TW202245738A - Curcuminoid-rich oil extract, its preparation method and uses for treating diseases associated with neuron injury - Google Patents

Abstract

Disclosed herein are curcuminoid-rich oil extract, its production method, and use thereof in treating diseases associated with neuron injury (e.g., dementia with Lewy bodies (DLB), Parkinson's disease (PD) and Alzheimer disease (AD)). According to preferred embodiments of the present disclosure, the curcuminoid-rich oil extract comprises curcuminoids dissolved in an edible oil, in which the curcuminoids consist of 25-30% (wt%) of bisdemethoxycurcumin, 25-30% (wt%) of demethoxycurcumin, and 40-50% (wt%) of curcumin.

Description

Oily extract rich in curcuminoids, its preparation method and use for treating diseases related to nerve damage

The present invention relates to the field of disease treatment, and in particular to a curcuminoid-rich extract, its preparation method and its use for treating diseases related to nerve damage (eg, dopamine nerve damage).

Turmeric, a member of the Zingiberaceae family, has underground rhizomes. Turmeric has been used in folk medicine for thousands of years to treat a variety of ailments such as inflammation, infectious diseases, and stomach, liver, and blood disorders.

Curcuminoids, such as curcumin, are natural polyphenolic compounds derived from turmeric (Curcuma longa), whose biological activities, including potential anticancer, diabetic and anti-inflammatory nutraceuticals, have been extensively studied.

The purpose of this disclosure is mainly to provide a curcuminoid-rich extract and its preparation method, wherein compared with the extracts prepared by conventional methods, the curcuminoid-rich extract of the present invention The bisdemethoxycurcumin in the extract is at least five times that of the extract produced by the prior art.

This Summary is intended to provide a simplified summary of the disclosure in order to provide the reader with a basic understanding of the disclosure. This summary is not an extensive overview of the disclosure and it is not intended to identify key/critical elements of the embodiments of the invention or to delineate the scope of the invention.

The present disclosure is about a preparation method for preparing a curcuminoid-rich oily extract, the obtained curcuminoid-rich oily extract, and its use for treating diseases related to nerve damage, Especially dopamine nerve damage.

The first aspect of the disclosure relates to a method for preparing a curcuminoid-rich oily extract. The method comprises extracting dried turmeric powder with oil to prepare the oily extract rich in curcuminoids, wherein the curcuminoids consist of 25-30% (weight %) of didemethoxycurcumin, 25- Composed of 30% (weight %) demethoxy curcumin and 40-50% (weight %) curcumin.

According to an embodiment of the present disclosure, the extraction is accomplished by the following steps: (i) mixing oil with dry turmeric powder; and (ii) heating the oil mixture in step (i) under water isolation under ultrasonic vibration.

According to an embodiment of the present disclosure, the oil suitable for mixing with the dry turmeric powder in step (i) is an edible vegetable oil. For example, the edible vegetable oils include, but are not limited to, coconut oil, corn oil, canola oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, sunflower oil, sesame oil, soybean oil and argan oil . In a preferred embodiment, the dry turmeric powder is extracted with olive oil.

According to the embodiment of the present disclosure, the water-isolated heating in step (ii) is heating at 50°C for 30 minutes, heating at 60°C for 30 minutes and heating at 80°C for 30 minutes to complete the heating of the oil mixture; During the process, ultrasonic vibration is performed at a frequency of about 25-35 kHz.

According to an embodiment of the present disclosure, the dried turmeric powder is produced by the following steps: (a) slice fresh turmeric rhizomes; (b) dry the slices of step (a) at 55° C.; and (c) Grinding the dried chips of step (b) into powder with a ball mill.

The second aspect of the disclosure relates to a curcuminoid-rich oily extract prepared by the method. The oily extract rich in curcuminoids comprises curcuminoids dissolved in oil, wherein the curcuminoids consist of 25-30% (weight %) of two demethoxy curcumin, 25-30% (weight %) ) of demethoxy curcumin and 40-50% (weight %) of curcumin.

The third aspect of the disclosure relates to a novel use of a curcuminoid-rich oily extract prepared by the method of the present invention, wherein it was found that the curcuminoid-rich oily extract can inhibit brain tissue and nerve damage Associated protein expression in brain tissue containing substantia nigra pars compacta (SNpc), striatum, hippocampus, and amygdala; thus, capable of treating related diseases.

According to an embodiment of the present disclosure, the curcuminoid-rich oily extract comprises curcuminoids dissolved in an oil, and the curcuminoids are composed of 25-30% (weight %) of didemethoxy Base curcumin, 25-30% (weight %) of demethoxy curcumin and 40-50% (weight %) of curcumin.

Diseases treated with the curcuminoid-rich oily extract of the present invention include, but are not limited to, dementia with Lewy bodies (DLB), Parkinson's disease (PD) or Alzheimer's disease (AD).

Suitable subjects for administering the curcuminoid-rich oily extract prepared by the method are mammals; preferably humans.

After referring to the following embodiments, those with ordinary knowledge in the technical field of the present invention can easily understand the basic spirit and other invention objectives of the present invention, as well as the technical means and implementation modes adopted by the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation aspects and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The description covers features of various embodiments as well as method steps and their sequences for constructing and operating those embodiments. However, other embodiments can also be used to achieve the same or equivalent functions and step sequences.

I. definition

Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those commonly understood and commonly used by those skilled in the art to which this invention belongs. In addition, the singular nouns used in this specification include the plural forms of the nouns, and the plural nouns used also include the singular forms of the nouns, unless the context conflicts with the context. Specifically, the singular word "a" used in the claims includes the plural unless otherwise specified herein. Furthermore, "at least one" and "one or more" used in the claims have the same meaning, including one, two, three or more.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the relative numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently inherently contain standard deviations resulting from their individual testing methodology. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specified value or range. Alternatively, the term "about" means that the actual value falls within acceptable standard error of the mean, as considered by one of ordinary skill in the art to which this invention pertains. Except for experimental examples, or unless otherwise expressly stated, all ranges, quantities, values and percentages used herein should be understood to be Those) are modified by "covenant". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the appended patent claims are approximate values and may be changed as required. At least these numerical parameters should be understood as the value obtained by applying the normal rounding method to the indicated effective digits. Herein, numerical ranges are expressed as being from one endpoint to another point or between two endpoints; unless otherwise stated, the numerical ranges stated herein are inclusive of the endpoints.

In this disclosure, the word "extract" includes crude extract and refined extract. Specifically, a crude extract is the product obtained by simple extraction by contacting selected plant parts with at least one extractant (ie, an oil). Optionally, one or more separation and/or purification treatments may be subsequently performed on the obtained crude extract to obtain a refined extract. Plant extracts may be in liquid form (eg, solutions, concentrates, distillates).

The term "weight percentage" (weight percentage; wt %) in this disclosure refers to a mixture comprising an ingredient, the ingredient (for example, the di-desmethoxycurcumin of the curcuminoid-enriched extract of the present invention) , demethoxy curcumin or curcumin) weight percent. Weight percent (wt %) is obtained by dividing the weight of the component by the total weight of the mixture, and is expressed as a percentage and/or a decimal.

In this disclosure, the term "treat" includes partial or complete prevention, improvement, alleviation and/or treatment of symptoms, secondary diseases or symptoms associated with nerve damage, especially dopamine nerve damage. The term "treat" as used in this specification also refers to the application or administration of the pharmaceutical compositions of the present disclosure to an individual suffering from neurological damage-related symptoms, secondary diseases or conditions, in order to achieve partial or complete Alleviate, slow down, cure the disease, delay the onset, inhibit the progression of the disease, reduce the severity of the disease, and/or reduce the occurrence of one or more symptoms, secondary diseases or conditions associated with nerve damage. "Treatment" can also be administered to individuals suffering from early signs or symptoms of these diseases, so as to reduce the occurrence of neurological damage-related symptoms, secondary diseases or symptoms. Treatment is "effective" when one or more symptoms or clinical markers are reduced. Alternatively, the treatment is "effective" when the progression of a symptom, secondary disease or condition is slowed or stopped.

"Effective amount" as used herein means an amount of an ingredient sufficient to produce the desired therapeutic response. An effective amount also means that the therapeutically beneficial effects of an ingredient outweigh its toxic or detrimental effects. An effective amount of a medicament does not necessarily cure a disease or a condition, but can delay, hinder or prevent the occurrence of the disease or condition, or alleviate symptoms associated with the disease or condition. A therapeutically effective amount may be divided into one, two or more doses and administered once, two or more times in an appropriate dosage form within a given period. The specific therapeutically effective amount depends on various factors, such as the particular condition to be treated, the physiological condition of the individual (e.g., the individual's weight, age, or sex), the type of individual being treated, the duration of treatment, concurrent therapy (if any) The nature of the compound and the specific formulation used and the structure of the compound or its derivatives. For example, a therapeutically effective amount may be expressed as the total weight of active ingredient, such as in grams, milligrams or micrograms; or as a ratio of the weight of active ingredient to body weight, such as milligrams per kilogram of body weight (mg/kg). Kilogram). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (for example, the didemethoxycurcumin, demethoxycurcumin or curcumin of the curcuminoid-rich oily extract of the present invention), such as Mo Ear concentration, weight concentration, volume concentration, weight molar concentration, molar fraction, weight fraction and mixing ratio. Those skilled in the art can calculate the human equivalent dose (human equivalent dose, HED) of the medicament (eg, the pharmaceutical composition of the present disclosure) based on the dose in the animal model. For example, those skilled in the art can follow the "Estimating the Maximum Safe Initial Dose of Adult Healthy Volunteers in Initial Clinical Treatment Test" (Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) to estimate the highest safe dose for human use.

"Individual" (subject) and "patient" (patient) are interchangeable terms in this disclosure, and refer to human beings, etc. who can be treated with the curcuminoid-rich oily extract and/or method of the present invention. animal. Unless otherwise indicated, the terms "subject" or "patient" refer to both males and females. Accordingly, the term "subject" or "patient" includes any mammal that can produce a therapeutic benefit after receiving the curcuminoid-rich oily extract or treatment method of the present invention. Exemplary "subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry . In an exemplary embodiment, the individual is a human.

II. Detailed Description of the Invention

(i) Method for preparing curcuminoid-rich extract

The first aspect of the disclosure relates to a method for preparing a curcuminoid-rich extract. The method comprises extracting turmeric with oil to obtain an oily extract rich in curcuminoids, wherein the curcuminoids are dissolved in the oil and the curcuminoids are removed from 25-30% (wt%) Composed of methoxy curcumin, 25-30% (weight %) demethoxy curcumin and 40-50% (weight %) curcumin.

According to an embodiment of the present disclosure, the rhizome of fresh turmeric is cut into slices with a thickness of about 0.2 cm, dried at 55°C, and then ground into powder, for example, with a ball mill.

Additionally or optionally, the finely ground powder can be filtered using a filter or sieve (about 400 mesh) to obtain a turmeric powder with a diameter of less than 38 μm.

To obtain the extract, the dried turmeric powder obtained is mixed with edible vegetable oil in a weight (gram) to volume (liter) ratio of 10:1 to 1:10, for example 10: 1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 9:2, 9:3, 9:4, 9:5, 9:6, 9:7, 9:8, 9:10, 8:3, 8:5, 8:6, 8:7, 8:9, 8:10, 7:2, 7: 3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 6:4, 6:5, 6:7, 6:8, 6:9, 6:10, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:10, 4:1, 4:3, 4:5, 4:6, 4: 7, 4:8, 4:9, 4:10, 3:1, 3:7, 3:8, 3:10, 2:9, 2:7, 1:7, 1:8, 1:9 and 1:10; preferably, the ratio of weight (grams) to volume (liters) is about 10:1. Edible vegetable oils suitable for use in this method include, but are not limited to, coconut oil, corn oil, canola oil, cottonseed oil, olive oil, palm oil, peanut oil, canola oil, sunflower oil, sesame oil, soybean oil, and nut oils Oil. In a preferred embodiment, the dried turmeric powder is extracted with olive oil.

Next, the oil mixture was extracted by means of water-proof heating. First, it was heated at 50°C for 30 minutes, then at 60°C for 30 minutes, and finally at 80°C for 30 minutes. During the heating process, it was oscillated by ultrasonic waves with a frequency of about 25-35 kHz, for example about 28 kHz. It should be noted that "water heating" refers to the indirect heating of dry turmeric powder oil mixture and oil (eg, olive oil). Specifically, the mixture is placed in the first container, and the first container is placed in the second container filled with a sufficient amount of water in advance, and then the second container is directly heated until the temperature in the second container reaches a preset temperature (eg, 50, 60 or 80°C), and then maintained for the aforementioned period (eg, 30 minutes). It is most important that the second container is continuously filled with water throughout the period of the soaker (ie, the volume of water in the container never drops to zero). Furthermore, during the water-isolated heating period, apply ultrasonic vibration frequencies of about 25-35 kHz, for example, 25, 26, 27, 28, 30, 31, 32, 33, 34 and 35 kHz, to the first container. In this disclosure, the obtained oily extract is named "ATG-235 solution".

(ii) The curcuminoid-rich extract of the present invention

Above-mentioned ATG-235 solution that makes with the present invention's method is rich in curcuminoids, wherein the curcuminoids in ATG-235 solution consists of 25-30% (weight %) of two demethoxycurcumin, 25- Composed of 30% (weight %) demethoxy curcumin and 40-50% (weight %) curcumin. In a preferred embodiment, the ATG-235 solution is composed of about 26.7% (weight %) of two demethoxy curcumin, about 28.8% (weight %) of demethoxy curcumin, and about 44.5% (% by weight) of curcumin; and curcuminoids in the turmeric extract prepared by known methods are 5.7% (% by weight) of two demethoxy curcumin, 24.1% (% by weight) ) of demethoxy curcumin, and 70.8% (% by weight) of curcumin. It should be noted that the ATG-235 solution prepared by the method of the present invention is rich in bis-demethoxy curcumin, and the ratio of curcumin is low. Compared with the extract prepared by the known method, the extract of the present invention The bis-demethoxycurcumin of the extract is five times higher than that of the extract of the prior art, and the amount of curcumin is only half of that of the extract of the prior art.

Depending on the purpose of implementation, one or more suitable pharmaceutically acceptable carriers or excipients can be used to prepare the curcuminoid-rich extract or ATG-235 solution of the present invention, and the pharmaceutical composition of the disclosure Forms suitable for oral and parenteral administration are prepared. In a pharmaceutical dosage form, the ATG-235 solution of the present invention can be administered to an individual alone or in combination with other known pharmaceutically active agents for treating nerve injury-related diseases. Different dosage forms suitable for each route of administration are well known to those skilled in the art. It should be noted that the optimal route of administration will vary with the type and severity of the disease or condition.

In certain embodiments, the ATG-235 solution of the present invention is administered in a liquid dosage form for parenteral administration, for example, injection administration, which includes but is not limited to subcutaneous, bolus injection, intramuscular, intraperitoneal and intravenous injection. ATG-235 solutions can be formulated in oily or aqueous vehicles, and can contain formulating agents and/or flavoring agents. The liquid dosage forms can be placed in sterile ampoules or vials. In certain embodiments, ATG-235 solutions of the invention are administered orally in liquid dosage form. Liquid dosage forms can be filled in soft gelatin capsules. For example, the liquid may comprise a solution, suspension, emulsion, microemulsion, precipitate or any desired liquid medium carrying the active ingredient of the ATG-235 solution of the present invention. Liquids can be designed to modify the active ingredients of the ATG-235 solutions of the present invention to form a drug-containing emulsion or released dispersion.

(iii) Use of the curcuminoid-rich extract of the present invention

One aspect of the present disclosure is the novel use of the curcuminoid-rich oily extract of the present invention, which was found to inhibit the expression of proteins associated with nerve damage in brain tissue comprising the substantia nigra pars compacta ( SNpc), striatum, hippocampus and amygdala; therefore, the oily extract rich in curcuminoids of the present invention can be used as a medicine or nutritional supplement for the treatment of diseases related to nerve damage.

According to an embodiment of the present disclosure, the curcuminoid-rich oily extract comprises curcuminoids dissolved in an oil, and the curcuminoids are composed of 25-30% (weight %) of didesmethoxy Composed of curcumin, 25-30% (weight %) of demethoxy curcumin and 40-50% (weight %) of curcumin.

According to the embodiment of the disclosure, the curcuminoid-rich oily extract or ATG-235 solution of the present invention is administered orally or parenterally to individuals suffering from diseases related to nerve damage ( For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more doses).

Diseases suitable for the treatment of the curcuminoid-rich oily extract of the present invention include, but are not limited to, dementia with Lewy bodies (DLB), Parkinson's disease (PD) or Alzheimer's disease (AD).

The subjects suitable for administering the oily extract rich in curcuminoids prepared by the method of the present invention are mammals, preferably humans.

In one embodiment, the individual is a mouse. In order to produce a therapeutic effect, the curcuminoid-rich oily extract of the present invention is administered to mice at about 1-100 mg/kg body weight per dose; preferably, about 10-90 mg/kg body weight per dose The curcuminoid-rich oily extract of the present invention; more preferably, administering the curcuminoid-rich oily extract of the present invention at about 50-70 mg per kilogram of body weight per dose is sufficient to treat nerve damage Individuals produce healing effects.

Those skilled in the art can calculate the human equivalent dose (human equivalent dose, HED) of the curcuminoid-rich oily extract of the present invention based on the animal model dose disclosed in this embodiment. Therefore, the effective dose of the ATG-235 solution of the present invention is suitable for administration to human individuals in the range of 0.08-8 mg/kg body weight per dose; preferably 0.8-8 mg/kg body weight per dose.

According to a specific preferred embodiment, the curcuminoid-rich oily extract of the present invention is administered daily for at least 7 days to the individual; for example, it is administered to the individual daily for at least 7, 8, 9, 10, 11, 12, 13, 14 or more days. In a specific embodiment, the curcuminoid-enriched oily extract of the present disclosure is administered to said individual daily for 14 days. It can be understood that those skilled in the art or clinical personnel can adjust the dose or administration regimen according to the physiological condition of the patient or the severity of the disease.

Depending on the desired purpose, the curcuminoid-rich oily extract of the present invention may be administered to an individual alone, or in combination with other treatments that have a therapeutic benefit on nerve damage. The curcuminoid-rich oily extract of the invention can be administered to the individual before, simultaneously with, or after administration of the other treatment.

A subject that can be treated using the curcuminoid-rich oily extracts and/or methods of the present invention is a mammal; for example, a human, mouse, rat, hamster, monkey, pig, dog, Cats, horses, sheep, goats, cows and rabbits. Preferably, the individual is a human.

A number of experimental examples are provided below to illustrate certain aspects of the present invention, so as to facilitate those skilled in the art to implement the present invention, and these experimental examples should not be considered as limiting the scope of the present invention. It is believed that one skilled in the art can, after reading the description presented herein, fully utilize and practice the present invention without undue interpretation. All publications cited here are considered as a part of this specification in their entirety.

Example

Materials and Methods

1- methyl -4- Phenyl — 1, 2, 3, 6- Tetrahydropyridine (1-methyl-4-phenyl-1 , 2 , 3 , 6-tetrahydropyridine, MPTP) induced neurotoxicity

C57BL/6 male mice (5 mice per cage, 12-hour light/dark cycle) were raised in an environment with an average constant temperature (25 ± 1.0°C) and a relative humidity of 60-70%, free access to food and water. All animal experiments were approved by the Animal Care and Use Committee. After one week of adaptation, the mice were randomly divided into four groups, including: (1) normal control group, (2) MPTP group, (3) MPTP + ATG-235 group, and (4) MPTP + hilligrin group, Among them, Hillichline was used as the positive control group of this experiment.

In order to establish the nerve injury model, mice were injected with intraperitoneal injection (ip) of PBS or MPTP (25 mg/kg) for 7 days; then mice were orally administered with olive oil, the ATG-235 solution of Example 2 (2 ml/ kg) or selegiline (1 mg/kg) for 14 consecutive days. All animals were sacrificed for subsequent analysis 15 days after the last dose of MPTP.

Histology, immunohistochemistry, and immunofluorescence analysis

Immediately after mouse sacrifice, brain tissue was collected. After the brain tissue was fixed with 4% paraformaldehyde and embedded in paraffin, the embedded tissue was cut into tissue sections with a thickness of 5 μm. Prior to immunostaining, tissue samples were deparaffinized with xylene and graded series of alcohols. Catalase is used to block the activity of endogenous peroxidases. The tissue samples were washed with distilled water, placed in Tris buffer containing 5% normal goat serum (containing 0.5% TWEEN ^® 20, pH 7.4 (TBS-T)), and reacted for 30 minutes. Afterwards, the tissue samples were mixed with anti-TH, anti-D1R, anti-M1R, anti-α-7R, anti-α-synuclein, anti-pTau S396, anti-pTDP43, anti-SIRT1, anti-PGC1, anti-UCP4 antibodies, and MITOTRACKER ^TM on Incubate for 2 hours at room temperature. Add secondary antibody (ALEXAFLUOR ^® 488, ALEXAFLUOR ^® 633, anti-mouse or anti-rabbit antibody) and incubate for 1 hour at room temperature. All antibodies were diluted with 2% non-immune goat serum in phosphate buffered saline (PBST) containing TWEEN ^® . The tissue samples were reacted with 3,30-diaminobenzidine (DAB) for 5-10 minutes, and treated with hematoxylin or 4',6-diamidino-2-phenylindole ( 4',6-diamidino-2-phenylindole, DAPI) stained nuclei.

Statistical Analysis

Data are presented as mean ± standard deviation (mean ± SE). The Mann-Whitney rank sum test and the Wilcoxon rank sum test were used to compare the data of different groups. P < 0.05 was considered statistically significant.

Example 1 Preparation of curcuminoid-rich oily extract of the present invention or ATG-235 the solution

The root of fresh turmeric (Taitung, Taiwan) is cut into 0.2 cm thin slices respectively. The slices were dried in an oven at 55° C., and then ground into powder by a ball mill. After the turmeric powder was filtered with 400 mesh, the dried and filtered turmeric powder was prepared, and the diameters were < 38 micrometers (μm). Take 20 grams of dried and filtered powder and 2 liters of olive oil and mix them in a flask, then extract by heating in a water-proof manner. The extraction step is a three-stage process, including (1) 50°C for 30 minutes (2) 60°C for 30 minutes (3) 80°C for 30 minutes to prepare the oily extract disclosed herein. It should be noted that in the step of "water heating", the flask is placed in a container with sufficient water in advance, and then the container is heated directly instead of directly heating the flask until the temperature in the container reaches the specified temperature. And maintain a certain preset period. Most importantly, the vessel needs to remain filled with water (i.e., the water level in the vessel must not drop to zero) throughout the baffle heating process. Furthermore, during the whole process of water heating and extraction, the flask was subjected to 28KHz ultrasonic vibration (generated by an ultrasonic oscillator (intensity: 4.1 kilowatts)). The obtained oily extract was named "ATG-235" solution, and then analyzed by liquid chromatography (LC/MS). For comparison with curcuminoids prepared by known conventional methods, dried and filtered curcuminoid powder (20 g) prepared by prior art was extracted with methanol followed by LC/MS analysis. The results are summarized in Table 1.

Table 1 summarizes the amount of curcuminoids present in the oily extracts of turmeric powder prepared by the method of the present invention and by conventional methods. Surprisingly, it has been found that the amount of bis-demethoxycurcumin contained in the oily extract of turmeric powder of the present invention is five times that of the bis-demethoxy-curcumin in the oily extract prepared by a known method; and turmeric The amount of prime was reduced to about 63%. Therefore, compared with the conventional method, the oily extract prepared in this example is rich in curcuminoids.

surface 1 Percentage of Curcuminoids in Turmeric Powder Oil Extract or Methanol Extract the compound Percentage (curcuminoids (mg) / extract (g)) The oily extract of embodiment 1 Methanol extract prepared by conventional method Didemethoxycurcumin 26.88% 5-15% Demethoxycurcumin 28.83% 20-27% curcumin 44.49% 60-70%

Example 2 ATG-235 Solution inhibits expression of proteins associated with nerve injury

As described in the materials and methods, MPTP was used to induce nerve injury in mice, and then treated with the ATG-235 solution in Example 1, Helichrine (as a positive control group) or buffer solution (as a negative control group) after MPTP For the treated mice, the expression levels of different nerve injury-related proteins in the brain tissue including the substantia nigra pars compacta (SNpc), striatum, hippocampus, and amygdala were determined. The results are illustrated in Figures 1 to 4, respectively.

The results in Figures 1 and 2 indicate that MPTP inhibits tyrosine hydroxylase (TH), type I dopamine Receptor (D1R), muscarinic acetylcholine type I receptor (M1R) and α7 nicotinic acetylcholine receptor (α-7R), but not α-synuclein expression quantity. The ATG-235 solution in Example 1 can reverse the MPTP-induced decrease in the expression of TH, D1R, M1R and α-7R, and has a better effect than Helrichline.

Furthermore, since the accumulation of tau S396 (pTau S396) and phosphorylated TDP43 (pTDP43) in the brain can cause neuropathy, the effect of ATG-235 solution on the expression of these two proteins was further determined. The results are shown in Figures 3 and 4.

The results showed that MPTP would increase the expressions of pTau S396 and pTDP43 in the substantia nigra pars compacta (SNpc), striatum, hippocampus and amygdala, respectively, which would be inhibited by the ATG-235 solution treatment of Example 1 (section 3 and 4). Furthermore, the ATG-235 solution has a better effect than Helrichline.

In summary, the results of this example can prove that the ATG-235 solution of Example 1 can achieve the effect of treating nerve damage by regulating the expression of related proteins to restore them to normal or similar levels.

Example 3 Example 1 Of ATG-235 Solution improves mitochondrial function in mice with nerve damage

Mitochondrial dysfunction is known to lead to nerve damage. Accordingly, this example will evaluate the effect of the ATG-235 solution of Example 1 on mitochondrial function in mice with nerve injury; the results are shown in Figures 5 to 7.

The data in Figures 5 to 7 show that compared with the control group, the expression levels of SIRT1, PGC1α and UCP4 in the hippocampus, striatum and substantia nigra compacta of mice treated with MPTP were respectively reduced. It can be known that IRT1, PGC1 and UCP4 are enzymes regulating mitochondrial biogenesis, respectively. Unexpectedly, administering the ATG-235 solution of Example 1 of the present invention to MPTP-treated mice can successfully restore the function of mitochondria. Furthermore, ATG-235 solution has more potential than Helrichline. In summary, the results shown in this example show that the ATG-235 solution of the present invention can improve the mitochondrial function in mice with neurotoxicity induced by MPTP.

Summarizing the above, the present disclosure provides a novel method for preparing a curcuminoid-rich oily extract (i.e., ATG-235 solution) in which 25-30% ( % by weight) of two demethoxy curcumin, 25-30% (weight %) of demethoxy curcumin and 40-50% (weight %) of curcumin. Furthermore, the oily extract rich in curcuminoids of the present invention can regulate the expression of different proteins related to nerve damage in individuals with nerve damage, and increase the function of mitochondria. Therefore, the oily extract rich in curcuminoids prepared by the method of the present invention can be used as a therapeutic agent for preventing and/or treating nerve damage, so as to improve the life span and quality of life of the individual.

Although the specific embodiments of the present invention have been disclosed in the above embodiments, they are not intended to limit the present invention. Those who have ordinary knowledge in the technical field of the present invention, without departing from the principle and spirit of the present invention, when Various alterations and modifications can be made to it, so the protection scope of the present invention should be defined by the appended patent scope.

In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows:

Figure 1 is a histogram of the results shown in Example 2 according to the present disclosure, which shows the effect of ATG-235 solution on (A) tyrosine hydroxylase (tyrosine hydroxylase; TH) in the substantia nigra pars compacta (SNpc) , (B) type 1 dopamine receptor (type 1 dopamine receptor; D1R), (C) muscarinic acetylcholine type 1 receptor (muscarinic acetylcholine type 1 receptor; M1R), (D) α7 nicotine acetyl Choline receptor (alpha-7 nicotinic acetylcholine receptor; α-7R), and (E) α-synuclein (α-synuclein) expression, each group n = 5, the results are expressed as mean ± SEM , the statistical significance of the difference between means was assessed by unpaired Student's t-test; * p < 0.05, normal control group vs. MPTP group; # p < 0.05, MPTP group vs. MPTP + ATG-235 group; ¥ P < 0.05, MPTP group is relative to MPTP+Selegiline (selegiline) group; Fig. 2 is a histogram according to the results shown in Example 2 of the present disclosure, which shows that ATG-235 solution is respectively effective for striatum (striatum) The influence of (A) TH, (B) D1R, (C) M1R, (D) α-7R) and (E) α-synuclein expression, each group n = 5, the results are mean ± SEM Performance, statistical significance of differences between means assessed by unpaired Student's t-test; * p < 0.05, normal control group vs. MPTP group; # p < 0.05, MPTP group vs. MPTP + ATG-235 group; ¥ P <0.05, the MPTP group is relative to the MPTP+Hilligrin group; Figure 3 is a histogram of the results shown in Example 2 according to the present disclosure, which shows that the ATG-235 solution is for (A) striatum, ( B) The effect of phosphorylated tau protein (phosphorylated tau; pTau) S396 expression in the substantia nigra pars compacta (SNpc), (C) hippocampus and (D) amygdala, n = 5 in each group, and the results are expressed as mean ± SEM Performance, statistical significance of differences between means assessed by unpaired Student's t-test; * p < 0.05, normal control group vs. MPTP group; # p < 0.05, MPTP group vs. MPTP + ATG-235 group; ¥ P <0.05, the MPTP group is relative to the MPTP+Hilligrin group; Figure 4 is a histogram of the results shown in Example 2 according to the present disclosure, which shows that the ATG-235 solution has effects on (A) striatum, ( B) Nigra The effect of phosphorylated 43-kDa TAR DNA-binding protein (pTDP43) expression in SNpc, (C) hippocampus and (D) amygdala, n = 5 in each group , the results are expressed as the mean ± SEM, and the statistical significance of the difference between the mean values was assessed by an unpaired Student's t test; * p < 0.05, normal control group vs. MPTP group; # p < 0.05, MPTP group vs. MPTP + ATG-235 group; ¥ P ＜0.05, MPTP group relative to MPTP+Hilligrin group; Fig. 5 is a histogram according to the results shown in Example 3 of the present disclosure, which depicts ATG-235 solution for ( A) Silent mating type information regulation 2 homolog 1 (sirtuin 1), SIRT1 expression in hippocampus, (B) striatum and (C) substantia nigra pars compacta (SNpc) The effect of each group n = 5, the results are presented as mean ± SEM, and the statistical significance of the difference between the mean values was evaluated by unpaired Student's t test; * p < 0.05, normal control group vs. MPTP group; # p < 0.05, the MPTP group is relative to the MPTP+ATG-235 group; ¥ P ＜0.05, the MPTP group is relative to the MPTP+Hilligrin group; Fig. 6 is a histogram of the results shown in Example 3 according to the present disclosure, and its plot Shows the effects of ATG-235 solution on the peroxisome proliferator-γ coactivator-1 (peroxisome proliferator- The effect of activated receptor (PPAR)-γ coactivator-1, PGC1) expression, each group n = 5, the results are expressed as mean ± SEM, and the statistical significance of the difference between the mean values is evaluated by unpaired Student's t test;* p < 0.05, normal control group vs. MPTP group; # p < 0.05, MPTP group vs. MPTP + ATG-235 group; ¥ P < 0.05, MPTP group vs. MPTP + Hilligline group; and based on Figure 7 Bar graphs of the results shown in Example 3 of the present disclosure showing ATG-235 solution for uncoupling in (A) hippocampus, (B) striatum, and (C) substantia nigra pars compacta (SNpc), respectively The impact of protein 4 (uncoupling protein 4, UCP4) expression, each group n = 5, the results are mean ± SE M performance, statistical significance of differences between means assessed by unpaired Student's t-test; * p < 0.05, normal control group vs. MPTP group; # p < 0.05, MPTP group vs. MPTP + ATG-235 group; ¥ P <0.05, MPTP group versus MPTP + Hillichline group.

Claims (10)

A method for preparing a curcuminoid-rich oily extract, comprising: extracting a dry turmeric ( Curcuma longa ) powder with an oil to obtain the curcuminoid-rich oily extract; wherein, The oily extract of the dried turmeric powder contains curcuminoids dissolved in the oil, and the curcuminoids consist of 25-30% (by weight) of bisdemethoxycurcumin, 25-30% (weight %) demethoxycurcumin (demethoxycurcumin) and 40-50% (weight %) curcumin (curcumin). The method as described in claim 1, wherein the extraction is accomplished by the following steps: (i) mixing the oil with the dry turmeric powder; and (ii) heating the oil mixture of the step (i) under ultrasonic vibration; Wherein the oil is an edible vegetable oil selected from the group consisting of coconut oil, corn oil, canola oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, sunflower oil, sesame oil, Soybean and Argan Oils. The method as described in claim 2, wherein in the step (ii), the water-isolated heating is sequentially heated at 50°C, 60°C and 80°C for 30 minutes to complete the heating of the oil mixture; and the ultrasonic The frequency of the oscillation is about 25-35kHz. The method as described in claim 2, wherein the dry turmeric powder is made by the following steps: (a) slice the rhizome of a fresh turmeric; (b) drying the slices of step (a) at 55°C; and (c) Grinding the dried chips of step (b) into powder with a ball mill. An oily extract rich in curcuminoids, which is used to treat a disease related to nerve damage, the extract comprises curcuminoids dissolved in an oil, wherein the curcuminoids consist of 25-30% (weight %) of two demethoxy curcumin, 25-30% (weight %) of demethoxy curcumin and 40-50% (weight %) of curcumin. The oily extract rich in curcuminoids as described in claim 5, wherein the oily extract rich in curcuminoids is made by the method described in any one of claims 1-4. The oily extract rich in curcuminoids as described in Claim 5, wherein the diseases related to nerve damage are dementia with Lewy bodies (DLB), Parkinson's disease (Parkinson's disease; PD) ) or Alzheimer's disease (Alzheimer disease; AD). A use of a curcuminoid-rich oily extract to prepare a medicine for treating a disease related to nerve damage, wherein the curcuminoid-rich oily extract contains curcuminoid In an oil, and the curcuminoids are composed of 25-30% (weight %) of two demethoxy curcumin, 25-30% (weight %) of demethoxy curcumin and 40-50% (weight %) %) of curcumin. The use as described in Claim 8, wherein the oily extract rich in curcuminoids is made from any one of Claims 1-4. The use as described in claim 8, wherein the disease related to nerve damage is dementia with Lewy bodies (DLB), Parkinson's disease (PD) or Alzheimer's disease (AD).

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