TW202019438A - Use of echinacoside as ghrelin receptor agonist - Google Patents

Abstract

The present disclosure is related to the use of echinacoside as a ghrelin receptor agonist for stimulating the secretion of growth hormone thereby ameliorating the diseases associated with ghrelin receptor of low activity.

Description

Use of echinacoside as a ghrelin receptor agonist

This disclosure relates to a hungerin receptor agonist. Specifically, this disclosure relates to the use of echinacoside as a hunger hormone receptor agonist to stimulate growth hormone secretion.

As far as normal human physiology is concerned, the secretion of human growth hormone will gradually decrease with age, and the reduction of growth hormone secretion is currently considered to be importantly related to the occurrence of metabolic and aging-related symptoms common in elderly people, such as triggering a sense of burnout , Loss of appetite, increased adipose tissue, decreased muscle and bone density, the occurrence of cardiovascular disease, increased risk of neurodegenerative diseases, decreased learning and cognitive ability, decreased immune system function, and poor sleep quality. Although the scientific community has not yet fully understood the causes and mechanisms of growth hormone secretion decreasing with age, there are clinical applications of growth hormone related to it, such as growth hormone supplementation can improve aging-related phenomena, including weight gain, increase muscle mass And strength, reduce total body fat, increase musculoskeletal density and increase glucose metabolism efficiency; in the central nervous system (CNS), growth hormone has the effect of improving sleep, improving cognitive ability and nerve cell survival; and supplementing is appropriate The dosage can alleviate the discomfort caused by acute related diseases and surgery. However, direct administration of growth hormone often causes related side effects, such as edema, joint swelling, joint pain, carpal tunnel syndrome, gynecomastia, etc., and may cause insulin resistance and increase the risk of diabetes.

At present, medical research believes that the above side effects may be caused by directly changing the frequency of human growth hormone secretion. In order to alleviate the above-mentioned side effects caused by the direct application of growth hormone, in recent years, the scientific community has been directed to regulate the hormone upstream of growth hormone: hunger ( ghrelin), or hungerin-like compounds with the same function as ghrelin (ie ghrelin receptor agonist), to develop drugs to achieve the regulation of growth hormone secretion, which is a pulse that stimulates growth hormone secretion instead of affecting secretion Frequency, and can be used to treat related diseases caused by insufficient growth hormone secretion, thereby reducing the problem of related side effects caused by the direct change of secretion frequency caused by the growth hormone.

Hungerin is a multi-peptide hormone composed of 28 amino acids. It is a G protein-couple receptor (GPCR): growth hormone secretagogue-1a receptor (GHS) -R1a) ligand. The growth hormone-secreting receptors are mainly distributed in the hypothalamus and pituitary gland. After ghrelin activates the growth hormone-secreting receptors, it mainly increases appetite through the hypothalamus and stimulates the anterior pituitary gland to release growth hormone and other physiological effects. In addition, hunger hormone also has a regulating function on body metabolism, energy balance and gastrointestinal function. Therefore, in view of the above-mentioned functions of ghrelin, the scientific community currently focuses on the development of hungerin-like compounds (ie ghrelin receptor agonists) with the same function as ghrelin as the treatment of diseases related to insufficient growth hormone secretion. It has been pointed out in the literature that in the aging human and animal models, the administration of hungerin and its oral synthetic derivatives have been found to stimulate appetite, increase the secretion of growth hormone secretion, reduce adipose tissue and increase the rate of bone turnover, while administering hungerin Agonists also have the effect of improving cardiovascular disease and learning and cognitive abilities.

Based on the above, it is currently believed that the treatment of these diseases through the mechanism of hunger receptor activation is a very promising treatment model. However, according to reports in the literature, currently the synthetic hunger receptor receptor agonist oral drugs still have the risk of causing slight edema, muscle pain, increased blood sugar, vomiting and diarrhea, so far no hunger receptor receptor agonist drugs without side effects have been found In the market, there is no hunger receptor agonist suitable for clinical treatment. In view of this, the related fields urgently need to develop hunger hormone receptor agonist drugs suitable for clinical use to treat the related diseases caused by insufficient growth hormone secretion.

The following presents a brief summary of the present invention to facilitate the reader's basic understanding of the present invention. This summary is not an extensive overview of the invention, nor is it critical or decisive for identifying the key elements of this disclosure, nor does it outline the scope of this disclosure. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

The present invention is based on the above purpose and found that molecular docking simulation studies using bioinformatics, the compound echinacoside and teaghrelin and ginkgoghrelin in the literature report, the three are In terms of binding to hungerin receptors, there are simulated molecular binding data similar to the binding of hungerin receptors (data not shown). Therefore, the inventors used this as a starting point to conduct experiments to verify that echinacoside can be combined with The ghrelin receptor binds and has the same regulatory pathway as ghrelin, which in turn regulates the secretion of growth hormone.

The first aspect of the present disclosure provides the use of echinacoside for the preparation of a medicament for activating hunger hormone receptors to stimulate growth hormone secretion, wherein the echinacoside acts as a hunger hormone receptor agonist Agent.

In one embodiment, the drug of the present disclosure further comprises an acteoside and/or a tubuloside A. In a specific embodiment, the content ratio of the echinacoside, the tube anthocyanin A and the verbascoside in the drug is about 20:2:3.

In another embodiment, the medicine of the present disclosure further includes chrysin, gingko hungin and/or emoghrelin.

In certain embodiments as required, the drug of the present disclosure may be a pharmaceutical composition or a food composition.

The second aspect of the present disclosure relates to a method for preventing and/or treating a condition related to low hunger receptor activity in a body.

According to some embodiments, the method includes administering an effective amount of the disclosed drug to the individual.

According to an embodiment of the present disclosure, the related condition of low hunger receptor activity is caused by insufficient secretion of growth hormone. For example, the related conditions of low hunger receptor activity include, but are not limited to, growth hormone deficiency, cachexia, anorexia, sepsis, multiple sclerosis, depression, functional dyspepsia, respiratory tract inflammation, Intestinal blockage, and its combination.

The details and descriptions of one or more embodiments of the present disclosure are set forth below. After referring to the following detailed description and patent application scope, the remaining features and advantages of the present disclosure will be apparent.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation form and specific embodiments of the present invention; however, this is not the only form for implementing or using specific embodiments of the present invention. The embodiments cover the features of multiple specific embodiments, as well as the method steps and their order for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps.

For ease of explanation, some terms used in the description, examples, and accompanying patent applications are collated here. Unless otherwise defined in this specification, the meanings of scientific and technical words used herein are the same as those understood and used by those with ordinary knowledge in the technical field to which the present invention belongs.

Without conflicting with the context, the singular nouns used in this specification cover the plural of the noun; and the plural nouns used also cover the singular of the noun.

Although the numerical ranges and parameters used to define the broader range of the present invention are approximate numerical values, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range. Or, the term "about" means that the actual value falls within the acceptable standard error of the average value, depending on the consideration of those with ordinary knowledge in the technical field to which the present invention belongs. Except for experimental examples, or unless clearly stated otherwise, all ranges, quantities, values, and percentages used herein can be understood (for example, to describe the amount of materials, length of time, temperature, operating conditions, quantity ratio, and other similarities All) have been modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent application are approximate values, and can be changed as required. At least these numerical parameters should be understood as the indicated significant digits and the values obtained by applying the general rounding method. Here, the numerical range is expressed from one end point to another segment point or between two end points; unless otherwise stated, the numerical range described herein includes end points.

The term "subject" or "individual" or "patient" refers to vertebrates. In some embodiments, the vertebrate refers to "mammal". The term "mammal" here refers to all members of the Mammalia including, but not limited to, humans; primates (such as monkeys and chimpanzees); household and agricultural animals (such as pigs, sheep, goats, cows) , Horses and cattle); sports animals, pets (such as cats, dogs and rabbits); rodents (such as mice, rats and guinea pigs). In certain embodiments, the mammal refers to a person who can be treated with the method of the invention of the present disclosure. Unless there is a specific indication of one of the genders, the term "individual" or "patient" means gender as both male and female.

The terms "application" and "administration" are used interchangeably in this disclosure, which refers to the administration of the drug of this disclosure to an individual in need of treatment.

The term "treating" or "treatment" in this disclosure refers to the administration or administration of the drug of this disclosure to an individual in which the individual has symptoms of a related disorder with low hunger receptor activity, or suffers from The tendency of illness; its purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or influence ( affect) The condition, symptoms, or tendency to get sick.

The term "effective amount" here refers to an agent (such as the drug of the present disclosure) in an amount sufficient to produce a desired therapeutic response. In other words, the term "effective amount" refers to an effective necessary dose to achieve the desired therapeutic or preventive effect. For therapeutic purposes, an effective amount also refers to the therapeutic benefit effect of an ingredient of a drug beyond the toxicity or harmful effects of the ingredient. The effective amount of the medicament does not necessarily cure the disease or condition, but it can delay, hinder or prevent the occurrence of the disease or condition, or it can alleviate the symptoms associated with the disease or condition. The therapeutically effective amount can be divided into one, two or more doses and administered once, twice or more times in a suitable dosage form within a specified period. The specific effective amount depends on various factors, such as the specific condition to be treated, the individual's physiological conditions (eg, the individual's weight, age, or gender), the type of animal being treated, the duration of treatment, and concurrent therapy (if any) The essence, the specific dosage form used, and the structure of the compound or its derivatives. The effective amount can be expressed in any suitable way. For example, the effective amount of the medicament can be expressed as the total weight of the drug (for example, grams, milligrams, or micrograms) or as the ratio of the weight of the drug to body weight (for example, a few milligrams per kilogram of body weight (mg/kg, mg/Kg)) . Alternatively, the effective amount of the agent may be expressed in terms of concentration, such as molar concentration, mass concentration, volume concentration, molarity, molar fraction, Weight fraction (mass fraction) and mixing ratio (mixing ratio). The appropriate dosage range is from 0.01 mg to 100.0 mg per kg of body weight. When it is understandable, a wide range of adjustments to the required dose are also expected, depending on the different efficacy due to the diverse composition and various routes of administration. For example, oral administration is expected to require higher doses for oral administration. Those skilled in the art can fully understand the dosage adjustment according to the rule of thumb. Specifically, the “effective amount” of the agent in the present disclosure refers to a dosage sufficient to alleviate or ameliorate the related diseases associated with low hunger receptor activity in the individual. A person skilled in the art may convert the dose obtained based on the experimental animal model into a human equivalent dose (HED) of a drug (such as the drug of the present disclosure). For example, a person skilled in the art may refer to the "estimating the maximum safe starting dose of an adult healthy volunteer in the initial clinical treatment test" published by the US Food and Drug Administration (FDA) Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) to estimate the safest dose for human use.

This disclosure is based at least in part on molecular docking bioinformatics simulations and unexpectedly discovered that echinacoside has a site similar to that of ghrelin or ginkgo hormone and other ghrelin receptor agonists that bind to ghrelin receptors (data (Not shown), using this as a starting point to conduct experiments to verify that echinacoside can actually bind to the hungerin receptor and has a role in promoting the expression of genes downstream of the hungerin receptor (such as GHS-R1a), where the downstream gene One is growth hormone. At the same time, it was found that the signal transmission path triggered by echinacoside after binding to the starving hormone receptor is the same as the signal transmission path triggered by the binding of GHRP-6 and GHS-R1a. In view of this, the present disclosure proposes a method for treating conditions related to low hunger hormone activity. This method uses echinacoside as an active ingredient and administers a drug containing echinacoside to an individual in need of treatment. Among them, the individual has a related disorder of low hunger hormone activity. When it is understandable, the present disclosure also covers the use of the above-mentioned active ingredient echinacoside for preparing a drug for activating hunger receptors, and the drug itself.

The first aspect of the present disclosure relates to the use of an echinacoside for the preparation of a medicament for activating hungerin receptors, wherein the echinacoside acts as a hungerin receptor agonist to activate hungerin The receptor, in turn, stimulates the secretion of growth hormone.

The echinacoside in this disclosure can be extracted from natural plants or synthesized, where the natural plants include, but are not limited to, Cistanche deserticola , Cistanche tubulosa , Cistanche salsa , Cistanche salsa var. albiflora or Cistanche sinensis . Preferably, the echinacoside from this disclosure is extracted from desert cistanche or cistanche tubulosa. According to a specific embodiment, the echinacoside is extracted from Cistanche tubulosa. In this embodiment, the Cistanche tubulosa was ground into a powder, and the powdered product was extracted by boiling, and then processed by steps such as filtration and concentration to prepare the echinacoside of the present disclosure.

Generally speaking, distillation, recrystallization, extraction, sublimation, separate precipitation, column chromatography (such as gel chromatography and protein fast performance liquid chromatography (FPLC)), thin layer Chromatography and other methods for purification. In a specific embodiment, the echinacoside of the present disclosure is purified by FPLC.

The extraction product (ie, echinacoside) can be analyzed (or identified) using any method well known to those of ordinary skill in the art to which this invention belongs. For example, chemical analysis (such as titration analysis, gravimetric analysis, and microspectral analysis), spectral analysis (such as optical spectroscopy, atomic spectroscopy, and molecular spectroscopy), mass spectrometry, nuclear magnetic resonance spectroscopy, and chromatographic analysis (such as column color Layer analysis: ion exchange chromatography, gel chromatography, affinity chromatography and high performance liquid chromatography (HPLC), or thin layer chromatography), infrared spectroscopy, or Combination (for example, liquid chromatography mass spectrometry (LC-MS)). In one embodiment, HPLC is used to analyze the composition of the extracted product. In another embodiment, LC-MS is used to analyze the composition of the extracted product.

(I)According to an embodiment of the disclosure, the echinacoside in the disclosure has the chemical structure of formula (I):

(I)

Optionally, the drug of the present disclosure further includes verbascoside and/or tube anthocyanin A. According to some embodiments, the drug of the present disclosure includes echinacoside, tube anthocyanin A and verbascoside, and the content ratio of the drug in the drug is about 20:0.01-50:0.01-50; preferably 20:1 -10:1-10; more preferably 20:2:3. In these embodiments, tube anthocyanin A and/or verbascoside can additively or synergistically increase the efficacy of echinacoside in activating the starving hormone receptor.

It is also not necessary that the medicines of the present disclosure further include tea hunger, gingko hunger and/or ginseng wuhan. According to some embodiments, the medicine of the present disclosure includes echinacoside, ginkgo bilobarin, and/or ginseng wuhansu. In these embodiments, Ginkgo bilobarine and/or ginseng wukongsu can add or multiply increase the efficacy of echinacoside in activating hunger receptors.

When conceivable, the drug of the present disclosure may further include other known ghrelin receptor agonists to increase the efficacy of echinacoside in stimulating growth hormone secretion, such as diltiazem or diltiazem analog ), tetrahydropyrazolopyridine derivative, 3-spirocyclic piperidine derivative, benzothiazole, adenosine, and amorelin ( alexamorelin, anamorelin, capromorelin, pyrazolopyridine (CP-464709), cortistatin-14 (cortistatin-14), examorelin, sea Hexarelin, growth hormone releasing peptide-1 (GHRP-1), growth hormone releasing peptide-3 (GHRP-3), growth hormone releasing peptide- 4 (growth hormone releasing peptide-4, GHRP-4), growth hormone releasing peptide-5 (GHRP-5), growth hormone releasing peptide-6 (GHRP-4) ), ibutamoren (MK-0677), ipamorelin (ipamorelin), macimorelin (macimorelin), prammorelin (pralmorelin), relamorelin (relamorelin), tamorelin (Tabimorelin), ulimorelin (ulimorelin), L-692585, LY-426410, LY-444711, SM-130,686, TZP-101, TZP-102, and combinations thereof.

When it is understandable, the medicine of the present disclosure can be formulated into a pharmaceutical preparation, wherein the pharmaceutical preparation is suitable for the desired mode of administration. Certain drugs formulated according to the present disclosure and the claimed inventive concept may be a single unit dosage form suitable for oral use by patients. Exemplary oral dosage forms include, but are not limited to, tablets, caplets; capsules (such as elastic gel capsules), cachets, tablets (troches), powders, suspensions Liquids (such as aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), dispersions, solutions, and elixirs. The scope of the present disclosure also covers these pharmaceutical preparations.

The second aspect of the present disclosure relates to a method for preventing and/or treating a condition related to low hunger receptor activity in a body. The method includes administering an effective amount of the disclosed drug to the individual. During the treatment period, the drug of the present disclosure can be administered to the individual at different time intervals and through different routes. For example, it can be administered orally or parenterally, intravenously, intravenously, injection subcutaneously or implantation subcutaneously, intramuscularly, intraspinal injection (Intrathecally), intraperitoneally, intracuteanously, intrasternally, intraarticularly, intracranially, intracranially, intralesionally, intrarectallyly ), intravaginally (intravaginally), intranasally (intranasally), gastrointestinal (intragastically), intratracheally (intratracheally), or intrapulmonarily (intrapulmonarily).

In this disclosure, the effective amount of the medicine is about 0.001 mg to 1,000 mg per kg of body weight per day; preferably, about 0.01 mg to 100 mg per kg of body weight per day; 10 mg. In a specific embodiment, the effective amount of the drug of the present disclosure is about 0.6 mg per kg of body weight per day.

According to an embodiment of the present disclosure, the related condition of low hunger receptor activity is caused by insufficient secretion of growth hormone. For example, the related conditions of low hunger receptor activity include, but are not limited to, growth hormone deficiency, cachexia, anorexia, sepsis, multiple sclerosis, depression, functional dyspepsia, respiratory tract inflammation, Intestinal blockage, and its combination.

The third aspect of the present disclosure provides a use of echinacoside for preparing food, drink or feed.

The content of the present disclosure containing, adding and/or diluting the aforementioned active ingredients in foods, beverages or feeds is very useful for the improvement or prevention of related diseases with low hunger receptor activity. The method of manufacturing food, beverages or feeds in this disclosure is not particularly limited, and only needs to follow the methods of compounding, conditioning or processing of general foods, beverages or feeds, as long as the foods, beverages or feeds obtained contain Adding and/or diluting the aforementioned active ingredients will achieve the purpose of this disclosure.

The type of food or beverage in this disclosure is not particularly limited. For example, it may be a cereal processed product (for example, wheat flour processed product, starch processed product, supplementary nutritious food processed product, noodles, macaroni, bread, wheat bran, rice noodles, winter flour, packaged cake) containing the aforementioned active ingredients ); processed oils and fats (such as plastic oil, salad oil, mayonnaise, sauce); processed soybeans (such as tofu, miso, natto); processed meat products (such as ham, pork, sausage); aquatic products (such as frozen Ground fish, fish cakes, bamboo wheels, fish meat yam balls, fish balls, dried bonito, fish eggs processed products, canned aquatic products, tenant boiled); dairy products (such as raw milk, cream cream, cheese, cream, cheese, condensed milk, Powder milk, ice cream); processed fruits and vegetables (such as pastes, jams, pickles, fruit drinks, vegetable drinks, mixed drinks); snacks (such as chocolate, biscuits, cakes, rice crackers); alcoholic beverages (such as Japanese sake, Chinese wine, wine, whiskey, shochu, vodka, brandy, gin, lye, beer, fruit wine, liqueur beverages); tea drinks (such as green tea, black tea, oolong tea, coffee, refreshing drinks, lactic acid drinks); flavoring Ingredients (such as soy sauce, sauce, vinegar, cooking wine); canned/bottled/bagged food (such as beef rice, kettle rice, red rice, curry rice, prepared food); semi-dry or concentrated food (such as liver paste, other Smeared food, buckwheat udon noodle juice, concentrated soup); dry food (such as instant noodles, instant curry, instant coffee, fruit juice powder, powder soup bag, instant miso soup, conditioning beverages, conditioning soups); Frozen foods (such as sukiyaki, steamed tea bowls, roasted eel, patties steak, siu Mai, dumplings, various frozen semi-processed foods); solid foods or liquid foods; agricultural and forestry processed products, animal processed products, aquatic processed products such as spices Wait.

The type of feed in this disclosure is not particularly limited. For example, it can be animal raw materials (such as fish meal, casein, octopus intestinal meal) containing the aforementioned active ingredients; plant raw materials (such as soybean meal, wheat flour, starch); microbial raw materials (such as yeast for feed); Animal fats (such as cod liver oil, squid liver oil); vegetable fats (such as soybean oil, rapeseed oil); artificial compound feed of other raw materials (such as vitamins, minerals, amino acids, anti-acidification agents), or minced fish The final feed for fish.

The fourth aspect of the present disclosure provides a method for screening a potential ghrelin modulator, which includes the following steps: (a) Input a GHS-R1a protein sequence into an operable molecular docking software and move In addition to the elastic protruding ring parts from V184 to N196 on the surface of the model; and (b) input a three-dimensional structure of the test compound into the software of the operable molecular docking; wherein, growth hormone-releasing peptide-6 (growth hormone-releasing peptide -6, GHRP-6) The three-dimensional structure occupies the site of the GHS-R1a is the active center area, the test compound can enter the active center area, is a potential regulator of hunger hormone.

A number of embodiments are presented below to illustrate certain aspects of the present invention, so that those with ordinary knowledge in the technical field to which the present invention pertains can implement the present invention, and these embodiments should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art, after reading the explanations presented here, can fully utilize and practice the present invention without excessive interpretation. All published documents cited herein are deemed to be part of this specification.

Materials and Methods

1. Experimental materials

(1) Source of Cistanche samples

Cistanche Tubulosa was purchased from Hetian Tianlishasheng Pharmaceutical Development Co., Ltd. (Hetian, Xinjiang); Desert Cistanche was purchased from Hongjie Natural Biotechnology Co., Ltd.

(2) Laboratory animals

Use male rats of the Sprague-Dawley (SD) strain purchased from BioLASCO Taiwan Co., Ltd and weighing about 250 to 300 grams. Before the experiment, every two animals were kept in a rat cage and placed in the animal room for one week. The experimental animals were free to eat and drink water. The laboratory rodent diet 5001 was used for the feed: protein 28.672%, lipid 13.384%, carbon water Compound 57.944%. The light period is 12 hours light and 12 hours dark, the temperature is 23±1°C, and the humidity is 60±10%. All tests are in compliance with and passed the specifications of the National ZTE University Laboratory Animal Care and Use Committee (IACUC) (IACUC number: 106-079).

2. Experimental methods

(1) Sample extraction steps and preparation

After crushing Cistanche deserticola and Cistanche Tubulosa with a grinder, take 25 grams of powder and mix with 500 ml of 50°C deionized water, mix in a 50°C water bath for 1 hour, repeat three times, and collect the extract (a total of 1500 ml ) After cooling, the insoluble impurities in the extract are removed by suction filtration, and then the volume is concentrated with a vacuum concentrator, part of which is dried into a powder with a freeze dryer, and the other part is filtered with a 0.45 micron filter membrane. The extract was added to the sample bottle to facilitate HPLC analysis.

(2) Analysis of sample composition

Using HPLC to analyze the components of Cistanche deserticola and Cistanche tubulosa, the chromatographic conditions are as follows: the chromatography column is Syncronis C18 (4.6 mm × 250 mm, 5 microns), the injection volume is 40 μl, the flow rate is 0.8 ml per minute, Detection wavelength is 330 nm, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 100% acetonitrile; elution gradient is: (1) 0 to 3 minutes, 14% mobile phase B, (2) 3 to 4 Minutes, 17% mobile phase B, (3) 4 to 15 minutes, 17% mobile phase B, (4) 15 to 20 minutes, 20% mobile phase B, (5) 20 to 50 minutes, 20% mobile phase B, (6) From 50 to 60 minutes, 14% mobile phase B is used for gradient elution.

(3) LC-MS identification of echinacoside

Using the electrospray ionization (ESI) negative ion mode of LC-MS, the sample circle is 5 ml, and the full scan scan range is the mass/charge ratio (mass-to-charge ratio, m/z) 400- 1000, relative collision energy is 35 electron volts (eV) and 45 electron volts; liquid chromatography conditions are as follows: the column is Syncronis C18 (4.6 mm × 250 mm, 5 microns), the flow rate is 0.8 per minute ML, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 100% acetonitrile; separation conditions are: (1) 0 to 24 minutes, 14% mobile phase B, (2) 24 to 25 minutes, 17% mobile phase B , (3) 25 to 36 minutes, 17% mobile phase B, (4) 36 to 37 minutes, 20% mobile phase B, (5) 37 to 80 minutes, 20% mobile phase B, (6) 80 to 90 minutes , 14% mobile phase B in order of gradient extraction.

(4) Purified echinacoside

The lyophilized powder of the initial extract is separated and purified by FPLC to obtain echinacoside. FPLC uses a column condensed with about 100 ml of gel Sephadex LH-20 colloidal solution, and purifies with a 10% methanol solution at a flow rate of 0.7 ml per minute, eluting the volume to 300 ml, and collecting each with an automatic sample collector The residual content, the absorbance value is 245 nanometers, according to the peak shown in the analysis chart, the specific residual content containing echinacoside is collected, dried with a vacuum rotary decompression concentrator, and dried into a powder with a freeze dryer for storage.

(5) Culture of primary cells of rat pituitary gland

In this experiment, male rats of living SD strain (N=6), weighing about 250 to 300 grams, were used as the animal source of primary pituitary cells. Rats were anesthetized by intraperitoneal injection with Shutai 50 (40 mg per kg of body weight). The animals were decapitated and the pituitary tissues were removed and washed with DMEM without DFBS. The tissues were cut into sterile tissues with a scalpel blade. The primary cells were separated by the shearing action of three different enzymes (as described below).

First, treat with 0.25% (w/v) DMEM containing type I collagenase without DFBS for 60 minutes, centrifuge for 5 minutes to remove the supernatant, and add 0.25% (w/v) trypsin-B Diamine tetraacetic acid was incubated for 15 minutes, followed by centrifugation for 5 minutes to remove the supernatant. Finally, the cell fluid was evenly mixed in 16 units of DNase I (DNase I) and filtered through a 100-micron cell filter screen. Finally, a single cell suspension was obtained, planted in a 96-well dish at a cell density of 4×10 ⁴ cells per well, and cultured in DMEM containing 10% DFBS at a culture temperature of 37°C and containing 5% CO ₂ . Wait two days for the cells to stabilize.

(6) Determination of growth hormone secretion

After culturing for two days, replace the culture medium with DMEM without DFBS, incubate for 90 minutes in the incubator, and then replace it with DMEM containing drugs in the cell culture plate according to the experimental group, and leave it in the incubator for 15 minutes and 30 minutes, respectively Take the cell culture fluid into a centrifuge tube, centrifuge at 1000g for 20 minutes at 4°C, and store the supernatant at -80°C for storage, and then test the growth hormone secretion by the rat growth hormone ELISA kit.

The experimental group of growth hormone secretion is: DMEM without DFBS is the control group, echinacoside (10 ^-5 to 10 ^-8 moles per liter; obtained from Cistanche tubulosa) is the drug group, and the known drug GHRP-6 (10 ^-7 moles per liter) is the positive control group.

In the starvation receptor antagonistic experiment, [D-Arg ¹ , D-Phe ⁵ , D-Trp ^7,9 , Leu11]-P substance (GHS-R1a antagonist, 0.5 micromole per liter) was used as the antagonist test Drugs; experimental grouping: pine cones with or without GHS-R1a antagonist (0.5 micromole per liter) without DFBS as a control group, with or without GHS-R1a antagonist (0.5 micromole per liter) Inulin (10 ^-5 moles of DMEM per liter) is the echinacoside drug test group, with or without GHS-R1a antagonist (0.5 micromoles per liter), GHRP-6 (10 ^-7 moles of DMEM per liter) is GHRP-6 drug test group.

(7) Operation of homology modeling and molecular docking

The human GHS-R1a protein sequence (AAI13548) was obtained from the website SWISS-Model as a molecular model for subsequent simulations. The crystal structure of β1 and β2 adrenergic receptors (PDB: 2YCY and 3PDS) and its related ligand cyanurindolol (Cyanopindolol) and FAUC50 binding conditions are used as the basis for binding to establish a binding model of GHS-R1a homology simulation. The related ligand structure has been removed before the simulation. By loop refinement (MODELER) module in the loop region (loop region) between the H186 to L210 position, the C116 and The C198 position is modified with a disulfide bond to facilitate molecular simulation and docking of the compound, and the elastic protruding ring at positions V184 to N196 on the surface of the hunger receptor model is removed, and the probability density function (probability) of the GHS-R1a structure established density function, PDF) The lowest total energy. All the above operations are calculated using the software platform Discovery Studio 3.5.

Obtain the three-dimensional structure of echinacoside and GHRP-6 from the website PubChem, and define the binding pocket by the pocket connected by the III-VII extracellular transmembrane region in the GHS-R1a structure, and use GHS -The binding position in R1a is defined by a sphere with a radius of 14Å. Simulated molecular docking of echinacoside and GHRP-6 with the receptor GHS-R1a, the program is simulated by the LibDock module in GEMDOCK and Discovery Studio 3.5, where the energy calculation of the relevant structure is optimized by the CHARMm force field Perform calculations to compare the molecular forces between echinacoside and GHRP-6, such as hydrogen bonds, ionic bonds, cationic-π bonds, and π-π bonds.

(8) Statistical analysis

All values are expressed as mean ± standard deviation. The statistical software system GraphPad Prism 6 was used to compare the difference between the control group and the dosing group with the T test. When p<0.05, there was a statistically significant difference.

Examples 1 Sample analysis and compound purification

(1) HPLC analysis of Cistanche tubulosa

Figure 1 shows the results of HPLC analysis of Cistanche tubulosa, which is presented with an absorbance value of 330 nm. According to the data in the published literature, it is preliminarily presumed that the echinacoside, tube anthocyanin A and verbascoside will have a peak signal at the residence time of about 27.33 minutes, 49.57 minutes and 50.18 minutes, respectively. Taking echinacoside, tube anthocyanin A and verbascoside as the total content, the area under the curve represents the respective content, showing that echinacoside, tube anthocyanin A and verbascoside respectively account for about 79.46 of the total content %, 7.83% and 12.71%, according to which, Echinacoside is the main compound of phenylglycoside in Cistanche tubulosa.

(2) Results of LC-MS identification of compounds

The compounds of Cistanche tubulosa were further identified by LC-MS. The molecular weight of the peak with a retention time of about 27.33 minutes is 786 Daltons. After the first negative ion scan mode (mass minus hydrogen atoms, [MH]), the 785 mass-to-charge ratio (786-1=785) is obtained. Signal (the left column of Figure 2A), and after the second scan, the signal of 623 mass-to-charge ratio (right column of Figure 2A) is obtained, and the mass-to-charge ratio of 162 is obtained from the above second scan results (785-623=162 ), the fragment is caffeic acid in the molecular structure of the wave peak (the right column of Figure 2A), and it is confirmed that the compound of the wave peak is echinacoside (molecular weight is 786 Dalton), which The chemical structure is shown in formula (I).

The molecular weight of the peak with a retention time of about 49.57 minutes is 828 Daltons. After the first scan in the negative ion scanning mode, a signal of 827 mass-to-charge ratio (828-1=827) is obtained (left column in Figure 2B), and After the second scan, the signals with mass-to-charge ratio of 665, 623 and 785 were obtained (right column of Figure 2B). The calculation result is as follows: the signal with mass-to-charge ratio of 665 (827-665=162), of which 162 The fragment is the fragment broken from the position marked 1 in the right column of Figure 2B; the signal of 623 mass-to-charge ratio (827-623=204), where the fragment of 204 mass charge ratio is the position marked 2 from the right column of Figure 2B The fragment obtained by breaking; the signal of 785 mass-to-charge ratio (827-785=42), of which the fragment of 42 mass-to-charge ratio is the fragment obtained by breaking from the position marked 3 in the right column of Figure 2B, and the fragment is broken from the molecule of the peak After that, the remaining molecule is echinacoside; according to the above results, it is confirmed that the compound of this peak is tube anthocyanin A.

The molecular weight of the peak with a retention time of about 50.18 minutes is 624 Daltons. After the first scan in negative ion scanning mode, a signal with a mass-to-charge ratio of 623 (624-1=623) is obtained (left column in Figure 2C), and the After the second scan, the signal of 461 mass-to-charge ratio (right column of Figure 2C) is obtained, and the fragment of 162 mass-to-charge ratio (623-461=162) is obtained from the result of the above second scan, and this fragment is the molecule of the peak The caffeic acid in the structure (right column of Figure 2C), based on this, the compound of this peak was identified as verbascoside.

(3) HPLC analysis of the composition difference between Cistanche samples

By HPLC analysis of Cistanche tubulosa and Cistanche deserticum aqueous extract component content analysis chart (Figure 3), it can be clearly found that the content of each compound in Cistanche tubulosa is much more than Cistanche deserticola. With the area under the curve representing the content of each compound, echinacoside accounted for about 51.56% of the aqueous extract of Cistanche tubulosa (Figure 3A), and about 13.96% of the aqueous extract of Cistanche deserticola (Figure 3B), so Subsequent purification and collection of echinacoside sources were all carried out with Cistanche tubulosa samples.

Basically, at the same concentration (10 mg per 500 microliters) of the aqueous extract of Cistanche deserticola and Cistanche deserticola, the effective ingredient content of Cistanche deserticola is far more than that of Cistanche deserticola. In desert Cistanche, Echinacein accounted for about 20.33% of the total content, Verbascoside accounted for about 21.81% of the total content; and in Cistanche Tubulosa, Echinacein accounted for about 58.08% of the total content, tube anthocyanin A accounted for the total content About 8.61%, verbascoside accounted for about 13.555% of the total content. Based on the above results, the content of Cistanche tubulosa was indeed higher than that of Cistanche deserticola, and echinacoside accounted for most of the two (Figure 3).

(4) FPLC purification and collection of echinacoside in Cistanche deserticola

The echinacoside in Cistanche tubulosa aqueous extract was purified by FPLC, and the absorbance value was detected at 254 nanometers. The purified analysis pattern was shown in Figure 4A, and the specific peaks in the analysis pattern were collected and confirmed by HPLC (Figure 4) B), in Figure 4B, small panels b to g are the extraction volume in Figure 4A, which is approximately in the range of 265 to 390 milliliters, and Figure 4B, small panels h to i are the reduction in Figure 4A. In the retention range of 680 to 725 ml, it can be found that the echinacoside will appear in the retention range of about 300 ml of the elution volume, and it will fall in the retention range shown in Figure 4, B, b and c Its purity is relatively high. After recovering these residues, it was concentrated under reduced pressure and drained, then dissolved in water, and finally freeze-dried to obtain echinacoside powder, which was confirmed by HPLC and its purity was more than 90% (Figure 3) C).

Examples 2 Rat pituitary gland Determination of growth hormone secretion from primary cells

Figure 5 illustrates the results of treating primary cells of rat anterior pituitary gland with echinacoside to stimulate growth hormone secretion. Echinacoside is used at a concentration of 10 ^-5 to 10 ^-8 moles per liter. The growth hormone secretion is dependent on the concentration of the drug at 15 minutes (Figure 5A) and 30 minutes (Figure 5B) In the second test, echinacoside (10 ^-5 to 10 ^-6 moles per liter) and GHRP-6 (10 ^-7 moles per liter) were significantly different from the control group, and were administered for 30 minutes At the time, the concentration of echinacoside at ^10-5 moles per liter stimulated the secretion of growth hormone up to 1.03 times of GHRP-6 ( ^10-7 moles per liter).

For an adult weighing 65 kg, the blood volume is about 5 liters (65 kg × 1/13 = 5 kg ≈ 5 liters), and the cell experiment results show that the effective dose of echinacoside is 10 per liter ^-5 moles, which is equivalent to about 39.3 mg of echinacoside (5 liters × 10 ^-5 moles per liter × 786 = 3.93 × 10 ^-2 grams = 39.3 mg). And every 50 grams of Cistanche tubulosa has about 20.1 grams of aqueous extract and 1 gram of Cistanche tubulosa aqueous extract is about 0.76 grams of echinacoside, so 1 gram of Cistanche tubulosa has about 0.3 grams of pine cones For chrysanthemum (0.76 g × 20.1 g/50 g = 0.3 g), for echinacoside which requires about 39.3 mg in the human body, the amount of Cistanche tubulosa required is 0.131 g (39.3 mg/0.3=0.131 G).

[D-Arg ¹ , D-Phe ⁵ , D-Trp ^7,9 , Leu ¹¹ ]-P is a GHS-R1a antagonist, and the concentration used in this experiment is 0.5 micromole per liter. Figure 6 illustrates the use of DFBS-free DMEM with or without GHS-R1a antagonist (control group), echinacoside with or without GHS-R1a antagonist ( ^10-5 mol DMEM per liter) (Echinacea Glycoside group) and GHRP-6 ( ^10-7 moles of DMEM per liter) with or without GHS-R1a antagonist (GHRP-6 group) after 15 minutes of treatment of primary cells of the anterior pituitary of rat pituitary, the results are shown in In the above three groups of treatments, GHS-R1a antagonists have the effect of inhibiting growth hormone secretion by cells. Furthermore, GHS-R1a antagonists can antagonize the effect of echinacoside or GHRP-6 on stimulating cells to secrete growth hormone. The results of the above data show that both echinacoside and GHRP-6 stimulate growth hormone secretion through activation of GHS-R1a.

Examples 3 To GEMDOCK Simulate molecular bonding

The molecular simulation software GEMDOCK was used for calculation, and the site of GHRP-6 binding to GHS-R1a was used as the active center region of GHS-R1a to test whether echinacoside could enter the active center region. According to the score of the GEMDOCK molecular simulation, the binding of the ligand to the receptor can be judged. The lower the score, the less energy is consumed, and the better the ability of the ligand to bind to the target receptor; otherwise, the higher the score , Which means that the ligand and target receptor require high energy consumption. Table 1 (shown below) shows the results of simulated combination of echinacoside, chrysophanol and ginkgo hungin with the active center area of GHS-R1a, showing that all three can enter the active center area of GHS-R1a. The chemical energy required for the tea hunger element is -176.65 kJ.mol ^-1 , including van der Waals -139.54 kJ.mol ^-1 and hydrogen bonding -37.1 kJ.mol ^-1 ; The energy is -153.96 kilojoules.mol ^-1 , including van der Waals-121.75 kilojoules.mol ^-1 and hydrogen bonding -21.59 kilojoules.mol ^-1 ; the chemical energy required for echinacoside is -132.35 kilojoules .Mol ^-1 , including van der Waals -111.82 kilojoules.mol ^-1 and hydrogen bonding -20.53 kilojoules.mol ^-1 ; the chemical energy required for anthocyanin A is -122.88 kilojoules.mol ^-1 , including Van der Waals -103.93 kilojoules mol ^-1 and hydrogen bonding -18.95 kilojoules mol ^-1 ; the chemical energy required for verbascoside is -120.35 kilojoules mol ^-1 , including van der Waals -93.65 kilojoules .Mol ^-1 and hydrogen bond -26.7 kilojoules.mol ^-1 . Table 1. GEMDOCK Comparison Energy Meter

Examples 4 Echinacoside and GHRP-6 Calculation of homology simulation and molecular docking simulation of hungerin receptor

The molecular simulation software Discovery Studio 3.5 was used to perform calculations to analyze the binding of GHRP-6, echinacoside, tube anthocyanin A and verbascoside to the active center of hunger receptors. As shown in Figure 7, the molecular simulation software simulates GHRP-6 (Figure 7A), Echinacein (Figure 7B), Tubularin A (Figure 7C), and Verbascoside (Figure 7D) into the hunger hormone receptor. The results of the body active center area show that all four can enter the active center area. Accordingly, the above-mentioned molecular docking simulation data proves that the echinacoside of the present disclosure can bind to the ghrelin receptor and acts as an effective non-peptide ghrelin analog.

To sum up, in the present disclosure, echinacoside can act as a hungerin receptor agonist by activating the hungerin receptor to further stimulate the growth hormone secretion. Therefore, this disclosure first proves that echinacoside has the activity of ghrelin receptor agonist, which can be extracted from Cistanche tubulosa, is a natural, safe and active compound that can be used to prevent and/or treat hunger hormone Related disorders of low receptor activity.

Although the above embodiments disclose specific examples of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs, without departing from the principle and spirit of the present invention, should Various changes and modifications can be made to it, so the scope of protection of the present invention shall be defined by the scope of the accompanying patent application.

no

After referring to the following detailed description, patent application scope and accompanying drawings, the disclosure and other features, forms and advantages will be more obvious and understandable, among which: Figure 1 is the HPLC obtained according to an embodiment of the disclosure Analysis chart, which illustrates the composition of the aqueous extract of Cistanche Tubulosa; Figure 2 is a LC-MS signal display diagram obtained according to an embodiment of the present disclosure, to confirm the composition of the aqueous extract of Cistanche Tubulosa, in which The second A is the analysis result of echinacoside, the second image B is the analysis result of tube anthocyanin A, and the second image C is the analysis result of verbascoside; Figure 3 is the HPLC analysis chart obtained according to one embodiment of the disclosure Among them, Figure 3A is the analysis map of desert Cistanche, Figure 3B is the analysis map of Cistanche tubulosa, and Figure 3C is the analysis map of echinacoside extracted from Cistanche tubulosa; Figure 4 is based on this disclosure The chromatographic analysis pattern obtained in one embodiment, in which Figure 4A is the FPLC analysis pattern of the aqueous extract of Cistanche tubulosa aqueous extract; Figure 4B is the analysis pattern of HPLC confirmation of the composition of the specific sample fraction, which is small Picture a is the analysis chart of the crude extract of Cistanche tubulosa aqueous extract. Panels b to g are the analysis maps of the sample extraction volume falling in the range of 265 to 390 ml, and panels h to i are the sample extraction volume. Analytical map of the remaining fraction falling between 680 and 725 milliliters; Figure 5 is a histogram drawn according to an embodiment of the present disclosure, which illustrates the specific treatment of primary pituitary cells of rats for 15 minutes (Figure Five A) or 30 minutes (Figure 5B), the secretion of growth hormone; all data (N=6) are expressed as mean ± standard error (mean ± standard error (mean ± SEM), T test) (T test) Compare the difference between the control group and the dosing group. When p<0.05 (marked with *), p<0.005 (marked with **) and p<0.001 (marked with ***), it means that there is Significant differences; Figure 6 is a histogram according to an embodiment of the present disclosure, which illustrates the secretion of growth hormone 15 minutes after the specific treatment of the primary pituitary cells of the rat; all data (N =6) All expressed as mean±standard deviation, compared with T test to compare the difference between groups, when p<0.05 (marked with *) means that there is a significant difference from the control group, while p<0.05 (marked with #) and p<0.005 (marked with ##) indicates that there is a significant difference between those with antagonists and those without antagonists in the same group; and FIG. 7 is the simulation analysis results of molecular docking according to an embodiment of the present disclosure. Seven A is the simulation analysis result of the molecular docking between GHRP-6 and GHS-R1a; Figure 7B is the simulation analysis result of the molecular docking between echinacoside and GHS-R1a; Figure 7C is the molecule of tube anthocyanin A and GHS-R1a The simulation analysis results of docking; Figure 7D is the simulation analysis results of the molecular docking of verbascoside and GHS-R1a.

Claims (10)

An echinacoside (echinacoside) is used for preparing a medicine for activating hungerin receptor, wherein the echinacoside is used as a hungerin receptor agonist. The use according to claim 1, wherein the medicine further comprises an acteoside and/or a tubeuloside A. The use according to claim 2, wherein the content ratio of the echinacoside, the tube anthocyanin A and the verbascoside in the medicine is 20:2:3. The use according to any one of claims 1 or 2, wherein the medicine further comprises teaghrelin, ginkgoghrelin and/or emoghrelin. The use according to claim 1, wherein the medicine is used to stimulate growth hormone secretion. The use according to claim 1, wherein the medicine is a pharmaceutical composition. The use according to claim 1, wherein the medicine is a food composition. The use as claimed in claim 1, wherein the medicament can be used to prevent and/or treat a disease related to a low level of hunger receptor in a body. The use according to claim 8, wherein the related condition of low hunger receptor activity is selected from growth hormone deficiency, cachexia, anorexia, sepsis, multiple sclerosis, depression, functional dyspepsia, Inflammation of the respiratory tract, intestinal blockage, and combinations of these. The use according to claim 8, wherein the related condition of low hunger receptor activity is caused by insufficient secretion of growth hormone.

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