KR101520643B1 - A Pharmaceutical Composition for the prevention and treatment of type 2 diabetes comprising concentrated Aloe vera gel - Google Patents

Abstract

The invention as aloe vera on the gel in a type 2 diabetes treatment a pharmaceutical composition for preventing and concentrated by an ultrafiltration method, specifically, a representative animal model of diabetes ^{BKS.Cg-m + / + Lepr db} / J homozygous diabetic (db / db ) is excellent in anti-diabetic effect, such as improving blood glucose and fat homeostasis in mice, and thus is useful for the treatment or prevention of metabolic disorders in humans, particularly, symptoms of type 2 diabetes or diabetes related diseases in which aloe vera gel is concentrated It can be used not only as an effective pharmaceutical composition but also as a health functional food for alleviating or improving type 2 diabetes.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of type 2 diabetes mellitus in which Aloe vera gel is concentrated,

The present invention relates to a medicinal composition for the prevention and treatment of type 2 diabetes mellitus in which aloe vera gel is concentrated by ultrafiltration. More specifically, the present invention relates to a medicinal composition for preventing and treating diabetes mellitus by removing the skin of aloe vera leaves and filling the parenchyma tissue After homogenization at a maximum speed in a homogenizer, the supernatant was taken out by centrifugation, the exclusion molecular weight of the membrane was adjusted to 50 to 200 kDa, and a zirconium dioxide (ZrO ₂ ) tubular ultrafiltration membrane was used to measure transmembrane pressure of 0.2-2.25 bar pressure drop (TMP), and ultrafiltration at a feed rate of 240 l / hr at a temperature of 15 ~ 25 ℃ to concentrate the aloe vera gel to maximize the amount and quality of the polysaccharide as an active ingredient. The aloe vera ultrafiltration concentrate (Sonde) 100 mg / kg per day by oral administration does not show hepatotoxicity as well as hypoglycemic effect aloe vera The present invention relates to a pharmaceutical composition for the prevention and treatment of type 2 diabetes, wherein the blood glucose level is lower than that of a gel (Processed Aloe vera Gel, PAG).

Diabetes mellitus is divided into Type 1 diabetes and Type 2 diabetes. Among them, type 1 diabetes is insulin-dependent diabetes mellitus, usually occurs before the age of 30 years, usually low body weight and dry people are insufficiently insufficient It is diabetes that occurs. This type of diabetes is most common in children aged 11 to 12 years, but it is less common among Asians than Westerners. Because type 1 diabetes is caused by the destruction of cells that secrete insulin from the pancreas, and insulin is absolutely insufficient, insulin injections are essential for the treatment of diabetes.

In contrast, type 2 diabetes accounts for more than 95% of diabetes globally and the incidence increases with age and obesity, and about half of patients are overweight and overweight. Therefore, the treatment of type 2 diabetes mellitus is insufficient, but insulin secretion ability of the pancreas remains, so it is possible to control glucose in the blood only by diet and exercise therapy, and if it is impossible to control blood glucose by this method The drug, oral hypoglycemic agent, or insulin injections are used to control glucose in the blood. In the world, studies are being focused on the development of a novel therapeutic agent for adult diabetes mellitus and the study of pathogenesis. Currently, oral and injectable therapeutic agents used for the treatment of type 2 diabetes include sulfonylurea (SU), metformin, (TZD), and insulin infusion. Although type 2 diabetics do not have to rely on insulin for survival, about one-third of patients require insulin for blood glucose control. It also has limitations in safety and toughness such as weight gain, hypoglycemia, and gastrointestinal side effects, which are obstacles to reaching the target blood glucose level.

As a suitable animal model of diabetes, the B6- Lep ^{ob / ob} (ob / ob) and BKS- Lepr ^{db / db} (db / db) mouse models are the most widely known. They are inherently unable to control appetite due to genetic mutations of leptin or leptin receptors, are over-consuming food, are continuously increasing in weight, and eventually develop a typical type 2 diabetes , It is widely used as the most appropriate model for the study of diabetes and the development of therapeutic agents. The two types of mouse strains described above are widely used as obese and diabetic animal models, since ob / ob mice have glucose levels of about 200-300 mg / dl in the blood and 300-600 mg / dl in the db / db mice ob / ob mice are obese model animals with slightly diabetic symptoms, and db / db mice are more severe models of diabetes mellitus. Therefore, when studying diabetes, db / db mice are more There is a tendency to use. Metformin, used as a comparative drug in the present invention, has been used as a treatment for type 2 diabetes since 1957, and is currently the most widely used in the world. Metformin lowers blood glucose levels but does not induce hypoglycemia nor promote insulin secretion. It does not affect body weight, has a slight weight loss effect, and has good effects on plasma lipids.

On the other hand, as a prior art related to a pharmaceutical composition for treating type 2 diabetes mellitus containing processed aloe vera gel, Patent No. 10-1127454 (Mar. 3, 2012) discloses a modified aloe vera gel (PAG) Wherein the content of the polysaccharide in the processed aloe vera gel is 20% to 30% based on the dry weight (weight cut-off, 3500 daltons) of the residual fraction in the dialysis net during dialysis against distilled water but this is of type 2 diabetes prevention or treatment a pharmaceutical composition for the initiation, characterized in that, and in db / db mice of 8-week-old final occurs a problem that sufficient reduction effect, such as blood glucose levels. Metformin, which is currently the most widely used drug for diabetes mellitus, has been shown to be effective in decreasing glucose levels in db / db mice at 8 weeks of age. However, metformin, an indicator of liver function test, The level of alanine aminotransferase (ALT) increases and thus toxicity appears.

Therefore, there is an urgent need to develop a novel pharmaceutical composition for prevention and treatment of diabetes, which has no side effects and has an increased efficacy.

Disclosure of the Invention The present invention has been made in order to solve the above problems, and it is an object of the present invention to provide a medicament composition for the prevention and treatment of type 2 diabetes, which is obtained by concentrating aloe vera gel excellent in antidiabetic effect such as improvement of blood glucose and fat homeostasis without hepatotoxicity Provided is the challenge.

The present invention relates to a method for removing aloe vera leaves by removing the skin of the aloe vera leaves and filling the parenchyma tissue, homogenizing at a maximum speed in a homogenizer, and then centrifuging the supernatant to remove excretion molecular weight of the membrane to 50 to 200 kDa (TMP) of 0.2-2.25 bar using a zirconium dioxide tubular ultrafiltration membrane and ultrafiltering at a feed rate of 240 l / hr at a temperature of 15-25 ° C to obtain an aloe vera gel concentrate The present invention also provides a pharmaceutical composition for the prevention and treatment of type 2 diabetes by concentrating the aloe vera gel.

The present invention also relates to a method for the prevention and treatment of type 2 diabetes mellitus in which the aloe vera gel concentrate contains 10 to 30% of acetylated polymannose, which is a high molecular polysaccharide, Lt; / RTI >

The present invention also relates to a method for the prevention and treatment of type 2 diabetes mellitus comprising concentrating the aloe vera gel, wherein the aloe vera gel concentrate has an average molar ratio of mannose to glucose of 15 to 30: Lt; / RTI >

The present invention also provides an aloe vera gel concentrate comprising an aloe vera gel concentrate as an active ingredient, wherein the aloe vera gel concentrate contains 10 to 30% of acetylated polymannose, which is a high molecular polysaccharide, And a health functional food for alleviating or improving the type 2 diabetes mellitus.

Finally, the present invention is characterized in that the aloe vera gel concentrate has an average molar ratio of mannose to glucose of 15 to 30: 1. The aloe vera gel concentrate for the alleviation or improvement of type 2 diabetes mellitus It provides health functional foods.

Type 2 diabetes prevention and treatment of a pharmaceutical composition for a concentrated Aloe Vera gel according to the invention in representative animal models of diabetes ^{BKS.Cg-m + / + Lepr db} / J homozygous diabetic (db / db) blood glucose in mice And lipid homeostasis. Therefore, it can be used as a composition effective for the treatment or prevention of human metabolic disorders, particularly, Type 2 diabetes or diabetes related diseases or symptoms. Furthermore, Aloe vera gel can be used as a relief or health functional food of type 2 diabetes which is enriched.

Figure 1 is a graph showing the effect of fasting glucose levels in type 2 diabetic db / db mice according to the present invention.
FIG. 2 is a graph showing the effect of hemoglobin on HbA1c in type 2 diabetic db / db mice according to the present invention.
FIG. 3 is a graph showing the effect on triglyceride in type 2 diabetic db / db mice according to the present invention. FIG.
Figure 4 is a graph showing the effect of AST and ALT levels in type 2 diabetic db / db mice according to the present invention.
FIG. 5 is a graph showing the effect of PPAR-γ mRNA gene expression in type 2 diabetic db / db mice according to the present invention.
FIG. 6 is a graph showing the effect of adiponectin mRNA gene expression in BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice of the type 2 diabetic animal model according to the present invention.

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown.

The present invention is characterized by a medicinal composition for the prevention and treatment of type 2 diabetes by concentrating aloe vera gel, which will be described in more detail as follows.

The pharmaceutical composition for the prevention and treatment of type 2 diabetes of the present invention comprising the aloe vera gel of the present invention is obtained by removing the skin of aloe vera leaves and filling the parenchyma tissue and homogenizing it at a maximum speed in a homogenizer . (TMP) of 0.2 to 2.25 bar using a zirconium dioxide tubular ultrafiltration membrane, a pressure of 240 L / hr at a temperature of 15 to 25 DEG C The amount and quality of the polysaccharide as an active ingredient was maximized by concentrating the aloe vera gel by ultrafiltration at the feed rate.

The aloe vera gel concentrate obtained by the ultrafiltration method is not commercially available at present and the aloe vera gel concentrate obtained by the ultrafiltration method contains 10 to 30% of acetylated polysaccharide (Accetylated Polymannose) . That is, the aloe vera gel concentrate showed a main peak at 2.0-2.2 ppm representing the acetyl group of the fingerprinting region of aloe vera polysaccharide, and a polysaccharide peak exhibiting different molecular species at 3-4 ppm indicating that aloe vera It exhibited polysaccharide properties similar to the typical commercial polysaccharides Acemannan or Aloverose. Thus, it was shown to be a polysaccharide moiety with acetyl groups containing a small amount of malic acid as compared with the chemical shift by the International Aloe Council (IASC) data base. In addition, in the FT-IR (Fourier transformed infra red ) analysis for functional analysis ^{IR (KBr) -1 (OH)} 3381cm as ^{_{ν max, 2922cm -1 (COOH,}} -NH-), 1735cm -1 (CH 3 COO) ^{, 1597cm -1 (-NHCO-), 1245cm} -1 (-CHOH) , and 1033cm ^- show ¹ (-CHOH) exhibited the characteristics of a typical polysaccharide. In particular, the carbonyl functional group and the COC stretch of acetylated sample, respectively observed in the 1748~1735 cm ^-1 and 1246~1235cm ^-1 was observed well acetylating properties.

As a result of examining the molecular weight distribution characteristics by HPGPC (high-performance gel permeation chromatography), it was found that a broad polymer region (1 peak) at an elution time of 19.55 to 19.72 minutes and a low molecular region at an elution time of 31.18 to 31.30 minutes (2 peaks) were observed. The weight average molecular weight of the polymer region was about 432 kDa (polydispersity = 1.24), while the low molecular weight region was mainly in the oligosaccharide range (830-838 Da) such as malonyl glucan.

Among them, the average molar ratio of mannose to glucose is 15 to 30: 1.

The initial aloe samples were obtained by removing the skin of aloe vera leaves and filling them with parenchyma tissue, homogenizing them at the maximum speed in a homogenizer, then centrifuging them at 8000 rpm for 30 minutes by centrifuge, taking the supernatant Aloe vera gel concentrate.

The initial solid content was 0.76%, and the effective polysaccharide content was 0.25-0.31 mg / g (absorbance at 540 nm) as measured by the congo red method, 2.3 times higher than that of the fresh leaf aloe gel . More specifically, the content of glucomannan, a marker compound of aloe vera gel, was about 1500 mg / l, which was 2.3 times higher than that of viable aloe gel, which was about 2.3 times higher than that of fresh aloe gel. Respectively. In addition, this polymer fraction has a helical structure closely related to the functional and biological activities of polysaccharides, showing a red shift of the absorption wavelength in the UV-Vis absorption spectrum in association with Congo red.

The ultrafiltration was carried out using a laboratory scale Tami UF system (Tami Industries SA, France). The Tamiya ultrafiltration system was operated with a 1 liter capacity feed tank, a feed pump, a filter cartridge, (ZrO ₂ , Tami Valisette module; MWCO = 50 to 200 kDa) having an outer diameter of 10 mm, a length of 250 mm and an area of 94 m ₂ was used as a filter, and ultrafiltration was performed in a batch concentration mode, . The sample solution was fed to the membrane module using a pump and was run at a flow rate of 1.8 / sec at 15-25 ° C and an intermembrane pressure differential (TMP) of 0.2-2.25 bar.

In the present invention, permeation of aloe using a tubular membrane with MWCO = 50 to 200 kDa is effective because the increase period of the permeate flow rate was longer, the maximum value of the permeate flux was higher and the decrease width of the maximum value was larger . Also, it is preferable to permeate using a zirconium dioxide tubular ultrafiltration membrane, and when using a planar ultrafiltration membrane, the permeation flux is lowered due to the high viscosity of the aloe, which is not effective.

Another embodiment of the present invention is a health functional food for the treatment of type 2 diabetes or diabetes related diseases or symptoms in which an aloe vera gel containing an aloe vera gel concentrate obtained by an ultrafiltration method as an active ingredient is concentrated.

In another embodiment of the present invention, the aloe vera gel concentrate obtained by the ultrafiltration method has an average molar ratio of mannose and glucose of 15 to 30: 1.

As used herein, the term " diabetes related disease or condition " includes hyperglycemia, hyperinsulinemia, hyperlipidemia, insulin resistance, glucose metabolism disorders, obesity, diabetic retinopathy, macular degeneration, cataract, diabetic nephropathy, glomerulosclerosis, diabetic neuropathy But are not limited to, symptoms due to disease, erectile dysfunction, skin or connective tissue disease, foot ulcers, metabolic acidosis and impaired glucose tolerance.

Oral compositions according to the present invention may further comprise inactive ingredients including pharmaceutically acceptable carriers, diluents, fillers, solubilizing or emulsifying agents and salts well known in the art.

In practical use, the pharmaceutical composition containing the aloe vera gel concentrate obtained by the ultrafiltration method of the present invention as an active ingredient can be combined with a pharmaceutical carrier according to conventional pharmaceutical preparation techniques. The carrier may take a wide variety of forms depending on the preparation desired for oral or parenteral administration.

The aloe vera gel concentrate obtained by the ultrafiltration method of the present invention may be added to foods to produce health functional foods for relieving type 2 diabetes, diabetes related diseases or symptoms. In this case, health functional food refers to foods that can be prepared in the form of tablets, capsules, powders, granules, liquids, and circles by using raw materials or ingredients having useful functions in the human body.

Hereinafter, the present invention relates to a medicinal composition containing an aloe vera gel concentrate obtained by an ultrafiltration method, wherein the blood glucose control effect on diabetes mellitus, diabetes and blood circulation improving effect is described in more detail based on the following examples I want to.

However, it is to be understood that the embodiments are provided so as to illustrate and explain the present invention, and the scope of the present invention should not be construed as being limited thereto.

After removing the skin of aloe vera leaves and filling the parenchyma tissue, it is homogenized at maximum speed in a homogenizer. The supernatant was centrifuged at 8000 rpm for 30 minutes using a centrifugal separator, and the exclusion molecular weight of the membrane was adjusted to 100 kDa. Using a zirconium dioxide (ZrO ₂ ) tubular ultrafiltration membrane, the intermembrane pressure difference (TMP) An aloe vera gel concentrate was prepared by ultrafiltration at a feed rate of 240 l / hr. Then, the aloe vera gel concentrate was freeze-dried or vacuum-dried to obtain a dried powder.

≪ Comparative Example 1 &

After removing the shell part of the aloe vera leaves, 1.5 g of the obtained gel and cellulase were reacted together at room temperature for 2 hours, and then the anthraquinone compound and other pigments were removed using an activated carbon column, and activated carbon After removing the column, it was dried to obtain aloe vera gel (Processed Aloe vera gel, PAG).

≪ Comparative Example 2 &

Metformin, the most widely used drug in the world, was used as a comparative dose group.

The following pharmaceutical preparations were prepared for the treatment or prevention of type 2 diabetes.

A mixture of 80 wt% of the Aloe Vera gel concentrate powder prepared in Example 1, 4 wt% of starch, 2 wt% of magnesium stearate, 10 wt% of crystalline cellulose, 4 wt% of methyl cellulose calcium, a mixture of the aloe vera gel concentrate powder, starch and methyl cellulose calcium After sterilized purified water is properly added and mixed uniformly, magnesium stearate is added to the dried product obtained by drying in a fluidized bed drier at 40 ° C to 60 ° C for 1 to 3 hours, mixed, and tableted by a conventional method to obtain tablets ≪ / RTI >

A health functional food for alleviating or ameliorating a type 2 diabetes or diabetes related disease or symptom was prepared with the following composition.

The mixture was uniformly mixed with 94.5% by weight of the aloe vera gel concentrate prepared in Example 1, 2% by weight of a concentrated concentrate, 3% by weight of liquid fructose, 0.2% of xanthan gum and 0.3% by weight of a complex gold extract, heated at 80 DEG C for 20 minutes, Functional foods were obtained.

<Preparation of Test Substance>

As a test group, aloe vera gel and metformin of Comparative Example 2 were suspended in physiological saline, and the final dose of 100 mg / kg per day was administered at 10 o'clock every morning at 0.2 o C using mouse sonde to 0.2 Ml orally.

&Lt; Experimental Example 1 > Diabetic model animal and experimental design

In the present invention, ^{BKS.Cg-m + / + Lepr db} / J mice (db / db mice) is globally the most widely used, because four-week-old male db / db mice as type II diabetic model Most important animals Were used as experimental groups. The db / db mice are over-fed with food because they can not control their appetite because the leptin receptor that mediates the signaling of leptin, which is an adipocyte-regulating hormone in the adipocyte, is mutated and gradually becomes obese, It is a model animal to accompany.

4-week-old male db / db mice were tested under specific pathogen-free growth conditions with a temperature of 71 ± 2 ° F, a humidity of 45 ± 11.0% and a 12-hour interval of contrast. Male db / db mice were given 3 weeks of age. After adjusting for 1 week in an animal breeding room, 7 mice were assigned to each experimental group and blood glucose measurements, dietary intake, and body weight changes were performed at 1 week intervals for a total of 8 weeks.

The aloe vera gel of Example 1 and Comparative Example 1 and the metformin of Comparative Example 2 were orally administered at a dose of 0.2 ml each day at 10:00 am and 16:00 pm using mouse sonde. A weekly blood glucose measurement is done every Wednesday at 09:00 am by removing the diet. (OneTOUCH @ Ultra, Johnson & Johnson Co., USA) at 15 o'clock after 6 hours of fasting. In addition, since the serum for the final biochemical analysis should be collected at a fasting time of 12 hours or more, autopsy of the experimental animals was carried out after the end of the 8-week breeding period and the blood glucose of the last 8 weeks was measured and after 2 days passed. All animals were anesthetized with ether for 12 hours after fasting. Blood was taken from the heart and placed in a serum separating tube. The blood of the serum separating tube was centrifuged at 3000 rpm for 20 minutes, and serum was collected and analyzed for biochemical indicators .

<Experimental Example 2> type 2 diabetic db / db Weight and Dietary Intake of Mice

3 weeks old type 2 diabetic db / db mice were matched with the basic feed (AIN-76A diet) for 1 week, and the aloe vera gel obtained from Example 1, Comparative Example 1 and Metformin obtained from Comparative Example 1 and Comparative Example 2, Orally. Dietary intake and body weight were measured and recorded every Wednesday at 09:00.

- Body weight

(1) Weight change: Measurement and recording at 9:00 every Wednesday

(2) Total weight gain: final weight - initial weight

(3) Average daily weight gain: Total weight gain ÷ Days

- Food intake

(1) Average Daily Dietary Intake: Total Dietary Intake ÷ Days

- Food efficiency ratio (FER)

(1) Dietary efficiency (FER): total weight gain ÷ total dietary intake

<Experimental Example 3> Lipid biochemical analysis of plasma of type 2 diabetic db / db mice

After 8 weeks of administration of the aloe vera gel obtained from Example 1, Comparative Example 1 and Metformin of Comparative Example 2, blood and serum for biochemical analysis must be collected from each experimental animal for 12 hours or more. Therefore, After 8 weeks of feeding, the blood glucose of the last 8th week was measured and after 2 days had elapsed. All animals were anesthetized with ether for 12 hours after the fasting, and the blood was taken from the heart and placed in a serum separating tube. The blood of the serum separating tube was centrifuged at 3000 rpm for 20 minutes and then serum was collected and analyzed for biochemical indicators Were used as a sample. (AST), alanine aminotransferase (ALT), and plasma triglyceride content were measured by a biochemical automatic analyzer (Hitachi-720, Hitachi Medical Co., ., Japan).

<Experimental Example 4> Insulin resistance index (IRI) analysis of type 2 diabetic db / db mice

After 8 weeks of administration of aloe vera gel obtained from Example 1 and Comparative Example 1 and metformin of Comparative Example 2, plasma and serum for biochemical analysis must be collected from each experimental animal for 12 hours or more before fasting. Therefore, After 8 weeks of feeding, blood glucose was measured at the last 8th week and 2 days later. All animals were anesthetized with ether for 12 hours, and the blood was taken from the heart and placed in a serum separation tube. The blood of the serum separation tube was centrifuged at 3000 rpm for 20 minutes, and serum was collected for biochemical indicator analysis And used as a sample. Insulin resistance index (IRI) was calculated by analyzing glucose and insulin in the separated serum.

o Insulin resistance index (HOMA-IR) = fasting plasma insulin (μU / ml) x fasting blood glucose concentration (mM) ÷ 22.5

<Experimental Example 5> Expression analysis of epididymal adipose tissue gene expression in type 2 diabetic db / db mice

After completion of the experiment, gene expression patterns in the epididymal adipose tissue extracted from each experimental animal were examined using real-time PCR amplification. For the adipose tissue surrounding the epididymis, the RNA was extracted from each tissue using RNAsol ^B (Tel-Test) solution and cDNA and real-time PCR analysis was performed using the One-step SYBR Green PCR kit (AB Science) Respectively.

500 ㎕ of RNAzol ^B was added to adipose tissue around the epididymis, and the tissue was ground with a homogenizer, and 50 클로 of chloroform (CHCl ₃ ) was added thereto and mixed again for 15 seconds. This was left on ice for 15 minutes, centrifuged at 13,000 rpm, and 200 μl of the supernatant was collected and mixed with 200 μl of 2-propanol in the same amount, followed by slow shaking, and then left on ice for 15 minutes. After centrifugation at 13,000 rpm, the cells were washed with 80% ethanol and dried in a vacuum pump for 3 minutes to extract RNA. The extracted RNA was dissolved in 20 μl of DEPC-treated distilled water and inactivated at a heating block of 75 ° C before it was used for first strand cDNA synthesis. The reverse transcription reaction was performed by reacting 3 ㎍ of prepared total RNA with DNase I (10 U / ㎕) 2 U / tube in a 37 캜 heating block for 30 minutes and then denaturing at 75 캜 for 10 minutes. 2.5 10 l 10 mM dNTPs mix, 1 μl of RNase inhibitor (20 U / μl), 1 μl of 100 mM DTT, 4.5 μl of 5 × RT buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl 2 ₂ ) was added, and then 1 μl of M-MLV RT (200 U / μl) was added again, and DEPC-treated distilled water was added to a final volume of 20 μl. After mixing 20 μl of the reaction mixture well, centrifugation was carried out at 2,000 rpm for 5 seconds. The first strand cDNA was synthesized by incubation in a 37 ° C heating block for 45 minutes, and then allowed to stand at 95 ° C for 5 minutes. After inactivation, the synthesized cDNA was used for polymerase chain reaction (PCR). Real-time quantitative PCR was performed using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, USA).

Gene expression was assessed using a TaqMan probe (FAM dye-labeled, ABi, USA) using the internal standard Mouse GAPDH probe set; Endogenous Control (VIC® / MGB Probe, Probe limited from Applied Biosystems, 4352339E) was used and reacted to a final concentration of 200 nM. Real-time quantitative PCR conditions were pre-denaturation at 50 ° C for 10 min, 94 ° C for 10 min, and 95 ° C for 40 min and 95 ° C for 1 min at 45 ° C for 0.15 min. G3PDH was used as an internal standard in the experimental group and the control group. Quantitative PCR of the target group

y = x (1 + e) n

x = starting quantity

y = yield

n = number of cycles

The relative quantitative (RQ) was calculated by calculating e = efficiency.

<Statistics Processing>

Results from various experiments were recorded as mean ± standard error, and significance was determined using Student's T-test.

<Experimental Result 1> Antidiabetic effect in BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice, a type 2 diabetic animal model

The anti-diabetic effect of Example 1 was observed in the db / db mice of the 4-week-old type, which is a type 2 diabetic model, and the aloe vera gel (PAG) of Comparative Example 1 and the positive control group of Comparative Example 2 Metformin was used. In Example 1 and Comparative Examples 1 and 2, 100 mg / kg / day was administered orally once a day for 6 weeks. Blood was collected from the tail of mice with a portable blood glucose meter once a week to measure fasting blood glucose Respectively. As shown in Fig. 1, blood glucose levels in the untreated control group were increased from 5. 3-fold to 5.8-fold in the 5-week period at 130.3 mg / dl at 4 weeks and 746.0 mg / dl at 8 weeks. At 8 weeks, the blood glucose level of the Example 1 administration group was significantly decreased by 37.6% (p <0.001) compared with the untreated negative control group. In contrast, Comparative Example 2 and Comparative Example 1 showed a 64.8% and 30.9% decrease in blood glucose levels, respectively, as compared with the untreated negative control group.

<Experimental Result 2> Effect on Dietary Efficacy in BKS.Cg-m + / + Leprdb / J mice, a Type 2 diabetic animal model

As shown in Table 2, when the FER was calculated using the feed intakes and the body weight gain after the test, the dietary efficiency was 19.2% in the Example 1 administration group compared to the untreated control group (P < 0.05). In the control group 1 and 2, there was no difference in the dietary efficiency between the untreated control group and the untreated control group. It is thought that this dietary efficiency change is due to the obesity inhibitory effect through weight reduction of Example 1.

<Experimental Result 3> Effect on HbA1c in BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice of the type 2 diabetic animal model

After 8 weeks of the experiment, the animals were anesthetized with ethyl ether in the fasting state (about 16 hours), the blood was collected from the heart with a 3 ml syringe, and then left at room temperature for 1 hour. After 10 minutes at 3000 rpm The serum was separated by centrifugation, and the frequency of HbA1c was measured with an EAJ-Glycerin hemoglobin analyzer. 2 shows the results of measurement of the frequency of glycated hemoglobin (HbA1c), which is proportional to the level of blood glucose change. The frequency of glycated hemoglobin of the untreated negative control group was 7.467%, and that of Example 1 was 5.567% (P <0.01). There was no statistically significant difference between the two groups. The frequency of glycated hemoglobin in the control group 2 was 4.967%, which was 33.4% lower than that in the untreated negative control group. However, the dose of the control group of Comparative Example 1 was 6.533%, which was 12.5% lower than that of the untreated negative control group, but was not significant. These results are almost the same as those of the plasma level. In Example 1, the frequency of glycated hemoglobin was reduced to twice as much as that of Comparative Example 1.

<Experimental Result 4> Effect on triglyceride in BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice, a type 2 diabetic animal model

At 8 weeks of age, the animals were anesthetized with ethyl ether in the fasting state (about 16 hours), the blood was collected from the heart using a 3 ml syringe, left at room temperature for 1 hour, and centrifuged at 3000 rpm for 10 minutes The serum was separated and serum triglyceride (TG) change was measured with a serum automatic meter. As shown in FIG. 3, the neutral fat level of the untreated negative control group was 171.0 ± 21.0 mg / dl, and that of the Example 1 administration group was 97.3 ± 12.3 mg / dl, which was a statistically significant decrease of 43.0% (P <0.01). The levels of triglyceride in the serum of the control groups 1 and 2 were 102.6 ± 20.1 (mg / dl) and 83.0 ± 7.3 (mg / dl), respectively, which were 40.0% (p <0.05) and 51.4% (p < 0.001).

<Experimental Result 5> Effect of ALT and AST on BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice, a type 2 diabetic animal model

After 8 weeks of age, the mice were anesthetized with ethyl ether in the fasting state (about 16 hours), the blood was collected from the heart using a 3 ml syringe, left at room temperature for 1 hour, and then incubated at 3000 rpm for 10 minutes Serum was separated by centrifugation, and serum ALT and AST levels were measured with a serum automatic meter. As shown in FIG. 4, the AST and ALT levels were not different from the untreated negative control group in Example 1 and Comparative Example 1, so that the hepatotoxicity was not observed. However, AST and ALT levels were increased by 76.4% and 34.4%, respectively, compared with the untreated negative control group in Comparative Examples 1 and 2, and hepatotoxicity was observed.

<Experimental Result 6> Effect on Insulin Resistance Index in BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice of the type 2 diabetic animal model

At 8 weeks of age, the mice were anesthetized with ethyl ether in the fasting state (about 16 hours), the blood was collected from the heart using a 3 ml syringe, and then left at room temperature for 1 hour. The blood glucose level was measured by separating the serum by centrifugation, and the insulin resistance index was analyzed by measuring the insulin level with a mouse insulin ELISA kit (SHIBAYAKI Co., Japan) (Table 3). As a result of calculating the insulin resistance index (IRI) by applying blood glucose and insulin levels, the insulin resistance index level of the untreated negative control group was 5,960 ± 209.3 (IRI), and that of Example 1 was 936.0 ± 97.7 IRI) showed a statistically significant decrease (p <0.001) compared with the untreated control group (84.2%). (P <0.001) and 86.7% (p <0.001), respectively, as compared with the untreated negative control group, and the insulin resistance index levels of the control group 1 and 2 were 1125.9 ± 93.6 (IRI) and 792.4 ± 35.1 Statistically significant decrease.

Therefore, the administration group of Example 1 showed an effect of inhibiting glucose in blood and improving the resistance to insulin, which is a problem of type 2 diabetes.

<Experimental Result 7> The effect of PPAR-mRNA gene expression on BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice, a type 2 diabetic animal model

PPAR-γ is mainly distributed in adipose tissue and smooth muscle cells, and acts as a transcription factor involved in adipocyte differentiation. The PPAR-γ ligand, 15-deoxy- △ ^12,14 -prostaglandin J2 and thiazolidinediones, It has been reported that the increase of insulin sensitivity in type 2 diabetic patients leads to a decrease in TNF-a production in adipocytes, a decrease in plasma leptin and free fatty acid concentration, and an increase in glucose receptor (GLUT-4). However, other insulin resistance modifiers, metformin, have been reported to be unaffected.

FIG. 5 shows the results of experimentsdb / db As a result of analysis of PPAR-γ mRNA gene expression by periepididymal adipose tissue in each experimental animal, the relative total amount (RQ) value of PPAR-γ mRNA gene expression to the non-treated negative control was 1 , The administration group of Example 1 was 4.803 ± 0.829 (RQ), which was 4.3 times higher than that of the untreated negative control group (p <0.001). (P <0.01) and 1.3 times (p <0.01), respectively, compared with the untreated negative control group, and the PPAR-γ mRNA gene expression changes of the control group 1 and 2 were 3.826 ± 0.801 <0.05).

Therefore, the administration group of Example 1 increased the expression of PPAR-gamma gene and improved the resistance to insulin, which is a problem of type 2 diabetes.

<Experimental Results 8> Effect of adiponectin mRNA gene expression on BKS.Cg-m ^{+ / +} Lepr ^{db / J} mice, a type 2 diabetic animal model

Adiponectin has been reported to be associated with PPAR-γ, thereby increasing insulin sensitivity, decreasing TNF-a production in adipocytes, decreasing plasma leptin, free fatty acid concentration, and increasing glucose receptor (GLUT-4).

Figure 6 shows the experimental db / db When adiponectin mRNA gene expression was analyzed by extracting periepididymal adipose tissue from each experimental animal in mice, when the relative total amount (RQ) value of adiponectin mRNA gene expression to untreated negative control was 1, The dose of Example 1 was 6.542 ± 1.128 (RQ), which was 6.8 times higher than that of the untreated negative control (p <0.001). The expression of adiponectin mRNA gene in the control group 1 was 4.337 ± 0.604 (RQ), which was 4.5 times higher than that of the untreated control group (p <0.001). However, the expression of adiponectin mRNA gene in the control group 2 was 1.543 ± 0.519 (RQ), which was 1.6 times higher than that of the untreated negative control group

Claims (5)

After removing the skin of the aloe vera leaves and filling the parenchyma tissue, the homogenized material was homogenized at a maximum speed in a homogenizer, followed by centrifugal separation to remove the supernatant. The exclusion molecular weight of the membrane was then adjusted to 100 kDa, and zirconium dioxide ZrO ₂ ) tubular ultrafiltration membrane at a feed rate of 240 L / hr at a temperature of 15 to 25 ° C and a ultrafiltration rate of 1 bar, and an aloe vera gel concentrate , The average molar ratio of mannose and glucose including the polymer polysaccharide Acetylated Polymannose is 15 to 30: 1, the insulin resistance index (IRI) reduction rate is 80% or more Wherein the aloe vera gel is concentrated.
delete delete After removing the skin of the aloe vera leaves and filling the parenchyma tissue, the homogenized material was homogenized at a maximum speed in a homogenizer, followed by centrifugal separation to remove the supernatant. The exclusion molecular weight of the membrane was then adjusted to 100 kDa, and zirconium dioxide ZrO ₂ ) tubular ultrafiltration membrane at a feed rate of 240 L / hr at a temperature of 15 to 25 ° C and a ultrafiltration rate of 1 bar, and an aloe vera gel concentrate , The average molar ratio of mannose and glucose including the polymer polysaccharide Acetylated Polymannose is 15 to 30: 1, the insulin resistance index (IRI) reduction rate is 80% or more Wherein the aloe vera gel is a concentrated form of a health functional food for relieving or improving type 2 diabetes.

5. The method of claim 4,
Wherein the aloe vera gel concentrate has an average molar ratio of mannose and glucose in the concentrate of 15 to 30: 1, wherein the aloe vera gel concentrate is a health functional food for the relief or improvement of type 2 diabetes mellitus.

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