Pseudolaric acids of Pseudolarix kaempferi (Pinaceae) have been known as diterpenoids with potent anti-fungal-, anti-microbial, and cytotoxic activities. In the present study, the five MeOH extracts were prepared from the five plant part (root bark, stem bark, leaf, the inner part of root, and cone) to find the relation between the concentration of pseudolaric acids and cytotoxicity. Pseudolaric acids B and C were isolated from the root bark of P. kaempferi to use them as standard compounds. The five extracts were tested on cytotoxicity against six cancer cell lines, A549 (lung), HCT116 (colon), MDA-MB-231 (breast), SNU638 (stomach), and SK-hep-1 (liver) by SRB assay, but against K562 (leukemia) by SRB- or MTT assay. HPLC quantification were performed on a Shisheido Capcell PAK C18 column (5 µm, 4.6 mm × 250 mm) using 254 nm wavelength. The cytotoxicity (IC₅₀, 0.36 µg/ml on K562 cell lines) of the root bark extract was potent and the content (101.1 mg/g extract) of pseudolaric acid B was very high in the root bark. These results suggest that the MeOH extract obtained from the root bark could be developed as the anti-cancer agent with a high quantity of pseudolaric acid B.
Cell Line ; Chromatography, High Pressure Liquid ; Diterpenes ; Pinaceae ; Plants*

OBJECTIVETo research molluscicidal effect activity, active components and stable passage of endophyte JJ18 from Pseudolarix amabilis and examine the possibility for practical application.

METHODMolluscicidal effect test was performed according to the immersion test method suggested by WHO.

RESULTImmersion test with JJ18 broth showed that the active components were extracellular moiety of the broth and that 10% concentration solution could kill nearly 90% snail immersed after 72 h, the salified broth has favourable thermostabily and photostability and showed that JJ18 has stable passage and its active components concentrate in the extract of n-butanol.

CONCLUSIONThe metabolite of endophyte JJ18 has activity for molluscicidal effect and potential for application.

Animals ; Bacteria ; chemistry ; growth & development ; Bacterial Proteins ; pharmacology ; Molluscacides ; chemistry ; pharmacology ; Pinaceae ; microbiology ; Polysaccharides, Bacterial ; pharmacology ; Snails ; drug effects

The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.
Animals ; Chromatography, High Pressure Liquid ; Diterpenes ; metabolism ; Glucosides ; metabolism ; Hydrolysis ; Mass Spectrometry ; Metabolic Networks and Pathways ; Pinaceae ; chemistry ; Rats

OBJECTIVETo research the constituents in needles of Pinus sylvestris.

METHODRepeated column chromatography and preparation HPLC are used for compound isolation, and their structures were elucidated on the basis of spectral data analysis.

RESULTSix compounds, pinifolic acid (1), 15-oxo-8 (17) -labden-18-oic acid (2) , 15-acetoxy-labd-8 ( 17)-en-18-oic acid (3), dehydroabietic acid (4), 7alpha-hydroxydehydroabietic acid (5), 7beta-hydroxydehydroabietic acid (6) were isolated from the needles of P. sylvestris.

CONCLUSIONCompound 3-6 were isolated from the needles of P. sylvestris for the first time, and compound 3 is a new natural product. The petroleum ether and EtOAc extracts showed significant cytotoxic effects to Hela and A549. Compounds 2, 4-6 revealed a positive distinction compared to the control group.

Antineoplastic Agents, Phytogenic ; chemistry ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Chromatography, High Pressure Liquid ; Diterpenes ; chemistry ; isolation & purification ; pharmacology ; Diterpenes, Abietane ; chemistry ; isolation & purification ; pharmacology ; HeLa Cells ; Humans ; Molecular Structure ; Pinus sylvestris ; chemistry ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Structure-Activity Relationship

OBJECTIVETo screen antifungal activity of endophytes from Pseudolarix kaempferi.

METHODEndophytes from P. kaempferi were separated. By means of microdilution method, antifungal active endophytes were fast screened by Pyricularia oryzae P-2b model, and activity of endophytes against pathogenic fungus was studied.

RESULT44.8% of endophytes showed activity against P. oryzae P-2b in Pseudolarix kaempferi. Among them JJ314, JJ323 introduced formation of characteristic beads and swellings on the growing hyphae, JJ324 inhibited the conidia germination. They all showed activity against Trichophyton rubrum, Cryptococcus neoformans and Candida albicans.

CONCLUSIONEndophytes from P. kaempferi are a potential resource for the development of antifungal agent.

Antifungal Agents ; isolation & purification ; pharmacology ; Candida albicans ; drug effects ; Cryptococcus ; drug effects ; Fungi ; chemistry ; classification ; isolation & purification ; Mitosporic Fungi ; drug effects ; Pinaceae ; microbiology ; Plants, Medicinal ; microbiology ; Symbiosis ; Trichophyton ; drug effects

The metabolic profile of pseudolaric acid B (PB) was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the specific metabolite of PB in plasma, urine, bile and feces using HPLC and HPLC-ESI/MS(n) after both oral and intravenous administration to rats, and almost no prototype was detected in all kinds of samples. The metabolic behaviors of PB orally administered in rats treated with antibiotics to eliminate intestinal microflora were identical with those in untreated rats, demonstrating that the metabolism of PB is independent of intestinal microflora. PB was stable in 48 h respective incubation with artificial gastric juice and artificial intestinal juice, suggesting that neither pepsin nor trypsin is in charge of metabolism of PB, and also demonstrating that PB is stable in both pH environments of gastric tract and intestinal tract. In vitro research on metabolism of PB in rat liver microsomes incubation revealed that little PB was metabolized and that the proposed metabolites were the demethoxy and demethoxydecarboxy products of the prototype. The amount of metabolites was extremely low compared with the prototype, indicating that liver microsomes are not responsible for the metabolism of PB either. PB was gradually metabolized into PC2 during 1 h in whole blood incubation in vitro, and the metabolic process showed dynamically dependent manner with incubation time. Once absorbed into blood, PB was quickly metabolized into PC2, accordingly, little prototype was detected in all kinds of samples. The metabolism was attributed to the rapid hydrolysis of C-19 ester bond by plasma esterase. These results clarified the metabolic pathway of PB for the first time, which was of great significance to identify the in vivo active form and interpret acting mechanism of the active compounds of P. kaempferi.
Administration, Intravenous ; Administration, Oral ; Animals ; Bile ; metabolism ; Diterpenes ; blood ; metabolism ; urine ; Esterases ; metabolism ; Feces ; chemistry ; Hydrolysis ; Male ; Metabolic Networks and Pathways ; Microsomes, Liver ; metabolism ; Pinaceae ; chemistry ; Plant Bark ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the mechanisms of pseudolaric acid B-induced apoptosis on A375-S2 cells.

METHODMTT, fluorescence microscope observation, DNA agarose gel electrophoresis and Western blot analysis wereused.

RESULTPseudolaric acid Binduces A375-S2 cell apoptosis in a time and dose-dependent manner. Apoptotic bodies and DNA ladder were observed in 5 micromol x L(-1) pseudolaric acid B-treated A375-S2 cells for 36 h. The expression of Bcl-2, Bcl-xL and ICAD was reduced time dependently, whereas the expression of Bax was increased.

CONCLUSIONThe major cause of pseudolaric acid B induced cytotoxicity on A375-S2 cells was apoptosis. Mitochondria proteins and ICAD might be involved in the apoptotic pathways of pseudolaric acid B-treated A375-S2 cells.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Cell Line, Tumor ; Diterpenes ; isolation & purification ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; Melanoma ; metabolism ; pathology ; Pinaceae ; chemistry ; Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; bcl-X Protein

Fragmentation behavior of diterpenoids was investigated by ESI/MSn and the qualitative analysis of diterpenoids in the bark of Pseudolarix kaempferi was performed using high-performance liquid chromatography/ multi-stage mass spectrometry (HPLC-ESI/MSn). The characteristic fragmentation behaviors of the diterpenoids are the cleavages of the lactone ring and C4-O bond. Furthermore, the eliminations of substituent groups at C-18, C-7 and C-8 can also be observed in the MS" (n = 3-4) spectra. For C-4 acetoxy subsititued diterpenoids, [M+Na-60]+ and [M-H-104] are the base peaks of MS2 spectra in the positive and negative ionization modes, respectively. For C-4 hydroxyl subsititued diterpenoids, [M+Na-44]+ and [M-H-62] are the base peaks of MS2 in the positive and negative ionization modes, respectively. For C-18 glucosylated or esterized diterpenoids, [M+Na-44]+ is the base peak of MS2 spectra in positive ionization mode. These fragmentation rules were successfully exploited in the identification of diterpenoids in methanol/water (6:4) extract of P. kaempferi by LC-MS in positive ionization mode. A total of 9 diterpenoids were identified or tentatively characterized, and one of them is reported here for the first time. The described method could be utilized for the sensitive and rapid qualitative analysis of P. kaempferi.
Chromatography, High Pressure Liquid ; methods ; Diterpenes ; analysis ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; analysis ; chemistry ; isolation & purification ; Molecular Structure ; Pinaceae ; chemistry ; Plant Bark ; chemistry ; Plants, Medicinal ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

Cedrus deodara (Pinaceae) has been used traditionally in Ayurveda for the treatment of central nervous system disorders. 3,4-bis(3,4-dimethoxyphenyl)furan-2,5-dione (BDFD) was isolated from heart wood of Cedrus deodara and was shown to have antiepileptic and anxiolytic activity. Thus, the present study was aimed to explore its anti-depressant effect and to correlate the effect with serotonin and nor adrenaline levels of brain. Albino mice were used as experimental animal. Animals were divided in to three groups; vehicle control, imipramine (30 mg/kg i.p.), BDFD (100 mg/kg i.p.). Tail suspension test (TST) and forced swim test (FST) was performed to evaluate antidepressant effect of BDFD. BDFD (100 mg/kg, i.p.) showed a significant decrease in immobility time when subjected to FST whereas immobility time was not significantly altered in TST. BDFD treatment increased serotonin and noradrenaline levels in the brain which is indicative of BDFD having possible atypical antidepressant action.
Animals ; Brain ; Cedrus* ; Central Nervous System Diseases ; Epinephrine ; Heart* ; Hindlimb Suspension ; Imipramine ; Mice ; Norepinephrine ; Rodentia* ; Serotonin ; Wood*

Upon studying the diversity of fungal endophytes associated with pine trees in Korea, many species of Penicillium were encountered. In this study, we report two species of Penicillium isolated from the needles of Pinus rigida. Based on ID region analysis, cultural and morphological characteristics, the two species were identified as Penicillium fellutanum and P. toxicarium, both of which are new to Korea.
Endophytes ; Korea ; Needles ; Penicillium ; Pinus