(1) Extracts from black carrot tissue culture as potent anticancer agents.

(2) Enhanced production of tropane alkaloids in transgenic Scopolia parviflora hairy root cultures over-expressing putrescine N-methyl transferase (PMT) and hyoscyamine-6β-hydroxylase (H6H).

(3) Taxus globosa S. cell lines: initiation, selection and characterization in terms of growth, and of baccatin III and paclitaxel production.

(4) Production of camptothecin in cultures of Chonemorpha grandiflora.

APPLICATION IN PLANT TISSUE CULTURE (CONT.,)

(5) Regeneration, in vitro glycoalkaloids production and evaluation of bioactivity of callus methanolic extract of Solanum tuberosum L.

(6) The influence of medium composition on alkaloid biosynthesis by Penicillium citrinum.

(7) Differential production of tropane alkaloids in hairy roots and in vitro cultured two accessions of Atropa belladonna L. under nitrate treatments.

(8) Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don.

APPLICATION IN PLANT TISSUE CULTURE (CONT.,)

(9) Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

(10) An endophytic taxol-producing fungus from Taxus media, Cladosporium cladosporioides MD2.

(11) Optimized nutrient medium for galanthamine production in Leucojum aestivum L. in vitro shoot system.

(12) Salinity stress enhances production of solasodine in Solanum nigrum L.

APPLICATION IN PLANT TISSUE CULTURE (CONT.,)

(13) Biosynthesis of hyperforin and adhyperforin from amino acid precursors in shoot cultures of Hypericum perforatum.

Plant Foods Hum Nutr. 2013 Sep

Black carrots contain anthocyanins. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin.

Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v).

Extracts applied to (human breast adenocarcinomas), (human colon adenocarcinoma), (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay.

The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38-46% at 6.25 μg/ml concentration for all calli and natural extracts. However, a significantly high IC50 value of 170.13 μg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells.

•In Vitro Cellular & Developmental Biology Plant . Aug2011, Vol. 47 Issue 4, p516-524. 9p.

scopolamine

Scopolia parviflora

Scopolia parviflora adventitious roots were metabolically engineered by co-expression of the two gene putrescine N-methyl transferase (PMT) and hyoscyamine-6β-hydroxylase (H6H) cDNAs with the aid of Agrobacterium rhizogenes.

The transformed roots developed into morphologically distinct S. parviflora PMT1 (Sp PMT1), S. parviflora PMT2 (Sp PMT2), and S. parviflora H6H (Sp H6H) transgenic hairy root lines.

Consequent to the introduction of these key enzyme genes, the production of the alkaloids hyoscyamine and scopolamine was enhanced.

Among the transgenic hairy root lines, Sp PMT2 line possessed the highest growth index.

The treatment of transgenic hairy roots with growth regulators further enhanced the production of scopolamine.

Thus, the results suggest that PMT1, PMT 2, and H6H genes may not only be involved in the metabolic regulation of alkaloid production but also that these genes may play a role in the root development.

Baccatin III

Biocell. 2010 Apr;34(1):1-6.

Taxus globosa

Of the initial six cell lines originating from explants of Taxus globosa, or Mexican yew (stem internode,leaves,meristematic tissue)

Three were selected for their microbial and oxidation resistance, two from leaves and the other from stem internode

A study of their behavior, both in terms of cell growth, and of baccatin III and paclitaxel production, was developed in suspension cultures with an initially standardized biomass (fresh weight 0.23 g/L) using modified Gamborg's B5 medium, and an elicitor (methyl jasmonate), on either the first or seventh day of culture, at several levels (0, 0.1, 1, 10, 100 microM).

In most of the conditions used, the three cell lines showed growth associated baccatin III production.

The cell line from stem internode was the highest producer of baccatin III using 1 microM elicitor, sampling at 10 days (p < or = 0.01, 6.45 mg/L).

This same line also had the highest biomass production (6.85 g/L, p < or = 0.01) at 10 days of culture but at the higher elicitor concentration of 10 microM.

All three cell lines did not produce paclitaxel under experimental conditions used.

camptothecin

Pharmacognosy Res. 2010 Sep;2(5):296-9. doi: 10.4103/0974-8490.72327.

Chonemorpha grandiflora

METHODS: Callus cultures of C. grandiflora were

raised on Murashige and Skoog's medium supplemented with 2, 4-D.

Stem with bark and callus were used for phytochemical analysis mainly the alkaloids.

Detection and identification of camptothecin was carried out using thin-layer chromatography (TLC), high-performance thin-layer chromatography, (HPTLC) and high-performance liquid chromatography (HPLC).

RESULTS: An important anticancer alkaloid,

camptothecin was detected in ethanolic extracts of stem with bark and callus cultures of C. grandiflora. camptothecin content was 0.013 mg/g in stem with bark and 0.003 mg/g in callus.

CONCLUSION: This is the first report on in vivo and in vitro

production of camptothecin in C. grandiflora. Camptothecin is known to occur only in six plant sources so, alternative sources for camptothecin are needed. Thus of C. grandiflora could be anew promising alternative source of camptothecin

Glycoalkaloid

Fitoterapia. 2010 Sep;81(6):600-6. doi: 10.1016/j.fitote.2010.03.001. Epub 2010 Mar 20.

Solanum tuberosum

Callus and differentiated shoots initiated from Solanum tuberosum L. on MS media containing BA, IAA, and Kin. Glycoalkaloids are produced in callus and shoots in concentrations higher than original tubers using HPLC.

Callus methanolic extract had promising anticancer activity with low IC(50) values against human carcinoma cell lines of breast, lymphoplastic leukemia, larynx, liver, cervix, colon, and brain.

IC(50) (microg/mL) were 2.7, 3.7, 6, 6.7, 10, 13.6, and 22.3 respectively.

Antioxidant capacity of the extract (76.4%) performed using ESR. Preliminary screening showed that the extract exhibited in vitro virucidal activity against Herpes simplex.

The extract possessed in vitro schistomicidal and fasciolicidal activity.

clavine ergot alkaloids

quinoline alkaloids

Prikl Biokhim Mikrobiol. 2010 Sep-Oct;46(5):572-6.

The fungus P. citrinum produces secondary metabolites, clavine ergot alkaloids (EA), and quinoline alkaloids quinocitrinines (QA) in medium with various carbon and nitrogen sources and in the presence of iron, copper, and zinc additives.

Mannitol and sucrose are most favorable for EA biosynthesis and mannitol is most favorable for QA.

Maximum alkaloid production is observed on urea.

Iron and copper additives in the medium containing zinc ions stimulated fungal growth but inhibited alkaloid biosynthesis.

The production of these secondary metabolites does not depend on the physiological state of culture, probably due to the constitutive nature of the enzymes involved in biosynthesis of these substances

Z Naturforsch C. 2010 May-Jun;65(5-6):373-9.

Atropa belladonna

Effects of nitrate concentrations (KNO3) on the production of two tropane alkaloids, hyoscyamine and scopolamine.

The growth of aerial parts and roots of two in vitro propagated accessions of Atropa belladonna and hairy roots were studied .

As hairy roots cultures are able to keep a stable production of alkaloids over long periods of subculturing, they are considered as an interesting option for the study of alkaloid biosynthesis.

A hairy roots culture of Atropa belladonna was established by transformation with Agrobacterium rhizogenes strain AR15834.

The results of our study showed that a rise in KNO3 concentration caused a decline in hairy roots growth, and had a remarkable effect on the alkaloid content.

The alkaloid concentrations obtained in the hairy roots were 3-20 times higher than that in the plants at 35 mM of KNO3.

Increasing the nitrate concentration in the medium of hairy roots also improved the hyoscyamine/scopolamine ratio, while it increased the scopolamine/hyoscyamine ratio in the studied plants

vincristine

Nat Prod Res. 2009;23(11):973-81. doi: 10.1080/14786410802131153.

Catharanthus roseus

Dimeric indole alkaloids are used extensively for cancer therapy.

Agrobacterium tumefaciens C58 strain was used for induction of shooty teratoma in Catharanthus roseus using epicotyl and stem node explants.

The transformed nature of shooty teratomas was confirmed by nopaline assay.

Growth kinetics of shooty teratomas depicted maximum growth during 21-24 days of culture.

Dimeric alkaloid vincristine in the transformed cultures was present at a concentration of 0.011 that was tenfold higher compared to untransformed control cultures

Baccatin III

J Plant Physiol. 2009 Nov 15;166(17):1950-4. doi: 10.1016/j.jplph.2009.05.001. Epub 2009 Jul 1.

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii.

The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1microM of l-phenylalanine (6.2gL(-1)).

Addition of 1microM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7microg L(-1)), which was not detected in the untreated control culture.

Supplementation with 100microM of l-phenylalanine together with 100microM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3microg L(-1) (control untreated culture) to 568.2microg L(-1), the highest paclitaxel content detected in the study.

The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8microg L(-1)) was observed when the medium was supplemented with 100microM of the precursor in combination with methyl jasmonate.

Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium

Curr Microbiol. 2009 Sep;59(3):227-32. doi: 10.1007/s00284-008-9270-1. Epub 2009 May 30.

Fermentation processes using taxol-producing fungi other than Taxus spp. may be an alternative way to produce taxol, which is an important antitumor agent used widely in the clinic setting.

In this study, a taxol-producing endophytic fungus strain MD2 was isolated from the inner bark of Taxus media.

Strain MD2 produced taxol when grown in potato dextrose liquid medium.

The fungal taxol-which was analyzed by ultraviolet, high-performance liquid chromatography and mass spectrometry-was shown to be identical to authentic taxol and 10-deacetylbaccatin III.

Further analysis with nuclear magnetic resonance (NMR) spectroscopy to show the chemical structure of the fungal taxol indicated that the fungal taxol produced an NMR spectrum identical to that of authentic taxol.

Strain MD2 was identified as Cladosporium cladosporioides according to morphology of the fungal culture, characteristics of the spores, and analysis of 18S rDNA sequence.

In addition, 10-deacetylbaccatin III-10-O-acetyl transferase gene of C. cladosporioides MD2 was cloned for the first time and was shown to share 99% identity with that of T. x media and 97% identity with that of T. wallichiana var. mairei.

galanthamineLeucojum aestivum

Z Naturforsch C. 2009 Mar-Apr;64(3-4):219-24.

The common effect of NH4+, NO3-, KH2PO4 and sucrose on the biosynthesis of galanthamine by a Leucojum aestivum shootculture was studied.

Polynominal regression models were elaborated for the description of the galanthamine biosynthesis as a consequence of variation of the investigated variables (NH4+ between 0.20 and 0.54 g/L; NO3- between 1.44 and 3.44 g/L; KH2PO4 between 0.10 and 0.24 g/L, and sucrose between 30.00 and 60.00 g/L).

Optimization procedures allowed us to establish the optimal concentrations of the investigated variables and to propose the modified MS nutrient medium, with 4.50 g/L KNO3, 0.89 g/L NH4NO3, 1.25 g/L (NH4)2SO4, 0.10 g/L KH2PO4 and 60 g/L sucrose, for the galanthamine production by a Leucojum aestivum shoot culture.

The proposed modified MS medium provided considerable increase of both the production yield and the relative content of the target alkaloid in the alkaloid mixture.

Chem Pharm Bull (). 2008 Jan;56(1):17-21.

Solanum nigrum

Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot derived leaves) of Solanum nigrum L. were cultured under salinity stress (0-150 mM NaCl) for enhanced production of solasodine, a steroidal alkaloid and an alternative to diosgenin, which is used as a precursor for the commercial production of steroidal drugs.

The role of plant growth regulators and various concentrations of NaCl during in vitro production of solasodine was studied.

The in vitro yield was compared with the yield from leaves of field grown plant.

Solasodine content was maximum (2.39 mg/g dry wt.) in regenerative callus when grown on medium added with 150 mM NaCl; followed by in vitro raised leaf of microshoot.

Quantitative estimation of solasodine was carried out using a new HPTLC method, which is validated for its recovery and precession.

The proposed HPTLC method showed a good linear relationship (r(2)=0.994) in 50-2000 ng/spot concentration ranges.

The data demonstrate that the solasodine production in cultures was growth dependent

Hypericum perforatum

Phytochemistry. 2007 Apr;68(7):1038-45. Epub 2007 Feb 20.

Hyperforin and adhyperforin contribute to the antidepressant effects of Hypericum perforatum.

The involvement of branched-chain amino acids in the biosynthesis of hyperforin and adhyperforin was demonstrated in H. perforatum shoot cultures.

L-[U-(13)C(5)]Valine and L-[U-(13)C(6)]isoleucine, upon administration to the shoot cultures, were incorporated into acyl side chain of hyperforin and adhyperforin, respectively.

Feeding the shoot cultures with unlabelled L-isoleucine at a concentration of 2mM induced a 3.7-fold increase in the production of adhyperforin.

The addition of 3mM L-threonine, a precursor of isoleucine, stimulated a 2.0-fold increase in the accumulation of adhyperforin.

The administration of L-valine at concentrations of 0-5mM had no stimulating effect on the hyperforin production in H. perforatum shoot cultures.

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