JP2018024649A - Compositions and methods for reducing ophthalmic oxidative stress and for improving dry eye, as well as safety and effective compositions and methods for inhibiting pancreatic lipase in mammals - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide safe and effective compositions and methods for inhibiting pancreatic lipase, reducing ophthalmic oxidative stress, and/or improving dry eye, in humans and other mammals.SOLUTION: The present invention provides a lutein-containing composition for improving ophthalmic oxidative stress and/or dry eye. The composition is used for increasing neutrophil rate in the blood for a subject of administration, reducing lymphocyte rate, increasing a neutrophil count, reducing ZTT, reducing creatinine, or increasing potassium. The composition contains lutein in an amount of 1 to 50% by weight. A method in which 10 to 2000 mg per day is administered to a subject for at least 8 weeks is also provided.SELECTED DRAWING: None

Description

  The present invention relates to a lutein-containing composition and method for reducing oxidative stress and / or improving dry eye in the eye. The present invention also relates to the effect of the lutein-containing composition changing the blood component of the subject. Furthermore, the present invention relates to a composition effective for safely and effectively inhibiting pancreatic lipase, its use, and a method for producing the composition.

  Lutein is originally present in the lens and macular region of the eye, and is a component that is abundant in green-yellow vegetables such as spinach and broccoli.

  In everyday life, light, especially blue light and ultraviolet rays, is particularly susceptible to active oxygen damage, but even when there is a risk of oxidative damage to eye cells due to light, this is prevented by antioxidant action. be able to.

  On the other hand, the digestion and absorption of dietary fat is regulated by the action of spleen triglyceride lipase (PTL) and a small protein cofactor that is required by pancreatic lipase to efficiently hydrolyze lipids in food. The The enzyme and cofactor are secreted from the spleen into the duodenum. PTL is a major lipase that hydrolyzes lipid molecules in food and converts triglycerides into monoglycerides and free fatty acids in the human duodenum. The resulting monomer (two molecules of free fatty acid and one molecule of 2-monoacylglycerol) is sealed in micelles and then absorbed into the lymphatic system by a special tube called the milk duct. Colipases belonging to the pancreatic lipase family bind to the non-catalytic domain of lipases, increasing stability and hydrophobicity of the binding site. Colipase suppresses the inhibitory effects of various food factors on the hydrolysis of long-chain triglycerides in food catalyzed by lipases.

  Blocking the mechanism of fat digestion and absorption in food has been utilized in the management of obesity. Inhibition of PTL activity by tetrahydrolipostatin (Orlistat / Xenical) and its dilutions, which can be purchased without a prescription under the brand name Alli, has been widely used in obesity pharmacotherapy. While products based on tetrahydrolipostatin have been clinically proven to be safe and effective, there are several known and potential side effects in their treatment.

  The main side effects of the drug are related to the gastrointestinal tract, fatty stool (oily loose stool containing excessive amounts of intestinal rot and intestinal gas due to unabsorbed fat reaching the large intestine), Includes stool incontinence, frequent bowel movements, and sudden bowel movements. The potential organ toxicity of tetrahydrolipostatin has been suggested by the US Food and Drug Administration (FDA). On May 26, 2010, the FDA approved a revised label containing new safety information for tetrahydrolipostatin-based medications for rarely reported liver injury events due to the use of this medication (non-patent literature). 1).

  The use of tetrahydrolipostatin leads to special cases of acute kidney injury. This is probably due to fat malabsorption resulting from excessive inhibition of pancreatic lipase. It has been found that due to fat malabsorption, fatty acid alkali salts (soap) are formed together with calcium, and the absorption of free oxalate increases, resulting in high oxalic aciduria (non-patent document 2).

  Since tetrahydrolipostatin can attenuate the absorption of fat-soluble vitamins and other fat-soluble nutrients, during treatment with tetrahydrolipostatin, vitamins A, D, E, K are used to prevent vitamin deficiencies that occur from that treatment. And a multivitamin supplement containing beta carotene should be taken once a day.

  Further, it is known that the therapeutic effect is insufficient in the weight loss mechanism of tetrahydrolipostatin. It includes increased appetite and time-dependent loss of drug effectiveness after weeks of treatment. In one study, satiety perception was significantly reduced in subjects receiving tetrahydrolipostatin, and at the same time circulating satiety hormones such as CCK, PYY and GLP-1 in the subject. Decreased significantly (Non-patent Document 3).

  Thus, current generation pancreatic lipase inhibitors have potentially serious side effects, and the effectiveness of the inhibitors tends to decrease over time. That is, the potential for weight loss is diminished by rapid growth tolerance and increased appetite.

http: // www. fda. gov / Drugs / DrugSafety / PostmarkedDrugSafetyInformationforPatients andProviders / ucm213038. htm Filippatos TD, Derdemesis CS, Gazi IF, Nakau ES, Mikhailidis DP, Elisaf MS. Orlistat-associated advice effects and drug interactions: a critical review. Drug Suf 2008; 31 (1): 53-65). Ellrichmann M, Kapelle M, Ritter PR, Holst JJ, Herzig KH, Schmidt WE, Schmitz F, Meier JJ Orlistat inhibition of intestinal lipase acutely increases appetite and attenuates postprandial glucagon-like peptide-1- (7-36) -amide-1 , Cholestokinin, and peptide YY contents. J Clin Endorinol Metab. 2008 Oct; 93 (10): 3995-8. Epub 2008 Jul 22. Badmaev V, Maged A, Conte A, Parker JE. Diterpene Forskolin (Coleus forskohlii, Benth.): A possible new compound for reduction of body weighting leaning body body. NutraCos Vol. 1, no. 2 (March / April); Godard MP, Johnson BA, Rich SRB Body composition and harmontal adaptations associated with forskolin consumption in overhead and oves. Obes Res2005 Aug; 13 (8): 1335-43. Duan RD, Erlanson-Albertsson C Evidence of a stimulus effect of cyclic AMP on pancreatic synthesis and rats. Scan J Gastroenterol. 1992 Aug; 27 (8): 644-8.

  It is an object of the present invention to provide compositions and methods for reducing oxidative stress in the eye and / or for improving dry eye. A further object of the present invention is to provide a composition having a useful effect on a blood component of a subject.

  It is also an object of the present invention to provide a method and composition for the safe and effective inhibition of pancreatic lipase in humans and other mammals, and a method for producing the composition.

  The present inventors have found that administration of a lutein-containing composition derived from marigold extract to a subject results in reduction of oxidative stress in the eye and improvement of dry eye. The present inventors have also found that the composition causes a significant change in the blood component to be administered.

  In addition, the present inventors also used Coleus forskori (also referred to as CF in this specification), Salacia reticulata (also referred to as SR in this specification), and sesame indicum (in this specification). It was also found that each of the extracts (also referred to as SI) has pancreatic lipase inhibitory activity. Furthermore, it discovered that the composition containing one or more of these extracts would inhibit pancreatic lipase safely and effectively, and completed the present invention.

  That is, the present invention provides a composition comprising a lutein-containing composition and one or more of a CF extract, an SR extract and an SI extract. The present invention also provides a method of administering the composition to humans and other mammals in a nutritionally and / or pharmacologically effective amount. The present invention further provides a method for producing the above composition.

  The present inventors have found that ocular oxidative stress is reduced by the lutein-containing composition of the present invention. Moreover, it discovered that dry eye was improved with the said lutein containing composition.

  Furthermore, as a result of intensive studies, the present inventors have found that in the blood of a subject administered with the lutein-containing composition of the present invention, an increase in neutrophil ratio, a decrease in lymphocyte ratio, an increase in neutrophil count, ZTT, It was found that a decrease in creatinine, a decrease in creatinine, or an increase in potassium occurred. That is, the lutein-containing composition of the present invention can prevent or treat diseases exhibiting a low neutrophil ratio, a high lymphocyte ratio, a low neutrophil count, a high ZTT value, a high creatinine concentration, or a low potassium concentration, or those diseases. Can be used to relieve symptoms caused by Such diseases include, for example, neutropenia, bacterial infection, viral infection, aplastic anemia, megaloblastic anemia, cirrhosis, lymphoma, acute / chronic lymphocytic leukemia, Graves' disease (Graves) Disease), Crohn's disease, tuberculosis, pertussis, mumps, chronic infections such as syphilis, chronic hepatitis, autoimmune hepatitis, cirrhosis, hepatocellular carcinoma, chronic inflammation, collagen disease, tuberculosis, myeloma, Acute / chronic nephritis, renal failure, uremia, pyelonephritis, kidney stones, cirrhosis, heart failure, primary aldosteronism, alkalosis, Graves' disease, Cushing syndrome, hyperthyroidism, and the like.

  That is, the present invention provides a composition comprising lutein. The present invention also provides a method of administering the composition to humans and other mammals in a nutritionally and / or pharmacologically effective amount.

  The present invention also provides a composition comprising one or more of a CF extract, an SR extract and an SI extract that are effective for safely and effectively inhibiting pancreatic lipase. By administering the composition of the present invention to a subject, the subject's visceral fat, liver fat, triglycerides, appetite, caloric intake, body fat percentage, body weight, waist circumference and / or blood lipid reduction and / or Prevention of rapid growth resistance is achieved.

It is a graph showing the inhibition rate of pancreatic lipase activity when CF extract (C. Forskohlii 98% or 40%), SR extract (Salacia) or SI extract (Sesamin 70%) is used alone. Inhibition rate of pancreatic lipase activity when SR extract (Salacia) and CF extract (C. Forskohlii 98% or 40%), or SR extract (Salacia) and SI extract (Sesamine 70%) are combined, respectively It is a graph showing.

  In one embodiment of the invention, the lutein-containing composition of the invention is administered in a nutritionally and / or pharmacologically effective amount to reduce ocular oxidative stress and / or improve dry eye. The

  In one embodiment, the lutein-containing composition of the invention has an increased neutrophil rate, decreased lymphocyte rate, increased neutrophil count, decreased ZTT, decreased creatinine in the blood of a subject, or Administered for potassium elevation.

  In one embodiment, lutein contained in the composition of the present invention can be extracted from marigold.

  In one embodiment, the composition of the present invention may contain lutein at a specific concentration. For example, the composition of the present invention contains lutein in an amount of 1% to 50% by weight, preferably 5% to 45%.

  In one embodiment, the compositions of the invention can be administered to a subject in the range of 10-2000 mg per day as lutein. Preferably, it can be administered to a subject in the range of 20 to 200 mg per day as lutein.

  The present invention also achieves safe and effective control of digestive fatty acids (liver fat), visceral fat (abdominal fat) and white (storage) fat of peripheral tissues that have accumulated excessively in organs in humans or other mammals. Accordingly, methods and compositions for inhibiting pancreatic lipase activity are provided. A method for producing the composition is also provided.

The composition of the present invention comprises a CF extract standardized for forskolin, an SR extract standardized to inhibit 50% of α-glucosidase and α-amylase (IC ₅₀ ), respectively, and SI standardized for sesamin. One or more extracts selected from the group consisting of extracts are included.

  In one embodiment, the composition of the present invention may contain one or more of CF extract, SR extract and SI extract at a specific concentration. For example, the composition of the present invention inhibits 50% of a CF extract, α-glucosidase, standardized to contain about 1% to 98% forskolin, at a concentration of 0.1-200 μg / mg, and Of SR extracts standardized to inhibit α-amylase by 50% at a concentration of 0.1-100 μg / mg and SI extracts standardized to contain about 40% -90% sesamin Contains one or more. The CF extract is more preferably standardized to contain forskolin in the range of 40% to 98%. The SR extract is more preferably standardized to inhibit α-glucosidase by 50% at a concentration of 0.5-150 μg / mg and inhibit α-amylase by 50% at a concentration of 0.5-50 μg / mg. Is done. The SR extract is even more preferably standardized to inhibit α-glucosidase by 50% at a concentration of 1-75 μg / mg and inhibit α-amylase by 50% at a concentration of 1-35 μg / mg. The SI extract is more preferably standardized to contain sesamin in the range of 60% to 80%. The preferred ratio (weight ratio) of these extracts is CF extract 2-30: SR extract 0.25-3: SI extract 0.025-0.3, more preferably CF extract 5 -15: SR extract 0.5-1.5: SI extract 0.05-0.15, more preferably CF extract 10: SR extract 1: SI extract 0.1.

  Here, the sesame extract may be extracted from any sesame. Examples include, but are not limited to, white sesame, black sesame, gold sesame, and tea sesame.

  The compositions of the present invention are preferably stored at room temperature and a relative humidity of about 60-80%. Further, it is more preferable to store at a relative humidity of about 70%.

  The compositions of the present invention may be topically administered orally, sublingually or parenterally, with oral administration being preferred. For oral administration, the compositions of the invention can be provided in the form of capsules, tablets, water dispersible powders, or beverages with suitable excipients. When used topically in cream or lotion form or when applied transdermally, the compositions of the invention are applied with a suitable excipient at a concentration ranging from 0.1% to 15%. The excipient may be any pharmaceutically acceptable excipient, and examples thereof include, but are not limited to, a colorant, a corrigent, a preservative, a stabilizer, and an antioxidant.

  In one embodiment of the invention, the composition of the invention is administered in a nutritionally and / or pharmacologically effective amount for reducing visceral fat (including liver fat) and body fat. This is because it results in continuous (preventing rapid growth resistance) and safe inhibition (mild inhibition of pancreatic lipase activity) of pancreatic lipase activity.

  The composition comprising one or more of the CF extract, SR extract and SI extract of the present invention is administered in a nutritionally and / or pharmacologically effective amount, for example, in the range of 10 to 2000 mg per day. can do. Preferably, it can be administered in the range of about 832.5 to 1665 mg. The daily dose is preferably administered twice or more, more preferably divided into 2 or 3 times. The administration is preferably performed 15 minutes to 1 hour before meal, more preferably 30 minutes before meal. In this case, the single dose is preferably 10 to 1000 mg / dose, more preferably about 277.5 mg to about 555 mg / dose.

  Moreover, the composition containing one or more of the CF extract, SR extract and SI extract of the present invention is preferably 5 to 100 mg / kg per body weight, more preferably 12 to An amount in the range of 25 mg / kg can be administered orally daily.

  Furthermore, the composition containing one or more of the CF extract, SR extract and SI extract of the present invention can be administered once a day, for example, as a maintenance dose. The maintenance dose is preferably in the range of 30.8 mg to 555 mg per day, more preferably in the range of 100 mg to 400 mg per day, and most preferably 200 mg to 300 mg per day.

  Also, in one embodiment, the composition of the present invention is a combination of an amount of sesame extract that modifies a biological response and a Coleus forskohlii extract or a Salacia reticulata extract. This can prevent excessive and non-physiological inhibition of pancreatic lipase activity and its potential side effects.

  In another embodiment, the composition of the present invention comprises a combination of a clinically effective amount of Coleus forscoli extract and a biologically negligible (asymptomatic) amount of Salacia reticulata extract. is there. Thereby, rapid growth tolerance is prevented.

  In the above-described embodiments, the preferred clinically effective amount of Coleus forskohlii extract ranges from 0.1 to 1000 μg / ml, more preferably from 1 to 100 μg / ml, and the biologically active Salacia reticulata extract Negligible (asymptomatic) amounts range from 0.01 to 100 μg / ml, more preferably from 0.1 to 10 μg / ml, and amounts that modify the biological response of sesame extract are 0.1 It is -1000 microgram / ml, More preferably, it is the range of 1-100 microgram / ml.

  By the administration method as described above, the composition of the present invention is provided to efficiently inhibit pancreatic lipase as safe without the disadvantages and side effects of current generation pancreatic lipase inhibitors. And it can prevent that the action mechanism of the composition of this invention declines in an old subject, and can prevent the inhibition of excessive and non-physiological pancreatic lipase activity.

  While not being bound by any particular logic, the mechanism of action of the compositions of the present invention is to inhibit the postprandial ghrelin response represented by a reduced appetite. Ghrelin is a hormonal substance associated with elevated plasma leptin levels. This combined action reduces pancreatic lipase production and secretion. The composition of the present invention also directly acts on pancreatic lipase to inhibit colipase and suppress activation of PTL enzyme.

  The present invention also includes a method for producing the composition of the present invention from Coleus forskori, Salacia reticulata and sesame.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Preparation of lutein-containing composition As a lutein-containing composition, a lutein-containing capsule containing 50.0 mg of lutein, 89.0 mg of neutral fatty acid triglyceride, and 11.0 mg of beeswax (total 150 mg) was prepared per capsule. .

2. Clinical Trial of Lutein-Containing Composition This study was conducted on Japanese men and women aged 30 to 49 who are aware of dry eyes. Specifically, there were 4 males, 11 females, and 41.6 ± 5.8 years old. Subjects took 2 capsules per day before breakfast and before dinner. The ingestion period was 8 weeks, and the following quantification and measurement were performed before and after ingestion.

  In order to evaluate the degree of oxidative stress, the oxidative stress substance (8-hydroxydeoxyguanosine) contained in the tears of both eyes attached to the test paper was quantified using Schirmer test paper.

  As an evaluation of dry eye, tear film break-up time (BUT) test was performed. Specifically, after the instillation of fluorescein, blinking was prohibited and the time until the tear film on the cornea was destroyed was observed with a slit lamp to measure tear retention.

For analysis of the test results, the measured values before intake and after 8 weeks of intake were compared using a corresponding t-test. The aggregation unit was the average value and the standard deviation.
Statistical analysis shall be performed by two-sided test. In statistical analysis, the significance level was 5%, and the two-sided test had a significant probability of less than 5% (p <0.050), with a significant difference of 5% to less than 10% (p <0.100) was determined to have a tendency difference. Statistical analysis was performed using Microsoft Excel 2007 and IBM SPSS ver 18.0.

In addition, blood tests were performed on the following items before taking the capsule and 8 weeks after taking the capsule.
Inspection item:
(I) Hematology examination: White blood cell count, red blood cell count, hemoglobin, hematocrit value, platelet count, MCV (average red blood cell volume), MCH (average red blood cell pigment content), MCHC (average red blood cell pigment concentration), white blood cell image (ii) live Chemical testing: AST (GOT), ALT (GPT), γ-GTP, ALP, LD (LDH), LAP, total bilirubin, direct bilirubin, indirect bilirubin, cholinesterase, ZTT, total protein, urea nitrogen, creatinine, uric acid, CK , Sodium, potassium, chlor, calcium, inorganic phosphorus, serum iron, serum amylase, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride (TG: neutral fat), free fatty acid, glucose

3. Results of clinical trials of lutein-containing compositions In this study, the effect of reducing the oxidative stress by ingesting lutein-containing compositions was verified by measuring the amount of 8-hydroxydeoxyguanosine (8-OHdG) secretion in tears. did. When stimulated by ultraviolet rays or blue light, the eye tissue is damaged and oxidative stress substances including 8-OHdG are generated. 8-OHdG is a DNA oxidative stress marker in the living body.

  8-OHdG significantly decreased from before ingestion of the lutein-containing composition to 8 weeks after ingestion (Table 1). The decrease in 8-OHdG showed the effect of reducing the oxidative stress of the lutein-containing composition.

  From this result, since lutein was accumulated in the ocular tissue by continuous ingestion of the lutein-containing composition and damage to the ocular tissue was prevented by absorbing ultraviolet rays and blue light, secretion of 8-OHdG, which is one of the oxidizing substances, was released. It was suggested that this could be suppressed.

  In the measurement of tear retention by tear film break-up time (BUT) test, BUT significantly increased from before ingestion of the lutein-containing composition to 8 weeks after ingestion (Table 1).

  As described above, by taking the lutein-containing composition of the present invention, an effect of reducing oxidative stress of the eye can be obtained, and an effect of improving the tear film destruction time (BUT), that is, an effect of improving dry eye can be obtained. It became clear.

Furthermore, regarding the blood test, Table 2 shows the results of statistical analysis comparing the measured values 8 weeks after the intake of the lutein-containing composition with the measured values before the intake.
The items in which significant differences were observed before and after ingestion of the lutein-containing composition of the present invention were neutrophil ratio (p = 0.006), lymphocyte ratio (p = 0.012), neutrophil count (p = 0.049), ZTT (p = 0.177), creatinine (p = 0.020), and potassium (p = 0.004). In addition, the neutrophil rate increased by 8.7% (56.4 → 61.3%) and the lymphocyte rate decreased by 10.9% (33.2 → 29.6%) compared to before intake. Neutrophil count increased by 13.4% (311.9 → 3530.4), ZTT decreased by 6.9% (7.6 → 7.1U), and creatinine decreased by 5.2% (0 .70 → 0.66 mg / dl), potassium increased by 5.9% (4.0 → 4.3 mEq / L).

4). Preparation of CF Extract Coleus forskohlii root powder was extracted with 2 volumes of water at 60 ° C for 2 hours. The used material was dried and extracted through supercritical fluid extraction (SCFE). The SCFE oil-containing resin was washed with twice the amount of 70% ethanol (sugar cane ethanol) at 50 ° C. for 1 hour. The mixture was allowed to stand for 18 hours, after which the clear liquid layer was separated by siphon from the top layer. The bottom layer was filtered through a Nutsche filter. The wet cake was dried under reduced pressure at 65 ° C. for 16 hours. The dried extract was ground through a 0.5 mm mesh and analyzed for forskolin content. The resulting product was mixed with maltodextrin and 3% Aerosil to obtain the desired forskolin concentration.

5. Preparation of CF Extract and SR Extract Mixture Next, CF extract of desired strength in the range of 10-98% was mixed with the extract of Salacia reticulata root.
Salacia reticulata root powder was extracted at 70-80 ° C. with 5 volumes of water. The water extraction was repeated three times and the resulting extract was filtered using a 5 micron filter cloth. The clear filtrate was allowed to stand for 8 hours and the top layer was separated by siphon and concentrated under reduced pressure at 80 ° C. to a concentration of 25-30% TDS (total evaporation residue). After being concentrated about half, it was then spray dried. The dried powder was ground through a 0.5 mm mesh.
Next, α-glucosidase inhibitory activity and α-amylase inhibitory activity were measured.

Measurement of α-glucosidase inhibitory activity α-glucosidase inhibitory activity for standardizing the SR extract was measured by the following method.

Principle α-glucosidase activity can be measured in vitro by measuring reducing sugar (glucose) resulting from hydrolysis of sucrose by α-glucosidase from rat small intestine.

Material α-Glucosidase was prepared as follows. Rats were sacrificed, the intestines were removed and cooled with ice-cold 80 mM phosphate buffer (pH 7.0). The intestine was cut open, the mucous membrane was peeled off with a glass rod, and homogenized with 4 volumes (volume / volume) of ice-cooled 80 mM phosphate buffer (pH 7.0). Sample tubes were cooled with ice during homogenization. Nuclei and cell debris were removed by centrifugation at 2000-4000 rpm for 10 minutes and the supernatant was aliquoted into 1.5 ml vials and stored at -20 ° C. This was used as an α-glucosidase enzyme solution. The protein content measured by the biuret method was about 0.5 g / dl. The biuret method was performed using the Total protein estimation kit (B-0211, Span diagnostics).
As the substrate, sucrose (RM3063, Himedia, India, stored at room temperature) dissolved in 80 mM phosphate buffer (pH 7.0) to 37 mM was used.
As a positive control, acarbose (Glucoby) (Bayer Pharma, India, stored at room temperature) dissolved in 80 mM phosphate buffer (pH 7.0) at 5 μg / ml was used.
The glucose reagent used was AGAPPE diagnostics 11208102, India.

Methods Vogel GH, Vogel WH (1997), Drug Discovery and Evaluation, Pharmalogic assay, Springer-Verlag: activity-modified α-Glucidase obtained by modifying the method described in Germany, 588-589. Briefly, 50 μl of enzyme solution was added to 250 μl of 80 mM phosphate buffer containing buffer, positive control or various concentrations of test samples to form a reaction mixture. The reaction mixture was mixed and preincubated for 30 minutes at 37 ° C. Substrate (37 mM sucrose) (500 μl) was added and incubated at 37 ° C. for 20 minutes. The reaction was stopped by holding the reaction mixture in boiling water for 2 minutes and then cooled. 250 μl of glucose reagent was added to 50 μl of the reaction mixture and incubated at 25 ° C. for 10 minutes. Absorbance at 510 nm was measured using a Versamax microplate reader manufactured by Molecular devices. As a control, a similar experiment was performed without a test sample.

The inhibition rate (%) was calculated by the following formula.
Inhibition rate (%) = (absorbance (control) −absorbance (test sample)) / absorbance (control) × 100
IC ₅₀ was calculated using log-probit analysis. If the sample was colored or not an aqueous solution, corrections for coloration and solvent were made appropriately.

Measurement of α-amylase inhibitory activity α-amylase inhibitory activity for standardizing the SR extract was measured by the following method.

Principle Pancreatic α-amylase hydrolyzes 2-chloro-4-nitrophenol α-D-maltotrioside (CNP-G3) to release 2-chloro-4-nitrophenol and 2-chloro-4 -Nitrophenol yields α-D-maltoside (CNPG2), maltotriose and glucose. The formation rate of 2-chloro-4-nitrophenol can be measured by absorbance at 405 nm.

Material α-Amylase (EC 3.2.1.1) uses Type VI-B derived from porcine pancreas purchased from Sigma, USA, 500,000 units (15.8 units / mg, solid at pH 6.9) (A3176) It was. The α-amylase was dissolved in 40 mM phosphate buffer (pH 6.9) and used to make 0.5128 units / ml.
As the substrate, a ready-made CNP-G3 reagent (2-chloro-4-nitrophenol α-D-maltotrioside, AMF060CH, Chema Diagnostica, Italy, stored at 2-8 ° C.) was used at a concentration of 2.3 mM.
As a positive control, acarbose (Glucoby) (Bayer Pharma, India, stored at room temperature) dissolved in 40 mM phosphate buffer (pH 6.9) at 2.5 μg / ml was used.

Method Gella FJ, Guern G, Vital R, Canada F, (1997), Determining of total and pancreatic α , Clinica Chimica Acta 259, 147-160 was modified to measure α-amylase inhibitory activity. Briefly, 30 μl of enzyme solution (0.5128 units / ml) was added to 60 μl of 40 mM phosphate buffer (pH 6.9) containing buffer, positive control or test sample to give a reaction mixture. The reaction mixture was mixed and preincubated at 37 ° C. for 10 minutes. 125 μl of substrate (2.3 mM CNP-G3) was added and incubated at 37 ° C. for 8 minutes. Absorbance at 405 nm was measured using a Versamax microplate reader manufactured by Molecular devices. As a control, a similar experiment was performed without a test sample.

The inhibition rate (%) was calculated by the following formula.
Inhibition rate (%) = (absorbance (control) −absorbance (test sample)) / absorbance (control) × 100
IC ₅₀ was calculated using a log-probit analysis. If the sample was colored or not an aqueous solution, corrections for coloration and solvent were made appropriately.

  Based on the method described above, the SR extract was standardized to inhibit α-glucosidase by 50% at a concentration of 1-75 μg / mg and inhibit α-amylase by 50% at a concentration of 1-35 μg / mg.

6). Preparation of a mixture of CF extract, SR extract and SI extract The mixture of CF extract and SR extract was then combined with an extract of sesame seeds.
Sesame seeds were washed with water, dried and extruded to collect the oil. The oil was filtered through a filter press and the clear oil was used for extraction. Sesame oil was extracted with ethanol (sugarcane ethanol) at 75-80 ° C. for 2 hours. It was left for 1 hour and the layers were separated. The top layer contained SI material, which was separated by viewing the glass portion through a bulb at the bottom of the extractor. The bottom layer was extracted three more times. The four extracts were concentrated under reduced pressure at 70 ° C. to a thick paste. The paste was left for 7 days to age. The aged material was mixed with 2 volumes of sugarcane ethanol and filtered through a Nutsche filter. The wet cake was washed with 1.5 volumes of ethanol and filtered. The resulting wet cake was mixed with 2 volumes of water, heated to 60 ° C. and filtered through a Nutsche filter. The wet cake was dried under reduced pressure at 65 ° C. The dried material was then dissolved in 21 volumes of ethanol and charcoalized. Based on dry weight, 5% activated carbon was added. The high flow bed was prepared using a 2 micron filter cloth. High flow filtration was repeated twice using a new high flow bed. The clear filtrate was concentrated under reduced pressure at 65 ° C. until it became a thick paste. Next, the high-viscosity paste was dried at 60 to 65 ° C. under reduced pressure for 3 to 4 hours. The dried material was ground to 0.5 mm and again dried at 70 ° C. under reduced pressure for 3 hours. The re-dried material was ground, sieved, checked for quality and packaged. The final product was standardized by HPLC to contain 70% sesamin.

7). Clinical trial of CF extract 15 volunteers (8 volunteers in the test diet group (average) on the Coleus forskori (CF) extract, a component of the present invention standardized to contain 10% forskolin Age 41 ± 4 years, average weight 77.5 ± 11.4 kg, average BMI 27.2 ± 0.9), 7 volunteers in control diet group (average age 36 ± 9 years, average weight 75.6 ± 7) A 6-week clinical trial with 3 kg, mean BMI 27.5 ± 1.4)) was conducted. These randomly selected test subjects took 250 mg × 2 capsules containing 10% CF extract or a corresponding 250 mg × 2 capsule of placebo 30 minutes before breakfast and 30 minutes before dinner. The total daily CF extract / placebo amount was 1000 mg (4 capsules), which was continued for 6 weeks. No changes were made to the diet or lifestyle during the course of this study.

8). Clinical Trial Results of CF Extract Body fat percentage The body fat percentage in the CF extract group was statistically significantly lower than baseline (P <0.05) when administration was completed. Body fat percentage in the placebo group was statistically significantly higher than baseline (p <0.05) when dosing was completed. The body fat percentage of the CF extract group before administration was 34.1 ± 4.2% and 33.5 ± 4.4% after administration, whereas in the placebo group, 35. 5% before administration. It was 0 ± 6.6% and 35.9 ± 6.5% after administration. At 6 weeks before and after the regimen, changes in fat mass in various parts of the body were measured with CT scans for 3 subjects in the CF extract group. Systemic body fat, subcutaneous fat and central abdominal fat were reduced compared to baseline at the completion of the study (see Table 3).

  Table 3 shows the amount of body fat in various body parts before and 6 weeks after the test in 3 subjects who received Coleus forskohlii extract (250 mg x 4 capsules / day). Represents the result of scanning.

Body weight Compared to baseline, the CF extract group showed decreased body weight at the end of 6 weeks and the placebo group showed increased body weight. The body weight before the test of the CF extract group was 77.5 ± 12 kg, and the body weight after the test was 76.6 ± 12.1 kg. The body weight of the placebo group was 75.5 ± 7.2 kg before the test and 76 ± 7.4 kg after the test.

Waist circumference The waist circumference in the CF extract group was numerically lower than the baseline. That is, it was 93.9 ± 7.8 cm before the test and 93.2 ± 8 cm after the test. On the other hand, in the placebo group, the waist circumference numerically increased at the end of the test compared to the baseline. That is, it was 92.1 ± 7.1 cm before the test and 92.4 ± 7.1 cm after the test. A pattern of change similar to the waist circumference was also recorded around the hips. That is, in the CF extract group, it is 101 ± 2.9 cm before the test and 100.1 ± 3 cm after the test, and in the placebo group, it is 100.2 ± 3.4 cm before the test and 100.4 ± 3. It was 3 cm.

Blood fat Laboratory data on blood fat showed the following trends in the CF extract and placebo groups. That is, the total cholesterol in the CF extract group was 200 ± 15 mg / dl, which was lower than the baseline value (213 ± 28 mg / dl). Total cholesterol in the placebo group increased from baseline (218 ± 38 mg / dl) to 221 ± 35 mg / dl. The triglyceride of the CF extract group was 137 ± 69 mg / dl before the test, but decreased to 120 ± 74 mg / dl after the test. In the placebo group, triglyceride levels increased from baseline values (98 ± 33 mg / dl) and were 109 ± 54 mg / dl after the test.

Caloric intake The effects of food on caloric intake when the CF extract was administered for 6 weeks were compared with the placebo group. In the placebo group, caloric intake increased by approximately 700 kcal at the end of 6 weeks. On the other hand, in the CF extract group, the calorie intake decreased by about 300 kcal. The difference in caloric intake between the CF extract group and the placebo group was statistically significant (p <0.05, see Table 4). A significantly reduced caloric intake in the CF extract group compared to the placebo group indicates the safe effect of the present invention. In view of the current generation of pancreatic lipase inhibitors (Non-Patent Document 3), which show a tendency to increase appetite, this can be said to be an unexpected effect.

  Table 4 presents the effect on appetite as measured by caloric intake in subjects who were administered Coleus forskohlii extract or placebo for 6 weeks.

9. Inhibition of pancreatic lipase in vitro The extracts of Coleus forskori, Salacia reticulata and sesame, which are constituents of the present invention, were evaluated in vitro for their ability to inhibit pancreatic lipase activity in the following manner.

(1) Test material The test substances included: Coleus forskori extract 98% (lot number: OL110048), Coleus forskori extract 40% (lot number: BAJ / COL40 / 110211), Salacia reticulata extract (lot number) : BAJ / SRE / 100816), white sesame extract 70% (lot number: BAJ / SME / 100220), Tarragon extract (lot number: 0708/908005). These were stored at room temperature.
Reagents include porcine pancreatic lipase (lot number: 020M1589V, SIGMA), MOPS (lot number: BCBB5948, SIGMA), EDTA · 2Na · 2H ₂ O (lot number: BCBB4814, SIGMA), Trizma (registered trademark) Base (lot) Number: 0001414448, SIGMA), calcium chloride · 2H ₂ O (lot number: BCBC3028V, SIGMA), N, N-dimethylformamide (lot number: 60796HM, SIGMA), 4-nitrophenylbutyrate (lot number: 029K5217V, SIGMA) ), Hydrochloric acid (lot number: KWM6937, Wako Pure Chemical Industries, Ltd.), Orlistat (lot number: 0422583-6, Cayman Chemical Company).
A multi-mode microplate reader Mithras LB940 (BERTHOLD TECHNOLOGIES GmbH & Co. KG) was used as a measuring instrument.

(2) Test method Reagent preparation method Reagents are reported by Lee et al. (Eun Mi Lee, Seung Sik Lee, Byung Yeup Chung, Jae-Young Cho, In Chul Lee, So Ra Jung, So Ra Ahn, So Ra J Pancreatic Lipase Inhibition by C-Glycosidic Flavors Isolated from Eremochloa ophioloids, Molecules 2010, 15, 8251-8259; B. Strukelj. 2 .. 05., Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies, J Lipid Res 46:.. 1512-1516 DOI 10.1194 / jlr.M500048-JLR200) was prepared as a reference. A Tris buffer solution of 100 mmol / L Tris-HCl (pH 6.8) containing 5 mmol / L calcium chloride and 1 mmol / L EDTA · 2Na was prepared. Porcine pancreatic lipase was suspended by adding 10 mmol / L MOPS aqueous solution to obtain a porcine pancreatic lipase solution of about 1000 units / mL. 850 μl of Tris buffer was added to 30 μl of porcine pancreatic lipase solution to prepare an enzyme solution. N, N-dimethylformamide was added to 4-nitrophenyl butyrate and dissolved to prepare a substrate solution of 40 mmol / L. N, N-dimethylformamide and Tris buffer were mixed at a ratio of 1: 4 to obtain a test substance preparation solution.

Test substance preparation method All test substances were dissolved in a test substance preparation solution to prepare a concentration of 1000 μg / mL, and then 10-fold diluted to prepare concentrations of 100, 10 and 1 μg / mL. These were used as specimens in the test, and only the test substance preparation solution was used as a control.

Measurement method The measurement was performed with reference to reports (1, 2) of Lee et al. And Lunder et al. Briefly, 100 μL sample (n = 2) and 880 μL enzyme solution were added to the microplate and incubated at 37 ° C. for 15 minutes. After completion of the incubation, 20 μL of the substrate solution was added, and the enzyme reaction was started at 37 ° C. After starting the enzyme reaction, the measurement of absorbance at 405 nm was continued every minute until 15 minutes later.

  As a result, the extent to which these three extracts inhibit pancreatic lipase activity was different. This fact provides new insights to safely and effectively inhibit pancreatic lipase activity.

Safe inhibition of pancreatic lipase A safe mechanism to prevent excessive inhibition of pancreatic lipase is a major feature of the method of the present invention. Sesame extract controls this mechanism in a dose-dependent manner to prevent the side effects of excessive pancreatic lipase inhibition. In in vitro experiments, sesame extract (Sesamine) alone showed a dual effect at low and high doses (see FIGS. 1 and 2).

  At low doses (0.1 μg / ml and 1 μg / ml), sesame extract is statistically significantly more efficient than Coleus forskori extract (C. Forskohlii) and Salacia reticulata extract (Salacia). Was inhibited. However, at higher doses (10 μg / ml and 100 μg / ml), the same doses of Coleus forskori extract and Salacia reticulata extract are statistically significantly more efficient than sesame extract. Inhibited.

  Unexpectedly, when 1 μg / ml sesame extract was added to 10 μg / ml 98% Coleus forskori extract, pancreatic lipase was inhibited by 9.6%. The sum of the inhibition rates obtained with each extract alone is in contrast to 23.4% (see FIG. 1, Table 5 and Table 6). This biological response that modifies the action of sesame extract is novel and useful in the sense of preventing excessive and non-physiological inhibition of pancreatic lipase activity and its potential side effects.

  Table 5 represents the in vitro dose-dependent inhibition of pancreatic lipase by the components of the present invention.

  Table 6 represents the inhibition of pancreatic lipase in vitro by the combination of components of the present invention.

Prevention of rapid growth resistance The extract of Coleus forskohlii and Salacia reticulata is used to prevent rapid growth resistance as well as the repeated use and time course of extracts of Coleus forskori and Salacia reticulata that inhibit pancreatic lipase activity. It works synergistically to the mechanisms that become resistant to decreased biological activity and aging. This fact is a new and useful finding for safely and effectively inhibiting pancreatic lipase activity.

Synergistic inhibition of pancreatic lipase The synergistic effect of the present invention is that the biologically inactive amount (1 μg / ml) of Salacia reticulata extract and the sub-optimal amount (10 μg / ml) of Coleus forskori extract Illustrated by an in vitro experiment for inhibition of pancreatic lipase in combination with (see Table 6). The additive inhibition rate of the CF extract and SR extract alone is only 10.8%, but these two have a synergistic effect and inhibit pancreatic lipase activity by 20.7%. did. Thus, it is clear that the combination of a biologically inert amount of Salacia reticulata and a sub-optimal amount of Coleus forskori provides an unexpected synergistic effect. This represents the inventiveness and usefulness of the present invention in weight management.

10. Results of clinical and in vitro studies Clinical evidence revealed in a 6-week study showed that CF extract reduced visceral fat and blood triglycerides. By combining this discovery with the in vitro results of inhibiting pancreatic lipase activity, it was first discovered that CF extracts inhibit pancreatic lipase. The discovery of the activity of a CF extract that inhibits pancreatic lipase is novel, especially when pancreatic tissue is incubated with diterpene forskolin, the rate of lipase and colipase synthesis increases rapidly (Non-Patent Document 6). Indicates the opposite.

Claims (6)

A lutein-containing composition for improving ocular oxidative stress and / or dry eye. The composition according to claim 1, comprising lutein in an amount of 1% to 50% by weight. Furthermore, it is used for increasing neutrophil ratio, decreasing lymphocyte ratio, increasing neutrophil count, decreasing ZTT, decreasing creatinine, or increasing potassium in the blood of the administration subject, Item 3. The composition according to Item 1 or 2. The composition according to any one of claims 1 to 3, wherein the composition is administered to a subject in the range of 10 to 2000 mg per day. 5. A composition according to any one of claims 1-4, characterized in that it is administered to a subject for at least 8 weeks. The composition according to claim 1, wherein the composition is administered orally.

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