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Olive derived cell culture and methods for preparing and using the same

a cell culture and olive technology, applied in the field of olive derived cell cultures, can solve the problems of bitterness of the final product, inability to perform large-scale processes by the same means, and inability to provide the natural ingredients along with the active ingredients of the synthetic process, so as to achieve the effect of reducing high blood pressure, maintaining normal haemostatic function, and beneficial to learning

Pending Publication Date: 2021-05-13
BIO HARVEST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0073]In further embodiments, the invention provides a method of prevention and / or treatment of risk factors for cardiovascular disease, development of atherosclerotic plaque, protection of LDL particles from oxidative damage, maintenance of normal blood HDL-cholesterol concentrations, decrease of high blood pressure and to maintain normal haemostatic function. Olive polyphenols were shown to have beneficial effects on learning and memory deficits found in ageing and diseases, such as those related to the overproduction of amyloid-beta peptide. In addition, olive polyphenols were shown to influence gut microbial balance by promoting growth of bacteria influencing lipid metabolism and inhibition of pathogenic bacteria. The method comprising administering an effective amount of a composition comprising composition in a form of a powder comprising olive fruit / leaf cells grown in vitro, whereby the olive cells are derived from one or more of cross section, olive pulp, olive seed, olive petiole or olive leaf.

Problems solved by technology

Since large scale processes cannot be performed by the same means as small scale processes, specific processes for the large scale production of materials must be designed, even if small scale processes exist.
However, the use of synthetic processes does not provide the natural ingredients along with the active ingredients, which sometimes contribute to the efficiency of the formulation.
However, when plants containing polyphenols, for example, are extracted, the final product may be bitter.
Also, only certain parts of the plant may be successfully extracted since only they contain the desired amounts of the active ingredients.
Small scale processes for the preparation of fruit cells are known in the art; however, large scale processes are more difficult to design since they tend to amplify the production of the primary metabolites, while minimizing the productions of the secondary metabolites.
Since active ingredients, such as polyphenols, are secondary metabolites their production in large-scale processes is complex.
However, it has been shown that the therapeutic effect of fruit extracts is dependent on species, location, year (annual climate), processing etc. and therefore reliance on natural fruits as a source of these regulatory compounds does not lead to a homogeneous or consistent supply of material.
Furthermore, fruits are often contaminated by residual fungicides, pathogens, pesticides and pollutants.

Method used

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  • Olive derived cell culture and methods for preparing and using the same
  • Olive derived cell culture and methods for preparing and using the same

Examples

Experimental program
Comparison scheme
Effect test

example 1

Generation of Olive Cell Lines (Calli) and Suspension Cultures in Erlenmeyer

1. Material and Methods

Plant Material

[0154]Olive cell culture was initiated from olive fruits (Olea europaea L.) of Nabali, Manzanilla, Souri and Barnea cultivars, including all leaves, petioles, fruits and kernels.

Establishment of Calli from Olive Explants

[0155]Olive plant parts: young leaves and their petriols, immature fruits (about 6 weeks from flowering) and kernels from immature olives were rinsed under running water and sterilized by agitation in 70% ethanol for three minutes and then 3% Na-hypochlorite solution for 20 minutes, followed by three washes in sterile water. Plant parts were dried under sterile conditions and further dissected into ˜0.5 cm sections.

[0156]Olive plant explants were plated on MS Murashige and Skoog medium basal medium (Table 1B) supplemented with sucrose and various combinations of the auxines 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA) and the cyto...

example 2

Expression Polyphenolic Compounds in Olive Cell Calli

Materials and Methods

[0163]Polyphenols extraction for HPLC analysis: Fresh olive Callus cell cultures were extracted for analytical determination of polyphenol content in the olive culture. About 1 gr of callus was harvested and kept at −20° C. for at least 16 h before analysis. The callus was extracted by 80% methanol / water solution in a ratio of 0.4 ml methanol per 1 gr of cells. The suspension was sonicated for 10 minutes at 30° C. in a sonicator. The solution was centrifuged and the supernatant was re-centrifuged, filtered through a 0.45 μm filter and used for the HPLC analysis.

[0164]Olive tissues (fruits and leaves) were crushed under liquid nitrogen and extracted as described above, the extract was analyzed by HPLC and used as a reference for olive polyphenols content.

[0165]Olive calli extract was analyzed by LC-MS. The method is described in: Food Chemistry 138 (2013) 1381-1391.

[0166]Olive calli samples were extracted in me...

example 3

Scale Up of Olive Culture in Bioreactors and Testing of the Total Amount of the Polyphenols, Including, Hydroxytyrosol, Tyrosol, Oleuropein, Verbascoside and Pinoresinol Content in Olive Cells Grown in Large Scale Bioreactors

Materials and Methods

[0172]Stage 1: cells are prepared and grown as described in example 1.

[0173]Stage 2: Small scale bioreactor

[0174]Small scale bioreactor culturing is performed by inoculating a 7 to 16 old day cell suspensions grown in the Erlenmeyer of Stage 1 into a 4-8 liter disposable bioreactor at 25 +5° C. The cells were grown in the suspension under continuous fluorescent light (1000 1×) in a growing medium containing modified MS supplemented with sucrose 1-6% and the Kinetin, 2,4,D, NAA, BA, 2iP and casein hydrolysate or a combination thereof

[0175](pH 4.0-6.0)-. The cells were sub-cultured every 5-21 days.

[0176]Stage 3: Large scale bioreactor

[0177]The cell suspensions grown in a small scale biorcactor were inoculated into a 30-50 liter disposable bior...

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Abstract

The present application describes a large scale process for the in vitro production of an olive cell culture.The application further describes a composition in a form of a powder comprising olive fruit / leaf cells grown in vitro and a method of treating metabolic syndrome disorders, such as, high cholesterol level, comprising administering an effective amount of the composition. The cell line callus culture of olive cells manufactured according to the process of the invention includes high level of hydroxytyrosoll, tyrosol, oleoropein and verbascoside.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of Ser. No. 16 / 072,886 filed Jul. 26, 2018, which is a NP application of PCT / IL2017 / 050098 filed Jan. 26, 2017, which claims priority to U.S. 62 / 287,453 filed Jan. 27, 2016. The contents of these applications are incorporated into this application in their entirety.FIELD OF THE INVENTION[0002]The invention is directed to olive derived cell cultures, a process for the large scale production of such cell cultures, as well as methods of using the same.BACKGROUND OF THE INVENTION[0003]Large scale processes are known in the art and are necessary for the industrial production of various materials. Since large scale processes cannot be performed by the same means as small scale processes, specific processes for the large scale production of materials must be designed, even if small scale processes exist.[0004]Nutraceuticals are sometimes prepared using synthetic processes that provide the desired active ingredi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K36/63A01H5/08A01H5/10A01H4/00A61P3/00A61K9/14C11B1/00C12N5/00
CPCA61K36/63A01H5/08A01H5/10A01H4/00C12M21/00A61K9/14C11B1/00C12N5/0025A61P3/00B65D88/00C12M23/14
Inventor HAGAY, YOHEVEDAZACHI, MALKIT
Owner BIO HARVEST
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