TWI531372B - Lotus seedpod extract and its use for manufaturing composition for inhibiting pparγ expression - Google Patents

Description

Lotus extract and its use for preparing a composition for inhibiting PPAR γ expression

The present invention relates to a lotus extract and a use thereof for preparing a composition for inhibiting PPAR γ expression, in particular to a lotus seed extract having inhibition of LDL oxidation, reduction of plasma triglyceride and cholesterol content, and reduction of foam cells The formation of the atherosclerosis is further achieved by the formation of the atherosclerosis; therefore, the lotus extract can be further applied to the preparation of health foods and food additives which are resistant to atherosclerosis.

According to the Department of Health, cardiovascular disease is the second leading cause of death among Chinese people, and atherosclerosis in cardiovascular disease is the leading cause of death. The occurrence of atherosclerosis is a complex and progressive pathogenic process, including lipid entry into the blood vessel wall, lipoprotein oxidation, foam cell formation and accumulation, vascular smooth muscle cell migration and proliferation. In the pathogenesis of atherosclerosis, the mechanism of atherosclerosis is a cluster of lipids and inflammatory cells, accompanied by vascular smooth muscle cell proliferation and extracellular interstitial fluid secretion caused by the endothelium. Fibrosis.

It is known that when the blood lipid component is too high, it is easy to cause damage to the endothelial cells, and the vascular lumen gap is increased, so that lipoproteins such as "low density lipoprotein" (low density lipoprotein) LDL) easily penetrates into the intima of the vascular lumen and accumulates in the intima of the vascular endothelium. The LDL in the vascular intima is easily affected by some endogenous oxidative enzymes in the vascular intima. The lipid peroxidation is modified to form "oxidative LDL (ox-LDL)", and these modified ox-LDL become difficult to penetrate the lumen of the vascular cavity due to the change in stereostructure. Leave the endometrium and accumulate. Therefore, ox-LDL deposited in the intima of the blood vessels affects the normal function of vascular endothelial cells, such as maintaining blood vessels, preventing blood loss, balancing the permeability of blood vessels, etc., and injured endothelial cells will become easily infected by blood. The adsorption of substances inside better affects its original function. Therefore, these damaged vascular endothelial cells will attract the accumulation of scavenger-monocytes in the blood, and the mononuclear cells will be transferred from the blood vessels to the intima of the vascular endothelial cells to remove These affect ox-LDL of vascular endothelial cells. When monocytes are transferred to the inner lining of the blood vessels, they differentiate and activate at the same time to form a macrophage with more scavenging ability. At this time, macrophages undergo unrestricted phagocytosis of excessively accumulated ox-LDL, and these cholesterol-rich LDL will gradually accumulate in macrophages, causing macrophages to form another pathological foam cell. The large accumulation of these foam cells in the lining of the blood vessels forms a so-called fatty streak, which is the earliest identifiable lesion in the atherosclerotic process. Then the fatty streaks slowly cause fibrosis and hardening of the blood vessels, forming a so-called The porridge-like plaque. It causes the obstruction situation to worsen, and the thrombus is a potential factor for stroke. All of the above-mentioned phenomena lead to complex and irreversible atherosclerotic plaques in the late arterial wall, which causes treatment difficulties. Therefore, the oxidative modification of LDL and the formation of foam cells play an important role in the early stage of atherosclerosis, so effectively preventing the occurrence of these two lesions will help slow the development of atherosclerosis. .

Nelumbo nucifera Gaertnero, commonly known as Lotus, is also known as "荷" and "lian" in Chinese. It is a perennial aquatic plant. In recent years, studies on plant lotus have focused on the analysis of functional components such as lotus root and lotus leaf, and the study of biological activity. However, there is very little scientific literature on Lotus seedpod. Lotus is the heart of the lotus, not to mention the lotus room. Lotus is a traditional Chinese medicinal material, which is bitter and warm. The Compendium of Materia Medica records that lotus can be used for qi stagnation, tonifying the spleen and stomach, stopping bleeding and removing blood stasis, clearing heat, reducing liver fire and stopping bleeding. However, the lotus seed is considered to be discarded in large quantities. In recent years, it has been confirmed in literature that lotus seeds are rich in proanthocyanidins, which are flavonoids. Further studies have shown that lotus procyanidins have antioxidant properties (J Agric Food Chem. 53, 2441-5, 2005; Behav Brain Res. 194, 100-7, 2008), anti-aging, and enhanced memory (J Gerontol A Biol Sci Med Sci. 65, 236-41) , 2010; J Gerontol A Biol Sci Med Sci. 65, 933-40, 2010; Rejuvenation Res. 14, 33-43, 2011), Immunoregulation (Food Chem Toxicol. 48, 3374-84, 2010) and Anticancer (Food Chem .122, 84-91, 2010). However, the impact of lotus seed on the occurrence of atherosclerotic lesions is still unknown, and no mechanism has been explored.

Accordingly, the inventors of the present invention are committed to the development of lotus seed, in order to give the lotus pen more practical value, and to develop a low-dose, high-efficiency medical and health care product, and to provide consumers with a better choice for improving atherosclerosis.

Nowadays, the inventors have made improvements in light of the above-mentioned deficiencies and have been assisted by their extensive professional knowledge and years of practical experience, and have developed the present invention accordingly.

The main object of the present invention is to provide a lotus extract and a use thereof for preparing a composition for inhibiting PPAR γ expression, which means that the lotus extract has excellent antioxidant activity and can inhibit oxidation of LDL and reduce triglyceride And cholesterol content, and inhibition of the formation of foam cells during atherosclerosis, to effectively reduce the role of atherosclerosis; therefore, lotus extract can be further applied to the development of anti-atherosclerotic health foods and food additives.

In order to achieve the above-mentioned object, the use of a lotus extract of the present invention for preparing a composition for inhibiting PPAR γ expression is to administer an effective dose (preferably, for example, 0.5% by weight to 1% by weight) of the lotus extract. The desired individual is preferably administered orally to the desired individual to inhibit PPAR gamma protein expression, wherein the lotus extract is obtained by the following steps: Step 1: providing a lotus material; Step 2: extracting with hot water The lotus material obtains a solution; the third step: filtering the solution to obtain a filtrate; and the fourth step: drying the filtrate to obtain a lotus seed extract; wherein the lotus extract extract inhibits oxidation of LDL, scavenges DPPH free radicals, and inhibits foam cell formation Inhibition of intracellular lipid accumulation, reduction of triglycerides and low-density lipoprotein cholesterol in the blood, or inhibition of arteriosclerotic plaque deposition, to improve the role of atherosclerosis; in addition, lotus extract can further inhibit PPAR γ and The performance of CD36 may increase LXR alpha and ABCA1 expression.

Another object of the present invention is to provide a composition for improving atherosclerosis comprising a lotus root extract and a food or pharmaceutically acceptable carrier, wherein the lotus extract is obtained by the following steps: Step 1: Providing a lotus material; step 2: extracting the lotus material with hot water to obtain a solution; step 3: filtering the solution to obtain a filtrate; and step 4: drying the filtrate to obtain a lotus seed extract; wherein the composition is an oral composition .

In one embodiment of the invention, the lotus extract may, for example, comprise at least 80% by weight total flavonoid content.

In an embodiment of the present invention, the lotus extract may include, for example, 2.8%-6.4% catechin, 2.7%-4.1% proanthocyanidins, 2.3%-3.3% coumaric acid, and 11.3%-13.7% table galls. Catechin.

Thereby, the lotus seed extract can be further applied to health foods, food additives or pharmaceutical compositions to achieve the effect of improving atherosclerosis.

(S1)‧‧‧Step one

(S2)‧‧‧Step 2

(S3) ‧ ‧ Step 3

(S4)‧‧‧Step four

First Figure: Flowchart of the steps of a preferred embodiment of the present invention

Figure 2A: HPLC chromatogram of seventeen polyphenol standards

Figure B: HPLC chromatogram of lotus extract

Figure A: Effect of lotus root extract on lipid peroxidation

Figure 3B: Effect of lotus extract on LDL oxidation

Third C: Effect of lotus root extract on DPPH free radical scavenging

Figure 4A: Macrophage toxicity test results of lotus extract

Figure 4B: Effect of lotus extract on foam cell formation

Figure 4 C: Effect of lotus extract on lipid content in macrophages

Figure 4D: Effect of lotus root extract on total cholesterol and triglyceride levels in macrophages

Figure 5: Effect of lotus extract on the expression of regulatory molecules of macrophage-adsorbed oxidized low-density lipoprotein

Figure 5: Effect of lotus extract on mRNA expression of regulatory molecules of oxidized low-density lipoproteins in macrophages

Figure 6: Effect of lotus extract on the deposition of arteriosclerotic plaque

Figure 7A: Schematic diagram of H&E tissue staining of the aortic arch

Figure 7B: CD68 immunohistochemical staining results of the aortic arch

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

The use of a lotus extract of the present invention for preparing a composition for inhibiting PPAR γ expression is to administer an effective dose of lotus seed extract to a desired individual (preferably orally administered to a desired individual) to inhibit PPAR γ protein expression; please refer to the first figure, the lotus extract is obtained by the following steps: Step 1 (S1): providing a lotus material; Step 2 (S2): extracting the lotus material with hot water to obtain a solution Step 3 (S3): filtering the solution to obtain a filtrate; and Step 4 (S4): drying the filtrate to obtain a lotus seed extract; the lotus extract can inhibit oxidation of LDL, scavenge DPPH free radicals, and inhibit foam cells (foam cells) use, inhibit intracellular lipid accumulation, reduce triglyceride and low-density lipoprotein cholesterol in the blood, or inhibit the deposition of arteriosclerotic plaque, to improve the use of atherosclerosis; further, the lotus extract can be By inhibiting the expression of PPAR γ (peroxisome proliferator-activated receptor-gamma) and CD36 (or class B scavenge receptors, SR-B), or increasing LXR α (liver-X receptor alph) a) and ABCA1 (ATP-binding cassette transporter A1), regulate the role of macrophages in the formation of atherosclerosis. It is to be noted that the preferred effective dose of the above-mentioned lotus seed extract to an individual animal can be, for example, 0.5% by weight to 1% by weight, and more than 1% can effectively improve the symptoms associated with atherosclerosis, but at a higher cost. And may cause higher cell death; while less than 0.5% of lotus extract, the effect of improving atherosclerosis may be less. In addition, the preferred effective dose of the lotus extract for in vitro testing may be, for example, 5 to 200 μg/ml, more preferably 10 to 100 μg/ml; and the use of the lotus root extract of more than 200 μg/ml can effectively improve atherosclerosis. Related symptoms, however, cost a relatively high cost and may result in higher cell death rates; while using a lotus root extract of less than 5 μg/ml, the effect of improving atherosclerosis may be less favorable.

Furthermore, the present invention provides a composition for improving atherosclerosis, which comprises a lotus seed extract and a food or a pharmaceutically acceptable carrier, and thus can be further applied to health foods and food additives; The system is prepared by the first step (S1) - step four (S4) as described above; the lotus extract may, for example, contain at least 80% by weight of total flavonoid content; or may contain lower polyphenol content: 2.8% - 6.4% catechins (catechin), 2.7% -4.1% proanthocyanidins (procyanidin), 2.3% -3.3% coumaric acid (ρ -coumaric acid), and 11.3% -13.7% table gallate (epigallocatechin, EGC) .

The above "drying" step may be, for example but not limited to, a freeze-drying treatment, a spray drying treatment, an evaporation treatment or a heat drying treatment; further, the "carrier" as used in the description means any safe and effective material suitable for use herein. It helps the lotus extract to be ingested into cells or tissues.

It is worth noting here that the inventor of the present invention applied for another application, "Lianpeng extract and its use for whitening", on the same day, which is not described in detail herein, and all of its contents are hereby incorporated by reference.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

Experiment 1: Preparation and composition analysis of lotus root extract (LSE)

First, the seedpod part (without lotus seeds) was taken from the lotus ( Nelumbo nucifera ) plant (the common lotus root in the Taiwanese countryside) to prepare the lotus seedpod extract (LSE). The extraction procedure was as follows: 100g of lotus seed, adding 4L of distilled water, heated to boiling with 95 °C hot water, boiled for 2 hours to obtain a solution, after cooling, filtered to obtain a filtrate, and then the filtrate was freeze-dried to obtain a powder of crude extract of lotus root. Finally, freeze-drying is used as a lotus seed extract (LSE) powder. In the subsequent experiments, LSE dry powder was dissolved in sterilized water as an experimental material.

(1) Total phenolic content assay

Take gallic acid (GA) (mg/kg) as the standard. After dissolving methanol, take different amounts, dilute with methanol to make up 1 ml, add 0.5 ml of 2N Folin-Ciocalteu phenol reagent and shake well. Shake well with 3 ml Na ₂ CO ₃ (200 g / L), and let stand for 15 minutes at room temperature, add 5 or 10 ml of deionized water, shake well, centrifuge at 1250 × g for 5 minutes, and measure at 725 nm. The absorbance value (methanol to zero), the absorbance value is the ordinate, the concentration is the abscissa, a standard curve is obtained, and the regression equation is calculated. The sample is soaked in a good concentration, and 0.1 ml of the sample is added with 0.9 ml of methanol. The following steps are the same as the standard, and the absorbance is measured according to the standard curve, and the phenol content is calculated according to the regression equation.

(2) Total flavonoid content assay

Rutin (mg/ml) was used as a standard. After dissolving methanol, take different volumes, dilute with 30% ethanol and make up the volume to 10 ml and shake well. Then, 0.3 ml of 1 M NaNO ₃ was added thereto, followed by shaking, and allowed to stand at room temperature for 6 minutes. After adding 0.3 ml of 10% AlNO ₃ , the mixture was shaken and allowed to stand at room temperature for 6 minutes. Finally, add 4 ml of 4% NaOH solution and mix well. Add 0.4 ml of deionized water to the mark and let stand for 15 minutes. The absorbance is measured at a wavelength of 510 nm (ethanol is returned to zero), the absorbance is an ordinate, and the concentration is an abscissa, and a standard curve is obtained. And calculate the regression equation. The sample is soaked in a good concentration, and 0.1 ml of the sample is added with 0.9 ml of methanol. The following steps are the same as the standard, and the absorbance is measured according to the standard curve, and the content of the flavonoid is calculated according to the regression equation.

(3) Total anthocyanin content assay

Anthocyanins are usually weakly acidic and form stable flavylium cation structures in an acidic environment. In this experiment, the pH difference method of Fuleki and Francis was used to accurately quantify the sample to be tested, and the volume V was obtained, and 2 ml of the sample was taken from the sample, and the pH was adjusted to 1.0 with a suitable concentration of concentrated hydrochloric acid or sodium hydroxide solution. Another pH is 4.5. The dilutions of the two dilutions were measured spectrophotometrically at 520 nm to obtain A ₁ (pH 1.0) and A ₂ (pH 4.5), and the total anthocyanin mg per 100 g of fruit was calculated by the following formula. number. Total anthocyanin content (mg anthocyanin/100mg extract): [(A1-A2)x F x MW x V x 100/ε x ω], where MW: anthocyanin molecular weight in delphinidin-3-diglycoside Molecular weight 518.5 calculation; V: total extract volume (mL); F: dilution factor; ε: anthocyanin molar absorption coefficient, delphinidin-3-diglycoside in 0.1% hydrochloric acid in methanol solution absorbance ε value 301.6 Calculation; ω: total weight of fruit (g).

(4) High performance liquid chromatography (HPLC) analysis

In order to standardize the LSE preparation, the experiment identified its polyphenol content by HPLC. Use 17 polyphenol standards: 1. Gallic acid (GA), 2. protocatechuic acid (PCA), 3.catechin, 4.procyanidin B2, 5.epicatechin, 6.caffeic acid, 7.epigallocatechin gallate (EGCG), 8.ellagic acid (EA), 9.rutin, 10. ρ -Coumaric acid, 11.Epigallocatechin (EGC), 12.ferulic acid (FA), 13.epicatechin gallate (ECG), 14.gossypin, 15.gossypetin, 16.quercetin, 17.naringenin. The analysis conditions were as follows: C-9 reverse-phase column (5 μm, Hypersil ODS, 200 mm X 2.1 mm), after filtering LSE (0.45 μm), 20 μl was injected into HPLC, and the mobile phase was A: 0.1% Formic acid; B: acetonitrile wit 0.1%formic acid; 0-5min is 90% A, 10% B; 5-14min is 70% A, 30% B; 14-19min is 60% A, 40% B; 19-23min is 40% A, 60 %B; 23-24 min is 10% A, 90% B; 24-33 min is 100% B, monitored at 285 nm and 345 nm.

Experiment 2: LSE inhibits the inhibition of LDL oxidation in vitro

(1) Lipid peroxidation test (TBARS formation assay)

A peroxide MDA (malondialdehyde) is formed during the oxidation of LDL, and a molecule of MDA forms a fluorescent polymer TBARS (thiobarbituric acid reactive substance) with two molecules of TBA (thiobarbituric acid). When the wavelength (Ex) is 532 nm and the emission wavelength (Em) is 600 nm, a specific fluorescence wavelength can be detected. A standard curve was prepared using TEP (1, 1, 3, 3-tetramethoxypropane) as a standard to quantify the extent to which LDL was oxidized. Copper ions (10 μM) were added to LDL (100 μg/ml) to induce LDL to form oxidized low-density lipoprotein (ox-LDL), and LSE was added at different concentrations, and placed in a 37 ° C water bath for 24 hours. The protein was denatured and precipitated by adding 25% TCA (trichloroacetic acid), and centrifuged at 10 ° C for 30 minutes at 10,000 rpm to remove the protein leaving only the lipid fraction, followed by the supernatant and then added. Volume of TBA solution reacts in the dark The mixture was heated at 95 ° C for 40 minutes, and after cooling, the amount of MDA production was measured at a specific wavelength of Ex/Em 532/600 using a Hitachi F-2000 fluorescence colorimeter.

(2) LDL peroxidation-relative electrophoretic mobility assay

When LDL is oxidized, the lysine, histidine, etc. in the amino acid sequence, the positively charged amino acid and the oxidized product MDA, hexanal, 4-hydroxynoneal (4-HNE) Binding causes a decrease in the positive charge on the surface of the LDL protein, resulting in an increase in negative charge. The change in the surface charge of the LDL protein can be observed by the relative distance of the LDL protein moving in the colloid to the positive electric field. Copper ions (10 μM) were added to LDL (100 μg/ml) to induce LDL to form ox-LDL, and then different concentrations of LSE were added and placed in a 37 ° C water bath for 24 hours. Using BACKMAN lipoprotein (LIPO) electrophoresis system, take a piece of prepared colloid, use template filter paper to absorb excess water, gently align the purple plate with c point, gently press the purple plate to attach it to the gel. 5 μl of each sample was injected into 10 wells, allowed to stand for 5 minutes, carefully placed on a purple plate with a long strip of filter paper, the excess sample was aspirated, the long filter paper and the purple plate were removed together, and the colloid was placed in the electrophoresis. On the trough, add an appropriate amount of electrophoresis solution (6.07g/500ml), electrophoresis at 100 volts for 30 minutes, then remove the colloid, fix it with a special clamp, and then place it in a fixed tank (180ml alcohol, 90ml water and 30ml glacial acetic acid). Mix), fix for 5 minutes, remove and dry (to be completely dry), put into the dyeing tank (staining solution: 165ml alcohol and 3 mlparagon Lipo stain, plus 135ml of water mixed), after the dye is evenly distributed, put Into the bleaching tank (decolorizing solution: 450ml of alcohol mixed with 550ml of water), after decolorizing 2 times, rinse with deionized water, and then dry.

(3) ApoB fragmentation assay (ApoB fragmentation assay)

When LDL is oxidized, the co-protein apoB protein (about 512 kDa) is decomposed into different size protein fragments by free radical attack, so the gradient of colloid analysis is used to observe the apoB accessory protein apoB fragmentation. Copper ions (10 μM) were added to LDL (100 μg/ml) to induce LDL to form ox-LDL, and then different concentrations of LSE were added and placed in a 37 ° C water bath for 24 hours. After the LDL was oxidized, a tracking dye was added and reacted at 100 ° C for 10 minutes, followed by cooling. On the other hand, 3 to 15% of the gradient colloid was prepared at a low temperature, and the LDL sample mixed with the trace dye was carefully injected into the colloid, and then electrophoresed (50 V, 20 mA, 13 hours). After electrophoresis, staining solution (1.25 g coomassie blue, 0.5 g amido black, 227 ml methanol, 46 ml acetic acid, 227 ml ddH ₂ O) was stained for 1 hour, and finally decolorized (40% methanol, 10% acetic acid, 50% ddH _{2 ).} O) Decolorize, decolorize until no staining solution remains in the background, remove and store after sealing.

(4) DPPH free radical scavenging effect

DPPH (1,1-diphenyl-2-picrylhydrazyl) exhibits a blue-violet color in a methanol solution and is a stable free radical which is often used as an antioxidant index test. The structure can produce a stable resonance structure. When the DPPH radical is removed, the color will be decolored from the original blue-purple to pale yellow; the higher the degree of decolorization, the higher the degree of DPPH radical removal. Moreover, the DPPH methanol solution has a specific absorbance at a wavelength of 517 nm, and the degree of DPPH radical scavenging can be represented by a decrease in absorbance at 517 nm. 0.1 ml of LSE was diluted with methanol to 4 ml, 1 ml of freshly prepared 0.2 mM DPPH in methanol was added, and the mixture was uniformly mixed and allowed to stand for 30 minutes, and then the change in absorbance was measured by a spectrophotometer at a wavelength of 517 nm. The formula is as follows: scavenging effects% = [1 - (A _sample at 517 nm - A _blank at 517 nm) / (A _control at 517 nm - A _blank at 517 nm)] X100.

Experiment 3: LSE inhibits the formation of foam cells

(1) Cytotoxicity assay

The macrophage J774A.1 (4×10 ⁵ cell/well) was cultured in a 24well culture dish, and after adding different concentrations of LSE for 24 hours, the old medium (including LSE) was removed, and new medium and MTT (0.5) were added. The reaction was carried out for 4 hours, the medium was removed, the crystals were eluted by adding isopropanol, and the change in absorbance was measured at a wavelength of 563 nm to calculate the cell survival rate (%).

(2) Foam cell formation assay

The formation of foam cells is characteristic of the development of early atherosclerosis, and a large amount of foam cell formation will accumulate in the intima of the blood vessels to form fatty plaques. Under the stimulation of ox-LDL, macrophages express a large number of scavenger receptors (SRs) and phagocytose ox-LDL into cells, thus inhibiting the formation of foam cells, which may be regarded as the key to reduce atherosclerosis. index.

Observing the formation of foam cells, the intracellular neutral fat was dyed red by the oil stain O (oil red O) and observed. J774A.1 cells (1×10 ⁵ cells/well) were treated with different concentrations of LSE and ox-LDL (50 μg/ml) after oxidization for 24 hours. The culture solution was removed and washed twice with PBS. The cells were fixed with 10% paraformaldehyde for 1 hour. The fixative was removed and washed twice with PBS, then infiltrated with 50% isopropanol for a while, covered with 60% oil red O stain, and after 15-20 minutes, the stain was removed and washed three times with PBS (15 Minutes/time). After completing the above steps, under the optical microscope, it is observed whether there is a fat red oil droplet in the cells, which is a foam cell.

(3) Extraction of intracellular lipids

Observe the effect of LSE on lipid content in foam cells. J774.1 cells (1×105 cells/well) were treated with different concentrations of LSE and ox-LDL (50 μg/ml) after oxidization for 24 hours, and the culture solution was removed and washed twice with PBS. The intracellular lipids were extracted with 0.5 ml of hexane:isopropanol (3:2, v/v), and the lipid extract was transferred to a glass centrifuge tube and air-dried, and then dissolved in 200 μl of isopropanol to obtain intracellular lipids. Extract.

(4) Determination of intracellular cholesterol content

The change in intracellular cholesterol content was measured using a commercially available enzyme assay reagent for measuring cholesterol content. 10 μl of the intracellular lipid extract was mixed with 1 ml of the reagent, reacted at room temperature for 10 minutes, and the change in absorbance was measured at a wavelength of 500 nm. The standard concentration of the known concentration was used as a control to calculate the intracellular cholesterol content. The formula is as follows: C = 200 × ΔA _sample / ΔA _STD [mg / dl].

(5) Determination of intracellular triglyceride content

The change in the intracellular triglyceride content was measured using a commercially available enzyme assay reagent for measuring the triglyceride content. 10 μl of the intracellular lipid extract was mixed with 1 ml of the reagent, reacted at room temperature for 10 minutes, and the change in absorbance was measured at a wavelength of 500 nm. The standard concentration of the standard was used as a control to calculate the intracellular triglyceride. The content is as follows: C = 200 × ΔA _sample / ΔA _STD [mg / dl].

Experiment 4: High cholesterol diet induces arteriosclerosis in New Zealand white rabbits

(1) Animal mode

This experimental mode group is summarized as follows: (a) normal feed feeding group, (b) high fat diet (HFD) feeding group, (c) HFD + 0.5 wt% LSE, (d) HFD + 1.0 wt% LSE, (e) 1.0wt% LSE (toxicity test group), the general group (control) was fed with normal feed, 2~5 groups were treated with HFD: 89.8% chow, 10% coconut oil and 0.2% cholesterol. Animal diet was recorded regularly during the experiment. As well as changes in body weight, and register the number of animal deaths. After 25 weeks of feeding, hyperlipidemia can be induced. The animals are sacrificed overnight and sacrificed to collect blood for analysis of blood fat, including total cholesterol, triglyceride, HDL, LDL, and the other part is taken from liver tissue to determine the total intrahepatic The concentration of lipid cholesterol and LDL. After sacrifice, the aortic arch portion of the experimental animals was removed, immersed in 10% neutral formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). In addition, the thoracic artery of the experimental animal was also carefully peeled off by the subject, and the body fat outside the lumen was carefully removed under a dissecting microscope, and then the vessel was cut open in a longitudinal manner, followed by staining with oil red O to observe the thoracic artery tube. Fat plaque in the inner wall of the cavity Deposition and distribution.

(2) Analysis of animal blood fat and liver fat

Before taking blood samples from New Zealand white rabbits and liver tissue, the animals were first fasted for 12-14 hours. After sampling, the following items were analyzed:

Triglyceride (TG): The enzyme is added to the quantitative serum and liver homogenate by the enzyme method, and the absorbance is measured by a spectrophotometer at 500 nm through a water bath at 22-25 ° C to calculate the TG concentration.

Total Cholesterol (TC): A coloring agent was added to the serum and liver homogenate quantified by the enzymatic colorimetric method. After a 37 ° C water bath, a red compound was formed, which was measured by a spectrophotometer at a wavelength of 500 nm. The absorbance value is converted to the TC concentration.

High-density lipoprotein cholesterol (HDL-C): using the principle of enzyme action and colorimetric determination, adding appropriate precipitant to the quantitative serum and liver homogenate, precipitating chylomicrons, VLDL, LDL, and then centrifuging for complete quantification The supernatant was added with a cholesterol measuring reagent, and the absorbance was measured at a suitable wavelength (500 nm) with a spectrophotometer, and the HDL-C concentration was converted.

Low-density lipoprotein cholesterol (LDL-C): using the principle of enzyme action and colorimetric determination, adding appropriate precipitant to the quantitative serum and liver homogenate, precipitating chylomicrons, VLDL, LDL, and performing centrifugation to determine the quantification The supernatant is added with a cholesterol measuring reagent, and the absorbance is measured at a suitable wavelength (500 nm) by a spectrophotometer. After calculation, the cholesterol value of the supernatant is deducted from the total cholesterol value to obtain an LDL-C concentration.

(3) Sample preparation by oil red O staining

A saturated solution of oil red O (300 mg/dl) was dissolved in 99% isopropanol as a preservation solution. After the configured oil red preservation solution and PBS were mixed at a ratio of 6:4, the mixture was allowed to stand for 10 minutes, and then centrifuged and filtered. Take the previous 10% neutral The formalin-fixed thoracic aorta was washed with PBS, soaked in 50% isopropanol for 5 minutes, soaked in oil red O for 3 minutes, then washed with 20% isopropanol for 1 minute, and finally treated with PBS. Excessive dye wash. If there is fat streak on the inner wall of the blood vessel, it will be dyed red by oil red O. After taking the photo, scan the photo into the computer and calculate the percentage of the area of the blood vessel with fat accumulation by computer program (Image Pro plus).

result

Results 1: Analysis and identification of lotus seed extract (LSE)

(1) Qualitative analysis

Please refer to Figure 2A. The X-axis represents the retention time (RT) and the Y-axis represents the absorbance. The retention time of 17 polyphenols standards by HPLC is sequentially presented. In addition, the lotus extract is added. Mixed with the standard product, the RT is consistent at several time points, and the result is obtained from the second B picture. Summary As shown in Table 1, the LSE contains four components, catechin, procyanidin B2, ρ- Coumaric acid, and epigallocatechin (EGC). It is 4.6%, 3.4%, 2.8% and 12.5%.

(2) Quantitative analysis

Referring to Table 1, the Folin-Ciocalteu method was used to determine LSE about 29.6 wt% of total polyphenol content; on the other hand, according to HPLC results: flavonoids (catechin+procyanidin B2+quercetin=20.5wt%) The proportion of LSE components is the highest. The results of analyzing the total flavonoid content by the Jia method showed that LSE yielded about 85.7 wt% of total flavonoids (total flavonoids). The anthocyanin extracted from the LSE has a purity of about 6.3 wt%. The above component identification test confirmed that LSE is indeed a flavonoid-rich extract with the highest ratio of EGC.

According to the preliminary quantitative experiment results, the flavonoid content of the lotus extract is 85.7 wt% higher than that of the lotus leaf extract (56 wt%) (J Agric Food Chem. 57, 5925-32, 2009), so it has become a chemopreventive substance. The potential.

Results 2: The effect of lotus root extract (LSE) on the inhibition of LDL oxidation in vitro and the effectiveness of scavenging free radicals

(1) Lipid peroxidation test

Please refer to the third panel A, the concentration of MDA produced as the final product of lipid peroxidation as an indicator of lipid peroxidation, treated with 10 μM copper ion (CuSO ₄ ) alone, in which the MDA concentration rapidly increased while adding simultaneously After reacting with different concentrations of LSE, it significantly reduces the formation of MDA. With the addition of the LSE 50 μg/ml group, the amount of MDA produced by 50% or more was remarkably lowered, indicating that LSE has an effect of suppressing lipid peroxidation induced by copper ions.

(2) Low-density lipoprotein surface charge change test

The change of the charge on the surface of the protein after LDL oxidation can be determined by the distance from the colloidal color band to the positive electric field, and the relative electrophoretic mobility (REM) of the control group is 1 cm. Referring to Figure 3B, the black histogram represents the electrophoretic mobility. After 10 hours of oxidation of LDL at 37 °C with 10 μM copper ion (CuSO ₄ ), the ribbon movement distance is about 3 more than the control group. After the reaction was simultaneously added with different concentrations of LSE, the REM returned to the LSE at a concentration of 100 μg/ml. From the above experimental results, it was revealed that the REM group had a lower REM than the positive control group (no added extract, but added with copper ion-inducing group), and showed inhibition and prevention of LDL oxidation.

(3) ApoB colipoprotein fragmentation test

Please continue to refer to the third B diagram. The gray histogram represents the apoB accessory protein apoB fragmentation. The LDL co-protein is induced by 10 μM copper ion (CuSO ₄ ) after LDL is oxidized at 37 ° C for 24 hours. ApoB is about 90% broken. After a simultaneous addition of different concentrations of LSE, there was about 48% cleavage (52% retention) at a concentration of apoB of LSE 50 μg/ml, and about 20% rupture (80% retention) of apoB at a concentration of 100 μg/ml. From the above experimental results, it was revealed that the group with LSE addition had a lower apoB cleavage than the positive control group, and it was confirmed that LSE has an effect of inhibiting LDL oxidation.

(4) DPPH free radical scavenging effect

From the third C chart, the DPPH scavenging effect is about 62% when the LSE concentration is 50 μg/ml, and the higher the LSE concentration, the stronger the ability to capture free radicals, and the concentration-dependent relationship.

Lipid peroxidation and LDL oxidation have been shown to be the causative factors in the development of atherosclerosis. The above results show that LSE has the ability to inhibit the oxidation of LDL, regardless of the protein oxidation modification or lipid peroxidation of LDL, from the results of various experiments. It can be seen that LSE can inhibit the oxidation of LDL at a concentration of 10-100 μg/ml. In the test for scavenging free radicals, it was found that LSE has a function of chelation of free radicals, indicating that it protects LDL from free radical attack by copper ion-promoting, and inhibits LDL oxidation. Since LSE has an effect of inhibiting oxidation of LDL, it can be expected to have an effect of inhibiting the occurrence of atherosclerosis.

Results 3: Lotus extract (LSE) inhibits foam cell production and intracellular lipids Material accumulation

(1) Cytotoxicity test

First, observe the sensitivity of J774A.1 macrophages to LSE to determine the dose of extract required for subsequent cell assays. Please refer to Figure 4A. The LSE requires a relatively large dose (IC50>200g/ml) to cause the lethal toxicity of macrophages, so the subsequent experiment used LSE non-toxic dose 10-50μg/ml.

(2) Foam cell generation test

As shown in the fourth B-picture, it was observed that the J774A.1 macrophage treated the ox-LDL which was first oxidized, and it was observed that a large amount of neutral fat accumulation was stained by the oil red O dye inside the cells. Phenomenon, compared to the normal group, but no red particles accumulate, it is known that the formation of foam cells is successfully induced by ox-LDL. However, under ox-LDL stimulation, the cells were treated with different concentrations of LSE at the same time, and the sharp decrease of red particles accumulated in macrophages was observed under a light microscope. From the results, it was inferred that the effect of foam cell formation was effectively reduced at the treatment of LSE 10-50 μg/ml. The experiment will further verify whether LSE reduces the foam cell production by regulating LDL metabolism.

(3) Analysis of cellular lipid content

According to the fourth C picture, under the induction of ox-LDL, the content of total cholesterol (TC) and cholesterol cholesterol (CE) in the cells increased by about 2 times and 2.7 times, respectively. The content is obtained by subtracting free cholesterol (FC) from the TC content; while under the protection of LSE, the excessive uptake of TC and CE by the cells is effectively reduced. Please refer to the fourth D picture, the same effect also appears in the intracellular triglyceride (TG) changes, ox-LDL induced the increase of TG in J774A.1 cells to 1.4 times that of the near normal group, and Under the treatment of LSE, the excessive uptake of the triglyceride by the cells was also inhibited, and it returned to the same phenomenon as the normal group under the action of LSE 50 μg/ml. This shows that LSE can It is effective enough to inhibit excessive intake of lipids by cells, thereby reducing the chance of foam cell formation.

(4) Discussion on the mechanism of foam cell formation

(4-a) Macrophage uptake of ox-LDL

The Cleaner Receiver (SRs) is an embedded membrane protein that has a unique binding capacity to modified low-density lipoproteins such as ox-LDL and acetylated low-density lipoprotein ac-LDL. The SRs mainly used by macrophages to recognize ox-LDL were confirmed to be type A SR (SR-A) or type B SR (SR-B or CD36). Expression mechanism CD36 in cells has been shown to be transcribed by the nuclear factor PPAR γ (peroxisome proliferator-activated receptor -gamma) regulated, PPAR γ ligands (Ligands) as BRL (rosiglitazone), TZD (thiazolidonedione ) and Both ox-LDL promote the expression of CD36. Therefore, the Western blot method was used to analyze the expression of CD36, SR-A and PPAR γ proteins in J774.1 cells accompanied by LSE treatment in the case of ox-LDL induction. The results are shown in Figure 5A. Under ox-LDL stimulation, the protein expression of CD36 and PPAR gamma increased; the performance of SR-A was not affected. However, the protein expression of CD36 and PPAR γ was significantly decreased in the group treated with LSE (10-50 μg/ml), indicating that LSE may affect the expression of CD36 by regulating the expression of PPAR γ .

(4-b) Metabolism of cholesterol in macrophages

There is a set of "cholesterol reverse transport" mechanism in the cells, which can remove the cholesterol from the surrounding cells to the liver. It must be HDL-mediated, and the cholesterol is collected from the surrounding tissue and transported to the liver for metabolism. The transport protein that transports the cholesterol in the surrounding cells from the cells to the outside of the cell is ABCA1. The cholesterol in the cells can be transported outside the cell to the HDL, and the cholesterol is transported to the liver by HDL. As shown in Figure 5A, ox-LDL can slightly promote LXR α and ABCA1 protein expression; however, LSE can significantly increase the trend of both protein expression. It was confirmed by experiments that increasing the expression of ABCA1 can slow down the transformation of macrophages into foam cells. This experiment further confirmed that ABCA1 can regulate its expression by the molecular mechanism of PPAR γ- LXR α . It is suggested that LSE may also promote reverse cholesterol transport by the molecular mechanism of LXR α -ABCA1. Please refer to Figure 5B again for the same results for LXR α and ABCA1 mRNA expression.

Results 4: Effect of lotus root extract (LSE) on arteriosclerosis induced by high cholesterol diet in rabbits

(1) Analysis results As shown in Table 2, after induction of rabbits with high cholesterol diet (HFD) for 25 weeks, serum TC of the induction group (HFD group) increased about 16 times compared with the normal group, but an additional LSE group, 0.5 wt Compared with the induction group, the %LSE group decreased by about 22.8%; the 1.0wt% LSE decreased by 32.2%, which is statistically significant. In terms of TG, the normal group was 48.67±12.86 mg/dL. Under the induction of high cholesterol diet, the induction group increased 1.9 times compared with the normal group and rose to 94.33±20.98 mg/dL. Under the administration of 0.5wt% LSE, the TG of the rabbit blood was 78.63±26.78mg/dL. When the dose was increased to 1.0wt% LSE, the TG of the test group was 55.25±12.57mg/dL, compared with the induction group. There is a downward trend. 0.5 wt% LSE inhibited blood lipid rise by 34.4%; while 1.0 wt% LSE inhibition was 86%. Also in the LDL-C project, the 0.5 wt% and 1.0 wt% LSE test groups had similar inhibition. For HDL-C, the normal group was 21.39±7.50 mg/dL, which increased to 88.04±10.65 mg/dL under the induction of cholesterol diet; while in the group given LSE, HDDL-C in the blood of 0.5 wt% LSE It is 93.64±17.61 mg/dL and has no statistical significance. When the HDL-C in the blood with a dose increase of 1.0 wt% LSE was 170.83 ± 49.82 mg / dL, there was a significant increase in the effect. Further analysis of risk factors, the ratio of LDL-C/HDL-C in the normal group was 1.49, the ratio of LDL-C/HDL-C in the induction group was 7.76, and the ratio of LDL-C/HDL-C in the experimental group was 0.5 wt%. The 1.0wt% LSE was 6.68 and 4.72, respectively, which also showed a downward trend compared with the induction group. In addition, 1.0wt% LSE given under normal diet as In the toxicity group, all the blood lipid determination values were similar to those in the normal group, indicating that LSE is non-toxic.

(2) Analysis of arteriosclerotic plaque

Results Referring to Figure 6, no animal atherosclerosis lesions were found on the surface of the aortic lining, animals in the normal control group and the toxic group (1.0 wt% LSE). In the cholesterol-inducing group (HFD), significant fatty plaque deposition was observed. After the LSE was 0.5 wt% and 1.0 wt% in the feed, the fatty plaque in the inner layer of the thoracic artery was significantly decreased. It can be seen that LSE can actually reduce the deposition of arteriosclerotic plaque in the in vivo test and can effectively delay the development of atherosclerosis.

(3) Pathological section observation

For the analysis results, please refer to Figure 7A. In the normal blood vessel wall, no foam cells were found and no displacement of vascular smooth muscle cells was found. In the induction group (HFD), there are obvious accumulations of foam cells on the pathological sections, and The thickness of the inner layer of the vein also became thicker and the phenomenon of displacement of vascular smooth muscle cells was found. In the group given LSE, the thickness of the inner layer of the aorta was significantly lower than that of the induction group, and the high dose of 1.0 wt% LSE group returned to The normal group is equivalent. In addition, please refer to the seventh B picture, the aortic arch part is also analyzed by immunohistochemical staining to detect the expression of macrophages CD68 in the tissue. No specific protein expression was found in the normal control vessel wall; in the induction group (HFD), it was found to have significant anti-CD68-specific staining, and the thickness of the inner layer of the artery also changed. Thick, in order to infer that macrophages phagocytose ox-LDL; LSE group has inhibition, this result can be used as a control for cell experiments.

According to the above effects, the lotus seed extract can form a composition alone or in combination with a food or a pharmaceutically acceptable carrier for use in the development of health foods and food additives to effectively improve atherosclerosis.

It can be seen from the above description that the present invention has the following advantages compared with the prior art:

1. The present invention utilizes cell and animal experiments for the first time to confirm that lotus seed extract (LSE) has excellent antioxidant activity and can inhibit LDL oxidation; in addition, LSE is also involved in regulating macrophage production of atherosclerosis due to oxidation of LDL. The process of hardening formation; therefore, LSE has the potential to be applied to medical health foods to improve the symptoms of atherosclerosis.

2. The lotus seed extract (LSE) of the present invention is directly obtained by boiling water extraction, and retains the original biological activity of the natural plant itself, not only reduces the residual enthalpy of the organic solvent, but also avoids the artificial oxidation of the synthetic chemical substance. The role of sexual injury.

3. The lotus seed extract (LSE) used in the present invention proves that the "rainper" portion which is often discarded and has no effect on improving cardiovascular disease; accordingly, it not only increases the use efficiency of the lotus plant, but also provides consumers with More options for slowing the symptoms of atherosclerosis related foods or health supplements.

In summary, the extract of the present invention and its use for preparing a composition for inhibiting PPAR gamma can indeed achieve the intended efficacy by the above-disclosed examples, and the present invention has not been disclosed. Prior to the application, Cheng has fully complied with the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

Claims (5)

A use of a lotus extract for the preparation of a composition for inhibiting the expression of PPAR gamma by administering an effective amount of a lotus extract to a desired individual to inhibit PPAR gamma protein expression, wherein the lotus extract is obtained by the following steps: Step 1: providing a lotus material; step 2: extracting the lotus material with hot water to obtain a solution; step 3: filtering the solution to obtain a filtrate; and step 4: drying the filtrate to obtain a lotus seed extract, Containing at least 80% by weight of total flavonoids. The use of a lotus extract as described in claim 1 for the preparation of a composition for inhibiting PPARγ expression, wherein the lotus extract further inhibits the expression of CD36 or increases the expression of LXRα and ABCA1. The use of a lotus extract as described in claim 1 for the preparation of a composition for inhibiting PPARγ expression, wherein an effective dose of 0.5 wt% to 1 wt% of the lotus extract is administered to a desired individual Inhibition of PPARγ expression. The use of a lotus extract as described in claim 1 for the preparation of a composition for inhibiting PPARγ expression, wherein the lotus extract is administered orally to the desired individual. The use of a lotus extract as described in claim 1 for the preparation of a composition for inhibiting PPARγ expression, wherein the lotus extract comprises catechin, procyanidin, and coumaric acid (ρ- Coumaric acid), and epigallocatechin (EGC).