Influence of different pre-treatment methods on isolation of extracts with strong antibacterial activity from lichen Usnea barbata using carbon dioxide as a solvent

SFE as a superior technique for isolation of extracts with strong antibacterial activities from lichen Usnea barbata L.

Environmental Factors Affecting the Content of Usnic Acid in the Lichen Mycobiont of Ramalina siliquosa

Polyphenols such as resveratrol, curcumin, (-)-epigallocatechin 3-gallate (EGCG) etc. have been shown to have anticancer properties. The anticancer activities of polyphenols include, but are not limited to, anti-oxidative effects, pro-apoptosis, DNA damage, anti-angiogenic effects, and immunostimulation. Lichens, symbiotic systems involving a fungus and an alga and/or cyanobacterium, are a rich source of polyphenols. Based on our previous research we have demonstrated that crude extracts of different lichen species affect cell proliferation, cell cycle dynamics, and cause apoptosis in Burkitt's lymphoma (Raji cells). This research examines the effects of a lichen-derived polyphenol compound, usnic acid, on human colon adenocarcinoma (HT-29) cells. We treated HT-29 cells with various concentrations (10, 30 and 50 µg/ml) of usnic acid for 24 hours. Although the viability was not affected by the treatment, there was a significant decrease in cell proliferation in dose-dependent manner. We also observed that after treatment, the morphology of treated cells was completely different from that of untreated control cells. Using transmission electron microscopy (TEM), we studied the morphological differences between control and treated HT-29 cells. The TEM micrographs clearly show that usnic acid damaged the mitochondria, reduced the amount of free ribosomes, and also distorted the shape of the nucleus. As the treatment of HT-29 with usnic acid reduced the rate of cell proliferation significantly, we investigated the effects of usnic acid on HT-29 cell cycle dynamics. Our results indicate that with the higher usnic acid concentrations (30 and 50 µg/ml) the cell cycle was stopped at the G0/G1 stage. These results further document the pattern of decreasing proliferation of HT-29 cells treated with usnic acid. With the distortion of the nucleus following exposure to usnic acid, we also investigated the effects of usnic acid on DNA the HT-29 cells. We performed a single cell gel electrophoresis (Comet Assay) to study the DNA damaging effects of various concentrations of usnic acid. Our results showed that there was a dose dependent increase in damage to the HT-29 cells' DNA resulting in 56.7% of the total DNA moving into the comet tail at 50 µg/ml concentration of usnic acid. These preliminary results suggest that lichen derived polyphenols could be a potential source of anticancer drug therapies. However, additional research is needed to further validate these findings. Citation Format: Gajendra Shrestha, Michael Xiao, Richard Robison, Larry L. St. Clair, Kim O'Neill. Lichen derived polyphenols as potential anticancer drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3220. doi:10.1158/1538-7445.AM2014-3220

본 연구에서는 송라과(Usneaceae)에 속하는 지의류의 한 종류인 송라(Usnea diffracta Vain)에서 추출된 usnic acid 화합물이 인체 대장암 세포에 미치는 항암효과 및 그 기전 에 대해 연구하였다. SW480 및 HCT116 세포에 대한 usnic acid의 세포 증식 억제 효과를 MTT assay를 통해 살펴본 결 과, 두 세포주 모두에서 시간 및 농도 의존적으로 세포 증식 억제효과가 있음이 드러났다. 특히 HCT116 세포보다 SW480 세포에 대한 usnic acid의 작용이 훨씬 더 강력함을 알 수 있었다. 세포의 생존율 감소가 세포 사멸인지 세포 괴사인지를 파악하기 위해 APOPercentage Apoptosis Assay kit를 이용하여 염색된 세포들을 현미경으로 관찰하였다. Usnic acid 48시간 처리 시 농도가 증가함에 따라 자주색으로 염색된 세포들이 많이 관찰되었다. 이를 통해 usnic acid 가 세포 사멸 과정을 통해 두 대장암 세포의 증식 억제에 영향을 주고 있음을 알 수 있었다. Usnic acid 농도 증가에 따른 세포 사멸 단계의 변화 양상을 파악하기 위해 이중 염색을 통한 유세포 분석을 진행하였다. 두 세포 모두에서 usnic acid 농도가 증가함에 따라 late apoptotic stage에 속한 세포 비율이 증가하였고, 두 세포간의 비교에서는 SW480 세포에서의 비율이 HCT116 세포에서의 비율보다 더 많음을 관 찰하였다. 세포 사멸에 관련된 인자들의 발현량 변화를 통 해 세포 사멸 경로를 규명하고자 관련 인자들에 대해 western blotting을 실시하였다. 미토콘드리아의 안정화에 영향을 미치는 Bcl-2와 Bax의 경우, usnic acid 농도가 증가함에 따라 Bcl-2의 발현량은 확연히 감소하였고, Bax의 발 현량은 증가함을 알 수 있었다. 양 세포간에서는 두 인자들 모두 SW480에서의 발현량의 변화가 HCT116세포에서의 변화보다 더 많음을 확인할 수 있었다. 단백분해 효소인 caspase의 경우 caspase-9, 3의 cleaved form 모두 다 대조군과 작은 농도의 usnic acid에서는 발현량이 거의 보이질 않았고, 30 μM 농도 처리 군에서는 발현량이 나타남을 확인 하였다. 대장암은 육류 섭취의 증가와 함께 우리나라에서 도 발병률이 증가되고 있는 호발성 암 중의 하나이다. 또한 여러 치료 방법에도 부작용 발생 등으로 치료법에 의문점 이 제시되는 바 천연물질을 이용하여 세포 사멸과 관련된 인자들을 조절함으로써 대장암을 치료하려는 노력이 증가 하고 있다. 송라는 예전부터 중국 등에서 차로 많이 음용되었던 한방약제로써, 이의 주성분인 usnic acid가 대장암 세 포에 항암효과가 있음이 본 연구에서 밝혀진 바 차 형태의 음용이 대장암의 억제에 도움이 될 것으로 생각된다.This study was carried out to investigate the effect of usnic acid on human colon cancer cells. Treatment with various concentrations of usnic acid for 24 and 48 h significantly inhibited the proliferation of SW480 and HCT116 cells. Usnic acid showed a stronger antiproliferative effect on SW480 than on HCT116. To ascertain the underlying mechanism of this antiproliferation by usnic acid, the observation of the stained cells by an inverted microscope was performed with an APOPercentage Apoptosis Assay kit. It was found that the number of purple-red stained cells in the SW480 and HCT116 cells increased in an usnic acid dose-dependent manner and that the discrepancy in the number of stained cells between the two cell types showed a similar trend to the results of the MTT assay. The double staining method was used for the characterization of the apoptotic stages according to the concentration of usnic acid. The results obtained with the FACS showed that the proportion of cells at the late apoptotic stage increased with increasing concentration of usnic acid in the SW480 and HCT116 cells. To elucidate mechanism of apoptosis, western blotting of the apoptosis related proteins was performed. It was found that the level of Bax was markedly increased and that of Bcl-2 was significantly decreased. Also it was revealed that usnic acid induced a cleaved form of caspase-3 and -9 at a high dosage level.

Clinal Variation in the Production of Usnic Acid in Cladonia subtenuis along Light Gradients

Usnic acid induces apoptosis in human gastric cancer cells through ROS generation and DNA damage and causes up-regulation of DNA-PKcs and γ-H2A.X phosphorylation

The ecologically important lichen Cladonia stellaris forms thick carpets in boreal forest floors. In addition to affecting temperature and water conditions in the soil underneath, the secondary metabolites formed by the lichen layer are of ecological interest. In this paper, we investigated the distribution of lichen acids in C. stellaris collected at different latitudes in Finland and developed methods to quantify the two optical enantiomers of usnic acid separately. The lichen extracts were analysed by high-performance liquid chromatography (HPLC) with UV and mass spectrometric (MS) detection and by gas chromatography with flame ionization (GC-FID) and MS detection. Usnic acid and perlatolic acid were quantified using GC-FID. The concentration of usnic acid in the top 20 mm of the lichen thallus ranged from 0·48–3·08% of dry weight, and that of perlatolic acid from 0·08–0·54%. The enantiomeric composition of usnic acid was determined using a chiral HPLC column coupled to an electrospray ionization-tandem mass spectrometer. (−)-Usnic acid was found to be the predominating enantiomer in all extracts; the proportion of (+)-usnic acid ranged from 0·4%–10·0%. Olivetoric acid methyl ester, diphenylmethanol, and 5-pentylresorcinol were identified, and several other olivetoric acid-type compounds were tentatively identified in the extracts.

The fruticose lichen Flavocetraria nivalis and the crustose lichen Ophioparma ventosa, both common in light-exposed arctic-alpine environments, were exposed to ultraviolet radiation (UVR) in growth chambers for 30 days. Treatment with visible light (PAR) served as control. Both species accumulate the UV-absorbing phenolic compound usnic acid in the upper cortex. The latter species also synthesises several UV-absorbing medullary compounds, among them divaricatic acid. The effects of treatment with UVR on the synthesis of these two compounds were investigated by analysing the compounds quantitatively by RP-HPLC. UV-exposed thallus tips of F. nivalis contained higher concentrations of usnic acid than those not grown under UVR. Both treatments had a positive effect on the synthesis of usnic acid in O. ventosa. An additional experiment with O. ventosa was performed by first storing samples in a low-light habitat for 1 year to obtain near-zero levels of phenolics, and thereby exposing the samples to UVR and PAR for 90 days. A rapid resynthesis of usnic acid was observed for both treatments. The amounts of divaricatic acid were highly variable in all groups, and were not correlated with usnic acid concentrations or treatments. A comparison of O. ventosa from three different habitat types showed that the highest usnic acid amounts were found in the habitat with the highest levels of solar radiation. Results indicate that the induction of usnic acid production by UVR depends on the species studied, and on how well acclimatised the lichen samples are to solar radiation before they are exposed to supplementary UVR. In lichens with an already well-developed internal screening capacity, like the population of F. nivalis, enhanced UVR need not induce further accumulation of usnic acid, but removal of UVR may induce a biodegradation of usnic acid. Results also indicate that PAR is just as important as UVR for triggering the resynthesis of usnic acid in shade-adapted lichens. Divaricatic acid seems to be of low importance for the UV-screening properties of O. ventosa.

Quantitative variations of usnic acid and selected elements in terricolous lichen Cladonia mitis Sandst., with respect to different environmental factors – A chemometric approach

Проведено исследование влияния напочвенных лишайников Flavocetraria cucullataи Certraria laevigataна химические, биохимические и микробиологические характеристики мерзлотных почв Центральной Якутии. Химический анализ не выявил значимых различий в показателях содержания гумуса, обменных катионов и pH в почвах под лишайниками F. cucullata и C. laevigata и на сопредельных с ними участках, лишенных растительного покрова. Выявлено, что основными вторичными метаболитами F. cucullata являлись усниновая, лихестериновая, протолихестериновая и алло-протолихестериновая кислоты, C. laevigata - фумарпротоцетраровая кислота. Хроматографический анализ почв выявил наличие только усниновой кислоты в образцах гумуса под F. cucullata и на сопредельных с ней участках. Показано, что по мере удаления от места произрастания F. cucullata наблюдалось понижение содержания усниновой кислоты и повышение содержания специфических соединений, которые могут являться биомаркерами грибов в почве. Анализ грибов и бактерий в почвах, выполненный с использованием культурального подхода, показал обилие быстрорастущих микромицетов и бактерий. По данным метагеномного анализа таксономическое разнообразие грибов в почвах было выше, чем по данным культуральных исследований, а преобладающей экологической группой были микоризные базидиомицеты. Показано, что эктомикоризные базидиомицеты в первую очередь подвержены воздействию лишайника F. cucullata. При этом аллелопатический эффект C. laevigataна почвенный микробиом выражен значительно слабее. Предположено, что синтезируемая F. cucullata усниновая кислота диффузно распространяется и накапливается в почве, избирательно подавляя рост эктомикоризных базидиомицетов. The influence of ground lichens Flavocetraria cucullata and Certraria laevigata on chemical, biochemical and microbiological characteristics of permafrost soils of Central Yakutia was studied. It was revealed that the values of the content of organic carbon, exchangeable cations and anions, and pH did not depend on the presence or absence of lichens on the soil surface. It was revealed that the main secondary metabolites of F. cucullata were usnic, lichesteric, protolichesteric and allo-protolichesteric acids, and C. laevigata - fumarprotocetraric acid. Chromatographic analysis of soils revealed the presence of only usnic acid in humus samples under F. cucullata and adjacent areas without vegetation. It was shown that as F. cucullata moved away from the place of growth, there was a decrease in the content of usnic acid and an increase in the content of specific compounds that could be biomarkers of fungi in the soil. Analysis of fungi and bacteria in soils, performed using the cultural approach, showed an abundance of fast-growing micromycetes and bacteria. According to the metagenomic analysis, the taxonomic diversity of fungi in soils was incomparably higher than according to the cultural studies, and mycorrhizal basidiomycetes were the predominant ecological group. Ectomycorrhizal basidiomycetes are primarily susceptible to the effect of lichen F. cucullata. The allelopathic effect of C. laevigata on the soil microbiome is much less pronounced. It is assumed that usnic acid synthesized by F. cucullata diffusely spreads and accumulates in the soil, selectively suppressing the growth of ectomycorrhizal basidiomycetes.

s. PHYTOPHARM 2012 M 68 Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] ТОМ 10/2012/2 (+)-USNIC ACID CONTENT AND ANTIOXIDANT ACTIVITY OF THREE LICHEN EXTRACTS © Koptina A. , Shcherbakova A. , Soldati F. , Gabdullin M. , Kanarskiy A. , Ulrich-Merzenich G. 3 Mari State Technical University, Yoshkar-ola, Russia Pharmaton SA, 6934 Bioggio, Switzerland Medical Clinic III, University of Bonn, Willhelmstr. 35–37, 53111 Bonn, Germany lichens produce a variety of secondary metabolites which belong to the depsides, depsidones, dibenzofurans and pulvinic acid derivative family. They proved to be a good source of natural antioxidants (1). Usnic acid uniquely found in many lichens has been reported to have analgesic, antimicrobial, anti-inflammatory, antiplatelet/antithrombotic, antiviral, cytotoxic/antiproliferative, gastroprotective, weight loss, and wound healing activity (2). We examined the (+)-usnic acid content and the anti-oxidative properties of the chloroform extract of lichen: Cladonia sylvatica (l.) hoffm., Evernia prunastri (l.) Ach. and Usnea barbata (l.) Wigg. (s. i.) were collected in Mari El Republic of Russian federation in June, 2011. Reverse phase hPlC analysis was carried out (Perkin Elmer Series 200 hPlC instrument; C18 column (C18; 25 cm ×4.6 mm, 5μm); UV/Vid detector; solvent: methanol-water-phosphoric acid (80:20:.9, v/v/v)). The sample injection volume was 20 μl and the flow rate 1.0 ml/min. A standard of (+)-usnic acid was used. The total antioxidant activity of the extracts was evaluated by the phosphomolybdenum method as described Manojlovic et al. (3). The chloroform extracts of C. sylvatica, E. prunastri and U. barbata showed contents of (+)-usnic acid of 8.91 ± 1.18, 5.68 ± 0.47 and 74.49 ± 8.64 % respectively. The antioxidant activities were: 277.1 ± 20.0, 171.1 ± 17.1 and 205.8 ± 7.2 mg of ascorbic acid (AA) per g of extract respectively; in comparison, the antioxidant activity of the (+)-usnic acid standard was determined as 154 ± 6.5 mg AA/g. We can report that U. barbata is a good source of (+)-usnic acid. Other secondary metabolites than (+)-usnic acid present in C. sylvatica and E. prunastri contribute to their high antioxidant activity and need to be further investigated.

Lichens are composed of symbiotic slow-growing organisms and are often exposed to extreme microenvironmental conditions, resulting in the production of unique secondary metabolites. One of the most commonly produced secondary metabolites is usnic acid, which is thought to be produced by two genes. The objectives of the present study were to compare polyketide synthase (PKS) gene expression and usnic acid concentration in Cladonia uncialis (L.) Weber ex F.H. Wigg., with two environmental factors. Seventy-five lichen samples were collected from three locations in Newfoundland, Canada, using a strip transect method (×5 transects, ×5 quadrats). Usnic acid concentration was measured using the liquid chromatography tandem mass-spectrometric method and gene expression of two PKS genes (methylphloracetophenone oxidase (MPAO) and methylphloracetophenone synthase (MPAS)) was examined using quantitative real-time PCR. The results showed that percent ground cover of C. uncialis was affected by soil pH level but not soil moisture, and usnic acid concentration was not affected by either soil pH or soil moisture. MPAO gene expression level was significantly affected by soil pH level but not soil moisture, and MPAS gene expression level was not affected by either soil pH level or soil moisture. There was no significant relationship between MPAS and MPAO gene expression levels and usnic acid concentration. These findings suggest that soil pH may be important for the production of usnic acid by C. uncialis but the genes involved require further study.

Microparticles will probably play a promising role in the future of chemotherapy. These polymeric delivery systems are capable of maximizing the therapeutic activity while reducing side effects of anti-cancer agents. Usnic acid (UA) is a secondary metabolite produced by lichens, which exhibits an anti-tumour activity. In this study, PLGA-microspheres containing usnic acid from Cladonia substellata were prepared by the double emulsion method, with or without PEG as stabilizer. The morphology of the microspheres was examined by optical and scanning electron microscopy. The in vitro kinetic profile of usnic acid loaded-microspheres was carried out by dissolution testing. The usnic acid content was analysed by HPLC. The cytotoxicity of free and encapsulated usnic acid was evaluated against HEp-2 cells using the MTT method. The anti-tumour assay was performed in mice against Sarcoma-180 tumour (UA 15 mg kg−1 weight body/day) during 7 days. Animals were then sacrificed and tumour and organs were excised for histopathological analysis. Microspheres presented a smooth spherical surface with a mean diameter of 7.02 ± 2.72 µm. The usnic acid encapsulation efficiency was ∼100% (UA 10 mg 460 mg−1 microspheres). A maximum release of 92% was achieved at the fifth day. The IC50 values for free and encapsulated usnic acid were 12 and 14 µg ml−1, respectively. The encapsulation of usnic acid into microspheres promoted an increase of 21% in the tumour inhibition as compared with the free usnic acid treatment. In summary, usnic acid was efficiently encapsulated into PLGA-microspheres and the microencapsulation improved its anti-tumour activity.

Objective To Explore the effect of usnic acid on the proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human hypertrophic scar fibroblasts (HSFBs) . Methods HSFBs were isolated and cultured in vitro, and their morphological features were analyzed. Different concentrations of usnic acid (0, 5, 10, 20, 50 μmol/L) were added to the HUVECs and HSFBs, control group was treated with Dimethyl sulfoxide (DMSO), after culture for 48 h, the proliferation activity of cells was detected with the MTS kit. The migration ability of two cells was analyzed by wound healing assay. The experimental data were analyzed by SPSS19.0 software, with P<0.05 as the statistical significance. Results Compared with the control group (100%), 5, 10, 20, 50 μmol/L usnic acid could significantly inhibit the proliferation of HUVECs, and the cell viability was reduced to (91.67±10.99)%, t=0.068, P>0.05; (40.68±8.87)%, t=5.280, P<0.01; (33.97±2.12)%, t=1.140, P<0.01; (20.39±0.84)%, t=8.280, P<0.01. respectively. Under the same experimental conditions, 20 μmol/L and 50 μmol/L usnic acid could inhibit the proliferation of HSFBs, cell viability was (76.16±11.03)% and (51.59±6.82)% respectively. 1, 5, 10 μmol/L usnic acid had no inhibitory effect on HSFBs cell proliferation. The migration of HUVECs was inhibited by 1, 10, 20 μmol/L of usnic acid (81.26±10.82, t=0.000, P<0.01; 71.81±3.96, t=3.780, P<0.01; 54.91±8.47, t=5.280, P<0.01). Meanwhile, usnic acid has no inhibition effect on HSFBs migration. Conclusion Usnic acid had the ability to inhibit HUVECs and HSFBs proliferation, and the ability to inhibit HUVECs migration. Key words: Usnic acid; Human umbilical vein endothelial cells; Human hypertrophic scar fibroblasts; Cell proliferation; Cell migration

Fungal metabolite isolated from Mycosphaerella nawae AM20 and its protective role in cerebral ischemia