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ObjectiveIn order to explore the changes of chemical constituents in Plantaginis Semen before and after stir-frying， ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF/MSE） was used to rapidly identify and semi-quantitatively analyze the differential components in Plantaginis Semen processed at different stir-frying time. MethodWaters ACQUITY UPLC BEH C18 column （2.1 mm×100 mm， 1.8 μm） was employed with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-1 min， 5%-10%B； 1-2 min， 10%-15%B； 2-10 min， 15%-20%B； 10-12 min， 20%-40%B； 12-13 min， 40%-100%B； 13-14 min， 100%-5%B； 14-15 min， 5%B）， the flow rate was 0.3 mL·min-1， the column temperature was 40 ℃， and the injection volume was 3 μL. Electrospray ionization （ESI） was applied for mass spectrometric analysis under positive and negative ion modes， and the scanning range was m/z 50-1 500. MarkerLynx 4.1 software was used to find the differential compounds， and the intensity of each ion peak in samples with different stir-frying time was compared to study the content variations of these compounds. ResultA total of 20 components with potential significant differences were found， among which 17 were identified and 3 were unknown， mainly including phenylethanoid glycosides， iridoid glycosides， alkaloids and others. After processing， the peak intensities of 7 compounds， such as sucrose， geniposidic acid， verbascoside and plantagoguanidinic acid A， in Plantaginis Semen decreased. The peak intensities of orobanchoside， dianthoside and plantain D increased first and then decreased during the stir-frying process. The peak intensities of 10 compounds （decaffeoylacteoside， calceolarioside A， isoacteoside， etc.） increased， and 9 of them were newly generated components. ConclusionThe content and composition of the chemical components in Plantaginis Semen changed significantly after stir-frying， which may be related to the reduction of laxative effect and the enhancement of antidiarrheal and diuretic activities of Plantaginis Semen after stir-frying.
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Objective:To compare the content change of 6 constituents in Plantaginis Semen from different habitats before and after salt processing. Methods:HPLC method was used to quantitatively analyze 6 ingredients in Plantaginis Semen and processed with salt including geniposidic acid, plantagoguanidinic acid, quercetin, kaempferol, verbascoside and isoverbascoside. Results:The geniposidic acid, plantagoguanidinic acid, quercetin, kaempferol, verbascoside and isoverbascoside were well separated. The linear ranges of which were 0.259 2-3.628 8 μg ( r=0.999 8), 0.054 3-0.760 5 μg ( r=0.999 6), 0.030 0-0.420 6 μg ( r=0.999 4), 0.055 6- 0.777 8 μg ( r=0.999 5), 0.287 0-4.018 0 μg ( r=0.999 8), 0.033 1-0.463 1 μg ( r=0.999 7), respectively. Average recovery rates were 98.68%, 98.46%, 98.87%, 98.99%, 98.34%, 98.75% ( n=6), respectively. There were mild differences in the contents of 6 ingredients of 8 batches of Plantaginis Semen from 5 different habitats. There were no obvious differences between the raw products and the products after salt process in Plantaginis Semen. The content of flavonoids, geniposidic acid and isoverbascoside in Plantaginis Semen were significantly increased after salt process, while the content of verbascoside was reduced. Conclusion:HPLC method to quantitatively analyze the 6 constituents in Plantaginis Semen before and after salt process could provided a reference for the quality change and the material basis for the efficacy of Plantaginis Semen before and after salt process.
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Plantaginis Semen is a traditional Chinese medicine （TCM） commonly used in China， which is one of the authentic medicinal materials in Jiangxi. It has great development prospects. However， the current research on Plantaginis Semen is not in-depth enough， mainly involving chemical components and pharmacological activities. There are few researches on processing and variety of Plantaginis Semen. In order to further develop and utilize the resources of Plantaginis Semen， we summarized 4 varieties that have been studied more at present， the processing contents of Plantaginis Semen in ancient and modern literature were consulted and sorted out， and its processing historical evolution were summarized. The influences of different processing technologies and methods on the chemical composition and pharmacological effects of Plantaginis Semen were analyzed， the possible processing mechanism was discussed. Meanwhile， and the quality evaluation methods of Plantaginis Semen varieties included in the 2020 edition of Chinese Pharmacopoeia were summarized. The author mainly analyzed the researches status of Plantaginis Semen and its decoction pieces in the three aspects of variety， processing and quality evaluation， and summarized its current major problems such as insufficient use and development of varieties， unclear processing mechanisms， and undetermined quality evaluation standards. And combined with the national standardization project of TCM to carry out the prospect and analysis for it， in order to solve the problems in the actual production and use of Plantaginis Semen， and provide reference for its further development， production of the high-quality decoction pieces， analysis of the processing mechanism， and establishment of the quality control system.
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Objective:To study the protective effect of polysaccharides from Plantaginis Semen （PSP） against renal injury in rats with membranous nephropathy （MN） and its influence on the gut microbiota to provide a theoretical basis for the further investigation of PSP in the treatment of MN. Method:The MN model was induced by tail vein injection of cationic bovine serum albumin （C-BSA， 3.5 g·L<sup>-1</sup>） in rats with a modeling period of seven weeks. At the 4th week of modeling， the model rats were divided into a model group， a positive drug group （benazepril hydrochloride， 10 mg·kg<sup>-1</sup>·d<sup>-1</sup>）， a PSP high-dose group （PSP-H， 800 mg·kg<sup>-1</sup>·d<sup>-1</sup>）， a PSP medium-dose group （PSP-M， 400 mg·kg<sup>-1</sup>·d<sup>-1</sup>）， and a PSP low-dose group （PSP-L， 200 mg·kg<sup>-1</sup>·d<sup>-1</sup>） according to the random number table， with 10 in each group. Ten healthy rats were assigned to the normal control group. The rats in the normal control group and the control group received an equal amount of physiological saline by gavage， and those in the groups with drug intervention were administered correspondingly，once a day，for consecutive four weeks. The pathological changes of rat kidney and colon tissues were observed by optical microscopy. The enzyme-linked immunosorbent assay （ELISA） was used to detect the content of tumor necrosis factor-<italic>α </italic>（TNF-<italic>α</italic>） and interleukin-1<italic>β </italic>（IL-1<italic>β</italic>） in the serum and colon tissues. The immunohistochemistry （IHC） was used to detect the protein expression of TNF-<italic>α </italic>and IL-1<italic>β </italic>in renal tissues. The 16S rRNA sequencing method was used to investigate the effect of PSP on the gut microbiota in MN rats. Result:Compared with the normal control group， the model group showed enlarged glomeruli， thickened basement membrane， atrophied colonic gland， increased TNF-<italic>α</italic> and IL-1<italic>β</italic> in the serum and colon tissues （<italic>P</italic><0.05）， and elevated protein expression of TNF-<italic>α</italic> and IL-1<italic>β </italic>（<italic>P</italic><0.01）. Compared with the model group， the positive drug group and the PSP-H group displayed shrunk glomerular capsules， relieved basement membrane thickening， and neatly arranged colonic mucosa in colon tissues， while the PSP-M and PSP-L groups were inferior in improving renal tissues and colon tissues. Additionally， the PSP-H and PSP-M groups showed declining TNF-<italic>α</italic> and IL-1<italic>β</italic> in the serum and colon tissues （<italic>P</italic><0.05） and dwindled protein expression of TNF-<italic>α</italic> and IL-1<italic>β </italic>in the renal tissues （<italic>P</italic><0.01）. No significant difference was observed in the PSP-L group. Compared with the normal control group， the model group showed increased abundance of Firmicutes and decreased abundance of Bacteroidetes. After PSP intervention， the abundance of Firmicutes was decreased， while that of Bacteroidetes was increased， and such changes were predominant in the PSP-H group. Conclusion:PSP can effectively alleviate renal injury， reduce the expression of inflammatory factors， regulate the structure of gut microbiota， and improve the damaged intestinal barrier of MN rats.
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Objective:To optimize the processing technology of salt-processed products of Plantaginis Semen with the specific process parameters, and verify the obtained processing technology by pharmacodynamic research, so as to provide experimental basis for the standardized production and quality control of this decoction pieces. Method:Taking composite score of appearance character score, dry extract yield and contents of three components (geniposidic acid, acteoside and isoacteoside) as index, the analytic hierarchy process (AHP)-criteria importance through intercrieria correlation (CRITIC) mixed weighting method was used to determine the weight coefficient of each index. Based on single factor tests, the response surface method was used to investigate the effects of frying time, frying temperature, salt amount and water amount on the processing technology of salt-processed products of Plantaginis Semen, and the processing technology was verified by diuretic experiment with furosemide tablets as the positive drug (administration dose of 0.01 g·kg-1). Result:The weight coefficients of geniposidic acid content, acteoside content, appearance character score, isoacteoside content and dry extract yield were 0.319, 0.193, 0.207, 0.273 and 0.008, respectively. The optimal process parameters were as following：fried at 150-180 ℃ for 10 min (obtained from the single factor tests), 100 g of Plantaginis Semen sprayed evenly with 2 g of salt (2 g of salt dissolved in 20 mL of water), and fried at 150-180 ℃ for 15 min. Compared with the blank group, both of the raw products group and the salt-processed products group could significantly increase the secretion of urine volume (P<0.01), but the excretion of Na+ in the urine of rats in the salt-processed products group was significantly higher than that in the raw products group (P<0.05). Conclusion:The optimized processing technology is simple and feasible, which can provide reference for standardizing the industrial production of salt-processed products of Plantaginis Semen. At the same time, combined with inherent quality and appearance of the salt-processed products, and verified by pharmacodynamic test, the obtained results are reasonable and reliable, which can be used for quality control of this decoction pieces.
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Objective:To observe Plantaginis Semen's mechanism in treating diarrhea by observing the effect on inflammatory factors in serum and mRNA and protein expressions of aquaporin4 (AQP4) in colon tissue of diarrhea rats. Method:Senne Folium was orally administered to duplicate diarrhea rats. Sixty male SD rats were randomly divided into normal group, model group, hydrochlorothiazide group (9 mg·kg-1), and low, middle, and high-dose Plantaginis Semen groups (0.95, 1.9, 3.8 g·kg-1). Senne Folium (20 mL·kg-1) was intragastrically administered in 5 groups in the morning, except for normal group that was orally given the same dose of distilled water. In the afternoon, each treatment group was orally given the corresponding drugs, while normal group and model group were orally given the same dose of distilled water. The loose stool rate, average degree of loose stool, and diarrhea index were compared according to fecal traits and stool times after 14 days of treatment. The serum and colon tissue were collected to detect the contents of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) in serum. Hematoxylin-eosin (HE) staining was used to observe the pathological morphological changes of colon tissue, and quantiative Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to detect the mRNA and protein expressions of AQP4 in colon tissue. Result:In the model group, the loose stool rate, average degree of loose stool, and diarrhea index were significantly increased (P<0.01), apoptosis and necrosis were observed in the epidermal cells of colonic mucosa, telangiectasia and congestion in lamina propria were obvious, and a few neutrophils were infiltrated, and the contents of TNF-α, IL-6 and CRP in serum increased (P<0.05, P<0.01), the mRNA and protein expressions of AQP4 significantly decreased (P<0.01). Compared with the model group, the loose stool rate, average degree of loose stool, and diarrhea index were significantly decreased in low, middle, and high-dose Plantaginis Semen groups (P<0.01), the apoptosis and necrosis of epidermal cells, telangiectasia and hyperemia and neutrophil infiltration in colonic mucosa were obviously improved, and the contents of TNF-α and CRP in serum significantly decreased (P<0.05, P<0.01), the mRNA and protein expressions of AQP4 increased （P<0.05, P<0.01）. Conclusion:Plantaginis Semen has a better antidiarrheal effect, and its mechanism may be related to inhibition of inflammatory reaction, repair of pathological damage of colonic mucosa, up-regulation of AQP4 expression and promotion of water and fluid metabolism.
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Objective:To establish a HPLC fingerprint detection method of Plantaginis Semen, and analyze the samples from different producing areas in Jiangxi province by combining with chemical pattern recognition method, and the contents of five ingredients in Plantaginis Semen were determined. Method:A total of 34 batches of Plantaginis Semen medicinal materials were detected by HPLC. The similarity evaluation was carried out by the 2012 edition of similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine. The chromatographic peak information was used as the data source, and three chemical pattern recognition methods were used to comprehensively analyze the quality of this medicinal herb. Quantitative analysis was performed on the 5 active components, including geniposidic acid, plantamajoside, acteoside, galuteolin and isoacteoside. Result:The similarities between Plantaginis Semen samples from different producing areas in Jiangxi province were >0.86. Orthogonal partial least squares-discriminant analysis (OPLS-DA) could distinguish samples from different producing areas, and be used to determine the chemical components, which had strong correlation with the quality of Plantaginis Semen. The contents of 5 active components in samples from different producing areas were different to some degree, especially in the content of plantamajoside. Conclusion:The established HPLC fingerprint of Plantaginis Semen has strong characteristics, combined with chemical pattern recognition method, it can effectively evaluate the quality of Plantaginis Semen and distinguish its producing areas.
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AIM To compare the diuretic effects of Descurainiae Semen (DS),Coicis Semen (CS) and Plantaginis Semen (PS),and to observe their mechanical similarities and differences.METHODS Metabolic cage method was applied to investigating the diuretic effects of DS (2.34 g/kg),CS (7.00 g/kg) and PS (3.50 g/kg),whose diuretic mechanisms were studied by cryoscopic method,enzyme method,ion selective electrode method,ELISA and Western blot.RESULTS DS,CS and PS obviously increased saline-loaded rats' urine volume (P ＜ 0.05) and reduced their body weight (P ＜ 0.05) after administration for 7 h,which exhibited no significant effects on urine creatinine (Ucr),serum creatinine (Scr) and blood urea nitrogen (BUN)(P ＞ 0.05).DS showed its diuretic effect mainly by lowering the levels of serum Na +,atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP),pulmonary AQP3,renal AQP1 and AQP2;CS showed its diuretic effect mainly by reducing the levels of serum Na +,Cl-,ANP,pulmonary AQP3,gastric AQP3,renal AQP1 and AQP2;PS showed its diuretic effect mainly by decreasing the levels of serum Na + and Cl-,pulmonary AQP3,gastric AQP3,renal AQP1 and AQP2.CONCLUSION Three medicinal materials have significant diuretic effects without obvious renal harm.DS categorized as a medicinal plant of lung channel and tropism has a great effect on netriuretic peptide system,CS categorized as a medicinal plant of spleen channel and tropism has a great effect on gastric AQP3,and PS categorized as a medicinal plant of renal channel and tropism has a great effect on renal AQPs.
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OBJECTIVE: To establish an HPLC method to simultaneously determine the contents of geniposidic acid, caffeic acid, acteoside and isoacteoside in Plantaginis Semen formula granules, in order to provide basis for studying its quality standards. METHODS: An HPLC method was developed. Kinetex C18 column (2.1 mm×100 mm, 2.6 μm)was used and eluted with mobile phase of 0.5% acetic acid-acetonitrile at the flow rate of 0.3 mL·min-1. The wavelength was set at 239 nm for geniposidic acid, 325 nm for caffeic acid, and 330 nm for acteoside and isoacteoside. The column temperature was maintained at 30℃. RESULTS: The good linear relationships between the concentration and peak area were in the range of 0.292 1-2.92 1 μg for geniposidic acid, 0.003 4-0.033 6 μg for caffeic acid, 0.047 6-0.476 μg for acteoside and 0.102 7-1.027 μg for isoacteoside (r≥0.999 8), respectively. The average recoveries were 99.32%, 98.62%, 98.23% and 98.51% with RSDs of 1.47%, 1.36%, 1.62% and 1.53%, respectively. CONCLUSION: The method is simple, feasible and reproducible and can be used for the quality control of Plantaginis Semen formula granules.
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AIM To study the phenols from Plantaginis Semen.METHODS The 65％ and 95％ ethanol extracts of Plantaginis Semen were isolated and purified by macroporous resin,silica,ODS,Sephadex and preparative column,then the structures of obtained compounds were identified by physicochemical properties and spectral data.RESULTS Nine compounds were isolated and identified as (+)-(7R,7'R,8S,8'S)-neo-olivil (1),erythro-guaiacylglycerol-β-ferulic acid ether (2),eriodictyol (3),luteolin (4),chrysoeriol (5),hydroxytyrosol (6),4-(3,4-dihydroxyphenyl)-(E)-3-buten-2-one (7),ferulic acid (8),5,7-dihydroxychromone (9).CONCLUSION Compounds 2-3,6-7 and 9 are isolated from genus Plantago for the first time,compound 5 is first obtained from this plant.
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The chemical constituents of Plantaginis Semen with hypoglycemic effect was investigated in this paper. The previous results of the in vivo hypoglycemic effect showed that 60% ethanol extract of Plantaginis Semen decreased the levels of FBG and improved the glucose tolerance in high fat diet(HFD)-induced diabetic C57BL/6 mice. Then, in the present study, the above potential bioactive extract was separated and purified by silica gel, ODS, Sephadex LH-20 column chromatography, medium pressure liquid chromatography(MPLC)and preparative HPLC. The structures of isolated compounds were identified by physicochemical properties and spectral analyses. Eight compounds were obtained and identified as 4, 4a, 5, 7a-tetrahydro-7-(hydroxymethyl)cyclopenta[c]pyran-3(1H)-one(1), iridolactone(2), pedicularislacton(3), rehmaglutin C(4), geniposidic acid(5), p-hydroxylphenylglycerol(6), 1, 2-benzenediol-4-(2-hydroxyethyl)(7), and 3-buten-2-one-4-[3-(β-D-glucopyranosyloxy)-4-hydroxyphenyl](8). Among them, compounds 1-5 were iridoids, and 6-8 were phenolic acids. Compound 1 was a new natural product, and compounds 2-4, 6 and 8 were isolated from the Plantaginaceae family for the first time.
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This study was aimed to explore a new method to identify the original plant of Plantaginis Semen and its adulterants by the ITS2 regions. The second internal transcribed spacer (ITS2) of ribosomal DNA was amplified and sequenced by bidirectional sequencing of PCR products. Sequence assembly and consensus sequence generation were performed by using CodonCode Aligner. The ITS2 secondary structure was predicted using ITS2 database and web-sites. The phylogenetic tree was constructed by MEGA5. The results showed that the maximum intraspecific K2P dis-tance of Plantago asiatica was 0.009 9, while the minimum interspecific K2P distance was 0.497 6; the maximum in-traspecific K2P distance of P. depressa was 0.005 2, while the minimum interspecific K2P distance was 0.519 1. The ITS2 secondary structure showed that P. asiatica and P. depressa can be differentiated obviously from its adulterants. Different samples of P. asiatica and P. depressa were gathered together and can be distinguished from its adulterants by NJ tree. It was concluded that the ITS2 sequence was able to identify original plant of Plantaginis Semen and its adulterants correctly. It provided a new method for the identification of original plant of Plantaginis Semen.
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Objective: To establish the fingerprint of Plantaginis Semen and to determine the two index contents of geniposidic acid and verbascoside. Methods: The chromatographic separation was performed on a C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase was a mixture of acetonitrile-0.5% acetic acid aqueous solution in gradient elution. The flow rate was 1.0 mL/min, and the detection wave length was 254 nm. Similarity evaluation system was used in data analysis. Results: The HPLC fingerprint of Plantaginis Semen showed 13 characteristic peaks with high similarity, and contents of geniposidic acid and verbascoside were 0.794 3% and 0.664 2%, respectively. Conclusion: The method that combining the determination of index contents with fingerprint analysis is timesaving and could be used for the quality control of Plantaginis Semen. It could provide a reference for the further research on the quality control of formular granule with Plantaginis Semen extracts.