Triterpene Saponins from the Roots of Ilex asprella

Six new triterpene saponins, ilexasprellanosides A–F ( 1 – 6 , resp.), together with eleven known compounds were isolated from the roots of Ilex asprella. The new saponins were characterized as ursa‐12,18‐dien‐28‐oic acid 3‐O‐β‐D ‐xylopyranoside ( 1 ), 19α‐hydroxyursolic acid 3‐O‐β‐D ‐(2′‐O‐acetylxylopyranoside) ( 2 ), 19α‐hydroxyursolic acid 3‐O‐β‐D ‐glucuronopyranoside ( 3 ), 3β,19α‐dihydroxyolean‐12‐en‐23,28‐dioic acid 28‐O‐β‐D ‐glucopyranoside ( 4 ), 19α‐hydroxyoleanolic acid 3‐O‐β‐D ‐(2′‐O‐acetylxylopyranoside) ( 5 ), 19α‐hydroxyoleanolic acid 3‐O‐β‐D ‐glucuronopyranoside ( 6 ). The structures of the new compounds were elucidated by analysis of their spectroscopic data and chemical degradation. Compounds 2, 4 , oleanolic acid 3‐O‐β‐D ‐glucuronopyranoside, 3‐β‐acetoxy‐28‐hydroxyurs‐12‐ene, and pomolic acid showed significant cytotoxic activities against human tumor cell line A549 (IC₅₀ values of 1.87, 2.51, 1.41, 3.24, and 5.63 μM , resp.).     

Phytochemical Investigation and Cytotoxic Evaluation of the Components of the Medicinal Plant Ligularia atroviolacea

A phytochemical investigation of the roots of Ligularia atroviolacea resulted in the isolation of 24 compounds including seven new eremophilanoids named eremophila‐3,7(11),8‐triene‐12,8;14,6α‐diolide ( 1 ), 3β‐(angeloyloxy)eremophil‐7(11)‐en‐12,8β‐olid‐14‐oic acid ( 2 ), 1α‐chloro‐10β‐hydroxy‐6β‐(2‐methylpropanoyloxy)‐9‐oxo‐7,8‐furoeremophilane ( 3 ), (10βH)‐8‐oxoeremophila‐3(4),6(7)‐diene‐12,14‐dioic acid ( 4 ), (10αH)‐8‐oxoeremophila‐3(4),6(7)‐diene‐12,14‐dioic acid ( 5 ), 8β‐[eremophila‐3′,7′(11′)‐diene‐12′,8′α;14′,6′α‐diolide]eremophila‐3,7(11)‐diene‐12,8α;14,6α‐diolide ( 6 ), and ligulatrovine A ( 7 ), eleven known eremophilanoids, 8 – 18 , four steroids, one glucose derivative, and one fatty acid. The structures of these compounds were elucidated by spectroscopic methods including 2D‐NMR experiments. The structure of 3 was also established by an X‐ray diffraction study. The in vitro cytotoxicity evaluation of selected compounds was performed on seven cultured tumor cell lines, i.e., KB, BEL‐7404, A549, HL‐60, HeLa, CNE, and P‐388D1. The preliminary taxonomy of this species was also discussed, and the possible biogenesis of a dimer possessing a new noreremophilanoid type skeleton, 7 , is presented in a preliminary form.     

Determination of the Enantiomeric Purity of the Antiasthmatic Drug Montelukast by Means of 1H NMR Spectroscopy

In order to define an enantioselective nuclear magnetic resonance (NMR) method for the antiasthmatic drug montelukast, a series of nine easily available products were evaluated as NMR chiral solvating agents (CSAs): D‐dibenzoyltartaric acid, D‐ditoluoyltartaric acid, (+)‐camphorsulfonic acid, (S)‐BINOL, (S)‐3,3’‐diphenyl‐2,2’‐binaphthyl‐1,1’‐diol, (R)‐3,3'′‐di‐9‐anthracenyl‐1,1'′‐bi‐2‐naphthol, (R)‐3,3'′‐di‐9‐phenanthrenyl‐1,1'′‐bi‐2‐naphthol, Pirkle's alcohol, and (?)‐cinchonidine. It was proved that most of the studied agents constitute diastereomeric complexes with both drug enantiomers in CD₂Cl₂ or CDCl₃ solutions, thus permitting the direct ¹H NMR detection of the unwanted S‐enantiomer, even at levels of 0.75%. (?)‐Cinchonidine was found to be the more convenient CSA in terms of NMR enantiodiscrimination power and ease of experimental requirements. The final method was validated and applied to the fast monitoring of the optical purity of montelukast “in‐process” samples, circumventing the need for tedious and slower analytical procedures like enantioselective chromatography or capillary electrophoresis. In addition, a method for the enantiopurity control of the commercial drug (montelukast sodium salt) was also established using (S)‐BINOL as NMR CSA. Chirality 25: 780–786, 2013. © 2013 Wiley Periodicals, Inc.     

Cytotoxic Steroidal Saponins from the Rhizomes of Tacca integrifolia

Three new steroid saponins (3β,25R)‐spirost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[β‐D ‐glucopyranosyl‐(1→4)‐6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 1 ), (3β,22R,25R)‐26‐(β‐D ‐glucopyranosyloxy)‐22‐hydroxyfurost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 3 ), and (3β,22R,25R)‐26‐(β‐D ‐glucopyranosyloxy)‐22‐hydroxyfurost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[β‐D ‐glucopyranosyl‐(1→4)‐6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 5 ), as well as the new pregnane glycoside (3β,16β)‐3‐{[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranosyl]oxy}‐20‐oxopregn‐5‐en‐16‐yl (4R)‐5‐(β‐D ‐glucopyranosyloxy)‐4‐methylpentanoate ( 6 ), were isolated from the rhizomes of Tacca integrifolia together with two known (25R) configurated steroid saponins (3β,25R)‐spirost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 2 ) and (3β,22R,25R)‐26‐(β‐D ‐glucopyranosyloxy)‐22‐methoxyfurost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 4 ). The cytotoxic activity of the isolated compounds was evaluated in HeLa cells and showed the highest cytotoxicity value for compound 2 with an IC₅₀ of 1.2±0.4 μM . Intriguingly, while compounds 1 – 5 exhibited similar cytotoxic properties between 1.2±0.4 ( 2 ) and 4.0±0.6 μM ( 5 ), only compound 2 showed a significant microtubule‐stabilizing activity in vitro.     

Cytotoxic Compounds from the Leaves of Garcinia polyantha

A new compound, named banganxanthone C (=12‐(1,1‐dimethylprop‐2‐en‐1‐yl)‐5,10‐dihydroxy‐9‐methoxy‐2‐methyl‐2‐(4‐methylpent‐3‐en‐1‐yl)‐2H,6H‐pyrano[3,2‐b]xanthen‐6‐one; 4 ), together with five known compounds, were isolated from the leaves of Garcinia polyantha. The structures of the compounds were elucidated on the basis of 1D‐ and 2D‐NMR spectroscopy. Among the known compounds, two were xanthones, one was a pentacyclic triterpene, one sterol, and one benzophenone derivative. Isoxanthochymol ( 2 ) and 4‐[(2E)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl]‐1,5,8‐trihydroxy‐3‐methoxy‐9H‐xanthen‐9‐one ( 3 ) exhibited significant antiproliferative activity against the leukemia cell line TPH‐1 with IC₅₀ inhibition values of 1.5 and 2.8 μg/ml, respectively. The cytotoxic activity was found to be related to apoptosis induction.     

Cytotoxic Oleanane‐Type Saponins from the Leaves of Albizia anthelmintica Brongn.

Two new oleanane‐type saponins: β‐d ‐xylopyranosyl‐(1 → 4)‐6‐deoxy‐α‐l ‐mannopyranosyl‐(1 → 2)‐1‐O‐{(3β)‐28‐oxo‐3‐[(2‐O‐β‐d ‐xylopyranosyl‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐yl}‐β‐d ‐glucopyranose ( 1 ) and 1‐O‐[(3β)‐28‐oxo‐3‐{[β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐arabinopyranosyl‐(1 → 6)‐2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐yl]β‐d ‐glucopyranose ( 2 ), along with two known saponins: (3β)‐3‐[(β‐d ‐Glucopyranosyl‐(1 → 2)‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐oic acid ( 3 ) and (3β)‐3‐{[α‐l ‐arabinopyranosyl‐(1 → 6)‐[β‐d ‐glucopyranosyl‐(1 → 2)]‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐oic acid ( 4 ) were isolated from the acetone‐insoluble fraction obtained from the 80% aqueous MeOH extract of Albizia anthelmintica Brongn . leaves. Their structures were identified using different NMR experiments including: ¹H‐ and ¹³C‐NMR, HSQC, HMBC and ¹H,¹H‐COSY, together with HR‐ESI‐MS/MS, as well as by acid hydrolysis. The four isolated saponins and the fractions of the extract exhibited cytotoxic activity against HepG‐2 and HCT‐116 cell lines. Compound 2 showed the most potent cytotoxic activity among the other tested compounds against the HepG2 cell line with an IC₅₀ value of 3.60μm . Whereas, compound 1 showed the most potent cytotoxic effect with an IC₅₀ value of 4.75μm on HCT‐116 cells.     

Changes in the Proliferation and Differentiation of Neonatal Mouse Pink‐Eyed Dilution Melanocytes in the Presence of Excess Tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild‐type at the pink‐eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum‐free primary culture, with or without the addition of L‐Tyr. Incubation with added L‐Tyr inhibited the proliferation of P/P melanocytes in a concentration‐dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L‐Tyr stimulated the proliferation of p/p melanoblasts–melanocytes derived from 0.5‐day‐old mice, but inhibited their proliferation when derived from 3.5‐ or 7.5‐day‐old mice. L‐Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5‐, 3.5‐ and 7.5‐day‐old p/p mice, but L‐Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole‐2,3,5‐tricarboxylic acid as well as the pheomelanin marker, 4‐amino‐3‐hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6‐dihydroxyindole‐2‐carboxylic acid, as well as the contents of pheomelanin and its precursor, 5‐S‐cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L‐Tyr increased the content of eumelanin and pheomelanin two‐ to threefold in cultured cells and media derived from 0.5‐, 3.5‐ and 7.5‐day‐old mice. These results suggest that the proliferation of p/p melanoblasts–melanocytes is stimulated by L‐Tyr, and that the differentiation of melanocytes is induced by L‐Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

Developments in the synthesis of the antiplatelet and antithrombotic drug (S)‐clopidogrel

S ‐(+)‐Methyl 2‐(2‐chlorophenyl)‐2‐(6,7‐dihydrothieno[3,2‐c ]pyridin‐5(4H )‐yl)acetate, also known as (S )‐clopidogrel, is marketed under the trade names Plavix and Iscover. It is a potent thienopyridine‐class of antithrombotic and antiplatelet drug (antiaggregant). Among the two available stereoisomers of clopidogrel, for pharmaceutical activities only the S ‐enantiomer is applicable, as no antithrombotic activity is observed in the R ‐enantiomer and causes political upheavals and social turmoil in animal experiments. Worldwide sales of Plavix amounted to $6.4 billion yearly, which ranks second. Attributed to the increased demand of (S )‐clopidogrel drug, it provoked the synthetic community to devise facile synthetic approaches. This review aims to summarize the synthetic methods of (S )‐clopidogrel drug reported in the literature. The present review discusses the pros and cons of each synthetic methodology, which would be beneficial to the scientific community for further developments in the synthetic methodologies for (S )‐clopidogrel. In addition, the compilation approach of literature‐reported synthetic strategies of (S )‐clopidogrel in one platform is advantageous, supportive, and crucial for the synthetic community to elect the best synthetic methodology of (S )‐clopidogrel and to create new synthesis ideas.     

Behavioral responses for evaluating the attractiveness of specific tea shoot volatiles to the tea green leafhopper,Empoaca vitis

Abstract The tea green leafhopper, Empoasca vitis Göthe, is one of the most serious insect pests of tea plantations in mainland China. Over the past decades, this pest has been controlled mainly by spraying pesticides. Insecticide applications not only have become less effective in controlling damage, but even more seriously, have caused high levels of toxic residues in teas, which ultimately threatens human health. Therefore, we should seek a safer biological control approach. In the present study, key components of tea shoot volatiles were identified and behaviorally tested as potential leafhopper attractants. The following 13 volatile compounds were identified from aeration samples of tea shoots using gas chromatography‐mass spectrometry (GC‐MS): (E)‐2‐hexenal, (Z)‐3‐hexen‐1‐ol, (Z)‐3‐hexenyl acetate, 2‐ethyl‐1‐hexanol, (E)‐ocimene, linalool, nonanol, (Z)‐butanoic acid, 3‐hexenyl ester, decanal, tetradecane, β‐caryophyllene, geraniol and hexadecane. In Y‐tube olfactometer tests, the following individual compounds were identified: (E)‐2‐hexenal, (E)‐ocimene, (Z)‐3‐hexenyl acetate and linalool, as well as two synthetic mixtures (called blend 1 and blend 2) elicited significant taxis, with blend 2 being the most attractive. Blend 1 included linalool, (Z)‐3‐hexen‐1‐ol and (E)‐2‐hexenal at a 1 : 1 : 1 ratio, whereas blend 2 was a mixture of eight compounds at the same loading ratio: (E)‐2‐hexenal, (Z)‐3‐hexen‐1‐ol, (Z)‐3‐hexenyl acetate, 2‐penten‐1‐ol, (E)‐2‐pentenal, pentanol, hexanol and 1‐penten‐3‐ol. In tea fields, the bud‐green sticky board traps baited with blend 2, (E)‐2‐hexenal or hexane captured adults and nymphs of the leafhoppers, with blend 2 being the most attractive, followed by (E)‐2‐hexenal and hexane. Placing sticky traps baited with blend 2 or (E)‐2‐hexenal in the tea fields significantly reduced leafhopper populations. Our results indicate that the bud‐green sticky traps baited with tea shoot volatiles can provide a new tool for monitoring and managing the tea leafhopper.     

New Thymoquinol Glycosides and Neuroprotective Dibenzocyclooctane Lignans from the Rattan Stems of Schisandra chinensis

Three new lignans ( 1 – 3 ), together with four new thymoquinol glycosides ( 4 – 7 ), were isolated from 70%‐EtOH extract of the rattan stems of Schisandra chinensis. The structures of 1 – 7 were elucidated by detailed spectroscopic analyses, and these new compounds were identified as pinobatol‐9‐O‐β‐d ‐glucopyranoside ( 1 ), 1,2,13,14‐tetramethoxydibenzocyclooctadiene 3,12‐O‐β‐d ‐diglucopyranoside ( 2 ), 3,7‐dihydroxy‐1,2,13,14‐tetramethoxydibenzocyclooctadiene 12‐O‐β‐d ‐glucopyranoside ( 3 ), thymoquinol 2‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 4 ), thymoquinol 2‐O‐α‐d ‐arabinofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 5 ), thymoquinol 5‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 6 ), and thymoquinol 5‐O‐α‐d ‐arabinofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 7 ). The neuroprotective activity of 1 – 7 was evaluated on PC12 cells with neurotoxicity induced by amyloid‐beta 1 – 42 (Aβ_{1 – 42}). Compounds 2 and 3 showed protecting activity against Aβ‐induced toxicity in PC12 cells.     

The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum has previously been shown to involve the sequential production of (E)‐ and (Z)‐p‐hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild‐type and mutant sorghum or transiently transformed Nicotiana benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)‐p‐hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)‐p‐hydroxyphenylacetaldoxime into the corresponding geometrical Z‐isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme, but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsis thaliana cannot convert the (E)‐p‐hydroxyphenylacetaldoxime to the (Z)‐isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the (E)‐p‐hydroxyphenylacetaldoxime into an S‐alkyl‐thiohydroximate with retention of the configuration of the E‐oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z‐oximes but not E‐oximes. The CYP79‐derived E‐oximes may play an important role in plant defense.     

Association of the variants and haplotypes in the DOCK7, PCSK9 and GALNT2 genes and the risk of hyperlipidaemia

Little is known about the association between the single nucleotide polymorphisms (SNPs) and haplotypes of the dedicator of cytokinesis 7 (DOCK7), pro‐protein convertase subtilisin/kexin type 9 (PCSK9) and polypeptide N‐acetylgalactosaminyltransferase 2 (GALNT2) and serum lipid traits in the Chinese populations. This study was to determine the association between nine SNPs in the three genes and their haplotypes and hypercholesterolaemia (HCH)/hypertriglyceridaemia (HTG), and to identify the possible gene–gene interactions among these SNPs. Genotyping was performed in 733 HCH and 540 HTG participants. The haplotype of C‐C‐G‐C‐T‐G‐C‐C‐G [in the order of DOCK7 rs1168013 (G>C), rs10889332 (C>T); PCSK9 rs615563 (G>A), rs7552841 (C>T), rs11206517 (T>G); and GALNT2 rs1997947 (G>A), rs2760537 (C>T), rs4846913 (C>A) and rs11122316 (G>A) SNPs] was associated with increased risk of HCH and HTG. The haplotypes of C‐C‐G‐C‐T‐G‐C‐C‐A and G‐C‐G‐T‐T‐G‐T‐C‐G were associated with a reduced risk of HCH and HTG. The haplotypes of G‐C‐G‐C‐T‐G‐C‐C‐A and G‐C‐G‐C‐T‐G‐T‐C‐G were associated with increased risk of HCH. The haplotypes of C‐T‐G‐C‐T‐G‐C‐C‐G, G‐C‐A‐C‐T‐G‐C‐C‐G and G‐C‐G‐C‐T‐G‐C‐C‐A were associated with an increased risk of HTG. The haplotypes of G‐C‐G‐C‐T‐G‐T‐C‐A and G‐C‐G‐T‐T‐G‐T‐C‐G were associated with a reduced risk of HTG. In addition, possible inter‐locus interactions among the DOCK7, PCSK9 and GALNT2 SNPs were also noted. However, further functional studies of these genes are still required to clarify which SNPs are functional and how these genes actually affect the serum lipid levels.     

Effect of glypican‐1 gene on the pulp cells during the reparative dentine process

GPC‐1 (glypican‐1) is a cell surface heparan sulfate proteoglycan that acts as a co‐receptor for heparin‐binding growth factors and members of the TGF‐β (transforming growth factor beta‐1) family. The function of cell‐surface proteoglycans in the reparative dentine process has been under investigation. Gpc‐1 was detected with similar frequency as tgf‐β1 in the cDNA library using mRNA from the odontoblast‐like cell‐enriched pulp of rat incisors. The aim of this study was to test our hypothesis that gpc‐1 may be related to reparative dentine formation. We examined the expression of this gene during the reparative dentine process, as well as the effect of gpc‐1 on odontoblast‐like cell differentiation using siRNA (small interfering RNA) to down‐regulate gpc‐1 expression. Immunohistological examination showed that GPC‐1 was expressed in pulp cells entrapped by fibrodentine and odontoblast‐like cells as well as TGF‐β1. The mRNAs for gpc‐1, ‐3 and ‐4, except for gpc‐2, were expressed during odontoblast‐like cell differentiation in pulp cells. The relative levels of gpc‐1 mRNA were increased prior to the differentiation stages and were decreased during the secretory and maturation stages of pulp cells. Down‐regulation of gpc‐1 expression resulted in a 3.9‐fold increase in tgf‐β1 expression in pulp cells and a 0.3‐fold decrease in dspp (dentine sialophosphoprotein) expression compared with control. These results suggested that gpc‐1 and tgfβ‐1 expression are necessary for the onset of differentiation, but should be down‐regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc‐1 expression in odontoblast‐like cells is associated with the early differentiation but not with the formation of reparative dentine.     

Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

After cultivation on (R,S)‐2‐(2,4‐dichlorophenoxy)propionate, two α‐ketoglutarate‐dependent dioxygenases were isolated and purified from Delftia acidovorans MC1, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R‐enantiomer of substituted phenoxypropionate derivatives: the K_m values were 55 μM and 30 μM, the k_cat values 55 min^–1 and 34 min^–1 with (R)‐2‐(2,4‐dichlorophenoxy)propionate [(R)‐2,4‐DP] and (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S‐enantiomers with K_m values of 49 μM and 22 μM, and k_cat values of 50 min^–1 and 46 min^–1 with (S)‐2‐(2,4‐dichlorophenoxy)propionate [(S)‐2,4‐DP] and (S)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4‐dichlorophenoxyacetate: K_m = 123 μM, k_cat = 36 min^–1) with significant activity. As the second substrate, only α‐ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K_i values of 3 mM with (R)‐2,4‐DP and 1.5 mM with (S)‐2,4‐DP. The reaction was strictly dependent on the presence of Fe²⁺ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).     

Heterologous expression of the immunomodulatory protein gene from Ganoderma sinense in the basidiomycete Coprinopsis cinerea

Aims: FIP‐gsi, a fungal immunomodulatory protein found in Ganoderma sinense, has antitumour, anti‐allergy and immunomodulatory activities and is regulated by the fip‐gsi gene. In this study, we aimed to express the fip‐gsi gene from G. sinense in Coprinopsis cinerea to increase yield of FIPs‐gsi. Methods and Results: A fungal expression vector pBfip‐gsi containing the gpd promoter from Agaricus bisporus and the fip‐gsi gene from the G. sinense was constructed and transformed into C. cinerea. PCR and Southern blotting analysis verified the successful integration of the exogenous gene fip‐gsi into the genome of C. cinerea. RT‐PCR and Northern blotting analysis confirmed that the fip‐gsi gene was transcribed in C. cinerea. The yield of the FIP‐gsi protein reached 314 mg kg^?1 fresh mycelia. The molecular weight of the FIP‐gsi was 13 kDa, and the FIP‐gsi was capable of hemagglutinating mouse red blood cells, but no such activity was observed towards human red blood cells in vitro. Conclusions: The fip‐gsi from G. sinense has been successfully translated in C. cinerea, and the yield of bioactive FIP‐gsi protein was high. Significance and Impact of the Study: This is the first report using the C. cinerea for the heterologous expression of FIP‐gsi protein and it might supply a basis for large‐scale production of the protein.     

Triterpenoids from the Roots of Actinidia chinensis

Two new triterpenoids, 1 and 2 , were isolated from the hepatoprotective AcOEt fraction of the roots of Actinidia chinensis, together with eight known 12‐en‐28‐oic acids of oleanane or ursane type, 3 – 10 . The two new compounds were elucidated as 2α,3β‐dihydroxyurs‐12‐en‐28,30‐olide ( 1 ) and 2α,3β,24‐trihydroxyurs‐12‐en‐28,30‐olide ( 2 ), on the basis of spectroscopic (IR, NMR, and MS) analyses. The chemotaxonomic significances of some triterpenoids were also discussed.