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,3β,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.

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-β-D-glucopyranosylcampesterol (1) and 6-O-palmitoyl-3-O-β-D-glucopyranosyl-β-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 OPEN ACCESSMolecules 2014, 19 8680 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.

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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.

Background: Stanhopea hernandezii was collected from natural habitat in Mexico for its beautiful fragrant flowers. Biotechnological strategies of propagation may satisfy the market demand and are useful for conservation programs. Hypothesis: Vigorous seedlings of S. hernandezii can be produced in vitro by asymbiotic seed germination techniques and the addition of chitosan to the culture medium in the temporary immersion system (RITA®) and in semi-solid medium systems. Methods: The first step was the in vitro germination of seeds obtained from a mature capsule of wild plants, followed by multiplication via adventitious protocorm induction known as protocorm-like bodies, using plant growth regulators. For this purpose, we utilized Murashige and Skoog (MS) basal medium amended with 0.5 mg/L ?-Naphthaleneacetic acid, combined with different concentrations of 6-Benzylaminepurine (1, 3, and 5 mg/L). The following step comprised the growth and development of protocorms to obtain plantlets in RITA® flasks containing 250 mL of liquid MS medium combined or not with different chitosan concentrations (5, 10, 15, 20, and 25 mg/L). Results: The results showed that media supplemented with 5, 10, and 15 mg/L chitosan concentrations enabled the obtaining of a larger biomass with a range of 40-48 seedlings/RITA® and an average height of 13 mm. The last step was the development from seedlings into plantlets, the latter being, vigorous and achieving up to 100 % survival after 12 weeks of ex vitro cultivation. Conclusion: This paper describes an efficient process of asymbiotic germination and mass propagation of S. hernandezii, a vulnerable orchid species endemic to Mexico.

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 β-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.

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