CN114306439B - Loquat leaf villus extract and application thereof - Google Patents
Loquat leaf villus extract and application thereof Download PDFInfo
- Publication number
- CN114306439B CN114306439B CN202210184202.0A CN202210184202A CN114306439B CN 114306439 B CN114306439 B CN 114306439B CN 202210184202 A CN202210184202 A CN 202210184202A CN 114306439 B CN114306439 B CN 114306439B
- Authority
- CN
- China
- Prior art keywords
- extract
- villus
- lps
- glucoside
- induced
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to a loquat leaf villus extract and application thereof, belonging to the technical field of medicine preparation. The invention provides an application of loquat leaf villus in preparing a medicament for preventing and/or treating inflammation. The eriobotrya japonica leaf villus extract can inhibit NO release induced by LPS, inhibit inflammatory factor release induced by LPS, inhibit up-regulation expression of inflammatory related genes induced by LPS, and inhibit gene expression and protein phosphorylation of Jak2 and Stat3 induced by LPS, so that the treatment of the LPS-induced inflammation is realized.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to a loquat leaf villus extract and application thereof.
Background
Loquat (Eriobotrya japonica (thunder.) Lindl) is a plant of Eriobotrya genus of Rosaceae family, and its fruits, flowers and leaves can be used as medicine for treating diseases. Loquat is native in China, and the earliest record of loquat is Shanglin Fu Lin of Western Han Sima Xiangru in the past century before the male. According to the description of the outline of materia medica, "wood Gao Zhang is more, fat branches and leaves are longer and longer than donkey ears, yellow hairs are on the back, the aesculus pubescens is lovely, the aesculus pubescens is not withered in four hours, cheng Dong is white, the aesculus pubescens is in effect until three months, the aesculus pubescens is as big as a pill, the aesculus pubescens is yellow as yellow apricot, the skin and the meat are very thin when the aesculus pubescens is cooked, and the nuggets are as big as taro chestnut and yellow brown. At present, china is the most dominant loquat producing country in the world, and the producing areas are mainly distributed in three areas of the provinces of the south of the Yangtze river, namely Fujian Pu field, zhejiang pond perch and Jiangsu Dongting mountain.
Loquat She Gezhi, which has gray brown fluff densely grown on the back of the leaf, has bitter and slightly pungent taste and slightly cold nature, and enters lung and stomach meridians. In the application process of the traditional loquat leaf, the back fluff of the loquat leaf, namely the loquat leaf fluff, is brushed off, and the loquat leaf flesh is singly used as medicine, but the functional activity of the loquat leaf fluff is unknown.
Disclosure of Invention
The invention aims to provide a loquat leaf villus extract and application thereof. The invention discovers the anti-inflammatory activity of the loquat leaf villus, and the loquat leaf villus extract can realize the improvement and treatment of inflammation.
The invention provides an application of loquat leaf villus in preparing a medicament for preventing and/or treating inflammation.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for preventing and/or treating inflammation.
Preferably, the inflammation comprises LPS-induced inflammation.
Preferably, the inflammation comprises inflammation of the liver.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for inhibiting NO release induced by LPS.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for inhibiting the release of inflammatory factors induced by LPS; the LPS-induced inflammatory factor comprises one or more than two of IL-1 beta, IL-6 and TNF alpha.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for inhibiting up-regulated expression of inflammatory related genes induced by LPS; the LPS-induced inflammation-related genes comprise one or more than two of iNOS, IL-6, IL-1 beta and TNF alpha.
The invention also provides application of the loquat leaf villus extract in preparing medicines for inhibiting gene expression and protein phosphorylation of Jak2 and Stat3 induced by LPS.
The invention also provides a loquat leaf and villus extract for preventing and/or treating inflammation, which comprises the following components: quinic acid, uridine, crotonic acid, 4-O-glucosyl-4-hydroxybenzoic acid, 3-caffeoylquinic acid, p-coumaric acid-4-O-glucoside, procyanidin B2, cherry glycoside, quercetin-3-O-sophoroside, quercetin-3-O-rutin, quercetin-3-O-glucoside, naringenin-6-C- (2 "-O-acetyl) -glucoside, naringin, hesperidin, neohesperidin, hesperetin-7-O-glucoside, isorhamnetin-3-O-glucoside, hesperenol, naringenin-6-C- (2 ',4', 6" -O-triacetyl) -glucoside, sweet flavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxyflavone, hesperetin and phthalic anhydride.
The invention also provides a preparation method of the loquat leaf and villus extract, which comprises the following steps: pulverizing folium Eriobotryae fuzz to obtain pulverized folium Eriobotryae fuzz; mixing pulverized folium Eriobotryae villi with solvent, extracting, and filtering to obtain filtrate; removing solvent from the filtrate to obtain folium Eriobotryae villus extract.
The invention provides an application of loquat leaf villus in preparing a medicament for preventing and/or treating inflammation. The invention discovers the anti-inflammatory activity of the loquat leaf villus, and the loquat leaf villus extract can realize the improvement and treatment of inflammation. The test result shows that the loquat leaf villus extract can inhibit NO release induced by LPS, inhibit inflammatory factor release induced by LPS, inhibit up-regulation expression of related genes of the inflammation induced by LPS, and inhibit gene expression and protein phosphorylation of Jak2 and Stat3 induced by LPS, so that the treatment of the inflammation is realized, and the treatment of the inflammation induced by LPS is particularly realized.
Drawings
FIG. 1 is a liquid phase diagram of a loquat leaf villus extract provided by the invention;
FIG. 2 is a graph showing the effect of different concentrations of Eriobotrya japonica leaf villus extract on the cell viability of L02 cells;
FIG. 3 is a graph showing the result of inhibiting the NO release capacity of L02 cells induced by LPS by loquat leaf and villus extracts with different concentrations;
FIG. 4 is a graph showing the results of inhibition of LPS-induced L02 cell inflammatory factor release by different concentrations of Eriobotrya japonica villus extracts;
FIG. 5 is a graph showing the result of inhibiting the LPS-induced expression of related inflammatory factor genes by using loquat leaf villus extracts with different concentrations;
FIG. 6 is a graph showing the result of inhibiting the expression of Jak2 and Stat3 genes induced by LPS by using the eriobotrya japonica leaf villus extract provided by the invention;
FIG. 7 is a graph showing the results of inhibition of LPS-induced expression and phosphorylation of Jak2 and Stat3 proteins by loquat leaf villus extract provided by the present invention;
FIG. 8 is a graph showing the results of inhibition of LPS-induced hepatic inflammatory factor release in mice by different concentrations of Eriobotrya japonica extract;
FIG. 9 is a graph showing the result of inhibiting the LPS-induced expression of mouse liver-related inflammatory factor gene by using the eriobotrya japonica villus extract;
FIG. 10 is a graph showing the result of inhibiting LPS-induced expression of mouse liver Jak2 and Stat3 genes by using the eriobotrya japonica leaf villus extract provided by the invention;
FIG. 11 is a graph showing the results of inhibition of LPS-induced expression and phosphorylation of mouse liver Jak2 and Stat3 proteins by loquat leaf villus extract provided by the present invention.
Detailed Description
The invention provides an application of loquat leaf villus in preparing a medicament for preventing and/or treating inflammation. In the present invention, the inflammation preferably includes LPS-induced inflammation. In the present invention, the inflammation preferably includes inflammation of the liver. The embodiment proves that the loquat leaf villus has anti-inflammatory activity, can treat inflammation, and can treat inflammation induced by LPS preferably.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for preventing and/or treating inflammation. In the present invention, the inflammation preferably includes LPS-induced inflammation. In the present invention, the inflammation preferably includes inflammation of the liver. The extraction method of the loquat leaf villus extract is not particularly limited, and water extraction or organic solvent extraction are all within the protection scope of the application. In the present invention, the loquat leaf and villus extract preferably comprises a loquat leaf and villus ethanol extract. The Eriobotrya japonica leaf villus extract has anti-inflammatory activity, can treat inflammation, and preferably can treat inflammation induced by LPS.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for inhibiting NO release induced by LPS. The extraction method of the loquat leaf villus extract is not particularly limited, and water extraction or organic solvent extraction are all within the protection scope of the application. In the present invention, the loquat leaf and villus extract preferably comprises a loquat leaf and villus ethanol extract. The examples prove that the eriobotrya japonica leaf villus extract can inhibit the NO release induced by LPS in liver cells or livers.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for inhibiting the release of inflammatory factors induced by LPS; the LPS-induced inflammatory factor comprises one or more than two of IL-1 beta, IL-6 and TNF alpha. The extraction method of the loquat leaf villus extract is not particularly limited, and water extraction or organic solvent extraction are all within the protection scope of the application. In the present invention, the loquat leaf and villus extract preferably comprises a loquat leaf and villus ethanol extract. The embodiment proves that the eriobotrya japonica leaf villus extract can inhibit the release of inflammatory factors induced by LPS in liver cells or livers.
The invention also provides application of the loquat leaf villus extract in preparing a medicament for inhibiting up-regulated expression of inflammatory related genes induced by LPS; the LPS-induced inflammation-related genes comprise one or more than two of iNOS, IL-6, IL-1 beta and TNF alpha. The extraction method of the loquat leaf villus extract is not particularly limited, and water extraction or organic solvent extraction are all within the protection scope of the application. In the present invention, the loquat leaf and villus extract preferably comprises a loquat leaf and villus ethanol extract. The embodiment proves that the eriobotrya japonica leaf villus extract can inhibit the up-regulated expression of the inflammation related genes induced by LPS in liver cells or livers.
The invention also provides application of the loquat leaf villus extract in preparing medicines for inhibiting gene expression and protein phosphorylation of Jak2 and Stat3 induced by LPS, the extraction method of the loquat leaf villus extract is not particularly limited, and water extraction or organic solvent extraction are all within the protection scope of the application. In the present invention, the loquat leaf and villus extract preferably comprises a loquat leaf and villus ethanol extract. The embodiment verifies that the loquat leaf villus extract can inhibit the gene expression and protein phosphorylation of Jak2 and Stat3 induced by LPS in liver cells or livers, namely the loquat leaf villus extract can inhibit the expression of Jak2 genes and the phosphorylation of Jak2 proteins induced by LPS in liver cells or livers, and can inhibit the expression of Stat3 genes and the phosphorylation of Stat3 proteins induced by LPS in liver cells or livers.
The invention also provides a loquat leaf and villus extract for preventing and/or treating inflammation, which comprises the following components: quinic acid, uridine, crotonic acid, 4-O-glucosyl-4-hydroxybenzoic acid, 3-caffeoylquinic acid, p-coumaric acid-4-O-glucoside, procyanidin B2, cherry glycoside, quercetin-3-O-sophoroside, quercetin-3-O-rutin, quercetin-3-O-glucoside, naringenin-6-C- (2 "-O-acetyl) -glucoside, naringin, hesperidin, neohesperidin, hesperetin-7-O-glucoside, isorhamnetin-3-O-glucoside, hesperenol, naringenin-6-C- (2 ',4', 6" -O-triacetyl) -glucoside, sweet flavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxyflavone, hesperetin and phthalic anhydride.
The invention also provides a preparation method of the loquat leaf and villus extract, which comprises the following steps: pulverizing folium Eriobotryae fuzz to obtain pulverized folium Eriobotryae fuzz; mixing pulverized folium Eriobotryae villi with solvent, extracting, and filtering to obtain filtrate; removing solvent from the filtrate to obtain folium Eriobotryae villus extract. The loquat leaf extract is preferably frozen before crushing. The method of the present invention is not particularly limited, and conventional pulverization methods known to those skilled in the art may be employed. In the present invention, the solvent preferably includes ethanol. In the invention, the mass and solvent volume ratio of the crushed loquat leaf villus is preferably 1: (8-12) mg/mL, more preferably 1:1mg/mL. In the present invention, the extraction method preferably includes an ultrasonic extraction method. The ultrasonic extraction according to the invention is preferably carried out using an Shanghai department ultrasonic cleaner (model SK 5200G). In the invention, the working frequency of the ultrasonic wave is preferably 35kHz, and the power is preferably 200W. In the present invention, the extraction time of the ultrasonic extraction is preferably 55 to 65 minutes, more preferably 60 minutes. The method for removing the solvent according to the present invention preferably includes a rotary evaporation method, and the condition of rotary evaporation is preferably 37℃at 80rpm. After removal of the solvent, the present invention preferably utilizes a Eppendorf concentrator plus vacuum centrifuge system to dry the extract powder by vacuum centrifugation at 35 ℃.
The following examples are provided to illustrate the invention in further detail, including but not limited to, the following examples.
Example 1
The extraction method comprises the following steps:
cleaning folium Eriobotryae, air drying at room temperature, brushing down the back fluff of folium Eriobotryae with a brush, quick-freezing with liquid nitrogen, standing at-80deg.C, and grinding fluff into powder with a sample grinder. Dissolving the powder in ethanol, and performing ultrasonic-assisted extraction by using an Shanghai department ultrasonic cleaner (model SK 5200G), wherein the working frequency is 35kHz, the power is 200W, and the feed-liquid ratio is 1:10 (mg: mL) for 60min. After the extract was filtered, the organic phase was removed by rotary evaporation (37 ℃,80 rpm), and the extract powder was obtained by using a vacuum centrifugal system.
The detection method comprises the following steps:
the powder was dissolved in chromatographic methanol for substance detection. Substance detection was performed using UPLC-MS/MS as follows:
the UPLC detection conditions are as follows: ACQUITY UPLC HSS T3 (1.8 μm, 2.1X100 mm) was used as stationary phase; the mobile phase was water containing 1% formic acid (mobile phase a) and acetonitrile containing 1% formic acid (mobile phase B). The gradient elution procedure was: 0-20 min, 5-95% B; 20-22 min,95% B; 22-23 min, 95-5% B; 23-25 min,5% B. The scanning wavelength was 254nm, the flow rate was 0.3mL/min, the column temperature was 50deg.C, and the sample volume was 2. Mu.L.
Mass spectrometry conditions: UPLC-Triple-TOF 5600+ time-of-flight liquid chromatography-mass spectrometry: positive and negative ion scanning mode; scanning range: m/z is 100-1500; atomizing gas (GS 1): 55psi; atomizing gas (GS 2): 55psi; curtain gas (CUR): 35psi; ion source Temperature (TEM): 600 ℃ (positive) 550 ℃ (negative); ion source voltage (IS): 5500V (positive) -4500V (negative); primary scanning: de-clustering voltage (DP): 100V; focus voltage (CE): 10V; and (3) secondary scanning: and acquiring mass spectrum data by using a TOF MS-Product Ion-IDA mode, wherein CID energy is 40+/-20 eV, and performing mass axis correction by using a CDS pump before sample injection to ensure that the mass axis error is less than 2ppm.
The detection results are shown in FIG. 1 and Table 1, and FIG. 1 shows a liquid phase diagram of the Eriobotrya japonica leaf villus extract. The loquat leaf villus extract contains the following components: quinic acid, uridine, crotonic acid, 4-O-glucosyl-4-hydroxybenzoic acid, 3-caffeoylquinic acid, p-coumaric acid-4-O-glucoside, procyanidin B2, cherry glycoside, quercetin-3-O-sophoroside, quercetin-3-O-rutin, quercetin-3-O-glucoside, naringenin-6-C- (2 "-O-acetyl) -glucoside, naringin, hesperidin, neohesperidin, hesperetin-7-O-glucoside, isorhamnetin-3-O-glucoside, hesperenol, naringenin-6-C- (2 ',4', 6" -O-triacetyl) -glucoside, sweet flavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxyflavone, hesperetin and phthalic anhydride.
TABLE 1 analysis of the ingredients of the Eriobotrya japonica leaf villus extract
Example 2
Inhibition of Nitric Oxide (NO) release induced in human liver cells L02 by Eriobotrya japonica leaf villus extract
The experimental method comprises the following steps:
this example is directed to evaluating the induction of NO release by loquat leaf and villus extract on Lipopolysaccharide (LPS) using human liver cell line L02 as a subjectIs effective in inhibiting the inhibition of the action of (a). L02 cells were cultured in a constant temperature and humidity incubator (37℃C, 5% CO) using RPMI-1640 complete medium (containing 10% fetal bovine serum) 2 ). Cells in logarithmic growth phase were grown at 5X 10 4 The density of each hole is inoculated in a 96-well plate, after 24 hours of culture, fresh culture medium is replaced, loquat leaf villus extracts (Loquat Greenleaf Hair, LGH) with different concentrations are dissolved in dimethyl sulfoxide (DMSO) and then are cultured for 24 hours, then 0.1 mug/mL of LPS is added, the culture is incubated for 12 hours (NO LPS is added in a control group), and the NO content in the culture medium is measured according to the NO detection kit.
The experimental results are shown in fig. 2 and 3, wherein fig. 2 shows the effect of different concentrations of the eriobotrya japonica leaf villus extract on the cell viability of the L02 cells, and fig. 3 shows the inhibition effect of different concentrations of the eriobotrya japonica leaf villus extract on the NO release capacity of the L02 cells induced by LPS.
As shown in FIG. 2, in the experimental concentration range (12.5-200. Mu.g/mL of loquat leaf extract), the loquat leaf extract (LGH) has no cytotoxicity to L02 cells, and the cell activity is above 90%. In contrast, when LPS and LGH were co-treated, 200. Mu.g/mL of the Eriobotrya japonica extract had a remarkable proliferation inhibition effect on cells, and therefore, in the subsequent experiments, concentrations of 100. Mu.g/mL and below were selected. As shown in fig. 3, LGH alone had NO significant effect on NO release from cells, whereas LPS could significantly induce NO release (6.05 μg/mL), exhibiting an inflammatory induction effect, and when cells were subjected to LGH pretreatment, NO in the medium was significantly reduced, especially 100 μg/mL pretreatment, NO concentration in the medium was reduced to 1.51 μg/mL, 75% as compared to NO concentration induced by LPS alone. The above results demonstrate that the eriobotrya japonica leaf villus extract has the capability of remarkably inhibiting the NO release induced by LPS.
Example 3
Inhibition of LPS-induced inflammatory factor release in human liver cell L02 by Eriobotrya japonica leaf villus extract
The experimental method comprises the following steps:
this example is directed to evaluating the inhibition of LPS-induced inflammatory factor release by Eriobotrya japonica leaf villus extract using human liver cell line L02 as a subject. The cell culture and treatment method is shown in example 1, and the detection method of the cytokines interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF alpha) is carried out by an Elisa method, and the detection method is a commercial kit (Abcam company).
The experimental results are shown in fig. 4, and fig. 4 shows the inhibition effect of different concentrations of loquat leaf and villus extracts on the release capacity of LPS-induced L02 cell inflammatory factors; wherein, control: a control group; LPS: lipopolysaccharide intervention group; 25. 50, 100 are three concentrations of loquat leaf villus extract treatment groups (all under LPS intervention), respectively.
As shown in fig. 4, LPS significantly induced the release of cytokines, and LPS treatment promoted up-regulation by 11.53-fold, 6.89-fold and 5.23-fold, respectively, for IL-1 β, IL-6 and tnfα, compared to the control group, to 928.80pg/mL, 967.32pg/mL and 409.28pg/mL. In the experimental concentration range (25-100 mug/mL), the loquat leaf villus extract (LGH) remarkably inhibits the release of LPS (LPS) to cell inflammatory factors and shows a gradient dosage effect. In particular, the pretreatment of LGH at 100. Mu.g/mL controls the inflammatory cytokines at 423.52pg/mL (IL-1β), 249.89pg/mL (IL-6), 113.77pg/mL (TNF. Alpha.). The results show that the eriobotrya japonica leaf villus extract has the capability of remarkably inhibiting the release of cell inflammatory factors induced by LPS.
Example 4
The folium Eriobotryae villosa extract has effect of inhibiting LPS-induced up-regulation expression of inflammation related gene in L02 cells
This example is directed to evaluating the inhibition of LPS-induced up-regulation of inflammation-related genes by Eriobotrya japonica leaf villus extract using human liver cell line L02 as a subject. L02 cells were 1X 10 per well 6 The density of individual cells was plated on 6-well plates. Total RNA was extracted using Trizol reagent, reverse transcribed using Invitrogen kit, and then tested in a real-time PCR system following standard quantitative PCR procedure, the Q-PCR total system was configured as follows at 50. Mu.L: 10 mu L SYBR Green1 dye, 1 mu L forward primer, 1 mu L reverse primer, 1 mu L dNTP, 2 mu L Taq polymerase, 5 mu L cDNA of the sample to be detected and ddH 2 O30. Mu.L. The solution was mixed at the bottom of the flick tube and centrifuged briefly at 6000 rpm. The reaction procedure is 93 ℃ for 2min of pre-denaturation; 40 cycles at 93℃1min,55℃1min,72℃ 1 min; 72 ℃,7min extends. qRT-PCR primers were as follows:
β-actin：
5'-GTGGCCGAGGACTTTGATTG-3' (Forward primer, SEQ ID NO. 1)
5'-AGTGGGGTGGCTTTTAGGATG-3' (reverse primer, SEQ ID NO. 2);
iNOS:
5'-CAGGACCACACCCCCTAGGA-3' (Forward primer, SEQ ID NO. 3)
5'-AGCCACATACCGAGCCATGC-3' (reverse primer, SEQ ID NO. 4);
IL-1β：
5'-GTGTCCTGCGTGTTGAAAGAT-3' (Forward primer, SEQ ID NO. 5)
5'-GAGTTGGGCATTGGTGTAGAC-3' (reverse primer, SEQ ID NO. 6);
IL-6：
5'-ACCCCTGACCCAACCACAAAT-3' (Forward primer, SEQ ID NO. 7)
5'-AGCTGCGCAGAATGAGATGAGTT-3' (reverse primer, SEQ ID NO. 8);
TNFα：
5'-CACAGTGAAGTGCTGGCAAC-3' (Forward primer, SEQ ID NO. 9)
5'-GGCGATTACAGACACAACTCC-3' (reverse primer, SEQ ID NO. 10)
Beta-actin was used as a control. By comparison (2) -ΔΔCT ) The relative levels of gene expression were calculated by the method.
The experimental results are shown in fig. 5, and fig. 5 shows the inhibition effect of different concentrations of loquat leaf villus extract on the gene expression of related inflammatory factors induced by LPS.
As shown in fig. 5, compared with the control group, the LPS induction significantly induced the expression of inflammatory factor related genes iNOS, IL-1 β, IL-6, tnfα, indicating that the LPS induced the expression of inflammatory factors by up-regulating the expression of inflammatory factor genes. The eriobotrya japonica leaf villus extract can inhibit the expression of the inflammatory factor related genes by LPS and has a dosage effect. For the expression of genes such as iNOS, IL-1 beta, IL-6, TNF alpha and the like, the inhibition rate of 100 mug/mL loquat leaf villus extract treatment reaches 97.91%, 97.23%, 97.42% and 83.42%. The results show that the loquat leaf villus has the function of obviously inhibiting the inflammatory factor gene expression induced by LPS.
Example 5
Eriobotrya japonica leaf villus extract has effects of inhibiting LPS-induced Jak2 and Stat3 gene expression and protein phosphorylation in L02 cells
The experimental method comprises the following steps:
this example is directed to evaluating the inhibition of LPS-induced Jak2, stat3 gene expression and protein phosphorylation by Eriobotrya japonica leaf villus extracts using human liver cell line L02 as a subject. L02 cells were 1X 10 per well 6 The density of individual cells was plated on 6-well plates. Total RNA was extracted using Trizol reagent, reverse transcribed using Invitrogen kit, and then tested in a real-time PCR system following standard quantitative PCR procedures, qRT-PCR primers were as follows:
β-actin：
5'-GTGGCCGAGGACTTTGATTG-3' (Forward primer, SEQ ID NO. 1)
5'-AGTGGGGTGGCTTTTAGGATG-3' (reverse primer, SEQ ID NO. 2);
Jak2:
5'-AGCCTATCGGCATGGAATATCT-3' (Forward primer, SEQ ID NO. 11)
5'-TAACACTGCCATCCCAAGACA-3' (reverse primer, SEQ ID NO. 12);
Stat3:
5'-ACCAGCAGTATAGCCGCTTC-3' (Forward primer, SEQ ID NO. 13)
5'-GCCACAATCCGGGCAATCT-3' (reverse primer, SEQ ID NO. 14);
beta-actin was used as a control. By comparison (2) -ΔΔCT ) The relative levels of gene expression were calculated by the method. Protein expression and phosphorylation levels were determined using the Elisa kit from Abcam, according to standard procedures of the specification.
The experimental results are shown in FIG. 6 and FIG. 7, wherein FIG. 6 shows the inhibitory effect of the Eriobotrya japonica leaf villus extract on the LPS-induced expression of Jak2 and Stat3 genes, and FIG. 7 shows the inhibitory effect of the Eriobotrya japonica leaf villus extract on the LPS-induced expression and phosphorylation of Jak2 and Stat3 proteins (p- "in the figure represents" phosphoated ", i.e., phosphorylated).
As shown in fig. 6, compared with the control group, the induction of LPS significantly induced the expression of Jak2 and Stat3 genes of the inflammatory regulation pathway, whereas the eriobotrya japonica villus extract can significantly inhibit the induction of Jak2 and Stat3 genes by LPS, and exhibits a dose effect, wherein the inhibition rate of 100 μg/mL of eriobotrya japonica villus extract treatment reaches 94.61% (Jak 2) and 67.70% (Stat 3). As shown in fig. 7, LPS significantly induced Jak2 protein expression to 17.03-fold compared to the control, whereas LGH treatment did not significantly modulate Jak2 protein expression under LPS intervention, but LGH treatment significantly inhibited the phosphorylation of Jak2 protein by LPS and was dose-responsive, suggesting that LGH inhibited the functional induction of Jak2 protein by LPS by decreasing phosphorylation of Jak2 protein. For Stat3 protein, LPS induced significantly its expression and phosphorylation levels, whereas LGH treatment could significantly inhibit LPS induction, all exhibiting dose effects. I.e., LGH has a significant effect on protein phosphorylation of both Jak2 and Stat 3. Phosphorylation is a characteristic indicator of protein-initiated expression. LGH differs in the mechanism of action of Jak2 and Stat3, but both inhibit LPS-induced activation of protein action. The results show that the loquat leaf villus has the effect of obviously inhibiting the expression of Jak2 and Stat3 genes and the phosphorylation of protein of an inflammation related pathway induced by LPS.
Example 6
The folium Eriobotryae villosa extract has inhibiting effect on inhibiting LPS-induced inflammatory factor accumulation in L02 cells induced by LPS in mouse liver
The experimental method comprises the following steps:
this example was conducted to evaluate the inhibitory effect of Eriobotrya japonica leaf villus extract on LPS-induced release of liver inflammatory factor in mice, using C57BL/6J mice as a subject. Male 6-week-old C57BL/6J mice are fed in an SPF environment, feed and drinking water are freely obtained, the feeding temperature is 22+/-2 ℃, and the light-dark period is 12 hours respectively. Mice were divided into 5 groups, control group (feeding water, intraperitoneal injection of physiological saline), model group (feeding water, intraperitoneal injection of LPS, dose of 0.25mg/kg, duration 7 d), LGH low dose group (after lavage 25mg/kg of LGH for 21d, lavage was kept while intraperitoneal injection of 0.25mg/kg of LPS was continued for 7 d), LGH dose group (after lavage 50mg/kg of LGH for 21d, lavage was kept while intraperitoneal injection of 0.25mg/kg of LPS was continued for 7 d), LGH high dose group (after lavage 100mg/kg of LGH for 21d, lavage was kept while intraperitoneal injection of 0.25mg/kg of LPS was continued for 7 d), 10 each group. The experiment starts for 28d from the beginning of gastric lavage, the mice are killed after cervical dislocation, the livers are collected, quick frozen by liquid nitrogen, and the mice are ground in a sample grinder for later use. Inflammatory factors IL-1 beta, IL-6, TNF alpha were detected using the Abcam company Elisa kit.
The experimental results are shown in fig. 8, and fig. 8 shows the inhibition effect of different concentrations of loquat leaf and villus extracts on the release capacity of LPS-induced liver inflammatory factors of mice.
As shown in fig. 8, intraperitoneal injection of LPS significantly induced accumulation of mouse liver inflammatory factors, with LPS treatment promoting up-regulation by 8.68-fold, 6.54-fold and 7.59-fold, respectively, for the three inflammatory factors IL-1 beta, IL-6 and tnfα, compared to the control group, reaching 1637.00pg/mL, 1590.93pg/mL and 1423.01pg/mL. In the experimental concentration range (25-100 mg/kg bw d), the eriobotrya japonica leaf villus extract (LGH) remarkably inhibits the accumulation of cell inflammatory factors induced by LPS in the liver of the mice. The results show that the loquat leaf villus extract has the capability of obviously inhibiting the accumulation of mouse liver inflammatory factors induced by LPS.
Example 7
The folium Eriobotryae villosa extract has effect in inhibiting LPS-induced up-regulation expression of inflammation related gene in mouse liver
The experimental method comprises the following steps:
the present example aims at evaluating the inhibition of LPS-induced gene up-regulation expression of liver inflammation related genes of mice by loquat leaf villus extract in C57BL/6J mice. The mice were kept and treated in the same manner as in example 6. Total liver RNA was extracted using Trizol reagent, reverse transcribed using Invitrogen kit, and then tested in a real-time PCR system following standard quantitative PCR procedures.
qRT-PCR primers were as follows:
β-actin：
5'-GGCTGTATTCCCCTCCATCG-3' (Forward primer, SEQ ID NO. 15)
5'-CCAGTTGGTAACAATGCCATGT-3' (reverse primer, SEQ ID NO. 16);
iNOS:
5'-GTTCTCAGCCCAACAATACAAGA-3' (Forward primer, SEQ ID NO. 17)
5'-GTGGACGGGTCGATGTCAC-3' (reverse primer, SEQ ID NO. 18);
IL-1β：
5'-GCAACTGTTCCTGAACTCAACT-3' (Forward primer, SEQ ID NO. 19)
5'-ATCTTTTGGGGTCCGTCAACT-3' (reverse primer, SEQ ID NO. 20);
IL-6：
5'-TAGTCCTTCCTACCCCAATTTCC-3' (Forward primer, SEQ ID NO. 21)
5'-TTGGTCCTTAGCCACTCCTTC-3' (reverse primer, SEQ ID NO. 22);
TNFα：
5'-CCCTCACACTCAGATCATCTTCT-3' (Forward primer, SEQ ID NO. 23)
5'-GCTACGACGTGGGCTACAG-3' (reverse primer, SEQ ID NO. 24)
Beta-actin was used as a control. By comparison (2) -ΔΔCT ) The relative levels of gene expression were calculated by the method.
The experimental results are shown in fig. 9, and fig. 9 shows the inhibition effect of the eriobotrya japonica leaf villus extract on the LPS-induced liver-related inflammatory factor gene expression of mice.
As shown in fig. 9, intraperitoneal injection of LPS induced significant induction of expression of inflammatory factor related genes iNOS, IL-1 β, IL-6, tnfα compared to the control group, indicating that LPS induced expression of inflammatory factors by up-regulating expression of inflammatory factor genes. The eriobotrya japonica leaf villus extract can inhibit the expression of the inflammatory factor related genes by LPS and has a dosage effect. For the expression of genes such as iNOS, IL-1 beta, IL-6, TNF alpha and the like, the inhibition rate of 100mg/kg loquat leaf villus extract treatment reaches 98.20 percent, 93.99 percent, 98.89 percent and 92.48 percent. The results show that the loquat leaf villus has the function of obviously inhibiting the inflammatory factor gene expression induced by LPS.
Example 8
Eriobotrya japonica leaf villus extract has effects of inhibiting LPS-induced Jak2 and Stat3 gene expression and protein phosphorylation in mouse liver
The experimental method comprises the following steps:
this example aims at evaluating the inhibition of LPS-induced Jak2, stat3 gene expression and protein phosphorylation by Eriobotrya japonica leaf villus extracts in C57BL/6J mice. The mice were kept and treated in the same manner as in example 6. Total liver RNA was extracted using Trizol reagent, reverse transcribed using Invitrogen kit, and then tested in a real-time PCR system following standard quantitative PCR procedures. qRT-PCR primers were as follows: protein expression and phosphorylation levels were determined using the Elisa kit from Abcam, according to standard procedures of the specification.
β-actin：
5'-GGCTGTATTCCCCTCCATCG-3' (Forward primer, SEQ ID NO. 15)
5'-CCAGTTGGTAACAATGCCATGT-3' (reverse primer, SEQ ID NO. 16);
Jak2:
5'-TTGTGGTATTACGCCTGTGTATC-3' (Forward primer, SEQ ID NO. 25)
5'-ATGCCTGGTTGACTCGTCTAT-3' (reverse primer, SEQ ID NO. 26);
Stat3:
5'-CAATACCATTGACCTGCCGAT-3' (Forward primer, SEQ ID NO. 27)
5'-GAGCGACTCAAACTGCCCT-3' (reverse primer, SEQ ID NO. 28);
the experimental results are shown in fig. 10 and 11, wherein fig. 10 shows the inhibitory effect of the eriobotrya japonica leaf villus extract on the LPS-induced expression of the mouse liver Jak2 and Stat3 genes, and fig. 11 shows the inhibitory effect of the eriobotrya japonica leaf villus extract on the LPS-induced expression and phosphorylation of the mouse liver Jak2 and Stat3 proteins.
As shown in fig. 10, compared with the control group, the induction of the LPS by intraperitoneal injection significantly induced the expression of the Jak2 and Stat3 genes in the inflammatory regulation pathway, and the induction of the Jak2 and Stat3 genes by LPS can be significantly inhibited by a certain concentration of the eriobotrya japonica extract, wherein the inhibition rate of 100mg/kg of the eriobotrya japonica extract treatment reaches 95.32% (Jak 2) and 66.29% (Stat 3). As shown in fig. 11, LPS significantly induced protein expression and phosphorylation levels of Jak2 and Stat3 compared to the control group, suggesting that LPS may induce inflammation in mouse liver by both pathways, whereas oral administration LGH may significantly reduce protein expression and phosphorylation levels of Jak2 and Stat 3. The results show that the loquat leaf villus has the effect of obviously inhibiting the expression of Jak2 and Stat3 genes and the phosphorylation of protein of an inflammation related pathway induced by LPS.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> university of Zhejiang
<120> loquat leaf villus extract and application thereof
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
agtggggtgg cttttaggat g 21
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gagttgggca ttggtgtaga c 21
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
acccctgacc caaccacaaa t 21
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
agctgcgcag aatgagatga gtt 23
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
ggcgattaca gacacaactc c 21
<210> 11
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
agcctatcgg catggaatat ct 22
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
taacactgcc atcccaagac a 21
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
accagcagta tagccgcttc 20
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ggctgtattc ccctccatcg 20
<210> 16
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ccagttggta acaatgccat gt 22
<210> 17
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gttctcagcc caacaataca aga 23
<210> 18
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
<210> 19
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
gcaactgttc ctgaactcaa ct 22
<210> 20
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
atcttttggg gtccgtcaac t 21
<210> 21
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tagtccttcc taccccaatt tcc 23
<210> 22
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ttggtcctta gccactcctt c 21
<210> 23
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ccctcacact cagatcatct tct 23
<210> 24
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
<210> 25
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ttgtggtatt acgcctgtgt atc 23
<210> 26
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
atgcctggtt gactcgtcta t 21
<210> 27
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
<210> 28
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
Claims (5)
1. The application of folium Eriobotryae villosa extract in preparing medicine for preventing and/or treating inflammation is provided; the solvent for extracting the loquat leaf villus extract is ethanol; the loquat leaf villus extract comprises the following components: quinic acid, uridine, crotonoside, 4-O-glucosyl-4-hydroxybenzoic acid, 3-caffeoylquinic acid, p-coumaric acid-4-O-glucoside, procyanidin B2, cherry glycoside, quercetin-3-O-sophoroside, quercetin-3-O-rutin, quercetin-3-O-glucoside, naringenin-6-C- (2 ' -O-acetyl) -glucoside, naringin, hesperidin, neohesperidin, hesperetin-7-O-glucoside, isorhamnetin-3-O-glucoside, hesperenol, naringenin-6-C- (2 ' ',4' ',6' ' -O-triacetyl) -glucoside, sweet orange flavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxy flavone, hesperetin and phthalic anhydride.
2. The use according to claim 1, wherein the inflammation is LPS-induced inflammation.
3. The use according to claim 1, wherein the inflammation is inflammation of the liver.
4. An extract of loquat leaf and villus for preventing and/or treating inflammation, wherein the solvent for extracting the extract of loquat leaf and villus is ethanol; the loquat leaf villus extract comprises the following components: quinic acid, uridine, crotonoside, 4-O-glucosyl-4-hydroxybenzoic acid, 3-caffeoylquinic acid, p-coumaric acid-4-O-glucoside, procyanidin B2, cherry glycoside, quercetin-3-O-sophoroside, quercetin-3-O-rutin, quercetin-3-O-glucoside, naringenin-6-C- (2 ' -O-acetyl) -glucoside, naringin, hesperidin, neohesperidin, hesperetin-7-O-glucoside, isorhamnetin-3-O-glucoside, hesperenol, naringenin-6-C- (2 ' ',4' ',6' ' -O-triacetyl) -glucoside, sweet orange flavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxy flavone, hesperetin and phthalic anhydride.
5. The method for preparing the loquat leaf and villus extract of claim 4, comprising the following steps: pulverizing folium Eriobotryae fuzz to obtain pulverized folium Eriobotryae fuzz; mixing pulverized folium Eriobotryae villi with solvent, extracting, and filtering to obtain filtrate; removing solvent from the filtrate to obtain folium Eriobotryae villus extract; the solvent is ethanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210184202.0A CN114306439B (en) | 2022-02-25 | 2022-02-25 | Loquat leaf villus extract and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210184202.0A CN114306439B (en) | 2022-02-25 | 2022-02-25 | Loquat leaf villus extract and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114306439A CN114306439A (en) | 2022-04-12 |
| CN114306439B true CN114306439B (en) | 2023-05-02 |
Family
ID=81030777
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210184202.0A Active CN114306439B (en) | 2022-02-25 | 2022-02-25 | Loquat leaf villus extract and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114306439B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116549478B (en) * | 2023-05-06 | 2024-03-19 | 云南中医药大学 | Pharmaceutical composition and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101313947A (en) * | 2008-05-28 | 2008-12-03 | 宁波大学 | Method for preventing fluff fly in folium eriobotryae process |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4974116B2 (en) * | 2005-06-27 | 2012-07-11 | 国立大学法人 鹿児島大学 | Foods and beverages and pharmaceuticals containing loquat leaf extract |
| TWI384993B (en) * | 2009-10-28 | 2013-02-11 | Jen Li Biotech Co Ltd | Loquat leaf cell extracts useful for hepatoprotection and/or reducing fat and preparation methods and uses of the same |
| CN102747068B (en) * | 2012-06-14 | 2015-07-15 | 浙江大学 | Method for regulating cloning and transcription of lignin metabolism-related ethylene response factors (ERFs) in Eriobotrya japonica Lindl. |
| CN103463227A (en) * | 2013-07-18 | 2013-12-25 | 苏州市天灵中药饮片有限公司 | Extraction and purification technology of folium eriobotryae flavones |
| CN107383932A (en) * | 2017-07-23 | 2017-11-24 | 李斌 | A kind of Preparation method and use of loguat leaf brown pigment |
| CN108686998A (en) * | 2018-05-23 | 2018-10-23 | 浙江国新中药饮片有限公司 | A kind of special brushing machine for the removal of loguat leaf villus |
-
2022
- 2022-02-25 CN CN202210184202.0A patent/CN114306439B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101313947A (en) * | 2008-05-28 | 2008-12-03 | 宁波大学 | Method for preventing fluff fly in folium eriobotryae process |
Non-Patent Citations (2)
| Title |
|---|
| Phyu Phyu Khine Zar,等.Anti-inflammatory effects and molecularmechanisms of loquat (Eriobotrya japonica) tea.journal of functional foods.2014,第523-533页. * |
| 葛金芳,等.枇杷叶三萜酸的抗炎作用.安徽医科大学学报.2007,第42卷(第42期),第174-178、214页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114306439A (en) | 2022-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102470155B (en) | Method for producing novel gynostemma pentaphyllum extracts having increased amounts of damulin A and damulin B and pharmaceutical composition for treating metabolic diseases using the same | |
| CN114306439B (en) | Loquat leaf villus extract and application thereof | |
| KR102206591B1 (en) | Composition for preventing, improving or treating of fibrosis | |
| KR101839721B1 (en) | pharmaceutical composition for prevention or treatment of allergic diseases including asthma or atopic dermatitis comprising the baicalein derivatives | |
| Atta et al. | New flavonoid glycoside and pharmacological activities of Pteranthus dichotomus Forssk. | |
| JP2015506962A (en) | Medicament containing root extract of dandelion (Taraxacum) plant for treating or preventing cancer and method for producing the same | |
| Együd | Studies on autobiotics: chemical nature of retine. | |
| KR20190081812A (en) | Composition for improving immune system comprising a complex extract of Corni fructus, Eriobotryae Folium, and Olive leaf | |
| AU2016244997B2 (en) | Extracts from plants of the Moringaceae family and methods of making | |
| KR20180055945A (en) | Composition for Improving hepatic function Including Extract of Salvia Militorhiza Bunge and Method for Preparation of the Same | |
| KR101887764B1 (en) | Process for Extracting of red bean shell having anti obesity and Compositions comprising the Extracts as an active ingredient | |
| KR20200081553A (en) | Composition for the prevention and improvement of Antitussive and Expectorant | |
| CN114588184B (en) | Herba Saururi extract, and preparation method and application thereof | |
| Cheng et al. | Adaptive immunoregulation of luteolin and chlorogenic acid in lipopolysaccharide-induced interleukin-10 expression | |
| CN111150754B (en) | Application of chestnut flower extract in preparation of food or anti-inflammatory drugs | |
| KR102149007B1 (en) | Method for kimchi using amtak baechu cabbage and uses thereof | |
| CN106309613B (en) | Chinese medicine with anti-inflammatory and anti-allergic effects and preparation method thereof | |
| CN114209728B (en) | Wild chrysanthemum active site with function of regulating epigenetic expression balance and resisting liver cancer as well as preparation method and application thereof | |
| CN114558061A (en) | Loquat leaf extract and preparation method and application thereof | |
| Hwang et al. | Doenjang extract has anticancer activity and induces apoptosis in AGS human gastric adenocarcinoma | |
| KR102071290B1 (en) | Use of the extract of aronia | |
| CN115177649A (en) | Dandelion extract and preparation method and application thereof | |
| CN108295104B (en) | Pure ambari hemp with anti-inflammatory activity and application thereof | |
| Shouaib et al. | Immunostimulating, proapoptotic and synergistic effects of propolis and its main constituent (chrysin) on the tumor growth and the cell sensitivity to ionizing radiation in mice | |
| KR20040067101A (en) | Flavones, isolated from Artemisia absinthium and their anti-inflammatory constituents containing bioflavonoids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |