Isolation, purification and characterisation of the biological activity of vegetable active substances.

Subsidised by ICEX Spain Exportation and Investment (España Exportación e Inversión) in the 2013 Invest in Spain invitation.

Code
1/2012-78
Started
January 01, 2013
Ended
December 31, 2013

Summary

The objective of this project was to isolate and purify biologically active compounds present in aloe, hemp and olive plants, due to their well-known therapeutic and healthy properties.

Compounds in Aloe vera of pharmaceutical, nutraceutical and dermocosmetic interest include anthraquinones/anthrones (Aloe emodin, aloetic acid, anthranol, aloines, A y B) and chromones (cinnamoyl, aloesol and methylaloediol glycosides, isoaloeresin D, isorabaichromone, neoaloesin A), which are present in the gel found in its leaves. These compounds stand out because of their antifungal and gastroprotective activity, as well as their use against psoriasis, to improve healing of wounds, to treat asthma and as mild anti-inflammatory agents.

The primary active substances found in hemp plants are non-psychoactive cannabinoids (CBD, CBG, CBC, CBDV, CBGV and CBCV), terpenes (β-caryophyllene, Pinene, Limonene, Eucalyptol, Menthol, Carene, Phellandrene, Myrcene, Ocimene, Terpinolene) and phenolic compounds (Cannflavins A and B, canniprene and denbinobine) present in the leaves and flowers of Cannabis sativa L., which present scientifically-proven therapeutic activity in a wide array of applications. At present, some countries such as Canada, Israel, Holland, a few states in the USA, and so on, accept the use of Cannabis sativa L. flowers or two of its principal cannabinoids (THC and CBD) to treat glaucoma and chemotherapy-induced vomiting, and as an analgesic in treatment of terminal cancer and multiple sclerosis.

Phenols mostly present in leaves and in residue from Olive fruit exploitation were isolated and purified in this project. Phenols are soluble in water, and as such are little soluble in a fatty matrix. Therefore, when oil separates from the aqueous fraction after olives are mashed, most phenolic compounds remain in the aqueous residue and low concentrations are found in the oil. Tyrosol, Hydroxytyrosol and Oleuropein stand out among these phenols, given their great impact on diminishing oxidative stress. The European Food Safety Authority (EFSA) recommends a consumption of 5 milligrams per day of Hydroxytyrosol.

Results

Fine-tuning of a pilot plant to purify the active substances of interest from Aloe vera, Hemp and Olive plants. This pilot plant has an isolation and purification system based on liquid chromatography-countercurrent chromatography (CCC). The basic principle of this system is that a liquid mobile phase flows through the liquid stationary phase, causing compounds to separate along an inert column, which is in continuous rotation to hold the stationary phase in place. The separation of the different compounds depends on the affinity of the compound of interest for each liquid phase. The instrumentation used is easily scalable and environmentally friendly.

CBD, CBDA, CBG and CBGA were purified from hemp, as shown in the following figure exhibiting the HPLC-DAD chromatograms with > 95% purity.

isolation-purification-1.jpg#asset:54

Oleuropein was purified from olive plant residue, waste or discarded material (such as leaves, two-phase olive mill pomace, source mass for lampante olive oil, fatty milled pits and dry pits obtained after lampante oil production). The HPLC-DAD chromatogram shows it was obtained at 40% purity, but it was recrystallised to obtain 90% purity.

isolation-purification-2.jpg#asset:53Alloin A was purified from residue from the Aloe industry, particularly leave husks, as shown in the HPLC-DED chromatogram, with 90% purity.

isolation-purification-3.jpg#asset:52A study was conducted of the bioactivity of the purified compounds obtained from industrial hemp, oil industry residues and Aloe vera exploitation. Promising results were found regarding the antioxidant activity of CBD and the anti-inflammatory effects of purified substances from hemp.

Collaborations

 - Research Group BIO304 “Immunopharmacology and Molecular Virology” at the University of Cordoba. Collaboration to evaluate the biological activity of at least 12 compounds from aloe, hemp and olive, in a standard battery of 10 in vitro tests to evaluate antioxidant and anti-inflammatory activity. Molecular targets related to obesity and metabolic syndromes were also evaluated.

- Research group FQM-227 “Analytical platform for metabolomics” at the University of Cordoba. Collaboration to identify possible biologically-active compounds obtained from aloe, hemp and waste from olive agri-food exploitation, evaluate such compounds previously isolated/purified by Phytoplant, and conduct a stability study of such compounds under different storage conditions.