AU2015101749A4 - Method of treating or preventing liver injury induced by acetaminophen - Google Patents
Method of treating or preventing liver injury induced by acetaminophen Download PDFInfo
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- AU2015101749A4 AU2015101749A4 AU2015101749A AU2015101749A AU2015101749A4 AU 2015101749 A4 AU2015101749 A4 AU 2015101749A4 AU 2015101749 A AU2015101749 A AU 2015101749A AU 2015101749 A AU2015101749 A AU 2015101749A AU 2015101749 A4 AU2015101749 A4 AU 2015101749A4
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- acetaminophen
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Landscapes
- Medicines Containing Plant Substances (AREA)
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Abstract
The present invention provides a method of treating or preventing a disease in a subject, which disease is a liver injury induced by acetaminophen, comprising the step 5 of: administering an effective amount of a composition comprising at least 15% by weight of flavonoids obtained from leaves of at least one species of Vaccinium (V.) based on the weight of the composition to the subject. Fig. 1A Fig. 1B Lig. 1 &4 Fig 1 D Fig. IC Fig. ID
Description
METHOD OF TREATING OR PREVENTING LIVER INJURY INDUCED BY ACETAMINOPHEN 5 TECHNICAL FIELD The present invention relates to a method of treating or preventing a disease in a subject, which disease is a liver injury induced by acetaminophen. The present invention provides a flavonoid-rich composition for use in such method. 10 BACKGROUND Acetaminophen, also known as paracetamol, is an active analgesic and antipyretic 15 substance frequently used for treating pain, fever or general symptoms associated with a cold in adults as well as in children. Various medicinal products contain acetaminophen as their active ingredient or as one of the active ingredients. A lot of these medicinal products are available without a prescription. 20 However, use of said active ingredient has been reported to be associated with severe and even fatal liver injuries. Said liver injuries are caused by the formation of a hepatotoxic metabolite due to the CYP P-450 meditated oxidative metabolism of acetaminophen. Said highly reactive metabolite known as NAPQI (N-acetyl-p benzoquinone iminine), in particular when excessively formed, can bind to 25 intracellular proteins leading to the liver injury. Excessive production can result from an overdose or a frequent use of an acetaminophen-containing medicinal product. Moreover, chronic consumption of alcohol can induce CYP P-45o activity and, thus, increase the rate of the formation of said metabolite even with therapeutic doses of acetaminophen. 30 Considering that overdose is frequent and may happen intentionally or non intentionally, the increasing number of medicinal products containing acetaminophen as well as the wide use of said active ingredient, there is a high and increasing number of patients suffering from a liver injury caused by this active ingredient. 35 Treatment options for treating acetaminophen-induced liver injury are limited and the injury can be fatal without further treatment. Hence, there is still a strong need for further treatment options or at least supportive treatment options, in particular those without further detrimental effects on the liver and/or a generally reduced risk for 40 severe side effects. Generally, plant materials as well as plants or respective components gained from plants are known to allow for treatment of various diseases and conditions while bearing a reduced risk for side effects even after frequent or long-term use. In view of 45 the rich medicinal plant resources available respective medicines can, moreover, usually be produced in a cost-effective way. Accordingly, treatment with plant materials or components derived therefrom is usually advantageous and having such treatment option or at least a supportive 50 treatment option for preventing and/or treating acetaminophen-induced liver injuries would, thus, be highly desirable. 1 SUMMARY OF THE INVENTION The present invention refers in an aspect to a method of treating or preventing a disease in a subject, which disease is a liver injury induced by acetaminophen, 5 comprising the step of: administering an effective amount of a composition comprising at least 15% by weight of flavonoids obtained from leaves of at least one species of Vaccinium (V.) based on the weight of the composition to the subject. The method according to the invention unexpectedly allows for an improvement of 10 acetaminophen-induced liver injury, in particular a significant decrease of liver markers such as ALT, AKT, MDA, and GSH-Px. The administration of the composition also advantageously increases GSH and CAT levels. Hence, the composition of the present invention is especially suitable for treating acetaminophen-induced liver injury. 15 BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A-iD are microphotographs showing histology of H&E dyes stained liver 20 tissues obtained from different treatments. Fig. 1A shows the liver tissue of mice being treated with 500mg/kg acetaminophen orally (i.e. disease model group). Fig. iB shows the liver tissue of mice being treated with 200mg/kg saline (i.e. normal control group). Fig. 1C shows the liver tissue of mice being treated with 500mg/kg acetaminophen orally and subsequently 200mg/kg flavonoid-rich composition 25 obtained from blueberry leaves (i.e. treatment group). Fig. 1D shows the liver tissue of mice being treated with 500mg/kg acetaminophen orally and subsequently 200mg/kg bifendate (i.e. positive control group). 30 DETAILED DESCRIPTION OF THE INVENTION Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs. 35 The present invention refers in a first aspect to a method of treating or preventing a disease in a subject, which disease is a liver injury induced by acetaminophen, comprising the step of: administering an effective amount of a composition comprising at least 15% by weight of flavonoids obtained from leaves of at least one species of Vaccinium (V.) based on the weight of the composition to said subject. In 40 embodiments, the method is for treating said liver injury. In other embodiments, the method is for preventing said liver injury. The composition can be a solid, semi-solid or liquid. The composition can be for example a tablet, a capsule, a balm, a cream, a powder, granules, a solution or a 45 dispersion, an ointment, a patch, a spray solution or a paste. The composition optionally comprises at least one excipient. The skilled person knows excipients and is able to select the suitable ones. In particular, the at least one excipient is selected from a solid or liquid carrier, more preferably, from ethanol, water or a mixture thereof. Most preferably, the excipient is 95% ethanol. Most preferably, the composition is a 50 pharmaceutical composition for treating a human subject with pharmaceutically acceptable excipients. 2 The composition can include further compounds which may contribute to a further improvement of the liver injury or prevention of the liver injury. Such compounds include for example phenolic acids. The composition may also include other therapeutic compounds, preferably therapeutic compounds which are used for 5 treating liver diseases, in particular for treating acetaminophen-induced liver injury, such as N-acetylcysteine (NAC) and bifendate. In other preferred embodiments, the composition is administered together with at least one other therapeutic compound such as NAC and bifendate, wherein the at least 10 one further therapeutic compound is administered before, along with or after the composition of the present invention. The term "liver injury" generally refers to a form of trauma sustained to the liver caused by external factors e.g. a foreign object hitting the liver, excessive intake of 15 alcohol or an intake of a drug. In the present invention, the liver injury is induced by acetaminophen. Preferably, the liver injury induced by acetaminophen is an acute liver injury with a sudden and usually fast proceeding onset of symptoms. In particular, the liver injury induced by acetaminophen results from at least one of an overdose of acetaminophen, a long-term intake of acetaminophen, a combined use of 20 alcohol and acetaminophen, and an intake of acetaminophen with a pre-existent liver disease. In particular embodiments of the present invention, the liver injury induced acetaminophen is an early stage liver injury. The early stage liver injury refers to a liver injury in which only a part of the liver is affected, and/or the symptoms are mild. 25 The subject suffering from the liver injury induced by acetaminophen has an increase of aminotransferase levels, in particular an increase of aminotransferase level of at least 2-fold, at least 5-fold, preferably at least 1o-fold compared with normal levels in healthy subjects. In particular, the aminotransferases serve as a liver marker and include at least one of alanine aminotransferase (ALT), aspartate aminotransferase 30 (AST), and alkaline phosphatase (ALP). Elevated levels of said liver markers indicate that there is an inflammation or necrosis in the liver. Preferably, the subject also has an increase of the levels of at least one of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase 35 (GSH-Px) in serum, preferably at least 2-fold compared to healthy subjects. Preferably, the serum level of at least one of catalase (CAT) and glutathione (GSH) in the subject is reduced by preferably at least 2-fold compared to healthy subjects. The composition comprises at least 15% by weight of flavonoids, as referenced as 40 "flavonoid-rich composition" herein, preferably at least 20% by weight of flavonoids, still more preferably at least 22 % by weight of flavonoids, and most preferably over 22% by weight of flavonoids. The term "flavonoids" refers to a group of compounds, present in various plants, 45 based on or derived from the following basic structure: 3 Preferably, the flavonoids include at least one of flavonols or derivatives thereof, or anthocyanins or derivatives thereof. Flavonols generally are based on or derived from the following structure: 5 Anthocyanins generally are based on or derived from the following structure: 10 "Derivatives" of the flavonols and anthocyanines include flavonols and anthocyanines having structural modification through for example hydroxylation, alkylation such as methylation, esterification such as acetylation, glycosylation such as glucosylation, glucuronidation, hydrogenation, or dehydrogenation. "Hydroxylation" refers to the presence of at least one additional OH-group. "Alkylation" refers to the presence of at 15 least one straight chain or branched Ci to C 3 alkyl-group, i.e. an alkyl group having 1to 3 carbon atoms, in particular of at least one methyl group, preferably attached to oxygen atoms in OH-groups, thus forming alkoxy- such as methoxy-substituents. "Esterification" refers to the presence of at least one alkylester, i.e. a carboxylic acid has been attached to an OH-group via an ester linkage. More specifically, esterification 20 refers to the presence of an alkanoyl-group attached to an OH-group forming an alkanoyloxy-group. "Alkanoyl-group" is a carbonyl group bonded to an alkyl, which alkyl can be saturated or unsaturated, i.e. can contain at least one double or triple bond and usually has not more than 12 carbon atoms. Preferably, esterification refers to "acetylation", i.e. the presence of at least one acetyl-group attached to oxygen atoms 25 in OH-groups, thus, forming acetoxy-groups. "Glycosylation" means presence of at least one carbohydrate-moiety in particular glucose-moiety (glucosylation) attached to an OH-group. Glucuronidation or glucuronosylation is the addition of at least one glucuronic acid-moiety to an OH-group. Hydrogenation refers to the presence of additional pairs of hydrogen atoms such as one additional pair of hydrogen atoms. 30 Dehydrogenation refers to the presence of an additional double bond. Preferably, the flavonols are selected from the group consisting of quercetin, syringetin, rhamnetin, kaempferol, rutin, and a combination thereof. The flavanol derivatives preferably include at least one of quercetin arabinoside and syringetin-3 35 galactoside. Preferably, the anthocyanins are selected from the group consisting of aurantinidin, delphinidin, cyanidin, europinidin, luteolinidin, pelargonidin, petunidin, peonidin, malvidin, rosinidin, and a combination thereof. The anthocyanin derivatives include 40 at least one of delphinidin 3-galactoside, delphinidin 3-glucoside, delphinidin 3 arabinoside, cyanidin 3-glucoside, petunidin 3-galactoside, peonidin 3-galactoside, malvidin 3-galactoside, malvidin 3-glucoside, malvidin 3-arabinoside, delphinidin 6 acetyl 3-glucoside, and malvidin 6-acetyl 3-glucoside. 4 The expression "effective amount" generally denotes an amount sufficient to produce therapeutically desirable results, wherein the exact nature of the result varies depending on the specific disorder which is treated. When the disorder is liver injury, 5 in particular an acetaminophen-induced liver injury, the result is usually a decrease of at least one liver marker selected from ALT, AST, ALP, SOD, MDA and GSH-PX. Preferably, at least one of said liver markers is reduced 2-fold, preferably is reduced 5 fold compared to untreated control. Preferably, when the disorder is an acetaminophen-induced liver injury, the result is usually an increase of at least one 10 liver marker selected from GSH and CAT. Preferably, at least one of GSH and CAT is increased 2-fold, preferably is increased 5-fold. The subject is preferably a mammal, more preferably a rodent or human, most preferably a human. 15 The effective amount of the composition is such that from about 100mg/kg to about 250mg/kg of the flavonoids are administered to the subject and wherein the subject is a rodent. Preferably, the effective amount of the composition is such that from about 200mg/kg of the flavonoids are administered to the subject and wherein the subject is 20 a rodent. In embodiments of the present invention, the effective amount of the composition is such that from about 5mg/kg to about 30mg/kg of the flavonoids are administered to the subject and wherein the subject is a human. 25 The at least one species of V. is preferably selected from the group comprising V. alaskaense, V. angustifolium, V. boreale, V. caesariense, V. corymbosum, V. constablaei, V. consanguineum, V. darrowii, V. elliottii, V.formosum, V.fuscatum, V. hirsutum, V. myrsinites, V. myrtilloides, V. operium, V. pallidum, V. simulatum, V. 30 tenellum, and V. virgatum. In preferred embodiments, the composition is obtained by steps comprising: (a) subjecting a mixture containing dried leaves of the species of V. and a first solvent to a first ultrasonic extraction; 35 (b) filtrating the mixture to obtain a first filtrate and a first solid residue; (c) adding a first aliphatic alcohol having a first concentration to the first solid residue and performing an extraction at a temperature from 30 to 70oC to obtain a first extracted solution; (d) concentrating the first extracted solution in order to reduce the volume to 40 lower than or at most 50% of the initial volume of the first extracted solution to obtain a concentrated first extracted solution; (e) adding a second aliphatic alcohol having a second concentration to the concentrated first extracted solution and performing a second ultrasonic extraction to obtain a second extracted solution; 45 (f) filtrating the second extracted solution to obtain a second filtrate and optionally a second solid residue; (g) drying the second filtrate to obtain a third solid residue; and (h) purifying the third solid residue, and optionally adding at least one excipient or optionally adding a further therapeutic compound to obtain the 50 composition; wherein the second concentration of the second aliphatic alcohol is higher than the first concentration of the first aliphatic alcohol. 5 The first solvent of step (a) is an organic solvent for example a solvent comprising aliphatic hydrocarbons. Preferably, the solvent is petroleum ether. The skilled person is aware of how to conduct an ultrasonic extraction in steps (a) and 5 (e) depending on desired products, raw materials, solvent used as well as side products produced during extraction. Similarly, the skilled person is aware of any common suitable method for conducting filtration in steps (b) and (f), in particular suction filtration can be used. 10 The first and second aliphatic alcohols of steps (c) and (e) can be independently selected from Ci-C6 alcohols, i.e. an alcohol of an alkane having 1 to 6 carbon atoms, for example, methanol, ethanol, propanol, butanol or the like. Most preferable, the first and second aliphatic alcohols are both ethanol including ethanol-water mixtures. 15 The first aliphatic alcohol preferably has a first concentration of at most 70% (v/v) and the second aliphatic alcohol preferably has a second concentration of at least 75% (v/v). More preferably, the first aliphatic alcohol has a first concentration of about 50 70% (v/v) and the second aliphatic alcohol preferably has a second concentration of about 75-95% (v/v). The first concentration and second concentration refer to 20 concentrations of the aliphatic alcohol in another solvent in particular water. In step (d), the first extracted solution is preferably concentrated such that the volume of it is reduced to 20% to 25% of the initial volume of the first extracted solution. In particular embodiments of the present invention, the second aliphatic alcohol is 25 ethanol, and is preferably added to the concentrated first extracted solution to obtain a solution having a concentration of 50-70% of ethanol. Preferably, the drying process in step (g) includes any common method for removing solvents such as at least one of the first solvent, the first and second aliphatic alcohols 30 which may be present in the third filtrate in the present invention. In particular, the method includes at least one of rotary evaporation and vacuum drying. The skilled person is aware of any common method to purify the third residue. In particular, ethanol precipitation, column chromatography in particular macroporous 35 resin column chromatography. The at least one macroporous resin can be any suitable resin such as AB-8. The skilled person is able to select an appropriate type of resin for the purification. The examples set out below further illustrate the invention. The preferred 40 embodiments described above and the drawing as well as examples given below represent preferred or exemplary embodiments and a skilled person will understand that the reference to those embodiments or examples is not intended to be limiting. 45 EXAMPLE EXAMPLE 1 Preparation of a flavonoid-rich composition from blueberry leaves 50 In this example, around 5.og of dried leaves of species of V. were mixed with petroleum ether as a first solvent in a weight-to-volume ratio of 1:20 in order to remove fatty acids from the dried leaves under ultrasonic extraction for 20 minutes followed by a first suction filtration. 6 The remaining solid residue was collected and then subjected to ethanol extraction with 50-70% ethanol at a volume ratio of 1:15-25. The extraction was carried out for at least three times at a temperature of 30-70 C. The resultant fluids were collected from 5 several extractions and combined. The collected fluids were then concentrated to 1/5 to 1/4 of the initial volume of the collected fluids. Corresponding volume of 75%-95% ethanol was then added into the concentrated fluid until the volume ratio of 50-70% ethanol was reached. A second ultrasonic 10 extraction was carried out on that mixture followed by a second suction filtration. The remaining solid residue was removed and the filtrate was concentrated to a volume of about 100 mL. The concentrated filtrate was subjected to rotary evaporation using a rotary 15 evaporator (R2oGB, Shanghai Xiafeng Co. Ltd.) to obtain a higher degree of concentration followed by vacuum drying at 70oC to obtain a solid residue. The solid residue was grinded to powder. The powder was further purified by using 95% ethanol for ethanol precipitation. Deionized water was used to mix with the evaporated solution obtained from the ethanol precipitation in order to prepare an aqueous 20 solution as an upper column solution for later macroporous resin separation. Flavonoids in the aqueous solution were adsorbed on the resin column during the AB 8 macroporous resin separation. The resin column was then washed with deionized water to remove the impurities until the eluent was clear. Lastly, a fresh 95% ethanol was used to remove the adsorbed flavonoids from the resins and the eluent was 25 collected. EXAMPLE 2 In vivo studies on the effects of the flavonoid-rich composition on the acetaminophen induced liver injury 30 Animal tests were conducted to evaluate the efficacy of the flavonoid-rich composition on treating or preventing the acetaminophen-induced acute liver injury. A total of 40 specific-pathogen-free (SPF) Kun Ming (KM) mice, with a weight of 35 20±2g, were obtained from Guangdong Medical Laboratory Animal Center (license number: SCXK (Guangdong) 2008-0002). The mice were divided randomly into four groups, i.e. n=lo in each group, after feeding them normally for 1 week at ambient conditions (22-26 0 C; 50-70% humidity). The mice were normally fed with SPF-grade feed. 40 SPSS software was used in statistical analysis to ensure no statistical discrepancy exists among the groups before treatment. In particular, SPSS12.0 was used to perform one-way ANOVA for statistical analysis. The experimental results are presented with mean values. P<o.05 is statistically significant. 45 Instruments used in the examples described herein include Multiskan GO Microplate Spectrophotometer (Thermo Scientific, US); a constant temperature water bath (Beijing Changyuan Experiment Equipment Factory); TC-12o Intelligent Program Controlled Automatic Tissue Processor, TB-718E Automatic Tissue Embedding Centre 50 (Hubei Taiva Medical Technology Co., Ltd); ILRM2235 tissue processor (Leica, Germany); GZX-9070MBE drying oven (Shanghai Boxun Industry & Commerce Co. Ltd Medical Equipment Factory); Multifuge XIR refrigerated centrifuge (Thermo Scientific, US); JA1003N digital electronic balance; XW-8oA vortex mixer (Shanghai 7 Jingke Industrial Co. Ltd.); Scientz-1oN lyophilizer (Ningbo Scientz Biotechnology Co., Ltd); and Olympus BX43 Biological Microscopes (Olymps, Japan). 5 EXAMPLE 2A Treatments on mice and sample collections 40 SPF mice were divided evenly and randomly into four groups, namely a disease model group, a treatment group, a positive control group and a normal control group. 10 The mice in the disease model group were treated with 500mg/kg of acetaminophen orally to induce an acute liver injury. The mice in the treatment group were treated with 500mg/kg of acetaminophen orally first and, after 12 hours, treated with the composition prepared according to the present invention such that 200mg/kg of flavonoids are orally administered. The mice in the positive control group were treated 15 with 500mg/kg of acetaminophen orally first and, after 12 hours, treated with 200mg/kg of bifendate orally, wherein the bifendate was purchased from Wuhanxin Jialing Biological Technology Co. Ltd. The mice in the normal control group were treated with 200mg/kg of saline orally once per day for 8 consecutive days. 20 All the mice were fasted for 12 hours before blood sample collection. The blood samples were collected at peri-orbital sinus of the mice. The collected blood samples were divided in two groups. The first group of blood samples was tested for liver markers' levels in the blood plasma. The second group of blood samples was centrifuged at 2,500 rpm for 15 minutes to isolate blood serum from each of the blood 25 samples. The blood serum of each sample was stored at 4 0 C until use. At the end of the test, all the mice were scarified by cervical vertebra dislocation and dissected to collect the liver samples. The liver samples were rinsed with saline and fixed in 10% formaldehyde solution for 20 hours. The fixed liver samples were then 30 dehydrated using an automatic dehydrating machine and embedded in paraffin wax for sectioning. Subsequently, the sectioned liver tissues were de-waxed and stained with haematoxyline and eosin dyes (H&E dyes) for histological observations. EXAMPLE 2B 35 Effect of the flavonoid-rich composition on ALK, AST and ALP levels in blood plasma The first group of blood samples was tested for the level of liver markers of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood plasma. AST and ALT are intracellular enzymes that distributed in liver with the greatest amount. 40 These two enzymes are used as liver markers to identify liver injury as their levels may be increased by liver inflammation or necrosis. The liver damage or injury may be resulted from different types of conditions. The mean values of ALT and AST levels of the four testing groups are set forth in Table 45 1. In view of the data, the AST and ALT levels in the disease model are significantly increased (P<o.oi), which indicating that the disease model was well established. The P-values of the ALT and AST levels in both treatment group and positive control group are larger than o.05 when compared against the respective levels in normal group, which indicating that there is no significant different in ALT and AST levels between 50 the normal control group, the treatment group and the positive control group. In other words, both of the flavonoid-rich composition used in the treatment group and the bifendate used in the positive control group exhibited protective effects on the livers. 8 When comparing the ALT and AST levels in the disease model group with the treatment group and the positive control group, the P values are smaller than 0.05. That is there is a significant difference between the disease model group and other three groups. 5 Table 1: ALT and AST levels in the blood plasma of mice (mean values, n=1o; * compared with the disease model group, P<o.05; ** compared with the disease model group, P<o.o1) Group ALT(U/L) AST(U/L) Normal control (N) 53-50* 159-00* Disease model (M) 1269.oo** 1383-00** Treatment (T) 71.00* 346.67* Positive control (C) 52.00* 254.33* 10 EXAMPLE 2C Effect of the flavonoid-rich composition on MDA, CAT, GSH, SOD and GSH-Px levels in blood serum The second group of blood samples was tested for the liver markers' levels in the blood 15 serum. In particular, levels of malondialdehyde (MDA), catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the blood serum were measured. Testing kits for MDA, CAT, GSH, SOD and GSH-Px were purchased from Nanjing Jiancheng Bioengineering Institute. 20 With reference to the results in Table 2, MDA and GSH-Px levels in the disease model group increase significantly (P<o.o1) while GSH and CAT expression levels decrease (P<o.o1). The SOD level in the disease model group does not show obvious changes (P>o.05). These results again prove that the disease model group was well established. 25 The MDA and GSH-Px levels in the treatment group and the positive control group are significantly reduced (P<o.o1) while GSH and CAT expression levels increase significantly (P<o.o1). The SOD level in these two groups do not show obvious changes (P>o.05). Also, the obtained markers results of the treatment group are similar to that of the positive control group. Therefore, the results prove that the 30 flavonoid-rich composition of the present invention is capable of protecting the liver as it lowers the MDA and GSH-Px levels while increasing the GSH and CAT levels. Table 2: MDA, GSH-Px, GSH, SOD and CAT levels in the blood serum of mice (mean values, n=1o; * compared with the disease model group, P<o.05; ** compared with 35 the disease model group, P<o.o1) Group MDA GSH-Px GSH SOD CAT (nmol/L) (pmol/L) (pmol/L) (U/ml) (U/ml) Normal control (N) 16.67 31.82 458.15 0.92 8.47 Disease model (M) 207.55** 50000** 197.37** 0.91 2.57** Treatment (T) 40.76** 206.67** 1350.80** 0.79 10.03** Positive control (C) 23.03** 60.00** 900-53 ** o.84 11.25** EXAMPLE 2D 9 Histologic studies on liver tissues The sectioned liver tissues were stained with H&E dyes for histological observations under microscope. Figs. 1A to 1D show the microscopic photos of the liver tissues of 5 four respective groups. With reference to Fig. 1A, the stained liver tissue of the disease model group reveals the presence of air bubbles and steatosis. The liver cells show significant cell swelling, self-destruction, and necrosis. There are inflammatory cells infiltrations at the periportal zones. The structures of the lobes are not clear. These results reflect that the disease model group was well established. 10 With reference to Fig. 1B, the normal control group shows liver tissue with liver cells having clear cell structure, absence of cell swelling, no steatosis and no necrosis. There are also no inflammatory cells infiltrations in the lobes and at the periportal zones. Regarding the treatment group, Fig. 1C shows a small part of the liver tissue with self 15 destruction, some cells swell. The level of necrosis is less severe than that in the disease model group. There is only a minor inflammatory cells infiltration. The structure of the lobe is relatively clear in the treatment group. Accordingly, the flavonoid-rich composition of the present invention exerts liver protection against liver injury, in particular the liver injury induced by acetaminophen. 20 Bifendate has a corresponding therapeutic effect against the liver injury. As shown in Fig. 1D, the liver tissue of the positive control group shows little cell self-destruction while only few cells have minor cell swelling. The cell necrosis is not significant in the positive control group and the inflammatory cells infiltration is not significant as well. 25 Moreover, the structures of the lobes are clear in the positive control group. 10
Claims (16)
1. A method of treating or preventing a disease in a subject, which disease is a liver injury induced by acetaminophen, comprising the step of: 5 administering an effective amount of a composition comprising at least 15% by weight of flavonoids obtained from leaves of at least one species of Vaccinium (V.) based on the weight of the composition to the subject.
2. The method according to claim 1, wherein the flavonoids include at least one of 10 flavonols or derivatives thereof, or anthocyanins or derivatives thereof.
3. The method according to claim 2, wherein the flavonols are selected from the group consisting of quercetin, syringetin, rhamnetin, kaempferol, rutin, and a combination thereof. 15
4. The method according to claim 2, wherein the anthocyanins are selected from the group consisting of aurantinidin, delphinidin, cyanidin, europinidin, luteolinidin, pelargonidin, petunidin, peonidin, malvidin, rosinidin, and a combination thereof. 20
5. The method according to claim 1, wherein the species is selected from the group comprising V. alaskaense, V. angustifolium, V. boreale, V. caesariense, V. corymbosum, V. constablaei, V. consanguineum, V. darrowii, V. elliottii, V. formosum, V. fuscatum, V. hirsutum, V. myrsinites, V. myrtilloides, V. operium, V. pallidum, V. simulatum, V. tenellum, and V. virgatum. 25
6. The method according to claim 1, wherein the subject is a mammal.
7. The method according to claim 1, wherein the effective amount of the composition is such that from about 100mg/kg to about 250mg/kg of the flavonoids 30 are administered to the subject and wherein the subject is a rodent.
8. The method according to claim 1, wherein the effective amount of the composition is such that from about 5mg/kg to about 30mg/kg of the flavonoids are administered to the subject and wherein the subject is a human. 35
9. The method according to claim 1, wherein the composition comprises at least 20% by weight of flavonoids based on the weight of the composition.
10. The method according to claim 1, wherein the composition further comprises 40 at least one excipient, wherein the excipient is ethanol, water or a mixture thereof.
11. The method according to claim 1 or claim 10, wherein the composition is obtained by steps comprising: (a) subjecting a mixture containing dried leaves of the species of V. and a first 45 solvent to a first ultrasonic extraction; (b) filtrating the mixture to obtain a first filtrate and a first solid residue; (c) adding a first aliphatic alcohol having a first concentration to the first solid residue and performing an extraction at a temperature from 30 to 70oC to obtain a first extracted solution; 50 (d) concentrating the first extracted solution in order to reduce the volume to lower than or at most 50% of the initial volume of the first extracted solution to obtain a concentrated first extracted solution; 11 (e) adding a second aliphatic alcohol having a second concentration to the concentrated first extracted solution and performing a second ultrasonic extraction to obtain a second extracted solution; (f) filtrating the second extracted solution to obtain a second filtrate and 5 optionally a second solid residue; (g) drying the second filtrate to obtain a third solid residue; and (h) purifying the third solid residue, and optionally adding at least one excipient to obtain the composition; wherein the second concentration of the second aliphatic alcohol is higher than 10 the first concentration of the first aliphatic alcohol.
12. The method according to claim 11, wherein the purifying step (h) is performed by using at least one macroporous resin. 15
13. The method according to claim 1, wherein the liver injury induced by acetaminophen is an acute liver injury.
14. The method according to claim 1, wherein the liver injury induced by acetaminophen results from at least one of an overdose of acetaminophen, a long-term 20 intake of acetaminophen, a combined use of alcohol and acetaminophen, and an intake of acetaminophen with a pre-existent liver disease.
15. The method according to claim 1, wherein the subject has an increase of at least one liver marker selected from ALT, AST, ALP, MDA and GSH-Px. 25
16. The method according to claim 1, wherein the administration of the composition leads to a decrease of at least one liver marker selected from ALT, AST, ALP, MDA and GSH-Px. 30 12
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109078038A (en) * | 2018-09-11 | 2018-12-25 | 南方医科大学 | The new opplication of blueberry leaf flavonoids |
| WO2020186683A1 (en) * | 2019-03-18 | 2020-09-24 | 广东药科大学 | Application of quercetin in preparation of drug for prevention and treatment of drug-induced liver injury |
| CN111939190A (en) * | 2020-08-23 | 2020-11-17 | 昆明理工大学 | Application of sparrow mouth tea or extract thereof in preparation of medicine or health product for preventing and treating acute liver injury |
| CN111939189A (en) * | 2020-08-23 | 2020-11-17 | 昆明理工大学 | Application of sparrow mouth tea or extract thereof in preparation of medicine or health-care product for preventing and treating hepatic fibrosis |
-
2015
- 2015-12-03 AU AU2015101749A patent/AU2015101749A4/en not_active Ceased
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109078038A (en) * | 2018-09-11 | 2018-12-25 | 南方医科大学 | The new opplication of blueberry leaf flavonoids |
| WO2020052196A1 (en) * | 2018-09-11 | 2020-03-19 | 南方医科大学 | Novel application of total flavones of blueberry leaves |
| WO2020186683A1 (en) * | 2019-03-18 | 2020-09-24 | 广东药科大学 | Application of quercetin in preparation of drug for prevention and treatment of drug-induced liver injury |
| CN111939190A (en) * | 2020-08-23 | 2020-11-17 | 昆明理工大学 | Application of sparrow mouth tea or extract thereof in preparation of medicine or health product for preventing and treating acute liver injury |
| CN111939189A (en) * | 2020-08-23 | 2020-11-17 | 昆明理工大学 | Application of sparrow mouth tea or extract thereof in preparation of medicine or health-care product for preventing and treating hepatic fibrosis |
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