Drug – bio-affecting and body treating compositions – Plant material or plant extract of undetermined constitution... – Containing or obtained from a tree having matured height of...
Reexamination Certificate
2002-03-08
2004-01-13
Tate, Christopher R. (Department: 1651)
Drug, bio-affecting and body treating compositions
Plant material or plant extract of undetermined constitution...
Containing or obtained from a tree having matured height of...
C424S774000, C514S885000
Reexamination Certificate
active
06676980
ABSTRACT:
TECHNICAL SCOPE OF THE INVENTION
This invention describes a procedure for obtaining stabilised vegetable extracts from
Olea europaea
by extraction from the leaves dried at less than 35° C., purification of the extract and evaporation of the extract. The extract obtained contains a high Oleuropein content and a high raw material yield.
This invention describes new therapeutic applications of
Olea europaea
extracts as an immunological agent, activating and proliferating T-lymphocytes, Natural Killer (NK) cells, monocytes and granulocytes in healthy humans, and increasing pro-inflammatory cytokines.
STATE OF THE ART
Oleuropein is a bitter glycoside that is found in the fruit, roots, bark and especially the leaves of
Olea europaea
. It is the active component of
Olea europaea
extracts, together with the flavonoids present in the extracts.
Methods for preparing vegetable extracts from
Olea europaea
leaves with pharmacological activity have been described in U.S. Pat. No. 5,714,150 and WO9614064.
U.S. Pat. No. 5,714,150 describes a procedure for extraction from
Olea europaea
leaves by maceration, using as an extractant an alcohol/water mix in an approximate proportion of 75%-25% at a temperature between 20-88° C., obtaining an extract with a Oleuropein content of 35%.
Patent application WO9614064 describes obtaining
Olea europaea
extracts with pharmacological activity using as an extractant water and/or water/alcohol solutions at a wide range of temperatures between 20-100° C., using dried leaves.
However, there is no document in the State of the Art describing the purification of the extract or drying the leaves at a low temperature.
According to the documents in the State of the Art, vegetable extracts from
Olea europaea
and Oleuropein have shown pharmacological properties, especially as anti-viral drugs.
The first medicinal use of this extract date from the early 1800's, when it was used in liquid form to treat malaria. Since then it has had many medical applications. Among the most significant effects described for this compound is its anti-infectious activity in the presence of a virus, although it is also effective against bacteria, fungi and some intracellular parasites. This anti-infectious activity against these pathogens has been related to a direct effect of the elenolic acid; in the case of viruses, the product's capacity to penetrate into the infected cells and directly inactive viral replication has been described, among others, either interfering, for example, with virus-critical amino-acids or, in the case of retroviruses, neutralising the production of reverse transcryptase or proteases. The many mechanisms used by Oleuropein to inactivate bacteria include a direct lytic effect on their external walls.
No document included in the State of the Art has shown the effect of
Olea europaea
extracts on the immune system, reinforcing Cellular Immunity and delayed Hypersensitivity in humans by the activation and proliferation of T-lymphocytes, Natural Killer (NK) cells, monocytes and granulocytes in healthy humans.
The CD16 membrane antigen, also known as a type II IgG Fc fragment receptor, is usually express in NK cells, granulocytes and macrophages. Most of the antibody-dependent cellular toxicity, or ADCC, corresponds to NK cells and a small population of cytotoxic T-lymphocytes that also express the CD16 marker, the Fc receptor that mainly binds human IgG1 and IgG3 complexes. The ADCC provides these cytotoxic cells with a mechanism with which, making use of the specificity of the antigen-antibody bond, they are able to direct or focus their cytotoxic activities. The CD16 present in the granulocyte and macrophage membrane also helps these cells in the phagocytosis. So a drug that increases this marker's expression in different cell populations can generate an activation of NK cells and a small population of cytotoxic T-lymphocytes, increasing their function as cytotoxic cells. The function of NK cells in the immune system is to defend the body from viral infections, eliminating virus-infected cells. The increased expression of CD16 in granulocytes and macrophages can reinforce the main function of these cells in the inflammatory process, which is the elimination of bacteria, fungi and other pathogens by phagocytosis.
On the other hand, for the cells in the immune system to function effectively requires contact with other cells or the extracellular matrix, so that they recognise the situation. The surface of the leukocytes not only possesses specific receptors that allow them to interact and become active in response to certain stimulants, but they also express a large number of molecules that are identified as adhesion molecules. These leukocyte molecules will act as receptors of ligands in other cells (in this case acting as counter-receptors) or amino-acid sequences present in different extracellular matrix proteins such as collagen, fibronectin, laminin and others.
Adhesion molecules also collaborate in cellular activation, sending co-activating signals to the cell interior. Depending on their structure, they can be classified into three general categories:
1.—The selectins
2.—Those that belong to the family of the integrins.
3.—Those that belong to the super-family of the immunoglobulins.
The CD11a and CD11b function-associated antigens are the alpha-chains of the LFA-1 and Mac-1 integrins, respectively. Both of them belong to the family of the &bgr;2 integrins. These membrane molecules are fundamental for the normal development of the inflammatory process, fundamentally mediating the final adhesion of the leukocytes to the vascular endothelium, the extravasation and the migration to the inflammatory foci. Their increased expression or new expression in the cellular membrane is usually a consequence of the release of pro-inflammatory cytokins such as IL-1, TNF-&agr;, IL-8 etc. In normal conditions, inflammation is a physiological phenomenon aimed at eliminating pathogen agents through phagocytosis and restoring the damaged tissue. The increased expression obtained by a drug in lymphocytes on the CD11a molecule and in lymphocytes and monocytes on the CD11b molecule, together with the increase in the plasmatic levels of pro-inflammatory cytokins such as IL-1&bgr; and IL-8, implies an activation of lymphocytes, monocytes and granulocytes that will finally result in a reinforcement of the inflammatory response. IL-8, which belongs to the family of the chemokins, also has the capacity to stimulate the movement of leukocytes (chemokinesis) and directed movement (chemotaxis), especially of neutrophils.
Moreover, the increased expression of CD25 membrane markers in monocytes and of CD69 in lymphocytes and monocytes produced by a drug, implies an activation of these populations, reinforcing the immune system with the administration of the drug.
The procedures for extraction from
Olea europaea
described in the State of the Art provide extracts with a high Oleuropein content, but these documents do not describe the Oleuropein yield from the raw material,
Olea europaea
leaves. Drying
Olea europaea
leaves at a low temperature leads to a greater extraction yield and obtains an extract with a high Oleuropein content, without losing the active components and maintaining all the pharmacological properties of the extract. Drying
Olea europaea
leaves at a temperature of less than 35° C. provides a raw material with a Oleuropein content of 5% compared with the 0.3% described in the State of the Art.
The use of water, and then membrane filtration, to obtain Olea extracts has the advantage of a greater level of selectivity in the extraction, eliminating the least polar components of the extract such as chlorophylls, polyphenols, fats and alkylphenols, with no pharmacological activity.
The action of the Oleuropein and the flavonoids present in
Olea europaea
extracts on the immune system by activating T-lymphocytes, Natural Killer cells, monocytes and granulocytes, reinforces the virus and bacteria elimination activity. This action is independent fr
Aviles Olmos Ginés
Diaz Alperi Joaquin
Pardo Zapata José
Quintanilla Almagro Eliseo
Sempere Ortells José
Asac Compañia de Biotecnologia e Investigacion S.A.
Flood Michele
Tate Christopher R.
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