Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.
Amino Acid Sequence ; Fallopia japonica/metabolism* ; Polyketide Synthases/chemistry* ; Acetone ; Mutagenesis, Site-Directed ; Flavonoids/metabolism* ; Acyltransferases/metabolism*

OBJECTIVE: Recent investigations have demonstrated that Polygonum perfoliatum L. can protect against chemical liver injury, but the mechanism behind its efficacy is still unclear. Therefore, we studied the pharmacological mechanism at work in P. perfoliatum protection against chemical liver injury. METHODS: To evaluate the activity of P. perfoliatum against chemical liver injury, levels of alanine transaminase, lactic dehydrogenase, aspartate transaminase, superoxide dismutase, glutathione peroxidase and malondialdehyde were measured, alongside histological assessments of the liver, heart and kidney tissue. A nontargeted lipidomics strategy based on ultra-performance liquid chromatography quadrupole-orbitrap high-resolution mass spectrometry method was used to obtain the lipid profiles of mice with chemical liver injury and following treatment with P. perfoliatum; these profiles were used to understand the possible mechanisms behind P. perfoliatum's protective activity. RESULTS: Lipidomic studies indicated that P. perfoliatum protected against chemical liver injury, and the results were consistent between histological and physiological analyses. By comparing the profiles of liver lipids in model and control mice, we found that the levels of 89 lipids were significantly changed. In animals receiving P. perfoliatum treatment, the levels of 8 lipids were significantly improved, relative to the model animals. The results showed that P. perfoliatum extract could effectively reverse the chemical liver injury and significantly improve the abnormal liver lipid metabolism of mice with chemical liver injury, especially glycerophospholipid metabolism. CONCLUSION Regulation of enzyme activity related to the glycerophospholipid metabolism pathway may be involved in the mechanism of P. perfoliatum's protection against liver injury. Please cite this article as: Peng L, Chen HG, Zhou X. Lipidomic investigation of the protective effects of Polygonum perfoliatum against chemical liver injury in mice. J Integr Med. 2023; 21(3): 289-301.
Animals ; Mice ; Polygonum/chemistry* ; Lipidomics ; Liver ; Lipids/pharmacology* ; Glycerophospholipids/pharmacology* ; Chemical and Drug Induced Liver Injury/metabolism*

This paper aimed to investigate the effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/peroxisome proliferator-activated receptor γ(PPARγ) pathway in arrhythmic rats. SD rats were randomly divided into a control group, a model group, a low-dose(20 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a medium-dose(40 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a high-dose(80 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a propranolol hydrochloride(2 mg·kg~(-1)) group, with 12 rats in each group. Except the control group, rats in other groups were prepared as models of arrhythmia by sublingual injection of 1 mL·kg~(-1) of 0.002% aconitine. After grouping and intervention with drugs, the arrhythmia, myocardial cells apoptosis, myocardial tissue glutathione peroxidase(GSH-Px), catalase(CAT), malondialdehyde(MDA), serum interleukin-6(IL-6), prostaglandin E2(PGE2) levels, myocardial tissue apoptosis, and Wnt/β-catenin/PPARγ pathway-related protein expression of rats in each group were measured. As compared with the control group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA levels in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels increased significantly in the model group, whereas the GSH-Px and CAT levels, and Bcl-2 and PPARγ protein expression levels in myocardial tissues reduced significantly. As compared with the model group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA leve in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels reduced in the drug intervention groups, whereas the GSH-Px and CAT levels and Bcl-2 and PPARγ protein expression levels in myocardial tissues increased. The groups of total flavonoids of buckwheat flower and leaf were in a dose-dependent manner. There was no significant difference in the levels of each index in rats between the propranolol hydrochloride group and the high-dose group of total flavonoids of buckwheat flower and leaf. The total flavonoids of buckwheat flower and leaf inhibit the activation of Wnt/β-catenin pathway, up-regulate the expression of PPARγ, reduce oxidative stress and inflammatory damage in myocardial tissues of arrhythmic rats, reduce myocardial cell apoptosis, and improve the symptoms of arrhythmia in rats.
Rats ; Animals ; PPAR gamma/metabolism* ; Fagopyrum/genetics* ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; beta Catenin/metabolism* ; Interleukin-6 ; Flavonoids/pharmacology* ; Propranolol/pharmacology* ; Ventricular Fibrillation ; Dinoprostone ; Wnt Signaling Pathway ; Plant Leaves/metabolism* ; Flowers/metabolism* ; Apoptosis ; Cardiac Complexes, Premature

To explore the changes and the reaction mechanisms between soil microecological environment and the content of secon-dary metabolites of plants under water deficit, this study carried out a pot experiment on the 3-leaf stage seedlings of Rheum officinale to analyze their response mechanism under different drought gradients(normal water supply, mild, moderate, and severe drought). The results indicated that the content of flavonoids, phenols, terpenoids, and alkaloids in the root of R. officinale varied greatly under drought stresses. Under mild drought stress, the content of substances mentioned above was comparatively high, and the content of rutin, emodin, gallic acid, and(+)-catechin hydrate in the root significantly increased. The content of rutin, emodin, and gallic acid under severe drought stress was significantly lower than that under normal water supply. The number of species, Shannon diversity index, richness index, and Simpson index of bacteria in the rhizosphere soil were significantly higher than those in blank soil, and the number of microbial species and richness index decreased significantly with the aggravation of drought stresses. In the context of water deficit, Cyanophyta, Firmicutes, Actinobacteria, Chloroflexi, Gemmatimonadetes, Streptomyces, and Actinomyces were the dominant bacteria in the rhizosphere of R. officinale. The relative content of rutin and emodin in the root of R. officinale was positively correlated with the relative abundance of Cyanophyta and Firmicutes, and the relative content of(+)-catechin hydrate and(-)-epicatechin gallate was positively correlated with the relative abundance of Bacteroidetes and Firmicutes. In conclusion, appropriate drought stress can increase the content of secondary metabolites of R. officinale from physiological induction and the increase in the association with beneficial microbe.
Rhizosphere ; Rheum ; Droughts ; Soil ; Catechin ; Emodin ; Bacteria/metabolism* ; Water/metabolism* ; Firmicutes ; Soil Microbiology

This study aimed to explore the efficacy and mechanism of combined rhein and emodin in the treatment of ulcerative colitis(UC) from the aspects of network pharmacology, animal inflammation improvement and molecular mechanism. Network pharmacology predicted that combined rhein and emodin acted on 52 potential targets, mainly participating in signaling pathways such as cancer, PI3 K/AKT, microRNAs in cancer and apoptosis. PI3 K/AKT signaling pathway has been reported to be closely related to UC, and the optimal candidate pathway for combined therapy. The UC mice model was established by dextran sodium sulfate, and then the modeled mice were randomly divided into control group, model group, rhein group, emodin group, rhein+emodin group and sulfasalazine group. After administration, compared with the conditions in model group, body weight, disease activity index(DAI) score, colon length, TNF-α, IL-6, IL-1β and myeloperoxidase(MPO) of mice in rhein+emodin group were improved(P<0.01); colonic mucosal injury was significantly reduced; the expression of p-PI3 K/PI3 K and p-AKT/AKT proteins were down-regulated(P<0.01). All the above indices were better than those in the rhein/emodin group alone. The Jin's Q-values of the effect of combined rhein and emodin on colon length, TNF-α, IL-6, IL-1β, MPO, p-PI3 K/PI3 K and p-AKT/AKT were all greater than 1.15, which indicated that there was obvious synergistic effect between rhein and emodin. In all, rhein and emodin have synergistic effect in the treatment of UC, and the mechanism may be related to the inhibition of PI3 K/AKT signaling pathway and the down-regulation of proinflammatory factors. They are the new components in the treatment of UC, which is worthy of attention.
Animals ; Anthraquinones ; Colitis, Ulcerative/metabolism* ; Colon ; Disease Models, Animal ; Emodin/pharmacology* ; Interleukin-6/metabolism* ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Rheum ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVE: To examine the antiplatelet and antithrombotic activity of Rumex acetosella extract. METHODS: Standard light aggregometry was used for platelet aggregation, intracellular calcium mobilization assessed using Fura-2/AM, granule secretion (ATP release) by luminometer, and fibrinogen binding to integrin α_IIbβ₃ detected using flow cytometry. Western blotting is carried out to determine the phosphorylation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt signaling. RESULTS: Rumex acetosella displayed the ability to inhibit platelet aggregation, calcium mobilization, granule secretion, and fibrinogen binding to integrin α_IIbβ₃. Rumex acetosella has also down-regulated MAPK and PI3K/Akt phosphorylation (all P<0.01). CONCLUSION Rumex acetosella extract exhibits antiplatelet activity via modulating GPVI signaling, and it may protect against the development of platelet-related cardiovascular diseases.
Blood Platelets/metabolism* ; Calcium/metabolism* ; Fibrinogen/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Phosphorylation ; Plant Extracts/pharmacology* ; Platelet Aggregation ; Platelet Aggregation Inhibitors/pharmacology* ; Platelet Glycoprotein GPIIb-IIIa Complex/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rumex/metabolism*

In this study, the rhizobacteria and actinomycetes of Polygonum multiflorum were screened for the strains with indole acetic acid(IAA)-producing capacity by Salkowski method, the siderophore-producing strains by Chrome Azurol S(CAS) assay, and the strains with inorganic phosphorus-solubilizing capacity by PKO inorganic phosphorus medium. The strains were identified by morphological identification, physiological and biochemical characteristics, and 16 S rDNA sequences. Furthermore, the effect of growth-promoting strains on the seed germination and development of P. multiflorum was tested. The results showed that among 196 strains, two strains F17 and F42 were found to be capable of producing IAA and siderophore and solubilizing inorganic phosphorus simulta-neously. For F17 and F42, the results are listed below: 38.65 and 33.64 mg·L~(-1) for IAA production, 0.85 and 0.49 for siderophore-producing capacities(A_s/A_r), and 1.35 and 1.70 for inorganic phosphorus-solubilizing capacities(D/d), respectively. Comprehensive analysis revealed that strains F17 and F42 were identified as Pseudochrobactrum asacharolyticum and Bacillus aryabhattai, respectively, and both could significantly promote the seed germination of P. multiflorum.
Bacillus ; Fallopia multiflora ; Germination ; Seeds ; Soil Microbiology

OBJECTIVE: To study the effects of buckwheat-oat-pea (BOP) composite flour [buckwheat ∶ oats ∶ peas=6 ∶ 1 ∶ 1 (quality ratio)] on blood glucose in diabetic rats. METHODS: In this study, 64 male Sprague-Dawley rats were divided into 8 groups by fasting blood glucose (FBG) and body weight: normal control group, model control group, metformin group, buckwheat group, oats group, BOP low-dose group (BOP-L), medium-dose group (BOP-M), and high-dose group (BOP-H). The rats in the normal control group were fed with normal diet, the rats in the model control group and metformin group were fed with a high-fat diet (HFD), and the rats in the buckwheat group, oats group, and BOP-L, BOP-M, BOP-H groups were fed with HFD containing 10% buckwheat flour, 10% oat flour, 3.3% BOP, 10% BOP, 30% BOP, respectively. The HFD in all the groups had the same percentage of energy from fat (45%). After 30 days, the rats fed with HFD received intraperitoneal injection of streptozotocin (30 mg/kg, once a week for two weeks) to establish diabetes mellitus. After the model was successful established, the rats were fed for another 28 days. During the study, the body weight, food intake/body weight (FI/BW) and water intake/body weight (WI/BW), food utilization rate, 24 h urine volume, FBG, glucose area under curve (GAUC) of oral glucose tolerance test were measured regularly. At the end of the study, the fasting serum glucose and insulin were measured, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. RESULTS: With the inducing of HFD and streptozotocin, compared with the normal control group, the rats in the model control group had higher FI/BW, WI/BW, 24 h urine volume, FBG, GAUC, HOMA-IR (P < 0.05), and lower body weight, food utilization rate (P < 0.05). Compared with the model control group, the rats in the three BOP groups all had higher body weight, food utilization rate (P < 0.05), and lower WI/BW, HOMA-IR (P < 0.05); the rats in the BOP-L and BOP-M groups had lower FI/BW, 24 h urine volume, FBG (P < 0.05), and the rats in the BOP-M group also had lower GAUC (P < 0.05). After the establishment of diabetes, there was no significant difference in blood glucose and the other indicators between the rats in the three BOP groups and the buckwheat group or the oats group (P>0.05). CONCLUSION The BOP had the effects of reducing blood glucose, insulin resistance and diabetic symptoms on diabetic rats, and had the value for further development and utilization.
Animals ; Avena ; Blood Glucose ; Diabetes Mellitus, Experimental ; Diabetes Mellitus, Type 2 ; Diet, High-Fat/adverse effects* ; Fagopyrum ; Insulin ; Insulin Resistance ; Male ; Peas ; Rats ; Rats, Sprague-Dawley

A detection method of ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was established to detect concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-β-D-glucoside in H9 c2 cells and applied to the pharmacokinetic study of Polygonum orientale extract in the cells. H9 c2 cells were treated with 100 μg·mL~(-1) P. orientale extract and then they and the corresponding nuclei, mitochondria and Golgi bodies were collected at the set time. After protein precipitation, UPLC-MS/MS was used to determine concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-β-D-glucoside in the whole cells and subcellular structures. Also, related pharmacokinetic parameters were calculated. The results showed that the peak time was 8 h for all these components. Orientin, vitexin, quercetin and isoorientin have high affinities to nuclei and mitochondria, while the affinity of kaempferol-3-O-β-D-glucoside is higher with mitochondria compared to nuclei. It is suggested that these chemical components of P. orientale may mainly act on nuclei or mitochondria to exert pharmacological effects of protecting cardiomyocytes.
Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Drugs, Chinese Herbal ; Polygonum ; Tandem Mass Spectrometry

To standardize the processing technology of Polygoni Multiflori Radix Praeparata and stabilize its quality, the similar change laws of Polygoni Multiflori Radix Praeparata with different processing methods and time were analyzed. The processing time of Polygoni Multiflori Radix Praeparata was studied at 24, 32, 40, 48 h, and 4 different processing methods were studied, namely stewed with black bean sauce, steamed, steamed with black bean sauce, and steamed with black bean sauce and rice wine. The content of stilbene glycosides and anthraquinones were determined by HPLC-DAD method. UV method was used to determine the content of polysaccharides, and HPLC-ELSD method was used to determine the monosaccharides and oligosaccharides. The comparative chart of content determination, cluster analysis and entropy weight TOPSIS model was used to find the similar change laws and time interval of different processing methods of Polygoni Multiflori Radix Praeparata. The results demonstrated that around 32 h, the content of nine components in Polygoni Multiflori Radix Praeparata with different processing methods had similar change laws, and the decoction pieces had a high quality, indicating that the four processing methods of Polygoni Multiflori Radix Praeparata are likely to be used as one type of decoction piece with the same name.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Glycosides ; Plant Roots ; Polygonum