High accumulation of tilianin in in-vitro cultures of Agastache mexicana and its potential vasorelaxant action.

Agastache mexicana has gained importance during the last decade as a natural source of bioactive compounds, mainly due to the antidiabetic, antihyperlipidemic, and vasorelaxant effects derived from its flavonoids, particularly tilianin. The goal of this work was to evaluate the production of tilianin during the in-vitro process of morphogenesis leading to plant regeneration and to investigate the vasorelaxant activity of its methanolic extracts. The cultures were established from nodal segments and leaf explants, inoculated on Murashige and Skoog (MS) media supplemented with various concentrations of benzyl aminopurine (BAP) alone or in combination with 2,4-Dichlorophenoxyacetic acid (2,4-D). Callus inductions were obtained in all treatments from both types of explants, but the presence of auxin was essential. Maximal shoot multiplication and elongation was achieved with 0.1 mg/l 2,4-D and 1.0 mg/l BAP from nodal- segment explants. Shoots were rooted in 75% MS medium and the plantlets were transferred to a greenhouse with 33% average survival. Analysis of tilianin production in methanolic extracts from calli (0.15-2.01 ± 0.06 mg/g dry weight), shoots (4.45 ± 0.01 mg/g DW), and whole plants (9.77 ± 0.02 mg/g DW) derived from in-vitro cultured nodal segments reveals that tilianin accumulation is associated with high cell differentiation and morphogenetic response to the plant-growth regulators. All of the extracts showed strong vasorelaxant activity, as compared to those of wild plant extracts. These results indicate that plant-tissue cultures of A. mexicana possess vast potential as a source of tilianin and other bioactive compounds.

A Cytotoxic and Anti-inflammatory Campesterol Derivative from Genetically Transformed Hairy Roots of Lopezia racemosa Cav. (Onagraceae).

The genetically transformed hairy root line LRT 7.31 obtained by infecting leaf explants of Lopezia racemosa Cav with the Agrobacterium rhizogenes strain ATCC15834/pTDT, was evaluated to identify the anti-inflammatory and cytotoxic compounds reported previously for the wild plant. After several subcultures of the LRT 7.31 line, the bio-guided fractionation of the dichloromethane-methanol (1:1) extract obtained from dry biomass afforded a fraction that showed important in vivo anti-inflammatory, and in vitro cytotoxic activities. Chemical separation of the active fraction allowed us to identify the triterpenes ursolic (1) and oleanolic (2) acids, and (23R)-2α,3ß,23,28-tetrahydroxy-14,15-dehydrocampesterol (3) as the anti-inflammatory principles of the active fraction. A new molecule 3 was characterized by spectroscopic analysis of its tetraacetate derivative 3a. This compound was not described in previous reports of callus cultures, in vitro germinated seedlings and wild plant extracts of whole L. racemosa plants. The anti-inflammatory and cytotoxic activities displayed by the fraction are associated to the presence of compounds 1-3. The present study reports the obtaining of the transformed hairy roots, the bioguided isolation of the new molecule 3, and its structure characterization.

Production of the anti-inflammatory compound 6-O-palmitoyl-3-O-ß-D-glucopyranosylcampesterol by Callus cultures of Lopezia racemosa Cav. (Onagraceae).

Lopezia racemosa Cav. is a plant used in Mexican traditional medicine to heal inflammatory diseases. From this plant we isolated the novel compound 6-O-palmitoyl- 3-O-ß-D-glucopyranosylcampesterol (1) and 6-O-palmitoyl-3-O-ß-D-glucopyranosyl-ß-sitosterol (2), previously reported to have cytotoxic activity on several cancer cell lines. We evaluated the anti-inflammatory activity of 1 in vivo by mouse ear edema induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and 57.14% inhibition was observed. The aim of our study was to obtain callus cultures derived from this plant species with the ability to produce the compounds of interest. Callus cultures were initiated on MS basal medium amended with variable amounts of naphthaleneacetic acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D), combined or not with 6-benzylaminopurine (BAP). Ten treatments with these growth regulators were carried out, using in vitro germinated seedlings as source of three different explants: hypocotyl, stem node, and leaf. Highest yield of 1 was observed on callus derived from leaf explants growing in medium containing 1.0 mg/L 2,4-D and 0.5 mg/L BAP. Selected callus lines produced less 1 than wild plants but the in vitro cultured seedlings showed higher production. So we conclude that it could be attractive to further investigate their metabolic potential.

Production of Two Isomers of Sphaeralcic Acid in Hairy Roots from Sphaeralcea angustifolia.

The Sphaeralcea angustifolia plant is used as an anti-inflammatory and gastrointestinal protector in Mexican traditional medicine. The immunomodulatory and anti-inflammatory effects have been attributed to scopoletin (1), tomentin (2), and sphaeralcic acid (3) isolated from cells in suspension cultures and identified in the aerial tissues of the wild plant. The hairy roots from S. angustifolia established by infecting internodes with Agrobacterium rhizogenes were explored to produce active compounds based on biosynthetic stability and their capacity to produce new compounds. Chemical analysis was resumed after 3 years in these transformed roots, SaTRN12.2 (line 1) produced scopoletin (0.0022 mg g-1) and sphaeralcic acid (0.22 mg g-1); instead, the SaTRN7.1 (line 2) only produced sphaeralcic acid (3.07 mg g-1). The sphaeralcic acid content was 85-fold higher than that reported for the cells in the suspension cultivated into flakes, and it was similar when the cells in suspension were cultivated in a stirring tank under nitrate restriction. Moreover, both hairy root lines produced stigmasterol (4) and ß-sitosterol (5), as well as two new naphthoic derivates: iso-sphaeralcic acid (6) and 8-methyl-iso-sphaeralcic acid (7), which turned out to be isomers of sphaeralcic acid (3) and have not been reported. The dichloromethane-methanol extract from SaTRN7.1 hairy root line had a gastroprotective effect on an ulcer model in mice induced with ethanol.

Spontaneous Regeneration of Plantlets Derived from Hairy Root Cultures of Lopezia racemosa and the Cytotoxic Activity of Their Organic Extracts.

A histological analysis was performed with the aim of elucidating the spontaneous regeneration process of the hairy root lines LRT 2.3 and LRT 6.4, derived from Lopezia racemosa leaf explants and genetically transformed with the Agrobacterium rhizogenes strain ATCC15834/pTDT. The analysis showed both lines regenerate via indirect somatic embryogenesis; LRT 6.4 also regenerated by direct organogenesis. The morphogenic characteristics of the regenerated plantlets from both lines showed the typical characteristics, described previously, including a higher number of axillary shoot formation, short internodes, and plagiotropic roots compared with wild-type seedlings. The regeneration process occurred without the addition of plant growth regulators and was linked to the sucrose concentration in the culture medium. Reducing the sucrose concentration from 3% to 2%, 1%, and 0.5% increased the regeneration rate in LRT 6.4; the effect was less pronounced in LRT 2.3. The cytotoxic activity of different organic extracts obtained from roots and shoots were evaluated in the cancer cell lines HeLa (cervical carcinoma), HCT-15 (colon adenocarcinoma), and OVCAR (ovary carcinoma). The hexane and dichloromethane extracts from roots of both lines showed cytotoxic activity against the HeLa cell line. Only the dichloromethane extract from the roots of PLRT 2.3 showed cytotoxic activity against the OVCAR cell line. None of the methanol extracts showed cytotoxic activity, nor the shoot extracts from any solvent.

Neuropharmacological and neuroprotective activities of some metabolites produced by cell suspension culture of Waltheria americana Linn.

Waltheria americana is a plant used in Mexican traditional medicine to treat some nervous system disorders. The aims of the present study were to isolate and determine the neuropharmacological and neurprotective activities of metabolites produced by a cell suspension culture of Waltheria americana. Submerged cultivation of W. americana cells provided biomass. A methanol-soluble extract (WAsc) was obtained from biomass. WAsc was fractionated yielding the chromatographic fractions 4WAsc-H2O and WAsc-CH2Cl2. For the determination of anticonvulsant activity in vivo, seizures were induced in mice by pentylenetetrazol (PTZ). Neuropharmacological activities (release of gamma amino butyric acid (GABA) and neuroprotection) of chromatographic fractions were determined by in vitro histological analysis of brain sections of mice post mortem. Fraction 4WAsc-H2O (containing saccharides) did not produce neuronal damage, neurodegeneration, interstitial tissue edema, astrocytic activation, nor cell death. Pretreatment of animals with 4WAsc-H2O and WAsc-CH2Cl2 from W. americana cell suspensions induced an increase in: GABA release, seizure latency, survival time, neuroprotection, and a decrease in the degree of severity of tonic/tonic-clonic convulsions, preventing PTZ-induced death of up to 100% of animals of study. Bioactive compounds produced in suspension cell culture of W. americana produce neuroprotective and neuropharmacological activities associated with the GABAergic neurotransmission system.