Comparative Studies on Essential Oil and Phenolic Content with In Vitro Antioxidant, Anticholinesterase, Antimicrobial Activities of Achillea biebersteinii Afan. and A. millefolium subsp. millefolium Afan. L. Growing in Eastern Turkey.

The potential applications of Achillea species in various industries have encouraged the examination of their phytochemical components along with their biological potential. In the present study, phenolic contents and essential oil compositions together with the in vitro biological activities of the aerial parts from Achillea biebersteinii Afan. and Achillea millefolium subsp. millefolium Afan. collected from Turkey were evaluated. Different solvent extracts (n-hexane, chloroform, methanol, water) were prepared and their antimicrobial, anticholinesterase, and antioxidant activities were studied. The LC-MS/MS results revealed the presence of 16 different phenolic compounds, including chlorogenic acid, rutin, quercetin, and luteolin glycosides, in methanolic extracts. According to GC-FID and GC/MS results, the primary components of the oils were identified as 1,8-cineole (32.5%), piperitone (14.4%), and camphor (13.7%) in A. biebersteinii and 1,8-cineole (12.3%) and ß-eudesmol (8.9%) in A. millefolium subsp. millefolium. The infusion and methanolic extracts of both species were found to be rich in their total phenolic content as well as their antioxidant and anticholinesterase activity. In contrast, the n-hexane and chloroform extracts of both species showed strong antimicrobial activity with MIC values ranging from 15 to 2000 µg/mL. Our findings suggest that the investigated Achillea species could be evaluated as potent natural agents, and further studies into the promising extracts are needed.

Identification of Mushroom and Murine Tyrosinase Inhibitors from Achillea biebersteinii Afan. Extract.

Growing scientific evidence indicates that Achillea biebersteinii is a valuable source of active ingredients with potential cosmetic applications. However, the data on its composition and pharmacological properties are still insufficient. This study aims to optimize the extraction procedure of the plant material, evaluate its phytochemical composition, and compare anti-tyrosinase potential of A. biebersteinii extracts obtained by various methods. In order to identify compounds responsible for the tyrosinase inhibitory activity of A. biebersteinii, the most active anti-tyrosinase extract was fractionated by column chromatography. The fractions were examined for their skin lightening potential by mushroom and murine tyrosinase inhibitory assays and melanin release assay. HPLC-ESI-Q-TOF-MS/MS analysis of the total extract revealed the presence of several phenolic acids, flavonoids, flavonoid glucosides, and carboxylic acid. Among them, fraxetin-8-O-glucoside, quercetin-O-glucopyranose, schaftoside/isoschaftoside, gmelinin B, 1,3-dicaffeoylquinic acid (1,3-DCQA), and ferulic acid were found in the fractions with the highest skin lightening potential. Based on obtained qualitative and quantitative analysis of the fractions, it was assumed that the caffeoylquinic acid derivatives and dicaffeoylquinic acid derivatives are more likely responsible for mushroom tyrosinase inhibitory activity of A. biebersteinii extracts and fractions. Ferulic acid was proposed as the most active murine tyrosinase inhibitor, responsible also for the reduced melanin release from B16F10 murine melanoma cells.

Chemical composition, insecticidal and biochemical effects of two plant oils and their major fractions against Aedes aegypti, the common vector of dengue fever.

In an attempt to reduce the massive application of the toxic chemical pesticides, essential oils (EOs) of Achillea biebersteinii and Juniperus procera were obtained through hydrodistillation and analyzed using (GC-FID) and (GC-MS). α-terpinene and p-cymene were detected as the major components in the EO of A. biebersteinii, while eugenol and ß-caryophyllene were the major constituents in the EO of J. procera. The plant EOs and major fractions act as considerable mosquitocides against Aedes aegypti L, the common transmitter of Dengue fever. The EOs and major fractions were tested at 6.25, 12.5, 25, 50 and 100 µl/l. Insect mortality was time and dose-dependent, and the adult stage was more sensitive than larvae. At a concentration of 50 µl/l, 24 post treatment larval and adult mortality ranged between (40.3 and 89.3%) and (51.4 and 95.6%), respectively. The LC50 values ranged between 12.2 and 70.1 µl/l against larvae and between 10.1 and 63.12 µl/l against adults. All of the crude EOs were more potent than their major fractions. Eugenol and ß-caryophyllene showed strong mosquitocidal activity than p-cymene and α-terpinene. The corrected percentage mortality was increased over time with all of the test materials. In terms of lethal time required to kill 50% of the population (LT50), a concentration of 100 µl/l of J. procera EO showed LT50 values of 2.3 and 1.7 h against larvae and adult, respectively. The EOs induced considerable inhibition of acetylcholinesterase activity, where J. procera crude oil (IC50 = 13.12mM) and eugenol (IC50 = 19.65mM) were the most potent. Results proved that the test plant oils and their major fractions could be developed as natural pest control agents to control A. aegypti.

Achillea millefolium L. and Achillea biebersteinii Afan. Hydroglycolic Extracts-Bioactive Ingredients for Cosmetic Use.

Studies on hydroglycolic (HG) extracts of Achillea biebersteinii (AB)-a less investigated representative of the genus-were performed to determine their potential for cosmetic applications compared to the well-known Achillea millefolium (AM). Three types of water:polyethylene glycol extracts (1:1, 4:1, 6:1 v/v) were obtained from both species and analyzed for their composition by high performance liquid chromatography coupled with mass spectrometry (HPLC-ESI-Q-TOF-MS) and assayed for their biological activities. The study led to the identification of 11 metabolites from different natural product classes with the highest share corresponding to 5-caffeoylquinic acid, axillarin, coumaroylquinic acid isomers and 3-caffeoylquinic acid. The highest antiradical capacity in DPPH and ABTS scavenging assays was shown for HG 4:1 of AB and AM extracts. HG 1:1 extracts from both species inhibited monophenolase and diphenolase activity of tyrosinase, whereas AB HG 4:1 extract showed significant monophenolase inhibition. The highest sun protection factor (SPF) was determined for AM HG 4:1 extract, equal to 14.04 ± 0.17. The AB extracts were cytotoxic for both human keratinocytes HaCaT and A375 melanoma, however HG 1:1 and 4:1 extracts were more cytotoxic for cancer than for noncancerous cells. In conclusion, AB HG 1:1 and 4:1 extracts display significant potential as active cosmetic ingredients.

Achillea biebersteinii extracts suppress angiogenesis and enhance sensitivity to 5-fluorouracil of human colon cancer cells via the PTEN/AKT/mTOR pathway in vitro

Objective: To investigate the antiproliferative, anti-angiogenic, and apoptotic effects of extracts of Achillea biebersteinii (ABE) and combined treatments of ABE with 5-fluorouracil (5-FU) on HT-29 cells.Methods: The effects of ABE, 5-FU, and combined treatments on the viability of HT-29 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Isobologram analysis was used to determine synergism between ABE and 5-FU. The apoptotic and anti-angiogenic effects were determined by cell death detection and human vascular endothelial growth factor ELISA method, respectively. Transcriptional and translational expressions of p53, Bax, Bcl-2, p38 MAPK, Akt, PTEN, and mTOR were also evaluated by real-time PCR and Western blotting analysis. Results: ABE decreased the viability of HT-29 cells in a dose-dependent manner. Combined treatment of hexane, chloroform, and methanol extracts of Achillea biebersteinii with 5-FU at IC50 doses decreased the cell viability to 26.0％, 19.1％, and 14.9％, respectively (P<0.001). Furthermore, ABE treatment alone and combination with 5-FU, induced apoptosis, significantly downregulated mTOR, Akt, Bcl-2 expression, upregulated p53, Bax, PTEN, p38 MAPK expression, and exhibited anti-angiogenetic effects. Conclusions: Our findings indicate that ABE shows synergism with 5-FU and inhibits the proliferation of HT-29 cells by inducing apoptosis and suppressing angiogenesis, which may provide biological evidence for further use of ABE in the treatment of colorectal cancer.

Achillea biebersteinii extracts suppress angiogenesis and enhance sensitivity to 5-fluorouracil of human colon cancer cells via the PTEN/AKT/mTOR pathway in vitro

To investigate the antiproliferative, anti-angiogenic, and apoptotic effects of extracts of Achillea biebersteinii (ABE) and combined treatments of ABE with 5-fluorouracil (5-FU) on HT-29 cells. Methods: The effects of ABE, 5-FU, and combined treatments on the viability of HT-29 cells were determined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Isobologram analysis was used to determine synergism between ABE and 5-FU. The apoptotic and anti-angiogenic effects were determined by cell death detection and human vascular endothelial growth factor ELISA method, respectively. Transcriptional and translational expressions of p53, Bax, Bcl-2, p38 MAPK, Akt, PTEN, and mTOR were also evaluated by real-time PCR and Western blotting analysis. Results: ABE decreased the viability of HT-29 cells in a dosedependent manner. Combined treatment of hexane, chloroform, and methanol extracts of Achillea biebersteinii with 5-FU at IC50 doses decreased the cell viability to 26.0%, 19.1%, and 14.9%, respectively (P0.001). Furthermore, ABE treatment alone and combination with 5-FU, induced apoptosis, significantly downregulated mTOR, Akt, Bcl-2 expression, upregulated p53, Bax, PTEN, p38 MAPK expression, and exhibited anti-angiogenetic effects. Conclusions: Our findings indicate that ABE shows synergism with 5-FU and inhibits the proliferation of HT-29 cells by inducing apoptosis and suppressing angiogenesis, which may provide biological evidence for further use of ABE in the treatment of colorectal cancer.

Antinociceptive action of Achillea biebersteinii methanolic flower extract is mediated by interaction with cholinergic receptor in mouse pain models.

Achillea biebersteinii is a perennial aromatic herb that grows in the Mediterranean area. The leaves of this plant are used in foods as bittering and appetizing agents. In folk medicine, it is used for the treatment of stomachache and abdominal pain. In this study, the analgesic effect of A. biebersteinii methanolic flower extract was tested in three pain models, namely: writhing, tail-flick and paw-licking (formalin) tests. A. biebersteinii extract inhibited abdominal cramps produced by acetic acid. The effect of A. biebersteinii was better than that of 70 mg/kg indomethacin. In tail flick, A. biebersteinii extract increased latency at 30 min and was as effective as 100 mg/kg diclofenac sodium. In formalin test, A. biebersteinii extracts decreased paw-licking and flinching response in early and late phases. Atropine blocked the action of A. biebersteinii extract (300 mg/kg) in the late phase of formalin test as well as in writhing and tail-flick tests. GC-MS analysis revealed that ascaridole and iso-ascaridole were the main constituents of A. biebersteinii flower extract. In conclusion, this study shows for the first time that the antinociceptive effect of A. biebersteinii is mediated by the cholinergic receptor.

Protective Role of Achillea biebersteinii Pretreatment on Dimethoate Induced Oxidative Stress in Guinea Pigs Liver.

OBJECTIVE: The present study investigated the influence of Achillea biebersteinii (Ab), a medicinal herb used widely in Yemeni's folk medicine as analgesic, antipyretic, against diarrhea and flatulence and for liver diseases, on the liver antioxidant potential of guinea pigs acutely intoxicated with dimethoate pesticide (DM). MATERIALS AND METHODS: Animals were administered with Ab aqueous extract (50 and 100 mg kg-1 b.wt., orally) or silymarin (100 mg kg-1 b.wt., orally) for 2 weeks followed by single acute DM administration (80 mg kg-1 b.wt., 1/4 of the LD50 orally). Data were analyzed by one-way ANOVA using SPSS. RESULTS: The results showed that the treatment with Ab extract significantly lowered the DM induced serum levels of hepatic marker enzymes (AST, ALT and ALP). Liver histopathology also showed that Ab extract reduced the incidence of lesions including the swelling of cells, lymphocytes infiltration, nucleus fragmentation and condensation and necrosis induced by DM treatment in guinea pigs. CONCLUSION: The results of this study suggest that Ab aqueous extract could protect the liver against DM-induced oxidative damage.

Differential allelopathic expression of different plant parts of Achillea biebersteinii.

Achillea biebersteinii (Asteraceae) is a perennial medicinal plant and has a wide distributional range in the Mediterranean region. The present study investigated the inhibitory effects of different plant parts of A. biebersteinii on germination characteristics and seedling growth of wild barley (Hordeum spontaneum). Water extracts were prepared by incubating separately five grams of dried powder of roots, stems, leaves and flowers of A. biebersteinii in 100 ml of distilled water for 24 h and distilled water was used as the control. The water extracts from different plant parts of A. biebersteinii differed in their effects on the germination and seedling growth of wild barley. Water extracts prepared from leaves and flowers were more suppressive to germination of wild barley than root and stem extracts. The maximum inhibition in radical and plumule growth of germinating caryopses and in root and shoot growth of greenhouse-grown wild barley was recorded for leaf extract followed by flower extract. The lowest Chl a, Chl b and total chlorophyll and protein contents were resulted after exposure to leaf extracts. According to these results, the inhibitory effects of different A. biebersteinii plant parts can be arranged in the order: leaf > flower > stem > root.

Phytochemical composition, protective and therapeutic effect on gastric ulcer and α-amylase inhibitory activity of Achillea biebersteinii Afan.

Three sesquiterpene lactones [two germacranolides (micranthin and sintenin) and one guaianolide (4ß,10α-dihydroxy-5ß,7ß,8ßH-guaia-1,11(13)dien-12,8α-olide)] and four derivatives of 3-methoxy flavones (santin, quercetagetin-3,6,3'-trimethyl ether, quercetagetin-3,6-dimethyl ether, and 5,7 dihydroxy 3,3',4'-trimethoxy flavone) were isolated from the ethyl acetate extract (EAE) of the aerial parts of Achillea biebersteinii Afan. (Asteraceae). Evaluation of protective and therapeutic effects of EAE against ethanol-induced gastric ulcer in rats was carried. Antiulcer activity evaluation was done through measuring ulcer indices, stomach acidity, gastric volume and lesion counts. Oxidative stress markers; malondialdehyde, glutathione and superoxide dismutase were also estimated. The work was extended to determine the histopathological assessment of the stomach. Gastric ulcer exhibited a significant elevation of the ulcer index and oxidative stress markers. The extract attenuated these increments and recorded protective and therapeutic effects against gastric ulcer. Hyperglycaemia increases the mucosal susceptibility to ulcerogenic stimuli and predisposes gastric ulceration. In vitro α-amylase inhibitory assay was applied to evaluate the post prandial antihyperglycaemia activity. The result showing that the EAE has the ability to reduce starch-induced postprandial glycaemic excursions by virtue of potent intestinal α-amylase inhibitory activity. These findings demonstrated the remarkable potential of A. biebersteinii as valuable source of antiulcer agent with post prandial hyperglycaemia lowering effect.

Experimental endometriosis remission in rats treated with Achillea biebersteinii Afan.: histopathological evaluation and determination of cytokine levels.

OBJECTIVE: The aim of the present study is to assess the beneficial effects of Achillea biebersteinii Afan. in the treatment of endometriosis in order to find scientific evidence for the folkloric use of this plant. STUDY DESIGN: Experimental endometriosis was induced in six-week-old female, nonpregnant, Sprague Dawley rats by suturing a 15mm piece of endometrium from uterine cornu into abdominal wall. After twenty-eight days, a second laparotomy was performed: the endometrial foci areas were measured and intra-abdominal adhesions were scored, and the abdomen was closed. Different groups then received n-hexane, ethyl acetate (EtOAc) and methanol (MeOH) extracts prepared from the aerial parts of A. biebersteinii, and a control group received inert material, administered per os once a day throughout the experiment. At the end of the treatment procedure all rats were sacrified and endometriotic foci areas and intra-abdominal adhesions were again evaluated and compared with the previous findings. The tissues were also histopathologically investigated. Moreover, peritoneal fluid was collected to detect tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) levels. Dunnett's test was used to determine the significance of differences between groups. In order to compare two groups Student's t test was used. RESULTS: Post-treatment volumes of endometrial foci were found to be significantly decreased, and no adhesion was detected, in the EtOAc extract treated group. The levels of TNF-α, VEGF and IL-6 also fell after the treatment with EtOAc extract. The therapeutic effect of the EtOAc extract of A. biebersteinii could be attributed to the flavonoid aglycones found in the extract. CONCLUSION: The EtOAc extract of A. biebersteinii appears to be a promising alternative for the treatment of endometriosis.