COMPOSITION FOR PREVENTING AND TREATING NON-ALCOHOLIC FATTY LIVER DISEASE
Inventors: Wei Zhang1’*, QingfengZhang2*,Shuying Gao2, Zhi-Hong Jiang1, Caiyun Wang1, Jianru Guo1 '’State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China. 2’ Faculty of Bio-engineering, Zhuhai Campus of Zunyi Medical College A portion of the disclosure of this patent document contains material, which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. [0001] The present invention relates to a composition for preventing and treating non-alcoholic fatty liver disease (NAFLD), and particularly relates to a composition comprising blueberry leaf flavonoids for preventing and treating NAFLD. The present invention also relates to methods for preparing the composition and using the composition in preventing and treating NAFLD. [0002] Bramble leaf, belonging to rose family Rosaceae, is a kind of leaf of [0003] Bramble leaf contains high content of flavonoids. Different plants have different contents of flavonoids and variations in pharmaceutical properties. Phytochemicals in terms of physiological and pharmaceutical uses have also drawn a lot of attentions from local and overseas scholars. More and more attentions are drawn to the preventive use of Chinese medicine as it is critical to ensure human health nowadays. Flavonoids are a kind of material which is widely sourced from nature and is capable of preventing and treating a wide variety of chronic diseases in human and animals. It is also believed to be one of the promising solutions for moderating malignant tumor. Corresponding literatures reported that content of flavonoids in bramble leaf is relatively higher than that in bramble fruit and seed. However, using bramble leaf in manufacturing of pharmaceutical product at industrial scale needs to be further developed. Among different bramble leaves described herein, blueberry leaf appears to have higher content of flavonoids, which is therefore a good candidate of anti-oxidant source in future for extracting highly effective and less toxic flavonoids in a cost-effective way. There are very few reports nowadays on blueberry leaf flavonoids. Most of the recent reports, on the other hand, focused on flavonoids extracted from other plants such as raspberry, [0004] Conventional methods for obtaining total flavonoids include organic solvent extraction, salt extraction, reflux extraction, soxhlet extraction, microwave and ultrasonic extraction methods. Purification is mainly via silicone gel electrophoresis, polyacrylamide gel electrophoresis, or macroporous resin separation. Ultrasonic as an advanced technology is nowadays widely used in botany because it provides a short-time, high-temperature, and cell-permeable approach for extracting substance from plant cells via disrupting the cell wall in order to increase the rate of extraction and yield of the substance being extracted. Because macroporous resin separation is cost-effective and outstanding in purification, it has become a research focus for purification of flavonoids in recent years. However, due to the fact that it is difficult to synthesize total flavonoids, a process that can obtain total flavonoids in higher yield and purity from a source which is rich in this compound is demanded. [0005] Non-alcoholic fatty liver, also called non-alcoholic fatty liver disease (NAFLD), is a kind of pseudo-alcoholic liver diseases. It is a clinically pathological disease that involves a change in liver histology which is similar to that in alcoholic liver disease but without any history of alcoholic abuse. According to severity and different clinical stages, the pathological changes of the liver disease can be transformed into simple fatty liver (SFL), nonalcoholic steatohepatitis (NASFI), fatty liver fibrosis and liver cirrhosis. Incidents of NAFLD have significantly increased in recent years. Some literatures reveal that total flavonoids from extract of some plants such as [0006] Accordingly, a first aspect of the present invention is to provide a method for extracting total flavonoids from blueberry leaves. [0007] In accordance with an embodiment of the present invention, the method includes the following steps: (a) using an organic solvent (e.g. petroleum ether) in a weight-to-volume ratio of 1:20 to remove fatty acids from dried blueberry leaves under a first ultrasonic extraction for certain time period; (b) after removing the fatty acids followed by a first suction filtration to collect the remaining solid, the solid is subject to extraction with 50-70% ethanol in a volume ratio of 1:15-20 at a temperature from 30 to 70°C; the extraction is repeated for three times; the extraction fluids are combined; (c) concentrating the extraction fluids into 1/5 to ℅ of the total volume, followed by adding corresponding volume of 75-95% ethanol until the volume ratio of ethanol in (b) is reached, then subject to a second ultrasonic extraction, followed by a second suction filtration, and then removing the residual solid and collecting the filtrate, the filtrate being further concentrated into a volume (e.g., 100 mL) or maintained at that volume, in order to obtain a crude extract of total flavonoids which is ready for further processing; (d) concentrating the crude extract of total flavonoids obtained from step (c) through rotary evaporation and collecting the concentrated solution, followed by vacuum drying at a temperature, grinding the remaining solid into powder, in order to become the total flavonoids; (e) using 95% ethanol for ethanol precipitation of the total flavonoids obtained from step (d), followed by using deionized water to prepare an aqueous solution with the evaporated solution as an upper column solution for macroporous resin separation, adsorbing active ingredients by the macroporous resin through macroporous resin separation; and (f) using deionized water to wash away impurities from the resin column until the flowthrough is not turbid, followed by using 95% ethanol to elute the adsorbed total flavonoids from the resin column, collecting the eluent, in order to obtain a purified total flavonoids from blueberry leaves. [0008] According to an exemplary embodiment of the present invention, the percentage yield of total flavonoids obtained from blueberry leaves by using the extraction method of the present invention is as high as 23% which is higher than that obtained by conventional methods from other natural sources. [0009] It should be understood that the extraction method disclosed in the present invention is not limited to blueberry leaf but also applicable to other species of bramble leaves which are rich in flavonoids that are useful for preventing and treating NAFLD. [0010] A second aspect of the present invention relates to a method for preventing and treating liver disease, more particularly, non-alcoholic fatty liver disease (NAFLD), by administering an effective amount of the total flavonoids prepared by the method of the present invention to a subject in needs thereof. [0011] In accordance with an embodiment of the present invention, the method for preventing and treating liver disease includes: (a) oral administration or intravenous administration of the total flavonoids of the present invention in a concentration of 20 mg/kg to 80 mg/kg to the subject in needs thereof where the subject is small animal, or in a concentration of 3 mg/kg to 15 mg/kg where the subject is human based on the dose translation formula in Reagan-Shaw et al. (2007); (b) the course of administration may last for 180 days or less. While the following steps may not be claimed, the method may optionally further include: (c) comparing weight of the subject before and after the course of administration; and (d) determining the content of the total flavonoids in the subject by analyzing the biological fluid of said subject during or after the course of administration through a high throughput method in order to determine the bioavailability of the total flavonoids and validate the clinical significance thereof. [0012] A third aspect of the present invention is to provide a composition comprising a therapeutically effective amount of total flavonoids obtained by the method of the present invention. In accordance with an embodiment of the present invention, the composition includes total flavonoids in a concentration of 20 mg/kg to 80 mg/kg for small animal or from 3 mg/kg to 15 mg/kg for human in a daily dosage. The course of administration of the composition may last for 180 days or less, depending on the progress and severity of the disease. The composition is preferably administered orally but other administration routes such as intravenous are also possible. The composition is useful for preventing and treating NAFLD. [0013] As disclosed herein, non-alcoholic fatty liver disease (NAFLD) includes but not limited to simple fatty liver (SFL), nonalcoholic steatohepatitis (NASH), fatty liver fibrosis and liver cirrhosis. [0014] Embodiments of the present invention are described in more detail hereinafter with reference to the drawings, in which: [0015] FIG. 1 is a flow chart illustrating the extraction method according to an embodiment of the present invention; [0016] FIG. 2A-E is microphotographs showing histology of liver tissue obtained from different groups of disease model after a 180-day time course: (A) 20mg/kg total flavonoids; (B) 40 mg/kg total flavonoids; (C) 80 mg/kg total flavonoids; (D) 0 mg/kg total flavonoids; (E) normal animal. [0017] FIG. 3A-C is chemical structure of different kudinosides: (A) kudionoside A; (B) kudinoside D; (C) kudinoside F. [0018] FIG. 4A-D is negative ion eletrospray mass spectrum of different kudinosides: (A) Ginsenoside Rbl (internal standard, I.S.); (B) kudinoside A; (C) kudinoside D; (D) kudinoside F. [0019] FIG. 5A-C is chromatogram of human plasma sample obtained from different groups: (A) a plasma sample spiked with kudinosides A and F at 10 ng/ml each and a blank plasma sample; (B) a plasma sample spiked with 10 ng/ml kudinoside D and a blank plasma sample; (C) a plasma sample spiked with 500 ng/ml I.S. and a blank plasma sample. [0020] The following descriptions are set forth as preferred examples. It will be apparent to those skilled in the art that modifications, including additions and/or substitutions, may be made without departing from the scope and spirit of the invention. Specific details may be omitted so as not to obscure the invention; however, the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation. [0022] FIG. 1 illustrates by means of a flow chart how total flavonoids are isolated from blueberry leaves according to the presently claimed extraction method and the experimental design testing efficacy of different concentrations of the total flavonoids on NAFLD disease model. In this example, 5.0 g of blueberry leaves are first dried (100). Petroleum ether in a weigh-to-volume ratio of 1:20 (101) is used to remove fatty acids from the dried leaves under ultrasonic extraction for 20 minutes followed by a first suction filtration (102). The remaining solid after suction filtration is collected and then subject to ethanol extraction (103). 50-70% ethanol at a volume ratio of 1:15-25 is used in 103 and said extraction is carried out for at least three times at a temperature of 30-70°C. Fluids collected from several times of ethanol extraction in 103 are combined and then concentrated into 1/5 to % of the total volume of the collected fluids. Corresponding volume of 75%-95% ethanol is then added into the concentrated fluids until the volume ratio of 50-70% ethanol in 103 is reached. A second ultrasonic extraction is carried out followed by a second suction filtration. The remaining solid after the second suction filtration is removed and only the filtrate is saved for further concentration into a volume of about 100 mL. This concentrated filtrate is a crude extract of total flavonoids (104). This crude extract of total flavonoids is then subject to rotary evaporation (105) by using a rotary evaporator (R20GB, Shanghai Xiafeng Co. Ltd.) for further concentration. The solution after concentration by rotary evaporator is collected and then subject to vacuum drying at 70°C. The remaining solid after the vacuum drying is then grinded to become powder form (106). The powder form of the total flavonoids is the total flavonoids extract. The total flavonoids extract obtained in 106 is further purified by using 95% ethanol for ethanol precipitation. Deionized water is used to mix with the evaporated solution obtained from the ethanol precipitation in order to prepare an aqueous solution as an upper column solution for later macroporous resin separation. Active ingredients (i.e., the total flavonoids) in the aqueous solution are adsorbed on the resin column during the AB-8 macroporous resin separation (107). The resin column is then washed by deionized water to remove the impurities until the eluent is not turbid. Lastly, another 95% ethanol is used to remove the adsorbed total flavonoids from the resin and collect the eluent. The eluent is the purified total flavonoids which is ready for use (108). [0023] In order to test the efficacy of the purified total flavonoids from blueberry leaves on treating NAFLD, an normal rats; Disease: Omg/kg flavonoids treated disease rats) are sacrificed by cervical vertebra dislocation and livers are dissected out, followed by washing in PBS. The washed livers are fixed in methanol and embedded in paraffin for sectioning. The liver tissue section is stained with H&E stain, dehydrated in alcohol followed by xylene to remove the wax, and then mounted with cover glass. The following data will be collected: weights measured at different time points during the course of treatment for different groups of rat will be compared; level of different markers in serum, e.g., total cholesterol (TC), three acyl glycerin (TG), high-density lipoprotein (HDL-c), low density lipoprotein (LDL-c), alanineaminotransferase (AST), aspertate aminotransferase (ALT), etc. will be determined; net weight of the liver dissected from different groups of rat will be measured; liver index will be calculated; histological change of the liver tissue from different groups of rat will also be studied (112). The data recorded will be statistically analyzed by using SPSS program (ver. 12.0). (113) Result will be expressed as x±s, where x is mean; s is standard deviation, and p<0.05 represents the p-value of the statistical significance. [0024] EXAMPLE 2-Results [0025] (A) Liver Anatomy: [0026] Not shown in any figures, livers dissected from normal rats appear to be in pale red, with shinny surface, smooth edges, and soft in texture. In comparison, livers from disease group appear to be relatively larger in size, more yellowish in color, with adhesions between lobes, and the sections thereof are fatty. [0027] (B) Body Weight, Liver Net Weight and Liver Index: [0028] Table 1 shows the physical data of rat sample from different treatment groups and their corresponding liver index (liver net weight/body weight). It is assumed that the initial body weight of all rats used in this study is more or less the same. As compared to normal group, body weight of the rats from the disease group is increased significantly (p<0.01); liver net weight and liver index are increased accordingly (p<0.01). As compared to disease group, body weight of the rats from different treatment groups (HLF1, HLF2 and HLF3) has no significant difference (p<0.05); liver net weight and liver index, however, are both decreased significantly (p<0.01). From these data, it demonstrates certain therapeutic effect of administration of the purified total flavonoids from blueberry leaves on the accumulation of fat in liver of NAFLD rat model. Note: Value expressed as χ±S ; **P<0.01 [0029] (C) Serum Marker Levels: [0030] Comparison of levels of different markers between different group of rats is shown in Table 2. From Table 2, expression level of TC, TG, ALT, AST, LDL-C in rats of the disease group is relatively higher than that of those in normal group; the deviation has statistical significance (p<0.05). On the other hand, HDL-C level in disease group is relatively lower than in normal group; the deviation also has statistical significance (p<0.05). [0031] (D) Clinical Pathology of Liver Tissue [0032] FIG. 2 shows the histological change of the liver tissue obtained from different groups by H&E staining of the paraffin section. From the microscopic images shown in FIG. 2, tissue sections from normal group (FIG. 2E) shows intact liver tissue. In FIG. 2E, the liver cells appear to be aligned in order with each other and propagated in radial pattern from the centre of the vein. Cell boundary of normal liver cells is clear. Position of the nucleus is right in the middle. Hepatic sinosoids are organized systematically. Portal area is clear. Cell vacuoles are rigid. In contrast, FIG. 2D shows that the cell morphology of liver tissue from disease group in the absence of administration with the total flavonoids preparations. From FIG. 2D, the cell size from the disease group is relatively larger than that from the normal group. The size also varies among different liver cells in the disease group. There are quite a number of empty lipid vesicles which can be found in the tissue section shown in FIG. 2D. Lipid of the liver cells from the disease group appears to be modified. Cell nucleus in some of these liver cells is not positioned in the middle but is away from the middle of the cell. Cell morphology of these cells appears to be in balloon-like shape. Disease groups treated with different concentrations of the purified total flavonoids from blueberry leaves according to the present invention show significant improvement in liver lipid deposition. In FIG. 2A-C, the number of lipid vesicles in the disease groups treated with the total flavonoids is comparatively smaller than that in the disease group without any treatment. Morphology of most cells from these total flavonoid-treated disease groups is substantially normal and structurally intact. The higher is the concentration of the total flavonoids, more organized the liver cells are aligned with each other, fewer lipid vesicles are present, and the extent of recruitment of the inflammatory cells is smaller. Among the three concentrations tested in the present invention, the highest concentration (i.e. 80mg/kg) of the total flavonoids has the most significant effect. Arrangement of hepatic cell cords in the group treated with the highest concentration of total flavonoids is more organized than that in the group treated with lower concentrations. The cell morphology is relatively clearer in the group treated with the highest concentration of the total flavonoids as compared to the lower concentration-treated groups. [0033] The disease model used in the present invention is a well-established disease model for testing drug candidates of lowering cholesterol thereby preventing liver disease. The animal model created in the present invention by feeding SD rats with a highcholesterol diet to affect their cholesterol metabolism mimics the pathological mechanism of NAFLD in human. Although there are quite a number of conventional products proved to be effective in modulating cholesterol level, they also possess liver toxicity, thereby worsening liver damage instead of improving the condition of cholesterol accumulation in liver. Thus, a pharmaceutical product capable of modulating the cholesterol level while liver is protected from potential attack by the toxicity is needed. The experimental data of the [0034] While the following example is not claimed, the principles disclosed hereinafter may be useful for validating the clinical significance of the presently claimed invention in future. [0036] A sensitive and selective high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the simultaneous determination of kudinoside A, kudinoside D and kudinoside F in human plasma is disclosed. Samples are prepared after protein precipitation and analyzed on a Cl8 column interfaced with a triple quadrupole tandem mass spectrometer. Negative electrospray ionization was employed as the ionization source. The mobile phase consists of Acetonitrile-water (35:65) at the flow rate of 0.3 mL/min. The analytes and internal standard Ginsenoside Rbl are both detected by using multiple reaction monitoring mode. The method is linear in the concentration range of 2.5-1000.0 ng/ml. The lower limit of quantification (LLOQ) is 2.5 ng/ml. The intra-and inter-day relative standard deviation across three validation runs over the entire concentration range is less than 12.4 %. The accuracy determined at three concentrations is within ±4.9 [0037] Below is detailed description of the high throughput analytical method and the result thereof: [0038] 1.1 Materials [0039] Kudinoside A (KA), Kudinoside D (KD), and Kudinoside F (KF) are isolated from plants Ilex Kudingcha by repeated open-column chromatography and preparative reverse-phase high pressure liquid chromatography. Their structures were determined by UV, MS,1H- and13 C-NMR, and by comparison with literature values. The purities of these compounds are all above 98% as determined by UPLC analysis. Ginsenoside Rbl purchased from Sigma (St. Louis, MO) is used as internal standard (I.S). HPLC-Mass grade methanol, acetonitrile and formic acid are purchased from Anaqua Chemical Supply (Houston, TX, USA). Ultrapure water is obtained from a Milli-Q Gradient water system (Millipore, Bedford, MA, USA). [0040] 1.2 Instrumentation [0041] The chromatographic system used in this example consists of an Agilent 1200 HPLC series, including a binary pump (Model G1312A), a vacuum degasser (Model G1379B), an autosampler (Model G1329A) and a column oven (Model G1316A). The mass spectrometer is an Applied Biosystems Sciex 4000 Q-trap® mass spectrometer (Applied Biosystems Sciex, Foster, CA,USA). Data acquisition is carried out by Analyst 1.4.2® software on a DELL computer. [0042] 1.3 LC-MS Conditions [0043] The chromatographic separation is achieved on a ZORBAX SB-C18 column (100 mm x2.1 mm i.d., 3.5 μm, Agilent, Palo Alto, CA, USA). The mobile phase is acetonitrile-water, (35:65, and v/v) at the flow rate of 0.3 mL/min. The column temperature is maintained at 30°C. [0044] After chromatographic separation, the mobile phase is directly introduced into the mass spectrometer via an electrospray ionization (ESI) source operating in the negative mode. Quantification is performed using multiple reaction monitoring (MRM) of the transitions of m/z 925.5->m/z 59 for KA, m/z 907.5-mi/z 745.5 for KD, m/z 925,5->m/z 59 for KF, m/z 1107.6->m/z 58.9 for Ginsenoside Rbl (internal standard, I.S), respectively, with a dwell time of 100 msec. [0045] In order to optimize all the MS parameters, a standard solution (O.lμg/ml) of the analyte and I.S. is infused into the mass spectrometer. Some mass spectrometer parameters are identical for all analytes. The curtain gas reaches 15 psi. The ionspray voltage is set at 4500 V and the temperature at 700°C. The nebulizer gas (GS1) and turbo gas (GS2) are both 60 psi. The declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) are optimized for each analyte. The declustering potentials are set at -165,-160,-165 and -155 V for KA, KD, KF and I.S., respectively. The values of the collision energy are -120,-52,-120 and -130 V for KA, KD, KF for and I.S, respectively. The collision cell exit potentials are -7,-20,-7 and 7 V for KA, KD, KF and I.S, respectively. [0046] 1.4 Sample Preparation [0047] The plasma is prepared by removing protein through a precipitation method. In a 1.5 mL centrifuge tube an aliquot 100 μL of human plasma is spiked with 10 μL of Ginsenoside Rbl solution (internal standard, 5 μg/mL). After vortexing, 400 μL of methanol is added to the tubes and the tubes are vortex mixed for 3 minutes. After centrifugation at 14,000 rpm for 15 minutes in a refrigerated Microcentrifuge (Labnet International, Woodbridge, NJ, USA) at 4°C, 5 μL of clear supernatant fluid is injected into the FIPLC-MS/MS system for KA, KD and KF. The prepared samples are kept in room temperature until injection. [0048] 1.5 Preparation of Standard and Quality Control Samples [0049] Stock solutions of KA, KD and KF are prepared in methanol at the concentration of 1,000 μg/mL. Stock solution of I.S. is prepared in methanol at the concentration of 500 μg/mL and diluted to 1 μg /mL with methanol. Calibration curves are prepared by spiking the appropriate standard solution in 0.1 mL of blank plasma. Effective concentrations in plasma samples are 2.5, 5, 10, 25, 50, 100, 250, 500 and 1,000 ng/mL. The quality control (QC) samples are separately prepared in blank plasma at the concentrations of 10, 100 and 500 ng/mL. The spiked plasma samples (standards and quality controls) are then treated following the “Sample preparation” procedure on each analytical batch along with the unknown samples. [0050] 1.6 Method Validation [0051] Plasma samples are quantified using the ratio of the peak area of each analyte to that of I.S. as the assay parameter. Peak area ratios are plotted against analyte concentrations and standard curves are in the form of y= [0052] To evaluate linearity, plasma calibration curves are prepared and assayed in duplicate on three separate days. The accuracy and precision are also assessed by determining QC samples at three concentration levels on three different validation days. The accuracy is expressed by (mean observed concentration-theoretical concentration)/ (theoretical concentration) χl00% and the precision by relative standard deviation (RSD %)• [0053] Absolute recoveries of KA, KD and KF at three QC levels are determined by assaying the samples as described above and comparing the peak areas of KA, KD, KF and I.S. with those obtained from direct injection of the compounds dissolved in the supernatant of the processed blank plasma. [0054] 2.1 Mass Spectrometry [0055] Because KA, KD, KF and I.S. have numerous hydroxyl groups (FIG. 3), the negative ionization mode is initially chosen. The Q1 full scan spectra of KA, KD, KF and I.S. are dominated by protonated molecules [M-H] and no significant solvent adduct ions and fragments ions are observed. In the product spectra of [M-H] ions for KA, KD, KF and I.S., when the CID energy is increased, more fragment ions are observed, while the response of [M-H] is lowered significantly. When the CID energy is set at -120, -52, -120 and -130V, respectively, the main fragment ion from KA, KD, KF and I.S. shows the highest MS response (FIG. 4). [0056] 2.2 Preparation of plasma samples [0057] Sample preparation is a critical step for accurate and reliable HPLC-MS/MS assays. The most widely employed biological sample preparation methodologies currently are liquid-liquid extraction (LLE), protein precipitation (PPT), and solid-phase extraction (SPE). The PPT is a simpler and more time-saving method. Methanol, acetonitrile and acetone are all tested for extraction, and methanol is finally adapted because of its high extraction efficiency and stability. [0058] 2.3 Method validation [0059] 2.3.1 Specificity [0060] The specificity tests the ability of the method to differentiate and quantifies the analyte in the presence of other endogenous constituents in the sample and to detect potential interferences. The chromatographs of human blank plasma and the same plasma spike at the 10 ng/ml of KA, KD and KF are presented in FIG. 5. There is no significant interference or ion suppression from endogenous substances observed at the retention times of the analytes. Typical retention times for KA, KD, KF and I.S. are about 3.63 min, 4.27 min, 2.13 min and 2.11 min, respectively. [0061] 2.3.2 Linearity of calibration curves and lower limits of quantification Standard curves are performed in triplicate for each analyte in plasma. In all cases the regression coefficient is >0.99. KA, KD and KF curves are linear over a range of 2.5-1000 ng/ml with a weighting on 1/x2. Typical standard curves are/=0.0118, C',+0.0111 for KA;/=0.033, C/+0.016 for KD; and/=0.0089, [0063] The LLOQ is defined as the lowest concentration on the calibration curve for which an acceptable accuracy of ±15% and a precision below 15% are obtained. The present LC-MS/MS method offered an LLOQ of 2.5 ng/ml for KA, KD and KF in 0.1 ml plasma sample. This is sensitive enough to investigate the pharmacokinetic behaviors of KA, KD and KF, to establish the relationship between dose and pharmacological effect in humans. [0064] 2.3.3 Precision and accuracy [0065] Table 3 summarizes the intra-and inter-day precision and accuracy for KA » KD and KF evaluated by assaying the QC samples. The precision is calculated by using oneway ANOVA. In this assay, the intra-run precision is 12.4 % or less, and the inter-run precision is 11.5 [0067] To evaluate the absolute matrix effect (ME), i.e., the potential ion suppression or enhancement due to co-eluting matrix components, five different batches of blank plasma are extracted and then spiked with the analyte at three QC concentrations. The corresponding peak areas of the analyte in spiked plasma post-extraction (A) are then compared to those of the aqueous standards in mobile phase (B) at equivalent concentrations. The ratio (A/Bχl00) is defined as the ME. A ME value of 100 % indicates that the responses for KA, KD and KF in the solution and in the plasma extracts are the same and that no absolute ME is observed. A value of >100% indicates ionization enhancement and a value of<100 % indicates ionization suppression. The assessment of the relative ME is made by a direct comparison of the analyte peak area values between different lots (sources) of plasma. The variability in the values, expressed as RSD [0068] The results show that there is no absolute ME in this study. The variability is acceptable, with RSD values <13.8 % at different concentrations of KA > KD, KF and internal standard indicating that the relative ME for the analytes are minimal. [0069] 3.1.5 Recovery and stability [0070] The recovery of KA, KD and KF, determined at three concentrations (10, 100, 500 ng/ml), are 89.32±6.3 %, 97.33±3.5 %, 97.12±5.9 % for KA, 98.50±5.8 %, 94.89±4.3 %, 92.83±3.4 % for KD and 84.61±11.57 %, 82.59±4.6 %, 102.5±5.9 % (n=5) for KF, respectively. [0071] The stabilities of QC samples at three different concentrations (10, 100, 500 ng/ml) in the whole blood prepared according to the above-mentioned method are tested by short-term stability (room temperature) assays. KA, KD and KF are stable for 8 hours at different concentrations (10, 100, 500 ng/ml) ranging from 93.55 % to 107.0 %, from 85.19 % to 113.6 % and from 87.45 [0072] In conclusion, the proposed method of analysis provides a sensitive and specific assay for determination of KA, KD and KF in human plasma. The simple protein precipitation procedure and short HPLC-MS/MS run time can allow a large number of samples to be analyzed. This method is shown to be suitable for analysis of KA, KD and KF in human plasma samples collected for pharmacokinetic, bioavailability or bioequivalence studies in humans and mice. The results prove that the method is rapid, sensitive and highly selective. Not limited to KA, KD and KF, this analytical method may also be useful for determining quantitatively other active ingredients in remained in human/animal body such as the presently claimed total flavonoids isolated and purified from blueberry leaves. [0073] The foregoing descriptions of the present invention have been provided for the purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent to the practitioner skilled in the art. [0074] The embodiments are chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention for various embodiments and with various modifications that are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the following claims and their equivalence. [0075] REFERENCES [1] Chuanhe TANG & Zhiying PENG, “Survey On The Physiological Functions Of Soybean Isoflavone” 17. 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Seong, [17] J.L. Song, Y. Qian, G.J. Li, X. Zhao, [18] S. Fan, Y. Zhang, N. Hu, Q. Sun, X. Ding, G. Li, B. Zheng, M. Gu, F. Huang, Y.Q. Sun, Z. Zhou, X. Lu, C. Huang, G. Ji, [19] M.A. Ouyang, C.R. Yang, Z.L. Chen, H.Q. Wang, [20] W.J. Zuo, H.F. Dai, J. Chen, H.Q. Chen, Y.X. Zhao, W.L. Mei, X. Li, J.H. Wang, Field Code •' Field Code L. Li, L.J. Xu, G.Z. Ma, Y.M. Dong, Y. Peng, P.G. Xiao, J Nat Med, 67 (2013) 425-437. [22] W.J. Zuo, H.F. Dai, Y.B. Zeng, H. Wang, H.Q. Chen, J.H. Wang, J Asian Nat Prod Res, 14(2012)308-313. [23] J. Zheng, L. Tang, X.D. Xian, S.X. Zhou, H.M. Shi, Y. Jiang, Y.Q. Gu, G. Liu, P.F. Tu, Planta Med, 75 (2009) 1410-1414. [24] L. Li, Y. Peng, G. Ma, C. He, Y. Feng, Q. Lei, P. Xiao, Phytochem Anal, 23 (2012) 677-683. [25] J.Q. ZHANG, Y.F. TAN, H.L. LI, M.S. LIU, C.L. FAN, W.C. YE, Chinese Pharmaceutical Journal, 9 (2010) 007. [26] M.A. Ouyang, H.Q. Wang, Z.L. Chen, C.R. Yang, Phytochemistry, 43 (1996) 443-445. [27] M.A. Ouyang, C.R. Yang, Z.J. Wu, J Asian Nat Prod Res, 3 (2001) 31-42. [28] Shannon Reagan-Shaw et al. “Dose Translation From Animal To Human Studies Revisited”, The FASEB Journal, 2007, 22, 659-661. The present invention relates to a composition for preventing and treating non-alcoholic fatty liver disease (NAFLD), and particularly relates to a composition comprising blueberry leaf flavonoids for preventing and treating NAFLD. The present invention also relates to methods for preparing the composition and using the composition in preventing and treating NAFLD. 100 102 104 HLFI, HLF2, HLF3 Normal, Disease Docket No.: UM1137AU00 1. A composition for preventing and treating non-alcoholic fatty liver disease comprising an effective amount of total flavonoids isolated from blueberry leaves. 2. The composition of claim 1, wherein said effective amount of total flavonoids is from 20 mg/kg to 80 mg/kg to said subject when said subject is a small animal, or is from 3 mg/kg to 15mg/kg to said subject when said subject is human. 3. A method for isolating the total flavonoids of claim 1 from blueberry leaves, said method comprising: (a) using petroleum ether in a weight-to-volume ratio of 1:20 to remove fatty acids from dried blueberry leaves under a first ultrasonic extraction for about 20 minutes; (b) after removing the fatty acids in (a), a first suction filtration is carried out and the remaining solid is collected for extraction with 50-70% ethanol in a volume ratio of 1:15-20 at a temperature from 30 to 70°C; the extraction is repeated for three times; the extraction fluids are then combined; (c) concentrating the combined fluids in (b) into 1/5 to % of the total volume thereof, followed by adding corresponding volume of 75-95% ethanol until the volume ratio of the ethanol in (b) is reached, then subject to a second ultrasonic extraction, followed by a second suction filtration, and then removing the remaining solid and collecting the filtrate, the filtrate being further concentrated into about 100 mL or maintained at about 100 mL, in order to obtain a crude extract of total flavonoids; (d) concentrating the crude extract of total flavonoids obtained in (c) by rotary evaporation and collecting the concentrated solution, followed by vacuum drying at about 70°C to obtain the remaining solid, grinding the remaining solid into powder form, in order to become the total flavonoids; (e) using 95% ethanol for ethanol precipitation of the total flavonoids obtained from (d), followed by using deionized water to prepare an aqueous solution with the evaporated solution as an upper column solution for macroporous resin separation, adsorbing the total flavonoids by the macroporous resin during the macroporous resin separation; and (f) using deionized water to wash away impurities from the resin column until the flow-through is not turbid, followed by using 95% ethanol to elute the adsorbed total flavonoids from the resin column, collecting the eluent, in order to obtain purified total flavonoids from the blueberry leaves. 4. A method for preventing and treating non-alcoholic fatty liver disease comprising administering the composition of claim 1 to a subject in needs thereof. 5. The method of claim 4, wherein said non-alcoholic fatty liver disease comprises simple fatty liver (SFL), nonalcoholic steatohepatitis (NASH), fatty liver fibrosis and liver cirrhosis.COMPOSITION FOR PREVENTING AND TREATING NON-ALCOHOLIC FATTY LIVER DISEASE
*Wei. Zhang, and Qingfeng Zhang contributed equally to this patent.
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FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
SUMMARY OF THE INVENTION
BRIEF DESCRIPTION OF THE DRAWINGS
DETAILED DESCRIPTION OF THE INVENTION
[0021] EXAMPLE 1 - Method for Preparing Purified Total Flavonoids from Blueberry Leaves and
Group No. of Samples Body Weight (g) Liver Net Weight (g) Liver Index (%) Normal 12 336.87±11.81 9.50±0.82 2.82i0.40 Disease 12 356.00i25.83** 13.49il.23*’ 3.79i0.18** HLF1 12 344.68±37.5 9.72il.4θ“ 2.82i0.20*‘ HLF2 12 294.24±36.69 8.18il.94" 2.78i0.30** HLF3 12 278.54±40.65 7.66il.48** 2.75i0.30** Group No. of Samples TC TG HDL-C Markers LDL-C ALT AST Normal 12 1.40i0.20 0.42i0.06 1.82i0.23 0.36i0.19 49.00i7.04 119.40il8.12 Disease 12 1.93±0.66 1.77±1.88 0.68±0.12 2.86±0.36 64,60±8.47 177.40±29.44 HLF1 12 1.83±0.32“ 1.56±1.21* 0.72±0.12** 1.62±0.21* * 61.40±7.15 170.20±24.5" HLF2 12 1.66±0.48” 1.32±0.87‘* 1.01±0.14** 1.21±0.32” 58.18±6.75 154.20±21.45“ HLF3 12 1.51±0.37** 0.92±0.15*‘ 1.65±0.1l“ 0.63±0.14‘* 52.26±7.23 128.04±20.34** [0035] EXAMPLE 3 - Quantification Of Kudinosides Using A Fligh Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometric Method
Table 3: Accuracy and precision for the analysis of KA, KD and KF in human plasma (in pre-study validation,
10.0 10.27 8.1 10.1 2.6 KA 100.0 95.76 4.3 6.8 -4.2 500.0 507.6 2.6 6.4 1.5 10.0 9.88 7.8 9.7 -1.2 Table 4: Recovery and Matrix effect data for KA, KD and KF in five different lots of human plasma (n=5)
RSD (%) 10.0 93.99 13.0 89.32 6.3 KA 100.0 98.10 6.3 97.33 3.5 500.0 95.91 7.4 97.12 5.9 10.0 98.86 9.8 98.58 5.8 KD 100.0 102.9 9.0 94.89 4.3