Chaga mushroom extract induces autophagy via the AMPK-mTOR signaling pathway in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaga mushrooms (Inonotus obliquus) are commonly used in traditional treatments in Eastern Europe and Asia due to their diverse pharmacological effects, including anti-tumor and immunologic effects. Thus, many cancer patients take Chaga mushrooms as a complementary medicine, even during chemotherapy or radiotherapy. However, few studies have investigated the effects or molecular targets of Chaga mushrooms in breast cancer. AIM OF THE STUDY: Herein, we examined the anticancer effects of Chaga mushrooms in different types of breast cancer cell lines, and explored the underlying molecular mechanism to better understand their effects and benefits. MATERIALS AND METHODS: Chaga mushroom extract (CME) was prepared by extracting Chaga mushrooms with 70% ethanol. The cytotoxic effects of CME were assessed by MTT assay and protein expressions were evaluated by western blotting. To evaluate in vivo anti-tumor effects of CME, CME (2 g/kg) was orally administered to 4T1 tumor-bearing BALB/c mice every other day over 30 days (15 administrations), and tumor sizes were measured. Silica gel column chromatography was used to fractionate CME, and major constituents responsible for cytotoxic effects of CME were identified by 1H/13C-NMR and LC-MS. RESULTS: CME inhibited the proliferation of 4T1 mouse breast cancer cells in a dose and time-dependent manner. The expression of LC3 and phosphorylation of AMPK were increased by CME, while the phosphorylation of mTOR, S6, and S6K1 were suppressed, suggesting that CME induced autophagy by activating AMPK and inhibiting mTOR signaling pathways. Consistent with its observed cytotoxic effect in vitro, CME effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition, inotodiol and trametenolic acid were identified as the major constituents responsible for the cytotoxic effects of CME on breast cancer cells. Moreover, inotodiol and trametenolic acid-enriched fractions both exhibited cytotoxic effects regardless of breast cancer cell subtypes and did not interfere with the cytotoxic effects of conventional drugs. CONCLUSIONS: Taken together, Chaga mushroom extract induced autophagy by activating AMPK and inhibiting the mTOR signaling pathway. Our data suggest Chaga mushrooms may be a beneficial complementary medicine for breast cancer patients.

Nutritional Component and Chemical Characterization of Chinese Highland Barley Bran Oil.

The nutritional composition and chemical properties of the Chinese highland barley bran oil were characterized in this study. The barley bran oil extracted with solvent possessed relatively high acid value and peroxide value, indicating that the oil should be further refined before using. The fatty acid composition of the oil showed that the content of unsaturated fatty acids was 80.12 g/100 g, in which the content of polyunsaturated fatty acids was as high as 60.41 g/100 g. The overall triacylglycerol profile showed that the oil contained 27 TAGs including 21 regioisomers. Major TAGs included LLL (21.08 g/100 g), PLL (19.27 g/100 g), LLO (12.24 g/100 g), and LLLn (12.17 g/100 g). The total unsaponifiable matter of the oil reached up to 10.74 g/100 g oil. The total phytosterol content reached 7.90 g/100 g oil, in which ß-sitosterol was the most predominant, with the content of 5.69 g/100 g oil. Other important sterols included campesterol (1.32 g/100 g oil), lanosterol (0.70 g/100 g oil) and stigmasterol (0.19 g/100 g oil).

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

Lanostane-type triterpenes from the sclerotium of Inonotus obliquus (Chaga mushrooms) as proproliferative agents on human follicle dermal papilla cells.

Chaga mushrooms, the sclerotium of Inonotus obliquus, have been used in Mongolia as a traditional hair shampoo to maintain healthy hair. Bioassay-guided fractionations of the extract of Chaga mushrooms using a proliferation assay on human follicle dermal papilla cells (HFDPCs) gave five lanostane-type triterpenes (1-5), whose structures were identified by spectroscopic evidence. Among these, lanosterol (1), inotodiol (3), lanost-8,24-diene-3ß,21-diol (4), and trametenolic acid (5) demonstrated proproliferative effects on HFDPCs more potent than minoxidil, an anti-alopecia agent, used as the positive control. The lanostane-type triterpenes (1, 3, 4, and 5) appeared to be potential candidates of new agents possibly used for hair-care with a stimulative effect on hair growth.

A targeted strategy for analyzing untargeted mass spectral data to identify lanostane-type triterpene acids in Poria cocos by integrating a scientific information system and liquid chromatography-tandem mass spectrometry combined with ion mobility spectrometry.

Rapid structural identification of natural compounds in the crude extract of traditional Chinese medicine by conventional liquid chromatography-mass spectrometry is complex and challenging. In particular, it is difficult to distinguish and identify structural isomers. In this work, we proposed a novel strategy that combines a typical ultrahigh-performance liquid chromatography-multidimensional mass spectrometry approach and the post-processing UNIFI scientific information system to rapidly identify lanostane analogs and isomers in Poria cocos. First, this strategy requires setting up a high-resolution key MS database and an in-house compound library. Then, ultrahigh-performance liquid chromatography coupled with high-resolution tandem data-independent mass spectrometry and ion mobility mass spectrometry was used to acquire untargeted multidimensional mass spectral data. Finally, a new and reliable multidimensional MS analytical workflow was developed to targeted filter the acquired data based on an in-house compound library via the UNIFI™ software. As result, a total of 121 lanostane-type triterpene acids were identified by high-resolution molecular mass, fragment ions, and collision cross-section values. Eight triterpene acids were unambiguously identified by comparing the retention time and MS/MS data with those of reference compounds. Three compounds were detected and reported for the first time based on their neutral losses, characteristic ions, and fragmentation pathways compared with those of known compounds. We anticipate that such an analytical approach can be extended to rapidly screen and characterize other herbal medicine compounds with multiple isomers.

Detection of Ganoderic Acid A in Ganoderma lingzhi by an Indirect Competitive Enzyme-Linked Immunosorbent Assay.

Ganoderma is a genus of medicinal mushroom traditionally used for treating various diseases. Ganoderic acid A is one of the major bioactive Ganoderma triterpenoids isolated from Ganoderma species. Herein, we produced a highly specific monoclonal antibody against ganoderic acid A (MAb 12 A) and developed an indirect competitive ELISA for the highly sensitive detection of ganoderic acid A in Ganoderma lingzhi, with a limit of detection of 6.10 ng/mL. Several validation analyses support the accuracy and reliability of the developed indirect competitive ELISA for use in the quality control of Ganoderma based on ganoderic acid A content. Furthermore, quantitative analysis of ganoderic acid A in G. lingzhi revealed that the pileus exhibits the highest ganoderic acid A content compared with the stipe and spore of the fruiting body; the best extraction efficiency was found when 50 % ethanol was used, which suggests the use of a strong liquor to completely harness the potential of Ganoderma triterpenoids in daily life.

[Dynamic accumulation of three main triterpenic acids in submerged cultivation mycelium of Poria cocos].

OBJECTIVE: To study the dymamic accumulation of triterpenic acids production in submerged cultivation mycelium of Poria cocos. METHOD: Liquid culture method of P. cocos was established and RP-HPLC was applied to determine the contents of three main triterpenic acids dehydrotumulosic acid (DTA), 3-epi-dehydrotumulosic acid (eDTA) and polyporenic acid C (PAC) in submerged cultivation mycelium P. cocos at different culture stages and the contents were compared with cultivated P. cocos. The HPLC method is as follows, column: Plastisil ODS (4.6 mm x 250 mm, 5 microm); mobile phase: ACN/0.5% phosphate (80:20); flow rate: 1.0 mL . min-1; detective wavelength: 242 nm. RESULT: The maximum biomass occurred at the 8th d after inoluctation, however, the contents and yield of three compounds increased till the 17th day. The contents of three compounds were 1. 2% (DTA), 0. 42% (eDTA) and 1.0% (PAC) at the 17th day after inoculation, which were significantly higher than that in cultivated material [0.2% (DTA), 0. 12(eDTA) and 0. 16% (PAC) ]. Furthermore, a correlation analysis between the content ratios of three independent compounds was carried out. The results showed that DTA negatively correlated with eDTA and PAC, with R2 of 0. 857 6 and 0. 971 7, respectively, which suggested the role of DTA as an important intermediate in the biosynthesis of triterpenic acids in P. cocos. CONCLUSION: The sum content of three main terpenoids in submerged cultivation mycelium P. cocos was 5. 55 times as that in cultivated material, which strongly suggested the possibility of fermentation in the production of medicinally important triterpenic acids in the future.

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the determination of five ganoderic acids in Ganoderma lucidum and its related species.

The present paper describes a novel, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of ganoderic acids C(2), B, A, H, D in Ganoderma lucidum and its related species. Ganoderma samples were prepared using simple ultrasonic extraction. Chromatographic separation was carried out on an Agilent Zorbax XDB C(18) column (250 mm × 4.6 mm i.d., 5µm) with an isocratic mobile phase consisting of acetonitrile, water and formic acid (42:58:0.5, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operating in negative and positive ionization mode via a single within-run polarity switching. Quantitation of five ganoderic acids was performed using selective reaction monitoring (SRM) mode. The intra- and inter-day precision was less than 6.2% and the accuracy ranged from 90.0% to 105.7%. The limit of quantification (LOQ) was 20.0-40.0 ng/mL and the limit of detection (LOD) was 3.0-25.0 ng/mL. With this method, low levels of ganoderic acids in the fruiting bodies of Ganoderma sinense and Ganoderma applanatum were accurately quantified for the first time. Importantly, the method allows unequivocal quantification of the five ganoderic acids in the spores and fruiting bodies of Ganoderma lucidum, whereas the previously published methods have lacked the capability. The method presented will be a powerful tool for quality control of Ganoderma lucidum and its related species.

Separation and determination of four ganoderic acids from dried fermentation mycelia powder of Ganoderma lucidum by capillary zone electrophoresis.

Ganoderic acids (GAs) were bioactive secondary metabolites produced by a traditional mushroom Ganoderma lucidum. We describe a simple and efficient method for the separation and quantitative determination of four GAs, namely Ganoderic acid T (GA-T), Ganoderic acid Mk (GA-Mk), Ganoderic acid Me (GA-Me) and Ganoderic acid S (GA-S) from dried triterpene-enriched extracts of G. lucidum mycelia powder by capillary zone electrophoresis (CZE). Under the optimum conditions, the four GAs reached the baseline separation in 9 min with Glycyrrhetinic acid (GTA) as internal standard. The four GAs and internal standard (GTA) were detected at a wavelength 245 nm. All calibration curves showed good linearity (r(2)>0.9958) within test ranges. Limit of detection (LOD) and limit of quantification (LOQ) were less than 0.6 and 1.8 microg/mL, respectively. The relative standard deviation (R.S.D.) values of precision and recoveries were less than 5% and recoveries ranged from 91.4% to 103.6%. This was the first report on simultaneous determination of the four GAs and the results provided a firm basis for the trace analysis of GAs in dried fermentation mycelia powder of G. lucidum with high accuracy.

[RP-HPLC simultaneous determination of five triterpenoid acids in different parts of Poria cocos by UV wavelengths switch].

To establish a method for simultaneous determination of dehydrotumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid and pachymic acid in Poria, a RP-HPLC method detected by UV wavelengths switch had been developed, including 210 nm (48-55 min) for pachymic acid and 241 nm (0-48 min) for dehydrotumulosic acid, polyporenic acid C, 3-epi-dehydrotumulosic acid, dehydropachymic acid, separately. The system consisting of a Kromasil C18 column (250 mm x 4.6 mm, 5 microm) and a mixture of acetonitrile and 0.05% phosphate acid as the mobile phase was adopted; The flow rate was 1.0 mL x min(-1). The linear response range was 30.5-610.0 microg x mL(-1) (r = 0.999 6) for dehydrotumulosic acid, 12.66-253.2 microg x mL(-1) (r = 0.999 5) for polyporenic acid C, 2.99-59.7 microg x mL(-1) (r = 0.999 7) for 3-epi-dehydrotumulosic acid, 6.13-122.5 microg x mL(-1) (r = 0.999 5) for dehydropachymic acid and 11.3-226.0 microg x mL(-1) (r = 0.9995) for pachymic acid. The average recoveries of these compounds were 98.5% (RSD = 1.9%), 99.4% (RSD = 1.7%), 97.9% (RSD = 1.2%), 96.7% (RSD = 2.5%) and 97.9% (RSD = 2.3%), respectively. The method is simple, accurate and reproducible for quality control of Poria.

Modulation of cholesterol metabolism by the green tea polyphenol (-)-epigallocatechin gallate in cultured human liver (HepG2) cells.

Epidemiological and animal studies have found that green tea is associated with lower plasma cholesterol. This study aimed to further elucidate how green tea modulates cholesterol metabolism. When HepG2 cells were incubated with the main green tea constituents, the catechins, epigallocatechin gallate (EGCG) was the only catechin to increase LDL receptor binding activity (3-fold) and protein (2.5-fold) above controls. EGCG increased the conversion of sterol regulatory element binding protein-1 (SREBP-1) to its active form (+56%) and lowered the cellular cholesterol concentration (-28%). At 50 microM, EGCG significantly lowered cellular cholesterol synthesis, explaining the reduction in cellular cholesterol. At 200 microM EGCG, cholesterol synthesis was significantly increased even though cellular cholesterol was lower, but there was a significant increase seen in medium cholesterol. This indicates that, at 200 microM, EGCG increases cellular cholesterol efflux. This study provides mechanisms by which green tea modulates cholesterol metabolism and indicates that EGCG might be its active constituent.

Cimicifuga species identification by high performance liquid chromatography-photodiode array/mass spectrometric/evaporative light scattering detection for quality control of black cohosh products.

Black cohosh has become one of the most important herbal products in the US dietary supplements market. It is manufactured from roots and rhizomes of Cimicifuga racemosa (Ranunculaceae). Botanical identification of the raw starting material is a key step in the quality control of black cohosh preparations. The present report summarizes a fingerprinting approach based on high performance liquid chromatography-photodiode array/mass spectrometric/evaporative light scattering detection (HPLC-PDA/MS/ELSD) that has been developed and validated using a total of 10 Cimicifuga species. These include three North American species, Cimicifuga racemosa, Cimicifuga americana, Cimicifuga rubifolia, and seven Asian species, Cimicifuga acerina, Cimicifuga biternat, Cimicifuga dahurica, Cimicifuga heracleifolia, Cimicifuga japonica, Cimicifuga foetida, and Cimicifuga simplex. The chemotaxonomic distinctiveness of the HPLC fingerprints allows identification of all 10 Cimicifuga species. The triterpene glycoside cimigenol-3-O-arabinoside (3), cimifugin (12), and cimifugin-3-O-glucoside (18) were determined to be suitable species-specific markers for the distinction of C. racemosa from the other Cimicifuga species. In addition to identification, the fingerprint method provided insight into chemical interconversion processes occurring between the diverse triterpene glycosides contained in black cohosh. The reported method has proven its usefulness in the botanical standardization and quality control of black cohosh products.

[Studies on quality specification standards for Saposhnicovia divaricata (Turcz.) Schischk].

OBJECTIVE: To work out a qualitative-quantitative standard for Saposhnikovia divaricata. METHOD: TLC and HPLC were adopted. RESULT: Four chromone compounds were identified by TLC, and the contents of two chromone compounds were determined by HPLC. CONCLUSION: Prim-O-glucosylcimifugin, 4'-O-beta-D-glucosyl-5-O-methylvisamminol, sec-O-glucosylhamaudol and cimifugin should be present simultaneously in S. divaricata. The contents of prim-O-glucosylcimifugin and 4'-O-beta-D-glucosyl-5-O-methylvisamminol in S. divaricata should not be lower than 0.12% respectively.

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: modulating effect of retinoic acid.

Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 microM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.

Three new lanostanoids from Ganoderma lucidum.

Three new lanostanoids--ganodermenonol (1), ganodermadiol (2), and ganodermatriol (3) [isolated as its triacetate derivative (3a)]--were isolated from the MeOH extract of Ganoderma lucidum, together with ergosterol and its peroxide. The new compounds were identified as 26-hydroxy-5 alpha-lanosta-7,9(11),24-trien-3-one (1), 5 alpha-lanosta-7,9(11),24-triene-3 beta, 26-diol (2), and 5 alpha-lanosta-7,9(11),24-triene-3 beta, 26,27-triol (3) by their respective spectral data.