Optimization of Ziziphora clinopodioides L. essential oil nanoencapsulation in chitosan nanocomplex by response surface methodology.

Nano-encapsulation of essential oils, a specific area of interest, can help overcome challenges associated with their commercial use. This study aimed to evaluate the effect of different concentrations of chitosan, Ziziphora clinopodioides L. essential oil (ZcEO), and Sodium-Tri Polyphosphate (TPP), both individually and in interaction, on several properties of EO-loaded chitosan nanoparticles. These properties include particle size (PS), zeta potential (ZP), and encapsulation efficiency (EE) using a two-stage emulsion-ionic gelation approach. The optimization of the parameters was done by response surface methodology using Box-Behnken design. The chemical composition of ZcEO was analyzed as well. The primary compounds in ZcEO were found to be pulegone (29.24 %), 1,3-dimethyl-2-(2-methylpropylidene) imidazolidine (9.05 %), piperitenone (6.65 %), thymol (5.38 %), and carvacrol (5.27 %). The PS ranged from 117.33 to 4934.1 nm, the ZP varied from -1.1 to -30.83 mV, and the EE spanned from 31.74 to 87.04 %. The results showed that an increase in the initial EO content led to a decrease in PS and ZP, but an increase in EE. Moreover, increasing the TPP concentration resulted in an enhancement in PS, ZP, and EE, whereas increasing the Chs concentration led to a slight increase in PS, ZP, and EE. Furthermore, the results of this study proved the interaction effect of different parameters on the responses investigated. Under optimized conditions, the optimal concentrations of chitosan, ZcEO and TPP were attained at 6.768, 6.078, and 7.595 mg/mL respectively. This resulted in a PS of 117.331 nm, a ZP of -20.949 mV, and an EE of 75.385 %. In conclusion, the results suggest that adjusting the concentrations of Chs, EO, and TPP is an effective approach to controlling the properties of NPs and optimizing their performance.

Development of biomaterials based on chitosan-gelatin nanofibers encapsulated with Ziziphora clinopodioides essential oil and Heracleum persicum extract for extending the shelf-life of vacuum-cooked beef sausages.

The aims of the current study were to encapsulate Ziziphora clinopodioides essential oil (ZEO, 0%, 0.15%, and 0.25%) and Heracleum persicum extract (HPE, 0%, 0.25%, and 0.5%) into the chitosan-gelatin (CH-GE) nanofibers through the electrospinning process to improve the shelf-life of vacuum-cooked beef sausages through 70 days of refrigerated storage. Scanning electron microscopy indicated that all nanofibers appeared thin, well-defined, smooth, and possessed uniform thread-like fibers without any beads or nodule formations. The Fourier transform infrared spectroscopy study confirmed the molecular interaction between encapsulated compounds and CH-GE nanofibers. The X-ray diffraction analysis of nanofibers showed an increase in crystallinity after incorporating ZEO and HPE into the polymer. Treated sausages with CH-GE-ZEO 0.25%-HPE 0.25% and CH-GE-ZEO 0.25%-HPE 0.5% showed significantly lower microbial population and lipid oxidation than the control group during the experiment period (P < 0.05). Sausages formulated with designated CH-GE nanofibers had better microbial, chemical, and sensory properties compared to sausages treated with pure ZEO/HPE during refrigerated storage. The findings also showed that treated sausages with CH-GE-ZEO 0.25%-HPE 0.5% had the highest color, odor, texture, and overall acceptability after 70 days of refrigerated storage conditions. Therefore, this treatment could be applicable for the prolonged storage conditions during cooked beef sausage production.

The Anti-atherosclerosis Mechanism of Ziziphora clinopodioides Lam. Based On Network Pharmacology.

We investigated the mechanisms underlying the effects of Ziziphora clinopodioides Lam. (ZCL) on atherosclerosis (AS) using network pharmacology and in vitro validation.We collected the active components of ZCL and predicted their targets in AS. We constructed the protein-protein interaction, compound-target, and target-compound-pathway networks, and performed GO and KEGG analyses. Molecular docking of the active components and key targets was constructed with Autodock and Pymol software. Validation was performed with qRT-PCR, ELISA, and Western blot.We obtained 80 components of ZCL. The network analysis identified that 14 components and 37 genes were involved in AS. Then, 10 key nodes in the PPI network were identified as the key targets of ZCL because of their importance in network topology. The binding energy of 8 components (Cynaroside, α-Spinasterol, Linarin, Kaempferide, Acacetin, Genkwanin, Chrysin, and Apiin) to 4 targets (MMP9, TP53, AKT1, SRC) was strong and <-1 kJ/mol. In addition, 13 of the 14 components were flavonoids and thus total flavonoids of Ziziphora clinopodioides Lam. (ZCF) were used for in vitro validation. We found that ZCF reduced eNOS, P22phox, gp91phox, and PCSK9 at mRNA and protein levels, as well as the levels of IL-1ß, TNF-α, and IL-6 proteins in vitro (P < 0.05).We successfully predicted the active components, targets, and mechanisms of ZCL in treating AS using network pharmacology. We confirmed that ZCF may play a role in AS by modulating oxidative stress, lipid metabolism, and inflammatory response via Cynaroside, Linarin, Kaempferide, Acacetin, Genkwanin, Chrysin, and Apiin.

Chemical Profiling and Antifungal Activity of Ziziphora clinopodioides Leaf Essential Oil against the Pathogen Verticillium dahliae.

Essential oils (EOs) are often used as natural antifungal agents to control the growth of phytopathogenic fungi. The aim of this study was to determine the effect of Ziziphora clinopodioides leaf EO against Verticillium dahliae, a pathogenic fungus of cotton. Gas chromatography-mass spectrometry (GC/MS) analysis revealed the presence of 15 compounds of the total of extracted oil, which was consisted of 98.79 % monoterpenes and 0.61 % sesquiterpenes. The major constituents were pulegone (62.17 %), isomenthone (18.42 %), l-menthone (5.55 %) and piperitenone (3.99 %). The mycelial growth of Verticillium dahliae was completely inhibited at 0.24 µL/mL air under vapor phase condition. Considerable morphological variations were also observed in the fungal sclerotia at the contact phase at 3 µL/mL. This study demonstrated for the first time that Z. clinopodioides EO can effectively inhibit the growth of V. dahliae, implying that it has the potential to be explored as an antifungal agent against Verticillium Wilt of cotton.

Antibacterial and Antioxidant Activity of Ziziphora clinopodioid Lam. (Lamiaceae) Essential Oil.

The genus Ziziphora belongs to medicinal plants. It is often used as a stomach tonic, carminative, antimicrobial, and expectorant; the extracted essential oils can be used as a second line of defence against pathogens. This study aimed to determine the antioxidant activity of essential oils of Z. clinopodioides as well as antibacterial activity against foodborne pathogens (Bacillus sp., Escherichia coli, Staphylococcus aureus and Pseudomonas sp.). The antibacterial activity of Z. clinopodioides essential oil was determined using the microdilution (M.D.) method in the nutritional broth medium and the agar disk diffusion assay. The result demonstrated that essential oil exhibit solid antibacterial properties against both gram-positive and gram-negative bacteria. Sequentially regarding MIC and MBC values, Escherichia coli was a higher level of resistance to the essential oil compared to Bacillus sp. Our findings suggested that the essential oil of Z. clinopodioides could be used as an antibacterial agent. The total antioxidant capacity of Z. clinopodioides leaves was assessed as ascorbic acid equivalents per gram of the leaves essential oil extract. The total antioxidant capacity was determined using ascorbic acid (y=0.1185x + 49.508, R2=0.3877). While the result of Z. clinopodioides was (y=0.1372x + 40.032, R2=0.4503).

The variability of phenolic constituents and antioxidant properties among wild populations of Ziziphora clinopodioides Lam.

In this study, phenolic derivatives and antioxidant activities of fourteen Ziziphora clinopodioides populations, as well as LC-MS/MS analysis of three specific flavonoids were evaluated. Generally, high contents of phenolic derivatives were found in shoot extracts compared to roots. LC-MS/MS, a powerful analytical technique, was employed for the identification and quantify the individual flavonoids in Z. clinopodioides populations' extracts, in a quantity order of quercetin > rutin > apigenin. Scavenging activity by DPPH and FRAP was performed, and accordingly, in the shoot, the highest values for the DDPH were 4.61 ± 0.4 and 7.59 ± 0.26 µg ml- 1 in populations 1 and 13, respectively, and for the FRAP were 328.61 ± 5.54 and 292.84 ± 2.85 mg g DW- 1, in populations 6 and 1 respectively. Multivariate analysis results of the principal component analysis indicated the amount of polyphenols to be useful indicators in differentiating the geographical localities which explain 92.7% of the total variance. According to the results of hierarchical cluster analysis, the studied populations could be separated into two groups in that the contents of phenolic derivatives and antioxidant activities of different plant parts. Both shoot and root samples were well discriminated with the orthogonal partial least squares discriminant analysis (R2X: 0.861; Q2: 0.47) model. The validity of the model was confirmed by using receiver operating characteristic curve analysis and permutation tests. Such data make an important addition to our current knowledge of Ziziphora chemistry and are decisive in the identification of germplasms with a homogeneous phytochemical profile, high chemical content and bioactivity. The present results could also be helpful for the potential application of Z. clinopodioides in different kinds of industries as natural antioxidants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01283-y.

[Mechanism of total flavonoids of Ziziphora clinopodioides in improving atherosclerosis by regulating PI3K/Akt/mTOR pathway].

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.

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.

ICP-OES assessment of trace and toxic elements in Ziziphora clinopodioides Lam. from Iran by chemometric approaches.

The aim of the study was to evaluate the concentrations of some essential and toxic elements (including Ca, K, Mg, Na, Fe, Cu, Mn, Zn, Co, Mo, Pb, Ni, Cr, and Cd) in Ziziphora clinopodioides Lam. (endemic Iranian herb) from 14 different regions by ICP-OES (inductively coupled plasma-optical emission spectrometry) method followed by multivariate statistical analyses. The analytical performances were assessed as the limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. Multivariate analysis (CA, PCA and HCA) showed the elemental distribution in the roots were higher than the shoots and significant element was Ca between regions. Three principal components (PCs) explained 77.94% of the total variance. They were as follows: PC1 with Cu, Zn, K, Cr, Ni, and Mn; PC2 with Na, Ca, Pb, and Fe; and PC3 with Mg. Hierarchical cluster analysis indicated four groups of Z. clinopodioides samples from the 14 regions based on their trace and toxic element levels. These chemometric approaches with multivariate analysis enable researchers to understand and quantify the relationship between the variables in a data set, and the analysis considers more than one factor. The concentrations of Cu, Na, Mn, Zn, and Pb in most Z. clinopodioides samples were below the WHO (world health organization) limit for herbal medicines (10, 51,340, 200, 50, and 10 µg g-1 respectively), while Fe and Ca levels were higher than allowed (15 and 614 µg g-1 respectively). However, the WHO limit for Mg, K, Co, and Mo in medicinal plants has not yet been determined. The results of this study confirmed that different parts of Z. clinopodioides can be used as an important source for human nutrition due to its essential mineral elements.

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.

Phytochemical composition and in vitro safety evaluation of Ziziphora clinopodioides Lam. ethanolic extract: Cytotoxicity, genotoxicity and mutagenicity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: The application of the herb Ziziphora clinopodioides Lam. in folk medicine and as a food additive has been recommended due to its many claimed bioactivities. Regardless of the plant benefits, its safety considerations are largely unknown. AIM OF THE STUDY: The aim of the present research was to determine the chemical compositions and cytotoxicity, genotoxicity, and mutagenicity potentials of the ethanolic extract of Ziziphora clinopdioides Lam. (EEZC). MATERIALS AND METHODS: GC-MS and LC-MS analysis were used for chemical composition determination. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion dye assays were used for cytotoxicity and the Comet assay was employed for genotoxicity assessment on human blood lymphocytes. Also, the Ames Salmonella/microsome test was carried out for the evaluation of mutagenicity. RESULTS: Pulegone was the main component of the n-hexane fraction. Different phenolic acids and flavonoids were detected by LC-MS. The cytotoxicity study indicated a conspicuous decline in human lymphocyte viability ranging from 52% to 100% as showed by the MTT assay and 67% up to 100% by the trypan blue assay, at 1 and 10 mg/mL, respectively. The Comet assay results revealed a dose dependent genotoxicity, in so much as 90% and 98% of the cells were screened as damaged at concentrations of 5 and 10 mg/mL, respectively. An incidence rate of 8% and 13% of grade 4 damage was observed at 5 and 10 mg/mL, respectively. Additionally, the DNA damage index (DI) was elevated dose-dependently by a rising concentration of the extract, wherein the DI at 10 mg/mL concentration was 2.22, which was 22 times greater than that of negative control, and even more than positive control. The Ames test exhibited no signs of mutagenicity for neither Salmonella typhimurium TA98 nor TA100 strains, accompanied or unaccompanied by S9 metabolic activation. CONCLUSION: Results indicated a dose-dependent cytotoxicity and genotoxicity potential of the EEZC on human lymphocytes, suggesting that this plant should be used with caution by consumers, even in the food and pharmaceutical industries. Since the plant usage in daily life continues to increase due to its ever growing phytotherapical and phytonutritional properties, it may pose a health risk by its high concentration's uptake. Although no mutagenicity of this extract was observed in this study, further research is recommended to clarify the mutagenic risks of this herb.

The effect of hydroalcoholic extract of Ziziphora clinopodioides L. on spatial memory and neuronal density of hippocampal CA1 region in rats with sporadic Alzheimer's disease.

OBJECTIVE: Alzheimer's disease is a neurodegenerative disorder associated with gradual loss of cognitive and memory abilities. It was shown that the hippocampus is one of the first structures in the brain that is affected by the disease. Ziziphora clinopodioides (Z. clinopodioides) is a member of Lamiaceae family and contains various substances. MATERIALS AND METHODS: In this experimental study, 72 adult male Wistar rats were used for behavioral and histopathologic studies. They were divided into nine groups included: control, negative control (Alzheimer), positive control (Alzheimer's treated with rivastigmine), aCSF (artificial cerebrospinal fluid) + ziziphora extract with doses of 200,400, and 600 mg/kg, and STZ (stereptozotocine)+ziziphora extract in 200,400,600 mg/kg doses. The injury was created with bilaterally intraventricular injection. The spatial memory was studied by passive avoidance test and neuronal density was evaluated by dissector method. To examine the histopathological lesions, Congo red and toluidine blue staining were done. Data were analyzed by ANOVA Minitab software. RESULTS: The memory index (neuronal density and passive avoidance test results) showed a significant decrease in negative control group compared to control (p≤0.001). Treatment with the hydroalcoholic extract at the doses of 400 and 600 mg/kg showed a significant increase in memory index in rats with Alzheimer's disease (p≤0.001). The effect of 200 mg/kg extract was not significantly different from that of the negative control group. The results of histological analysis indicated beta-amyloid plaques formation in the control group as compared to the negative control group while treatment with the extract at the doses of 400 and 600 mg/kg, significantly reduced beta-amyloid plaques formation. CONCLUSION: These findings suggest that the extract of Z. clinopodioides can improve Alzheimer's condition and alleviate memory and histopathologic damages; also, it decreases beta-amyloid plaques and apoptosis in CA1 region of the hippocampus.

Chemical Composition and Role of Opioidergic System in Antinociceptive Effect of Ziziphora Clinopodioides Essential Oil.

INTRODUCTION: Ziziphora Clinopodioides has been used widely for various therapeutic purposes in Iranian folk medicine. The current study aimed to determine interaction of antinociceptive effect of the Essential Oil of Ziziphora Clinopodioides (EOZC) and opioidergic system in male rats using formalin test. METHODS: Sixty-four male Wistar rats were divided into eight groups. The groups 1 to 7 were injected with normal saline, vehicle (Tween-80, 0.5%), 10, 20, 40 mg/kg of the EOZC, morphine (5 mg/kg) and naloxone (2 mg/kg), respectively. Thirty minutes later, the formalin test was performed by intraplantar injection of formalin (50 µL, 2%). In group 8, naloxone (2 mg/kg) was injected 15 min before injection of EOZC (20 mg/kg), followed by formalin at 15 min later. The formalin test was done as time spent for licking and biting of the injected paw. Formalin induced a biphasic pain reaction. The chemical composition of EOZC was identified using Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: EOZC (10, 20, and 40 mg/kg) dose dependently and morphine (5 mg/kg) reduced pain responses in the both phases of pain (P<0.05). Naloxone (2 mg/kg) alone had no effect on the severity of pain (P>0.05) but pretreatment with naloxone inhibited EOZC-induced antinociception activity (P<0.05). Based on the GC-MS results, EOZC comprised 65.22% carvacrol, 19.51% thymol, 4.86% p-cymene and 4.63% γ-terpinene. CONCLUSION: These results demonstrate that EOZC has antinociceptive effect and this effect might mediate via opioidergic pathways.

Chemical compositions, antioxidant and antimicrobial properties of Ziziphora clinopodioides Lam. essential oils collected from different parts of Iran.

The aims of the present study were to investigate chemical compositions, antioxidant and antimicrobial properties of Ziziphora clinopodioides essential oils (ZEOs) collected from four provinces in western Iran (Ilam, Lorestan, Kermanshah and Kurdestan). Carvacrol was the most abundant constituent in the flower, stem and leaf oil samples of Ilam, Lorestan and Kermanshah regions by 73.12-74.29%, 66.47-66.89% and 65.11-65.32%, respectively. The most abundant components in Kurdestan sample were thymol (55.32-55.60%), followed by γ-terpinene (24.45-24.56%), p-cymene (10.21-10.25%) and α-terpinene (2.75-2.77%). The ZEO inhibited the growth of Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157:H7, Bacillus subtilis, Bacillus cereus and Staphylococcus aureus at MIC values between 0.03 and 0.04%. Kermanshah oil sample had a higher 1,1-diphenyl-2-picrylhydrazyl radical scavenging (0.30-0.31 mg/ml), ability to prevent the bleaching of ß-carotene (0.09-0.1 mg/ml), ferric reducing power (0.40-0.42 mg/ml) and thiobarbituric acid (0.004-0.006 Meq of malondialdehyde/g) values than that of ZEOs from Ilam, Kurdestan and Lorestan. The strong in vitro antimicrobial and antioxidant activities supports the traditional use of ZEO in the treatments of gastrointestinal diseases.

Antibacterial Effects of Garlic Extracts and Ziziphora Essential Oil on Bacteria Associated with Peri-Implantitis.

INTRODUCTION: Peri-implantitis is an inflammatory process, characterized by loss of bone around implants and bleeding on probing. Colonization of bacteria in the pocket created around the implant is one of the most important aetiologic factors involved. Although antibiotics are routinely used and are effective agents against this bacterial infection, due to the side effects and drug resistance of these medications, researchers are more interested in using herbal derivatives. There are relatively limited number of studies in this respect. AIM: To evaluate and compare the antibacterial effects of the extracts of Allium sativum and Ziziphora clinopodioides essential oil on S. aureus and P. aeruginosa. MATERIALS AND METHODS: In this in vitro study conducted at Tabriz University of Medical Sciences between March 2016 and July 2016, aqueous and methanolic extracts of garlic and ziziphora essential oil were prepared and then their effects on one standard strain of P. aeruginosa and two standard strains of S. aureus and 18 clinical strains, (nine strains of P. aeruginosa and nine strains of S. aureus) which had been isolated from wound and blood cultures, were evaluated using the reference broth macro dilution method and disk diffusion technique. Data were evaluated with descriptive statistical techniques and t-test for independent groups, using SPSS 17. RESULTS: Aqueous and methanolic extracts of garlic did not exhibit inhibitory effects on S. aureus and P. aeruginosa. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for ziziphora essential oil against S. aureus were 0.25 and 0.5 µg/mL, respectively. The mean diameter of growth inhibition zone for S. aureus in the ziziphora essential oil group (0.25µg/mL) and Vancomycin group (10 µg/mL) were 26.09±5.8 and 14.63±1.20 mm respectively (p<0.001). Growth inhibition zone for P. aeruginosa were observed in only one (35 mm) and three cases (12.5±3.4 mm) in the ziziphora essential oil group (0.5 µg/mL) and Nalidixic acid group (10 µg/mL) respectively. CONCLUSION: The essential oil of ziziphora exhibited a favourable antibacterial effect on S. aureus; therefore, its extract can be used as an effective antimicrobial agent alone or in association with other antibiotics in the treatment of infections due to S. aureus.

The properties of chitosan and gelatin films incorporated with ethanolic red grape seed extract and Ziziphora clinopodioides essential oil as biodegradable materials for active food packaging.

The aim of this study was to improve different characteristics including antibacterial, antioxidant, physical and mechanical properties of chitosan (Ch) and gelatin (Ge) films by incorporating Ziziphora clinopodioides essential oil (ZEO; 0 and 1% v/w) and ethanolic grape seed extract (GSE; 0 and 1% v/w). The main compounds of the ZEO were carvacrol (65.22%) and thymol (19.51%). According to our findings, addition of aforementioned materials could improve total phenolic content, antibacterial and antioxidant activities, thickness and also water vapor barrier property. ZEO and GSE reduces swelling index, tensile strength, puncture force and puncture deformation of Ch and Ge films. Pure Ch and Ge films had slightly yellow and white appearances, respectively, while films incorporated with GSE in combination with ZEO had grey appearances. This study indicated the some benefits of addition of ZEO and GSE into Ch and Ge films and their potentials for application as biodegradable active packaging.

Effect of PLA films containing propolis ethanolic extract, cellulose nanoparticle and Ziziphora clinopodioides essential oil on chemical, microbial and sensory properties of minced beef.

This study was conducted to examine the effects of polylactic acid (PLA) film containing propolis ethanolic extract (PE), cellulose nanoparticle (CN) and Ziziphora clinopodioides essential oil (ZEO) on chemical, microbial and sensory properties of minced beef during storage at refrigerated temperature for 11days. The initial total volatile base nitrogen (TVB-N) was 8.2mg/100g and after 7days reached to 29.1mg/100g in control, while it was lower than 25mg/100g for treated samples. At the end of storage time in control samples peroxide value (PV) reached to 2.01meqperoxide/1000g lipid, while the values for the treated samples remained lower than 2meqperoxide/1000g lipid. Final microbial population decreased approximately 1-3logCFU/g in treated samples compared to control (P<0.05). Films containing 2% ZEO alone and in combination with different concentrations of PE and CN extended the shelf life of minced beef during storage in refrigerated condition for at least 11days without any unfavorable organoleptic properties.

The antibacterial effect of Ziziphora clinopodioides essential oil and nisinagainst Salmonella typhimurium and Staphylococcus aureus in doogh, a yoghurt-based Iranian drink.

Doogh is the most popular and commonly consumed yoghurt-based Iranian drink. The aim of this study was to investigate the antibacterial effects of Ziziphora clinopodioides essential oil (ZEO) at 0.10 and 0.20% concentrations, nisin at 250 and 500 IU mL-1, and their combination against Salmonella typhimurium and Staphylococcusaureus in doogh during storage at 4 ˚C for  9 days. Nine batches were studied as follows: control: no ZEO or nisin added, A: 0.10% ZEO, B: 0.20% ZEO, C: 250 IU mL-1 nisin, D: 500 IU mL-1 nisin, E: 0.10% ZEO + 250 IU mL-1 nisin, F: 0.10% ZEO + 500 IU mL-1 nisin, G: 0.20% ZEO + 250 IU mL-1 nisin and H: 0.20% ZEO + 500 IU mL-1 nisin. Based on gas-chromatography and mass spectrometry, carvacrol (65.22%), thymol (19.51%), p-cymene (4.86%) and É£-terpinene (4.63%) were the major components of ZEO. The populations of S. typhimurium and S.aureus in samples treated with all concentrations of the ZEO and nisin were kept below 1 log CFU mL-1 on day 5 of storage, while the count of S. typhimurium and S.aureus was found as 2.72 ± 0.02 and 2.21 ± 0.00 log CFU mL-1 on day 5 for untreated samples, respectively. The ZEO separately and in combination with nisin, was very effective against these two common food-borne pathogens. The ZEO alone and in combination with nisin could be considered as a potential strong antimicrobial agent that can be used for the growth inhibition of aforementioned bacteria in food products especially doogh.

Antifungal activity of essential oil of Ziziphora clinopodioides and the inhibition of aflatoxin B1 production in maize grain.

The aim of this study was to determine the antifungal effect of the essential oil obtained from Ziziphora clinopodioides L on two fungi species including Aspergillus flavus and Aspergillus parasiticus using microdilution method. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined for the essential oil at 10 different concentrations (i.e. 25,000, 12,500, 6250, 3125, 1562.5, 781.25, 390.625, 195.31, 97.65, and 48.82 µg/ml). Finally, the effect of the essential oil at six levels (6250, 3125, 1600, 800, 400, and 196 µg/ml) was investigated on the growth and activity of A. flavus and A. parasiticus, and also toxin production of these species in maize at 0.97 aw and 25°C after 29 days. Aflatoxin B1 (AFB1) content was assayed by enzyme linked immuno-sorbent assay technique. Results showed that essential oil of Z. clinopodioides was found more effective on A. parasiticus than A. flavus in both in vitro and in vivo conditions. Z. clinopodioides oil exhibited the same MIC value in the liquid medium against all fungal strains (48.82 µg/ml), while it showed different activity against A. flavus and A. parasiticus with MFC values of 781.25 and 390.625 µg/ml respectively. Under storage condition in maize, AFB1 production was significantly (p < 0.05) repressed at the concentration of 6250 µg/ml for A. flavus and 6250 and 3125 µg/ml for A. parasiticus. At the lower concentrations, the AFB1 production increased gradually. The results of the present study indicated that the essential oil of Z. clinopodioides had significant antifungal activity (p < 0.05); therefore, it can be used as an antifungal agent in the food and medicinal industries.

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.