De novo transcriptome sequencing of Rhododendron molle and identification of genes involved in the biosynthesis of secondary metabolites.

BACKGROUND: Rhododendron molle (Ericaceae) is a traditional Chinese medicinal plant, its flower and root have been widely used to treat rheumatism and relieve pain for thousands of years in China. Chemical studies have revealed that R. molle contains abundant secondary metabolites such as terpenoinds, flavonoids and lignans, some of which have exhibited various bioactivities including antioxidant, hypotension and analgesic activity. In spite of immense pharmaceutical importance, the mechanism underlying the biosynthesis of secondary metabolites remains unknown and the genomic information is unavailable. RESULTS: To gain molecular insight into this plant, especially on the information of pharmaceutically important secondary metabolites including grayanane diterpenoids, we conducted deep transcriptome sequencing for R. molle flower and root using the Illumina Hiseq platform. In total, 100,603 unigenes were generated through de novo assembly with mean length of 778 bp, 57.1% of these unigenes were annotated in public databases and 17,906 of those unigenes showed significant match in the KEGG database. Unigenes involved in the biosynthesis of secondary metabolites were annotated, including the TPSs and CYPs that were potentially responsible for the biosynthesis of grayanoids. Moreover, 3376 transcription factors and 10,828 simple sequence repeats (SSRs) were also identified. Additionally, we further performed differential gene expression (DEG) analysis of the flower and root transcriptome libraries and identified numerous genes that were specifically expressed or up-regulated in flower. CONCLUSIONS: To the best of our knowledge, this is the first time to generate and thoroughly analyze the transcriptome data of both R. molle flower and root. This study provided an important genetic resource which will shed light on elucidating various secondary metabolite biosynthetic pathways in R. molle, especially for those with medicinal value and allow for drug development in this plant.

Sesamin from Cuscuta palaestina natural plant extracts: Directions for new prospective applications.

The aim of this study is to disclose the potential bioactive components of Cuscuta palaestina, a native parasitic natural plant of flora palaestina and to open direction towards new prospective application. GC-MS analysis identified 18 components in the methanolic extract of C. palaestina for the first time. The most appealing among them are Sesamin and two other phytosterols (Campesterol and Stigmasterol), all of which are documented in the scientific literature for their anticancer activity. Quantitation of Sesamin extracted from C. palaestina by HPLC-PDA with the use of three organic solvents showed that the Sesamin content in the methanolic extract was the highest. Following the disclosure of Sesamin presence in C. palaestina, we raised the question of whether it is produced naturally in C. palaestina or acquired from the host plant. The quantitation of Sesamin in C. palaestina was performed while being with five different host plants, and was compared with the amount of Sesamin in C. palaestina grown alone. The findings reveal that Sesamin is an endogenous secondary metabolite in C. palaestina. Thus, further studies are required to prove if C. palaestina can be used as an alternative source of anticancer phytochemicals, mainly Sesamin, and if proteins in the Sesamin production pathway could be valid biological targets for the development of novel and selective pesticides for control/ eradication of C. palaestina and maybe some other Cuscuta species. As well, the findings from this study raise a big question of whether inferring Sesamin production in C. palaestina could reduce its attack ability to host plants.

Phytochemical and biotechnological studies on Schisandra chinensis cultivar Sadova No. 1-a high utility medicinal plant.

In the presented work, raw materials (fruits and leaves) and in vitro biomass of a highly productive Schisandra chinensis Sadova No. 1 cultivar (SchS) were evaluated for the production of therapeutically useful schisandra lignans (SL). In vitro cultures of SchS were initiated, followed by extensive optimization studies focused on maximizing secondary metabolite production, with the aim of establishing a sustainable source of SL. Different cultivation systems (agar, agitated, bioreactor), experiment times (10, 20, 30, 40, 50 and 60 days) and plant growth regulators (6-benzyladenine-BA and 1-naphthaleneacetic acid-NAA, from 0 to 3 mg/l) in Murashige-Skoog (MS) medium were tested. Moreover, an elicitation procedure was applied to bioreactor-grown microshoots in order to increase SL production. Validated HPLC-DAD protocol enabled to detect fourteen SL in the extracts from in vitro and in vivo materials. The main compounds in the in vitro cultures were as follows: schisandrin (max. 176.3 mg/100 g DW), angeloylgomisin Q (max. 85.1 mg/100 g DW), gomisin A (max. 71.4 mg/100 g DW) and angeloylgomisin H (max. 67.0 mg/100 g DW). The highest total SL content (490.3 mg/100 g DW) was obtained in extracts from the biomass of agar cultures cultivated for 30 days on the MS medium variant containing 3 mg/l BA and 1 mg/l NAA. This amount was 1.32 times lower than in fruit extracts (646.0 mg/100 g DW) and 2.04 times higher than in leaf extracts (240.7 mg/100 g DW). The study demonstrated that SchS is a rich source of SL, thus proving its value for medical, cosmetic and food industry.

Justicidin B: A Promising Bioactive Lignan.

Adverse effects and drug resistance to the current onchopharmacologicals have increased the demand for alternative novel therapeutics. We herein introduce justicidin B, an arylnaphthalen lignan isolated from different plant origins, especially Justicia, Phyllanthus, Haplophyllum and Linum species. This cyclolignan exhibits a wide array of biological properties ranges from piscicidal to antifungal, antiviral and antibacterial activities. Activity against Trypanosoma brucei makes justicidin B a potential antiprotozoal agent for the treatment of neglected tropical diseases. Pharmacological properties like antiplatelet, anti-inflammatory and bone resorption inhibition have been also attributed to justicidin B. This compound is a potent cytotoxic substance on several cell lines, especially chronic myeloid and chronic lymphoid leukemia. Pharmacological values, natural variation, as well as biotechnological production of justicidin B by plant cell, tissue and organ culture are also described in this review. Chemical characteristics and chromatographic methods to identify justicidin B and its biosynthetic pathway have been discussed. Different approaches to the total synthesis of justicidin B are compared. This review would shed light on the role of justicidin B as an intriguing natural compound and provides a chance to optimize conditions for industrial applications.

Hinokinin, an emerging bioactive lignan.

Hinokinin is a lignan isolated from several plant species that has been recently investigated in order to establish its biological activities. So far, its cytotoxicity, its anti-inflammatory and antimicrobial activities have been studied. Particularly interesting is its notable anti-trypanosomal activity.

Molecular cloning and functional characterization of two divergent 4-coumarate†:†coenzyme A ligases from Kudzu (Pueraria lobata).

As part of the efforts to understand isoflavonoid metabolism in Pueraria lobata at the molecular level, the cDNAs encoding two divergent 4-coumarate : coenzyme A ligases (4CLs, designated Pl4CL1 and Pl4CL2, respectively) were isolated from P. lobata roots. Sequence analysis revealed that Pl4CL1 had an N-terminal extension of twenty-one amino acid residues compared to Pl4CL2. Phylogenetic analysis showed that Pl4CL1 and Pl4CL2 fell into angiosperm Class II and Class I, respectively. Through in vitro biochemical assays, both Pl4CLs were found to have the capacity to utilize 4-coumarate and trans-cinnamate as substrates, while neither of them could convert sinapate. Pl4CL2 had a broader substrate specificity than Pl4CL1. The affinity of Pl4CL1 for 4-coumarate was 2.6-fold higher than that of Pl4CL2 (with the Km values of 3.5 µM and 9.1 µM, respectively). Combining the dataset including gene expression profiles, metabolites measurements, and biochemical properties, our results indicated that Pl4CL1, just as other angiosperm Class II 4CLs, might play a role in isoflavone biosynthesis in P. lobata, while Pl4CL2 belongs to angiosperm Class I, and may function as a housekeeping enzyme concerning lignification.

[Advances in studies on aryltetralin lactone lignans].

Natural aryltetralin lactone lignans existed in the plants of family Berberidaceae, Acanthaceae, Burseraceae, Verbenaceae, Euphorbiaceae, etc. Due to the antineoplastic and antiviral properties, it has become a hot research topic in medicinal chemistry. This review covers extraction and isolation, biosynthesis, plant origin, and structure and spectral characteristics of natural aryltetralin lactone lignans. It will provide a useful reference for the intensive studies and rational utilization of aryltetralin lactone lignans.

Chemoenzymatic total synthesis of hyperiones A and B.

The first asymmetric total synthesis of hyperiones A and B, two norlignans from Hypericum chinense, has been accomplished following a chemoenzymatic approach. Key features of this synthesis include the lipase-catalyzed enantioselective desymmetrization of a prochiral allenic diol and a silver-mediated cycloisomerization of the resulting axially chiral product to furnish the furan core structure. Two alternative pathways, a ruthenium-catalyzed redox isomerization on the one side and a platinum-catalyzed hydrogenation on the other, are described to finally obtain the desired norlignans.

Production of lignans in calluses of Schisandra chinensis.

Calluses were induced from leaves of Schisandra chinensis Baillon (Schisandraceae). Murashige-Skoog (MS) and Woody Plant (WP) media were used for the induction, in full and half strength (1/2 MS or 1/2 WP) salt formulations. Test media were solidified with 0.25% gelrite and supplemented with 2% sucrose and various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), 3-indolebutyric acid (IBA), and 6-benzylaminopurine (BAP). Optimal conditions for callus induction and growth were found to be 1/2 MS medium containing 0.02 mg/l Kin and 0.2 mg/l 2,4-D. Chloroform extracts of all induced calluses contained gomisin A and F as major components. Gomisin A and F contents of calluses that were cultured under the optimal conditions mentioned above were highest compared to the calluses incubated with other combinations of plant hormones and media. Subculture, by repeated transfer of cultured calluses to fresh medium, caused no decrease in the production of gomisin A and F. Optimal conditions for lignan production were found to be 1/2 MS medium supplemented with 0.05 mg/1 Kin and 0.2 mg/l 2,4-D. Under these conditions, gomisin A and gomisin F contents were 0.05 and 0.04% of callus dry weight, respectively.

Pinoresinol-lariciresinol reductases with opposite enantiospecificity determine the enantiomeric composition of lignans in the different organs of Linum usitatissimum L.

Lignans in higher plants represent an ideal class of natural products to be investigated for the origin of stereochemical diversity since chiral lignans occur in pure enantiomeric form as well as in enantiomeric mixtures. Seeds of Linum usitatissimum contain 8S, 8'S-(+)- and 8R, 8'R-(-)-secoisolariciresinol [SS-(+)- and RR-(-)-secoisolariciresinol, respectively] as diglucosides (SS- and RR-secoisolariciresinol diglucosides) whereas aerial parts of flowering L. usitatissimum accumulate only lignans derived from RR-(-)-secoisolariciresinol. Pinoresinol-lariciresinol reductase (PLR) catalyzes two early steps in lignan biosynthesis. Up to now, only a cDNA encoding a PLR ( PLR-LU1) which is enantiospecific for the conversion of 8S, 8'S-(-)-pinoresinol (SS-pinoresinol) via 8S, 8'S-(-)-lariciresinol (SS-lariciresinol) to SS-(+)-secoisolariciresinol was cloned. Here we present the cloning of a cDNA encoding a RR-pinoresinol-RR-lariciresinol reductase ( PLR-LU2) from the leaves of L. usitatissimum which converts only RR-pinoresinol to RR-secoisolariciresinol. In leaves and stems of L. usitatissimum accumulating the 8R, 8'R-enantiomers of lignans, only PLR-LU2 was transcriptionally active. Both PLR-LU1 and PLR-LU2 transcripts were observed in seeds and contribute to the synthesis of SS- and RR-secoisolariciresinol, respectively. Thus, the enantiomeric composition of lignans in the organs of L. usitatissimum appears to be determined by the relative action of two PLRs with opposite enantiospecificities rather than by a single enzyme of low enantiospecificity.

Influence of lignans depletion on murine mammary gland morphology.

Different studies have focused on the effects of phytoestrogens-supplemented diets on mammary gland morphogenesis and breast cancer risk; however, particular dieting behaviors and food choices may result in a reduction of the natural source of phytoestrogens. The evaluation of a reduced phytoestrogens intake effect by depletion without modifying other dietary ingredients is hard. Since lignans, the largest contributors to phytoestrogens intake in Western diets, are metabolized into bioactive compounds by gut bacteria, long-term antibiotic treatments, inducing intestinal microflora disruption, may reduce enterolactone availability. To elucidate the effect of phytoestrogens lack on mammary tissue morphogenesis, female FVB mice were treated with gentamicin or metronidazole/ciprofloxacin from the age of 6 to 7 wk. After 21 wk, enterolactone urine levels were 120.07 +/- 20.5 ng/ml in untreated mice, 30.4 +/- 24.46 ng/ml in metronidazole/ciprofloxacin-treated mice, and 3.29 +/- 4.38 ng/ml in gentamicin-treated mice. Histological analysis revealed no significant alterations of mammary morphology in metronidazole/ciprofloxacin-treated mice, whereas gentamicin-treated mice showed increase of ducts number and duct-tree branching vs. controls. These findings indicate that normal mammary tissue size and shape are maintained even in the presence of low levels of lignans and suggest that only a complete depletion of these compounds induced significant alterations of mammary gland structure.

Production of honokiol and magnolol in suspension cultures of Magnolia dealbata Zucc.

Honokiol and magnolol, important anxiolytic and anti-cancer agents, have been produced in cell-suspension cultures of the endangered Mexican plant Magnolia dealbata Zucc. In vitro cultures of the plant were established, and the accumulation of honokiol and magnolol in callus and cell-suspension cultures was measured. Leaf samples were the best explants for callus establishment and metabolite production, and Murashige and Skoog medium supplemented with 1 mg/L 2, 4-dichlorophenoxyacetic acid and 1 mg/L kinetin yielded 2.3 mg/g of honokiol and 5.9 mg/g of magnolol. Bacterial and fungal contamination was inhibited with a multiple-step tissue sterilization procedure. Oxidation was inhibited with 1 g/L activated charcoal. Cell-suspension batch cultures derived from friable callus obtained from leaves of this species were grown for 30 days in shaker flasks containing Murashige and Skoog medium. Throughout the growth cycle, honokiol and magnolol levels, fresh and dry weight, and sucrose uptake were determined. The effects of carbon source concentration on biomass accumulation and the synthesis of bioactive compounds were studied. By using 3 mL of inocula supplemented with 3% (w/v) sucrose, maximum yields of honokiol (8.1 mg/g) and magnolol (13.4 mg/g) were obtained after 25 days. These yields were 300% and 382%, respectively, of the yields of honokiol and magnolol obtained from field-grown plants.

Aryltetralin-lignan formation in two different cell suspension cultures of Linum album: deoxypodophyllotoxin 6-hydroxylase, a key enzyme for the formation of 6-methoxypodophyllotoxin.

Suspension cultures initiated from two different Linum album seedlings accumulate either podophyllotoxin (PTOX, 2.6 mg/g DW) or 6-methoxypodophyllotoxin (6MPTOX, 5.4 mg/g DW) as main lignans. Two molecules of coniferyl alcohol are dimerized to pinoresinol which is converted via several steps into deoxypodophyllotoxin (DOP) which seems to be the branching point to PTOX or 6MPTOX biosynthesis. DOP is hydroxylated at position 7 to give PTOX by deoxypodophyllotoxin 7-hydroxylase (DOP7H). In contrast, 6MPTOX biosynthesis is achieved by DOP hydroxylation at position 6 to beta-peltatin by the cytochrome P450 enzyme deoxypodophyllotoxin 6-hydroxylase (DOP6H). The following methylation to beta-peltatin-A-methylether is catalyzed by beta-peltatin 6-O-methyltransferase (betaP6OMT) from which 6MPTOX is formed by hydroxylation at position 7 by beta-peltatin-A-methylether 7-hydroxylase (PAM7H). DOP6H and betaP6OMT could be characterized in protein extracts from cell cultures of L. flavum and L. nodiflorum, respectively, and here in L. album for the first time. DOP7H and PAM7H activities could not yet be detected with protein extracts. Experiments of feeding DOP together with inhibitors of cytochrome P450 depending as well as dioxygenase enzymes were performed in order to shed light on the type of DOP7H and PAM7H. Growth parameters and specific activities of enzymes from the phenylpropane as well as the lignan specific biosynthetic pathway were measured during a culture period of 16 days. From the enzymes studied only the DOP6H showed a differential activity sustaining the hypothesis that this enzyme is responsible for the differential lignan accumulation in both cell lines.

[Content comparison of pinoresinol diglucoside in original and reborn bark of Eucommia ulmoides].

OBJECTIVE: To evaluate the reborn barks quality, antihypertensive effective component of pinoresnol diglucoside (PDG) in three times reborn barks were determined. METHODS: A YWG C18 column (10 microm, 250 x 4.6 mm) was used with a mobile phase of methanol-water (30:75) and flow rate of 1.0 (ml/min). The detective wave-length was set at 277nm and the column temperature at room temperature. RESULTS: PDG in the first reborn barks are slightly higher than the original ones, and in the second reborn barks are similar with the barks before girdling (the fist reborn barks), but in the third reborn barks, PDG are much lower than the barks before girdling (the second reborn barks). CONCLUSION: In order to ensure reborn barks quality, we suggest that the girdling bark regeneration can be made two times, the time between the first and the second girdling is not less than five years. PDG in the third reborn barks should be enhanced.

Lignans from cell suspension cultures of Phyllanthus niruri, an Indonesian medicinal plant.

Cell suspension cultures of Phyllanthus niruri were used to study the lignan profiles and biosynthesis. Suspension cultures yielded two lignans: the new cubebin dimethyl ether (1) and urinatetralin (2), a new lignan from P. niruri, but reported earlier from P. urinaria. This is the first report of cell suspension cultures of P. niruri that successfully produce lignans. Feeding 0.5 mM ferulic acid or 0.5 mM caffeic acid, being early precursors of lignan biosynthesis, resulted in an increase up to 0.7 mg g(-1) DW of 1 (control value 0.1 mg g(-1) DW) and up to 0.3 mg g(-1) DW of 2 (control value 0.2 mg g(-1) DW). Comparison of the lignan profiles of cell suspensions, callus cultures, aerial plant parts, roots, and seeds showed significant differences.

Dietary lignins are precursors of mammalian lignans in rats.

The mammalian lignans enterolactone (ENL) and enterodiol, commonly found in human plasma and urine, are phytoestrogens that may contribute to the prevention of breast cancer and coronary heart disease. They are formed by the conversion of dietary precursors such as secoisolariciresinol and matairesinol lignans by the colonic microflora. The identification of lignins, cell-wall polymers structurally related to lignans, as precursors of mammalian lignans is reported here for the first time. In study 1, rats were fed rye or wheat bran (15% diet) for 5 d. Untreated brans and brans extracted with solvents to remove lignans were compared. ENL was estimated in urine samples collected for 24 h by time-resolved fluoroimmunoassay. ENL urinary excretion was reduced from 18.6 to 5.3 nmol/d (n=8; P<0.001) when lignans were removed from rye bran and from 30.5 to 6.2 nmol/d (P<0.001) when they were removed from wheat bran. These results suggest that lignins, embedded in the cell wall and retained in the bran during solvent extraction, account for 26-32% of the ENL formed from cereal brans. In study 2, rats were fed a deuterated synthetic lignin (0.2% diet) together with wheat bran (15%) for 3 d. The detection of deuterated ENL by LC-tandem MS in urine (20 nmol/d) clearly confirms the conversion of lignin into mammalian lignans. More research is warranted to determine the bioavailability of lignins in the human diet.

A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris.

In the roots of Anthriscus sylvestris 12 different lignans were detected. Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A. sylvestris. In the cell suspension cultures, which were initiated for this study, trace amounts of deoxypodophyllotoxin could be detected. With these cell suspension cultures we carried out feeding experiments using deoxypodophyllotoxin, yatein and, anhydropodorhizol. Yatein had a toxic effect on the cell cultures and was, like anhydropodorhizol, not converted into any detectable product. Deoxypodophyllotoxin, in contrast, was converted into podophyllotoxin, yielding significantly higher concentration than measured in whole plants.

Beta-peltatin 6-O-methyltransferase from suspension cultures of Linum nodiflorum.

S-Adenosyl-L-methionine:beta-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme transfers a methyl group from S-adenosyl-L-methionine to the only free OH-group of beta-peltatin in position 6 thus forming beta-peltatin-A methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 degrees C. The enzyme activity is strongly inhibited by MnSO(4), FeCl(3), FeSO(4) and ZnSO(4) as well as S-adenosyl-homocysteine. Mg(2+) and EDTA did not influence the methylation of beta-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and beta-peltatin and apparent K(m)-values of 15 microM and 40 microM, respectively, were determined for these substrates. Substrate inhibition was observed for beta-peltatin. No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:beta-peltatin 6-O-methyltransferase. Highest specific activities of beta-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12.

Mammalian lignan formation in rats fed a wheat bran diet.

The dietary origin of lignan phytoestrogens is still poorly understood more than 20 years after their discovery in human urine. Their level in urine has been associated with the consumption of dietary fiber. This paper reports the study of the excretion of enterolactone, assayed by a time-resolved fluoroimmunoassay, in rats fed a diet supplemented with 15% wheat bran, one of the main sources of fiber in Western countries. Enterolactone excretion regularly increased during the two weeks of the diet to reach a value of 45 nmol/day. The level of excretion also increased upon preadaptation to ferulic acid, structurally related to secoisolariciresinol, an established precursor of enterolactone in flaxseeds, and decreased upon preadaptation to potato starch rich in fiber. These results show that the formation of lignans from wheat bran is influenced by the diet, possibly because of an adaptation of the colonic microflora.

Biosynthesis of podophyllotoxin in Linum album cell cultures.

Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-(13)C]3',4'-dimethoxycinnamic acid, [2-(13)C]3',4'-methylenedioxycinnamic acid (MDCA), [2-(13)C]3',4',5'-trimethoxycinnamic acid, [2-(13)C]sinapic acid, [2-(13)C]- and [2,3-(13)C(2)]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/wo.1007/s00425-002-0834-1.