KR20190065581A - Pharmaceutical composition for preventing and treating kidney disease use extract containing mealworm and hippocampus abdominalis, health functional food composition - Google Patents

Abstract

The present invention confirms that an extract of mealworm and Hippocampus coronatus enhances kidney function by reducing a blood urea nitrogen (bun) content, a blood creatinine (cr) content, a number of necrotic tubular cells and defending oxidative stress. Therefore, the present invention can be usefully used as a pharmaceutical composition having a use for preventing and treating kidney disease and a health functional food for improving kidney function.

Description

Pharmaceutical composition for preventing and treating kidney disease use extract containing mealworm and hippocampus abdominalis, health functional food composition}

The present invention relates to a pharmaceutical composition for preventing or treating renal toxicity caused by vancomycin or a health functional food composition for improving it, containing a mixed extract of heightened aphids and hippocampus as an active ingredient.

As consumption of proteins such as meat increases, interest in new sources of animal protein that can satisfy this demand is increasing. Of the more than 10 million insect species on Earth, about 1,500 edible insect species have been proposed as dietary protein sources for humans and animals. Kosaeae, which means “savory caterpillar,” is a larva of the brown mealworm belonging to the Mealworm family. Kosoae contains 56.58% protein and 28.20% fat. It has a high protein and unsaturated fatty acid content compared to vegetable protein, so it has an excellent effect on preventing cardiovascular diseases and is also supplied as hospital food. It is also a highly nutritious food with a lot of amino acids (leucine 4.5%, valine 3%, isoleucine 2.5%), and its energy contribution is 379 ~ 573 kcal / 100g, satisfying about 1/4 of the daily energy required by adults. These high love birds have a high ratio of protein to mass, and above all, the ratio of recoverable stored energy to energy required to grow is overwhelming. Besides, it has powerful bacteria. A research result was reported that the strong bacteria found in the stomach of Ko-Ae-ae decomposes paper and Styrofoam and converts them into organic matter, growing just like the animals given normal feed. They are already used for food in China, South America and Southeast Asia, and international organizations such as the Food and Agriculture Organization have announced policies to encourage insect consumption as a way to combat hunger and ensure food security. Therefore, high fertility, high-efficiency organic matter conversion, and low breeding cost will attract attention as a new alternative food when the current meat production system cannot be maintained due to population explosion and ecosystem destruction. Most likely.

The seahorse, a globally protected species according to the International Union for Conservation of Nature's Red List and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), is a collective term for fish belonging to the genus Hippocampus. “Hippocampus” is derived from the ancient Greek words “Hippo” meaning “horse” and “Kampos” meaning “sea monster”. In monogamous seahorses, males are known to give birth, and the female lays eggs in a brood pouch on the male's abdomen, which the male tends to and incubates. In addition, until the offspring can become independent, they raise them in their own nursery bag and give birth to 30 to 70 fry that have grown to about 0.7 cm. The color of the body is brown, and the length of the body connected by the small corrugated plate that protects the body is about 6 ~ 10cm. Seahorses mainly live in shallow waters of uncontaminated subtropical seas, but it is difficult to find seahorses in coastal areas of Korea due to water temperature and polluted coastal environment. However, recently, the only large-scale seahorse farm in Korea has been established in Jeju Island, and it is expected to be a new breed of fishermen for export to China as well as medicinal and health supplement production using seahorses. In addition, the 'Soan seahorse', which was discovered for the first time in the world in 2012 in Wando-gun, Jeollanam-do, is a new candidate species, with a survival rate close to 70% through artificial breeding, so artificial breeding was successful. Afterwards, it is planned to be released in the leech horse colony in Soan Island. As a result, it will be possible to stably maintain the domestic seahorse population, which is a protected species.

Looking at the information reported in the literature on the efficacy of the hippocampus as the most used drug so far, all parts except the intestines are used for medicinal purposes, and the main ingredients are arginine, aspartic acid, alanine, glycine, proline, and glutamic acid. It contains amino acid and contains abundant inorganic elements such as calcium, magnesium, potassium, sodium, iron, zinc, manganese, copper, chromium, and zinc, including taurine with high medicinal value. The characteristics of the medicine are sweet and salty, and the nature of the medicine is warm and returns to the liver and kidneys, so it is widely known as a summary of nourishing the kidneys (補腎壯陽), early blood (調气活血), and sangyeolsojong (散結消腫).

Vancomycin is an antibiotic isolated from Streptomyces orientalis, a glycopeptide antibiotic that has been used to treat methicillin-resistant staphylococcus aureus (MRSA) infection for more than 50 years. The bactericidal action appears quickly and the development of bacterial resistance is not remarkable. Since it is not absorbed from the digestive tract, it is administered intravenously and has a long duration of action. Side effects include skin rash and hypersensitivity reaction at the time of fever, but the main ones are auditory nerve disorder and kidney disorder. The reported incidence of vancomycin-induced nephrotoxicity ranges from 5 to 25%. Although the exact mechanism of vancomycin-induced nephrotoxicity is unknown, it is known that oxidative stress and hypoxia may be involved in the pathogenesis. In addition, vancomycin is secreted into the renal proximal tubule, so it is hypothesized that this is the site of vancomycin-induced nephrotoxicity.

The purpose of this study was to investigate the protective effects of a mixture of extracts of height love and hippocampus against acute renal failure, renal injury and oxidative stress induced by vancomycin.

Example 1

high love Preparation of hippocampus mixed extract

The method of extracting the mixture of high love and hippocampus spirits was extracted by immersing 500 g of dried and crushed hippocampus powder and 500 g of hippocampus powder in 10 folds of 70% fermented ethanol at room temperature for 24 hours. After sifting through a sieve once after extraction, the extract was filtered using filter paper (whatman No. 1). Ethanol was removed by concentration under reduced pressure (ratary evaporator N-1200, Eyela, Japan), and the final volume was 1L, which was then used as a sample. At this time, the final concentration is 0.5 g/ml.

Example 2

Experimental animals and sample administration

Twenty-six male Sprague Dawley rats (5 weeks old, about 180-200 g) were acclimatized for one week, and then randomly divided into groups as follows. control (n=8); Distilled water was orally administered for 2 weeks, and saline was injected intraperitoneally for the last 3 days. vancomycin-treated group (n=9); Distilled water was orally administered for 2 weeks, and 400 mg/kg of vancomycin was injected intraperitoneally once a day at 5 ml/kg for the last 3 days. The hippocampus mixed alcohol extract + vancomycin administration group (n=9) was orally administered 10 ml/kg of the hippocampus mixed alcohol extract, and for the last 3 days, vancomycin was intraperitoneally injected 1 hour after administration of the administered substance. . The breeding environment was maintained at 12 h light/dark cycle, 22 ± 2 ℃ temperature, and 50 ± 10% humidity. Commercial food (Purina Inc., Gyeonggi-Do, Korea) and water were supplied ad libitum to all animals. All animals were weighed twice a week, sacrificed 24 hours after the last administration, and fasted from all food except water for 12 hours before sacrifice. Specific sample administration conditions are shown in Table 1 below.

Table 1

Example 3

high love Verification of abnormal findings in kidney weight by administration of hippocampus mixed extract

1 shows the results of measuring the weight change of kidney tissue after vancomycin treatment in an animal model of acute nephrotoxicity. The kidney weight of the acute nephropathy control group (vancomycin group) was 1.9 g, an increase of 58.3% compared to that of the normal group. could

Figure 1.

Example 4

high love hippocampus mixed in the extract extract Verification of abnormal findings of renal function by

In order to evaluate renal toxicity, the improvement effect of the hippocampus mixed extract prepared in Example 1 on renal toxicity by vancomycin was confirmed by measuring blood urea nitrogen (BUN) and creatinine levels as follows.

A) Test method

On the final sacrifice day, all experimental animals were fasted for 12 hours or more, blood was collected from the abdominal aorta, left at room temperature for about 1 hour, and then centrifuged at 3,000 rpm to separate serum. after

Blood BUN and creatinine contents were measured using a commercial kit, respectively.

B) Experiment results

In the acute renal failure animal model, the blood BUN and creatinine measurement results after vancomycin treatment are shown in FIG. 2 . It was confirmed that the administration of vancomycin increased the blood BUN concentration by about 2.9 times to 35 mg/dL, and the BUN level was significantly suppressed by 77.1% compared to the vancomycin-induced BUN level when the hippocampus mixed extract was administered. was able to confirm

Compared to the normal group, the serum creatinine level in the acute renal failure control group, vancomycin, was 0.54 mg/dL, a 2.4-fold increase was observed.

Figure 2.

Example 5

high love Verification of morphological abnormality of kidney tissue by administration of hippocampus mixed extract

To evaluate the morphological changes in renal tissue, Hematoxylin-Eosin (H&E) staining of renal tissue and Periodic acid-Schiff (PAS) staining were performed to see the basement membrane of renal cortex.

A) Test method

On the final day of sacrifice, the left kidney excised from the test animal was fixed in 10% neutral formalin and sectioned through alcohol dehydration, xylene, and paraffin infiltration, followed by Hematoxylin-Eosin (H&E) staining, tissue observation, and optical microscopy. Observed. Periodic acid-Schiff (PAS) staining was oxidized with 0.5% periodic acid, followed by color development with Schiff reagent, and nuclear staining was performed with Harris hematoxylin, followed by tissue observation and observation under an optical microscope.

B) Experiment results

In order to evaluate the morphological changes of the kidney, the values were normalized and proceeded. The evaluation items for morphological changes in the kidney include tubular dilation, tubular cell desquamation, tubular vacuolation, interstitial inflammation, interstitial edema, and cast, which were measured and averaged as follows.

grade 0 : normal

grade 1: focal, mild

grade 2: moderate

grade 3: severe

As shown in FIG. 3, tubular dilation, cell desquamation, vacuolation, interstitial inflammation, interstitial edema, and cast formation were confirmed in the vancomycin group compared to the normal group in the kidney of the acute renal failure animal model. This was recognized as a typical case of acute renal failure characterized by focal degeneration including tubular epithelial necrosis and desquamation. However, it was observed that these changes were improved in the group administered with the mixed extract of the algae hippocampus. Table 2 is a result of quantifying and quantifying histopathological acute renal toxicity based on the results of FIG. 3.

Figure 3.

3

3

Table 2.

The results of FIG. 4 show the results of PAS staining of kidney tissue. As a result of observing histological changes, the normal group showed a clear and dark PAS-positive reaction on the brush border of the basement membrane and proximal tubule. On the other hand, in the vancomycin group, the PAS-positive reaction of the basement membrane was weakened and the brush border of the proximal tubule was lost, resulting in a reduced PAS-positive reaction. The brush border loss of the proximal tubule was observed to be less in the group administered with the ALA hippocampus mixed extract than in the vancomycin group.

Figure 4.

Example 6

high love Inflammation of kidney tissue by administration of hippocampus mixed extract and proximal tubular Damage remediation verification

A) Test method

Tumor necrosis factor alpha (TNF-α) immunostaining was performed to observe inflammation in the renal cortex. Incubation was performed at 37°C for 30 minutes with 0.1% trypsin for antigenic revitalization, incubation with 3% H2O2 in methanol for 30 minutes at room temperature to inhibit endogenous enzyme activity, and 3% bovine serum albumin in 1.5% normal to block non-specific reactions. Incubation was performed for 1 hour at room temperature with goat serum in 1x PBS (pH 7.4), and TNF-α (1:200 in 3% bovine serum albumin in 1.5% normal goat serum in 1x PBS (pH 7.4; ab66579, abcam) was used as the primary antibody. ) was used to proceed overnight at 4 ° C., the secondary antibody was incubated with biotinylated goat anti-rabbit IgG for 30 minutes at room temperature, and visualized with 3,3-diaminobenzidine tetrahydrochloride Harris hematoxylin was used for counterstaining.

N-Acetyl-β-D-Glucosaminidase (NAG) immunostaining was performed to observe damage to the proximal tubule of the kidney. After boiling with 1x citrate buffer (pH 6.4) for 10 minutes at low power using a microwave for antigenic revival, it was cooled for 30 minutes in a microwave, and 0.5% H2O2inmethanol at room temperature for 30 minutes to inhibit endogenous enzyme activity. Incubation was performed for 20 minutes at room temperature with 3% bovine serum albumin in 1.5% normal goat serum in 1x PBS (pH 7.4) to block non-specific reactions, and NAGLU (1:2000 in 3% bovine serum) as the primary antibody. Albumin in 1.5% normal goat serum in 1x PBS (pH 7.4); ab217671, abcam) was used for incubation at 4°C for 30 minutes, and secondary antibody was incubated for 30 minutes at room temperature using biotinylated goat anti-rabbit IgG. , 3,3-diamino-

Visualized with benzidine tetrahydrochloride. Harris hematoxylin was used as a counterstain.

B) Experiment results

As shown in Figure 5, the expression of TNF-α in the kidney tissue of the acute renal failure animal model by vancomycin was significantly increased when compared to the normal group. On the other hand, it was observed that the expression of TNF-α was decreased in the group administered with the mixed extract of the algae hippocampus. Fig. 6 shows the experimental results of confirming functional damage of renal tubules by checking the expression of NAG, which is one of the enzymes present in renal tubules. It was observed that the expression of NAG in the renal tubule was increased in vancomycin compared to the normal group, whereas the expression of NAG was decreased in the group administered with the hippocampus mixed extract.

Figure 5.

Figure 6.

Example 7

Verification of improvement in apoptosis of renal tissue by administration of mixed extract of alpine hippocampus

A) Test method

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to see apoptosis of proximal tubular cells in the renal cortex.

The paraffin-sectioned kidneys were deparaffinized, and then incubated with 0.1% trypsin in 1x PBS (pH 7.4) at 37°C for 10 minutes to revive antigenicity. To inhibit endogenous enzymes, after incubation with 3% H2O2 in methanol at room temperature for 30 minutes, enzyme solution and label solution were mixed and dispensed, incubated at 37 ° C for 1 hour, and convent POD was added and incubated at 37 ° C for 30 minutes. For visualization, 3,3-diaminobenzidine tetrahydrochloride was used as a substrate and Harris hematoxylin was used as a counterstain. Scoring was performed by TUNEL positive cell counting. Five fields of view were randomly observed at 400 times magnification and TUNEL positive cells were counted, and the total number of nuclei was expressed as a percentage.

B) Experiment results

As shown in FIG. 7 , TUNEL-positive cells were seen in the distal tubular nucleus, and it was confirmed that the number of TUNEL-positive cells increased in the vancomycin-treated group compared to the normal group. On the other hand, it was observed that the number of TUNEL-positive cells was decreased in the group administered with the mixed extract of the algae hippocampus. The graph of FIG. 8 shows the number of TUNEL-positive cells counted based on the results shown in FIG. 7 and expressed as a percentage.

Figure 7.

Figure 8.

Example 8

Verification of Malondialdehyde (MDA) level change in renal tissue by administration of mixed extracts of heightened hippocampus

A) Test method

To measure the degree of lipid peroxidation, MDA kit (sta-330, Cell Biolabs, INC.) was used. To measure the MDA of the tissue, the tissue was homogenized with 100 mg/ml in 1x PBS (pH 7.4), centrifuged at 4°C, 10,000 g, and 5 minutes, and 6 ul 100x BHT was added to 600 ul supernant. Stored at -80°C for later analysis. 100 ul SDS lysis solution was added to the MDA standard and sample, and after thoroughly mixing, incubation was performed at room temperature for 5 minutes, and then 250 ul Thiobarbituric acid (TBA) reagent was added to the standard and sample, followed by air at 95℃ for 60 minutes. Incubation was performed in a dry oven. After cooling to room temperature on ice for 5 minutes, the samples were centrifuged at 3000 rpm for 15 minutes. To prevent interference between hemoglobin and hemoglobin derivatives, 300 ul of the supernatant was put into another Eppendorf tube, 300 ul of n-buthanol was added, vigorously vortexed for 1 minute, and centrifuged at 10,000 g for 5 minutes. 200 ul each of the supernatant of the standard and sample was placed in a 96 well plate, and the absorbance was measured at 532 nm.

B) Experiment results

MDA is a metabolite produced during fat peroxidation and is used as an indicator of oxidative stress. The results of measuring the MDA level by collecting kidney tissue are shown in FIG. 9 . Compared with the normal group, the MDA level increased in the vancomycin-treated group. In the group administered with the hippocampus mixed extract, it was confirmed that the MDA level decreased compared to the group treated with vancomycin alone.

Figure 9.

In the present invention, it was confirmed that the mixed extract of P. aphid and hippocampus reduced blood urea nitrogen (BUN) content, blood creatinine (Cr) content, and the number of necrotic tubular cells, and improved kidney function by defending against oxidative stress. Therefore, the present invention can be usefully used as a pharmaceutical composition having a use for preventing and treating kidney disease and a health functional food for improving kidney function.

was to confirm the protective effect of a mixture of extracts of height love and hippocampus against acute renal failure, renal damage and oxidative stress induced by vancomycin.

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