KR20090094614A - An extract of Nelumbo nucifera's leaves for preventing or treating diabetic complication - Google Patents

Abstract

A pharmaceutical composition containing crude extract of Nelumbo nuciferas leaves or flavonoid isolated from the same is provided to suppress aldose reductase, prevent or treat complication of diabetes. A pharmaceutical composition for preventing or treating complication of diabetes comprises crude extract of Nelumbo nuciferas leaves, non polar solvent-soluble extract or flavonoid compound isolated from the extracts as an active ingredient. The flovonoid compound is one among the chemical formulas 1-5. The flavonoid compound of the chemical formula 1 is quercetin.

Description

Pharmaceutical extracts and health foods having an antidiabetic and therapeutic effect containing extracts extracted from lotus leaf {An extract of Nelumbo nucifera's leaves for preventing or treating diabetic complication}

The present invention relates to a pharmaceutical composition and health food having a diabetic prophylactic and therapeutic effect containing an extract extracted from lotus leaf, and more particularly, containing a lotus leaf crude extract, a non-polar solvent soluble extract or a compound of flavonoids isolated therefrom. It relates to a pharmaceutical composition for preventing or treating diabetic complications and health foods by inhibiting the dose reducing element.

Diabetes mellitus is one of the representative chronic adult diseases, with at least 2.5 million diabetics in Korea, estimated to be about 5% of the total population. In advanced countries, the number of diabetic patients is increasing rapidly each year, and in Korea, the number of patients is expected to increase gradually as the standard of living is improved and the lifestyle is westernized.

Insulin, a polypeptide hormone, is produced from beta cells in the island of Langerhans in the pancreas and is the hormone most cells in the body need to use glucose.

Diabetes is a metabolic disorder in which glucose metabolism in somatic cells increases and blood glucose levels increase, and excess glucose is excreted in the urine as glucose increases in the blood.

Insulin, a pancreatic hormone, maintains normal glycemic function when balanced in the body with hormones that act against insulin, such as the pituitary gland, the adrenal glands, and the thyroid gland.

However, insulin secretion is insufficient or insulin secretion is almost normal, but there is a relatively high number of anti-hormones, insulin deficiency causes diabetes.

Since diabetes is more dangerous than complications per se, the biggest goal in the treatment of diabetes today is to inhibit the development or progression of diabetic complications.

Complications are long-term symptoms of diabetes, usually chronic complications occurring after 10 to 15 years, and the typical chronic complications include diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy.

Diabetic neuropathy is a disorder of the nervous system due to diabetes, which is caused by disorders of the peripheral nerves, loss of the keyboard, paralysis of motor nerves, autonomic nerve disorders, etc., resulting in tingling, burning, painful, and sexual dysfunction. It can also cause your stool to go away.

Diabetic nephropathy is a microvascular complication caused by sclerotic lesions of renal glomerular capillaries.

Elevated blood pressure also acts to exacerbate diabetic nephropathy, with about 5% of people who have diabetes for 10 to 15 years or more.

Diabetic retinopathy is a microvascular complication that is a serious complication that causes blindness in diabetics. Recent studies on the relationship between diabetes control and complications have shown that intensifying insulin therapy can significantly reduce the incidence of diabetes complications.

Diabetic complications vary and are caused by hyperglycemia, such as peripheral neuropathy, retinopathy, nephropathy, cataracts, and corneal disease.The mechanisms include polyol pathway abnormalities and excess sugar and protein binding in the blood. Can be mentioned.

The polyol pathway is one of the glucose metabolic pathways and glucose is converted into sorbitol by aldose reductase.

Aldose reductase at steady state is very low in substrate affinity for glucose and rarely produces sorbitol, but in diabetic state, the glucose pathway in elevated insulin-insensitive tissues such as the lens, peripheral nerve, and retina increases. By generating a large amount of sorbitol, the accumulation of sorbitol in each of these tissues is known to cause diabetic cataracts, peripheral neuropathy, diabetic retinopathy and the like.

In the hyperglycemic state of diabetes, sugar carbonyl group and protein amino group combine to produce AGEs, which accumulate in the body and cause diabetic complications such as diabetic retinopathy, nephropathy, cataracts and atherosclerosis. Done.

As a result, aldose reductase and final glycation inhibitors have attracted attention as agents for the prevention and treatment of diabetic complications such as cataracts, and several of these inhibitors have been reported to improve diabetic complications in animal and clinical trials. Rather, active studies have been conducted to identify aldose reductase and final glycation end product inhibitors from natural products.

Thus, it has been reported that flavonoids, tannins, coumarins, essential oils and the like have an inhibitory effect on aldose reductase activity prepared from various tissues of cattle, rats or humans.

However, the inhibitory effect of the crude extract of lotus leaf on aldose reductase has not been reported, and there has been no report on the effects of diabetes and its complications.

Accordingly, the present invention is to solve the above-mentioned disadvantages and problems of the prior art, by inhibiting aldose reductase containing a compound of lotus leaf crude extract, non-polar solvent soluble extract or flavonoids separated therefrom, diabetic It is an object of the present invention to provide a pharmaceutical composition and a health food to enable the prevention or treatment of complications.

The above object of the present invention comprises diabetic complications by containing any one or more of the compounds of lotus leaf crude extract extracted from lotus leaf, nonpolar solvent soluble extract extracted from lotus leaf and flavonoids compounds isolated from the crude extract or nonpolar solvent soluble extract of lotus leaf It is achieved by pharmaceutical compositions and health foods that have a prophylactic or therapeutic action.

In addition, the object of the present invention is achieved by the pharmaceutical composition and health food, characterized in that the non-polar solvent soluble extract is an extract extracted from lotus leaf using any one or more of dichloromethane, ethyl acetate and butanol.

Pharmaceutical compositions and health foods having an anti-diabetic and therapeutic effect containing extracts extracted from lotus leaf of the present invention is a pharmaceutical composition and health foods that can prevent or treat diabetic complications by inhibiting aldose reductase with extracts extracted from lotus leaves Has the effect of providing.

The lotus (Nelumbo nucifera Gaertn.) Is a perennial aquatic plant belonging to the family Nymphaeceae, and is known as a paleozoic plant widely distributed in Korea and Japan, including eastern Asia in tropical and temperate regions, mainly in India and China. Yan has been used as a traditional folk medicine in China and India for a long time, and it is also used as food or medicine in Korea.

Traditionally, the leaves are called the lower lobe and have been used for fever and detoxification. Seeds and flesh have been used for tonic, hemostatic drugs, nocturnal enuresis, and gynecological diseases. Kim, Young-Bae, Herbal Medicines (KHP), pp500, 2000; Kim, Chang-Min, Chinese Medicine Dictionary, pp5956-5959, 1998).

Compounds of crude extract, nonpolar solvent soluble extract and flavonoids isolated therefrom from the lotus leaf of the present invention can be obtained as follows.

The lotus leaf crude extract of the present invention is powdered by pulverizing the lotus leaf after lyophilization, the volume of water, methanol, ethanol and butanol of about 3 to 20 times, preferably about 3 to 5 times the dry weight of the lotus leaf Lower alcohol or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably about 0.5 hour to 2 days at an extraction temperature of 20 to 100 ° C, preferably 20 to 70 ° C, preferably with ethanol It is preferably obtained by extracting from 1 to 5 times, preferably 3 times consecutively by extraction methods such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 1 hour to 1 day, and then filtered under reduced pressure and rotating the filtrate under vacuum Concentrated under reduced pressure at 20 to 100 ℃, preferably 40 to 70 ℃ with a concentrator can be obtained crude extract of lotus leaf soluble in water, lower alcohol or a mixed solvent thereof.

The lotus leaf crude extract is suspended in water, and then extracted with a solvent in the order of dichloromethane, ethyl acetate, n-butanol to obtain a lotus leaf non-polar solvent soluble extract of the present invention, preferably ethyl acetate soluble fraction, More specifically, dichloromethane: water: ethanol may be mixed with a crude extract of lotus leaf, that is, lotus leaf ethanol extract in a ratio, preferably 10: 9: 1, to obtain a dichloromethane fraction and a water-soluble fraction. Ethyl acetate can be added to the soluble fraction to obtain ethyl acetate soluble fraction and water soluble fraction, and finally the water soluble fraction can be extracted with butanol to obtain butanol soluble fraction and water soluble fraction.

Ethyl acetate soluble fraction, which is a nonpolar solvent, is a dichloromethane: methanol mixed solvent, preferably a 6: 1 to 1: 1 mixed solvent of dichloromethane: methanol at a constant volume per hour, preferably at a rate of 1,000 ml. The chromatography can be carried out to obtain six fractions from F1 to F6.

The ethyl acetate soluble fraction is an RP-18 gel using MPLC (Medium Pressure Preparative Liquid Chromatography, Yamazen Co., Osaka, Japan) to isolate and identify compounds represented by the formulas of 1, 2, 3, 4, and 5, respectively. can do.

<Structure Formula>

Chemical Formula 1. Quercetin

Quercetin 3-O-β-D-glucopyranoside: R 1 = Glc

Quercetin 3-O-β-D-glucuronopyranoside: R 1 = Gln

Quercetin 3-O-β-D-galactopyranoside: R 1 = Gal

Quercetin 3-O-α-L-rhamnopyranosyl- (1 → 6) -β-D-glucopyranoside: R1 = Rha- (1 → 6) -Glc

The present invention provides a method for separating and purifying crude extracts, nonpolar solvent soluble extracts and compounds of flavonoids separated therefrom from lotus leaves.

The present invention provides a pharmaceutical composition exhibiting a prophylactic and therapeutic effect against diabetic complications containing the compound of the lotus leaf extract obtained by the above method and the flavonoids isolated therefrom.

To investigate the efficacy of the crude extract of lotus leaf obtained above or the flavonoid compounds isolated therefrom as a pharmaceutical composition showing the prophylactic and therapeutic effects against diabetic complications, the aldose reductase inhibitory activity and the final glycation product inhibitory activity were investigated. As a result, it was confirmed that the compound of the lotus leaf crude extract or flavonoids isolated therefrom exhibits a prophylactic and therapeutic effect against diabetic complications.

In another aspect, the present invention provides a pharmaceutical composition exhibiting a prophylactic and therapeutic effect against the diabetic complications obtained from the lotus leaf crude extract obtained by the above method or a compound of flavonoids separated therefrom as an active ingredient.

In addition, the lotus leaf crude extract of the present invention or a compound of flavonoids isolated therefrom can be used as a pharmaceutical composition showing a prophylactic and therapeutic effect against diabetic complications.

The lotus leaf crude extract of the present invention or a compound isolated therefrom has little toxicity and side effects, and thus can be used safely for long-term use for prophylactic purposes.

The application amount and application method of the pharmaceutical composition comprising the lotus leaf crude extract of the present invention and a compound separated therefrom may vary depending on the formulation and the purpose of use.

<Example 1> Preparation of lotus leaf crude extract

 The lotus leaf used in the present invention was purchased and used in Muan, Jeonnam. After lotus leaf lyophilized, 300 g of the powder obtained by grinding was put into 3 l of ethanol, and refluxed and extracted at 70 ° C. three times at regular intervals (12 h, 6 h, 3 h) for cooling under reduced pressure and filtered under a reduced pressure (Watman, USA). The filtrate extract was obtained by removing the ethanol at 40 ℃ in a vacuum rotary concentrator, 53.7 g of the crude extract of the lotus leaf as an extracted residue, of which 2 g was used as a sample for screening inhibitory activity.

Example 2 Preparation of Lotus Leaf Dichloromethane Soluble Extract

2 liters of dichloromethane was added to the water-soluble layer obtained by dissolving the crude product obtained in Example 1 in 2 liters of water, and then fractionated 3 to 4 times to obtain 2 liters of water-soluble fractions and 2 liters of dichloromethane-soluble fractions. The methane soluble fraction was dried to give 16.1 g of dichloromethane soluble extract, of which 2 g was used as a sample for screening inhibitory activity.

Example 3 Preparation of Lotus Leaf Ethyl Acetate Soluble Extract

2 liters of ethyl acetate was added to 2 liters of the water-soluble fraction obtained in Example 2, followed by mixing 3 to 4 times to obtain 2 liters of water-soluble fractions and 2 liters of ethyl acetate-soluble fractions, and then dried the ethyl acetate fractions to obtain ethyl acetate. Soluble extract 3.7g was obtained and used as a sample, of which 2g was used as a sample for screening inhibitory activity.

Example 4 Preparation of Lotus Leaf Butanol Soluble Extract

2 L of butanol was added to 2 L of the water-soluble fraction obtained in Example 3, followed by mixing 3 to 4 times to obtain 2 L of water-soluble fraction and 2 L of butanol-soluble fraction, and then the water-soluble fraction and butanol-soluble fraction were dried. Soluble extract and 6.9 g of butanol soluble extract were obtained and used as a sample, of which 2 g was used as a sample for screening inhibitory activity.

Example 5 Isolation of Flavonoid Compounds

3.7 g of the lotus leaf ethyl acetate extract of <Example 3> was developed as a developing solvent using a mixed solvent of dichloromethane: methanol 6: 1 to 1: 1 (Kiesel gel 60, 230-400 mesh, Merck, Germany) column chromatography was carried out and divided into six lower fractions (EF01-EF06).

EF01 (1.14 g) in the lower fractions was subjected to MPLC with RP-18 gel (LiChroprep RP-18, 40-63 μm, Merck, Darmstadt, Germany) with 90% methanol to separate compound 1 (25 mg).

Compounds 2 (450 mg) and 4 (66 mg) were separated by MPLC using ERP03 and EF04 mixed fractions (2.78 g) with 75% methanol using RP-18 gel.

Compounds 3 (320 mg) and 5 (25 mg) were separated by MPLC of EF05 (0.89 g) and EF06 (0.86 g), respectively. Each compound was determined by measuring NMR to determine the structure, and the results are as follows.

Name of substance: Quercetin

Appearance of substance: Amorphous yellow powder.

Molecular formula of substance: c ₁₅ h ₁₀ o ₇

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 6.19 (1H, d, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H , d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 12.47 (1H, broad singlet, 5-OH).

¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 175.9 (C-4), 163.9 (C-7), 161.7 (C-5), 156.5 (C-9), 148.5 (C-4 ′) , 146.8 (C-2), 144.8 (C-3 '), 133.3 (C-3), 121.6 (C-6'), 121.0 (C-1 '), 116.2 (C-5'), 115.2 (C -2 '), 104.1 (C-10), 98.2 (C-6), 93.5 (C-8).

Name of substance: Quercetin 3-O-β-D-glucopyranoside

Appearance of substance: Amorphous yellow powder.

Molecular formula of substance: c ₂₁ h ₂₀ o ₁₂

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 5.47 (1H, d, J = 7.3 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 ′), 12.62 (1H, brs, 5-OH).

¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 100.9 (C-1 "), 98.8 (C-6), 93.7 (C-8), 77.6 (C-5"), 76.5 (C-3 "), 74.3 (C-2 ″), 70.0 (C-4 ″), 60.9 (C-6 ″).

Name of substance: Quercetin 3-O-β-D-glucuronopyranoside

Appearance of substance: Amorphous yellow powder.

Molecular formula of substance: c ₂₁ h ₁₈ o ₁₃

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 12.30 (1H, s, 5-OH), 9.72 (1H, brs, H-3 ′), 8.10 (1H, s, H-2), 7.40 (1H, dd, J = 2.0, 8.4Hz, H-6 ′), 6.82 (1H, d, J = 8.6Hz, H-5 ′), 6.34 (1H, d, J = 1.8Hz, H-8) , 6.15 (1H, d, J = 2.1 Hz, H-6), 5.26 (1H, d, J = 6.5 Hz, H-1 ″).

¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.4 (C-4), 172.6 (C-6 ″), 165.2 (C-7), 160.9 (C-5), 157.2 (C-9) , 156.5 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.9 (C-3), 120.9 (C-1 '), 120.5 (C-6'), 117.6 (C -5 '), 115.4 (C-2'), 103.5 (C-10), 102.7 (C-1 "), 99.0 (C-6), 93.8 (C-8), 76.5 (C-3"), 74.3 (C-5 ″), 74.0 (C-2 ″), 71.8 (C-4 ″).

Name of substance: Quercetin 3-O-β-D-galactopyranoside

Appearance of substance: Amorphous yellow powder.

Molecule Of Material: C ₂₁ H ₂₀ O ₁₂

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 5.41 (1H, d, J = 7.7 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 '), 12.62 (1H, brs, 5-OH).

¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 101.7 (C-1 "), 98.7 (C-6), 93.7 (C-8), 75.8 (C-5"), 73.1 (C-3 "), 71.2 (C-2 ″), 67.9 (C-4 ″), 60.2 (C-6 ″).

Name of substance: Quercetin 3-O-rutinoside

       (Quercetin 3-O-α-L-rhamnopyranosyl- (1 → 6) -β-D-glucopyranoside)

Appearance of substance: Amorphous yellow powder.

Molecular formula of substance: C ₂₇ H ₃₀ O ₁₆

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 12.64 (1H, s, 5-OH), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 6.42 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 2.1 Hz, H-6), 5.47 (1H, d, J = 7.3 Hz, H-1 "), 4.40 (1H, brs, H-1 ''), 0.98 (3H, d, J = 6.2 Hz, H-6 '').

¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.3 (C-4), 164.2 (C-7), 161.2 (C-5), 156.3 (C-9), 156.1 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 120.9 (C-1'), 116.2 (C-5 '), 115.2 (C- 2 ′), 104.1 (C-10), 101.1 (C-1 ″), 100.6 (C-1 ′ ′ ′), 98.7 (C-6), 93.6 (C-8), 77.5 (C-3 ″) , 75.8 (C-5 ″), 74.2 (C-2 ′ ′), 71.8 (C-4 ′ ′ ′), 70.6 (C-4 ′ ′), 70.3 (C-3 ′ ′ ′), 69.9 (C -2 ″), 68.3 (C-5 ′ ′ ′), 66.9 (C-6 ″), 17.2 (C-6 ′ ′ ′).

Experimental Example 1 Determination of Total Phenolic Components of Crude Extracts

The total phenolic component of the crude extract of the soft area was determined using the Folin-Ciocalteu method, and was modified by a method devised by Iqbal et al. (Iqbal et al., J. Food Comp. Anal. , 19, pp 544-551, 2006).

250 μl of the sample at a concentration of 0.4 to 2 mg / ml and 750 μl of the Pauline-Siocalto reaction reagent were added at room temperature for 5 minutes, and then ₂ ml of 7.5% Na ₂ CO ₃ was added.

It was then diluted with deionized water to a total volume of 7 ml.

The reaction solution was left in the dark for 1 hour to allow the reaction to occur, and then absorbance was measured at 765 nm using a UV / Visible spectrophotometer (Ultrospec 2100pro, Amersham Biosciences, USA). All experiments used gallic acid (gallic acid, Sigma, St. Louis, MO, USA) as the calibration standard (CS), and each result was expressed as the equivalent of gallic acid per gram of dry extract [gallic acid equivalent (GAE), mg / g of each extract or fraction].

As a result, as shown in Table 1, the total phenolic content of 177.71 ± 1.23mg / g extract was the highest in the lotus leaf crude extract.

Soft area yield(%) Total Phenolic Content (mg / g extract) Lotus leaf 15.5 177.71 ± 1.23 Soft water 12.4 83.39 ± 0.52 Lotus root 12.0 92.65 ± 0.72 Year 35.3 41.00 ± 0.20 Lotus root 17.3 21.61 ± 0.35

Yield: Percent of ethanol extract in 300 g of each raw sample.

Experimental Example 2 Quantification of Total Phenolic Components of Lotus Leaf Fractions

 Quantitative determination of the total phenolic components of the lotus leaf fraction was carried out using the Pauline-Siocalto method, which was measured by modifying the method devised by Iqbal et al.

250 μl of the sample at a concentration of 0.4 to 2 mg / ml and 750 μl of the Pauline-Siocalto reaction reagent were added at room temperature for 5 minutes, and then ₂ ml of 7.5% Na ₂ CO ₃ was added.

It was then diluted with deionized water to a total volume of 7 ml.

The reaction solution was left in the dark for 1 hour to allow the reaction to occur, and the absorbance was measured at 765 nm using a UV / Visible spectrophotometer. All experiments used gallic acid as CS, with each result expressed in equivalents of gallic acid per gram of dry extract.

As a result, as shown in Table 2, the total phenolic content of the lotus leaf ethyl acetate soluble fraction was 418.19 ± 2.35mg / g fraction, indicating the highest total phenolic content among the lotus leaf fractions.

Fraction yield(%) Total Phenolic Content (mg / g fraction) Lotus leaf dichloromethane soluble fraction 29.9 59.87 ± 0.78 Lotus leaf ethyl acetate soluble fraction 6.8 418.19 ± 2.35 Lotus leaf butanol soluble fraction 12.9 250.61 ± 1.98 Lotus Leaf Water Soluble Fraction 50.3 74.19 ± 0.46

Yield: percent of each solvent fraction in lotus leaf extract.

Experimental Example 3 Determination of Total Flavonoid Components of Crude Extracts

Determination of total flavonoid components of the soft spot crude extract was determined by modifying the colorimetric method devised by Iqbal et al. (Iqbal et al., J. Food Comp. Anal., 19, pp544-551, 2006).

1 ml of the sample at a concentration of 0.1 to 2 mg / ml was diluted with 4 ml of deionized water, 0.3 ml of 5% NaNO ₂ was added thereto, and left at room temperature for 5 minutes.

0.3 ml of 10% AlCl ₃ was added and allowed to stand for 6 minutes, and then 2 ml of 1 M NaOH was added. Deionized water was added and mixed so that the total volume was 2.4 ml, and the absorbance was measured at 510 nm using a UV / Visible spectrophotometer. All experiments used (+)-catechin (Sigma, St. Louis, MO, USA) as CS, and each result was expressed in equivalents of (+)-catechin per gram of dry extract [CE (( +)-catechin equivalent), mg / g of each extract or fraction].

As a result, as shown in Table 3, the total flavonoid content of the crude extract of lotus leaf was 125.61 ± 1.14 mg / g extract, indicating that the highest content of flavonoid was found in the lotus leaf of the lotus leaf.

Kite area yield(%) Total Flavonoid Content (mg / g extract) Lotus leaf 15.5 125.61 ± 1.14 Soft water 12.4 50.29 ± 0.78 Lotus root 12.0 82.93 ± 0.34 Year 35.3 18.91 ± 0.56 theater 17.3 8.45 ± 0.09

Experimental Example 4 Determination of Total Flavonoid Components of Lotus Leaf Fractions

Quantification of the total flavonoid components of the lotus leaf fraction was measured by modifying the colorimetric method devised by Iqbal et al.

1 ml of the sample at a concentration of 0.1 to 2 mg / ml was diluted with 4 ml of deionized water, 0.3 ml of 5% NaNO ₂ was added thereto, and left at room temperature for 5 minutes.

0.3 ml of 10% AlCl ₃ was added and allowed to stand for 6 minutes, and then 2 ml of 1 M NaOH was added.

Deionized water was added and mixed so that the total volume was 2.4 ml, and the absorbance was measured at 510 nm using a UV / Visible spectrophotometer.

All experiments used (+)-catechin as CS and each result was expressed as equivalent of (+)-catechin per gram of dry extract.

As a result, as shown in Table 4, the total flavonoid component content of the lotus leaf ethyl acetate soluble fraction was 342.66 ± 2.05 mg / g fraction, indicating that the lotus leaf ethyl acetate soluble fraction had the highest total flavonoid content.

Fraction yield(%) Total flavonoid content (mg / g fraction) Lotus leaf dichloromethane soluble fraction 29.9 55.80 ± 0.66 Lotus leaf ethyl acetate soluble fraction 6.8 342.66 ± 2.05 Lotus leaf butanol soluble fraction 12.9 231.35 ± 2.12 Lotus Leaf Water Soluble Fraction 50.3 52.15 ± 0.40

Experimental Example 5 Aldose Reductase (AR) Inhibitory Activity of Crude Extracts

Preparation of enzyme source for measuring rat lens aldose reductase inhibitory activity of the soft extract of soft region was used by Hayman and Kinoshite (Hayman S. and Kinoshite IH, J. Biol. Chem., 240, pp877-882, 1965). The method was modified.

The lens was isolated from the eye of the rat, and homogenized by adding a certain amount (0.5 ml of sodium phosphate buffer (pH 6.2) per lens) according to the wet weight.

After centrifugation at 10,000 g for 20 minutes at 4 ℃, the supernatant was taken and used as the enzyme source.

Put 621μl potassium phosphate buffer (pH 7.0), 90μl enzyme, 90μl coenzyme NADPH (1.6 mM), 9μl sample dissolved in DMSO, and finally add 90μl substrate DL-glyceraldehyde (50 mM) to 1.5ml quartz cuvette. The total reaction solution was 900 μl and the absorbance reduction rate of NADPH was measured by measuring a spectrophotometer at 340 nm for 4 minutes.

The control group was measured by adding DMSO instead of the sample, and the positive control group was measured for each concentration using quercetin and camphorol (quercetin, kaempferol: Sigma, St. Louis, MO, USA).

% Of rat lens aldose reductase inhibitory activity was calculated by Equation 1 below and statistically processed using a regression equation.

% Inhibition = 1- (absorbance of sample-absorbance of control) / standard absorbance × 100

As a result, the table as shown in 5, except for yeonjayuk lotus root, lotus leaf, yeonsim, stations, IC ₅₀ value of 8.8 ± 0.12, 34.3 ± 0.16, 36.0 ± 0.72 and 48.3 ± 0.86μg / Al higher dose reductase inhibitory activity in ml Showed this.

Kite area RLAR (μg / ml) Lotus leaf 34.3 ± 0.16 Soft water 48.3 ± 0.86 Lotus root ＞ 100 Year 36.0 ± 0.72 Lotus root 8.8 ± 0.12 Quercetin 1.92 ± 0.03

Experimental Example 6 Aldose Reductase (AR) Inhibitory Activity of Crude Extracts and Fractions of Lotus Leaf

Preparation of enzyme source for measuring rat lens aldose reductase inhibitory activity of the crude leaf extract and each solvent fraction was carried out by modifying the method used by Hayman and Kinoshite.

The lens was extracted from the eye of the rat, and homogenized by adding a certain amount (0.5 ml of sodium phosphate buffer (pH 6.2) per lens) according to the wet weight.

After centrifugation at 10,000 g for 20 minutes at 4 ℃, the supernatant was taken and used as the enzyme source.

Into a 1.5 ml quartz cuvette add 621 μl potassium phosphate buffer (pH 7.0), 90 μl enzyme, 90 μl coenzyme NADPH (1.6 mM), 9 μl of sample dissolved in DMSO, and finally add 90 μl of DL-glyceraldehyde (50 mM) substrate. The reaction solution was 900 μl and the absorbance reduction rate of NADPH was measured by measuring a spectrophotometer at 340 nm for 4 minutes.

The control group was measured by adding DMSO instead of the sample, and the positive control group was measured by concentration using quercetin and camphorol.

% Of rat lens aldose reductase inhibitory activity was calculated by Equation 2 below and statistically processed using a regression equation.

% Inhibition = 1- (absorbance of sample-absorbance of control) / standard absorbance × 100

As a result, as described lotus leaf ethyl acetate soluble fraction and the butanol-soluble fraction of the IC ₅₀ value of 2.40 ± 0.11, 3.26 ± 0.10 μg / ml in the control compound, quercetin, as shown in Table 6 (the IC ₅₀ value of 1.92 ± 0.03μg / ml) Strong aldose reductase inhibitory activity was shown to be comparable with that of caffeferol (IC ₅₀ value of 2.47 ± 0.10 μg / ml).

In particular, lotus leaf ethyl acetate soluble fraction showed stronger aldose reductase inhibitory activity than butanol soluble fraction.

Crude extracts and fractions RLAR (μg / ml) Lotus leaf crude extract 36.97 ± 0.16 Lotus leaf dichloromethane soluble fraction 47.51 ± 0.86 Lotus leaf ethyl acetate soluble fraction 2.40 ± 0.11 Lotus leaf butanol soluble fraction 3.26 ± 0.10 Lotus Leaf Water Soluble Fraction ＞ 100 Quercetin 1.92 ± 0.03 Camperol 2.47 ± 0.10

Experimental Example 7 HPLC Quantitative Analysis of Flavonoid Components Isolated from Lotus Leaf

Ethyl acetate soluble fraction of lotus leaf was subjected to repeated column chromatography using kiesel gel and RP-18 gel to give five flavonoid compounds: quercetin, quercetin 3-glucoside, quercetin 3-gluronide, quercetin 3-galactoside Quercetin 3-leutinoside was isolated.

HPLC analysis was performed for quantitative analysis of the active ingredient in the lotus leaf ethyl acetate soluble fraction and butanol soluble fraction.

Chromatography is a Dionex HPLC system Dionex Co., Germering, Germany), a quatenary gradient pump (Model P680A) with an on-line vacuum degasser, an automatic sample injector (Model ASI-100), and a 4-channel multi UV-Vis detector Model 170 U) was used.

Analysis of flavonoid components from lotus leaf extracts and fractions was performed using guard columns (2.0 × 40 mm, 3μm, Phenomenex, Torrance, CA, USA) and C-18 columns (2.0 × 150 mm, 3μm, Phenomenex, Torrance, CA, USA). Quantitative analysis was performed.

In the mobile phase used in HPLC, mobile phase A used deionized water containing 0.1% formic acid, and mobile phase B used acetonitrile containing 0.1% formic acid, respectively.

Gradient condition of mobile phase is 10% B from 0 to 10 minutes, 10 → 20% B from 10 to 40 minutes, 20% B from 40 to 42 minutes, 60% B from 42 to 45 minutes, from 45 minutes Up to 60 minutes, 10% B was used, the flow rate was 0.2 ml per minute, and the injection volume was 5 μl injection of a solution of 2 mg of sample dissolved in 1 ml of methanol.

UV wavelength was analyzed at 340 nm. Five relative amounts were obtained from Equation 3 below.

Relative amount of compound = (peak area of each compound / total area for all peaks in each plant) × 100

As a result, as shown in Fig. 1, Fig. 2 and Table 8, it was confirmed that these compounds were the main component of the quercetin 3-glucoside in the lotus leaf ethyl acetate soluble fraction, 32.1% and quercetin 3-gluronide 30.4%, and the quercetin 3-gal Lactobacillus was found to be 4.3%, quercetin 2.3%, and quercetin 3-lutinoside 0.3%.

 In the lotus leaf butanol soluble fraction, quercetin 3-gluronide accounted for 38.8%, confirming that it was the main component.

In this case, (a) in FIG. 1 shows HPLC chromatograms of lotus leaf butanol soluble fraction, and (b) shows HPLC chromatograms of lotus leaf acetate soluble fraction. In addition, 1 to 5 shown in the graph of Fig. 1 is' 1: quercetin 3-lutinoside, 2: quercetin 3-galactoside, 3: quercetin 3-glucoside, 4: quercetin 3-gluronide, 5: quercetin ',' And '*' represents 'unknown peak'.

Quercetin 3-lutinoside Quercetin 3-galactoside Quercetin 3-glucoside Quercetin 3-gluconide Quercetin Ethanol extract 0.40% 0.66% 3.50% 10.13% 0.36% Ethyl acetate fraction 0.32% 4.25% 32.08% 30.40% 2.26% Butanol fraction 1.42% 1.00% 5.54% 38.83% 0.44%

Experimental Example 8 Aldose Reductase (AR) Inhibitory Activity of Flavonoid Components Isolated from Lotus Leaf

Preparation of enzyme source for measuring rat lens aldose reductase inhibitory activity of flavonoid component isolated from ethyl acetate soluble fraction of lotus leaf was carried out by modifying the method used by Hayman and Kinoshite.

The lens was extracted from the eye of the rat, and homogenized by adding a certain amount (0.5 ml of sodium phosphate buffer (pH 6.2) per lens) according to the wet weight.

After centrifugation at 10,000 g for 20 minutes at 4 ℃, the supernatant was taken and used as the enzyme source.

Put 621μl potassium phosphate buffer (pH 7.0), 90μl enzyme, 90μl coenzyme NADPH (1.6mM), 9μl sample dissolved in DMSO, and finally add 90μl substrate DL-glyceraldehyde (50mM) to 1.5ml quartz cuvette. The total reaction solution was 900 μl and the absorbance reduction rate of NADPH was measured by measuring a spectrophotometer at 340 nm for 4 minutes.

The control group was measured by concentration by adding DMSO instead of the sample.

% Of rat lens aldose reductase inhibitory activity was calculated by Equation 4 below and statistically processed using a regression equation.

% Inhibition = 1- (absorbance of sample-absorbance of control) / standard absorbance × 100

As a result, as shown in Table 8, among the flavonoid components separated from the lotus leaf ethyl acetate soluble fraction, quercetin-3-rutinoside showed the strongest aldose reductase inhibitory activity with an IC ₅₀ value of 2.49 ± 0.04 μM. Next, quercetin (IC ₅₀ value is 5.54 ± 0.15 μM), quercetin-3-gluronide (IC ₅₀ value is 13.59 ± 0.06 μM), quercetin-3-galactoside (IC ₅₀ value is 19.30 ± 0.00 μM), Quercetin-3-glucoside (IC ₅₀ value of 20.83 ± 0.51 μM) showed aldose reductase inhibitory activity.

compound RLAR (μM) Quercetin 5.54 ± 0.15 Quercetin 3-glucoside 20.83 ± 0.51 Quercetin 3-gluronide 13.59 ± 0.06 Quercetin 3-galactoside 19.30 ± 0.00 Quercetin 3-lutinoside 2.49 ± 0.04

Experimental Example 9 Inhibitory Activity on the Production of Final Glycation Products (AGEs)

Determination of the final glycation end product production inhibitory activity of the crude extract was performed by modifying the method used by Vinson (Vinson J. A., J. Nutr. Biochem., 7, pp659-663, 1996).

AGE reaction reagents were used in 50 mM sodium phosphate buffer (pH 7.4) with 0.02% sodiumazide (10 mg / ml bovine serum albumin (Sigma, St. Louis, MO, USA)), 0.2 M fructose and 0.2 M glucose Make it put

The sample was dissolved in 10% DMSO, 50 μl of the reaction sample was added to 950 μl of the AGE reaction reagent, and then incubated at 37 ° C. for 7 days.

After incubation, the fluorescence intensity of the reaction product was measured with a spectrofluorometric detector at excitation and emission wavelengths of 350 nm and 450 nm.

% Of the final glycation product inhibitory activity was calculated by Equation 5 below and statistically processed using a regression equation.

% Inhibition = 1- (absorbance of sample-absorbance of control) / standard absorbance × 100

As a result, as shown in Table 9, the lotus leaf and soft water in the soft parts had IC ₅₀ values of 110.5 ± 2.50 and 125.5 ± 7.80μg / ml, compared with aminoguanidine (IC ₅₀ value = 84.9 ± 0.51μg / ml) as a positive control. There was a potent inhibitory activity on the production of end glycosylated products.

Kite area AGEs (μg / ml) Lotus leaf 110.5 ± 2.50 Soft water 125.5 ± 7.80 Lotus root ＞ 200 Year ＞ 200 Lotus root ＞ 200 Aminoguanidine 84.9 ± 0.51

Experimental Example 10 Inhibitory Activity of Crude Extracts and Fractions of Final Glycation Products (AGEs)

The crude glycoside extract and fractions of the final glycation product production inhibition activity was measured by modifying the method used by Vinson.

The AGE reaction reagent is prepared by adding 10 mg / ml bovine serum albumin, 0.2 M fructose and 0.2 M glucose in 50 mM sodium phosphate buffer (pH 7.4) containing 0.02% sodiumazide to prevent bacterial growth.

The sample is dissolved in 10% DMSO, 50 μl of the reaction sample is added to 950 μl of the AGE reaction reagent, and then incubated at 37 ° C. for 7 days.

After incubation, the fluorescence intensity of the reaction product was measured with a spectrofluorometric detector at excitation and emission wavelengths of 350 nm and 450 nm.

% Of the final glycation product inhibitory activity was calculated by Equation 6 below and statistically processed using a regression equation.

% Inhibition = 1- (absorbance of sample-absorbance of control) / standard absorbance × 100

As a result, as shown in Table 10, the ethyl acetate soluble fraction of the lotus leaf fractions showed the strongest inhibitory activity of the final glycation product formation with an IC ₅₀ value of 28.18 ± 0.63 μg / ml, and aminoguanidine (IC ₅₀ value). = 45.51 ± 0.69 μg / ml).

Crude extracts and fractions AGEs (μg / ml) Lotus leaf crude extract 258.08 ± 0.64 Lotus leaf dichloromethane soluble fraction 568.38 ± 24.40 Lotus leaf ethyl acetate soluble fraction 28.18 ± 0.63 Lotus leaf butanol soluble fraction 140.04 ± 5.67 Lotus Leaf Water Soluble Fraction 358.92 ± 6.20 Aminoguanidine 45.51 ± 0.69

Although the present invention has been shown and described with reference to preferred embodiments as described above, it is not limited to the above-described embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

1 is a graph showing HPLC chromatograms of lotus leaf butanol soluble fraction and lotus leaf acetate soluble fraction.

Figure 2 Contents of quercetin 3-lutinoside, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-gluronide and quercetin in the lotus leaf dichloromethane soluble fraction, the lotus leaf ethyl acetate soluble fraction and the lotus leaf butanol soluble fraction This graph shows

Claims (6)

A crude leaf extract extracted from lotus leaf, a nonpolar solvent soluble extract extracted from lotus leaf, and any one or more compounds of the flavonoids isolated from the crude leaf extract or nonpolar solvent soluble extract as an active ingredient to prevent or treat diabetic complications Pharmaceutical composition. The method of claim 1, The compound of the flavonoids is any one of the following formula 1 to 5 pharmaceutical composition. <Formula 1> Name of substance: Quercetin Appearance of substance: Amorphous yellow powder. Molecular formula of substance: c ₁₅ h ₁₀ o ₇ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 6.19 (1H, d, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H , d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 12.47 (1H, broad singlet, 5-OH). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 175.9 (C-4), 163.9 (C-7), 161.7 (C-5), 156.5 (C-9), 148.5 (C-4 ′) , 146.8 (C-2), 144.8 (C-3 '), 133.3 (C-3), 121.6 (C-6'), 121.0 (C-1 '), 116.2 (C-5'), 115.2 (C -2 '), 104.1 (C-10), 98.2 (C-6), 93.5 (C-8). <Formula 2> Name of substance: Quercetin 3-O-β-D-glucopyranoside Appearance of substance: Amorphous yellow powder. Molecular formula of substance: c ₂₁ h ₂₀ o ₁₂ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 5.47 (1H, d, J = 7.3 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 ′), 12.62 (1H, brs, 5-OH). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 100.9 (C-1 "), 98.8 (C-6), 93.7 (C-8), 77.6 (C-5"), 76.5 (C-3 "), 74.3 (C-2 ″), 70.0 (C-4 ″), 60.9 (C-6 ″). <Formula 3> Name of substance: Quercetin 3-O-β-D-glucuronopyranoside Appearance of substance: Amorphous yellow powder. Molecular formula of substance: c ₂₁ h ₁₈ o ₁₃ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 12.30 (1H, s, 5-OH), 9.72 (1H, brs, H-3 ′), 8.10 (1H, s, H-2), 7.40 (1H, dd, J = 2.0, 8.4Hz, H-6 ′), 6.82 (1H, d, J = 8.6Hz, H-5 ′), 6.34 (1H, d, J = 1.8Hz, H-8) , 6.15 (1H, d, J = 2.1 Hz, H-6), 5.26 (1H, d, J = 6.5 Hz, H-1 ″). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.4 (C-4), 172.6 (C-6 ″), 165.2 (C-7), 160.9 (C-5), 157.2 (C-9) , 156.5 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.9 (C-3), 120.9 (C-1 '), 120.5 (C-6'), 117.6 (C -5 '), 115.4 (C-2'), 103.5 (C-10), 102.7 (C-1 "), 99.0 (C-6), 93.8 (C-8), 76.5 (C-3"), 74.3 (C-5 ″), 74.0 (C-2 ″), 71.8 (C-4 ″). <Formula 4> Name of substance: Quercetin 3-O-β-D- galactopyranoside Appearance of substance: Amorphous yellow powder. Molecule Of Material: C ₂₁ H ₂₀ O ₁₂ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 5.41 (1H, d, J = 7.7 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 '), 12.62 (1H, brs, 5-OH). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 101.7 (C-1 "), 98.7 (C-6), 93.7 (C-8), 75.8 (C-5"), 73.1 (C-3 "), 71.2 (C-2 ″), 67.9 (C-4 ″), 60.2 (C-6 ″). <Formula 5> Name of substance: Quercetin 3-O-rutinoside        (Quercetin 3-O-α-L-rhamnopyranosyl- (1 → 6) -β-D-glucopyranoside) Appearance of substance: Amorphous yellow powder. Molecular formula of substance: C ₂₇ H ₃₀ O ₁₆ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 12.64 (1H, s, 5-OH), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 6.42 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 2.1 Hz, H-6), 5.47 (1H, d, J = 7.3 Hz, H-1 "), 4.40 (1H, brs, H-1 ''), 0.98 (3H, d, J = 6.2 Hz, H-6 ''). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.3 (C-4), 164.2 (C-7), 161.2 (C-5), 156.3 (C-9), 156.1 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 120.9 (C-1'), 116.2 (C-5 '), 115.2 (C- 2 ′), 104.1 (C-10), 101.1 (C-1 ″), 100.6 (C-1 ′ ′ ′), 98.7 (C-6), 93.6 (C-8), 77.5 (C-3 ″) , 75.8 (C-5 ″), 74.2 (C-2 ′ ′), 71.8 (C-4 ′ ′ ′), 70.6 (C-4 ′ ′), 70.3 (C-3 ′ ′ ′), 69.9 (C -2 ″), 68.3 (C-5 ′ ′ ′), 66.9 (C-6 ″), 17.2 (C-6 ′ ′ ′). The method of claim 1, The non-polar solvent soluble extract is a pharmaceutical composition, characterized in that the extract extracted from the lotus leaf using any one or more of dichloromethane, acetylacetate and butanol. A crude leaf extract extracted from lotus leaf, a nonpolar solvent soluble extract extracted from lotus leaf, and any one or more compounds of the flavonoids isolated from the crude leaf extract or nonpolar solvent soluble extract as an active ingredient to prevent or treat diabetic complications Health food. The method of claim 4, wherein Compound of the flavonoids is a health food, characterized in that any one of the following formula (1) -5. <Formula 1> Name of substance: Quercetin Appearance of substance: Amorphous yellow powder. Molecular formula of substance: c ₁₅ h ₁₀ o ₇ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 6.19 (1H, d, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H , d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 12.47 (1H, broad singlet, 5-OH). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 175.9 (C-4), 163.9 (C-7), 161.7 (C-5), 156.5 (C-9), 148.5 (C-4 ′) , 146.8 (C-2), 144.8 (C-3 '), 133.3 (C-3), 121.6 (C-6'), 121.0 (C-1 '), 116.2 (C-5'), 115.2 (C -2 '), 104.1 (C-10), 98.2 (C-6), 93.5 (C-8). <Formula 2> Name of substance: Quercetin 3-O-β-D-glucopyranoside Appearance of substance: Amorphous yellow powder. Molecular formula of substance: c ₂₁ h ₂₀ o ₁₂ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 5.47 (1H, d, J = 7.3 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 ′), 12.62 (1H, brs, 5-OH). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 100.9 (C-1 "), 98.8 (C-6), 93.7 (C-8), 77.6 (C-5"), 76.5 (C-3 "), 74.3 (C-2 ″), 70.0 (C-4 ″), 60.9 (C-6 ″). <Formula 3> Name of substance: Quercetin 3-O-β-D-glucuronopyranoside Appearance of substance: Amorphous yellow powder. Molecular formula of substance: c ₂₁ h ₁₈ o ₁₃ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 12.30 (1H, s, 5-OH), 9.72 (1H, brs, H-3 ′), 8.10 (1H, s, H-2), 7.40 (1H, dd, J = 2.0, 8.4Hz, H-6 ′), 6.82 (1H, d, J = 8.6Hz, H-5 ′), 6.34 (1H, d, J = 1.8Hz, H-8) , 6.15 (1H, d, J = 2.1 Hz, H-6), 5.26 (1H, d, J = 6.5 Hz, H-1 ″). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.4 (C-4), 172.6 (C-6 ″), 165.2 (C-7), 160.9 (C-5), 157.2 (C-9) , 156.5 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.9 (C-3), 120.9 (C-1 '), 120.5 (C-6'), 117.6 (C -5 '), 115.4 (C-2'), 103.5 (C-10), 102.7 (C-1 "), 99.0 (C-6), 93.8 (C-8), 76.5 (C-3"), 74.3 (C-5 ″), 74.0 (C-2 ″), 71.8 (C-4 ″). <Formula 4> Name of substance: Quercetin 3-O-β-D-galactopyranoside Appearance of substance: Amorphous yellow powder. Molecule Of Material: C ₂₁ H ₂₀ O ₁₂ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 5.41 (1H, d, J = 7.7 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 '), 12.62 (1H, brs, 5-OH). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 101.7 (C-1 "), 98.7 (C-6), 93.7 (C-8), 75.8 (C-5"), 73.1 (C-3 "), 71.2 (C-2 ″), 67.9 (C-4 ″), 60.2 (C-6 ″). <Formula 5> Name of substance: Quercetin 3-O-rutinoside        (Quercetin 3-O-α-L-rhamnopyranosyl- (1 → 6) -β-D-glucopyranoside) Appearance of substance: Amorphous yellow powder. Molecular formula of substance: C ₂₇ H ₃₀ O ₁₆ ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 12.64 (1H, s, 5-OH), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 6.42 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 2.1 Hz, H-6), 5.47 (1H, d, J = 7.3 Hz, H-1 "), 4.40 (1H, brs, H-1 ''), 0.98 (3H, d, J = 6.2 Hz, H-6 ''). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 177.3 (C-4), 164.2 (C-7), 161.2 (C-5), 156.3 (C-9), 156.1 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 120.9 (C-1'), 116.2 (C-5 '), 115.2 (C- 2 ′), 104.1 (C-10), 101.1 (C-1 ″), 100.6 (C-1 ′ ′ ′), 98.7 (C-6), 93.6 (C-8), 77.5 (C-3 ″) , 75.8 (C-5 ″), 74.2 (C-2 ′ ′), 71.8 (C-4 ′ ′ ′), 70.6 (C-4 ′ ′), 70.3 (C-3 ′ ′ ′), 69.9 (C -2 ″), 68.3 (C-5 ′ ′ ′), 66.9 (C-6 ″), 17.2 (C-6 ′ ′ ′). The method of claim 4, wherein The non-polar solvent soluble extract is a health food, characterized in that the extract extracted from the lotus leaf using any one or more of dichloromethane, acetylacetate and butanol.

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