CN107998220B - Composition for reducing food intake, delaying hunger sensation, and controlling body weight - Google Patents
Composition for reducing food intake, delaying hunger sensation, and controlling body weight Download PDFInfo
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- CN107998220B CN107998220B CN201610951314.9A CN201610951314A CN107998220B CN 107998220 B CN107998220 B CN 107998220B CN 201610951314 A CN201610951314 A CN 201610951314A CN 107998220 B CN107998220 B CN 107998220B
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Abstract
The invention relates to a composition for reducing food intake, delaying hunger sensation and controlling weight, in particular to the composition which is prepared by mixing a tea flower extract and a gorse lineariifolia extract according to a certain proportion. The composition can inhibit the levels of neuropeptide Y and AGRP mRNA, and achieve the purposes of reducing food intake, delaying hunger sensation, and controlling body weight, especially reducing food intake of obesity or obesity-induced metabolic disorder disease patients, and can be used as oral medicine, health food, special medical formula food or functional nutritional food for weight control and management.
Description
Technical Field
The invention relates to a composition, in particular to a composition which achieves the effects of reducing food intake, delaying hunger sensation and controlling body weight by synergistically inhibiting the mRNA hormone level secretion of neuropeptide Y/agouti peptide gene-related protein (AGRP), and can be used for preventing and treating obesity and metabolic disorder diseases caused by obesity.
Background
With the change of human dietary structure and dietary habits, the incidence of obesity and metabolic disorders caused by obesity is increasing. Unfortunately, obese people are not adequately restrained from food, but they carelessly look to themselves fattened a day. Why do we face the same food 24 hours a day, do we differ in stature? We have been suggesting that obese people eat less and do more exercise, however, this approach does not really work.
Human beings have developed a complex energy balance regulating system in long-term struggle with hunger, and the energy intake and storage capacity is far greater than the energy consumption function. In modern times of extremely abundant substances, this energy balance mechanism has been a troubling cause of obesity in humans. Research data indicate that body weight is controlled by gastrointestinal hormones in humans. Hormone levels affect hunger and are also linked to areas of the brain that are pleasurable to perceive. The obesity patients become fat in the true phase of unbalanced secretion of gastric satiety/hunger hormone level, total eating of old hunger, out of control of diet and finally become fat. It should be noted that simply by simply reducing the amount of food intake, the food is quickly digested, becomes hungry soon after consumption, and the brain again signals ingestion. Therefore, the method for controlling the secretion level of relevant ingestion hormones, reducing the food intake and delaying the hunger sensation becomes the most powerful breakthrough scheme for overcoming obesity in human. Unfortunately, there are currently few compositions that are safe and effective for regulating gut hormone levels, reducing food intake, and delaying hunger.
Based on the level of gastrointestinal hormones, particularly food intake related neuropeptide hormones, the hormone plays a great role in the occurrence and development process of obesity, so the research on the role of the hormones in the pathogenesis of the obesity, particularly the aspects of reducing food intake, delaying hunger sensation and controlling body weight can provide certain scientific basis and prevention and treatment means for the prevention and treatment of the obesity and metabolic disorder diseases caused by the obesity. Antagonizing the expression of the hormone level of the food intake related neuropeptides is also becoming a new target for treating obesity and metabolic disorder caused by obesity.
More and more research results show that the hypothalamic energy balance regulation abnormality may be a central mechanism of obesity development, and is noteworthy about the level of food intake-related neuropeptide hormone, in which neuropeptide y (npy)/agouti gene-related protein (AGRP) hormone secreted from the same neuron of its arcuate nucleus has the effects of appetite enhancement and energy consumption inhibition.
Neuropeptide Y (NPY) is a 36 amino acid polypeptide widely distributed in the central and peripheral nervous systems, a strong stimulator of food intake, one of the most potent pro-food neuropeptide hormones, and increasing NPY levels can lead to excessive food intake. Research shows that the NPY hormone level in the hypothalamus influences the nutritional state of the organism and is an important factor for long-term regulation of energy balance. NPY is present in higher concentrations in the hypothalamus and is secreted mainly by the arcuate nucleus (ARC) neurons, which extend to the paraventricular and dorsal-medial nuclei, both of which are rich in NPY immunoreactive nerve endings. Under conditions of hunger, diabetes and strenuous exercise, NPY stimulates food intake and efficient storage of energy, mainly manifested by hyperphagia and reduced thermogenesis in brown adipose tissue, which are of great importance for restoration of energy balance and survival. Thus, the regulation of energy metabolism by NPY neurons may be in that they are able to significantly perceive a reduction in body energy storage and restore energy balance by stimulating feeding and attenuating thermogenesis. Activation of the neuropeptide Y Y5 receptor has been shown to result in overabundance of appetite, and therefore NPY Y5 receptor antagonists may be useful in treating obesity caused by overabundance of appetite, in promoting weight loss or in preventing weight gain.
The Agouti Gene Related Protein (AGRP) is a polypeptide consisting of 50 amino acid residues. Moussa et al found that a yellow mouse was obese due to gene mutation of Agouti peptide (Agouti), and had symptoms of high food intake, insulin resistance, hyperglycemia, and the like. Agouti is a peptide hormone produced by hair follicle cells, and has the effect of inhibiting the production of melanin by pigment cells caused by alpha-MSH. alpha-MSH has an inhibitory effect on feeding and is an endogenous ligand for the central melanocortin receptor (MCR). Agouti acts mainly on the MC1 receptor to exert a blocking effect, AGRP is mainly expressed in the middle of the hypothalamic arcuate nucleus (ARC), has high affinity for MC3R and MC4R, and is a competitive antagonist for α -MSH. Increased AGRP mRNA fasted state expression; transgenic mice overexpressing AGRP are obese but the skin color is not altered; intracerebroventricular injection of AGRP results in mice feeding and weight gain, in a dose-effect relationship.
While NPY and AGRP are food preference signals, their mechanisms of action are not the same. NPY acts directly through NPY receptors, whereas AGRP antagonizes the effects of α -MSH primarily by competitively binding the melanocortin receptors MC3R and MC 4R. Since NPY and AGRP are produced by the same neuron as ARC, factors that can affect synthesis and release of NPY can also affect AGRP. Therefore, inhibition of NPY/AGRP mRNA expression is also becoming a new target for reducing food intake, delaying hunger sensation, and controlling weight concerns.
Tea flower (Tea blossom) is a flower of Camellia sinensis (L.) o.kuntze of Camellia in the family of theaceae, is widely planted in southern and central provinces in china, and is approved as a new food material by the Ministry of health in 2013, 1 month and 4 days (No. 1 of Ministry of health in 2013). The tea flower extract has been found to have the effect of controlling body weight, and is mainly related to inhibiting pancreatic lipase activity, inhibiting fat absorption, improving fat metabolism and inhibiting the increase of blood neutral fat level. In patent CN105685995, "a composition for reducing weight", a composition containing navy bean extract, tea flower extract, green tea extract and inulin, animal experiments prove that the composition can inhibit weight gain of obese model rats, reduce fat accumulation in body of obese model rats, and reduce food utilization rate of obese model rats, which suggests that the composition has a function of reducing weight, but the test result proves that the composition has no significant effect on food intake and total caloric intake of animals. CN 104366468, "a weight-reducing meal replacement composition and its preparation method", provides a meal replacement composition containing isolated soy protein, milk protein, whey protein, water-soluble dietary fiber, tea flower extract, crystalline fructose and malto-oligosaccharide, wherein data of satiety test indicates that the hunger sensation is reduced after eating the composition, and the highest proportion of hunger felt after 5 hours is 12.32% (example 6), wherein the tea flower extract accounts for 0.62%, the water-soluble dietary fiber accounts for 17.8%, and the total protein accounts for 57.7% (isolated soy protein accounts for 8.9%, milk protein-casein accounts for 4.4%, whey protein accounts for 44.4%); the lowest proportion of hunger felt after 5 hours was 6.31% (example 2) with tea flower extract 0.18%, water-soluble dietary fiber 13.4%, total protein 58% (soy isolate 22.3%, milk protein-casein 22.3%, whey protein 13.4%). In a 30-day diet meal replacement test, the average body weight of example 2 was reduced by 9kg, and the average body fat rate was reduced by 6.22%; the average body weight of example 6 was reduced by 5kg and the body fat percentage was reduced by 2.69%. In examples 1, 3,4 and 5, the ratio of the tea flower extract was 0.09%, 0.89%, 0.45% and 0.36%, the average body weight was reduced by 4.2kg, 2.9kg, 4.9kg and 3.2kg, and the average body fat percentage was reduced by 2.13%, 5%, 3.09% and 3.17%, respectively. 2 human clinical test evidences all indicate that the proportion of the tea flower extract and the water-soluble dietary fibers in the composition is increased, and the expected dose-effect related effects on satiety and weight and body fat rate reduction are not presented, on the contrary, the proportion of the soybean protein isolate and the milk protein (casein) is increased, the strong satiety effect is generated, the weight and body fat rate is obviously reduced, and the composition is supposed to generate the weight reduction effect mainly related to the satiety generated by the synergy of the soybean protein isolate and the milk protein (casein). Therefore, although the camellia sinensis flower extract has certain effects of delaying gastric emptying time and delaying hunger sensation, clinical tests show that the camellia sinensis flower extract has the curative effects of satiety, hunger sensation delay and weight and body fat rate reduction and cannot meet the requirement of better weight control.
The maidenhair, also known as Rooibos (Rooibos or red bush tea), is derived from leguminous (Leguminosae) plants in south Africa (Latin school name: Aspalathus Linearis (Brum. f.) R.Dahlgren), and is eaten by leaves and thin stems in a brewing manner, and is approved as a new food material by Ministry of health 6 and 26 days 2014 (publication No. 12 in 2014). Patent CN105248769, a process for preparing an instant powder of Rooibos tea, discloses that Rooibos tea contains no caffeine and is rich in various flavonoids, and modern pharmacological studies show that the tea has effects in resisting oxidation, controlling diabetes and its complications, and delaying aging. In patent CN104127594, "plant extract composition for preventing and controlling diabetes mellitus" mentioned that roiibos are rich in antioxidants, tea polyphenol content is higher than that of green tea, and has special effects on resisting gene mutation, cancer, inflammation and viruses, animal experiments prove that the composition containing dietary fiber, grape seed extract and south african black tea extract provided by the invention has significant effects on lowering fasting blood glucose and postprandial blood glucose for 2 hours, but the glucose tolerance value is reduced and has no significant difference compared with that of a model control group. CN 101765433, "an extract of Bush for treating diabetes", discloses that Bush tea has the functions of relieving insomnia, nervous tension, stomach spasm and allergy, and contains flavonoids with strong antioxidant and free radical scavenging activities, and has anticancer and anti-arteriosclerosis potential. The black bush tea water extract provided by the invention has the function of controlling blood sugar, the active component of the extract is a flavonoid compound Aspalathin (3 '-C-f 1-D-glucopyranosyl-2', 3,4,4 ', 6' -pentahydroxydihydrochalcone), and a monkey animal experiment proves that oral administration of 1-2.5mg/kg of red bush tea water extract 7D can effectively reduce the blood sugar of a nonhuman primate (Chlorocebus aethiops), and the sparrow cord extract can be used for controlling blood sugar. No study and report of the sparrow's broom extract on obesity are found.
Disclosure of Invention
Modern people are becoming fatter, our diets are rich and even too plentiful, and the problem of solving the metabolic disorder diseases caused by obesity is urgent. The present inventors have conducted studies to screen plant extracts or plant extract compositions that can inhibit the expression of NPY/AGRP mRNA hormone levels, synergistically reduce food intake, delay hunger, and control body weight, using the NPY/AGRP mRNA expression level as a target. The present inventors have surprisingly found that a camellia sinensis extract and a gorse lineariifolia extract can effectively synergistically inhibit the expression of NPY/AGRP mRNA hormone levels, have synergistic effects of reducing food intake, delaying hunger sensation, and controlling body weight, and show more excellent effects than the camellia sinensis extract or the gorse lineariifolia extract used alone, thereby completing the present invention.
The invention aims to provide a composition for reducing food intake, delaying hunger sensation and controlling body weight, which is used for preventing or treating obesity and metabolic disorder diseases caused by obesity. The medicinal composition is prepared by compounding a tea flower extract and a gorse canadensis flower extract according to a certain proportion.
The composition can synergistically inhibit the secretion of mRNA hormone level of neuropeptide Y/agouti peptide gene-related protein (AGRP), and achieves the effects of reducing food intake, delaying hunger sensation and controlling body weight.
The tea flower of the present invention is a flower selected from the Camellia sinensis plant Camellia sinensis of the Camellia genus of the Camellia family, and the maidenhair is a leaf and a thin stem selected from the leguminous (Leguminosae) plant of south africa (latin scientific name: Aspalathus Linearis (brum. f.) r. dahlgren).
In one embodiment of the invention, the composition is prepared from tea flower extract and sparrow linearis extract as raw materials, wherein the weight ratio of the tea flower extract to the sparrow linearis extract is 1-20: 20-1.
In one embodiment of the invention, the content of total saponins in the tea flower extract is not less than 2.0%, and the content of Chakasaponin II is not less than 1.0%; the content of total flavone in the extract of Genistis lineariifolia is not less than 2.0%, and the content of Aspalathin is not less than 0.1%.
In one embodiment of the invention, the weight ratio of the tea flower extract and the sparrow velveteen extract of the composition is preferably 1-5: 5-1.
In one embodiment of the invention, the total saponins of camellia sinensis flower is not less than 50.0%, Chakasaponin II is not less than 20.0%, the total flavonoids of gorgonia lineariifolia is not less than 50.0%, and Aspalathin is not less than 2%.
The determination of the total saponins of tea tree flowers is carried out by taking Chakasaponin II (shown in figure 1 and figure 2) with the purity of 92-98% prepared by repeatedly separating and refining the total saponins of tea tree flowers through column chromatography as a reference substance, detecting according to a conventional HPLC external standard quantitative method (C18 chromatographic column 4.6 x 260 mm; mobile phase: methanol: trifluoroacetic acid (65: 35V/V); wavelength 230 nm; column temperature 40 ℃ and flow rate 1.0ml/min) of a person skilled in the art, and calculating the content of the total saponins of tea tree flowers and the content of the Chakasaponin II contained in a tea tree flower extract according to the total area of saponin peaks; taking rutin as a reference substance, detecting and calculating the content of the sparteinia lineata total flavonoids according to a conventional ultraviolet-visible light spectrophotometry (Jianya Nu et al. determination of the total flavonoids in health food, physicochemical inspection-chemical breakdown, 2003,39(5): 307;) by a person skilled in the art, repeatedly separating and refining the sparteinia lineata total flavonoids by column chromatography to obtain Aspalathin with the purity of 92% -98% as a reference substance (see figures 4 and 5), and detecting and calculating the content of the Aspalathin in the sparteinia lineata extract according to a conventional HPLC gradient elution and external standard quantitative method (Chashenghua et al. New resource food, namely doctor tea HPLC fingerprint spectrum research, modern traditional Chinese medicine research and practice, 2010, 24 (3): 69-71) by the person skilled in the art.
It is another object of the present invention to provide a method for preparing the composition of the present invention. In one embodiment of the present invention, the preparation method comprises the steps of extracting and purifying the tea flower and the maidenhair flower with an extraction solvent selected from the group consisting of water and ethanol.
In one embodiment of the invention, the preparation method specifically comprises 1) crushing tea flowers, extracting with 6-15 times of water or ethanol with different concentrations for 1-3 times at 25-100 ℃ for 0.5-24 hours each time, filtering, combining filtrates, concentrating under reduced pressure, wherein the pressure can be 1.2-25.5kpa, the temperature is 40-50 ℃, or concentrating with a membrane, the pore diameter of the membrane can be 0.2 μm, the membrane in the membrane separation treatment adopts a nanofiltration membrane with the molecular weight cutoff of 100-1000 Dal, the total saponins of tea flowers can be cut off, water separation is facilitated, the tea flower extract is concentrated, the working temperature in the membrane concentration process is controlled below 40 ℃, and the total saponins of tea flowers are prevented from being damaged due to high heat; spray drying at air inlet temperature of 120-; 2) pulverizing herba Caraganae Sinicae, extracting with 6-15 times of water or ethanol of different concentrations for 1-3 times at 25-100 deg.C for 0.5-24 hr, filtering, mixing filtrates, and concentrating under reduced pressure at 40-50 deg.C under 1.2-25.5kpa or membrane pore size of 0.2 μm. The membrane in the membrane separation treatment adopts a nanofiltration membrane with the molecular weight cutoff of 100-1000 Dal, can cut off the sparrow general flavone to facilitate water separation, concentrates the sparrow general flavone extraction solution, controls the working temperature below 40 ℃ in the membrane concentration process to prevent the sparrow general flavone from being damaged due to high heat, and performs spray drying, wherein the air inlet temperature can be 120 plus 160 ℃, the air outlet temperature can be 70-100 ℃, or performs freeze drying to obtain the sparrow extract; 3) mixing the tea flower extract and the broom cypress extract uniformly according to the weight ratio of 1-20:20-1, adding a proper amount of pharmaceutically acceptable auxiliary materials, and mixing uniformly to obtain the tea flower extract.
Based on that tea flowers and gorse canary are new food raw materials approved by the Ministry of health, the tea is brewed. In consideration of food safety, the functional food prepared by using the composition of the tea flower extract and the sparrow leucomelas extract as raw materials is preferably extracted by water, and the sparrow leucomelas extract does not contain caffeine, so that the functional food is good in safety, and not only suitable for adults, but also suitable for weight control of obese children and prevention and treatment of metabolic disorder diseases caused by obesity.
In one embodiment of the present invention, the composition of the present invention is preferably prepared as follows: 1) pulverizing tea flower, extracting with 8-12 times of water or ethanol of different concentrations for 1-3 times at 25-100 deg.C for 0.5-24 hr, filtering, mixing filtrates, concentrating under reduced pressure (pressure of 1.2-25.5kpa, temperature of 40-50 deg.C or membrane concentration, membrane pore diameter of 0.2 μm, and membrane separation treatment using nanofiltration membrane with molecular weight cutoff of 100-1000 Dal to intercept total saponin of tea flower, facilitate separation of water or ethanol solution, and concentrating tea flower extract solution, wherein the working temperature during membrane concentration is controlled below 40 deg.C. Preventing the total saponins of tea tree flower from being damaged due to high heat, passing the concentrated solution through a pretreated D-101 macroporous resin column, eluting with deionized water, discarding the eluate, eluting with 50-90% ethanol, collecting the eluate, recovering ethanol, and drying to obtain tea tree flower extract; 2) pulverizing the sparrow velutina, extracting for 1-3 times with 8-12 times of water or ethanol with different concentrations at 25-100 ℃ for 0.5-24 hours each time, filtering, combining the filtrates, concentrating under reduced pressure at a pressure of 1.2-25.5kpa at 40-50 ℃ or concentrating with a membrane having a pore size of 0.2 μm, wherein the membrane in the membrane separation treatment is a nanofiltration membrane with a molecular weight cutoff of 100-1000 Dal, and can cut off the sparrow velutina total flavonoids, so that water or ethanol solution can be conveniently separated, and the sparrow velutina extract solution can be concentrated. Controlling the working temperature below 40 ℃ in the membrane concentration process to prevent the total flavonoids of the gordon malachite from being damaged due to high heat), passing the concentrated solution through a pretreated AB-8 macroporous resin column, eluting with deionized water, discarding the eluent, eluting with 50-90% ethanol, collecting the eluent, and recovering ethanol to obtain the gordon malachite extract; 3) uniformly mixing the tea flower extract and the gorse canadensis flower extract according to the weight ratio of 1-5:5-1, adding a proper amount of pharmaceutically acceptable auxiliary materials, and uniformly mixing to obtain the tea flower extract.
The preparation method of the pharmaceutical composition can be carried out by adopting a conventional method in the pharmaceutical field and using conventional pharmaceutical excipients. For example, the tea flower extract and the broom cypress extract which are compounded according to a certain proportion are mixed with any one or more than one pharmaceutically commonly used acceptable carriers or auxiliary materials by a conventional method and then are prepared into various oral dosage forms. Such carriers as diluents, anticaking agents, and the like. Specifically, the carrier is, for example, resistant starch, maltodextrin, resistant dextrin, microcrystalline cellulose, hydroxypropylmethylcellulose, aerosil, or the like. According to the requirements, the composition of the invention is used as an active ingredient, other acceptable raw materials and auxiliary materials are added, and the composition is prepared into a medicament, health food, formula food for special medical purposes or functional nutritional food preparation suitable for oral administration by using a conventional technology in pharmaceutics, and can be any one of the following formulations: powder, granules, tablets, hard capsules, oral liquid, suspension and the like, and is used for achieving the purpose of preventing and treating obesity and metabolic disorder diseases caused by the obesity through weight control.
Drawings
FIG. 1 chemical structure of active ingredient Chakasaponin II of tea flower extract
FIG. 2 shows HPLC chromatogram of tea flower saponin Chakasaponin II control, with detection wavelength of 230nm and retention time of 37.856 min.
FIG. 3 HPLC chromatogram of tea flower extract 3 of example 1, with detection wavelength of 230nm and retention time of 38.003min as Chakasaponin II peak.
FIG. 4 shows the chemical structure of Aspalathin as the active ingredient of the extract of Genistis linearis
FIG. 5 shows HPLC chromatogram of Aspalathin control of Genistis sinica Linne extract, with detection wavelength of 278nm and retention time of 44.952.
FIG. 6 HPLC chromatogram of the extract 3 of Genistis leucomae of example 2, with detection wavelength of 278nm and retention time of 44.998min as Aspalathin peak.
Detailed Description
Example 1: preparation method of tea flower extract
1) Tea flower extract 1: pulverizing tea flower, extracting with 15 times of water at 30 deg.C for 3 times, each for 3 hr, centrifuging, filtering, mixing filtrates, concentrating under reduced pressure (12kpa,45 deg.C) to density of 1.1g/ml, and spray drying (air inlet 140 deg.C, air outlet 80 deg.C, material liquid temperature 70 deg.C) to obtain tea flower extract 1. With Chakasaponin II as a reference substance, the content of total saponins in tea flowers is 3.6 percent and the content of Chakasaponin II is 1.6 percent by HPLC (high performance liquid chromatography) determination;
2) tea flower extract 2: pulverizing tea flower, extracting with 6 times of 50% ethanol under reflux at 85 deg.C for 3 times, each for 1 hr, centrifuging, filtering, mixing filtrates, concentrating under reduced pressure (12kpa,45 deg.C) to density of 1.1g/ml, and spray drying (air inlet 140 deg.C, air outlet 80 deg.C, material liquid temperature 70 deg.C) to obtain tea flower extract 2. By taking Chakasaponin II as a reference substance and adopting an HPLC method for determination, the content of the total saponin of the tea flowers is 8.6 percent, and the content of the Chakasaponin II is 4.0 percent;
3) tea flower extract 3: crushing tea flowers, leaching for 3 times at 40 ℃ by using 10 times of water, each time for 24 hours, carrying out centrifugal filtration, combining filtrates, concentrating the filtrate to 10% of the original volume by using a membrane, wherein the membrane aperture is 0.2 mu m, the membrane in the membrane separation treatment adopts a nanofiltration membrane with the molecular weight cutoff of 100-1000 Dal, the total saponin of the tea flowers can be cut off, the water separation is convenient, the tea flower extract solution is concentrated, the working temperature in the membrane concentration process is controlled below 40 ℃, the total saponin of the tea flowers is prevented from being damaged due to high heat, and freeze drying is carried out to obtain a light brown tea flower extract 3 (see figure 3). By taking Chakasaponin II as a reference substance and adopting an HPLC method for determination, the content of the total saponin of the tea flowers is 2.2 percent, and the content of the Chakasaponin II is 1.2 percent;
4) tea flower extract 4: crushing tea flowers, extracting for 3 times at 80 ℃ by using 8 times of water, each time for 3 hours, carrying out centrifugal filtration, combining filtrates, concentrating the mixed filtrates to 10% of the original volume by using a membrane, wherein the membrane aperture is 0.2 mu m, a nanofiltration membrane with the molecular weight cutoff of 100-1000 Dal is adopted, the working temperature in the membrane concentration process is controlled below 40 ℃, and the cutoff solution is a tea flower extract concentrated solution. Passing the concentrated solution through pretreated D-101 macroporous resin column, eluting with deionized water, discarding eluate, eluting with 50% ethanol, collecting eluate, recovering ethanol, and drying under reduced pressure to obtain tea flower extract 4. With Chakasaponin II as a reference substance, the content of total saponins in tea flowers is 52.0 percent and the content of Chakasaponin II is 20.8 percent by HPLC (high performance liquid chromatography) determination;
5) tea flower extract 5: pulverizing tea flower, extracting with 70% ethanol under reflux for 2 times, soaking in 12 times of 70% ethanol at 25 deg.C overnight (12 hr), and extracting under reflux at 80 deg.C for 1.5 hr; adding 10 times of 70% ethanol, and reflux-extracting at 80 deg.C for 1 hr. Centrifuging, filtering, mixing filtrates, concentrating under reduced pressure (12kpa,45 ℃) until the density is 1.1g/ml, passing the concentrated solution through a pretreated D-101 macroporous resin column, eluting with deionized water, removing impurities such as saccharides until the molish reaction is negative, discarding the eluent, eluting with 50% ethanol, collecting the eluent, recovering ethanol, and drying under reduced pressure to constant weight to obtain the tea flower extract 5. The content of total saponins in tea plant flowers is 80.0% and the content of Chakasaponin II is 40.0% by HPLC (high performance liquid chromatography) with Chakasaponin II as a reference substance.
Example 2: preparation method of Genistis lineariifolia extract
1) Extract of malachite linearis 1: pulverizing herba Caraganae Sinicae, extracting with 15 times of water at 30 deg.C for 3 times, each for 3 hr, centrifuging, filtering, mixing filtrates, concentrating under reduced pressure (12kpa,45 deg.C) to density of 1.1g/ml, and spray drying (air inlet 140 deg.C, air outlet 80 deg.C, material liquid temperature 70 deg.C) to obtain herba Caraganae Sinicae extract 1. Rutin is used as a reference substance, and the content of the total flavonoids in the broom cypress leaves is 6.5 percent by adopting an ultraviolet spectrophotometry; by taking Aspalathin as a reference substance and adopting an HPLC method for determination, the content of Aspalathin is 0.26%;
2) extract of malachite linearis 2: pulverizing flos Caraganae Sinicae, reflux-extracting with 6 times of 60% ethanol at 85 deg.C for 3 times, each for 3 hr, centrifuging, filtering, mixing filtrates, concentrating under reduced pressure (12kpa,45 deg.C) to density of 1.1g/ml, and spray-drying (air inlet 140 deg.C, air outlet 80 deg.C, material liquid temperature 70 deg.C) to obtain flos Caraganae Sinicae extract 2. Rutin is used as a reference substance, and ultraviolet spectrophotometry is adopted to determine that the total flavone of the linear broom is 10.8%; by taking Aspalathin as a reference substance and adopting an HPLC method for determination, the content of Aspalathin is 0.43 percent;
3) extract of malachite linearis 3: crushing the sparrow vellica, leaching for 3 times by 10 times of water at 40 ℃, each time for 24 hours, centrifuging and filtering, combining filtrates, concentrating the membrane to 10% of the original volume, wherein the membrane aperture is 0.2 mu m, the membrane in the membrane separation treatment adopts a nanofiltration membrane with the molecular weight cutoff of 100-1000 Dal, the membrane can cut off the sparrow vellica total flavonoids so as to facilitate water separation, concentrating the sparrow vellica extract solution, controlling the working temperature below 40 ℃ in the membrane concentration process so as to prevent the sparrow vellica total flavonoids from being damaged due to high heat, and freeze-drying to obtain the sparrow vellica extract 3 (see figure 6). Rutin is used as a reference substance, and the content of the total flavonoids in the broom cypress leaves is 3.0 percent by adopting an ultraviolet spectrophotometry; by taking Aspalathin as a reference substance and adopting an HPLC method for determination, the content of Aspalathin is 0.12%;
4) extract of malachite linearis 4: crushing the sparrow velutina, leaching for 3 times at 80 ℃ by using 8 times of water, each time for 3 hours, centrifuging, filtering, combining filtrates, concentrating the filtrate by using a membrane to 10% of the original volume (the membrane aperture is 0.2 mu m, a nanofiltration membrane with the molecular weight cutoff of 100-1000 Dal is adopted, the working temperature in the membrane concentration process is controlled below 40 ℃, and the cutoff solution is the sparrow velutina extract concentrated solution). Passing the concentrated solution through pretreated AB-8 macroporous resin column, eluting with deionized water, discarding eluate, eluting with 60% ethanol, collecting eluate, recovering ethanol, and drying under reduced pressure to obtain extract 4. Rutin is used as a reference substance, and the content of the total flavonoids in the broom cypress is 54.6 percent by adopting an ultraviolet spectrophotometry; by taking Aspalathin as a reference substance and adopting an HPLC method for determination, the content of Aspalathin is 2.2%;
5) extract of malachite linearis 5: pulverizing herba Caraganae Sinicae, reflux-extracting with 85% ethanol for 2 times, soaking in 12 times of 85% ethanol at 25 deg.C overnight (12 hr), and reflux-extracting at 80 deg.C for 1.5 hr; adding 8 times of 85% ethanol, and reflux-extracting at 80 deg.C for 1 hr. Combining the extracting solutions, carrying out centrifugal filtration, combining filtrates, concentrating the filtrate under reduced pressure to 0.2-0.8 g (crude drugs)/ml, passing the concentrated solution through a pretreated AB-8 macroporous resin column, eluting with deionized water, removing impurities such as saccharides and the like until the molish reaction is negative, discarding the eluent, eluting with 60% ethanol, collecting the eluent, recovering ethanol, and drying under reduced pressure to constant weight to obtain the maidenhair extract 5. Rutin is used as a reference substance, and the content of the total flavonoids in the broom cypress is 80.0 percent by adopting an ultraviolet spectrophotometry; aspalathin was 3.8% by HPLC using Aspalathin as a control.
Example 3: preparation of the compositions of the invention
The environmental humidity is controlled below 40%, the temperature is controlled at 18-25 ℃, the tea flower extract and the gorse linearis extract (in table 1, the tea flower extract n from example 1 is called tea n for short, and the gorse linearis extract n from example 2 is called line n for short) are respectively crushed and then sieved by a 40-100 mesh sieve, the samples are weighed according to the compounding proportion of the composition shown in table 1, a proper amount of carriers (such as pharmaceutically acceptable auxiliary materials such as resistant starch, maltodextrin, resistant dextrin, microcrystalline cellulose, hydroxypropyl methyl cellulose, aerosil and the like), the tea flower extract, the gorse linearis extract and a proper amount of carriers (such as pharmaceutically acceptable auxiliary materials such as resistant starch, maltodextrin, resistant dextrin, microcrystalline cellulose, hydroxypropyl methyl cellulose, aerosil and the like) are crosswise added into a mixer, the mixing time is 5-30 min, the rotating speed is 20-30 r/min, sieving the mixed powder with 20-40 mesh sieve, and quantitatively packaging. The water content is less than 5 percent, and the microbial limit meets the regulation.
TABLE 1 extracts and compounding ratios contained in the compositions of the invention
Example 4 preparation of powders, granules, tablets, hard capsules, oral liquids, suspensions of the pharmaceutical compositions of the invention
Powder preparation: the environmental humidity is controlled to be below 40%, the temperature is controlled to be 18-25 ℃, the raw and auxiliary materials are crushed, sieved and weighed, a proper amount of auxiliary materials capable of being accepted by the medicinal materials, the composition of the embodiment 3, other compound raw materials and a proper amount of auxiliary materials capable of being accepted by the medicinal materials are alternately added into a mixer, the mixing time is 5-30 min, the rotating speed is 20-30 r/min, the mixed powder is sieved by a 20-40 mesh sieve, and the mixture is quantitatively packaged. The water content is less than 5 percent, and the microbial limit meets the regulation.
Granules: the composition of the embodiment 3, other compound raw materials and a proper amount of auxiliary materials are taken. Mixing, spraying, granulating, drying, sieving, grading, and packaging.
And (3) tablet preparation: further tabletting the obtained granule, drying, and making into tablet.
Hard capsule preparation: encapsulating the obtained granule to obtain capsule.
Oral liquid: taking the composition of the embodiment 3, other water-soluble compound raw materials and a proper amount of auxiliary materials, stirring and dissolving in water, quantitatively filling and sterilizing.
Suspension: taking the composition of the embodiment 3, other compound raw materials, an appropriate amount of auxiliary materials such as an emulsifier and the like, stirring and dissolving in water, shearing at a high speed, homogenizing at a high pressure, quantitatively filling, and sterilizing.
EXAMPLE 4 Effect of the compositions of the invention on NPY/AGRP mRNA hormone level expression, rat body weight variation and food intake
The effect of the composition of the present invention on the expression of NPY/AGRP mRNA hormone levels was examined using a high-fat diet obese rat model.
Assuming a normal control group, a high-fat diet model group, a composition 11 low dose group (tea 4, 67.5 mg/(kg. d) + line 4, 67.5 mg/(kg. d)) prepared according to example 3, a composition 11 medium dose group (tea 4, 135 mg/(kg. d) + line 4, 135 mg/(kg. d)) a composition 11 high dose group (tea 4, 270 mg/(kg. d) + line 4, 270 mg/(kg. d)), a tea flower extract group (tea 4, 135 mg/(kg. d)), a delavay flower extract group (line 4, 135 mg/(kg. d)) and a composition 10 medium dose group (tea 3, 135 mg/(kg. d) + line 3, 432 mg/(kg. d)) prepared according to example 3, wherein tea 4 (using the tea flower extract 4 of example 1), line 4 (using the delavay flower extract 4 of example 2), and, Tea 3 (using tea flower extract 3 of example 1), and string 3 (using string broom extract 3 of example 2. the dosage of the composition administered is expressed in terms of the amount of tea flower extract and string broom extract administered per kg per day to rats, and as described above and below, the tea flower extract is referred to as "tea" and the string broom extract is referred to as "string".
The subjects were Sprague-Dawley rats, gavaged daily, food intake recorded, and body weight measured before and at the end of the trial.
The results show that the expression of the hypothalamus NPY and AGRP mRNA of the rats is increased after the rats are fed with high-fat diet, the expression of the hypothalamus NPY and AGRP mRNA is reduced after the rats fed with the high-fat diet are fed with the composition, and in addition, the composition obviously reduces the food intake and inhibits the weight increase and is obviously superior to the single administration of the total saponins of tea plant flowers and the total flavonoids of broom cypress flowers. It is concluded that the composition can obviously reduce the expression of hypothalamus NPY and AGRP mRNA, and inhibit the relation of central nerves to suppress hypothalamus appetite signals, thereby achieving the effects of reducing food intake and weight and providing a feasible basis for searching effective obesity prevention and treatment measures.
1 materials and methods
1.1 materials
Healthy male SD rats, 10 days after weaning, body weight (100 ± 2g), animals and basal diet (3500 kcal/kg caloric value) were provided by the fourth department of medical and institutional animal center, certification: SCXK (military) 2002-.
The high-fat feed is prepared by self, and the formula comprises the following components: every 100g of the feed contains 12g of egg yolk powder, 10g of lard oil, 0.2g of pig bile salt, 8g of casein, 15g of milk powder, 0.1g of salt, 0.4g of yeast powder, 58g of basic feed and 4346kcal/kg of calories.
The Trizol reagent, the reverse transcription kit and the PCR reaction kit are provided by Invitrogen, Bao bioengineering Dalian Co., Ltd and Bao bioengineering Dalian Co., Ltd, respectively. The 18s ribosomal RNA, NPY and AGRP primers were synthesized by Bao bioengineering Daizian GmbH.
1.2 methods
The SD rats were 80, randomly grouped into 10 groups, and divided into 8 groups. The normal control group was given basal diet, and the other groups were given high-fat diet. The preparation method of the medicine comprises the following steps: dissolved with 0.9% physiological saline to the desired concentration.
The administration route is as follows: and (5) performing intragastric administration. Dose capacity: 5ml/kg body weight.
The administration time is as follows: 2 times daily; the drug was administered at 9 am and 5 pm for 8 weeks. The normal control group and the high fat diet group were administered with 0.9% physiological saline 2 times per day by gavage, and the remaining groups were sequentially administered with gavage at the dose, and the dose was maintained until the 8 th week. Daily food intake (g) was recorded for each rat and body weight was measured before and at the end of the experiment. When the experiment is finished at the end of 8 weeks, the hypothalamus of the rat is taken, and NPY and AGRP are detected by a real-time fluorescence quantitative polymerase chain reaction method (RT-PCR).
1.3RNA extraction and real-time fluorescence quantification
The rats in the experimental group were treated at the end of week 8, and the hypothalamus was removed after craniotomyAnd (4) preserving in a refrigerator at the temperature of minus 80 ℃. Trizol decomposes hypothalamic tissue, extracts RNA according to the instructions of the reagents, and measures the concentration and purity of total RNA using a nucleic acid protein content meter. Calculating the amount of total RNA required by reverse transcription, adding corresponding reagents according to the instructions of a reverse transcription kit, and carrying out reverse transcription under the reaction conditions: total RNA was transcribed into cDNA at 37 ℃ for 15min and 85 ℃ for 5 s. The contents of NPY, AGRP and 18s ribosomal RNA were determined using an RG-3000RT-PCR instrument. The fluorescence used for real-time fluorescence quantification is SYBR Premix Ex TaqTM, the volume of the reaction solution is 20 mul, and sequentially SYBR Premix Ex Taq 10 mul, two primers 0.4 mul, template 1.6 mul and deionized water 7.6 mul. The primer sequences for NPY, AGRP and 18s ribosomal RNA are as follows: NPY: p1 is TGTGAAACCAGTCTGCCTGT, P2: CAACGACAACAAGGGAAATG, respectively; AGRP: p1 is ACGGTGGGCCCTTTATTAG, P2: GGACACAGCTCAGCAACATT, respectively; 18s ribosomal RNA: p1 is TTCGGAACTGAGGCCATGAT, P2: CGAACCTCCGACTTTCGTTC, performed in two steps, PCR pre-denaturation 95 ℃ for 10s, PCR reaction: 95 ℃ for 5s, 60 ℃ for 30s, repeating 40 cycles (NPY) or 45 cycles (AGRP). Results analysis adopted 2-△△CTThe method comprises obtaining △ CT by subtracting target gene (NPY or AGRP) from housekeeping gene (18 s ribosomal RNA) and selecting a CT value from control group, calibrating △ CT, obtaining △△ CT value by subtracting calibrated CT from △ CT, and calculating 2-△△CTThe numerical value is the relative quantitative content of the objective gene.
1.4 statistical methods
The experimental data were first analyzed for differences between overall means using one-way ANOVA variance, and then multiple comparisons between means were tested using Post-Hoc-Tests LSD. All statistical treatments were statistically processed using SPSS statistical software.
2 results
2.1 Effect of the composition on the expression of NPY and AGRP mRNA in the hypothalamus of obese rats on high fat diet
As shown in table 2, rat hypothalamus NPY and AGRP mRNA expression was increased after administration of high fat diet compared to normal control group. Both NPY and AGRP mRNA expression were significantly reduced to different extents following administration of high, medium and low dose of composition 11 compared to the high fat diet model group; after the tea flower extract 4 is singly administered, the NPY and AGRP mRNA expression is obviously reduced, the reduction range is equivalent to that of the low-dose composition 11, but the reduction range is not as much as that of the medium-dose and high-dose composition 11 administration group; the gorse linearis extract 4 group showed a downward trend but no statistical difference. The composition is obviously superior to the single administration of the tea flower extract or the maidenhair linearis extract, and the combination of the tea flower extract and the maidenhair linearis extract shows obvious synergistic inhibition effect on the expression of the hypothalamus NPY and AGRPmRNA of the high-fat diet obese rat.
TABLE 2 Effect of composition 11 on hypothalamic NPY and AGRP mRNA expression after 8 weeks of high fat diet obese rat study
(
P < 0.01vs normal control group; # p < 0.05vs high-fat diet model group; # p < 0.01vs high fat diet model group)
As shown in table 3, compared with the high fat diet model group, the NPY and AGRP mRNA expressions were significantly decreased, but the decrease was significantly lower than that of the composition 11 group after the administration of the composition 10 dose, i.e., the composition 11, in which the effective components of the extract including the total saponins of camellia sinensis and the total flavonoids of rosa laevigata, had a stronger effect of down-regulating the hypothalamus NPY and AGRP mRNA expressions, and had significant differences compared with the composition 10. In addition, the efficacy of the dose group in the composition 10 is equivalent to that of the tea flower extract 4 (see table 2) used alone, but the content of the tea flower saponin contained in the composition 10 is significantly lower than that of the tea flower extract 4, which suggests that the combined broom cypress extract 3 has a significant synergistic effect.
TABLE 3 Effect of different compositions on hypothalamic NPY and AGRP mRNA expression after 8 weeks of high fat diet obese rat study
(
P < 0.05vs high-fat diet model group, p < 0.01vs high-fat diet model group; # p < 0.05 composition 10vs composition 11)
2.2 Effect of the composition on weight Change in high fat diet obese rats
As shown in table 4, the body weight of the rats in the model group and each of the administration groups significantly increased after the administration of the high fat diet compared to the normal control group, and the body weight of the rats in the model group continued to increase after 8 weeks after the administration of the high fat diet compared to the normal group. The body weight of rats in the high, medium and low dose composition 11 groups was significantly reduced to different extents after 8 weeks of administration, compared to the high fat diet model group; after single administration of tea flower extract 4, the body weight of the rat is obviously reduced, but the reduction range is less than that of 3 dose groups of composition 11; the gorse linearis extract 4 group showed a downward trend but no statistical difference. The composition is obviously superior to the single administration of the tea flower extract or the maidenhair herb extract, and the combination of the tea flower extract and the maidenhair herb extract shows a remarkable synergistic inhibition effect on the weight reduction of the high-fat diet obese rats.
TABLE 4 Effect of composition 11 on body weight Change before and after 8 weeks of high fat diet obese rat test
(
P < 0.001vs normal control group; # p < 0.05vs high-fat diet model group; # p < 0.01vs high fat diet model group; # p < 0.001vs high fat diet model group)
As shown in table 5, compared with the high fat diet model group, the weight of the rats was significantly reduced after 8 weeks of administration of the composition 10, but the reduction degree was significantly lower than that of the composition 11, i.e., the composition 11, in which the effective ingredients of the extract include camellia sinensis total saponin and sparrow broom total flavonoids were greatly increased, had a stronger effect on weight loss of the rats, had a larger reduction range, and had significant differences compared with the composition 10. In addition, the efficacy of the dose group in the composition 10 is equivalent to that of the tea flower extract 4 (see table 4) used alone, but the content of the tea flower saponin contained in the composition 10 is significantly lower than that of the tea flower extract 4, which suggests that the combined broom cypress extract 3 has a significant synergistic effect.
TABLE 5 Effect of different compositions on the body weight Change before and after 8 weeks of high fat diet obese rats
(
P < 0.05vs high-fat diet model group, p < 0.01vs high-fat diet model group, p < 0.001vs high-fat diet model group; # p < 0.05 composition 10vs composition 11)
2.3 Effect of the composition on Total food intake in high fat diet obese rats
As shown in table 6, the total food intake of the model group rats was significantly increased after 8 weeks of administration of the high fat diet, compared to the normal control group. The total food intake of rats was significantly reduced to different extents after 8 weeks of administration of the high, medium and low dose of composition 11, compared to the high fat diet model group; after 8 weeks of single administration of the camellia sinensis flower extract 4, the total food intake of the rats is remarkably reduced, and the reduction range is equivalent to that of the low-dose composition 11, but is not as much as that of the medium-dose and high-dose composition 11 administration groups; the gorse linearis extract 4 group showed a downward trend but no statistical difference. The composition is obviously superior to the single administration of the tea flower extract or the sparrow leucomelas extract, and the combination of the tea flower extract and the sparrow leucomelas extract shows obvious synergistic inhibition effect on the reduction of the total food intake of the high-fat diet obese rat.
TABLE 6 Effect of composition 11 on Total food intake after 8 weeks of high fat diet obese rat study
(
P < 0.01vs normal control group; # p < 0.05vs high-fat diet model group; # p < 0.001vs high fat diet model group)
As shown in table 7, compared with the high fat diet model group, the total food intake of rats after 8 weeks of administration of the composition 10 was significantly reduced, but the degree of reduction was significantly lower than that of the composition 11, i.e., the composition 11, in which the effective ingredients of the extract include total saponins of camellia sinensis and total flavonoids of rosa laevigata, had a stronger effect of reducing the food intake of rats, and had significant differences compared with the composition 10. In addition, the efficacy of the dose group in the composition 10 is equivalent to that of the tea flower extract 4 (see table 6) used alone, but the content of the tea flower saponin contained in the composition 10 is significantly lower than that of the tea flower extract 4, which suggests that the combined broom cypress extract 3 has a significant synergistic effect.
TABLE 7 Effect of different compositions on Total food intake after 8 weeks of high fat diet obese rats
(
P < 0.05vs high-fat diet model group, p < 0.01vs high-fat diet model group; # p < 0.05 composition 10vs composition 11)
In the experiment, the weight of the rats is obviously increased after the administration of high-fat diet, the calorie intake is obviously increased, and the NPY and AGRP mRNA expression in hypothalamic plasma dissolving tissues is obviously increased, which indicates that the increase of the weight and the food intake of the high-fat diet obese rats is related to the high expression of the NPY and the AGRP mRNA. In the experiment, after the high fat diet obese rat is fed with the composition, the weight and the total food intake are reduced, the expression of NPY and AGRP mRNA is reduced, the composition is supposed to play the roles of reducing the weight and reducing the total food intake by reducing the expression of NPY and AGRP mRNA of hypothalamus, and the composition is obviously superior to the single medicine of the tea flower extract and the broom cypress extract, and the effect of synergistically inhibiting the expression of NPY and AGRP mRNA is shown after the tea flower extract and the broom cypress extract are jointly used. In addition, test results show that the compound composition has better curative effect after the content of total saponins contained in the tea flower extract and the content of total flavonoids contained in the broom cypress extract are improved.
Example 5 human feeding trial test
Tea flower and broom cypress are new food raw materials approved by the Ministry of health, and the tea flower extract (example I, tea flower total saponin 3) and the broom cypress total flavone (example I, broom cypress extract 3) used in the test are both extracted by water, so that the safety is better. Based on the above animal test, the dosage of the medium dosage composition 10 (tea 3, 135 mg/(kg. d) + line 3, 432 mg/(kg. d)) was selected and calculated as the human dosage (tea 3, 150mg/(70 kg. d) + line 3, 480mg/(70 kg. d)) based on the 1/10 dose. The dosage of tea is 3, 50 mg/time plus 3, 160 mg/time, calculated according to 1 time before three meals a day.
The BMI value is 25-35 (BMI is less than or equal to 24 and less than or equal to 27, overweight; BMI is less than or equal to 27 and less than or equal to 30, mild obesity; BMI is less than or equal to 30 and less than or equal to 35, moderate obesity), adults aged 20-58 years serve as the study subjects, and the study subjects are in male and female halves, and the results of the pre-test examination of all the subjects are good in health (euglycemia) and voluntarily participate in the experiment. In 2 months, under the condition of normal diet, 30 experimental group subjects take 50mg of tea flower extract 3, 160mg of sparrow velveteen extract 3 and a composition of the two (composition 10) 15 minutes before three meals in the morning, in the middle and at night, and the weight, the waist circumference, the BMI value and the body fat rate of the experimental group subjects are measured regularly for 10 persons in each group, and the blood sugar value is tested after the experiment. After 15 days of the human body trial test, the subjects participated in quantitative standard lunch, the food intake rate was measured and the number and the ratio of hunger sensation generated 3h, 5h and 8h after meal were evaluated, and 10 persons were taken as normal control group.
1. Effect of composition 10 on adult quantitative Standard postprandial Total food intake and hunger
As shown in Table 8, the hunger sensation was generated by 80% of the normal group after 3 hours. Compared with a normal control group, the total food intake of the group taking the composition 10 15 minutes before a meal is greatly reduced, and the hunger feelings generated in 5 hours and 8 hours respectively account for 10 percent and 40 percent; the total food intake of the tea flower extract group (tea 3) taken alone is remarkably reduced, but the reduction amplitude is remarkably lower than that of the composition 10 group (p is less than 0.001), and the hunger feeling rates of 5h and 8h respectively account for 30% and 70%; the total food intake of the group with the extract of Genistis linearis (line 3) taken alone was slightly lower than that of the normal group, but there was no statistical difference, and the hunger was observed at rates of 80% and 100% in 5h and 8h, respectively. The composition is obviously superior to the single administration of the tea flower extract or the sparrow leucomelas extract, and the combination of the tea flower extract and the sparrow leucomelas extract has obvious synergistic inhibition effect on reducing the intake and delaying hunger sensation.
TABLE 8 composition 10 of the present invention quantifies the standard postprandial total food intake rate and postprandial hunger sensation development time test results
(
P < 0.05vs normal control group; p < 0.001vs normal control group; composition group with # p < 0.001 vs)
2. Effect of composition 10 on changes in adult body weight and body fat Rate
As shown in table 9, the composition 10 taken 15 minutes before meal significantly reduced the body weight, BMI value, waist circumference, and body fat percentage after 8 weeks; the weight, BMI value, waistline and body fat rate of the tea flower extract group (tea 3) which is taken alone are all reduced, but the reduction range is obviously lower than that of the composition group (p is less than 0.001); the weight, BMI value, waist circumference and body fat rate of the group of the maidenhair extract (line 3) taken alone slightly decreased, but the changes were not obvious. The above groups have no influence on blood glucose level. The composition is obviously superior to the single administration of the tea flower extract or the sparrow leucomelas extract, the combination of the tea flower extract and the sparrow leucomelas extract shows obvious synergistic inhibition effect on the reduction of weight, BMI value, waist circumference and body fat rate, and the safety is good.
TABLE 9 Effect of inventive composition 10 on BMI, body weight, waist circumference, body fat percentage, and blood glucose in adults after 8 weeks of trial
(
Composition group of 0.001 vs. p
The human body test results show that the weight, BMI value, waist circumference and body fat rate of a subject taking the composition of the tea flower extract and the sparrow leucomelas extract are all obviously reduced, and blood sugar value is not abnormally changed, so that the combination of the tea flower extract and the sparrow leucomelas extract has obvious synergistic effect, and the weight control of the subject is obviously related to the obvious reduction of food intake of the subject and the obvious time delay of hunger sensation.
The above-described embodiments are intended to illustrate the substance of the present invention, but are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the invention.
Claims (6)
1. The application of a composition of a tea flower extract and a maidenhair flower extract in preparing a product for weight control is characterized in that the composition is prepared by taking the tea flower extract and the maidenhair flower extract as raw materials, wherein the weight ratio of the tea flower extract to the maidenhair flower extract is 1-20: 20-1; the content of total saponins in the tea flower extract is not less than 2.0%, and the content of Chakasaponin II is not less than 1.0%; the content of the total flavone in the gordon europaea extract is not less than 2.0 percent, and the content of Aspalathin is not less than 0.1 percent; the preparation method of the composition comprises the following steps:
1) pulverizing tea flower, extracting with 6-15 times of water or ethanol of different concentrations for 1-3 times at 25-100 deg.C for 0.5-24 hr each time, filtering, mixing filtrates, concentrating under reduced pressure or membrane concentrating, and spray drying or freeze drying to obtain tea flower extract;
2) pulverizing flos Caraganae Sinicae, extracting with 6-15 times of water or ethanol of different concentrations for 1-3 times at 25-100 deg.C for 0.5-24 hr each time, filtering, mixing filtrates, concentrating under reduced pressure or membrane concentrating, spray drying or freeze drying, and drying to obtain flos Caraganae Sinicae extract;
3) mixing the tea flower extract and the broom cypress extract uniformly according to the weight ratio of 1-20:20-1, adding a proper amount of pharmaceutically acceptable auxiliary materials, and mixing uniformly to obtain the tea flower extract.
2. Use according to claim 1, wherein the weight ratio of tea flower extract to broom cypress extract is preferably 1-5: 5-1.
3. The use as claimed in claim 2, wherein the camellia sinensis flower extract has a total saponin content of not less than 50% and contains Chakasaponin II of not less than 20%; the content of the total flavone in the gordon malachitum extract is not less than 50%, and the content of Aspalathin is not less than 2%.
4. Use according to claim 3, preferably prepared as follows:
1) pulverizing tea flower, extracting with 8-12 times of water or ethanol of different concentrations for 1-3 times at 25-100 deg.C for 0.5-24 hr, filtering, mixing filtrates, concentrating under reduced pressure or membrane concentrating, passing the concentrated solution through pretreated D101 macroporous adsorbent resin column, eluting with deionized water, discarding eluate, eluting with 50-90% ethanol, collecting eluate, recovering ethanol, and drying under reduced pressure to obtain tea flower extract;
2) pulverizing flos Caraganae Sinicae, extracting with 8-12 times of water or ethanol of different concentrations for 1-3 times at 25-100 deg.C for 0.5-24 hr each time, filtering, mixing filtrates, concentrating under reduced pressure or membrane concentrating, passing the concentrated solution through pretreated AB-8 macroporous adsorbent resin column, eluting with deionized water, discarding eluate, eluting with 50-90% ethanol, collecting eluate, recovering ethanol, and drying under reduced pressure to obtain flos Caraganae Sinicae extract;
3) mixing the tea flower extract and the broom cypress extract uniformly according to the weight ratio of 1-5:5-1, adding a proper amount of pharmaceutically acceptable auxiliary materials, and mixing uniformly to obtain the tea flower extract.
5. The use according to any one of claims 1 to 4, wherein said composition synergistically inhibits secretion of neuropeptide Y/agouti gene-related protein (AGRP) mRNA hormone levels to achieve the effects of reducing food intake, delaying hunger sensation, and controlling body weight.
6. Use according to any one of claims 1 to 4, wherein the product is an oral medicament, nutraceutical, food formulation for special medical use or functional nutritional food for weight management, characterized in that the composition according to any one of claims 1 to 4 is mixed with pharmaceutical excipients to make an oral preparation, which may be any one of powder, granule, tablet, hard capsule, oral liquid, suspension.
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