CN113694158A - Application, extraction and determination method of raw sharpleaf galangal fruit and salt sharpleaf galangal fruit extracts - Google Patents
Application, extraction and determination method of raw sharpleaf galangal fruit and salt sharpleaf galangal fruit extracts Download PDFInfo
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Abstract
The invention relates to the technical field of plant extraction, in particular to application, extraction and determination methods of alpinia oxyphylla and alpinia oxyphylla extracts, wherein the alpinia oxyphylla and alpinia oxyphylla extracts can be applied to preparation of a medicine for treating diabetic nephropathy, renal fibrosis and renal tubular injury can be treated by regulating and controlling the expression of transforming growth factors beta 1 and alpha-smooth muscle actin, and large intestine flora can be regulated and controlled by promoting the generation of short chain fatty acids SCFAs.
Description
Technical Field
The invention relates to the technical field of plant extraction processes, in particular to application, extraction and determination methods of extracts of raw sharpleaf galangal fruit and salt sharpleaf galangal fruit.
Background
Diabetic Nephropathy (DN) is one of the serious microvascular complications of diabetes. At present, the molecular pathogenesis of DN is not completely understood, and as a typical metabolic disease, the occurrence and development processes include not only hemodynamic abnormalities and metabolic abnormalities, but also various pathological phenomena such as inflammatory reaction, oxidative stress, Renin Angiotensin System (RAS) activation and intestinal flora imbalance.
The occurrence of diabetic nephropathy is often accompanied by the development of hyperlipidemia, and dyslipidemia is manifested by an increase in Total Cholesterol (TC), Triglycerides (TG), low-density lipoproteins (LDL-C), and a decrease in high-density lipoproteins (HDL-C). Abnormal metabolism in the body induces diabetic nephropathy, and as the condition of diabetic nephropathy becomes worse, inflammatory cytokines are secreted and active oxygen production increases, resulting in tissue damage and renal fibrosis. Studies have shown that the TGF- β signaling pathway plays a key role in epithelial-mesenchymal transition (EMT) of DN and tubulointerstitial fibrosis. In diabetic nephropathy, transforming growth factor-beta 1 (TGF-beta 1) is an important factor promoting fibrosis, which mediates renal fibrosis and glomerulosclerosis. The kidney of DN produces increased TGF- β 1, thereby increasing the expression and secretion of extracellular matrix proteins (ECM) and leading to renal fibrosis. Alpha-smooth muscle actin (alpha-SMA) is closely related to kidney fibrosis, is expressed in myofibroblasts and is an important marker protein for kidney tissue fibrosis, and researches show that in the kidney of a diabetic nephropathy patient, the alpha-SMA is increased in expression, extracellular matrix is accumulated, and the apoptosis of renal tubular epithelial cells is increased, so that the kidney injury is caused.
Changes in the composition and metabolic activity of the intestinal flora lead to the imbalance of the intestinal flora, and are closely related to the generation and development of DN, but the specific mechanism is not clarified. Recent studies have found that the gut flora digests dietary fiber in humans and produces Short Chain Fatty Acids (SCFAs) for the host. SCFAs are the primary energy source of the intestinal epithelium and provide additional energy to the host. SCFA can reduce inflammation by inhibiting the activity of inflammatory cells. The SCFA acetate can inhibit histone deacetylase activity of T cells and regulate oxidative stress of different immune cells, indicating that acetate has a good anti-inflammatory effect.
Alpinia oxyphylla (a. oxyphylla) is a traditional Chinese medicine of Alpinia genus of zingiberaceae family, and sharpleaf galangal fruit is a dried fruit of Alpinia oxyphylla, is one of four southern Chinese medicines and is mainly distributed in Guangxi, Hainan, Guangdong and the like in China. The sharpleaf galangal fruit is widely used clinically from ancient times to present, is recorded in numerous Chinese medicine books in China, has warm property and pungent taste, and has the functions of warming kidney, securing essence, reducing urination, warming spleen, stopping diarrhea and absorbing saliva. In the traditional medicinal experience of sharpleaf galangal fruit, the traditional recognition that the raw product enters spleen, is stir-baked with salt and enters kidney, and is uncooked and cooked, namely the raw product is pungent, warm and dry, mainly enters spleen channels, has the effects of warming spleen to stop diarrhea and collecting saliva and is commonly used for abdominal pain, vomiting and diarrhea and other symptoms caused by deficiency-cold of spleen and stomach is formed; stir-baked with salt for its mild and pungent nature, mainly entering kidney meridian, mainly warming kidney to secure essence and reduce urination, and it is mainly used in Xiiquan Wan. The fructus alpiniae oxyphyllae mainly contains sesquiterpenes, diphenylheptanes, flavonoids and the like, has pharmacological actions of resisting oxidation, resisting inflammation, reducing blood sugar and the like, and researches show that the fructus alpiniae oxyphyllae can increase the level of plasma insulin by regulating and controlling the blood sugar concentration. However, no report is found on the application of the alpinia oxyphylla extract in treating and preventing diabetic complications DN at present, and the action mechanism of the alpinia oxyphylla extract on each signal path in DN is not known.
Disclosure of Invention
In view of the above, the invention provides an application, extraction and determination method of the extracts of fructus alpiniae oxyphyllae and fructus alpiniae oxyphyllae.
The technical scheme of the invention is realized as follows:
application of extracts of raw fructus Alpinae Oxyphyllae and salt fructus Alpinae Oxyphyllae in preparing medicine for treating diabetic nephropathy is provided.
Further discloses the application of the extracts of the raw sharpleaf galangal fruit and the salt sharpleaf galangal fruit in preparing the medicines for treating the renal fibrosis and the renal tubular injury.
Further illustrates the application of the extracts of the raw sharpleaf galangal fruit and the salt sharpleaf galangal fruit in preparing the medicines for regulating the expression of the transforming growth factor-beta 1 and treating the kidney fibrosis and the renal tubular injury.
Further indicates that the applications of the extracts of the raw sharpleaf galangal fruit and the salt sharpleaf galangal fruit in preparing the medicines for regulating and controlling the expression of alpha-smooth muscle actin and treating renal fibrosis and renal tubular injury.
Further discloses the application of the raw sharpleaf galangal fruit and the salt sharpleaf galangal fruit extract in preparing the medicine for regulating the coliform group.
Further illustrates the application of the alpinia oxyphylla and the alpinia oxyphylla extract in the preparation of the medicine for promoting the generation of short chain fatty acid SCFAs and regulating and controlling coliform group bacteria.
A method for extracting fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae comprises the following steps:
(1) pulverizing salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae, and sieving with 40-60 mesh sieve to obtain salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae powder;
(2) respectively adding 7-9 times of 95% ethanol solution into salt fructus Alpinae Oxyphyllae powder and fructus Alpinae Oxyphyllae powder, heating and reflux-extracting for 2 times, each time for 1.5-2.5 hr, filtering, mixing filtrates, rotary evaporating to recover ethanol, and oven drying at 68-72 deg.C to obtain salt fructus Alpinae Oxyphyllae extract and fructus Alpinae Oxyphyllae extract.
The prepared extracts of the raw sharpleaf galangal fruit and the salt sharpleaf galangal fruit are measured by a UPLC-MS/MS method combining high performance liquid chromatography and mass spectrometry.
Further illustratively, the chromatographic conditions are: the chromatographic column is Phenomenex kinetex 2.6um XB-C18; the mobile phase is 0.2 per mill of formic acid water-0.2 per mill of formic acid acetonitrile by mass concentration, the flow rate is 0.40mL/min, the column temperature is 40 ℃, and the detection wavelength is 260 nm; the injection volume is 5 μ.
Further illustratively, the mass spectrometry conditions are: an ion source: an electrospray ion source; the detection mode is as follows: a multiple reaction monitoring mode; adopting a positive ion mode; source ejection voltage: 5500/-4500V; ion source temperature: 550 ℃; curtain gas: 30 psi; atomizing: 55psi, heating gas: 55psi, nitrogen gas was used throughout.
Compared with the prior art, the invention has the beneficial effects that:
1. the fructus Alpinae Oxyphyllae extract can be used for treating diabetic nephropathy by regulating and controlling the expression of transforming growth factor-beta 1 and alpha-smooth muscle actin, and treating renal fibrosis and renal tubule injury, so as to delay occurrence of diabetic nephropathy and protect kidney injury.
2. Experiments show that the extracts of Alpinia oxyphylla and Alpinia oxyphylla can obviously reduce the unidentified-Enterobacteriaceae bacteria level of STZ-induced diabetic rats, and simultaneously improve the acetate, propionate and butyrate levels and the abundance levels of Roembuz bacteria and unidentified _ Prevotella. The fructus Alpiniae Oxyphyllae extract can effectively reduce the abundance of intestinal bacteria in Bacteroides and Actinomycetes, and the fructus Alpiniae Oxyphyllae extract can reduce the abundance of intestinal bacteria in Bifidobacterium. Thereby indicating that the extracts before and after the fructus alpiniae oxyphyllae is roasted with salt are used for preparing the medicine for regulating and controlling the coliform group.
3. According to the method, the active ingredients of the alpinia oxyphylla and the alpinia oxyphylla extract are determined by adopting a UPLC-MS/MS method combined with high performance liquid chromatography-mass spectrometry, the detection method is quick, efficient and high in sensitivity, the method can be used for simultaneously determining multiple ingredients of the alpinia oxyphylla medicinal material, the main ingredients of the method are nootkatone, nootkatone A, nootkatol and the like, and reference is provided for overall quality control and clinical application of the alpinia oxyphylla medicinal material in the future.
4. The alpinia oxyphylla and the alpinia oxyphylla extract can effectively regulate and control the expression level of Scr, BUN, TG, TC and LDL-C, increase the content of HDL-C, improve renal function injury and reduce vascular injury, can be effectively used for preparing a medicament for treating diabetic nephropathy, realizes effective treatment of diabetic complications, and provides an important basis for expanding the clinical application of the alpinia oxyphylla extract.
Drawings
FIG. 1 shows the urine protein content of rats in each group for 24 hours in the example of the present invention;
FIG. 2 is a pathological section of kidney of rats in each group according to the example of the present invention, wherein a represents a normal group; b-a model set; c-positive group; d-salted fructus alpiniae oxyphyllae group; e-raw fructus alpiniae oxyphyllae group;
FIG. 3 shows the expression of TGF- β 1, a protein involved in immunohistochemical analysis of kidney tissues in each group according to an example of the present invention; wherein a-normal group; b-a model set; c-positive group; d-salted fructus alpiniae oxyphyllae group; e-raw fructus alpiniae oxyphyllae group;
FIG. 4 shows the expression of TGF- β 1 and α -SMA in various groups of kidney tissues in accordance with embodiments of the present invention;
FIG. 5 is a comparison of alpha diversity indices for each group in an embodiment of the present invention, wherein a is the normal group; b is a model group; c is a salt group; d is an intelligence development group;
FIG. 6 is a graph comparing the SCFAs content of each group of short chain fatty acids in examples of the present invention, wherein A-acetic acid; b-propionic acid; c-butyric acid.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1-extraction method of raw and salted fructus alpiniae oxyphyllae extracts, comprising the steps of:
(1) pulverizing salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae, and sieving with 40-mesh sieve to obtain salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae powder;
(2) respectively adding 7 times of 95% ethanol solution into the fructus Alpinae Oxyphyllae powder and fructus Alpinae Oxyphyllae powder, heating and reflux-extracting for 2 times, each time for 1.5 hr, filtering, mixing filtrates, rotary evaporating to recover ethanol, and oven drying at 68 deg.C to obtain fructus Alpinae Oxyphyllae extract and fructus Alpinae Oxyphyllae extract.
Example 2-extraction method of raw and salted fructus alpiniae oxyphyllae extracts, comprising the steps of:
(1) pulverizing salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae, and sieving with 60 mesh sieve to obtain salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae powder;
(2) respectively adding 9 times of 95% ethanol solution, heating and reflux-extracting for 2 times (2.5 hr each time), filtering, mixing filtrates, rotary evaporating to recover ethanol, and oven drying at 72 deg.C to obtain fructus Alpinae Oxyphyllae extract and fructus Alpinae Oxyphyllae extract.
Example 2 assay of extracts of raw Alpinia oxyphylla and salt Alpinia oxyphylla
The UPLC-MS/MS method combined with high performance liquid chromatography-mass spectrometry is adopted to effectively measure the contents of 9 chemical components in the extraction of the fructus alpiniae oxyphyllae and the fructus alpiniae oxyphyllae, the detection method is rapid, efficient and high in sensitivity, and the method can be used for simultaneously measuring multiple components of the fructus alpiniae oxyphyllae medicinal materials.
In the chromatographic analysis: the chromatographic column is Phenomenex kinetex 2.6um XB-C18100A (2.1mm × 50 mm); the chromatographic conditions are as follows: the chromatographic column is Phenomenex kinetex 2.6um XB-C18100A (2.1mm × 50 mm); the flow rate of the mobile phase is 0.40mL/min of 0.2 per mill formic acid water-0.2 per mill formic acid acetonitrile, the column temperature is 40 ℃, and the detection wavelength is 260 nm; sample volume 5 μ, gradient elution:
the mass spectrometry conditions were: an ion source: an electrospray ion source; the detection mode is as follows: a multiple reaction monitoring mode; adopting a positive ion mode; source ejection voltage: 5500/-4500V; ion source temperature: 550 ℃; curtain gas: 30 psi; atomizing: 55psi, heating gas: 55psi, nitrogen gas was used throughout.
The invention compares different chromatography columns in terms of their selection: phenomenex kinetex XB-C18100A (2.1mm X50 mm, 2.6 μm), Waters Xbridge C18(2.1mm X100 mm, 3.5 μm) and Agilent Poroshell 120SB-C18(4.6mm X50 mm, 2.7 μm) the results show that the Phenomenex kinetex XB-C18 column is superior to other columns in retention of chromatographic peaks and peak shape, and the analysis time to achieve baseline separation is shortened, efficiency is improved, so the column was selected.
In the selection of mobile phase systems, 3 mobile phase systems were examined: the experimental results prove that when the mobile phase is acetonitrile (containing 0.2 thousandth formic acid) -0.2 thousandth formic acid aqueous solution, the peak shapes and the peak emergence time of 9 components in the nootropic sample are superior to those of other mobile phases, so that the acetonitrile (containing 0.2 thousandth formic acid) -0.2 thousandth formic acid aqueous solution is selected as the mobile phase.
In the selection of mass spectrum conditions, the experiment tries to quantify 9 components to be detected in an MRM positive ion and negative ion switching mode, and finally selects a positive ion mode to detect the contents of nootropic ketone A, nootropic ketone B, myricetin, chrysin, nootropic alcohol, isoxaxanthin, kaempferol, nootkatone and apigenin-7, 4' -dimethoxy ether.
The invention determines the contents of 9 chemical components of nootkatone A, nootkatone, myricetin, isoxaxanthin, kaempferide, nootkatone B, nootkatol, chrysin and apigenin-7, 4' -dimethyl ether before and after the salt roasting of nootkatone. And the results of the content determination of 9 components of 4 batches of the medicinal materials for producing the intelligence and the salt intelligence are shown that the content of the nootkatone is the highest and reaches 1.525mg g-1～16.881mg·g-1Secondly 0.308mg g of nootropic alcohol-1～1.977mg·g-1Followed by nootropic ketonic methyl 0.145mg g-1～1.977mg·g-1The content of other components is low, and the content of flavonoids is less than 0.6mg-1。
Example 3 pharmacodynamic evaluation of Alpinia oxyphylla salt-roasted rats with diabetic nephropathy
1. Extraction of fructus Alpinae Oxyphyllae
Sun drying fructus Alpinae Oxyphyllae, pulverizing, and selecting fructus Alpinae Oxyphyllae and shell portion. Respectively placing 500g of raw fructus Alpinae Oxyphyllae and salt fructus Alpinae Oxyphyllae powder in a 10L round-bottom flask, adding 8 times of 95% ethanol, heating and reflux-extracting for 2 times, each time for 2 hr, filtering, mixing filtrates, rotary evaporating to recover ethanol, and oven drying in oven at 70 deg.C to obtain extract. 43.12g of raw fructus alpiniae oxyphyllae crude extract and 44.36g of salt fructus alpiniae oxyphyllae crude extract are respectively obtained.
2. Model making and grouping of DN rats
Male SD rats, weighing 200g-230g, SPF grade, all mice were housed for one week in 12h light/dark cycle acclimatized feeding at 25 ℃ room temperature and 50% -60% relative humidity, without adverse reactions, and the rats were subjected to a single intraperitoneal injection of 1.0mL of a solution of Streptozotocin (STZ) freshly dissolved in 0.1M citrate buffer (pH 4.5) at 50 mg/kg. One week after injection, blood was taken from the tip of the tail of the rat to measure the blood glucose level. Rats with blood glucose levels above 250mg/dL (13.88mmol/L) were considered diabetic rats. Blood glucose and 24h urine protein were monitored periodically weekly thereafter, and mice that continued to develop proteinuria were considered DN-modelled.
The modelled DN rats were randomly divided into 5 groups of 7 rats each. Namely a normal group (ND), a model group (STZ), a positive drug group (STZ-Met) of 40mg/kg, a salt fructus Alpinae Oxyphyllae group of 800mg/kg (SAOF), and a raw fructus Alpinae Oxyphyllae group of 800mg/kg (CAOF), wherein the normal group and the model group are administered with distilled water of the same volume, the positive group is administered with metformin of 40mg/kg for intragastric administration, and the raw fructus Alpinae Oxyphyllae group and the salt fructus Alpinae Oxyphyllae group are administered with raw fructus Alpinae Oxyphyllae extract and the salt fructus Alpinae Oxyphyllae extract of 800mg/kg for intragastric administration. Drinking and free diet were not restricted for the duration of the experiment for 4 weeks. Blood glucose levels and body weight were monitored weekly for each rat.
3. Observation index
(1) Detecting proteinuria for 24 h: after 4 weeks of administration, the rats were fasted for 12h, placed in metabolic cages to collect 24h proteinuria in the rats, and assayed for 24h proteinuria content. The 24h proteinuria was performed using Nanjing's kit to build Biotechnology Limited.
(2) Biochemical indexes in serum: the levels of urine nitrogen (BUN), creatinine (Scr), Total Cholesterol (TC), Triglyceride (TG), high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) in serum were determined using Nanjing Biotechnology Limited kit, and were performed strictly according to the instructions.
(3) Pathological tissues: after the experiment is finished, the right kidney tissue of the rat is dissected and taken out, dehydrated in a polychlorinated formaldehyde solution, embedded, sliced by 4 mu m, observed under an optical microscope after HE staining and described.
(4) Immunohistochemical detection of transforming growth factor-beta 1 (TGF-beta 1)
Paraffin section was dewaxed to water, kidney tissue section was placed in a repair box filled with citric acid antigen repair buffer (pH 6.0) for antigen repair in a microwave oven, the section was placed in 3% H2O2 solution, incubated at room temperature in the dark for 25min, the slide was placed in PBS (pH 7.4) and shaken and washed 3 times on a decolorizing shaker, and endogenous peroxidase was blocked 5min each time. 3% BSA was added dropwise to the tissue in the formation ring to cover the tissue uniformly, and the tissue was blocked at room temperature for 30 min. Primary antibody (rabbit anti-mouse TGF-. beta.1 monoclonal antibody) diluted appropriately (1: 100) was added dropwise thereto, incubated overnight at 4 ℃ and the slide was washed 3 times with shaking in PBS (pH 7.4) for 5min each in a decolorizing shaker. After the section was slightly spun dry, a secondary antibody (HRP-labeled) to the corresponding species was added dropwise to the ring to cover the tissue, and the mixture was incubated at room temperature for 50 min.
DAB color development: slides were washed 3 times 5min each time in PBS (pH 7.4) with shaking on a destaining shaker. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development. Counterstaining cell nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water. Dewatering and sealing: placing the slices in 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-xylene I for 5min, dehydrating, removing the slices from xylene, air drying, and sealing with neutral gum. Microscopic examination and image acquisition and analysis.
(5) Immunoblotting detection of kidney TGF-beta 1 and alpha-SMA expression
a. And (3) extracting total protein: extraction of total kidney protein from various groups of rats using a triumph bioassay: 50mg rat kidney tissue was minced in a 2mL centrifuge tube, 100. mu.L of BfferA and 100. mu.L of BuferB (17. mu.L of 10mMPMSF and 1. mu.L of proteolytic enzyme inhibitor were added to 1mL of BufferA before use), mixed well and placed on ice to homogenize for 1min, and left to stand for 30min to lyse the cells sufficiently. Centrifuging for 10min (4 deg.C, 3000rpm), and collecting supernatant to a new 2mL centrifuge tube to obtain total protein.
bca protein concentration assay: protein concentration was measured using BCA protein concentration assay kit (biosharp), and absorbance was measured at 562nm, and the protein concentration in the sample was calculated using absorbance as ordinate and BSA content as abscissa as a standard curve.
c. Protein quantification, denaturation and preparation of the upper samples: after protein concentration determination, diluting other samples to the same concentration by using precooled high-pressure steam sterilization water according to the minimum sample protein concentration, sealing the covers of all the samples by using sealing films, placing the samples in a constant-temperature water bath kettle at 90 ℃ for 8min to completely denature protein samples, adding 5 times of protein loading buffer SDS-PAGE loading buffer according to a ratio of 4:1 to prepare upper samples, subpackaging the upper samples in centrifugal tubes, cooling and placing the upper samples in a refrigerator at-80 ℃ for storage.
SDS-PAGE electrophoresis
SDS-PAGE electrophoresis separation gel was prepared according to the formulation in Table 1:
TABLE 1 preparation of solutions for electrophoretic separation gels
Preparing 10% separation gel according to the formula, and quickly adding ammonium persulfate and TEMED after the other solutions are prepared and uniformly shaking. And (3) sucking the separation glue by a liquid transfer gun to slowly pour the glue along one corner of the glass plate, avoiding generating air bubbles in the glue pouring process, determining the height of the glue pouring according to the height of the glue making comb, generally being not less than 2cm, adding a pure water seal layer after pouring, and waiting for the solidification of the separation glue.
SDS-PAGE electrophoresis concentrate was prepared according to the formulation of Table 2:
TABLE 2 solutions for preparing electrophoretic concentrates
5 percent of concentrated glue is prepared according to the formula, and finally TEMED is added and shaken up to be filled with glue. And (3) injecting the concentrated glue to the top of the glass plate, vertically inserting the glue making comb, avoiding generating bubbles during operation, and standing until the concentrated glue is solidified and the comb is pulled out.
Loading and electrophoresis: adding enough 1X electrophoresis buffer solution into the electrophoresis tank to submerge the glass plate, sucking a proper amount of sample and slowly adding the sample into the gel making hole, wherein the sample can be flushed out of the gel making hole due to too fast sample adding. Electrophoresis conditions: under the condition of room temperature, the electrophoresis is stopped when the electrophoresis is carried out at a constant voltage of 100V to be close to the bottom of the gel.
e. Film transfer: and soaking the PVDF membrane in an anhydrous methanol solution for activation for 1 h. And pouring the membrane transferring liquid into an empty tray to soak the membrane transferring clamp, the spongy cushion, the rolling rod and the membrane transferring filter paper, and slightly rolling the rolling rod back and forth after the membrane transferring liquid wets the filter paper to remove bubbles in the filter paper. Carefully, the separation gel after electrophoresis is smoothly scraped off and placed on a membrane-rotating filter paper, and the separation gel is gently rolled back and forth by a rolling rod to remove bubbles. The PVDF membrane was placed on the upper layer of the separation gel, and the air bubbles were removed. And (5) covering the filter paper and the spongy cushion, and closing the rotary die clamp. The clip is placed in the transfer tank so that the black side of the clip faces the black side of the tank and the white side of the clip faces the red side of the tank. Film transferring conditions: under the condition of ice bath, the film is rotated for 40min at 200 mA.
f. Blocking and immune response: transferring the PVDF membrane subjected to membrane conversion into 5% skimmed milk sealing solution with the front side facing upwards, and sealing for 2h by slowly shaking a shaking table at room temperature. After blocking was complete, the PVDF membrane was washed twice with 1 XTBST solution. Adding primary anti-dilution TGF-beta 1(1:500, ab92486, Abcam, UK) and alpha-SMA (1:500, Abcam, USA) and incubating in a freezer at constant temperature of 4 ℃ for 24h, recovering the primary antibody the next day, washing PVDF membrane with TBST for 6 times, shaking quickly by a shaking table and washing for 8 min/time. Adding horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) secondary antibody diluent, incubating the secondary antibody for 2H by shaking the shaking table slowly at room temperature, recovering the secondary antibody, washing the PVDF membrane for 3 times by TBST after recovery, and washing by shaking the shaking table quickly for 8 min/time.
g. And (3) developing: developing the PVDF film by using a chemiluminescence method to prepare developing solutions BeyoECL Star A solution and B solution according to the ratio of 1: 1, uniformly mixing, putting the membrane with the protein surface upwards on a high-sensitivity chemiluminescence imaging system, uniformly adding the membrane on the surface of the PVDF membrane by using a 200uL liquid-transferring gun for exposure and development, storing a picture in a computer, and determining proper exposure intensity and time according to the sample amount and the difference between different proteins.
Image J grey value analysis: grey value analysis was performed on the protein conditions of the developed pictures using Image J software.
4. Results of the experiment
(1) The urine protein content of diabetic nephropathy rats was significantly increased (p <0.05) compared to normal rats. Treatment with the raw and salted Alpinia oxyphylla groups significantly reduced urinary protein levels (p <0.05) compared to the model group, and the results are shown in FIG. 1.
(2) Effects of renal function:
as shown in Table 3, after 4 weeks of gavage, the levels of Scr, BUN, TG, TC, LDL-C were significantly increased and the level of HDL-C was decreased in the model group, as compared with the normal group. Compared with the model group, the plasma of the raw sharpleaf galangal fruit group has significantly reduced Scr, BUN, TG, TC and LDL-C and increased HDL-C level, and has statistical significance (P <0.05 and P < 0.01). The contents of BUN, TG, TC and LDL-C in the sharpleaf galangal fruit group are obviously reduced compared with the model group, and the statistical significance is achieved (P is less than 0.05, and P is less than 0.01).
Table 3 comparison of rat kidney function parameters (mean ± SD, n ═ 7)
(3) Pathological tissue analysis
Compared with the normal group, the diabetic nephropathy rats gradually lose weight, do not move and have more urination. As shown in fig. 2, pathological section of rat kidney, glomerular morphological structure of normal group has no obvious abnormality; the renal tubules are arranged closely, the morphological structure of epithelial cells is not obviously abnormal, and inflammatory cell infiltration is not obviously seen. In the model group, individual tubular necrosis calcifications were visible in the tissues; a small amount of renal tubular epithelial cell particle degeneration was seen. The morphological structure of the glomerulus tissue of the metformin group is not obviously abnormal, and the morphological structure of the glomerulus tissue of the raw fructus alpiniae oxyphyllae group is not obviously abnormal; no significant inflammatory cell infiltration was seen. The salt alpinia oxyphylla tissue can be seen with a small amount of degeneration of renal tubular epithelial cell particles and a small amount of shedding of the brush border of the renal tubules.
(4) Immunohistochemical staining of TGF-. beta.1 in each group
The expression of TGF-beta 1 related to the kidney tissue fibrosis is detected by an immunohistochemical staining method. The expression of TGF-. beta.1 was upregulated in the model group compared to the normal group (p < 0.01). TGF- β 1 levels in the kidney were significantly reduced in figure 3 (P <0.05) following treatment with 800mg/kg of raw and salt sharpleaf galangal fruit extract.
(5) Expression of TGF-beta 1 and alpha-SMA in rat kidney tissue
Compared with the normal group, the content of TGF-beta 1, alpha-SMA protein in the kidney tissue of the rat in the model group is obviously increased; compared with the model group, the kidney tissue TGF-beta 1, alpha-SMA protein content of the rats in the intelligence-growing and salt intelligence-developing groups is obviously reduced (P < 0.01). The results are shown in FIG. 4.
The experimental result shows that the expression of TGF-beta 1 protein in the kidney tissue of diabetic nephropathy is obviously increased, the expression of alpha-SMA in the kidney of a diabetic nephropathy patient is increased, extracellular matrix is accumulated, and the apoptosis of renal tubular epithelial cells is increased, so that the renal injury is caused, and DN rats treated by CAOF and SAOF obviously inhibit the up-regulation of TGF-beta 1 and alpha-SMA, which indicates that CAOF and SAOF may have the potential effect of relieving DN renal fibrosis. In conclusion, the treatment of the sharpleaf galangal fruit extract can reduce the levels of Scr, BUN, TG, TC and LDL-C of diabetic nephropathy rats, increase the content of HDL-C and improve renal function injury, and the sharpleaf galangal fruit treatment can reduce renal fibrosis and renal tubular injury by regulating the expression of TGF-beta 1 and alpha-SMA, thereby delaying the occurrence of diabetic nephropathy.
Example 4 Effect of fructus Alpinae Oxyphyllae roasted with salt on intestinal flora of DN rat
1. To assess whether the CAOF and SAOF extracts modulate the composition of DN rat intestinal flora, rat fecal samples were analyzed by 16S rRNA high throughput sequencing in this experiment. The abundance and diversity of the intestinal flora was analyzed using alpha diversity. In general, the alpha diversity index includes chao1, ACE, observed _ specs, and shannon indices. Among them, the chao1, ACE, observed _ species index is used to reflect the abundance of intestinal flora, and the Shannon index is used to reflect the species diversity.
The results are shown in FIG. 5. Compared with the STZ group, the indexes of chao1, ACE, observed _ species and Shannon of the SAOF group are obviously improved, which indicates that the diversity and the richness of the intestinal flora of the salt intelligence-improving group are higher than those of the normal group. Compared with the STZ group, the CAOF group and the SAOF group have higher chao1, ACE and observed _ species indexes, which indicates that the abundance of intestinal flora of rats in the administration group is obviously increased. The Shannon index of the intelligence-developing group is lower than that of the model group, which indicates that the diversity of intestinal flora of the diabetic nephropathy rats is obviously increased.
2. Gas Chromatography (GC) detection of short chain fatty acids
(1) Extraction of SCFAs from rat feces
Taking 200mg of fresh excrement, adding 4mL of deionized water, uniformly mixing and shaking, standing at room temperature for 20min, centrifuging at 4 ℃ and 10000rpm for 15min, transferring supernatant to a new EP tube, filtering by a 0.45 mu m filter, taking 900uL of supernatant and 100uL of 100 mu g/mL 2-butyl acetate to mix and prepare a sample, and processing on a sample machine for GC detection.
(2) Gas chromatography detection conditions
The column was HP-FFAP (30 m.times.0.25 mm.times.0.25 μm); the injection port temperature is 280 ℃, the split ratio is 5:1, and the programmed temperature program: the initial temperature is 100 ℃, and the temperature is kept for 3 min; heating to 160 deg.C at 5 deg.C/min, and maintaining for 3 min; the temperature is increased to 190 ℃ at a speed of 2 ℃/min. The detector is a Flame Ionization Detector (FID) and the temperature is 300 ℃. The injection volume was 2. mu.L.
(3) Determination of SCFAs content of short-chain fatty acid
The contents of acetic acid, propionic acid and butyric acid in the normal group, the model group and the administration group have obvious statistical difference after being compared pairwise, and the contents of acetic acid, propionic acid and butyric acid in the model group are lower than those in the normal group and the administration group. Compared with the model group, the content of acetic acid, propionic acid and butyric acid in the sharpleaf galangal fruit salt group is increased and has statistical significance (P <0.05), and the content of propionic acid and butyric acid in the raw sharpleaf galangal fruit group is also increased, while the increase of the acetic acid content is not significant and has no statistical significance. The content of SCFAs in the fructus alpiniae oxyphyllae is acetic acid, butyric acid and propionic acid in turn. The results are shown in Table 4 and in FIG. 6.
TABLE 4 determination of the content of short-chain fatty acids SCFAs
In conclusion, the analysis shows that the diabetic nephropathy causes a certain degree of intestinal flora imbalance, the proportion of bifidobacteria in the ND group is obviously increased, and the concentrations of short-chain fatty acid acetic acid, propionic acid and butyric acid are reduced.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The application of the extracts of the raw sharpleaf galangal fruit and the salt sharpleaf galangal fruit is characterized in that the extracts of the raw sharpleaf galangal fruit and the salt sharpleaf galangal fruit are applied to the preparation of the medicine for treating diabetic nephropathy.
2. The use of the extract of Alpinia oxyphylla and Alpinia oxyphylla as claimed in claim 1, wherein the extract of Alpinia oxyphylla and Alpinia oxyphylla is used for preparing the medicine for treating renal fibrosis and renal tubular injury.
3. The use of the extracts of Alpinia oxyphylla and Alpinia oxyphylla as claimed in claim 2, wherein the use of the extracts of Alpinia oxyphylla and Alpinia oxyphylla for preparing the medicine for regulating the expression of transforming growth factor-beta 1 and treating renal fibrosis and renal tubular injury.
4. The use of the extracts of Alpinia oxyphylla and Alpinia oxyphylla according to claim 2, wherein the extracts of Alpinia oxyphylla and Alpinia oxyphylla are used for preparing the medicine for regulating the expression of alpha-smooth muscle actin and treating renal fibrosis and renal tubular injury.
5. The use of the extracts of Alpinia oxyphylla and Alpinia oxyphylla as claimed in claim 1, wherein the use of the extracts of Alpinia oxyphylla and Alpinia oxyphylla in preparing medicine for regulating coliform group is provided.
6. The use of the extract of Alpinia oxyphylla and Alpinia oxyphylla as claimed in claim 5, wherein the extract of Alpinia oxyphylla and Alpinia oxyphylla is used for preparing the medicine for promoting the generation of SCFAs, short chain fatty acids, and regulating the flora of large intestine.
7. A method for extracting Alpinia oxyphylla and Alpinia oxyphylla extract as claimed in any one of claims 1 to 6, comprising the steps of:
(1) pulverizing salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae, and sieving with 40-60 mesh sieve to obtain salt fructus Alpinae Oxyphyllae and fructus Alpinae Oxyphyllae powder;
(2) respectively adding 7-9 times of 95% ethanol solution into salt fructus Alpinae Oxyphyllae powder and fructus Alpinae Oxyphyllae powder, heating and reflux-extracting for 2 times, each time for 1.5-2.5 hr, filtering, mixing filtrates, rotary evaporating to recover ethanol, and oven drying at 68-72 deg.C to obtain salt fructus Alpinae Oxyphyllae extract and fructus Alpinae Oxyphyllae extract.
8. The method for determining the extracts of Alpinia oxyphylla and Alpinia oxyphylla prepared by the method according to claim 7, wherein the determination is carried out by UPLC-MS/MS method combined with high performance liquid chromatography-mass spectrometry.
9. The method for determining the extracts of Alpinia oxyphylla and Alpinia oxyphylla prepared by the extraction method according to claim 7, wherein the chromatographic conditions are as follows: the chromatographic column is Phenomenex kinetex 2.6um XB-C18; the mobile phase is 0.2 per mill of formic acid water-0.2 per mill of formic acid acetonitrile by mass concentration, the flow rate is 0.40mL/min, the column temperature is 40 ℃, and the detection wavelength is 260 nm; the injection volume is 5 μ.
10. The method for determining the extracts of Alpinia oxyphylla and Alpinia oxyphylla prepared by the extraction method according to claim 7, wherein the mass spectrometry conditions are as follows: an ion source: an electrospray ion source; the detection mode is as follows: a multiple reaction monitoring mode; adopting a positive ion mode; source ejection voltage: 5500/-4500V; ion source temperature: 550 ℃; curtain gas: 30 psi; atomizing: 55psi, heating gas: 55psi, nitrogen gas was used throughout.
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