation of ligustrazine. The permeation-enhancing effect of the permeation enhancers is as follows: propylene glycol > reconstituted essential oil > Tween 80 > Span 80 > unreconstituted essential oil > oleic acid.

[0239] Compared with the volatile oil before reconstitution, the drug permeation ratio of the reconstituted perilla leaf volatile oil was 1.378 (p < 0.01), and the steady-state permeation rate increased from 0.55 μg·cm -2 ·h -1 increased to 1.88 μg·cm -2 ·h -1 , the cumulative penetration amount increased by 3.305 times.

[0240] Table 15 Penetration parameters of permeation enhancers on ligustrazine

[0241] Permeation enhancer (PE) <![CDATA[J ss (μg·cm -2 ·h -1 )]]> <![CDATA[Q 8h (μg·cm -2 )]]> ER Perilla frutescens essential oil before reconstruction 0.55±0.09 4.27±0.65 0.417 Reconstructed Perilla frutescens volatile oil 1.88±0.24 14.12±1.51 1.378 Oleic acid 0.44±0.04 3.27±0.32 0.319 Propylene glycol 2.19±0.24 15.0±1.94 1.463 Twain 80 1.37±0.09 10.38±0.75 1.013 Span 80 1.44±0.15 10.28±1.01 1.003 blank 0.85±0.11 10.25±0.72 1

[0242] 4. Result analysis.

[0243] This experiment used ex vivo rat abdominal skin, whose barrier properties are comparable to those of human skin, and has certain guiding significance for human medication. In the in vitro transdermal permeation test, the selection of model drugs can quickly and effectively determine the permeation-enhancing ability of permeation enhancers. This study selected four active ingredients of traditional Chinese medicine commonly used to treat chronic diseases, including rutin, luteolin, ferulic acid, and ligustrazine as model drugs. It can fully reflect the changes in the permeation-enhancing ability of the components of perilla leaf volatile oil before and after reconstruction, and selected six commonly used permeation enhancers (menthol, Span 80, Tween 80, isopropyl myristate, oleic acid, propylene glycol) to compare with the perilla volatile oil before and after reconstruction.

[0244] The results show that the permeation-enhancing effect of the reconstructed perilla volatile oil is significantly improved compared to the perilla volatile oil before reconstruction; compared with several other common permeation enhancers, the reconstructed perilla volatile oil also has a stronger permeation-enhancing ability. The reconstructed perilla volatile oil is a complex mixture with a richer structure compared to other single permeation enhancers. The main component of the reconstructed perilla leaf volatile oil, 1-(3-furyl)-4-methyl-1-pentanol, has a head-to-tail structure similar to that of intercellular lipids, which enables it to insert into the tightly packed intercellular lipids in the stratum corneum, disrupting the orderly arrangement of the lipid bilayer, and greatly improving the transdermal permeation-promoting effect. A large number of studies have shown that the terpenoid compounds contained in volatile oils are important components of traditional Chinese medicine volatile oils that play a role in promoting transdermal penetration. Caryophyllene and (Z,E)-α-farnesene in the reconstructed perilla leaf volatile oil are terpene compounds. Linalool belongs to the terpene alcohol class, and it can work together with 1-(3-furyl)-4-methyl-1-pentanol to enhance the transdermal penetration effect of perilla leaf volatile oil.

[0245] In vitro studies on the transdermal penetration enhancement effects of rutin, luteolin, ferulic acid, and ligustrazine have shown that reconstituted perilla leaf volatile oil exhibits superior transdermal penetration compared to both unreconstituted perilla leaf volatile oil and currently used chemical penetration enhancers, demonstrating promising application prospects in transdermal drug delivery systems. While perilla cultivation has a history of over 2,000 years in my country, research on its use as a penetration enhancer is still in its infancy. These experimental data demonstrate that reconstituted perilla leaf volatile oil exhibits reduced toxicity, high penetration enhancement activity, and potential use as a penetration enhancer, particularly in the preparation of transdermal drug delivery formulations.

[0246] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0247] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A use of a reconstructed perilla leaf volatile oil in the preparation of a penetration enhancer and / or a drug, characterized in that: The dosage form of the drug is a transdermal preparation. The reconstituted perilla leaf volatile oil comprises a perilla ketone reduction product, wherein the perilla ketone reduction product is 1-(3-furyl)-4-methyl-1-pentanol. The perilla ketone reduction product is shown in the following formula I: 。 2. The use according to claim 1, characterized in that The perilla leaf volatile oil further comprises at least one of 1-octen-3-ol, linalool, caryophyllene, or (Z,E)-α-farnesene.

3. The use according to any one of claims 1-2, characterized in that The component-reconstructed perilla leaf volatile oil is obtained by reducing the perilla leaf volatile oil with sodium borohydride.

4. A penetration enhancer, characterized in that The invention relates to a perilla leaf volatile oil reconstructed with the components for use according to any one of claims 1 to 3.

5. A drug, characterized in that The volatile oil of perilla leaves reconstructed with the components used in any one of claims 1 to 3 is in the form of a transdermal preparation.

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