WO2022142324A1 - Gardenia extract and application thereof - Google Patents

Abstract

A gardenia extract, and use thereof in the preparation of a composition for improving the component, exercise capacity and senile dementia of a subject.

Description

Gardenia extract and its application

This application claims the priority of the Chinese patent application filed on December 31, 2020 with application number 202011632257.0, which is hereby incorporated by reference in its entirety.

technical field

This article relates to gardenia flower extracts, and also to novel uses of gardenia flower extracts to improve body composition and/or exercise capacity and/or symptoms of Alzheimer's disease.

Background technique

Gardenia jasminoides, also known as Mountain Gardenia, is a small evergreen shrub belonging to the Rubiaceae Gardenia genus. Gardenia is born in the mountains and has strong adaptability, mainly distributed in tropical and subtropical regions.

Gardenia fruits are rich in pigment components and are widely used as food additives or industrial dyes. Gardenia fruit is also a traditional Chinese medicine, which has the effect of promoting gallbladder and anti-inflammatory, and is clinically used to treat cholecystitis, jaundice hepatitis, etc.

Gardenia (referring to its flowers, the same below) is white or yellow and has a strong aroma. Gardenia is a precious natural spice, and the active ingredients it contains are used as high-grade spice in various products, which have the effect of clearing away heat and purging fire, treating fever, reducing stress, moisturizing and moisturizing the skin. According to research, the main components of gardenia volatile oil are linalool, jasmine lactone, farnesene, β-geranene, methyl benzoate, basilene, etc.

Still, other new uses for gardenias remain to be developed.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a gardenia extract prepared by the steps of:

1) take fresh gardenia, crush and ultrasonically treat in water for 1-2h;

2) extracting the sonicated mixture of step 1) with ether; and

3) removing ether to obtain the gardenia flower extract.

In another aspect, provided herein is a method of improving body composition or athletic performance in a subject comprising administering to the subject an effective amount of a gardenia extract.

In some embodiments, the Gardenia extract is an organic solvent (eg, ethanol, ether) extract of Gardenia. In other embodiments, the gardenia flower extract is an extract obtained from gardenia flower by CO ₂ supercritical extraction. In other embodiments, the gardenia extract is a gardenia extract prepared by the steps described above.

In some embodiments, the subject is a mammal, preferably a human, including healthy humans such as athletes, bodybuilders, dieters. In other embodiments, the subject is a patient, such as an Alzheimer's patient, preferably an early-stage Alzheimer's patient. In other embodiments, the subject is a person in need of muscle rehabilitation following muscle damage.

In some embodiments, wherein the body composition includes fat content or muscle-to-fat ratio, the exercise capacity includes muscle strength, exercise responsiveness, spontaneity, and/or exercise balance. Said improving body composition in a subject includes reducing fat content and/or increasing muscle to fat ratio. Said improving exercise capacity includes increasing muscle strength, endurance, motor response, locomotor activity and/or motor balance.

In some embodiments, the gardenia extract is administered to the subject by aromatherapy.

In another aspect, provided herein is the use of a gardenia flower extract as a health care product for improving body composition or athletic ability of a subject. In other words, provided herein is a method of improving body composition or exercise capacity in a subject for non-medical purposes, comprising administering to the subject a gardenia extract.

In some embodiments, the Gardenia extract is an organic solvent (eg, ethanol, ether) extract of Gardenia. In other embodiments, the gardenia flower extract is an extract obtained from gardenia flower by CO ₂ supercritical extraction. In other embodiments, the gardenia extract is a gardenia extract prepared by the steps described above.

In some embodiments, the subject is a mammal, preferably a human, including healthy individuals such as athletes, bodybuilders, dieters.

In some embodiments, wherein the body composition includes fat content or muscle-to-fat ratio, the exercise capacity includes muscle strength, exercise responsiveness, spontaneity, and/or exercise balance. Said improving body composition in a subject includes reducing fat content and/or increasing muscle to fat ratio. Said improving exercise capacity includes increasing muscle strength, endurance, motor response, locomotor activity and/or motor balance.

In some embodiments, the nutraceutical is administered to the subject by aromatherapy.

In some embodiments, the nutraceutical is a weight loss formulation.

In some embodiments, the nutraceutical is in the form of an inhalable formulation.

In another aspect, provided herein is the use of a gardenia extract in the manufacture of a medicament for improving body composition or exercise capacity in a subject.

In some embodiments, the Gardenia extract is an organic solvent (eg, ethanol, ether) extract of Gardenia. In other embodiments, the gardenia flower extract is an extract obtained from gardenia flower by CO ₂ supercritical extraction. In other embodiments, the gardenia extract is a gardenia extract prepared by the steps described above.

In some embodiments, the subject is a patient, such as an Alzheimer's patient, preferably an early-stage Alzheimer's patient. In other embodiments, the subject is a person in need of muscle rehabilitation following muscle damage.

In some embodiments, wherein the body composition includes fat content or muscle-to-fat ratio, the exercise capacity includes muscle strength, exercise responsiveness, spontaneity, and/or exercise balance. Said improving body composition in a subject includes reducing fat content and/or increasing muscle to fat ratio. Said improving exercise capacity includes increasing muscle strength, endurance, motor response, locomotor activity and/or motor balance.

In some embodiments, the gardenia extract is administered to the subject by aromatherapy.

In some embodiments, the medicament is in the form of an inhalable formulation.

In another aspect, provided herein is a method of ameliorating symptoms in a subject with Alzheimer's disease, comprising administering to the subject an effective amount of a gardenia extract.

In some embodiments, the gardenia extract is administered to the Alzheimer's subject by aromatherapy.

In another aspect, this article provides the use of a gardenia extract in the preparation of a medicament for improving the symptoms of Alzheimer's disease.

In some embodiments, the medicament is in the form of an inhalable formulation.

In some embodiments, the symptoms include loss of olfactory sensitivity, loss of olfactory discrimination, anxiety, and/or deposition of amyloid plaques in the hippocampus of the brain.

In some embodiments, the gardenia flower extract is an organic solvent extract of gardenia flower or an extract obtained by supercritical extraction of gardenia flower with CO ₂ , preferably a gardenia flower extract prepared by the steps described above thing.

Provided herein are gardenia extracts that can significantly improve body composition and/or exercise capacity and/or Alzheimer's symptoms in subjects by aromatherapy.

Description of drawings

Figure 1 shows the effect of aromatherapy intervention on body weight in mice. Over time, the weight of mice in each group increased slowly, and there was no significant difference between the groups. The experimental data are expressed as mean.

Figure 2 shows the effect of aromatherapy intervention on body composition in mice. (A) Changes in fat content after aromatherapy intervention. (B) Changes in the ratio of muscle to fat content after aromatherapy intervention. Experimental data are presented as mean ± standard error

means; compared with the experimental group, *P<0.05.

Figure 3 shows the effect of aromatherapy intervention on body weight in dementia model mice. (A) Effects of aromatherapy interventions on body weight in the short and medium term over time. (B) Body weight observation after the short- and mid-term aromatherapy intervention. (C) Effects of long-term aromatherapy intervention on body weight over time. (D) Observation of body weight after long-term aromatherapy intervention. Experimental data are presented as mean or mean ± standard error

Indicates; compared with the model group, *P<0.05, **P<0.01.

Figure 4 shows the effect of aromatherapy intervention on body composition in dementia model mice. (A) Effects of long-term aromatherapy intervention on muscle mass. (B) Effects on fat content after the end of a long-term aromatherapy intervention. Experimental data are presented as mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 5 shows the effect of aromatherapy intervention on mouse grip. (A) After 7 days of aromatherapy intervention, the maximum and mean values of the grasping force of mice were observed. (B) After 15 days of aromatherapy, the maximum and mean values of the grasping force of mice were observed. Experimental data are presented as mean ± standard error

means; compared with the model group, **P<0.01, ***P<0.001.

Figure 6 shows the effect of aromatherapy intervention on motor reaction time after water entry in mice. Experimental data are presented as mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 7 shows the effect of aromatherapy intervention on self-swimming in mice. (A) After 15 days of aromatherapy intervention, the swimming distance of mice self-swimming was observed. (B) After 15 days of aromatherapy, the swimming speed of self-swimming mice was observed. Experimental data are presented as mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 8 shows the effect of aromatherapy intervention on weight-bearing swimming in mice. (A) After 15 days of aromatherapy intervention, the swimming distance of mice in weight-bearing swimming was observed. (B) After 15 days of aromatherapy, the swimming speed of mice in weight-bearing swimming was observed. Experimental data are presented as mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 9 shows the effect of aromatherapy intervention on spontaneous motor ability in dementia model mice. (A) Spontaneous movement distance after short- and mid-term aromatherapy intervention. (B) Spontaneous motor speed after short and mid-term aromatherapy intervention. (C) Spontaneous movement distance after long-term aromatherapy intervention. (D) Spontaneous motor speed after short- and mid-term aromatherapy intervention. Experimental data are presented as mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 10 shows the effect of aromatherapy on motor balance in dementia model mice. (A) Residence time of mice on the rotarod 1 hour after aromatherapy intervention. (B) Residence time of mice on the rotarod 3 hours after aromatherapy intervention. Experimental data are presented as mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 11 shows the effect of aromatherapy on hindlimb blood flow velocity in dementia model mice. The experimental results were the blood flow velocity of the hindlimbs of the mice in each group after 6 months of aromatherapy. Experimental data are presented as mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 12 is a schematic diagram of the olfactory maze experiment.

Figure 13 is a schematic diagram of an olfactory recognition experiment.

Figure 14 shows the effect of aromatherapy intervention on olfactory sensitivity in dementia model mice. Experimental data are presented as mean or mean ± standard error

Indicates; compared with the model group, *P<0.05, **P<0.01.

Figure 15 shows the effect of aromatherapy on the ability of olfactory recognition in dementia model mice. Experimental data are presented as mean or mean ± standard error

Indicates; compared with the model group, *P<0.05, **P<0.01.

Figure 16 shows the effect of aromatherapy intervention on anxiety behavior in dementia model mice. (A) Observation of movement distance in the middle region. (B) Observation of movement time in the middle region. Experimental data are presented as mean or mean ± standard error

Indicates; compared with the model group, *P<0.05.

Figure 17 shows the effect of aromatherapy on the deposition of the pathological product, amyloid plaques, in the brains of dementia model mice. (A) Statistics of total area of amyloid plaques in the brain. (B) Immunofluorescence staining of amyloid plaques (green) and blood vessels (red) in the hippocampus of the brain. Experimental data are presented as mean or mean ± standard error

Indicates; compared with the model group, *P<0.05, **P<0.01.

Detailed ways

Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art.

"Gardenia extract" refers to a mixture obtained by contacting a liquid reagent (water or organic matter) with gardenia so that at least part of the chemical components in gardenia enter the liquid reagent. The gardenia can usually be pulverized prior to contacting the liquid agent to provide sufficient contact with the liquid agent. In order to fully extract or speed up the extraction, heating, microwave or ultrasonic treatment can also be used in the extraction process. In some embodiments herein, the liquid reagent used is an organic solvent such as diethyl ether or ethanol. In this case, after the ether or ethanol is volatilized, an oily gardenia extract including various components can be obtained. It has an aromatic odor and may also be called "gardenia essential oil". Gardenia extract itself is volatile, and some components can enter the air when it is in an unsealed state.

"Body composition" as used herein refers to the body composition of a subject, including the content of muscle, bone, water, fat, etc. in the body, especially the fat content (ratio of fat weight to body weight). In some cases, body composition also refers to the relative amount of muscle and fat in the body, ie, the ratio of muscle to fat mass in the body. Thus, when referring to "improving body composition", it refers to a decrease in body fat content and/or an increase in the muscle-to-fat ratio.

"Aromatherapy", also known as "aroma intervention" or "fumigation", refers to subjecting a subject to a volatile preparation (essential oil, such as the gardenia extract above) Co-located within a spatial range such that a subject can receive at least a portion of the volatile formulation by inhalation. Aromatherapy can be done in a variety of ways, for example, fumigation (using an incense lamp), sniffing (dropping the volatile preparation on a fabric such as a handkerchief and inhaling it near the nostrils, or nasal drops, nasal strips, etc.) such as dripping or sticking to the inside of the nose), wearing a sachet, etc. It is known in the art that some nebulizing devices can also be used to facilitate the subject to absorb the essential oil components. It should be pointed out that when aromatherapy is mentioned, it does not mean that it is necessarily used for the treatment of a specific disease, but can also be used to improve the health status of the subject, such as reducing fat content or increasing muscle strength, with Bodybuilding or health effects.

As used herein, "nutraceutical" refers to a non-pharmaceutical formulation that improves the condition of a subject. When the gardenia extract is used as a health product or mixed with other components as a health product (such as a slimming preparation), it means that it is administered to a healthy subject as a non-pharmaceutical preparation, involving the non-therapeutic use of the gardenia extract .

As used herein, "drug" refers to a pharmaceutical formulation that improves the condition of a subject. When the gardenia flower extract is used as a pharmaceutical product or mixed with other components, it means that it is administered as a pharmaceutical preparation to a subject suffering from a certain disease or at risk of a certain disease, involving the gardenia flower extract therapeutic use.

As used herein, "subject" refers to the subject to which various chemical or pharmaceutical agents are applied, including various vertebrates, such as mammals and birds, preferably primates, and more preferably humans. The term is often used interchangeably with "individual," "subject," "subject," and the like. A subject can be a patient or a healthy individual.

When referring to the amount of gardenia extract to be used, "effective amount" refers to an amount sufficient to cause the desired biological or medical effect in a subject, which can generally be determined by those skilled in the art according to the individual subject. circumstances, such as weight, age, physical condition and other factors to determine. When determining an "effective amount", consideration should not only be given to the concentration of the gardenia extract to which the subject is exposed per administration, but also the duration of the subject's exposure to the gardenia extract.

In the art, it is known to those skilled in the art that the extract of gardenia has various functions, such as anti-inflammatory, antipyretic, sedative, liver protection and other effects. During the research process, the inventor unexpectedly found that the gardenia extract can also improve the body composition and motor function of experimental animals.

In some embodiments, provided herein is the use of a gardenia extract to reduce body fat content (eg, weight loss) in a subject. The subject can be a healthy animal or human, or an obese population. This was achieved by having the subjects inhale the gardenia extract. For example, when the subject is an animal (eg, rat, dog, cat, pig, cow, sheep), the gardenia extract can be placed in a feedlot or shed; when the subject is a human, it can be placed in a residential place , office or sports venues place gardenia extract. When the gardenia extract is in an unsealed state, some components can be volatilized into the air and inhaled by the subjects to implement aromatherapy. The aromatherapy practice period is usually 7 days or longer, such as 15 days, 30 days, 3 months, 6 months or longer. Preferably, the subject is an Alzheimer's disease (AD) patient, more preferably an early-stage Alzheimer's patient.

In other embodiments, provided herein is the use of a gardenia extract to increase the ratio of muscle fat content in a subject (eg, bodybuilding or health care). The subject can be a healthy animal or human, or an obese population. This was achieved by having the subjects inhale the gardenia extract. For example, when the subject is an animal (such as a mouse, dog, cat, pig, cow, sheep, monkey), the gardenia extract can be placed in a feedlot or shed; when the subject is a human, it can be placed in the Place gardenia extract in residential, office or sports venues. When the gardenia extract is in an unsealed state, some components can be volatilized into the air and inhaled by the subjects to implement aromatherapy. The aromatherapy practice period is usually 7 days or longer, such as 15 days, 30 days, 3 months, 6 months or longer. Preferably, the subject is an senile dementia patient, more preferably an early senile dementia patient.

In other embodiments, provided herein is the use of a gardenia extract to increase muscle strength in a subject. The subject can be a healthy animal or human population (eg, an athlete), or a patient in need of increased muscle strength (eg, a patient recovering from a muscle injury). This was achieved by having the subjects inhale the gardenia extract. For example, when the subject is an animal (eg, rat, dog, cat, pig, cow, sheep), the gardenia extract can be placed in a feedlot or shed; when the subject is a human, it can be placed in a residential place , office or sports venues place gardenia extract. When the gardenia extract is in an unsealed state, some components can be volatilized into the air and inhaled by the subjects to implement aromatherapy. The aromatherapy practice period is usually 7 days or longer, such as 15 days, 30 days, 3 months, 6 months or longer. Preferably, the subject is an senile dementia patient, more preferably an early senile dementia patient.

In other embodiments, provided herein is the use of a gardenia extract to increase exercise capacity in a subject. The motor capacity includes, but is not limited to, motor response capacity, spontaneity, and/or motor balance. The subject can be a healthy animal or human population (such as an athlete), or a patient in need of increased exercise capacity (such as a patient recovering from a muscle injury). This was achieved by having the subjects inhale the gardenia extract. For example, when the subject is an animal (eg, rat, dog, cat, pig, cow, sheep), the gardenia extract can be placed in a feedlot or shed; when the subject is a human, it can be placed in a residential place , office or sports venues place gardenia extract. When the gardenia extract is in an unsealed state, some components can be volatilized into the air and inhaled by the subjects to implement aromatherapy. The aromatherapy practice period is usually 7 days or longer, such as 15 days, 30 days, 3 months, 6 months or longer. Preferably, the subject is an senile dementia patient, more preferably an early senile dementia patient.

In other embodiments, provided herein is the use of a gardenia extract to improve symptoms in Alzheimer's patients. These symptoms include, but are not limited to, loss of olfactory sensitivity, loss of smell recognition, anxiety, and/or deposition of amyloid plaques in the hippocampus of the brain. This was achieved by having the subjects inhale the gardenia extract. For example, when the subject is an animal (eg, rat, dog, cat, pig, cow, sheep), the gardenia extract can be placed in a feedlot or shed; when the subject is a human, it can be placed in a residential place , office or sports venues place gardenia extract. When the gardenia extract is in an unsealed state, some components can be volatilized into the air and inhaled by the subjects to implement aromatherapy. The aromatherapy practice period is usually 7 days or longer, such as 15 days, 30 days, 3 months, 6 months or longer. Preferably, the subject is an early-stage Alzheimer's patient.

In other embodiments, provided herein is the use of a gardenia flower extract in the manufacture of a nutraceutical for improving body composition or athletic performance in a subject. In some other embodiments, provided herein is the use of a gardenia extract in the manufacture of a medicament for improving body composition or athletic performance in a subject. The gardenia flower extract provided herein can be used directly as a health product or medicine, or can be formulated into an inhalable preparation together with other commonly used pharmaceutical carriers. If necessary, some substances that promote or reduce volatilization can be added to the gardenia extract.

The inventors also expect that the gardenia extract can improve the sports performance of subjects (such as athletes), or improve the labor ability of manual workers.

In view of the fact that the gardenia extract provided in this paper is extracted by organic solvent, the inventor believes that the main component should be a fat-soluble component, and combined with its volatility, it can be considered that the above-mentioned use or effect of the gardenia extract is Acts through one or more of these volatile lipid-soluble molecules.

In addition, although the inventor believes that the gardenia extract is inhaled into the body of the subject, it does not rule out that some components will penetrate into the blood and tissue of the subject through the skin or mucous membranes.

The present invention is further described below through specific embodiments.

Example 1 Preparation of gardenia extract by ether extraction

In this example, the gardenia flower extract was prepared by the ether extraction method.

Take 100-300g of fresh gardenia, crush and put it in a volatile oil extractor, add 5-8 times the amount of deionized water, set the ultrasonic power to 100-200W, ultrasonically grind for 1-2h; add 8-30 times anhydrous ether to extract ; After extraction, use an appropriate amount of anhydrous sodium sulfate to dry, evaporate the ether to obtain about 0.5-3mL of light yellow essential oil with aromatic odor, protect from light, seal, and store at 4°C.

Example 2 The effect of gardenia extract aromatherapy on body composition of wild-type mice

In this example, the effect of aroma intervention on the body composition of wild-type mice was evaluated through a body composition analysis experiment.

2.1 Materials

Experimental animals: wild-type C57 mice, male, purchased from Speifu (Beijing) Biotechnology Co., Ltd. Animal experiments strictly abide by the requirements of the Animal Ethics Committee of Beijing University of Traditional Chinese Medicine, the ethics code is BUCM-4-2020093002-3096.

Aromatic agent: Gardenia extract prepared in Example 1.

Instruments: nuclear magnetic resonance (MRI) fat measuring instrument (Huijia Biotechnology (Shanghai) Co., Ltd.), electronic balance.

2.2 Methods

Grouping and aromatherapy intervention: There are three groups in total, including the wild-type group (without aromatherapy intervention), the aromatherapy intervention group for 7 days, and the aromatherapy intervention group for 15 days, with 12 animals in each group, 6 animals/cage. Aroma intervention 1 time / day, 2 hours / time. Aroma intervention method: During the aroma intervention process, the mouse feed was withdrawn. The size of the rat cage is 39cm×18cm×18cm, and each rat cage is independently ventilated. 3-5uL of gardenia extract is dropped on the plastic sheet, spread out evenly, and attached to the inner wall of the rat cage. During the aromatherapy intervention, remain quiet throughout. The wild-type control group did not use gardenia extract for aromatherapy.

Determination of body mass and body composition: On the day after the completion of the aromatherapy intervention, the body weight of the mice was measured using an electronic balance, and the fat and muscle content of the mice were detected using a fat meter.

2.3 Results

During the aromatherapy intervention, the mice in each group were lively, in good mental state, with smooth fur, and no obvious adverse reactions were found. The weight of mice in each group increased slowly; there was no fluctuation in body weight in the aroma group, indicating that aromatherapy has certain safety (Figure 1).

Further body composition analysis found that compared with the wild-type group, the fat content of the mice was significantly reduced after the aromatherapy 7-day intervention and the aromatherapy 15-day intervention (Fig. 2A); The ratio of muscle-to-fat content in mice significantly increased after the day of intervention (Fig. 2B). The results suggest that aromatherapy interventions can reduce fat content and promote an increase in muscle-to-fat ratio.

Example 3 The effect of aromatherapy of gardenia extract on body composition of dementia model mice

In this example, the effect of aromatherapy intervention on the body composition of dementia model mice was evaluated through a body composition analysis experiment.

3.1 Materials

Experimental animals: Transgenic animals APP/PS1 double-transgenic mice (senile dementia model mice) and littermate wild-type mice, male, purchased from the Institute of Model Animals, Nanjing University, experimental animal license number: SCXK (Su) 2018 -0008, SYXK (Su) 2018-0027. Animal experiments strictly abide by the requirements of the Animal Ethics Committee of Beijing University of Traditional Chinese Medicine, the ethics code is BUCM-4-2019121002-4080.

Aromatic formula and reagents: the gardenia flower extract prepared in Example 1.

Instruments: nuclear magnetic resonance (MRI) fat measuring instrument (Huijia Biotechnology (Shanghai) Co., Ltd.), electronic balance.

3.2 Methods

Grouping and aroma intervention: Short and medium-term intervention experiments were divided into wild-type normal group, transgenic model group, aroma intervention group for 1 month, aroma intervention group for 1.5 months, aroma intervention group for 3 months; long-term intervention experiments were divided into wild type normal group, transgenic model group group, aromatherapy intervention group for 6 months. In the short-term and mid-term intervention experiments, aromatherapy intervention was performed once a day, 2 hours/time; in long-term intervention experiments, aromatherapy intervention was performed once every 4 days, 2 hours/time. There were 6 mice in each group. The specific intervention method is the same as that in Example 2.

Determination of body weight and body composition: On the next day after the completion of the aromatherapy intervention, the weight of the mice was measured with an electronic balance, and the fat and muscle content of the mice was measured with a fat meter.

3.3 Results

During the aromatherapy intervention, the mice in each group were lively, in good mental state, with smooth fur, and no obvious adverse reactions were found. The weight of mice in each group increased slowly; there was no fluctuation in body weight in the aroma group, indicating that aromatherapy has certain safety (Figure 3). In addition, in the long-term aromatherapy intervention experiment, we found that the weight of mice in the transgenic model group increased significantly; compared with the transgenic model group, aromatherapy intervention for 6 months could significantly reduce the weight gain (Figure 3D).

Further body composition analysis found that compared with the normal group, the muscle content of the mice in the model group had no significant change, but the fat content increased significantly; compared with the model group, aromatherapy intervention did not affect the muscle content, but significantly reduced the fat content (Fig. 4), indicating that fat increase is one of the important reasons for the weight gain of mice in the model group, and aromatherapy can improve fat levels.

Example 4 The effect of gardenia extract aromatherapy on exercise ability of wild-type mice

In this example, behavioral experiments were used to evaluate the improvement effect of aromatherapy on the exercise capacity of wild-type mice.

4.1 Materials

Experimental animals: wild-type C57 mice, male, purchased from Speifu (Beijing) Biotechnology Co., Ltd. Animal experiments strictly abide by the requirements of the Animal Ethics Committee of Beijing University of Traditional Chinese Medicine, the ethics code is BUCM-4-2020093002-3096.

Aromatic formula and reagents: the gardenia flower extract prepared in Example 1.

Instruments: Grip detector, water tank laser multi-speckle imaging (Leica, Germany).

4.2 Methods

Grouping and aromatherapy intervention: wild-type group, aromatherapy intervention group for 7 days, aromatherapy intervention group for 15 days, 12 animals in each group. Aroma intervention 1 time / day, 2 hours / time. The specific aroma intervention method is the same as that in Example 2. The following experiments were performed on the day following the completion of the aroma intervention.

Grip test: Turn on the grip tester and set it to zero. Open the MiniTR software, select "General" - "Processing Method" - "Tension". To start the measurement, place the mouse on the grip net and quickly pull the mouse to leave the grip net. Each mouse was measured 3-5 times.

Self-swimming experiment: The water tank is 80cm long, 60cm wide, and 30cm deep. The water temperature is kept constant at 25±2°C. The mice are gently placed in the center of the water surface, and the camera and timing are performed at the same time. After 10 minutes of recording, the camera stops. Change the animal and continue the experiment. Computer software was used to analyze the movement distance and speed of the mice in the water, and the observed time from the mice entering the water to the start of swimming was recorded as the movement reaction time.

Weight-bearing swimming experiment: the water tank is 80cm long and 60cm wide and the water depth is 30cm, the water temperature is kept constant, 25±2℃, and a camera is set 150cm above the water tank. A metal weight of 6% of the body weight of the mouse was suspended from the tail, and the mouse was gently placed in the center of the water surface, and video acquisition was started at the same time. The swimming distance and speed of the mice were recorded within 5 min. When the nostrils sank into the water for 5 s, the recording was stopped and the animal was replaced.

Statistical analysis: Experimental data are presented as mean ± standard error

Graphpad statistical software was used, and one-way ANOVA test was used to compare the significant differences between groups. The LSD test was used for homogeneity of variance, and the Dunnett's T3 test was used for heterogeneity of variance. P<0.05 indicated statistical significance.

4.3 Results

As shown in Figure 5-8, after 7 days or 15 days of aromatherapy intervention, the aromatherapy intervention group was significantly larger than the wild-type normal group (no aromatherapy intervention group) in terms of maximum gripping force and average gripping power, which suggested that aromatherapy intervention could enhance normal Muscle strength in mice. After 15 days of aromatherapy intervention, the aromatherapy intervention group was significantly better than the wild-type normal group in terms of motor reaction time, self-swimming distance and self-swimming speed into the water, suggesting that aromatherapy intervention can improve the exercise capacity of normal mice. After 15 days of aromatherapy intervention, both the weight-bearing swimming distance and the weight-bearing swimming speed were significantly greater in the aromatherapy intervention group than in the wild-type normal group, which further suggested that the aromatherapy intervention could improve the exercise capacity of normal mice.

Example 5 The effect of gardenia extract aromatherapy on the motor ability of dementia model mice

In this example, behavioral experiments were used to evaluate the improvement effect of aromatherapy on the motor ability of dementia mice.

5.1 Materials

Experimental animals: transgenic APP/PS1 double transgenic mice and littermate wild-type mice, male, purchased from the Institute of Model Animals, Nanjing University, experimental animal license number: SCXK (Su) 2018-0008, SYXK (Su) 2018-0027. Animal experiments strictly abide by the requirements of the Animal Ethics Committee of Beijing University of Traditional Chinese Medicine, the ethics code is BUCM-4-2019121002-4080.

Aromatic formula and reagents: Gardenia extract prepared in Example 1, Avertin (10g tribromoethanol mixed with 10ml tert-amyl alcohol, diluted to 1.2% with sterile distilled water, Beijing Hanlongda Company), Fluorescein isothiocyanate- dextran (FITC-dextran, purchased from SIGMA, Cat. No. BCBV4422).

Instruments: nuclear magnetic resonance (MRI) fat measuring instrument (Huijia Biotechnology (Shanghai) Co., Ltd.), fatigue rotarod instrument (Hong Kong Youcheng Biotechnology Co., Ltd.), two-photon microscope TCS SP5 (German Leica Company).

5.2 Methods

Grouping and aroma intervention: Short and medium-term intervention experiments were divided into wild-type normal group, transgenic model group, aroma intervention group for 1 month, aroma intervention group for 1.5 months, aroma intervention group for 3 months; long-term intervention experiments were divided into wild type normal group, transgenic model group group, aromatherapy intervention group for 6 months. In the short- and mid-term intervention experiments, aromatherapy intervention was performed once a day, 2 hours/time; in the long-term intervention experiment, aromatherapy intervention was performed once every 4 days, 2 hours/time. There were 6 mice in each group. The aroma intervention method is the same as that in Example 2. Unless otherwise stated, the following experiments were performed on the day following the completion of the aromatherapy intervention.

Open field experiment: To test the spontaneous motor ability and anxiety of mice, the open field box is an opaque box of 50x50x50cm, and the bottom surface is divided into 25 squares of 10x10cm on average. During the experiment, mice in each group were placed in the center of the bottom plate of the open field box, and the camera was turned on to record for 5 minutes. The walking distance (cm), time (s) and average speed (cm/s) of the mice in the entire open field and the middle area (9 grids in the middle) were recorded. After each experiment, wipe the bottom of the open field box with clean water.

Rotarod test: to detect exercise tolerance in mice. First let the mice adapt to the rotarod environment, place the mice on a stationary rotarod for 3 min, then adjust the speed to 5 r/min, and let the mice acclimate on the rotarod for 3 min. During the experiment, the rotational speed of the rotor was increased from 4 r/min to 40 r/min at a constant speed within 5 min. Rotarod test was performed 1h and 3h after the mouse aromatherapy intervention. When the mouse fell from the rotarod, the time (s) that the mouse stayed on the rotarod and the dropping time were recorded. Rotate bar speed (r/min), measure 3 times, and take the average value.

Hindlimb blood flow detection: The microvascular blood flow velocity of the hindlimbs of mice was observed by laser confocal microscope. The anesthetic Avertin was used to anesthetize the mice at a dose of 0.6 mL/20 g, and then the hind limbs were depilated with a depilatory cream. After injecting 200ul FITC-dextran from the mouse ocular venous sinus with an insulin syringe, place the mouse on a laser confocal microscope, make the skin of the hind limb depilation close to the lens, look for the blood flow of capillaries (less than 8um in diameter), and record the blood flow speed. The blood flow rate of each vessel is the average of the blood flow at three locations on that vessel.

Statistical analysis: Experimental data are presented as mean ± standard error

Graphpad statistical software was used to compare the significant differences between groups by one-way ANOVA test, in which LSD test was used for homogeneity of variance, and Dunnett's T3 test was used for heterogeneity of variance. P<0.05 indicated statistical significance. .

5.3 Results

The open field test was used to detect the locomotor activity of mice. In the short-term and mid-term aromatherapy intervention experiments, it was found that there was no significant difference in the spontaneous movement distance and movement speed of the mice in each group (Figure 9A and 9B). Long-term aromatherapy intervention experiments found that the spontaneous movement distance and movement speed of mice in the model group were significantly lower than those of wild-type mice, but aromatherapy intervention significantly increased the spontaneous movement distance and movement speed (Figure 9C and 9D).

Since long-term aromatherapy intervention can improve the spontaneous motor ability of dementia mice, we used the rotarod experiment to further observe the motor balance ability of mice. After aromatherapy intervention, the dwell time of mice on the rotarod was measured after 1 hour and 3 hours, respectively. The study found that compared with the wild type, the residence time of the model group was significantly reduced; aromatherapy intervention could significantly increase the residence time of dementia mice on the rotarod (Figure 10).

Next, using two-photon detection technology, we found that the aromatherapy intervention could significantly increase the blood flow velocity in the hindlimbs of mice (Fig. 11).

These findings suggest that long-term aromatherapy intervention can effectively improve the spontaneous motor ability and motor balance in dementia mice.

Example 6 The effect of gardenia extract aromatherapy intervention on the symptoms of dementia model mice

6.1 Materials

Experimental animals: Transgenic animals APP/PS1 double-transgenic mice (senile dementia model mice) and littermate wild-type mice, male, purchased from the Institute of Model Animals, Nanjing University, experimental animal license number: SCXK (Su) 2018 -0008, SYXK (Su) 2018-0027. Animal experiments strictly abide by the requirements of the Animal Ethics Committee of Beijing University of Traditional Chinese Medicine, the ethics code is BUCM-4-2019121002-4080.

Aromatic formula and reagents: the gardenia flower extract prepared in Example 1.

Instruments: open field box, camera, Smart analysis software.

6.2 Methods

Grouping and aroma intervention: Short- and mid-term intervention experiments were divided into wild-type normal group, transgenic model group, aroma intervention group for 1 month, aroma intervention group for 1.5 months; long-term intervention experiments were divided into wild-type normal group, transgenic model group, aroma intervention group for 6 months Group. In the short-term and mid-term intervention experiments, aromatherapy intervention was performed once a day, 2 hours/time; in long-term intervention experiments, aromatherapy intervention was performed once every 4 days, 2 hours/time. There were 6 mice in each group. Aroma intervention method: During the aroma intervention process, the mouse feed was withdrawn. The size of the rat cage is 39cm×18cm×18cm, and each rat cage is independently ventilated. 3-5uL of gardenia extract is dropped on the plastic sheet, spread out evenly, and attached to the inner wall of the rat cage. During the aromatherapy intervention, remain quiet throughout. The wild-type control group did not use gardenia extract for aromatherapy.

Olfactory Sensitivity Test: Observe the olfactory sensitivity of mice. Before the start of the formal experiment, the feeding conditions of the mice were changed to 0.5 g feed/mice/day, with free water for a total of 3 days, during which the status of the mice was monitored every day. As shown in Figure 12, use 4 pieces of opaque cardboard to separate a small space from the 4 corners of the 50×50×50cm open field, one side is close to the wall of the open field, and the other side is set aside for the mice to pass through. gap. A piece of 0.5 g mouse chow for the test was randomly placed behind a cardboard. The mice were placed in the middle of the open field, and they were free to search for food. At the same time, the camera equipment above the open field was turned on to record the activities of the mice within 5 minutes. After the observation, the feed residue and mouse excrement were removed, and the bottom surface of the open field box was wiped with 75% ethanol to prevent the interference of mouse odor.

Detection of olfactory recognition ability: Observe the olfactory recognition ability of mice. The experiment is divided into two parts, training and testing. Training: As shown in Figure 13, a circular container with a diameter of 5cm and a height of 3cm is placed in the four corners of the open field (50×50×50cm), and the bottoms of the four containers are respectively placed with star anise, garlic, 10 μl of ethanol, and 5 μl of acetic acid , the container is filled with clean corn bran (bedding for mouse feed). A piece of 0.5g mouse feed was placed in a container with star anise, and the mice were placed in the middle of the open field to freely search for food, and were trained for 5 consecutive days, once a day. The first day of training, the feed is placed on the surface of the litter; the second day of training, 1/2 of the feed is buried in the litter; the third day of training, the whole feed is buried in the litter; the fourth day of training, 1/2 of the feed is buried In litter; the fifth day of training, the feed was entirely buried in the litter. 24 hours after the training, the test is carried out. Test: The whole feed was buried in anise litter, and the mice were placed in the middle of the open field to move freely and search for feed. The activity of the mice within 5 minutes was observed, and the time when the mice turned to the feed and grasped and gnawed with their forepaws was recorded. After the observation, the feed residue and mouse excrement were removed, and the bottom surface of the open field box was wiped with 75% ethanol to prevent the interference of mouse odor.

Open field test: observe the anxiety of mice. The open field is an opaque cube box of 50 × 50 × 50 cm, and the bottom of the box is divided into 25 squares of 10 × 10 cm with 4 black lines horizontally and vertically. During the experiment, the mice were placed in the square in the middle of the open field to observe the free movement of the mice in the open field within 5 minutes. After the observation, the feed residue and mouse excrement were removed, and the bottom surface of the open field box was wiped with 75% ethanol to prevent the interference of mouse odor. Use Smart software to analyze the movement distance and time of mice in the middle 9 grid area of the open field.

Detection of pathological product amyloid plaques: Frozen brain slices (10 μm thick) were prepared, equilibrated at room temperature for 5 min, washed three times with PBS buffer, 5 min each time, and shaker at 120 rpm/min. Dye with 0.5% thioflavin solution for 8 min, wash twice with 50% ethanol solution, 5 min each time, and shake at 120 rpm/min. Mount the slides with water-soluble mounting medium and store at 4°C in the dark. Observed and photographed under an inverted fluorescence microscope.

6.3 Results

The olfactory maze test can detect the olfactory sensitivity of mice. The shorter the time for the mice to search for food, the higher the olfactory sensitivity. The experimental observation found that compared with wild-type mice, the model group mice (APP/PS1 transgenic mice) had a significantly increased time to find food, indicating a loss of olfactory sensitivity. After short- and mid-term aromatherapy intervention, the time to search for food was significantly reduced, indicating that aromatherapy intervention can effectively improve olfactory sensitivity (Figure 14).

The olfactory recognition experiment can detect the olfactory recognition ability of mice, that is, the ability of mice to distinguish different odors. The shorter the time for mice to search for food, the stronger their olfactory recognition ability. The experimental observation found that compared with the wild-type mice, the model group mice (APP/PS1 transgenic mice), the time to find food was significantly increased, indicating the loss of olfactory recognition ability. After long-term aromatherapy intervention, the time to search for food was significantly reduced, indicating that aromatherapy intervention can effectively improve the ability of olfactory recognition (Figure 15).

The open field test can detect the anxiety behavior of mice, and the longer the distance or time the mice move in the middle area of the open field, the less anxious behavior. The experimental observation found that compared with wild-type mice, the movement distance and movement time of the model group mice were significantly reduced in the middle area, indicating that the anxiety behavior of the model mice was more significant. After long-term aromatherapy intervention, the movement distance (Figure 16A) and movement time (Figure 16B) of the mice in the middle area of the open field were significantly increased, indicating that aromatherapy intervention can effectively improve the anxiety behavior of model mice.

Thioflavin staining was used to observe the changes of pathological product amyloid plaques in the brains of dementia mice. The experimental observation found that there was no amyloid plaque deposition in the hippocampus of the wild-type mice; the model group and the aroma intervention group had amyloid plaque deposition in the brain. Compared with the model group, the total area of amyloid plaques in the brain was significantly reduced after aromatherapy (Figure 17A). Further observation found that compared with the model group, the aromatherapy intervention group found that the plaque structure was loose and the blood vessels around the plaque were abundant (Figure 17B). effect.

Claims (16)

1. Gardenia extract, which is prepared by the following steps:

   1) take fresh gardenia, crush and ultrasonically treat in water for 1-2h;

   2) extracting the sonicated mixture of step 1) with ether; and

   3) removing ether to obtain the gardenia flower extract.
2. A method of improving body composition or exercise capacity in a subject comprising administering to the subject an effective amount of a gardenia flower extract.
3. The method of claim 2, wherein the gardenia extract is administered to the subject by aromatherapy.
4. Application of gardenia flower extract as a health product for improving body composition or athletic ability of subjects.
5. Use of a gardenia flower extract in the preparation of a medicament for improving body composition or exercise ability of a subject.
6. The method according to claim 2 or 3 or the application according to claim 4 or 5, wherein the gardenia flower extract is the organic solvent extract of gardenia flower or the gardenia flower obtained by _CO supercritical extraction Extract, preferably the gardenia extract of claim 1.
7. The method of claim 2 or 3 or the application of claim 4 or 5, wherein the subject is a mammal, preferably a human, including healthy people, such as athletes, bodybuilders, dieters; patients, For example, senile dementia patients, preferably early senile dementia patients, or muscle rehabilitation personnel.
8. The method of claim 2 or 3 or the use of claim 4 or 5, wherein the body composition includes fat content or muscle-to-fat ratio, and the exercise capacity includes muscle strength, exercise responsiveness, locomotor activity, and / or motor balance ability.
9. The application according to claim 4, wherein the health care product is a slimming preparation.
10. The application according to claim 4 or 5, wherein the health product or medicine is in the form of an inhalable preparation.
11. A method of improving symptoms in a subject with Alzheimer's disease, comprising administering to the subject an effective amount of a gardenia extract.
12. The method of claim 11, wherein the gardenia extract is administered to the Alzheimer's subject by aromatherapy.
13. The method of claim 11 or 12, wherein the symptoms include loss of olfactory sensitivity, loss of olfactory discrimination, anxiety and/or amyloid plaque deposition in the hippocampus of the brain.
14. The method according to any one of claims 11-13, wherein the gardenia extract is an organic solvent extract of gardenia or an extract obtained from gardenia by CO ₂ supercritical extraction, preferably claim 1 The gardenia flower extract.
15. The application of gardenia flower extract in the preparation of medicine for improving the symptoms of Alzheimer's disease.
16. 16. The use of claim 15, wherein the medicament is in the form of an inhalable formulation.

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