Plant Milking Technology-An Innovative and Sustainable Process to Produce Highly Active Extracts from Plant Roots.

We have used an original technology (Plant Milking Technology) based on aeroponic cultivation of plants associated with the gentle recovery of active ingredients from roots. Extraction of bioactive molecules was achieved by soaking the roots, still attached to the living plants, into a nontoxic solvent for a 2 h period. This nondestructive recovery process allows using the same root biomass for successive harvesting dates, in a recyclable way. We have applied this technology to Morus alba L. (mulberry tree), an emblematic tree of the Traditional Chinese Medicine (TCM). Trees were aeroponically grown in large-scale devices (100 m2) and were submitted to nitrogen deprivation to increase the content in active molecules (prenylated flavonoids). The Plant Milking technology applied to Morus alba L. allowed to produce an extract enriched in prenylated compounds (18-fold increase when compared to commercial root extract). Prenylated flavonoids (moracenin A and B, kuwanon C, wittiorumin F, morusin) presented a high affinity for the aged-associated collagenase enzyme, which was confirmed by activity inhibition. In accordance, M. alba extract presents efficient properties to regulate the skin matrisome, which is critical during skin aging. The benefits have been especially confirmed in vivo on wrinkle reduction, in a clinical study that involved aged women. Plant Milking technology is an optimal solution to produce active ingredients from plant roots, including trees, that meet both customer expectations around sustainability, as well as the need for an efficient production system for biotechnologists.

A simple SDS-PAGE protein pattern from pitcher secretions as a new tool to distinguish Nepenthes species (Nepenthaceae).

PREMISE OF THE STUDY: Carnivorous plants have always fascinated scientists because these plants are able to attract, capture, and digest animal prey using their remarkable traps that contain digestive secretions. Nepenthes is one of the largest genera of carnivorous plants, with 120 species described thus far. Despite an outstanding diversity of trap designs, many species are often confused with each other and remain difficult to classify because they resemble pitchers or of the occurrence of interspecific hybrids. METHODS: Here, we propose a new method to easily distinguish Nepenthes species based on a SDS PAGE protein pattern analysis of their pitcher secretions. Intraspecific comparisons were performed among specimens growing in different environmental conditions to ascertain the robustness of this method. KEY RESULTS: Our results show that, at the juvenile stage and in the absence of prey in the pitcher, an examined species is characterized by a specific and stable profile, whatever the environmental conditions. CONCLUSIONS: The method we describe here can be used as a reliable tool to easily distinguish between Nepenthes species and to help with potential identification based on the species-specific protein pattern of their pitcher secretions, which is complementary to the monograph information.

A rapid and efficient method for isolating high quality DNA from leaves of carnivorous plants from the Drosera genus.

Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 µg/g of fresh weight to 35.29 µg/g depending of the species of Drosera, with a A260/A280 ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h.

Screening of DHFR-binding drugs by MALDI-TOFMS.

The class of antimetabolite chemotherapeutical agents has been used to treat cancers in humans for almost 50 years and gives significant results by binding dihydrofolate reductase (DHFR), a key enzyme in DNA synthesis. Therefore, finding new active compounds inhibiting DNA synthesis through their binding to DHFR is of prime interest. The aim of this work is to describe a protocol designed to study the binding of compounds to DHFR. This screening protocol involves matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) detection of target-bound compounds. Firstly, a screening protocol is developed and proves to be a simple, fast, and specific method to characterize the binding capability of a compound. Secondly, the possibility of determining the relative affinities of DHFR-binding compounds by comparing MALDI-TOFMS data is discussed. A ratio is calculated for a compound X such as R(X) = A.I.(denaturation)(X)/A.I.(direct)(X) (where AI(direct) and AI(denaturation) are the average absolute intensities of a binding compound X before and after denaturation of DHFR). It is shown that by using this protocol, one can characterize the strength of the binding of different compounds. These two strategies are then applied to screen green tea (Camellia sinensis) extracts for DHFR-binding compounds, and epigallocatechin gallate is shown to be an active compound with a relative affinity between those of pyrimethamine and methotrexate.

Tubulin-binding drug screening by MALDI-TOFMS.

Despite a large amount of drugs available to treat cancer, none is totally satisfactory with respect to its tolerance or side effects. It is very important to discover new compounds that exhibit specific features such as binding to proteic targets. Given the clinical successes of the poisons of the mitotic spindle chemotherapeutic agent class, it is often considered that tubulin represents one of the best cancer targets identified so far, and it seems likely that discovering new drugs of this class will significantly improve the range of active chemotherapeutic agents. The aim of this work is to present the new screening test that has been developed in our laboratory in order to study the binding of compounds to tubulin. We have developed a screening protocol involving three sampling strategies before the MALDI-TOFMS analysis. The three strategies give very accurate and reproducible results and could therefore possibly be used in screening campaigns. We have also proved that no unspecific binding can provide a loss of specificity of the test. Our protocol presents all the requirements for being a useful tool to screen the binding of compounds to tubulin.