Mechanism of total flavonoids of Ziziphora clinopodioides in improving atherosclerosis by regulating PI3K/Akt/mTOR pathway / 中国中药杂志

The present study observed the regulatory effect of total flavonoids of Ziziphora clinopodioides on autophagy and the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathways in ApoE~(-/-) mice and explored the mechanism of total flavonoids of Z. clinopodioides against atherosclerosis(AS). ApoE~(-/-) mice were fed on a high-fat diet for eight weeks to induce an AS model. The model mice were randomly divided into a model group, a positive control group, and low-, medium-and high-dose groups of total flavonoids of Z. clinopodioides, while C57BL/6J mice fed on a common diet were assigned to the blank group. The serum and aorta samples were collected after intragastric administration for 12 weeks, and the serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein-cholesterol(LDL-C), and high density lipoprotein-cholesterol(HDL-C) were detected by an automatic biochemical analyzer. The serum expression levels of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), matrix metalloproteinase-2(MMP-2), and matrix metalloprotei-nase-9(MMP-9) were detected by enzyme-linked immunosorbent assay(ELISA). Oil red O staining was used to observe the aortic plaque area in mice. Hematoxylin-eosin(HE) staining was used to observe the aortic plaque and pathological changes in mice. The expression of P62 and LC3 in the aorta was detected by the immunofluorescence method. The protein expression of LC3Ⅱ/Ⅰ, Beclin-1, P62, p-PI3K, p-Akt, and p-mTOR in the aorta of mice was detected by Western blot. The results showed that compared with the blank group, the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2 and MMP-9 in the model group were significantly increased(P<0.01 or P<0.05), the content of HDL-C was decreased(P<0.05), intra-aortic plaque area was enlarged(P<0.01), the expression of LC3 in the aorta was significantly down-regulated, P62 expression was up-regulated(P<0.01 or P<0.05), the expressions of LC3Ⅱ/Ⅰ and Beclin-1 in the aortic lysate were significantly down-regulated, and the expressions of p-PI3K, p-Akt, p-mTOR and P62 were significantly increased(P<0.01). The medium-and high-dose groups of total flavonoids of Z. clinopodioides could reduce the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2, and MMP-9 in AS model mice(P<0.01 or P<0.05), and increase the content of HDL-C(P<0.01 or P<0.05). The aortic plaque area of mice after middle and high doses of total flavonoids of Z. clinopodioides was significantly reduced(P<0.01), the content of foam cells decrease, and the narrowing of the lumen decreased. The total flavonoids of Z. clinopodioides significantly increased the expression of LC3 in the aorta and the expression of LC3Ⅱ/Ⅰ and Beclin-1 in the lysate, and decreased the expression of P62 in the aorta and the expression of p-PI3K, p-Akt, p-mTOR and P62 in the lysate(P<0.01 or P<0.05). The results showed that the total flavonoids of Z. clinopodioides could improve the content of blood lipids and inflammatory factors, and reduce the generation of foam cells and plaques in aortic tissue, and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.

The study of the biological activities of Ziziphora clinopodioides

Abstract The aim of the current study was to determine the chemical constituents of essential oil and to study the antibacterial and antioxidant activities of essential oil and the extracts obtained from the raw material of Ziziphora wild growing in the floras of Armenia and Artsakh cultivated in the hydroponic conditions. The essential oils were obtained by the method of hydro-distillation. The determination of the essential oil constituents were performed by the GC-MS method. Agar disk diffusion method was used to study the antimicrobial activity of essential oils. The antioxidant activity determination was carried out DPPH test by the spectrophotometric method, at the same time IC50 was determined. The highest values of the essential oils yield (1.25 ± 0.01%) and IC50 13.83±0.218(x10-5)g/l) were received for the plant cultivated in hydroponic conditions. For the first time in the above studied samples, by the method of GC-MS more than 70 components were revealed. The results of the study showed that essential oils of Ziziphora exhibit antimicrobial activity and the extracts revealed relatively expressed antioxidant activity. The study results show the future prospects of the use of Ziziphora not only as the source of flavonoids and essential oils, but also antimicrobial and antioxidant agents.

Xintahua water extract improves the disease indexes of atherosclerosis model rats / 中国临床药理学与治疗学

AIM: To investigate the effect of water extract of Ziziphora clinopodioides Lam (WEZ) on improving atherosclerosis model rats. METHODS: Sixty SD rats were randomly selected and 50 rats of them were randomly selected for atherosclerosis model with high-fat emulsion and vitamin D3, and then randomly divided into atherosclerosis (AS) model group, low, medium, high WEZ group and the positive control group. After 8 weeks of drug intervention, the plasma of each group of rats was collected to detect total cholesterol (TC), triglyceride (TG), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and trimethylamine oxide (TMAO) level. The correlation between plasma TMAO and TNF-α and IL-6 levels in AS rats was detected. The aortic tissue-embedded sections of rats in each group were taken out to compare the aortic plaque area/aortic lumen area (PA/LA) ratio. RESULTS: Compared with the blank control group, the serum TC and TG levels of the AS model group increased significantly, and the difference was statistically significant (P<0.01). Compared with the AS model group, the serum TC and TG levels of the middle and high dose WEZ group and the positive control group were significantly decreased, and the difference was statistically significant (P<0.05). Compared with the blank control group, the serum TNF-α and IL-6 levels in the AS model group increased significantly, and the difference was statistically significant (P<0.01). Compared with the AS model group, the serum TNF-α and IL-6 levels of the high-dose WEZ group and the positive control group were significantly decreased, and the difference was statistically significant (P<0.05). Compared with the blank control group, the plasma TMAO level of rats in the AS model group increased significantly, and the difference was statistically significant (P<0.01). Compared with the AS model group, the plasma TMAO levels of rats in the high-dose WEZ group and the positive control group decreased significantly, and the difference was statistically significant (P<0.05). The analysis of the correlation between TMAO level and TNF-α/IL-6 level showed that TMAO level was positively correlated with TNF-α level (P=0.001, r=0.673), and positively correlated with IL-6 level (P=0.002, r= 0.646). Compared with the blank control group, the PA/LA ratio of the AS model group increased significantly, and the difference was statistically significant (P<0.01). Compared with the AS model group, the PA/LA ratio of rats in the medium and high dose WEZ group and the positive control group decreased significantly, and the difference was statistically significant (P<0.05). CONCLUSION: WEZ may regulate TMAO levels, down-regulate TNF-α and IL-6 levels, and reduces TC and TG levels, thereby improving AS, but its mechanism still needs further study.

Multi-elemental analysis of Ziziphora clinopodioidesfrom different regions, periods and parts using atomic absorption spectrometry and chemometric approaches

ABSTRACTIn this study, ten trace elements in Ziziphora clinopodioidesLam., Lamiaceae, from different regions, periods and parts in Xinjiang were determined by atomic absorption spectrometry following microwave-assisted acid digestion. The decreasing sequence of elements levels was K > Ca > Mg > Fe > Cu > Zn > Na > Mn > Cd > Pb. Chemometric approaches, such as correlation analysis, principal component analysis, and hierarchical cluster analysis were applied to classify Z. clinopodioides according to its elements contents. Principal component analysis revealed 83.51% of the variance with the first four principal component variables. Hierarchical cluster analysis indicated five groups from the eighteen regions, and the result of classification can correspond to the geographical distribution for the most regions. Variation in the elements exhibited a decreasing trend, but of different types in the studied periods. Elemental contents distributed in leaves were higher than those in flowers and stems. Therefore, chemometric approaches could be used to analyze data to accurately classify Z. clinopodioides according to origins. This study provided some elemental information on chemotaxonomy, diversity, changing pattern, distribution, and metabolism of Z. clinopodioides at spatial and temporal levels, and could be used as a reference of planting and quality standards.

Vasodilatation Effects of the Alcohol Extract from Uighur Medicine Ziziphora clinopodioides on Isolated Rat Thoracic Aorta Vessel Rings / 中国药房

OBJECTIVE:To study the vasodilatation effects of the alcohol extract from Uighur medicine Ziziphora clinopodioi-des(EEZ)on isolated rat thoracic aorta vessel rings. METHODS:Isolated rat thoracic aorta vessel rings were prepared. There was a group with intact vessel ring endothelium and a group with vessel ring endothelium removed in the test. After preshrinking the ves-sel rings with phenylephrine(PE),EEZ of 100,300,500,700,900 and 1 100 mg/L was added gradually,concentration-vasodila-tation curve was drawn and maximal vasodilatation rate (Emax) and median effective concentration (EC50) were calculated. For the group with vessel ring endothelium removed,after the vessel ring was pretreated with EEZ at EC50 in calcium-free solution or calci-um-free high-potassium solution,CaCl2 of 0.4,0.8,1.2,1.6,2.0 and 2.4 mmol/L was added gradually,and calcium concentra-tion-tension curve was drawn;following the pretreatment of the vessel ring with EEZ at EC50,1 μmol/L PE was added and shrink tension was recorded and vasoconstriction percentages was calculated. RESULTS:EEZ had vasodilatation effect on the vessel ring preshrunk with PE in a concentration and smooth muscle-dependent manner. The group with intact vessel ring endothelium and the group with vessel ring endothelium removed respectively had Emax of (58.18 ± 16.23)% and (73.54 ± 17.21)%,and EEZ EC50 of 773.27 mg/L. For calcium-free high-potassium solution,EEZ could cause the calcium ion-vasoconstriction curve to move to the right obviously;for calcium-free solution,EEZ could inhibit the vasoconstriction caused by PE. CONCLUSIONS:EEZ has vasodi-latation effects by a mechanism which may be related to inhibiting calcium influx and intracytoplasmic calcium release through the inhibition of voltage-dependent calcium channels(VDCCs),and thus interfering intracytoplasmic calcium ion balance.

Chemical constituents from Ziziphora clinopodioides / 中草药

Objective: To investigate the chemical constituents of the Ziziphora clinopodioides. Methods: The compounds were isolated and purified by means of various chromatographic techniques and their structures were identified on the basis of NMR spectral data. Results: Twenty-one known compounds were isolated from the ethanol extract of the dry stems of P. amarus and their structures were identified as protocatechuic acid (1), picein (2), rosmarinic acid (3), cafferic acid (4), luteolin (5), 4', 5, 7-trihydroxy isoflavone (6), irisdichotototin D (7), pinocembrin-7-O-rutinoside (8), acacetin-7-O-rutinoside (9), 4-hydroxy-3-methoxy-acetophenone-4-O-β-D-apiofuranosy-(1→6)-β-D-glucopyranoside (10), baicalein (11), kaempferide (12), chrysin-7-O-rutinoside (13), quercetin (14), apigenin (15), ploybotrin (16), 1-O-p-coumaroylglycerol (17), 5-(hydroxymethyl)-2-furancarboxaldehyde (18), acacetin (19), 6, 8-di-C-β-D-glucosylchrysin (20), and dibutyl phthalate (21). Conclusion: Compounds 5-8, 10-12, 16-18, and 20-21 are first obtained from this plant for the first time.

Identification of Essential Oil ofZiziphora Clinopodioides Lam. in Xinjiang by Infrared Spectroscopy and Clustering Analysis / 中国中医药信息杂志

Objective To analyze and compare the essential oil ofZiziphora clinopodioides Lam. from different regions in Xinjiang by infrared spectroscopy;To provide a reference for its identification and quality control.Methods Characteristic absorption peaks ofZiziphora clinopodioides Lam. essential oil from 18 regions in Xinjiang Uygur autonomous region were identified, compared and analyzed according to peaks’ shapes, positions and intensities by infrared spectroscopy, second derivative infrared spectroscopy and clustering analysis.Results Differences exist in the characteristic absorption peaks according to peaks’ shapes, positions and intensities. And then the samples from 18 regions can be divided into four categories.Conclusion This method was direct, simple, fast and convenient. Combining with clustering analysis, It could provide a reliable evidence for identification and quality control ofZiziphora clinopodioides Lam..

HPLC fingerprint analysis on Wei Medicine Ziziphora tenuior and Ziziphora clinopodioides / 中草药

Objective: To establish HPLC fingerprint for the analysis on Ziziphora tenuior and Z. clinopodioides and the determination of hyperin, rutine, and pulegone in eight batches of Z. tenuior and 22 batches of Z. clinopodioides synchronously. Methods: The HPLC method was used with Kromasil C18 column (250 mm × 4.6 mm, 5 μm), and a mixture of methanol-0.08% HCOOH was used as mobile phase with gradient elution. The HPLC fingerprint for eight batches of Z. tenuior and 22 batches of Z. clinopodioides was established. Results: The HPLC fingerprints of the two species were obviously different, so two modes were established for two species, respectively. The eight batches of Z. tenuior and 22 batches of Z. clinopodioides were classified into two groups based on the result of principal component, hierarchical cluster, and similarity analyses. Hyperin and pulegone were the common compositions; quercetin (161.06-213.22 μg/g) was founded in Z. tenuior, but not in Z. clinopodioides; the contents of hyperin (174.15-802.24 μg/g), pulegone (77.43-353.45 μg/g) of Z. clinopodioides were higher than those of Z. tenuior (49.72-204.33 and 36.19-93.29 μg/g). Conclusion: Z. tenuior and Z. clinopodioides have the different fingerprint, which could provide the evidence for their quality control, species identification, and classification.

The potential inhibitory effect of cuminum cyminum, ziziphora clinopodioides and nigella sativa essential oils on the growth of Aspergillus fumigatus and Aspergillus flavus

The goals of this study were to evaluate the effectiveness of Cuminum cyminum, Ziziphora clinopodioides and Nigella sativa essential oils to inhibit the growth of Aspergillus fumigatus and A. flavus and to evoke ultrastructural changes. The fungi were cultured into RPMI 1640 media in the presence of oils at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 mg/ml in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods with shaking for 48 h at 28ºC. Conidial and mycelial samples exposed to 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml for 5 days in 2 percent yeast extract granulated plus 15 percent Saccharose media were processed for transmission electron microscopy (TEM). Based on broth dilution methods, C. cyminum and to a lesser extent Z. clinopodioides oils exhibited the strongest activity against A. fumigatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/ml, while the oil from N. sativa exhibited relatively moderate activity against two above fungi with MIC90 ranging from 1.5 to 2 mg/ml. The main changes observed by TEM were in the cell wall, plasma membrane and membranous organelles; in particular, in the nuclei and mitochondria. These modifications in fungal structure were associated with the interference of the essential oils with the enzymes responsible for cell wall synthesis, which disturbed normal growth. Moreover, the essential oils caused high vacuolation of the cytoplasm, detachment of fibrillar layer of cell wall, plasma membrane disruption and disorganization of the nuclear and mitochondrial structures. Aspergillus fumigatus and A. flavus growth inhibition induced by these oils were found to be well-correlated with subsequent morphological changes of the fungi exposed to different fungistatic concentrations of the oils. Our results show the anti-Aspergillus activities of C. cyminum, Z. clinopodioides and N. sativa essential oils, which strengthens the potential use of these substances as anti-mould in the future.