L-serine having reduced heavy metal content, pharmaceutical composition thereof, method for preparing same, and use thereof

Abstract

The present invention relates to L S-serine having a reduced heavy metal content, a pharmaceutical composition thereof, a method for preparing same, and a use thereof. The L-serine having a reduced heavy metal content, according to the present invention, may be used for preventing, alleviating, or treating neurological diseases or metabolic diseases.

Description

L-serine with reduced heavy metal content, pharmaceutical composition thereof, method for preparing the same, and use thereof

[0001] The present invention relates to L-serine with reduced heavy metal content, a pharmaceutical composition thereof, a method for preparing the same, and a use thereof.

[0002] Heavy metals are non-biodegradable and chemically heavy metals with a specific gravity of 4.0 or higher. While iron, zinc, and copper are essential substances for the human body, lead, mercury, cadmium, and arsenic are not only harmful to living organisms but also accumulate in the body without being metabolized, causing various serious diseases, including cancer. In particular, lead, mercury, cadmium, and arsenic have various adverse effects on metabolic activities throughout the body, including the central nervous system, peripheral nervous system, circulatory system, lungs, kidneys, liver, blood, digestive system, and reproductive system. In particular, exposure of the nervous system to harmful heavy metals can lead to serious and irreversible conditions, such as degenerative neurological diseases. Fetuses and children are especially vulnerable to heavy metal exposure, and can be seriously affected during their neural development, leading to lifelong developmental disabilities or mental / neurological symptoms. Until now, the correlation between heavy metals, especially Class 1 heavy metals such as lead, mercury, cadmium, and arsenic, and neurological and metabolic diseases has been studied and reported.

[0003] In the literature [J Child Neurol, 2002, 19, 692-695, Clin Chem 1981, 27, 879-881], the content of harmful heavy metals in the hair of children with normal development and children with autism spectrum disorder was measured, and it was observed that the content of cadmium and lead was significantly higher in children with autism spectrum disorder than in children with normal development.

[0004] In the literature [Journal of Trace Elements in Medicine and Biology, 2021], the effect of neurotoxic metals (lead, mercury, cadmium, nickel, manganese) as environmental factors on autism spectrum disorder was studied, and it was confirmed that children are more vulnerable because they have immature and inefficient metabolic and detoxification processes, and their immune systems are particularly weak in infancy, making them unable to effectively remove toxic substances, and that autism symptoms are alleviated with anti-heavy metal treatment.

[0005] L-serine is a non-essential amino acid and an endogenous substance, and is used in various ways, such as in foods, health functional foods, food additives, and intravenous drugs. The present inventors conducted research on a health functional food composition containing L-serine as an active ingredient for preventing or improving neurological diseases through Patent No. 2019-0046017, and subsequently conducted a phase 2 clinical trial and confirmed that when administered to children 11 years of age or younger for 24 weeks at a dose of 400 mg / kg, it showed a therapeutic efficacy of approximately 40% without any specific side effects. The clinical results conducted by the present inventors confirmed that the therapeutic effect was maximized when L-serine was administered at a high dose for a long period of time.

[0006] In other clinical trials in humans, long-term administration of 400 mg / kg / day L-serine for up to 2 years was shown to be safe and improve Charcot-Marie-Tooth Neuropathy Score (CMTNS) in patients with hereditary sensory and autonomic neuropathy type 1 (HSAN1), and oral L-serine at doses up to 15 g / day twice daily for 6 months was reported to be safe and effective in slowing the progression of the disease in patients with amyotrophic lateral sclerosis (ALS). In addition, oral administration of L-serine at concentrations up to 400 mg / kg / day for 10 and 52 weeks in HSAN1 patients was found to reduce the level of neurotoxicity by reducing 1-deoxySL (dSL), which induces neurodegeneration. In addition, in a phase 2 clinical trial targeting ALS patients, L-serine was found to be safe in ALS patients at very high doses of up to 15 g twice a day for 6 months, and in a trial observing the efficacy and adverse reactions of L-serine in pregnant women and their children with a genetic factor for congenital microcephaly, it was reported that maternal L-serine supply during pregnancy improved brain growth in fetuses with 3-PGDH deficiency, and administration of L-serine to children after birth (birth to 4 years old) prevented neurological symptoms, and no adverse reactions were observed in pregnant women, fetuses, or newborns.

[0007] Publication No. US 20180027781 discloses a method for treating neurometabolic diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), Progressive Supranuclear Palsy (PSP), Lewy Body Dementia (LBD), Amyotrophic Lateral Sclerosis / Parkinsonism Dementia Complex (ALS / PDC), Huntington's disease (HD), Pick's disease, or Frontotemporal Dementia (FTD) by taking L-serine at a daily dose of up to 750-1,000 mg / kg.

[0008] Publication patent KR 10-2022-0124007 discloses a pharmaceutical composition for preventing or treating liver fibrosis containing L-serine as an active ingredient, and discloses that L-serine can be used in combination with losartan to prevent and treat liver disease.

[0009] However, when using L-serine as an active ingredient in pharmaceuticals or health functional foods, if taken in high doses for a long period of time, the use of L-serine managed by a simple daily allowance limit may reduce the original efficacy due to the nature of heavy metals that accumulate without being biodegraded, and especially in children and infants, it may cause serious illness. In particular, L-serine intended for use in subjects with neurological or metabolic diseases needs to be managed below the standard allowable value for Class 1 heavy metals.

[0010] For example, if we apply the EU heavy metal allowance (Table 1) of Omega 3, widely known as a health functional food, to the dosage of L-serine used in phase 2 clinical trials for the treatment of patients with autism spectrum disorder, if a 40 kg patient with autism spectrum disorder aged 2-11 years took L-serine for 24 weeks, the amount of each group 1 heavy metal while taking 16 g of L-serine per day would be 1.6 μg, and the maximum amount of group 1 heavy metals that can accumulate for 24 weeks would be 1,152 μg, meaning that a significant amount of heavy metals would accumulate in the body. Furthermore, if we consider the possible routes of ingestion through food, drinking water, beverages, etc., the situation requires even more urgent management.

[0011] EU standard omega-3 heavy metal content allowable standardHeavy metal contentArsenic (As) 0.1 (μg / g)Lead (Pb) 0.1 (μg / g)Cadmium (Cd) 0.1 (μg / g)Mercury (Hg) 0.1 (μg / g)

[0012] The daily heavy metal exposure amount of L-serine calculated above is in accordance with the permitted daily exposure (PDE) of Class 1 heavy metals of concern for toxicity recommended by the Ministry of Food and Drug Safety in Table 2 below. However, considering that L-serine is used as a drug or health functional food in high doses for a long period of time, an excessive amount of Class 1 heavy metals may accumulate in the body and cause effects at the effective dose.

[0013] Acceptable daily exposure levels for metal impurities Metal classification (grade) Oral PDE (μg / day) Parenteral PDE (μg / day) Inhalation PDE (μg / day) Cadmium (Cd) 1522 Lead (Pb) 1555 Arsenic (As) 115152 Mercury (Hg) 13031

[0014] Accordingly, the inventors of the present invention have examined various methods for removing heavy metals from L-serine, but theoretically, methods frequently used for removing heavy metals, such as precipitation in the form of hydroxides or sulfides, ion exchange, adsorption treatment using activated carbon, electrochemical treatment, solvent extraction, and evaporation, have the disadvantages of being expensive to process industrially, incomplete removal depending on the item, being restricted by pH conditions or limited to high concentrations, or generating harmful sludge or by-products.

[0015] Specifically, precipitation with sulfides or hydroxides carries the risk of exothermic reactions when used in mass purification methods. In particular, L-serine, which has a chiral carbon, may experience a switch in optical activity due to exothermic reactions under strong acid or strong alkaline conditions. In addition, after heavy metal removal, equipment may be damaged during filtration and hygroscopicity may reduce the yield. In addition, additional costs arise due to the cost of the neutralization process and wastewater treatment. On the other hand, when L-serine is dissolved in water and stirred with an ion exchange resin, the water solubility of L-serine decreases, requiring a large amount of purified water. In addition, precipitation may form at the point where the ion exchange resin and L-serine meet during ion exchange, reducing the ion exchange effect. In addition, the recycling of the ion exchange resin is not easy. On the other hand, the use of activated carbon increases the risk of contamination of the reactor and leakage during filtration. Therefore, it is rarely stirred with the purified product industrially. In addition, to prevent a decrease in yield, the dissolved solvent must be removed again, which entails a lot of time and money.

[0016] Therefore, the inventors of the present invention can obtain a primary heavy metal removal effect by maximizing the water solubility of existing L-serine through appropriate pH control in the solution phase, and can significantly reduce heavy metals compared to the existing one simply by passing it through activated carbon and / or ion exchange resin, and can expect a significant economic saving effect by obtaining the desired L-serine as a solid precipitate only by pH control in the solution phase.

[0017] Accordingly, the inventors of the present invention have developed a method for economically producing and / or purifying L-serine with a reduced content of class 1 heavy metals for use in the prevention, treatment or improvement of neurological or metabolic diseases.

[0018] The purpose of the present invention is to provide L-serine with reduced heavy metal content, which can exhibit increased efficacy and reduced side effects compared to existing L-serine when used for the prevention, improvement or treatment of neurological or metabolic diseases, and to provide a purification method for economically reducing the heavy metal content.

[0019] To achieve the above purpose, the present invention provides L-serine of the following chemical formula 1 having a reduced content of heavy metals.

[0020] The present invention also provides a pharmaceutical composition for preventing or treating a neurological disease or metabolic disease, comprising the L-serine.

[0021] The present invention also provides a health functional food for preventing or improving neurological or metabolic diseases, comprising the L-serine as an active ingredient.

[0022] The present invention also provides a method for reducing the heavy metal content of L-serine, comprising the following steps.

[0023] (a) A step of dissolving L-serine of the following chemical formula 1 in water;

[0024] (b) a step of adjusting the pH of the L-serine aqueous solution obtained in step (a) with an acid;

[0025] (c) passing the pH-adjusted L-serine solution through activated carbon and / or ion exchange resin; and

[0026] (d) A method for producing L-serine with reduced heavy metal content, comprising a step of neutralizing the L-serine aqueous solution obtained in step (c) while gradually lowering the temperature.

[0027] [Chemical Formula 1]

[0028]

[0029] A method for producing L-serine with reduced heavy metal content, wherein the heavy metal is at least one selected from the group consisting of cadmium, arsenic, lead and mercury.

[0030] The present invention maximizes the effect of L-serine, which can be used to prevent and treat diseases selected from the group consisting of developmental disorders, developmental delay, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, emotional disorders, sleep disorders, and movement disorders, and to prevent and treat fatty liver, non-alcoholic steatohepatitis, abdominal obesity, hypertriglyceridemia, low HDL (high density lipoprotein) cholesterolemia, hypertension, dysglycemic syndrome, type 2 diabetes, cardiovascular disease, myocardial infarction, and angina, and can minimize the side effects of class 1 heavy metals that can accumulate due to long-term administration of high doses.

[0031] Figure 1 shows the inhibitory effect on mitochondrial damage according to the difference in the content of class 1 heavy metals using L-serine obtained by Example 1 of the present invention.

[0032] Figure 2 shows the effect of drugs on the survival of animals when L-serine and unpurified L-serine obtained by Example 1 of the present invention were administered to an Alzheimer's disease model for long-term use for 9 months.

[0033] Figure 3 confirms the protective effect of the drug against neurotoxicity of L-serine and unpurified L-serine obtained by Example 1 of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0035] The present invention relates, in one aspect, to a method for producing L-serine with reduced heavy metal content.

[0036] Embodiments of the present invention include:

[0037] (a) A step of dissolving L-serine of the following chemical formula 1 in water;

[0038] (b) a step of adjusting the pH of the L-serine aqueous solution obtained in step (a) with an acid;

[0039] (c) passing the pH-adjusted L-serine solution through activated carbon and / or ion exchange resin; and

[0040] (d) A method for producing L-serine with reduced heavy metal content, comprising a step of neutralizing the L-serine aqueous solution obtained in step (c) while gradually lowering the temperature.

[0041] [Chemical Formula 1]

[0042]

[0043] A method for producing L-serine with reduced heavy metal content, wherein the heavy metal is at least one selected from the group consisting of cadmium, arsenic, lead and mercury.

[0044] In the present invention, “L-serine” is a type of amino acid that constitutes proteins, is a monoamide of glutamic acid, and means an L-amino acid having a structure represented by the following chemical formula 1.

[0045] [Chemical Formula 1]

[0046]

[0047] In the present invention, step (a) is a step of dissolving L-serine in water.

[0048] (a) The amount of water used in step may be about 6-30 times the weight of L-serine used, preferably about 5-20 times, and most preferably about 4-10 times, but is not limited thereto. If too much water is used, handling during the process becomes difficult, and the amount of wastewater discarded after the process increases, so dissolving it in a minimal amount is important for economic efficiency. In addition, when the temperature is increased, some of the L-serine is converted to D-serine, so it is necessary not to increase the temperature above room temperature.

[0049] In the present invention, step (b) is a step of adjusting the pH of the L-serine aqueous solution obtained in step (a) with acid.

[0050] Adjusting the pH of an L-serine aqueous solution to acidic increases its water solubility by more than five times compared to neutral L-serine, not only significantly reducing the process volume but also increasing the water solubility of heavy metals that are difficult to remove due to low water solubility in neutral conditions, thereby improving the removal rate. In other words, by appropriately adjusting the pH in the solution, the water solubility of existing L-serine can be increased, thereby enhancing the removal rate of heavy metals.

[0051] (b) In step (b), the pH is adjusted with acid, and the adjusted pH can be about pH 1.0-5.0, preferably about pH 2.0-4.5, most preferably about pH 2.5-4.0, but is not limited thereto. On the other hand, if the pH is too low, the amount of acid used increases, which reduces economic feasibility, and there is a risk of corrosion of equipment used in the process, such as reactors or filters made of metal and weak to acid, and a risk of additional heavy metal inflow due to this. In addition, a large amount of alkaline solution is required in the subsequent neutralization process, which significantly reduces the economic feasibility of the process.

[0052] In the present invention, any acid known in the art may be selected and used as long as it does not deviate from the purpose of the present invention. Specifically, the acid may be at least one selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, p-toluenesulfonic acid, fumaric acid, citric acid, succinic acid, salicylic acid, oxalic acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, tartaric acid, sulfuric acid, and mandelic acid. Preferably, the acid is hydrochloric acid, but is not limited thereto.

[0053] The acid can be used by adding it directly to the solution or by dissolving it in a certain amount of water.

[0054] In the present invention, step (c) is a step of passing the L-serine solution with adjusted pH through activated carbon and / or ion exchange resin.

[0055] In the present invention, the activated carbon may be an amorphous, spherical or spherical activated carbon having a density (g / cm3) of about 0.2-2.5, and amorphous activated carbon is most preferred, but is not limited thereto. The weight of the activated carbon may be about 1 / 100 to 1 / 10 of the weight of the L-serine to be purified, and preferably about 2 / 100-5 / 100.

[0056] In the present invention, all types of ion exchange resins can be used, including strongly acidic, weakly acidic, weakly basic, and strongly basic. The weight of the ion exchange resin is about 1-10 times the weight of the L-serine to be purified, preferably about 2-8 times, and most preferably about 3-5 times, but is not limited thereto. For example, the solution can be passed through activated carbon and / or ion exchange resin or stirred together. When passing through activated carbon and / or ion exchange resin, a Nutche or pressure filter having an appropriate diameter can be used so that the solution containing dissolved L-serine can pass through with sufficient contact. After use, the ion exchange resin can be recovered by filtration, washed with purified water, dried, and reused.

[0057] In the present invention, either activated carbon or ion exchange resin may be used in step (c), or both activated carbon and ion exchange resin may be used. If the heavy metal content exceeds 50 ppm after activated carbon treatment, ion exchange resin may be used as a secondary purification to remove the heavy metals. When using them sequentially, it is preferable to use activated carbon first.

[0058] In the present invention, step (d) is a step of neutralizing the L-serine aqueous solution obtained in step (c) while gradually lowering the temperature.

[0059] In the above step (d), neutralization generates heat of neutralization. If the temperature is too high, some of the L-serine will not be identical to the original crystalline form, and discoloration may occur. Therefore, the reaction temperature should be as low as possible, preferably below room temperature, and most preferably below 15°C, but is not limited thereto. For example, neutralization can be carried out by gradually lowering the temperature to about 10 to 15°C.

[0060] In the present invention, any base known in the art may be selected and used, as long as it does not deviate from the purpose of the present invention. Specifically, the base may be at least one selected from the group consisting of potassium hydroxide, calcium hydroxide, potassium carbonate, calcium carbonate, sodium hydroxide, sodium carbonate, sodium phosphate, and potassium phosphate, most preferably sodium hydroxide and potassium hydroxide, but is not limited thereto.

[0061] The base can be used in an amount equivalent to the acid used in step (b) above, and the base can be added directly to the solution or dissolved in a certain amount of water.

[0062] In the present invention, after step (d), a step (e) of performing pressure filtration and drying the obtained solid may be additionally included.

[0063] Specifically, in step (e), any filtration method possible in industrial production, such as depressurization filtration, centrifugation, and filter dryer, may be used, and the drying temperature may be about 20-80°C, preferably about 30-70°C, and most preferably about 40-60°C, using a vacuum or hot air drying method, but is not limited thereto. However, caution should be exercised as discoloration may occur if the drying temperature is too high.

[0064] In the present invention, “heavy metal” or “class 1 heavy metal” is selected from the group consisting of cadmium, arsenic, lead, and mercury.

[0065] According to the method of the present invention, the content of the heavy metal can be reduced to about 76 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, or 55 ppm or less per 1 g of L-serine, and preferably, can be reduced to about 50 ppm or less.

[0066] In another aspect, the present invention relates to L-serine, wherein the content of cadmium, arsenic, lead and mercury is each 50 ppm or less per 1 g of L-serine.

[0067] L-serine manufactured according to the above method of the present invention may be L-serine having a content of cadmium, arsenic, lead and mercury of 50 ppm or less per 1 g of L-serine.

[0068] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a neurological disease or metabolic disease, comprising L-serine as an active ingredient, wherein the content of cadmium, arsenic, lead and mercury is 50 ppm or less per 1 g of L-serine.

[0069] The term "treatment" as used herein refers to any act of improving or beneficially changing the symptoms of a disease by administering the composition according to the present invention, the term "prevention" refers to any act of suppressing or delaying a disease by administering the composition according to the present invention, and the term "improvement" refers to any act of improving the bad state of a disease by administering or ingesting the composition of the present invention to a subject.

[0070] The disease targeted for "treatment," "prevention," or "improvement" of the present invention may be a neurological disease or a metabolic disease. Specifically, the neurological disease may be selected from the group consisting of developmental disorders, developmental delay, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, emotional disorders, sleep disorders, and movement disorders, and the metabolic disease may be selected from the group consisting of fatty liver, non-alcoholic steatohepatitis, abdominal obesity, hypertriglyceridemia, low HDL (high-density lipoprotein) cholesterolemia, hypertension, dysglycemic syndrome, type 2 diabetes, cardiovascular disease, myocardial infarction, and angina.

[0071] The pharmaceutical composition of the present invention may be administered to a subject or individual via any route of administration, and may be a pharmaceutical composition for oral administration or injection. Preferably, the pharmaceutical composition of the present invention may be administered orally or intravenously.

[0072] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of such pharmaceutically acceptable carriers include carriers for oral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Furthermore, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose, and glycols, and may further comprise stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers known in the art may be selected and used.

[0073] The pharmaceutical composition of the present invention may further include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0074] An effective dosage may generally range from about 50 to about 1000 mg / kg body weight per day, either in single or divided doses, preferably from about 100 to about 800 mg / kg body weight per day, and most preferably from 200 to 400 mg / kg body weight per day. Dosage levels below the lower end of this range may be appropriate, depending on age, species, and the disease or condition being treated. Higher doses may be used as long as no adverse side effects occur. Higher doses may be divided into several smaller doses for administration throughout the day. Appropriate dosages can be determined by methods known in the art.

[0075] In the present invention, the content of L-serine in the pharmaceutical composition of the present invention may be about 60% to 100% by weight.

[0076] In another aspect, the present invention relates to a health functional food composition for preventing or improving neurological or metabolic diseases, comprising L-serine as an active ingredient, wherein the content of cadmium, arsenic, lead and mercury is 50 ppm or less per 1 g of L-serine.

[0077] The health functional food according to the present invention may be used together with other foods or food additives in addition to those containing the L-serine as an active ingredient, and may be appropriately used according to a conventional method.

[0078] In the present invention, the amount of active ingredient mixed may be appropriately determined depending on the intended use, such as preventive, health, or therapeutic treatment. For example, the content of L-serine in the health functional food composition of the present invention may be about 60% to 100% by weight.

[0079] The effective dosage of compounds contained in health functional foods may be used in accordance with the effective dosage of the aforementioned therapeutic agent. However, for long-term consumption for health and hygiene purposes or for health management purposes, the dosage may be lower than the aforementioned range. Furthermore, amounts exceeding the aforementioned range may be used as long as there are no safety concerns.

[0080] There are no specific restrictions on the types of health functional foods, and examples thereof include, but are not limited to, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0081] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0082] In another aspect, the present invention is a method for preventing or treating a neurological disease or metabolic disease in a subject, comprising administering to the subject L-serine having a content of cadmium, arsenic, lead and mercury of 50 ppm or less per 1 g of L-serine.

[0083] In the present invention, the term "subject" is used synonymously with an individual, and the subject may be a mammal, for example, a human, a rat, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.

[0084] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0085] In another aspect, the present invention is a use of L-serine having a content of cadmium, arsenic, lead and mercury of 50 ppm or less per 1 g of L-serine, for producing a medicament for preventing or treating a neurological disease or metabolic disease.

[0086] Any description that overlaps with the pharmaceutical composition of the present invention described above is omitted.

[0087] Hereinafter, preferred examples are presented to help understand the present invention, but the following examples are only illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and technical idea of ​​the present invention, and it is also natural that such changes and modifications fall within the scope of the appended patent claims.

[0088] Example

[0089] Example 1

[0090] At room temperature, place 4.0 kg of L-serine (Hunan Furui, China) and 20 L of purified water in a reactor and stir for 10 minutes. Adjust the pH to approximately 3.3-3.5 with concentrated hydrochloric acid (Daejung Chemicals) and confirm complete dissolution. After stirring for 1 hour, place a celite pad, place 150 g of amorphous activated carbon (Samjeon Pure Chemicals) on the celite pad, and filter under reduced pressure, being careful not to damage the laminated surface. Check the content of Class 1 heavy metals in the filtered solution using an ICP analyzer. Place 12 kg of TRILITE® CLR-10 (purchased from Samyang Corporation) on the celite pad, add the obtained solution, and filter under reduced pressure. The ion exchange resin was recovered, the temperature of the obtained solution was lowered to 10-15℃, the pH was adjusted to 6-7 with 6N-NaOH, and the mixture was slowly stirred at 20-40 rpm for 3 hours, filtered under reduced pressure, and the obtained solid was washed with 2 L of purified water. L-serine was obtained in a yield of 91% by vacuum drying at 50℃.

[0091] Example 1 Grade 1 heavy metal content Before purification (a) After activated carbon purification (1st, b) After ion exchange resin purification (2nd, c) Mercury content in L-serine 72 ppm 46 ppm 39 ppm mL - Lead content in serine 86 ppm 56 ppm 27 ppm mL - Cadmium content in serine 69 ppm 30 ppm 22 ppm mL - Arsenic content in serine 76 ppm 51 ppm 13 ppm

[0092] Example 2

[0093] At room temperature, place 4.0 kg of L-serine (Hunan Furui, China) and 20 L of purified water in a reactor and stir for 10 minutes. Adjust the pH to approximately 3.3-3.5 with concentrated hydrochloric acid (Daejung Chemicals) and confirm complete dissolution. After stirring for 1 hour, place a celite pad, place 120 g of amorphous activated carbon (Samjeon Pure Chemicals) on the celite pad, and filter under reduced pressure, being careful not to damage the laminated surface. Check the content of Class 1 heavy metals in the filtered solution using an ICP analyzer. Place 10 kg of TRILITE® CLR-10 (purchased from Samyang Corporation) on the celite pad, add the obtained solution, and filter under reduced pressure. The ion exchange resin was recovered, the temperature of the obtained solution was lowered to 10-15℃, the pH was adjusted to 6-7 with 6N-NaOH, and the mixture was slowly stirred at 20-40 rpm for 3 hours, filtered under reduced pressure, and the obtained solid was washed with 2 L of purified water. L-serine was obtained in 88% yield by vacuum drying at 50℃.

[0094] Example 2 Grade 1 heavy metal content Before purification (a) After activated carbon purification (1st, b) After ion exchange resin purification (2nd, c) Mercury content in L-serine 72 ppm 55 ppm 39 ppm mL - Lead content in serine 86 ppm 66 ppm 47 ppm mL - Cadmium content in serine 69 ppm 37 ppm 42 ppm mL - Arsenic content in serine 76 ppm 50 ppm 18 ppm

[0095] Example 3

[0096] At room temperature, place 4.0 kg of L-serine (Hunan Furui, China) and 20 L of purified water in a reactor and stir for 10 minutes. Adjust the pH to approximately 3.3-3.5 with concentrated hydrochloric acid (Daejung Chemicals) and confirm complete dissolution. After stirring for 1 hour, place a celite pad, place 150 g of amorphous activated carbon (Samjeon Pure Chemicals) on the celite pad, and filter under reduced pressure, being careful not to damage the laminated surface. Check the content of Class 1 heavy metals in the filtered solution using an ICP analyzer. Place 12 kg of TRILITE® CLR-8 (purchased from Samyang Corporation) on the celite pad, add the obtained solution, and filter under reduced pressure. The ion exchange resin was recovered, the temperature of the obtained solution was lowered to 10-15℃, the pH was adjusted to 6-7 with 6N-NaOH, and the mixture was slowly stirred at 20-40 rpm for 3 hours, filtered under reduced pressure, and the obtained solid was washed with 2 L of purified water. L-serine was obtained in a yield of 91% by vacuum drying at 50℃.

[0097] Example 3 Grade 1 heavy metal content Before purification (a) After activated carbon purification (1st, b) After ion exchange resin purification (2nd, c) Mercury content in L-serine 72 ppm 46 ppm 37 ppm - Lead content in serine 86 ppm 52 ppm 32 ppm - Cadmium content in serine 69 ppm 44 ppm 26 ppm - Arsenic content in serine 76 ppm 55 ppm 37 ppm

[0098] Example 4

[0099] At room temperature, place 4.0 kg of L-serine (Hunan Furui, China) and 20 L of purified water in a reactor and stir for 10 minutes. Adjust the pH to approximately 3.3-3.5 with concentrated sulfuric acid (Daejung Chemicals) and confirm complete dissolution. After stirring for 1 hour, place a celite pad, place 150 g of amorphous activated carbon (Samjeon Pure Chemicals) on the celite pad, and filter under reduced pressure, being careful not to damage the laminated surface. Check the content of Class 1 heavy metals in the filtered solution using an ICP analyzer. Place 12 kg of TRILITE® CLR-10 (purchased from Samyang Corporation) on the celite pad, add the obtained solution, and filter under reduced pressure. The ion exchange resin was recovered, the temperature of the obtained solution was lowered to 10-15℃, the pH was adjusted to 6-7 with 6N-NaOH, and the mixture was slowly stirred at 20-40 rpm for 3 hours, filtered under reduced pressure, and the obtained solid was washed with 2 L of purified water. L-serine was obtained in 80% yield by vacuum drying at 50℃.

[0100] Example 4 Grade 1 heavy metal content Before purification (a) After activated carbon purification (1st, b) After ion exchange resin purification (2nd, c) Mercury content in L-serine 72 ppm 33 ppm 19 ppm - Lead content in serine 86 ppm 76 ppm 47 ppm - Cadmium content in serine 69 ppm 40 ppm 42 ppm - Arsenic content in serine 76 ppm 51 ppm 43 ppm

[0101] As can be seen from the content of class 1 heavy metals in Examples 1 to 4, it can be confirmed that the content of class 1 heavy metals in L-serine is greatly reduced when both activated carbon and ion exchange resin are used.

[0102] Below, the effect of L-serine obtained in Example 1 on neurological or metabolic diseases of class 1 heavy metals was confirmed through experimental examples.

[0103]

[0104] Experimental Example 1

[0105] HT22 cells were cultured in DMEM medium containing 10% FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin at 5% CO2 and 37°C. 5 × 10 cells were seeded in 6-well plates. 5 Cells were seeded at a concentration of 10 cells / well and cultured for 20 hours at 37°C and 5% CO2. When the cell confluency reached 80%, the cells were pretreated with the drug and cultured for 4 hours. 20 μM DMNQ was treated together with the drug and cultured for 2 hours. After checking the cell status, 5 μM JC-1 was added to the medium, and staining was performed for 30 minutes at 37°C and 5% CO2. The cells were collected in an e-tube by treating with 0.25% trypsin-EDTA, washed twice with PBS, and analyzed with Attune NxT.

[0106] We investigated the concentration-dependent effects of L-serine, a heavy metal that affects mitochondria, which is involved in both neurological and metabolic diseases. DMNQ induces oxidative stress, resulting in excessive production of ROS, which induces mitochondrial membrane potential depolarization, leading to mitochondrial dysfunction and apoptosis. Mitochondria generate an electrochemical proton gradient across the mitochondrial membrane, which produces ATP. When mitochondria are damaged and apoptosis occurs, mitochondrial permeability pores open, reducing the mitochondrial transmembrane potential (membrane potential depolarization). The mitochondrial membrane potential difference, an indicator of cell death, was measured using JC-1 staining. JC-1 is a red complex that enters the mitochondria in normal cells due to changes in mitochondrial membrane potential, and when apoptosis occurs due to mitochondrial membrane potential depolarization, it remains in the cytoplasm and turns green. 4 hours before DMNQ treatment, unpurified L-serine, primary purified L-serine, and secondary purified L-serine were treated to neurons, and then DMNQ was treated for 2 hours and 30 minutes. The degree of neuronal protection was measured by measuring the mitochondrial membrane potential difference through JC-1 staining.

[0107] As a result, the L-serine treatment groups showed an inhibitory effect on mitochondrial damage (green: 81.4±4.2%) caused by DMNQ depending on the content of class 1 heavy metals (green; 0.5 mM-32.1±1.52%, 1 mM-18.7±11.1%, 2.5 mM-1.5±0.23%). In particular, it was confirmed that the secondary purified L-serine, in which all class 1 heavy metals were less than 50 ppm, showed a similar level of inhibitory effect on damage as the control group (see Fig. 1).

[0108]

[0109] Experimental Example 2

[0110] In order to measure the extent to which the heavy metal content of L-serine affects neurons when taken long-term, 5XFAD (Tg6799; five familial mutation), an animal model of Alzheimer's disease, was used. L-serine was administered long-term for 9 months from the early stage of the disease (3 months of age) to 12 months of age, and the effect of the drug on the survival of the animals was confirmed. Specifically, WT & 5XFAD mice (purchased from The Jackson Lab) administered saline and WT & 5XFAD mice administered L-serine were divided into groups of 12 each. Saline and 500 mg / kg crude L-serine and secondary purified L-serine were orally administered 5 days a week for 9 months, and the survival rate (%) was measured by observing the mice daily after administration, which was expressed as the survival rate.

[0111] As a result, it was confirmed that the survival rate of the secondary purified L-serine treatment group was significantly higher than that of the non-purified L-serine treatment group (80% compared to 58%) (see Fig. 2).

[0112]

[0113] Experimental Example 3

[0114] The cerebral cortex of mouse fetuses was isolated from the mother and transferred to HBSS solution, minced, and treated with 0.5% trypsin-EDTA in a 37°C water bath for 20 minutes. An equal volume of culture media was added, and the cells were mechanically separated using a pipet. Single cells were isolated using a 40 μm cell strainer. After centrifugation at 2,000 rpm for 5 minutes, the supernatant was removed and resuspended in culture media.

[0115] <Plate 제작>

[0116] Preparation of PEI solution - A 50% PEI solution was prepared by mixing borate buffer and PEI in a 1:1 ratio, diluted to 0.1% PEI solution using borate buffer, and then filtered using a 0.22 μm filter.

[0117] Laminin Preparation - Laminin stock stored at -20°C was thawed at 4°C and diluted with serum-free medium (20 μg / ml). 40 μl of 0.1% PEI was added to each well of a 24-well MEA plate and incubated in a 37°C, 5% CO2 incubator for 60 minutes. The 24-well MEA plate was washed three times with sterile deionized water, and 50 μl of laminin solution was added per well and incubated overnight at 37°C, 5% CO2 in a cell incubator.

[0118] <Primary cortical neuronal cultures>

[0119] After removing the laminin solution from the 24-well MEA plate, 1.0×10 prepared primary neuron cells were seeded 5Cells were seeded at a concentration of 10 cells / well. The cells were cultured at 37°C and 5% CO2, with neuronal media containing 2% B-27 plus supplement, 100 units / mL penicillin, and 100 μg / mL streptomycin replaced every 3-4 days until DIV (Day In Vitro) 14-18. The optimal state of neuronal network electrical activity was confirmed at DIV 14-18 using the Maestro Edge system (Axion Biosystems, GA). Using the Maestro Edge system, neuronal electrical activity was measured every 30 minutes for 15 minutes, and 0.1 mM unpurified L-serine was pretreated with the same amount of secondary purified L-serine for 4 hours. After 4 hours of drug pretreatment, neuronal electrical activity was measured four times at 30-minute intervals during 2 hours of treatment with 20 μM DMNQ, and the data were analyzed using a neural metric tool and an axis metric plotting tool.

[0120] To determine how the level of heavy metals in the first group affects the neuroprotective effect of L-serine, a multi-electrode array (MEA) was used. MEA cultures neurons on electrodes for several weeks to form a network, and then measures the electrical signals, or action potentials, generated by the neurons. This method can simultaneously measure cell responses to external stimuli or environmental changes from as many cells as there are electrodes. It records the action potentials of the entire neural network in vivo and in vitro, and is widely used in drug screening by detecting corresponding voltage changes spontaneously or in response to stimulation or treatment.

[0121] Primary neuronal cultures were cultured in 24-well MEA plates for approximately 15 days, and action potentials were measured and compared by treating test substances at concentrations that did not affect cell viability when electrical signals activated sporadic bursts and synchronous network bursts.

[0122] As a result, when DMNQ was treated at a concentration that did not affect cell viability in primary neurons (b), active electrodes (spikes and network bursts) were significantly reduced compared to the control group (a). In the case of the unrefined L-serine treatment group (c), there was a change in the active electrodes reduced by DMNQ, but the effect was not clear. On the other hand, in the case of the DMNQ + secondary purified L-serine treatment group (d), it was confirmed that the active electrodes in the untreated state were maintained even after DMNQ treatment. From this, it can be confirmed that L-serine has a protective effect against DMNQ-induced neurotoxicity, and that the protective effect is increased by a decrease in the content of Class 1 heavy metals in L-serine (see Fig. 3).

[0123]

[0124] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the claims and their equivalents.

Claims

1. (a) A step of dissolving L-serine of the following chemical formula 1 in water; (b) a step of adjusting the pH of the L-serine aqueous solution obtained in step (a) with an acid; (c) passing the pH-adjusted L-serine solution through activated carbon and / or ion exchange resin; and (d) a method for producing L-serine with reduced heavy metal content, comprising a step of neutralizing the L-serine aqueous solution obtained in step (c) while gradually lowering the temperature; [Chemical Formula 1] A method for producing L-serine with reduced heavy metal content, wherein the heavy metal is at least one selected from the group consisting of cadmium, arsenic, lead and mercury.

2. A method for producing L-serine with reduced heavy metal content, wherein the content of each heavy metal in paragraph 1 is 50 ppm or less per 1 g of L-serine.

3. A method for producing L-serine with reduced heavy metal content, wherein the pH is adjusted to 1 to 5 in step (b) of paragraph 1.

4. A method for producing L-serine with reduced heavy metal content, wherein in step (b), the pH is adjusted with one or more acids selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, p-toluenesulfonic acid, fumaric acid, citric acid, succinic acid, salicylic acid, oxalic acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, tartaric acid, sulfuric acid, and mandelic acid.

5. A method for producing L-serine with reduced heavy metal content, wherein in step (d) of paragraph 1, the temperature is lowered to 10 to 15°C.

6. A method for producing L-serine with reduced heavy metal content, wherein in step (d), the process is performed by neutralizing with at least one base selected from the group consisting of potassium hydroxide, calcium hydroxide, potassium carbonate, calcium carbonate, sodium hydroxide, sodium carbonate, sodium phosphate, and potassium phosphate.

7. A method for producing L-serine with reduced heavy metal content, further comprising the step of performing (e) reduced pressure filtration and drying the obtained solid after step (d) in paragraph 1.

8. L-serine with cadmium, arsenic, lead and mercury contents of less than 50 ppm per 1 g of L-serine.

9. A pharmaceutical composition for the prevention or treatment of neurological or metabolic diseases, comprising L-serine of Article 8 as an active ingredient.

10. A pharmaceutical composition according to claim 9, wherein the neurological disease is selected from the group consisting of developmental disorders, developmental delay, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, emotional disorders, sleep disorders, and movement disorders.

11. A pharmaceutical composition according to claim 9, wherein the metabolic disease is selected from the group consisting of fatty liver, non-alcoholic steatohepatitis, abdominal obesity, hypertriglyceridemia, low HDL (high density lipoprotein) cholesterol, hypertension, abnormal glucose metabolism syndrome, type 2 diabetes, cardiovascular disease, myocardial infarction, and angina pectoris.

12. A pharmaceutical composition according to claim 9, wherein the content of L-serine in the composition is 60 to 100 wt%.

13. A pharmaceutical composition according to claim 9, wherein the composition is administered orally or intravenously.

14. A health functional food composition for preventing or improving neurological or metabolic diseases, containing L-serine of Article 8 as an effective ingredient.

15. In paragraph 14, a health functional food composition selected from the group consisting of a neurological disorder, developmental delay, cognitive decline, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, Parkinson's disease, Huntington's disease, emotional disorder, sleep disorder, and movement disorder.

16. A health functional food composition in claim 14, wherein the metabolic disease is selected from the group consisting of fatty liver, non-alcoholic steatohepatitis, abdominal obesity, hypertriglyceridemia, low HDL (high density lipoprotein) cholesterol, hypertension, abnormal glucose metabolism syndrome, type 2 diabetes, cardiovascular disease, myocardial infarction, and angina pectoris.

17. A health functional food composition in claim 14, wherein the content of L-serine in the composition is 60 to 100 wt%.

18. A method for preventing or treating a neurological disease or metabolic disease in a subject, comprising the step of administering L-serine of clause 8 to the subject.

19. Use of L-serine of paragraph 8 for manufacturing a medicament for preventing or treating a neurological disease or metabolic disease.

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