Cytotoxic Lignans Research Articles

Antimicrobial activity and cytotoxicity of endophytic fungi associated with four medicinal plants from Sudan

In this study four medicinal plants, used in Sudan to cure different types of health problems, were selected for isolation of endophytic fungi, fungi reside within the plant host tissues without causing disease symptoms. A total of 20 distinct endophytic fungi species were isolated from surface- sterilized parts of the plants, Kigelia africana, Cymbopogon proximus, Balanites egyptiaca and Solenostemma argel. Nine of the isolated fungi were identified according to their microscopic and macroscopic characteristics. The remaining strains which did not have spores or have sterile mycelium were given codes as unidentified species (UID). The ethyl acetate extracts of the pure isolated endophytic fungi were prepared from fungal material and culture media. Antimicrobial activity using agar diffusion well against different pathogenic microbes at 500 µg/ml concentration and MTT cytotoxicity bioassay using African green monkey kidney normal cell line (vero cell line) were performed for all extracts. The extracts were then screened for the presence of Taxol, a diterpene anticancer drug, using different chromatographic and spectroscopic techniques. The antimicrobial activity against the most common pathogenic bacteria and fungi revealed significant activity with inhibition zone range between 14- 37 mm compare to antimicrobial controls 17- 26 mm at 40µg/ml. The cytotoxicity assay results showed that the IC50 values were quite diverse, ranging from 3.5 μg/ml to more than 1000 μg/ml, whereas that of the positive control podophyllotoxin, a wellknown cytotoxic lignan, was 2.72 μg/ml. Taxol was detected in reference to standard one from the extract of unidentified fungi No. 10 of Kigelia africana,. Future work will focus on the bioassay guidedfractionation of potent bioactive extracts to isolate the active compounds responsible for their activities. Improvement of culture techniques and the application of genetic engineering to increase taxol production will be carried out, followed by isolation of pure Taxol from the active crude extract.

Anwulignan from the fruits of Schisandra chinensis and its cytotoxicity against human cancer cell lines

Background: The fruits of Schisandra chinensis have been used traditionally as the medicinal materials in East Asia. Their extracts have been reported to be cytotoxic to several cancer cells and phytochemical research has identified numerous cytotoxic lignans. Objectives: We reviewed for the effects of cytotoxic lignans against various human cancer cells. Materials and Methods: Solvent extraction of fruits in aqueous methanol and fractionation of the extract into H2O, n-BuOH, and EtOAc fractions (Fr) were carried out. Column chromatography (CC) of the non-polar EtOAc Fr, which indicated cytotoxicity to cancer cells, was performed to isolate the cytotoxic lignin. They were identified on the basis of the intensive spectroscopic interpretation of IR, 1D-, 2D-NMR, and mass spectrometry data. The cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell staining using Hoechst 32258 and propidium iodide. Results: Anwulignan was isolated and identified as a cytotoxic principal component. The IC50 value was calculated to be 22.01 ± 1.87, 156.04 ± 6.71, and 32.68 ± 2.21 μM for human stomach adenocarcinoma, human colon cancer (HT29), and human cervical cancer cells, respectively. Conclusion: Solvent extraction, systematic fractionation, and repeated CC for S. chinensis fruits led to the isolation of a lignin identified to be anwulignan based on various spectroscopic analyses. Anwulignan demonstrated very high cytotoxicity to most human cancer cell lines, and cell death was induced by apoptosis.

Cytotoxic compounds from the leaves and stems of the endemic Thai plant Mitrephora sirikitiae

Context Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders (Annonaceae) is a plant endemic to Thailand. Its constituents and their biological activities are unknown. Objective Isolation and identification of the compounds in the leaves and stems of M. sirikitiae and determination of their cytotoxicity. Materials and methods Methanol extracts of the leaves and stems of M. sirikitiae were separated by chromatography, and spectroscopic methods were used to determine the structures of the components. The cytotoxicity of the extracts and pure compounds was evaluated using the sulforhodamine B assay with several cell lines. The cells were treated with the compounds at concentrations of 0.16–20 µg/mL for 48 or 72 h. Results The investigation of the extracts of M. sirikitiae leaves and stems resulted in the isolation of a new lignan, mitrephoran, and 15 known compounds. Among these compounds, 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, ciliaric acid, 6-methoxymarcanine A, and stepharanine were isolated from this genus for the first time. The alkaloids liriodenine and oxoputerine exhibited strong cytotoxicity against all tested cells (IC50 values of 6.59–11.02 µM). In contrast, magnone A, 3′,4-O-dimethylcedrusin, and 6-methoxymarcanine A inhibited the growth of some of the tested cells (IC50 values of 2.03–19.73 µM). Magnone A and 6-methoxymarcanine A showed low toxicity for Hek 293 cells (IC50 >20 µM). Discussion and conclusions M. sirikitiae is a source of cytotoxic lignans and alkaloids. Among the cytotoxic compounds, magnone A and 6-methoxymarcanine A are potentially useful lead compounds for the further development of anticancer agents because of their selective inhibitory effects on cancer cell lines.

Increase in cytotoxic lignans production after smut infection in sugar cane plants

Smut infection alters the transcription of dirigent proteins (DIR) by sugarcane plants. Here, we show that these alterations are associated to an elevated production of cytotoxic lignans. Smut-resistant sugarcane varieties display a fivefold increase in pinoresinol and also produce elevated amounts of secoisolariciresinol. Conversely, smut-sensitive varieties do not produce pinoresinol or secoisolariciresinol upon infection, synthesizing instead small amounts of matairesinol. Our data indicate that commercial pinoresinol and secoisolariciresinol seem to prevent smut teliospore germination and sporidia release from sprouted teliospores. Consistently, we observed abundant morphological alterations of sporidia incubated in the presence of these lignans. However, commercial lignans do not block the development of the pathogen in a definitive way. Additional experiments demonstrate that only the extracts from healthy or smut-exposed resistant plants inhibit sporidia growth in vitro, indicating that a specific mixture of lignans from resistant plants is necessary to constitute an effective defense mechanism.

Dereplication of podophyllotoxin and related cytotoxic lignans in Hyptis verticillata by ultra-high-performance liquid chromatography tandem mass spectrometry.

Hyptis verticillata Jacq. (Lamiaceae) is a Mexican medicinal plant for the treatment of skin infections and illness affecting the respiratory and gastrointestinal systems. To associate the efficient resolution provided by ultra-high-performance liquid chromatography combined to the accuracy of a hybrid Fourier-transform (FT) mass spectrometer in order to dereplicate podophyllotoxin-type lignans in a plant extract. An ultra-high-performance liquid chromatography-photodiode array-high resolution electrospray ionisation tandem mass spectrometry (UHPLC-PDA-HRESI-MS/MS) method was applied in an Orbitrap hybrid FT spectrometer for dereplication of podophyllotoxin and related cytotoxic lignans in wild bushmint. This procedure included high-resolution mass values for positively charged ions [M+H]+ and [M+NH4 ]+ , MS/MS data, and comparison of UV maxima and retention times with pure compounds. Podophyllotoxin in addition to seven aryltetralins, four arylnaphthalenes, and one dibenzylbutyrolactone were dereplicated from the methanol extract in a short-time analysis (5min). 4'-O-Demethyl-dehydro-deoxypodophyllotoxin was identified as a new natural product. The applied UHPLC-MS/MS dereplication method is suitable for a rapid analysis of podophyllotoxin-type lignans and the resulting chemical fingerprinting could be valuable in quality control of herbal drugs and their phytopharmaceuticals.

Synthesis and evaluation of cytotoxic effects of hanultarin and its derivatives

One of the known cytotoxic lignans is (-)-1-O-feruloyl-secoisolariciresinol designated as hanultarin, which was isolated from the seeds of Trichosanthes kirilowii. In this Letter, we described the first synthesis of 1-O-feruloyl-secoisolariciresinol, 1,4-O-diferuloyl-secoisolariceresinol and their analogues. The cytotoxicities of these compounds were evaluated against several cancer cell lines. Interestingly, we found that the feruloyl diester derivative of secoisolariciresinol was the most active cytotoxic compound against all the cancer cells tested in this experiment. The IC(50) values of the1,4-O-diferuloyl-secoisolariceresinol were in the range of 7.1-9.8μM except one cell line. In considering that both ferulic acid and secoisolariciresinol are commonly found in many plants and have no cytotoxicity, this finding is remarkable in that simple covalent bonds between the ferulic acid and secoisolariciresinol cause a cytotoxic effect.

Phenolic compound production by Larrea divaricata Cav. plant cell cultures and effect of precursor feeding

This paper summarizes progress made in using Larrea divaricata Cav. cell cultures for the production of the cytotoxic lignan nordihydroguaiaretic acid (NDGA) and the phenylpropanoids p-coumaric acid, ferulic acid and sinapyl alcohol. In order to improve the biomass formation and production of these phenolic compounds, four precursors ( l-phenylalanine, cinnamic acid, ferulic acid, and sinapic acid) were fed to L. divaricata cell cultures. Feeding l-phenylalanine (0.5, 1 and 3 mM) resulted in an increase of NDGA of up to 301.35 ± 1.19, 285.23 ± 28.44 and 190.53 ± 19.50 μg/g DW. The addition of 0.5 μM cinnamic acid enhanced the cell culture growth, but not the NDGA production. When cinnamic acid (1 and 1.5 μM), ferulic acid (0.1, 0.5 and 1 mM) and sinapic acid (0.1, 0.5 and 1 mM) were added, the media became too toxic for the cells, and the production of NDGA and the phenylpropanoids was suppressed. The content of p-coumaric acid in the medium supplemented with 3 mM l-phenylalanine increased from 47.43 ± 9.01 to 1157.28 ± 47.79 μg/g DW, whereas the sinapyl alcohol content was not affected by any of the precursors tested, presenting similar values to the control medium.

CYTOTOXIC TRITERPENOIDS FROM Cornus kousa FRUITS

Cornus kousa Burg. (Cornaceae) is a small deciduous tree characterized by brilliant, colorful, and attractive flowers and fruits. It is widely distributed in the northern hemisphere, such as eastern Asia and eastern and northern parts of the United States. C. kousa is widely cultivated for ornamental purposes in eastern Asia. The fruits of this plant have been used as a hemostatic agent and for the treatment of diarrhea in Korean traditional medicine [1]. Additionally, immuno-regulatory properties for this fruit extract have been reported [2]. Some chemical constituents such as isoquercitrin, gallic acid, tannin, phenolics, and flavonoids [3] have been reported to be present in the leaves of C. kousa. Also, our previous phytochemical research on the fruits of this plant demonstrated the presence of steroids [4], cytotoxic lignans [5], and flavonoids [6]. Bioactivity-guided fractionation led to the isolation of cytotoxic triterpenoids from the fruits of C. kousa. When the methanol extracts of C. kousa were developed on silica gel TLC, pink and purple spots appeared after spraying with 10% H2SO4 solution and heating, indicating the presence of triterpenoids in the extracts. The methanol extract was fractionated into an EtOAc layer, an n-BuOH layer, and a H2O layer through solvent fractionation. The repeated silica gel (SiO2), octadecyl silica gel (ODS), and Sephadex LH-20 column chromatographies (c.c.) of EtOAc fractions yielded ten triterpenoids, compounds 1–10. The chemical structures of the triterpenoids were determined from spectroscopic analysis, including EI/FAB-MS, IR, PMR, 13C NMR, DEPT, and 2D NMR (COSY, HSQC, HMBC). The compounds were revealed to be ursolic acid (1) [7], lupeol (2) [8], taraxasterol (3) [9], betulinic acid (4) [10], betulinic aldehyde (5) [11], ursolic aldehyde (6) [7], arjunolic acid (7) [12], tormentic acid (8) [13], asiatic acid (9) [14], and 19 -hydroxyasiatic acid (10) [15]. Although these compounds were previously isolated from other plants, this is the first report of their isolation from C. kousa. All isolated compounds were evaluated for their cytotoxicity against human colon carcinoma (HCT-116), human breast carcinoma (MCF-7), human cervix carcinoma (HeLa), human ovary carcinoma (SK-OV-3), and human melanoma (SK-MEL-5) using the MTT assay. As shown in Table 2, some compounds inhibited the growth of human cancer cell lines. In conclusion, the findings of this study suggest that the methanolic extract of C. kousa, as well as its isolated triterpenoids, may prove useful for the treatment of human cancer. Plant Materials. The fruits of Cornus kousa Burg. (Cornaceae) were collected at the experimental farm at Kyung Hee University in August 2006 and identified by Prof. Dae-Keun Kim, College of Pharmacy, Woosuk University, Jeonju, Korea. A voucher specimen (KHU060907) was reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.

Hanultarin, a cytotoxic lignan as an inhibitor of actin cytoskeleton polymerization from the seeds of Trichosanthes kirilowii

Bioactivity-directed fractionation of extracts from the seeds of Trichosanthes kirilowii led to the isolation of (−)-1- O-feruloylsecoisolariciresinol (2), named hanultarin, In addition, four known lignans were also isolated, including (−)-secoisolariciresinol (1), 1,4- O-diferuloylsecoisolariciresinol (3), (−)-pinoresinol (4), and 4-ketopinoresinol (5). Their structures were elucidated on the basis of spectroscopic data. Compounds 2 and 3 exhibited strong cytotoxic effects against human lung carcinoma A549 cells, melanoma SK-Mel-2 cells, and mouse skin melanoma B16F1 cells with IC 50 ranges of 3–13 μg/mL. Compound 2 showed an inhibitory effect on the polymerization of the actin cytoskeleton in normal epidermal keratinocyte (HaCaT cells), suggesting unique biological properties of compound 2 compared to those of the other isolates.

Sesquiterpenoids and Cytotoxic Lignans from the Bark of Libocedrus chevalieri

Investigation of an EtOAc extract of the bark of Libocedrus chevalieri led to the isolation of a new cytotoxic lignan, 5-methoxy-4-epipodophyllotoxin (1), and three known podophyllotoxin analogues, 5-methoxypodophyllotoxin, 5-methoxypodophyllotoxin-4-O-beta-D-glucoside, and podophyllotoxin-4-O-beta-D-glucoside. Six sesquiterpenoids and a diterpenoid were also obtained. Of these, compounds 2-4 are new sesquiterpenoids, named libocedrines A-C, and 3beta-hydroxyilicic alcohol was isolated for the first time from a higher plant. Structures of the new compounds were determined on the basis of spectroscopic methods. Cytotoxicity of the isolated compounds against KB and L1210 cells and their effects on tubulin assembly were evaluated.

A novel cytotoxic lignan from Seseli annuum L.

A new tetrahydrofuranoid lignan named seselinone was isolated from the aerial parts of Seseli annuum, together with three known lignans (eudesmin, magnone A and hernone) and prenylated coumarin umbelliprenin. Seselinone and eudesmin showed cytotoxic activity against C6 rat glioma cell cultures.

Structural simplification of bioactive natural products with multicomponent synthesis: Dihydropyridopyrazole analogues of podophyllotoxin

A three-component condensation of 5-amino-3-methylpyrazole, tetronic acid, and various aromatic, heteroaromatic, and aliphatic aldehydes leads to the formation of dihydropyridopyrazole analogues of a cytotoxic lignan podophyllotoxin. This new heterocyclic scaffold-based library allows a drastic reduction of the structural complexity of the natural product with the retention of its potent cytotoxic properties. Similarly to podophyllotoxin, the presented analogues induce apoptosis in Jurkat cells.

Cytotoxic Compounds from Polygala vulgaris.

To search for antitumor agents from plants, we studied Polygala vulgaris since cytotoxic lignans are known to occur in some Polygala species. Preliminary data on plant petrol ether, chloroform, and methanol extracts from the roots and aerial parts, showed in vitro cytotoxic activity against the solid tumor LoVo cell line. Fractionation of the active extracts led to the isolation of three new compounds, a derivative of aucuparine and two xanthones, as well as a known methylsinapate. All compounds were tested for in vitro cytotoxic activity using two cell lines, LoVo and its strain, which express resistance to common antitumor agents.

Bioactivity of secoiridoid glycosides from Centaurium erythraea.

As part of our on-going search for bioactive compounds from Scottish plants, two secoiridoid glycosides, swertiamarin and sweroside, have been isolated from the aerial parts of Centaurium erythraea Rafn (Family: Gentianaceae) by reversed-phase preparative HPLC coupled with a photo-diode-array detector. The structures of these compounds were elucidated unambiguously by UV, FABMS and extensive 1D and 2D NMR spectroscopic analyses and also by comparing experimental data with literature data. Antibacterial, free radical scavenging activities and general toxicity of these glycosides have been assessed. Both compounds inhibited the growth of Bacillus cereus, Bacillus subtilis, Citrobacter freundii and Escherichia coli. While swertiamarin was also active against Proteus mirabilis and Serratia marcescens, sweroside inhibited the growth of Staphylococcus epidermidis. Swertiamarin and sweroside exhibited significant general toxicity in brine shrimp lethality bioassay and the LD50 values were 8.0 microg/ml and 34 microg/ml, respectively, whereas that of the positive control podophyllotoxin, a well known cytotoxic lignan, was 2.79 microg/ml. Chemotaxonomic implications of these compounds in the family Gentianaceae have also been discussed briefly.

Cytotoxic Compounds from Polygala vulgaris.

To search for antitumor agents from plants, we studied Polygala vulgaris since cytotoxic lignans are known to occur in some Polygala species. Preliminary data on plant petrol ether, chloroform, and methanol extracts from the roots and aerial parts, showed in vitro cytotoxic activity against the solid tumor LoVo cell line. Fractionation of the active extracts led to the isolation of three new compounds, a derivative of aucuparine and two xanthones, as well as a known methylsinapate. All compounds were tested for in vitro cytotoxic activity using two cell lines, LoVo and its strain, which express resistance to common antitumor agents.

Aspects of cytotoxic lignan biosynthesis in suspension cultures of Linum nodiflorum

Plant cell suspension cultures of Linum flavum, Linum nodiflorum and Linum album have been used to characterize the growth and production of cytotoxic lignans as well as to study the biosynthesis of these lignans. A cell culture of Linum nodiflorum accumulated up to 1.7% of the cell dry weight as 6-methoxypodophyllotoxin in only nine days of cultivation. The biosynthesis of podophyllotoxin and 6-methoxypodophyllotoxin follows the formation of the first aryltetralin lignan deoxypodophyllotoxin. Hydroxylation in position 7 by deoxypodophyllotoxin 7-hydroxylase leads to podophyllotoxin. Hydroxylation in position 6 by the cytochrome P450 monooxygenase deoxypodophyllotoxin 6-hydroxylase yields β-peltatin which is further methylated by S-adenosylmethionine:β-peltatin 6-O-methyltransferase to β-peltatin-A methylether and then hydroxylated to 6-methoxypodophyllotoxin. Both, podophyllotoxin as well as 6-methoxypodophyllotoxin are stored as glucosides in the vacuole. Certain enzymes of these transformations have been isolated and characterized from Linum cell cultures.

Deoxypodophyllotoxin 6-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum flavum involved in the biosynthesis of cytotoxic lignans.

Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrahydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degrees C. Deoxypodophyllotoxin is specifically accepted with an apparent Km of 20 microM and a saturation concentration of 200 microM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 microM NADPH; the apparent Km for this substrate is 36 microM.

The production of cytotoxic lignans by plant cell cultures.

Cytotoxic lignans derived from podophyllotoxin are currently used in cancer chemotherapy. Podophyllotoxin for semi-synthetic derivatization is isolated from the rhizomes of Podophyllum plants growing wild, some of which are counted as endangered species. An alternative source for podophyllotoxin or related lignans may in future be cell cultures derived from different plant species, such as Podophyllum spp or Linum spp. These cell cultures were shown to accumulate considerable amounts of podophyllotoxin or 5-methoxypodophyllotoxin. Optimization of the cell cultivation regime might lead to a renewable source of cytotoxic lignans for medicinal uses. This Mini-Review summarizes the attempts to establish plant cell cultures for the production of podophyllotoxin and related lignans and their optimization towards high levels of these target compounds. It also summarizes the results of studies on the biosynthesis of podophyllotoxin and 5-methoxypodophyllotoxin.

Glucosylation of cyclodextrin-complexed podophyllotoxin by cell cultures of Linum flavum L.

The glucosylation of the cytotoxic lignan podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: beta-cyclodextrin, gamma-cyclodextrin, dimethyl-beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin were used to improve the solubility of podophyllotoxin by complexation. Dimethyl-beta-cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM, using a podophyllotoxin/cyclodextrin ratio of 1:1. Growth parameters of the cell suspensions were not affected neither by the addition of cyclodextrins alone, nor when complexed podophyllotoxin was dissolved in the medium. The complexed lignan disappeared rapidly from the culture medium, within 24 h, under all experimental conditions. Almost simultaneously, between 73 and 100% of detectable podophyllotoxin was bioconverted into podophyllotoxin-beta-D-glucoside. A maximal bioconversion rate of 0.51 mmol l-1 suspension day-1 was calculated for the L. flavum cells growing in a medium which included the podophyllotoxin/dimethyl-beta-cyclodextrin complex at a final concentration of 1.35 mM.

The biotechnological production of podophyllotoxin and related cytotoxic lignans by plant cell cultures

The biotechnological production of podophyllotoxin and related cytotoxic lignans by plant cell cultures