CN111170977A - Plantain herb effective component for resisting diarrhea and application thereof - Google Patents
Plantain herb effective component for resisting diarrhea and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-diarrhea active ingredient of plantain and application thereof. The invention identifies the main chemical components of the ethyl acetate extraction part and the rest part of the plantain herb by ultra-high performance liquid chromatography-flight time mass spectrometry, thereby determining 14 monomer components of the effective part of the plantain herb, and then screens 3 anti-diarrhea effective components of scutellarein, caffeic acid and luteolin by a molecular docking technology. Through a castor oil-induced mouse diarrhea model test, the luteolin and the scutellarein have remarkable diarrhea resistance, but the diarrhea resistance effect of the caffeic acid is not obvious. The invention further provides application of luteolin or scutellarein separated from plantain in preparation of anti-diarrhea drugs.
Description
Technical Field
The invention relates to separation and identification of anti-diarrhea active ingredients of plantain and application thereof, belonging to the field of separation and application of the anti-diarrhea active ingredients of the plantain.
Background
Diarrhea is a disease characterized by an increased frequency of defecation, which is one of the causes of morbidity and mortality in developing countries, particularly in many young animals such as piglets and lactating calves. In addition, diarrhea is also a major cause of morbidity and ulceration in the elderly and in people infected with HIV. The physiological mechanisms that lead to diarrhea include increased intestinal motility, increased fluid volume in the intestinal lumen, and decreased absorption of water and electrolytes.
Na+/K+ATPases are present in the basolateral membrane of small intestine cells, providing the driving force for the active transport of many electrolytes. Inhibition of this intestinal enzyme may be critical to the regulation and absorption of Na + and K + in the gut and may lead to accumulation of intestinal fluid and thus to the induction of diarrhea (Rachmilewitz, D., F. Karmeli, and P. Sharon, secreted colon Na-K-ATPase activity in active intestinal activity, 1984.20(8): p.681-4.Yakubu, M.T. multidrug production, 1984.20 (8))And S.S.Salimon, antipathologic of aqueous extract of Mangifera L.leaves in great abilities of agriculture of Ethnopharmacology 2015.163: p.135-141.Yakubu, M.T., et al, antipathologic Activity of Musa paradisiac Sap in Wistar rates, event-base compatibility and Alternative Medicine 2015.). To date, there has been little drug development or research on Na+/K+-atpase as a target for the treatment of diarrhea; AQP 4Is an Aquaporin involved in the water metabolism in the intestinal tract, and if the expression of this protein Is inhibited, it leads to a disturbance in the absorption and secretion of water in the intestinal tract and promotes intestinal fluid accumulation-induced diarrhea (Zhang, D., et al, Aquaporin-4Is downward regulated in the basic Membrane of Ilium Epitheal Cells along with intestinal fluid accumulation, Escherichia coli-induced diarrhea in Rice, frontiers in Microbiology, 2018.8.); opioid receptors are involved in the regulation of intestinal motility, and drug binding to opioid receptors can inhibit intestinal motility and prolong intestinal content retention time, thereby alleviating diarrhea (Fujita, W.E., et al, Molecular characterization of intestinal motility and intestinal side effect targeting mu-delta opioid receptors: Biochemical Pharmacology,2014.92(3): p.448-456.). Therefore, the three proteins are likely to be target proteins of drugs for treating diarrhea. Currently, the pharmacotherapy of diarrhea is non-specific and generally aims to reduce the inconvenience caused by frequent defecation, dehydration and discomfort.
Although there are some effective drugs, such as chemical drugs, around the world, these drugs can induce adverse reactions, and natural plants have been the focus of recent attention due to their safety and availability. To avoid diarrhea, physicians and patients in many developing countries (e.g., china) still rely on traditional medications. To this end, the world health organization initiated a diarrhea control program involving the use of traditional herbs.
The plantain herb is a natural medicinal plant, has the advantages of rich resources, low price, containing a plurality of active ingredients and targets, low toxicity and difficult generation of drug resistance. Plantago asiatica contains some bioactive compounds, such as flavonoids, caffeic acid derivatives, iridoid glycosides, terpenoids, alkaloids and some organic Acids (Rossted, N.et. et. al., Chemotaxonomy of Plantago. Iridoid glycosides and caffeoyl phenolics. phytochemistry,2000.55(4): p.337-348.Taskova, R.et. et. Iridodperts of genus Plantago L.and the same systematic chemical design. C. Journal of biosciences,2002.57(1-2): p.42-50. grubic, R.J., Simulan et. S.S.HPLC-D.Action, Deteron.and 97. biological and chemical et. 97. biological of Plantago. and chemical et. 3. biological et. of Pasteur. 52. biological et. f.7. biological analysis, Pasteur. Biocide, et. 3. and chemical et. 3. biological). At present, the research on plantain at home and abroad mainly focuses on the aspects of antioxidation, anti-inflammation and antibiosis (Goncaves, S., et al., analytical activity and verbascoside content extracts from two uninhibited end plants and products,2015.65: p.198-202). Although the pharmacopoeia records the anti-diarrhea effect of plantain (the pharmacopoeia of the people's republic of China (2010) and is often used for folk treatment of diarrhea of piglets, no relevant literature reports exist and effective ingredients for the effect are unclear, so that the formulation of quality standards and the research of mechanisms are limited.
Disclosure of Invention
The main purpose of the invention is to separate the monomeric compound with anti-diarrhea activity from the plantain extract;
the other purpose of the invention is to apply the separated monomer compound to the preparation of the anti-diarrhea medicine;
the above object of the present invention is achieved by the following technical solutions:
the method comprises the following steps of firstly identifying the main chemical components of the ethyl acetate extraction part and the rest part of the plantain herb by ultra-high performance liquid chromatography-flight time mass spectrometry: in both positive and negative ion modes, most compounds can produce [ M + H [ + ] H]+Or [ M-H]-Molecular ion, then at 5X 10-6Accurately measuring the mass and isotope abundance ratio of ions by a high-resolution mass spectrometer within an error range; finally, the molecular Formula of the compound was determined based on the exact mass and isotopic abundance ratio by the Formula Finder function of the Peakview 2.0 software. Retention time and secondary mass spectral information were further confirmed for compounds with reference standard substancesThe structure of (1); for compounds for which a reference standard is not available, the fragment composition is first calculated from the exact mass of the fragment ion and the mass spectra of compounds of similar structure are studied by comparing the possible structural formulae given in the chemical database. The mass spectrum characteristics were analyzed by the Peakview 2.0 software and the structure of the compound was estimated in combination with the similarity of the secondary spectra. Finally, the invention determines 14 monomer components of the effective part of the plantain herb, and the monomers are mainly divided into 3 phenylethanoid glycosides and 3 triterpenes, 6 flavonoids and 2 organic acids; thereafter, 3 kinds of monomer components including scutellarein, caffeic acid and luteolin were selected by molecular docking technique for 3 kinds of proteins associated with diarrhea and further studied in the next step.
Castor oil is an effective laxative and is widely used to study symptoms associated with diarrhea and exercise in intestinal transit. The castor oil-induced diarrhea is attributable to ricinoleic acid, an active ingredient thereof. In the intestinal lumen, castor oil is hydrolyzed by lipase to ricinoleic acid, and anticoagulants of the intestinal mucosa interact with several endogenous mediators to induce diarrhea. Castor oil increases fluid secretion by adenylate cyclase as well as levels of cyclic adenosine monophosphate (cAMP). In addition, it reduces Na in the small intestine and colon+/K+-ATPase activity, preventing Na+And K+Re-absorption of (2). In order to investigate the antidiarrheal effect of 3 monomeric components or groups of monomeric components scutellarein, caffeic acid and luteolin isolated from Plantago asiatica, the present invention performed castor oil-induced diarrhea model experiments in mice. The test result shows that, among the three monomer components, luteolin and scutellarein have remarkable anti-diarrhea effect, but the anti-diarrhea effect of caffeic acid is not obvious.
In order to clarify the diarrhea-resisting action mechanism of luteolin and scutellarein, the invention carries out castor oil induced intestinal effusion test on rats. According to the experimental results, the volume and weight of the contents of the intestine were significantly reduced after treatment with luteolin and scutellarein compared to the model group, indicating that luteolin and scutellarein are involved in at least one of the major mechanisms associated with acute diarrhea, such as Na+/K+-inhibition of atpase activity, increasing intestinal peristalsis. Caused by castor oilDiarrhea is a change in intestinal homeostasis due to the formation of ricinoleic acid in the intestinal lumen. Na derived from ricinoleic acid+/K+Inhibition of the normal function of ATPase thus leads to changes in electrolyte permeability and contraction of the smooth muscle of the intestine, inducing severe diarrhoea. The results of this experiment show that luteolin and scutellarein significantly increase Na+/K+ATP enzyme activity, which indicates that both luteolin and scutellarein are mixed with Na+/K+ATPases have good binding capacity, which is consistent with molecular docking results.
Creatine Kinase (CK) is buffered by the temperature and spatial energy provided by the creatine kinase phosphate (CK/PCR) system to maintain cellular energy homeostasis, and is responsible for providing the correct Adenosine Triphosphate (ATP) ATPase function, such as sodium and potassium (Na)+/K+ATPase) and hydrogen (H + -ATPase) pumps, resulting in Na+，K+A change in ion level. To further elucidate whether luteolin and scutellarein improve Na indirectly+/K+-ATPase activity. The test results of the invention show that luteolin and scutellarein can improve creatine kinase activity reduction induced by castor oil and increase Na+And K+The concentrations of (A) indicate that luteolin and scutellarein can up-regulate creatine kinase and Na+/K+-ATPase activity, increased Na+And K+Thereby exerting an anti-diarrhea effect.
Na+/K+Results of-ATPase and creatine kinase Activity show that luteolin and scutellarein can significantly up-regulate the expression of relative gene levels of ckb and Atp1b3, which means that luteolin and scutellarein can change Na by regulating the gene expression level+/K+-the activity of atpase and creatine kinase to exert an anti-diarrheal effect.
In summary, luteolin or scutellarein isolated from plantain is found to regulate Na+/K+The activity of ATP enzyme and creatine kinase and the gene expression level thereof play the role of anti-diarrhea.
The invention provides an anti-diarrhea active component of plantain, which is a mixture of one or more than one monomer compounds selected from luteolin, scutellarin or caffeic acid according to any proportion; preferably, the plantain antidiarrheal active ingredient is any one monomer compound selected from luteolin and scutellarein or a mixture of luteolin and scutellarein in according to any proportion.
The luteolin, scutellarein or caffeic acid disclosed by the invention can be prepared by methods disclosed in the existing literatures, and can also be separated from plantain according to the methods provided in the specification.
For reference, the present invention provides a method for separating luteolin, scutellarein or caffeic acid from plantain herb, the method comprising:
separating and identifying the ethyl acetate part of the plantain herb by ultra-high performance liquid chromatography-time-of-flight mass spectrometry.
Wherein, the separation and identification method is preferably as follows: separation was performed on a Waters Acquity UPLC BEH C18 column, eluting with 0.1% formic acid and acetonitrile; time-of-flight mass spectrometry and electrospray ionization sources for positive and negative ion mode analysis; target search and non-target search are executed by Peakview 2.0/masterview1.0 or Markerview 1.2.1 software, and then the composition of the compound is determined by the accurate quality and isotope abundance ratio of the software target screening function; the structure of the isolated monomeric compounds is determined by analyzing the MS/MS fragments or comparing them to standard substances and references.
It is a further object of the present invention to provide an anti-diarrhea pharmaceutical composition consisting of a therapeutically effective amount of luteolin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; or comprises scutellarein with a therapeutically effective dose or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; or consists of caffeic acid or pharmaceutically acceptable salts thereof with effective dose in treatment and pharmaceutically acceptable carriers; or a mixture of more than one of luteolin, scutellarin or caffeic acid with a therapeutically effective amount according to any proportion and a pharmaceutically acceptable carrier.
Mixing pharmaceutically acceptable amount of luteolin, scutellarein or caffeic acid with pharmaceutically acceptable carrier or diluent, and making into any suitable pharmaceutical composition by conventional method; the compositions are generally suitable for oral administration and for administration by injection, as are other methods of administration; the pharmaceutical composition can be prepared into various preparation forms such as tablets, capsules, powder, granules, pastilles, suppositories or oral liquid and the like according to the conventional preparation method of pharmaceutical preparations. Depending on the method of administration, the pharmaceutical composition of the present invention may contain 0.1% to 99% by weight, preferably 10% to 60% by weight, of luteolin, scutellarin or caffeic acid monomeric compounds.
Drawings
FIG. 1 shows the molecular docking results of scutellarein, luteolin and caffeic acid with anti-diarrhea related proteins; (A) scutellarein and Na+/K+-ATPase binding, (B) luteolin in combination with Na+/K+-atpase binding, (C) scutellarein and opioid receptor binding (D) caffeic acid binding to AQP 4.
FIG. 2 is a graph showing the evaluation of the anti-diarrhea effect of scutellarein, luteolin and caffeic acid monomer components.
FIG. 3 pathological sections of mouse colon tissue; (A) luteolin group; (B) scutellarein group; (C) a caffeic acid group; (D)3 ingredient mixing groups; (E) negative control (ethanol) group; (F) a positive control; (G) blank group; (H) each group pathology was scored.
FIG. 4 comparison of luteolin and scutellarein treated groups in intestinal fluid, enzyme activity, ion concentration and enzyme gene levels compared to model groups; (A-B) the volume and weight of the intestinal effusion; (C-D) measurement of enzyme Activity in rat Small intestine; (E-F) measurement of ion concentration in rat small intestine; (G) measurement of enzyme Gene level in rat Small intestine.
FIG. 5 shows the results of the enterokinesia test in mice.
FIG. 6 shows the structural formula of scutellarein.
FIG. 7 structural formula of luteolin.
FIG. 8 shows the structural formula of caffeic acid.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Test example 1 separation and identification of anti-diarrhea active ingredient in plantain and anti-diarrhea action mechanism study test
1 test Material
The plantain is obtained from local farmers in the province of Heilongjiang, China, and is then classified, cleaned and cut into small pieces. The moisture content of the original sample was 8.0%. Luteolin, scutellarein and caffeic acid were purchased from Solarbio (purity ≥ 99%)
2 test method
2.1 ultra high performance liquid chromatography-time of flight mass spectrometry
The main chemical components of the effective parts (ethyl acetate part and the rest parts) of the plantain are identified by ultra performance liquid chromatography-time of flight mass spectrometry. Separation was performed on a Waters Acquity UPLC BEH C18 column (100mm × 2.1mm, 1.7 μm), eluting with 0.1% formic acid (a) and acetonitrile (B). Time-of-flight mass spectrometry and electrospray ionization (ESI) sources for analysis of positive and negative ion modes; target search and non-target search are performed by Peakview 2.0/masterview1.0 or Markerview 1.2.1 software, and then the composition of the compound is determined by the exact mass and isotopic abundance ratio of the software target screening function. Their structure is determined by analyzing the MS/MS fragments or comparing them with standard substances and references (Huo, J. -H., et al., Identification and characterization of major constraints and manufacturing methods using an ultra performance consistency and time-of-light mass spectrometry (UPLC-ESI-Q-TOF/MS). chip Journal of Natural Medicines,2018.16(7): p.525-545.).
2.2 molecular docking
2.2.1 homologous modeling
Structural information of target proteins is very important, and homologous modeling techniques are widely used to model proteins. Currently in the Protein Database (PDB), there is no available tertiary structure for the opioid and AQP4 receptors. Therefore, the successful construction of a 3D model of GS, the amino acid sequence of which was retrieved from the UNIPRO database (http:// www.uniprot.org), and a sequence similarity search of the PDB protein database was performed using the NCBI-BLAST server (http:/BLAST. NCBI. nlm. nih. gov /) in order to obtain the optimal template for homology modeling. EasyModeller4.0(Pourseif, M.M., et al, A novel B-and helper T-cell epitopes-based therapeutic vaccine against Echinococcus grandis. BioImages: BI,2018.8(1): p.39-52.) was used to generate a model that integrates the Modeller program at the back end and is believed to work well within the recognition range of 30-40% sequence similarity.
2.2.2 model optimization and evaluation
Protein models generated by using homologous modeling often produce unfavorable bond lengths, bond angles, twist angles, and contact points. It is therefore important to minimize the model energy, regularize the local key and corner geometry, and relax tight contacts on the geometric chain. To verify the accuracy of the predicted opioid and AQP4 receptor structures, protein structures were optimized and evaluated by the server. Submitting the GS structure to a Chon (http:// redshift. med. unc. edu/chiron/index. php) server for model optimization, submitting the optimized GS model to a structure analysis and verification server SAVES (http:// service. mbi. ula. edu/SAVES /), and verifying the model by using a PROCHECK [10] program for selection.
2.2.3CDOCKER molecular docking
CDOCKER (Li, K., et al., radial Design of novel phosphorus side 3-kinase Gamma (PI3Kgamma) Selective Inhibitors: adaptive estimation integration 3D-QSAR, Molecular Docking and Molecular Dynamics simulation & biology, 2019.) method based on CHARMM is used for Molecular Docking. CDOCKER is a protein ligand molecule docking program written in the CHARMM script in the discovery studio3.0(DS3.0) software. Among them, proteins (sodium potassium atpase, opioid receptor and AQP4 receptor) and mass-produced small molecules were subjected to hydrogenation and electric field treatment, and a molecular docking procedure (Cui, w. -q., et al., Discovery of functional Anti-invasive Therapy Targeting Glutamine synthesis in Chemistry,2019.7(381)) was performed using DS3.0 software.
2.3 test animals
Male or female Kunming mice (18-22 g) and Wistar rats (180-200 g) were obtained from the Harbin medical university animal center (Harbin, China). They were kept in plastic cages at 22 + -2 deg.C, with free access to food and water. The test was conducted according to the "Experimental animal management rules" (Chinese national Committee for science and technology, 1988) and approved by the department of health, China, in compliance with the NIH guidelines and the relevant provisions of the institutional animal Care and use Committee of university.
2.4 model test for diarrhea in mice
The effect of 3 monomers on castor oil induced diarrhea was studied. Kunming mice were fasted for 18 hours, allowed free access to water, and randomly divided into 8 groups (6 per group). Three single drug groups were orally administered scutellarein (1.75mg/kg), caffeic acid (332.5mg/kg) and luteolin (5mg/kg), respectively; the three monomer mixed groups take a mixture of 3 monomers orally according to a certain proportion; model group oral castor oil (10 mL/kg); the positive group was given loperamide hydrochloride (2 mg/kg); negative groups were given ethanol (10%). After 30 minutes, each mouse was given 0.2mL of castor oil (voucher sample No. 20160920) to induce diarrhea. Mice without drug and castor oil were used as a blank group. The animals were then individually placed in individual cages lined with grease proofing paper and the total number of defecations, number of loose stools and rate of loose stool inhibition were calculated and repeated three times per group. After 4 hours of observation, animals were euthanized and colon tissue was collected.
2.5 histopathology
The same mice as used for the castor oil induced diarrhea test were used, and the colons were collected, fixed in 10% formalin, and subjected to routine histological procedures of paraffin embedding and light microscopy, respectively. In addition, histopathological scores were performed, including edema, extent of injury, leukocyte infiltration, crypt abscesses and goblet cell loss (Lu, Y., et al, Cambog uppress series injury sodium-induced pathology by enhancing cell viability and function. British Journal of Pharmacology,2018.175(7): p.1085-1099.). In this grading system, 0-3 (0, 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; 3, severe inflammation) is used for the severity of the inflammation, such as the degree of injury (0, no injury; 1, mucosal injury; 2, mucosal and submucosal injury; 3, transmural injury). A scale of 0-4 was used (0, no damage; 1, one third of basal damaged; 2, two thirds of basal damaged; 3, only the surface epithelium was intact; 4, entire crypts and epithelial cells were lost). The total histopathological score was determined from the sum of the scores for each parameter to reflect the overall extent of damage for each sample.
2.6 rat intestinal effusion test
Model tests for castor oil-induced intestinal fluid accumulation in the reference (Lu, Y., et al., Cambogin suspensions treated with lipid-induced metabolism Treg cell stability and function. British journal of Pharmacology,2018.175(7): p.1085-1099.) were performed. Rats were fasted for 18 hours, had free access to water, and were randomized into 4 groups of 6 rats each. 1.75mg/kg of scutellarein and 5mg/kg of luteolin are orally taken in groups 1 and 2; groups 3,4 were orally administered with equal volumes of water. After one hour, all but group 4 received 1mL of castor oil. After 3 hours, the animals were euthanized and subjected to resection to cut the small intestine from the pylorus to the cecum. The intestinal contents were collected in a graduated tube and their volume and weight were measured. The calculation was performed as follows: the inhibition ratio (%) of intestinal fluid volume is [ (a-B)/a ] × 100, where a represents the average fluid volume after castor oil administration and B represents the average fluid volume after drug treatment.
2.7 Na in the Small intestine+/K+Determination of the Activity of ATP-ase and creatine kinase
The small intestine of each group of rats was collected, and intestinal supernatant was prepared according to the Na content of the rats+/K+Determination of Na by ATP enzyme ELISA kit+/K+ATPase Activity (Lu, Y., et al, Cambogin substrates and nucleic acids by enhancing Treg cell stability and function. British Journalof Pharmacology,2018.175(7):p.1085-1099.)。
Add 50. mu.L of standards at different concentrations to the standard wells, add 10. mu.L of test sample to the test sample wells, and then add 40mL of sample dilution. Blank wells were not reagent added. To each well 100 μ L HRP-conjugate reagent was added, covered with tape adhesive and incubated at 37 ℃ for 60 minutes. Each well filled with wash solution (400 μ Ι _) was drained and washed four times for a total of five times. In order to obtain good performance, the liquid must be completely removed at each step. To each well 50mL of chromogen solution a and chromogen solution B were added, then mixed gently and incubated at 37 ℃ for 15 minutes in the absence of light. Finally, 50 μ L of stop solution was added to each well. The optical density at 450nm (o.d.) was read in 15 minutes using a microtiter plate reader. In the Excel worksheet, standard concentrations were used as abscissa and OD values were plotted as ordinate. And (5) drawing a linear regression curve of the standard substance, and calculating the concentration value of each sample according to a curve equation. With Na+/K+ATP-ase similarity, creatine kinase assay was determined according to rat creatine kinase ELISA kit.
2.8 real-time quantitative PCR
The steps of total RNA extraction and cDNA synthesis were performed according to the manufacturer's instructions. To investigate the effect of luteolin and wild baicalein on the gene levels of Na +/K + -ATPase and creatine kinase, ckb and Atp1b3 (Table 2) were selected for real-time quantitative PCR assays. The reaction conditions are 95 ℃, 10min and 40 cycles; 95 ℃ for 15 s; 50-60 ℃ for 1 min. The assay was repeated 3 times.
2.9 Na in the Small intestine+And K+Determination of concentration
The sodium kit measures sodium ions. 0.1g of tissue was added to 1mL of deionized water and homogenized in an ice water bath, centrifuged at 3500rpm for 10 minutes, and the supernatant was taken for testing. The standard tube and the sample tube were each charged with 300. mu.L of the reagent, and the standard tube and the sample tube were charged with 20. mu.L of the standard solution and the supernatant, respectively, followed by rapid addition of 200. mu.L of the accelerator within 15 seconds and dilution with 1mL of ethanol. After mixing, the absorbance of each tube was measured at 620nm using a spectrophotometer.
A represents the OD of the measuring tube, B represents the OD of the standard tube, C represents the concentration of the standard, and D represents the dilution factor of the sample before the test. The determination of potassium ion is based on a potassium test kit. The wavelength was 450 nm.
2.10 intestinal peristalsis test in mice
Mice were fasted for 18 hours and allowed free access to water. The animals were divided into 4 groups of 6 animals each. Except group 4, all groups were orally administered castor oil. After 1 hour, scutellarein was orally administered (1.75mg/kg) to each animal in group 1, luteolin was orally administered (5mg/kg) to each animal in group 2, and the same volume of water was administered to each animal in groups 3 and 4. After 1 hour, the animals orally took a charcoal suspension (0.2 mL/animal) containing 10% activated carbon suspended in 5% gum arabic. After 30 minutes, the animals were euthanized and the small intestine was immediately isolated. The advance distance of the charcoal powder in the small intestine and the total length of the intestine were measured, and the advance rate of the charcoal powder represents the percentage of the advance distance of the charcoal powder relative to the total length of the small intestine.
2.11 data and statistical analysis
All experimental data are expressed as mean ± SD. Differences between the two groups were determined by Student's st-test using SPSS Statistics 17.0 and in addition, pictures were drawn using GraphPad Prism 5.0 software. P <0.05 is used to indicate statistically significant difference.
3 results of the test
3.1 ultra high performance liquid chromatography-time of flight mass spectrometry
According to Table 1, the effective fraction of herba plantaginis has 14 kinds of components including plantaginin, verbascoside, 6-hydroxy-luteolin, baicalin, ursolic acid, diosgenin, hispidoside, apigenin, scutellarin, caffeic acid, ferulic acid, luteolin, akebia saponin B and epimeddium A, which are identified by mass spectrometry. However, among many chemical components, the active ingredient or active ingredient group that plays a role is not known at all.
TABLE 1 results of the liquid quality measurement of Plantago asiatica
3.2 molecular docking
According to FIG. 1(A), scutellarein and Na+/K+-the TYP-43 amino acid, GLY-848 amino acid and TRP-32 amino acid of ATPase form stable hydrogen bonds; FIG. 1(B) shows luteolin in combination with Na+/K+-the TYR-43 amino acid and TRP-32 amino acid of atpase form stable hydrogen bonds; indicating scutellarein, luteolin and Na+/K+Good binding of ATPase; FIG. 1(C) shows that scutellarein forms stable hydrogen bonds with amino acids LYS-236, LYS-306, ASP-57, ASP-150, and TYR-151 of opioid receptors, indicating that scutellarein binds well to opioid receptors; FIG. 1(D) shows that caffeic acid forms stable hydrogen bonds with amino acids ASN-184, HIS-66, and GLY-65 of AQP4, indicating that caffeic acid binds well with AQP 4. Aiming at 3 proteins related to diarrhea, molecular docking is carried out on the 3 proteins and micromolecules such as scutellarein, luteolin and caffeic acid, and 3 monomer components are screened out and are luteolin, scutellarein and caffeic acid respectively.
3.3 anti-diarrhea effects of luteolin, scutellarein and caffeic acid
As can be seen from fig. 2A to C, the scutellarein, caffeic acid and luteolin groups were not different from the model group in terms of the time of primary diarrhea, but their differences from the control group were very significant except for caffeic acid in terms of loose stool count and total stool frequency. In addition, the shit inhibition rates of scutellarein, luteolin and caffeic acid groups were 24.55%, 54.55% and 9.09%, respectively. Therefore, luteolin and scutellarein with good effects are selected for further research.
3.4 histopathological analysis
To further verify the anti-diarrheal effect of the monomeric components, this experiment was performed by pathological section observation. As can be seen from the pathological section of fig. 3, compared with the control group, the intestinal epithelial cells of the model group are exfoliated, the glands and crypts are disappeared, the mucosal cell structure is seriously damaged, the submucosa is proliferated, the blood vessels are dilated, the thickness of the mucosa is obviously reduced, and the medicine group obviously relieves the symptom; wherein, the luteolin and the scutellarein have more obvious effects. According to the histopathological scoring results, the score of the model group is significantly higher than that of the blank group, and the score of the 3 monomer component groups is significantly lower than that of the model group, which shows that the scutellarein, the caffeic acid, the luteolin and the mixture thereof can significantly improve the colon tissue damage caused by the castor oil, wherein the scutellarein has the best effect.
3.5 changes in intestinal fluid
In FIGS. 4A-B, the volume and weight of the contents of the intestine were significantly reduced after treatment with luteolin and scutellarein as compared to the model groups. In addition, the intestinal content inhibition rates of luteolin and baicalein groups are respectively 25.94% and 31.88%. There was no significant difference between the two monomer groups.
3.6 Change in enzymatic Activity
FIGS. 4C-D show castor oil-induced diarrhea in rats versus uninduced rats (Na)+/K+ATP enzyme 115.299 + -5.74912U/g, creatine kinase 17.514 + -0.3442U/L) in comparison to Na+/K+Significant decrease (p) in ATPase (76.38. + -. 2.4278U/g) and creatine kinase activity (15.478. + -. 0.30831U/L)<0.05). Rats. However, luteolin and scutellarein significantly increased both enzyme activities (p)<0.05). Meanwhile, the luteolin has better binding capacity with Na +/K + -ATP enzyme, and the effect is better than that of scutellarein.
3.7 variation of enzyme Gene level
To investigate whether scutellarein and luteolin regulate Na by affecting gene expression+/K+ATPase and creatine kinase activity to exert anti-diarrheal effects, and real-time quantitative PCR was used to detect mRNA levels of ckb and Atp1b3 in intestinal tissue of drug-treated and untreated rats. As can be seen from FIG. 4(G), castor oil down-regulated the gene expression of ckb and Atp1b3, while luteolin and scutellarein up-regulated the levels of both genes. Among the regulation of these two genes, scutellarein has a better effect than luteolin.
3.8 Change in ion concentration
According to FIGS. 4E-F, there was a significant difference between the model group and the blank group, which means that the castor oil was reducedThe concentration of sodium and potassium ions in the small intestine is low. Meanwhile, luteolin and scutellarein increase Na+And K+And the effect is similar.
TABLE 2 primer sequences for real-time quantitative PCR analysis
3.9 changes in intestinal peristalsis
Changes in intestinal peristalsis are primarily assessed by the rate of advancement of the carbon powder. As shown in fig. 5, although the toner advancement rate was slightly decreased for the luteolin and scutellarein groups compared to the model group, there was no significant difference between them, suggesting that both monomers may not bind well to opioid receptors, which is consistent with the primary diarrhea time results.
Claims (10)
1. An anti-diarrheal monomer compound isolated from plantain, wherein the monomer compound is selected from luteolin, scutellarein or caffeic acid.
2. The anti-diarrheal monomeric compound according to claim 1, which is characterized in that: including acid addition salts of monomeric compounds.
3. A method for preparing the anti-diarrhea monomer compound of claim 1, comprising the steps of: separating and identifying the ethyl acetate extraction part of the plantain by ultra-high performance liquid chromatography-time-of-flight mass spectrometry to obtain the monomer compound.
4. The method of claim 3, wherein the separation and identification method comprises: separation was performed on a WatersAcquity UPLC BEH C18 column, eluting with 0.1% formic acid and acetonitrile; time-of-flight mass spectrometry and electrospray ionization sources for positive and negative ion mode analysis; target search and non-target search are executed by Peakview 2.0/masterview1.0 or Markerview 1.2.1 software, and the composition of the compound is determined by the accurate mass and isotope abundance ratio of the software target screening function; the structure of the isolated monomeric compounds is determined by analyzing the MS/MS fragments or comparing them to standard substances and references.
5. Use of the monomeric compound of claim 1 in the preparation of an anti-diarrhea medicament.
6. An anti-diarrhea pharmaceutical composition, which is characterized in that the pharmaceutical composition consists of a therapeutically effective amount of luteolin or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.
7. An anti-diarrhea pharmaceutical composition, which is characterized in that the pharmaceutical composition consists of scutellarein with a therapeutically effective dose or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
8. An anti-diarrhea pharmaceutical composition, which is characterized in that the pharmaceutical composition consists of caffeic acid or pharmaceutically acceptable salts thereof with effective treatment amount and pharmaceutically acceptable carriers.
9. An anti-diarrhea pharmaceutical composition, which is characterized in that the pharmaceutical composition consists of a mixture of more than one of luteolin, scutellarin or caffeic acid with a therapeutically effective amount according to any proportion and a pharmaceutically acceptable carrier.
10. The pharmaceutical composition according to any one of claims 7 to 9, which is prepared into any suitable pharmaceutical preparation according to a conventional formulation method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112843076A (en) * | 2021-01-30 | 2021-05-28 | 河南中医药大学第一附属医院 | Application of akebia quinata phenylethanoid glycoside B with CCR5 as target in preparation of anti-HIV-1 drugs |
| CN114788822A (en) * | 2022-05-27 | 2022-07-26 | 云南中医药大学 | Application of scutellarein in preparation of medicine for treating pancreatic cancer |
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