LU503834B1 - The use of a vegetable essential oil in an anti-inflammatory, antibacterial, free radical inhibiting and reactive oxygen species combating drug - Google Patents

Abstract

The present invention discloses the use of a vegetable essential oil in an anti-inflammatory, antibacterial, free radical inhibitory and reactive oxygen species combating drug, wherein the vegetable essential oil is forsythia flower essential oil; Forsythia flower essential oil has an anti-inflammatory effect in the target tissue and has an improving effect on contact dermatitis caused by TPA; forsythia flower essential oil has an inhibitory effect on LPS-induced NO in RAW 264.7 cells; Forsythia flower essential oil has a scavenging effect on reactive oxygen species in RAW264.7 cells; Forsythia flower essential oil prepared by the manufacturing process of the present invention has been shown to have anti-inflammatory, antibacterial, NO and reactive oxygen species inhibitory effects in animal and cell studies.

Description

The use of a vegetable essential oil in an anti-inflammatory, antibacterial, free radical inhibiting and reactive oxygen species combating agent

drug

Technical part

The present invention belongs to the technical field of the use of plant essential oils and relates in particular to the use of a plant essential oil

Oil as anti-inflammatory, antibacterial, nitric oxide radical (NO) inhibitory and reactive

Oxygen species removing drug.

Technology in the background

Essential oils (also called essential oils) are aromatic, oily liquids obtained from plant materials such as flowers, buds, seeds, leaves, fine twigs, bark, wood,

Fruits and roots are obtained. Essential oils or their components have long been known to have antibacterial, antiviral, insecticidal, anti-inflammatory and antioxidant effects. The continued popularity of essential oils in the cosmetic and pharmaceutical industries is due to their natural properties, both in

Regarding their health benefits as well as their aroma which is easily accepted.

Inflammation is the body's physiological response to irritation, which can be caused by both infection and tissue damage. The skin forms the

Interface between the external environment and the body and is the main barrier that protects the body from the invasion of irritants such as microbial pathogens.

Inflammatory skin diseases are often treated with antibiotics, glucocorticoids,

Treated with antihistamines and emollients to relieve itching, infection and inflammation. However, long-term treatment with such drugs can disrupt cytokine networks, inhibit lymphocyte function, reduce collagen synthesis and lead to a

lead to skin atrophy. Therefore, new treatments and medications that

Effectively suppressing the inflammatory response is being researched in detail.

The Korean forsythia (Forsythia koreana) belongs to the genus Forsythia in the

Myrtle family (Myrtaceae). During our research, we found that the essential oil extracted from its flowers has anti-inflammatory and decongestant properties

has properties. Korean forsythia blooms from March to April every year, has a long

Flowering time and a large yield and is now mainly used as a garden tree for greenery. Studies on Korean forsythia flower essential oil and its biological properties have not yet been published in the literature. The aromatic smell of the essential oil extracted from the flowers of Korean forsythia is more acceptable compared to the smell of the essential oil of Chinese medicine forsythia, and is more suitable for food and chemical applications, which is of great value for development.

Therefore, this paper discusses the application of a plant essential oil as anti-inflammatory, antibacterial, nitric oxide radical (NO) inhibiting and reactive

Oxygen species removing drug suggested.

Content of the invention

In order to solve the above technical problems, the present invention designs the application of a vegetable essential oil as an anti-inflammatory, antibacterial,

Nitric oxide radical inhibiting and reactive oxygen species removing drug, wherein forsythia flower essential oil has an anti-inflammatory effect in the target tissue and an improving effect on contact dermatitis caused by TPA; the ethereal&/909834

Forsythia flower oil has an inhibitory effect on LPS-induced NO in RAW 264.7 cells; The essential forsythia flower oil has a scavenging effect on reactive oxygen species

RAW264.7 cells.

In order to achieve the above-mentioned purpose, the invention presents the following technical aspects

Solution available:

The use of a plant essential oil as an anti-inflammatory, antibacterial, nitric oxide radical inhibitor and reactive oxygen species remover

Medicinal product, characterized in that the essential oil is a forsythia flower essential oil; Forsythia flower essential oil has an anti-inflammatory effect in the target tissue and an improving effect on contact dermatitis caused by TPA; the ethereal

Forsythia flower oil has an inhibitory effect on LPS-induced NO in RAW 264.7 cells; The essential forsythia flower oil has a scavenging effect on reactive oxygen species

RAW264.7 cells.

Preparation of forsythia flower essential oil as described:

For example, you put fresh forsythia flowers in a still, add distilled water, let it steep for 25-35 minutes and then boil in an electric heating jacket; the resulting water vapor is passed through a cooler and collected in a triangular flask; the distilled condensate is collected over a period of 4 hours, the essential oil is extracted with dichloromethane and at room temperature with anhydrous

dried sodium sulfate; You get a light yellow essential oil with a mild aroma

Taste;

Forsythia flower essential oil mainly includes the following components: n-

tetradecane (36.93%), n-heneikosan (13.94%), 1-dodecane (8.11%) and n-nonane (6.32%).

The essential forsythia flower oil has an anti-inflammatory, antibacterial and inhibiting effect

Nitric oxide radical inhibitor and removes reactive oxygen species;

In addition, the forsythia flower is the Korean forsythia flower.

The beneficial effects of the present invention are as follows:

The forsythia flower essential oil provided in the present invention has been verified through animal testing to have a significant improving effect on the by

having TPA-caused contact dermatitis; The forsythia flower essential oil provided in the present invention has been verified through cellular experiments to have an inhibitory effect on NO in cells, thereby preventing high levels of

NO cause host cell death and inflammatory tissue damage; the ethereal

Forsythia flower oil in the present invention has a scavenging effect on reactive

Oxygen species in cells and prevents tissue damage caused by excessive reactive

Oxygen species are caused in inflammation and worsen the inflammatory reaction; the essential forsythia flower oil according to the invention inhibits Salmonella enterica,

Escherichia coli, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes, with the strongest inhibitory effect on Bacillus cereus and Listeria monocytogenes.

Description of the attached drawings

In order to better illustrate the technical solutions of the embodiments of the invention, a brief description of the accompanying drawings follows

Description of the embodiments must be used.

Figure 1 shows a physical view of the forsythia flower of the present invention;

Figure 2 shows a schematic representation of the device for producing the essential oil. / 903834

forsythia flower oil of the present invention;

Figure 3 shows a schematic representation of the experimental results of the inhibitory

Effect of forsythia flower essential oil of the present invention on TPA-induced

ear swelling in BALB/c mice;

Figure 4 shows a schematic representation of the experimental results of the effect of the forsythia flower essential oil of the present invention on the TPA-induced

Water content in the ears of BALB/c mice;

Figure 5 shows a schematic representation of H&E stained sections of the present

Invention on TPA-induced mouse ear;

Figure 6 shows a schematic representation of the experimental results of the effect of the forsythia flower essential oil of the present invention on the TPA-induced increase

Epidermal and dermal thickness in BALB/c mice;

Figure 7 shows a schematic representation of the experimental results of the inhibitory

Effect of forsythia flower essential oil of the present invention on LPS-induced

NO production in RAW 264.7 cells;

Figure 8 shows a schematic representation of the experimental results of the effect of the

DCFH-DA method of the present invention to capture the living scavenging effect of the essential

Korean forsythia flower oil is reactive on LPS-induced production

detect oxygen species in RAW 264.7 cells;

Figure 9 shows a schematic representation of the experimental results of the effect of forsythia flower essential oil of the present invention on the viability of RAW 264.7 cells.

Detailed description

The technical solutions in embodiments of the invention are described below

In conjunction with the accompanying drawings, the embodiments of the invention are clearly and completely described.

Embodiment 1

With reference to the drawings shown in images 1 to 8;

Preparation of a forsythia flower essential oil as described:

For example, you put fresh forsythia flowers in a still, add distilled water, let it steep for 25-35 minutes and then boil in an electric heating jacket; the resulting water vapor is passed through a cooler and collected in a triangular flask; the distilled condensate is collected over a period of 4 hours, the essential oil is extracted with dichloromethane and at room temperature with anhydrous

dried sodium sulfate; You get a light yellow essential oil with a mild aroma

Taste.

Forsythia flower essential oil mainly includes the following components: n-

tetradecane (36.93%), n-heneikosan (13.94%), 1-dodecane (8.11%) and n-nonane (6.32%).

Embodiment 2

Gas chromatography-mass spectrometry analysis of the composition of the essential

Forsythia flower oil

GC-MS was used for the quantitative and qualitative analysis of the essential oils on a ZB-1MS capillary column with an ionization energy of 70 eV and a constant flow rate of 1 mL/min using helium as a carrier gas. The injector temperature was set at 220°C and the mass transfer tube temperature at 290°C. The oven/>03834 was set at 3°C/min from 50°C to 150°C, after 10 minutes of constant temperature the temperature is increased at a rate of 10°C/min to 250°C. 1 µl diluted essential

Forsythia flower oil (1/100, v/v, solvent dichloromethane) was manually added to the sample. The components were selected based on their retention indices on the ZB-1MS

Capillary column relative to the corresponding series of n-alkanes (C8 to C20) identified by matching to the NIST MS library.

Embodiment 3

Anti-inflammatory effect of forsythia flower essential oil

Five-week-old female BALB/c mice were housed in a climate-controlled room with a 12:12-hour light-dark cycle. The room temperature and humidity were automatically controlled at 23°C and 60%. They received standard laboratory diet and water ad libitum. Acclimatization took place at least 7 days before the experiment.

Effect of forsythia flower essential oil on TPA-induced

Inflammatory response in BALB/c mice

TPA was able to induce a skin inflammatory response in BALB/c mice, leading to ear skin thickening and edema. TPA was in the solvent

AOO dissolved and used as a trigger for skin inflammation. 20 µl of the test (1% or 10% essential oil dissolved in 50% ethanol) and control (50% ethanol) were applied to the inner and outer surfaces of the ears of mice, respectively, 30 minutes later 10 µl of TPA (4 mM) applied to the surface of the essential oil or ethanol-treated skin to induce an inflammatory response in the skin. Hydrocortisone, which is currently used to treat various inflammatory skin diseases, served as a positive control. Five mice were used in each group. The thickness of the same area of the earplugs was measured with a micrometer before TPA treatment (A) and 4 hours after

Sample treatment (B) measured. The increase in ear thickness was calculated as B - A.

After measuring the thickness of the mouse ears at the end of the experiment, the mice were

Ether anesthetized and 6 mm2 of tissue was removed by punching a hole in the center of the ear. Immediately after collection, the tissue was weighed on an electronic scale and defined as wet weight (W). The fabric was then dried in an oven to a constant weight, weighed and defined as dry weight (W'). The

Skin moisture content was calculated using the following formula: % water content = [(W -W')/W] x 100%

Histomorphological analysis of the mouse ear

Fresh tissue from mouse ears was collected, fixed in formalin, dehydrated, in

Embedded in paraffin and cut cross-sectionally into 5 µm thick pieces using a cutting machine.

They were then stained with hematoxylin and eosin (H&E). The tissue was observed under the light microscope, and the thickness of the epidermis and the dermis of the inflamed

Areas were measured.

Culture of RAW 264.7 cells

Mouse macrophage RAW 264.7 cells were used for in vitro anti-inflammatory effect test. The cells were grown in modified Dulbecco

Eagle medium cultured with 10% fetal bovine serum at 37°C in a 5% CO2 incubator.

Determination of NO content in LPS-induced RAW 264.7 cells 200 µl RAW 264.7 cells (1x10° cells/mL) were seeded in 96-well plates and 24

Incubated for hours so that the cells adhere to the wall. Cells were treated with samples of forsythia flower essential oil (100 µg/mL, 200 µg/mL) or vehicle for 4 hours, followed by induction with 100 ng/mL LPS for 24 hours. After LPS induction, 100 μL of the supernatant was transferred to another plate and an equal amount of Griess-5 reagent was added to each well. After 10 minutes of incubation at room temperature, the absorbance was measured at 540 nm using an enzyme marker. A standard curve was constructed using various concentrations of sodium nitrite and the NO

Concentration calculated for each well.

Reactive oxygen species assay in LPS-induced RAW264.7 cells

LPS induction of RAW264.7 cells was performed as described above. Forsythia flower essential oil was administered at a concentration of 50 µg/ml and 100 µg/ml. After LPS induction, RAW264.7 cells grown on the wall were collected and the cell culture medium was removed after serum-free medium diluted 1:1000 with DCFH-DA, then the appropriate volume of well-diluted DCFH-DA was added to the Cover cells and the plate was incubated at 37°C for 20 minutes.

Cells were washed three times with serum-free cell culture medium to remove residual DCFH.

DA to be removed sufficiently. The cells were observed with a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm).

Determination of antibacterial activity of Korean essential oil

Forsythia flowers

The in vitro antibacterial activity of forsythia flower essential oil was tested with a

Standard agar paper diffusion method determined. 0.1 mL of the suspension containing 10′ CFU/mL was evenly poured onto LB plates and dried for 5 minutes. Sterile

6 mm diameter pieces of filter paper were impregnated with 5 μL of forsythia flower essential oil or 5 μg of the positive control and placed on inoculated LB agar. The plates were left at room temperature for 30 minutes to allow the oil to diffuse into the agar and then incubated at 37°C for 24 hours, the inhibitory activity was monitored

Measurement of the diameter of the inhibition circle.

Study of cell viability

The viability of RAW 264.7 cells was determined using the MTT method. After determining the NO content in LPS-induced RAW 264.7 cells, 100 μl of supernatant was used for nitric oxide analysis and 20 μl of tetrazolium saline was added to each well

Original plate was added and incubated for 4 hours in a 5% CO2 incubator at 37°C. The

Medium was then discarded and 200 μL of DMSO was added to dissolve for 10 minutes.

The absorbance was then measured at 540 nm and the survival rate of the cells was calculated.

Results:

Chemical composition of forsythia flower essential oil

The essential oil of fresh Korean forsythia flowers contained approximately 0.53%, v/w. A total of 33 different compounds were detected by GC-MS analysis, accounting for 96.85% of the total oil fraction (Table 1). The oil contained 64.31% hydrocarbons, 13.78%

Alcohols, acids, esters and phenols, 17.67% aldehydes and ketones and 1.09% oxygenates

Diterpenes. The main components are n-tetradecane (36.93%), n-heneikosan (13.94%), 1-

dodecane (8.11%) and n-nonane (6.32%).

Table 1 Chemical composition of Korean forsythia essential oil em me ‘

Molecular

Compound compound “percentage|to

formula

identification

Ce [wee [en

Ce wee | (ww us ee (ue [ue we | le | © n-Tetradecane n-Tetracosane C24Hso 36.93 MS, RI 2,6,10,15- 2,6,10,15- Tetramethylheptadecane Cz1 H44 0.36 MS, RI

Tetramethylheptadecane [ue | ia kee | kes | [ee |__

Hexahydrofarnesyl

Phytonzid Ci3H360 MS, RI acetone

Ce Je [Tw cis-3-hexenyl iso-

Leaf alcohol isobutyrate CoH 150, MS, RI butyrate

Chloroformic acid-2- Chloroformic acid 2-

CoH17C102 0.17 MS, RI

Ethylhexyl ester ethylhexyl ester [me ew | [ew a Relative area (peak area in relation to the total peak area), b

Identification method MS: mass spectrometry, detection of mass spectral data against

data in the mass spectral library; RI: retention index, see Ref.

Anti-inflammatory effect of essential forsythia flower oil

Inhibition of TPA-induced skin inflammation in mice by essential oil

Forsythia flower oil

The first sign of skin irritation and local inflammation is often an increase in

Skin thickness due to edema. Inflammation of the skin is a complex process, the important indicator of which is edema, which is increased vascular permeability, swelling of the

Skin and a proliferation of epidermal keratin-forming cells. Figure 3 shows that topical application of low-dose (1% EO) and high-dose (10% EO) essential oils

Oiling significantly inhibited the edema in the ear. TPA stimulation resulted in an increase in

ear tip thickness by 0.21 mm, while treatment with 1% and 10% EO only increased ear thickness by 0.10 and 0.08 mm, respectively. 1% HC had a similar effect to 10% EO (0.08 mm

Increase in ear thickness). LUS03834

Forsythia flower essential oil also inhibited the TPA-induced increase in

Water content of the skin (Figure 4). 62.39% and 57.32% of the water content in the ear in the

Treatment with 1% or 10% EO differed significantly from the negative control (84.77%). 58.61% of the water content in the ear was achieved when treated with 1% HC. The

Results are shown in Figures 3 and 4, where the inhibitory effect of 10% Korean forsythia flower essential oil on TPA-induced skin inflammation was similar to that of 1% HC. These results suggest that Korean forsythia essential oil has potential anti-inflammatory effects.

Histomorphological examination of mouse ears

H&E-stained sections of the TPA-induced ear were then examined. As in

As shown in Figure 5, the ear treated with TPA experienced epidermal hyperplasia and massive infiltration of inflammatory cells in the skin region, accompanied by a

Destruction of the connective tissue (picture Sb). In histological comparison, both 1% and 10% EO treatment reduced epidermis, dermis thickness and infiltration

Inflammatory cells to some extent (Fig. 5d, e). These results suggest that Korean forsythia flower essential oil has anti-inflammatory effects in the

target tissues and demonstrate the soothing effect of essential forsythia flower oil on TPA-induced contact dermatitis.

Figure 5 Tissue sections of the skin of the mouse ear with epidermis, dermis and cartilage layers (magnification x100). a, blank control animals; b, TPA-treated skin with edema, epidermal

hyperplasia and dermal inflammatory cell infiltration; c, treatment with TPA and 1%

hydrocortisone; d, treatment with TPA and 1% forsythia flower essential oil; e, treatment with TPA and 10% forsythia flower essential oil.

In addition, the epidermal and dermal thicknesses of the TPA-induced

Mouse ear tissue measured. As shown in Figure 6, the thickness of the epidermis was 16.73 and 15.64 μm for the 1% and 10% EO treatments, both of which significantly reduced the epidermis thickness compared to the TPA treatment. The thickness of the dermis in the ear region was also significantly reduced for the 1% and 10% EO treatments. The thickness of the dermis was 219.13, 156.37, 165.70 and 270.60 µm for the 1% and 10% EO, 1% HC and TPA treatments.

Inhibition of NO in LPS-induced RAW 264.7 cells by the essential

Forsythia flower oil

Highly unstable NO radicals are the main mediators of tumor cell killing by macrophages. However, high concentrations of NO can lead to host cell death and inflammatory tissue damage. Therefore we have the effect of the etheric

Forsythia flower oil was examined for NO production in cell culture broth. The NO

Concentrations in unstimulated cells were 15.2 μM (Figure 7) compared to 41.43 μM in LPS-stimulated cells. Treatment with forsythia flower essential oil (100 and 200 pg/ml) had a significant inhibitory effect on NO production (33.03 and 23.58 μM, respectively).

Indomethacin (IM), a commonly used nonsteroidal anti-inflammatory

drug, reduced NO production to 15.2 μM.

Scavenging of reactive oxygen species in RAW264.7 cells by essential

Forsythienbliitensl

Reactive oxygen species include superoxide radicals, hydrogen peroxide, and downstream products such as peroxides and hydroxylates, which inhibit cell growth and dt&}03854

Cell proliferation, developmental differentiation, senescence and apoptosis as well as many other physiological and pathological processes. During the

In the inflammatory process, excess ROS often cause tissue damage, which in turn

worsen inflammatory reaction. Korean forsythia flower essential oil captures

ROS decreased in LPS-induced mouse macrophages RAW264.7, which increased with increasing dose (Figure 8). Furthermore, forsythia flower essential oil showed no significant cytotoxicity to RAW 264.7 cells at a concentration of 200 µg/mL (Figure 9).

Antibacterial effect of forsythia flower essential oil

The diameter of the inhibitory circles of forsythia flower essential oil against Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes ranged from 7.1 to 17.6 mm (Table 2). The results showed that the essential

Forsythia flower oil has the strongest inhibition against Bacillus cereus 22-1 and Listeria monocytogenes

ATCC 10943 showed.

Table 2 Antimicrobial activity of Korean forsythia flower essential oil

Diameter of inhibitor band (mm)

Tetracycline

Strain Forsythia Flower Oil | Streptomycin (5 (5 µL/disc) (5 µg/disc)

Hg/disc )

Salmonella enteritidis KCTC 12243 10.1+02 Eh

Escherichia coli ATCC 8739 7.1+04 11.0+0.8 Es

Staphylococcus aureus ATCC 6538 11.7+0.4 6.8+0.7 18.0£0.7

Bacillus cereus KCTC 14042 128 +1.1 123 + 0.7 me

Bacillus cereus 15-1 12.3 +14 12.4 + 0.6

Bacillus cereus 11-1 128+1.8 1L1+1.3

Bacillus cereus 22-1 176 41.1 18.6 + 0.8 me

Bacillus cereus 6-1 11.7+0.2 121+£03 Es

Bacillus cereus 19-1 123 +02 13.5 + 0.4 me

Listeria monocytogens ATCC 19118 10.0 + 0.7 Ee 16.5 + 0.7

Listeria monocytogens ATCC 19111 12.94 0.6 6.6 + 0.3 17.24 0.1

Listeria monocytogens ATCC 19166 102+09 64+01 —

Listeria monocytogens ATCC 19116 12.0+02 or 16.8+0.8

Listeria monocytogens ATCC 10943 17.9+ 0.6 20.6 + 0.5 22.0+0.1

Claims (4)

Claims LU503834 1. The use of a plant essential oil as an anti-inflammatory, antibacterial, nitric oxide radical inhibiting and reactive oxygen species removing drug, characterized in that the essential oil is a forsythia flower essential oil; Forsythia flower essential oil has an anti-inflammatory effect in the target tissue and an improving effect on contact dermatitis caused by TPA; forsythia flower essential oil has an inhibitory effect on LPS-induced NO in RAW 264.7 cells; Forsythia flower essential oil has a scavenging effect on reactive oxygen species in RAW264.7 cells. 2. The use of a vegetable essential oil as an anti-inflammatory, antibacterial, nitrogen oxide radical-inhibiting and reactive oxygen species-removing drug according to claim 1, characterized in that the essential forsythia flower oil is produced by the following steps: Fresh forsythia flowers are placed in a distillation apparatus, distilled water is added Add, let steep for 25-35 minutes and then cook in an electric heating mantle; the resulting water vapor is passed through a cooler and collected in a triangular flask; the distilled condensate is collected over a period of 4 hours, the essential oil is extracted with dichloromethane and dried at room temperature with anhydrous sodium sulfate; A light yellow essential oil with a mild aromatic taste is obtained. 3. The use of a vegetable essential oil as an anti-inflammatory, antibacterial, nitric oxide radical inhibiting and reactive oxygen species removing drug according to claim 1, characterized in that the forsythia flower essential oil mainly comprises the following components: n-tetradecane (36.93%), n-heneikosan (13.94%), 1-dodecane (8.11%) and n-nonane (6.32%). 4. The use of a vegetable essential oil as an anti-inflammatory, antibacterial, nitrogen oxide radical-inhibiting and reactive oxygen species-removing medicine according to one of claims 1 to 3, characterized in that the forsythia flower is the Korean forsythia flower (Forsythia koreana).