In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.

The Effect of Hydroalcoholic Extract of Quercus brantii and Artemisia aucheri Boiss Against Trichomonas vaginalis In vitro.

Objective: Trichomoniasis is the most common sexually transmitted protozoan infection worldwide. Metronidazole is widely considered as the drug of choice for treating of trichomoniasis but considering its potential side effects, we aimed to assess the therapeutic influences of hydro-alcoholic extracts of Quercus brantii and Artemisia aucheri Boiss as alternative medications against Trichomonas vaginalis (T. vaginalis). Methods: The trophozoites were cultured in TYI-S-33 medium at a density of 5x105 trophozoites/mL. Subsequently, they were incubated with varying concentrations of the plant extracts (32, 64, 125, 250, 500, and 1,000 µg/mL) and metronidazole (16, 32, 64, 125, 250, and 500 µg/mL), as the positive control. The number of trophozoites in each well plate was quantified after 2, 4, 6, 24, 48, and 72 hours using trypan blue staining. Finally, the viability of the parasite was assessed by vital methylene blue staining. Results: The hydro-alcoholic extracts of Q. brantii and A. aucheri Boiss at concentrations of 125, 250, 500, and 1,000 µg/mL demonstrated significant efficacy against the parasite. Our findings indicated that the minimum effective concentrations were 125 µg/mL and hydro-alcoholic extracts of Q. brantii and A. aucheri Boiss have the ability to effectively eliminate T. vaginalis after 48 and 72 hours of treatment. Conclusion: The findings of the present study showed that hydro-alcoholic extract of Q. brantii and A. aucheri Boiss can induce death in T. vaginalis. However, further complementary in vivo studies are needed to assess the components of these plants in the treatment of T. vaginalis.

Topical treatment of cutaneous leishmaniasis lesions using quercetin/ Artemisia-capped silver nanoparticles ointment: Modulation of inflammatory response.

Leishmaniasis is a major health issue that affects people all over the world, producing considerable morbidity and mortality in Asia, Africa, and the Americas, and existing treatments have significant side effects. Nowadays, the development of nanoscale materials such as biogenic silver nanoparticles has attracted much medical attraction. In this study, AgNPs were synthesized from leaf extract of Artemisia aucheri. Biosynthesized AgNPs were analyzed by UV-visible spectroscopy, dynamic light scattering and zeta potential, fourier transform infrared spectroscopy and field emission scanning electron microscopy. Biosynthesized AgNPs were examined for anti-leishmanial and antibacterial activity. The in vivo study was conducted by treating the L. major infected BALB/c mice with quercetin/ artemisia-capped silver nanoparticles ointment topically for 21 consecutive days. The in vitro and in vivo results showed that the ointment containig quercetin/artemisia-capped silver nanoparticles have the potential to decrease inflammatory responses and enhance wound healing with granulation tissue formation compared to the untreated group. Therefore, biogenic nanoparticles are safe, eco-friendly, and easy to synthesize and could be considered as an alternative regimen for treatment of L. major.

Antiviral effect of Artemisia aucheri aqueous extract on UL46 and US6 genes of HSV-1.

HSV-1 is associated with oral lesions. Recently, anti-herpetic activity of different plant species has been investigated. In this study, the effects of Artemisia aucheri aqueous extract on the HSV-1 virus-infected Vero cells were assessed. The highest cell viability occurred in plant aqueous extracts was with a concentration of 75 µg/mL, 1-2 h before viral infection. The IC50 of the aqueous extract of 24.7 µg/ml was calculated. Most percentage of infected cell inhibition (89.6%) was with the chloroform fraction in concentration of 75 µg/ml, and the least percentage of infected cell inhibition (21.7%) was in concentration of 12.5 µg/ml with the ethyl acetate fraction in comparison with untreated control. Moreover, Q-PCR results revealed that the expression of genes UL46 and US6 were significantly reduced in the presence of different treatments utilized in the experiment. In conclusion, the present study proposes that aqueous extracts of medicinal plant Artemisia aucheri have anti-viral property and may be considered as a remedy for HSV-1 treatment.

The Anticancer Properties of Artemisia aucheri Boiss Extract on HT29 Colon Cancer Cells.

BACKGROUND: Colon cancer is one of the most common cancers in the world, and efforts toward its treatment have not been completely successful. In recent years, more attention has been focused on herbal medicine (HM) due to their anticancer and cytotoxic properties. This study investigated the anticancer and antioxidant effects of Artemisia aucheri (A. aucheri) Boiss extract against HT29 colon cancer cells compared with HEK239 natural cells. METHODS: This study was performed on human HT29 colon cancer cells. Various doses of 0, 10, 100, 500, and 1000 µg/ml of A. aucheri extract were subjected to cells at specified time intervals. After treatment, the trypan blue test was employed to determine the viability of the cells. MTT and annexin tests were used to determine cell viability and the apoptosis induced by the extract. Malondialdehyde (MDA) testing was applied to investigate the antioxidant properties of the extract on fatty acids. Data analysis was performed using SPSS version 22. One-way ANOVA and paired comparison tests were employed for data analysis. RESULTS: The highest cytotoxicity effect of A. aucheri extract was observed at 1000 µg/ml (80.63 ± 3.66) being dose-dependent compared with the control in both cell lines (p < 0.001). Additionally, the survival rate of HT29 (IC50 = 57.88 µg/ml) and HEK (IC50 = 295 µg/ml) cancer cells decreased with increasing concentration of A. aucheri (the lowest cell viability was at 1000 µg/ml). Furthermore, the induction of membrane lipid peroxidation was significantly higher in HT29 compared with the control (p < 0.001). Another cytotoxic mechanism for the extract was the induction of apoptosis being significantly higher in HT29 colon cancer cells compared with the control group (p < 0.001). CONCLUSION: Cytotoxic effects of A. aucheri extract were dose-dependent. This HM exerted cytotoxic effects against HT29 cells through the induction of membrane lipid peroxidation and apoptosis.

Antioxidant Activity-guided Phytochemical Investigation of Artemisia aucheri Boiss.: Isolation of Ethyl Caffeate and a Spinacetin Glycoside.

Antioxidant activity of five different extracts (petroleum ether, dichloromethane, ethyl acetate, ethanol and ethanol-water) of Artemisia aucheri aerial parts was investigated by three various methods: ferrous ion chelating (FIC) assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and ß-carotene bleaching (BCB) test. Total phenolic contents (TPC) were measured by Folin-Ciocalteu method. The hydroethanolic extract exhibited the stronger inhibitory activity in BCB and FIC assays than the other extracts. Among the extracts analyzed, the ethyl acetate and ethanolic extracts exhibited the highest TPC and DPPH radical scavenging activity, respectively. Reversed phase vacuum liquid chromatography of ethanolic extract (with the highest extraction yield) produced five fractions (A to E) which were subjected to all antecedent experiments. The same sample (Fraction C) showed the highest TPC and DPPH radical scavenging activity while there were no statistically significant correlations between TPC and EC50 values of various antioxidant assays. Ethyl caffeate and a spinacetin glycoside were isolated from the most active fraction and their structures were established using spectroscopic analysis including NMR and MS.

In-vitro and in-vivo comparative effects of the spring and autumn-harvested Artemisia aucheri Bioss extracts on Leishmania major.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia aucheri Bioss contains flavonoid, coumarin and santonin with antioxidant, antimicrobial and antileishmanial effects. The current study was aimed to comparatively evaluate the effects of spring and autumn extracts of A. aucheri Bioss on Leishmania major both in-vitro and in-vivo conditions. METHODS: HPLC analysis was used to evaluate the percentages of compounds in spring and autumn extracts of A. aucheri. For in-vitro assay, the effect of different concentrations of spring and autumn extracts of A. aucheri was tested on L. major promastigotes and amastigotes. MTT and flow cytometry methods were used to evaluate the cytotoxicity and probable apoptosis of A. aucheri extracts on L. major promastigotes. On the other hand, for in-vivo assay, the extracts were used as ointments to treat lesions developed on BALB/c mice after 28 days post inoculation of L. major. The diameter of lesions and the survival rates of infected BALB/c mice were measured weekly for a period of two months. RESULTS: The HPLC analysis showed the substance Quercitrin was present in the spring A. aucheri extract but not in the autumn extract. The mean numbers of amastigotes in each treated macrophage with the spring and autumn A. aucheri extracts were 1.2 and 1.8 respectively, which showed statistically significant differences (P < 0.05). Flow cytometry revealed that the spring and autumn A. aucheri extracts caused about 32% and 3.78% apoptosis respectively. The inhibitory concentration (IC50) of spring and autumn A. aucheri extracts to amastigotes were determined to be 90 µg/mL and 183 µg/mL respectiovely. In-vivo, the diameter of lesions treated with the spring A. aucheri extract was significantly less (P < 0.05) compared to those treated with the autumn extract (2.6 and 7.8 mm respectively). Also, mice treated with spring A. aucheri extract had higher survival rates compared to control group. CONCLUSION: Given the above results, it can be concluded that spring A. aucheri extract has a greater fatality effect on L. major promastigotes in-vitro compared to the autum extract. In addition, the spring extract has stronger therapeutic effect on lesions caused by L. major in BALB/c mice than the autum extract.

Cytotoxic and Apoptogenic Sesquiterpenoids from the Petroleum Ether Extract of Artemisia aucheri Aerial Parts.

Different types of Artemisia aucheri extracts were reported to have various biological activities including a cytotoxic effect on some cancer cell lines. We investigated the antiproliferative activity of isolated sesquiterpenoids from petroleum ether extract of Artemisia aucheri (A. aucheri) aerial parts on SK-N-MC, MCF-7, and A2780 cell lines. Phytochemicals from the petroleum ether cold macerated extract were isolated using normal phase vacuum liquid chromatography and high pressure liquid chromatography (VLC and HPLC) and the structures of the components were determined by spectroscopic means. Cell viability was determined by 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Activation of caspases-3 and -9 was evaluated using a spectrophotometer. Mitochondrial membrane potential (MMP) was measured using rhodamine 123 fluorescent dye. Two tetrahydrofuran- type sesquiterpenoids, hydroperoxide of davanone (1) and hydroxydavanone (2) were isolated and characterized. Between these compounds, compound 1 exhibited more potent activity against the MCF-7, SK-N-MC and A2780 cell lines with IC50 values of 8.45 ± 0.81 µg/mL, 9.60 ± 1.32 µg/mL and 10.9 ± 2.03 µg/mL in A2780, MCF-7 and SK-N-MC cells, respectively. Compound 1 inhibited the growth of human cancer cells by induction of apoptosis. To the best of our knowledge, this is the first comprehensive study on cytotoxic and apoptotic mechanism of two davanone derivatives isolated from A. aucheri in human cancer cells. Overall, our data suggest that hydroperoxide of davanone (1) should be further studied in-vivo as a potential antitumor agent.

Sesquiterpene lactones from shoot culture of Artemisia aucheri with cytotoxicity against prostate and breast cancer cells.

Plant tissue culture is used to grow plant cells, tissues, or organs under sterile and determined conditions on culture media. It is alternative to traditional vegetative propagation, and is applied as an effective technology for the production of valuable secondary metabolites. The Artemisia aucheri (A. aucheri) was obtained from shoot culture grown on MS (Murashige and Skoog 1962) medium. Shade-dried aerial parts of in vitro grown A. aucheri (50 g) were extracted with dichloromethane-acetone (90:10). The extract was submitted for isolation to sephadex gel chromatography and preparative thin layer chromatography, which resulted in identification of one known eudesmanolide named artemin or 2,5-dihydroxy-12, 6-eudesmanolide-4(15)-en for the first time in this plant. In cell cytotoxicity test, artemin showed cytotoxic activity against DU-145,LNCaP prostate cancer, and MCF-7 breast cancer cells with IC50 values of 82.2 ± 5.6, 89.1 ± 6.3 and 111.5 ± 6.7 µM , respectively. Artemin was more active against prostate cancer cells with approximately same cytotoxicity against LNCaP androstane dependent cells and DU 145 which is androstane independent.

The combined effects of ethanolic extract of Artemisia aucheri and Artemisia oliveriana on biofilm genes expression of methicillin resistant Staphylococcus aureus.

BACKGROUND AND OBJECTIVES: One of the most important antibiotic-resistant bacteria is methicillin-resistant Staphylococcus aureus (MRSA) biofilm that has caused significant problems in treating the patients. Therefore, the aim of this study was to evaluate the levels of expression of genes involved in biofilm formation in MRSA (ATCC 33591) while being treated by a combination of Artemisia aucheri and Artemisia oliveriana. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) of ethanolic extract of A. aucheri and A. oliveriana and also the minimum inhibitory concentration of combination of both extracts were 512, 1024 and 256 µg/ml, respectively; then at concentrations lower than the MIC, expression levels of the desired genes were determined by Real Time PCR. RESULTS: Based on results, using a combination of two ethanolic extracts had a significant effect on expression of genes involved in biofilm formation in MRSA. The expression level of icaA at 4, 8, 16 h after being treated by herbal extracts of A. aucheri and A. oliveriana was 0.293, 0.121, 0.044, respectively. The expression level of icaD was 0.285, 0.097, 0.088, respectively, while that of ebps was 0.087, 0.042, 0.009 at 4, 8 and 16 h, respectively. CONCLUSION: This study provided evidence that ethanol extract of A. oliveriava and A. aucheri can inhibit the biofilm formation of S. aureus. As a traditional Iranian medicine, A. oliveriava and A. aucheri extracts have a potential antibiofilm formation against MRSA strains.

The Protective Effect of Different Extracts of Three Artemisia Species against H2O2-Induced Oxidative Stress and Apoptosis in PC12 Neuronal Cells.

BACKGROUND: Oxidative stress causes cell damage and is involved in many neurological diseases. The antioxidant properties of plant materials for the maintenance of health and protecting against different diseases stimulated scientist to investigate different herbs. Different Artemisia species have exhibited antioxidant activity. This study aims to investigate whether different Artemisia species could protect the PC12 cells against oxidative stress mediated by H2O2. METHODS: For this purpose, different extracts of three Artemisia species (Artemisia aucheri, Artemisia turanica, and Artemisia turcomanica) were prepared using petroleum ether, dichloromethane, ethyl acetate, ethanol, and Water: Ethanol mixture (1:1 volume ratio). The protective effect of the prepared extracts against H2O2-induced cytotoxicity and reactive oxygen species production were compared. The effect of treatment of PC12 cells with different extracts on total glutathione (GSH) level, caspase-3 activity, and mitochondrial membrane potential were also compared. RESULTS: The A. aucheri extracts could not rescue the PC12 cells from oxidative stress consequences. The A. turanica and A. turcomanica extracts were found potent in suppressing the toxicity and apoptosis of PC12 cells mediated by H2O2 and significantly antagonized the H2O2-induced GSH depletion. The hydroethanolic and ethyl acetate extracts of A. turanica and the petroleum ether and hydroethanolic extracts of A. turcomanica more efficiently suppressed cytotoxicity and loss of GSH caused by H2O2. CONCLUSION: This study shows the protective effects of Artemisia extracts on PC12 cell line and suggested that these species could be also considered as promising neuroprotective agents in treatment of different neurodegenerative diseases. SUMMARY: Artemisia turanica and Artemisia turcomanica extracts were found to potentially exert neuroprotective effect on PC12 cells. The results exhibited that the cytoprotective potential and anti-apoptotic mechanism of these species is not the same for different extracts, and suggested that based on the type of species and the type of solvents used in extraction, both intrinsic and extrinsic pathways could be included in the anti-apoptotic mechanism of these species. Abbreviations Used: GSH: Glutathion. ROS: Reactive Oxygen Species. GSSG: Glutathione disulfide. DCF-DA:2',7'-Dichlorofluorescin diacetate. FBS: Fetal Bovin Serum. MMP: Mitochondrial Membrane Potential. H-Et: Hydro-ethanolic. DCM: Dichloromethane. PE: Petroleum Ether. Et: Etanolic. EA: Ethyl Acetate.

Study of two-stage ohmic hydro-extraction of essential oil from Artemisia aucheri Boiss.: Antioxidant and antimicrobial characteristics.

The effect of two-stage ohmic-assisted hydrodistillation (TSOH) on the extraction and characteristics of essential oils (EOs) from the Artemisia aucheri Boiss. was studied, and the results were compared to conventional hydrodistillation (HD). According to the results, the yield of EOs obtained through TSOH was almost 30% higher than those extracted by HD in nearly one-quarter of a time used by the HD. Scanning electron micrographs of A. aucheri leaves showed almost complete eruption of EO glands and their surrounding area in TSOH extraction method, hence achieving higher yield. The components of the EOs obtained through TSOH were only slightly different from those of HD. GC/MS analysis indicated some differences in the quantity of the main components, too. The main components of EOs were identified as Thymol, Linalool, Geraniol, Camphor, and 1, 8-Cineole, Davana ether and Cis-Davanone. Thymol (~17%) and Cis-Davanone (~23%) were the highest quantity in the EOs extracted from TSOH and HD, respectively. The variation of antioxidant and antimicrobial activities of the EOs may be attributed to these differences in the percentage of the main components. The radical scavenging activity of the EOs obtained by TSOH was almost twice that of HD. Based on antimicrobial activity assays, the EOs were efficient against S. aureus (a Gram-positive), E. coli (a Gram-negative), and S. cerevisiae (yeast). However, the efficacy was higher in gram-positive than gram-negative bacteria and yeast. The results indicate TSOH has a potential to produce EOs from herbal plants at a faster rate, higher yield, being probably more efficient in terms of energy although having similar antimicrobial and antioxidant efficacy.

Physiological targets of salicylic acid on Artemisia aucheri BOISS as a medicinal and aromatic plant grown under in vitro drought stress.

BACKGROUND: Artemisia aucheri BOISS is a medicinal and aromatic plant, which is endemic to mountainous areas of Iran and surroundings. In this study, we investigated the alleviating effects of salicylic acid (SA) pretreatment (0.01 and 0.1 mM) on A. aucheri under in vitro drought stress induced by 2 and 4% polyethylene glycol (PEG/6000). RESULTS: Plants exposed to PEG stress showed higher levels of H2O2, MDA and electrolyte leakage compared with control. While SA pretreatment decreased these parameters under PEG stress significantly. The activity of CAT, POD, APX, SOD and GR positively changed with PEG and more induction in activity of antioxidant enzymes was observed in SA-pretreated plants under PEG stress. Furthermore, ASA, GSH and their redox ratios (ASC/DHA and GSH/GSSG) enhanced with SA pretreatments. Analysis of our data revealed that MDA, DHA and H2O2 were the best targets for SA under in vitro PEG treatment for A. aucheri plants. CONCLUSIONS: Salicylic acid as a signal molecule mitigated adverse effects of PEG-simulated drought stress on A. aucheri under in vitro condition by improving the activity of antioxidant enzymes. In addition, protective role of SA was also related to promotion of ascorbate-glutathione cycle.

A semipolar fraction of petroleum ether extract of Artemisia aucheri induces apoptosis and enhances the apoptotic response to doxorubicin in human neuroblastoma SKNMC cell line.

Artemisia is an important genus of Iranian flora whose potent anti-proliferative effect has been demonstrated previously on human cancerous cell lines. In the current study, further fractionation was carried out on the petroleum ether extract of A. aucheri and their cytotoxic effects were evaluated on three human cancer cell lines. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Real time polymerase chain reaction (RT-PCR) was used to evaluate the expression of apoptotic related genes. Activation of caspases and detection of intracellular doxorubicin (DOX) accumulation were evaluated using a spectrophotometer. Mitochondrial membrane potential (MMP) was measured using flow cytometry. The fraction NO-7 (F7) of petroleum ether extract showed the highest anti-proliferative effect, especially against SKNMC cells. Therefore, we focused on a description of the cytotoxic mechanism of the most potent fraction on SKNMC cells. The results indicated that F7 was able to induce apoptosis through MMP disruption, activation of caspases and increament of proapoptotic genes Bax and Smac/DIABLO. Moreover, our observation indicated that F7 is able to increase the cytotoxicity of DOX in SKNMC cells. The combination of F7+DOX significantly increased the intracellular accumulation of DOX. These results indicated that F7 induces apoptosis in SKNMC cells. Moreover, it might enhance the antitumor activity of DOX, through modulating the activity of multidrug resistant cancer cells and inducing apoptosis.

Evaluation of Effectiveness of Ethanolic Extract of Artemisia aucheri, Individually and in Combination with Chloroquine, on Chloroquine - Sensitive Strain of Plasmodium berghei in Sourian Mice.

BACKGROUND: Drug resistance in malaria parasites is extending in the world particularly in chemical synthesized drugs such as 4- aminoquinolines and aminoalcoholes. Employing herbal extracts is encouraged by WHO in the malarious areas. In this study, the effectiveness of ethanolic extract of Artemisia aucheri individually and in combination with chloroquine, has been considered against chloroquine - sensitive strain of Plasmodium berghei. METHODS: At the first stage, ED50 of A. aucheri and chloroquine on P. berghei was calculated using in vivo test. Then based on the ED50s combination of A. aucheri and chloroquine with ratios of 0/100,10/90,20/80,30/70,40/60,50/50,60/40,70/30,80/20,90/10 and100/0 were tested against the parasite. For evaluating the adverse effect of A. aucheri on the mice, for two weeks 1000mg/kg of the extract was daily employed and the mice were followed up for fifty days. RESULTS: ED50s for chloroquine and A. aucheri were 1.6mg/kg and 1000mg/kg respectively. The outcome of two drugs combination on the mice showed antagonistic effects on the chloroquine - sensitive strain of parasite. Two weeks daily administration of A. aucheri had no toxic effect on the mice. CONCLUSION: A. aucheri individually can be effective in reducing the parasite while in combination with chloroquine loses its property.

Antimicrobial Activity and Chemical Composition of Essential Oil From the Seeds of Artemisia aucheri Boiss.

BACKGROUND: Artemisia aerial parts are well known for antimicrobial activities including anti malaria. OBJECTIVES: This study was carried out to evaluate the antimicrobial activity and chemical composition of essential oil from the seeds of Artemisia aucheri Boiss (Asteraceae). MATERIALS AND METHODS: Essential oil was extracted from the powdered seeds of Artemisia aucheri by hydrodistillation. Antimicrobial activity against five bacterial species was tested using the disc diffusion method, and the chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: The essential oil of Artemisia aucheri seed showed activity against Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. The essential oil constituents identified by GC-MS were as follows: decane, ρ-cymene, 1,8-cineole, linalool, ρ-mentha-8-ol, triene, borneol, lavandulol, bornyl acetate, chrysanthenyl acetate, dehydro aromadenderene, and caryophyllene oxide. Most of these compounds are also found in the aerial parts of Artemisia aucheri. CONCLUSIONS: Variation in the compositions of essential oils from Artemisia aucheri, and thus variation in the antimicrobial activity of these oils, may be due to the plant parts used for essential oil prepration.