EP3829612A1

EP3829612A1 - English-daisy extract

Info

Publication number: EP3829612A1
Application number: EP18752122.4A
Authority: EP
Inventors: Rolf Sorg; Bettina SCHWARZINGER; Julian Weghuber; Tobias KÜHNE
Original assignee: PM International AG
Current assignee: PM International AG
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2021-06-09
Also published as: WO2020025137A1

Abstract

The invention relates to: a method for producing an extract from English daisies (Bellis perennis), comprising: a) providing a material to perform extraction on, produced from English daisies; b) performing extraction on the material to perform extraction on by means of an aqueous solvent in order to obtain an extract; an extract obtainable by said method; a composition comprising an extract obtainable by said method; and the use of said extract as a food supplement and/or drug, in particularly for reducing blood sugar peaks and/or for inhibiting intestinal glucose reabsorption and/or for weight reduction, and for use in the treatment or prophylaxis of diabetes mellitus and/or hyperglycemia and/or obesity.

Description

Daisy extract

description

The present invention relates to a process for producing an extract from daisies (Bellis perennis) and an extract obtainable by this process. Furthermore, the invention relates to a composition containing the extract according to the invention and the use of

composition according to the invention as a dietary supplement and / or medicament. The invention also relates to the production of a composition according to the invention, in particular a food supplement.

Diabetes mellitus is a group of metabolic diseases that lead to hyperglycemia. People with type 2 diabetes mellitus (T2DM) represent the main group of patients with diabetes (90%) worldwide. The global prevalence of diabetes has increased significantly in 2014 with more than 400 million people affected in 2014. Diabetes caused 1.5 million deaths in 2012 and another 2.2 million

Deaths from increased risks for cardiovascular and other diseases. Apart from health problems, diabetes threatens the economies of all nations. The absolute global costs of diabetes and its consequences are high and will increase significantly by 2030. The share of costs in global gross domestic product is therefore enormous.

The main features of T2DM are hyperglycemia, insulin resistance and

Obesity. In addition, T2DM is also associated with hyperlipidemia and high blood pressure. This combination is called a metabolic syndrome, which is a high risk factor for cardiovascular diseases. For this reason, there is a great demand for approaches to prevent and treat the health problems associated with T2DM.

The previously known glucose lowering pharmaceuticals include

Insulin sensitizers such as glitazone, inhibitors of hepatic gluconeogenesis such as metformin, or kidney sodium glucose co-transporters (SGLT1 / 2) inhibitors such as dapagliflozin. However, the use of these drugs is associated with serious side effects such as diarrhea, nausea, abdominal pain, edema, congestive heart failure, glycosuria followed by urinary tract infections and possibly bladder cancer.

There is therefore a need to provide further agents which lower the blood glucose level and do not have the disadvantages of the pharmaceuticals known from the prior art.

The object of the present invention is to provide a method for producing an extract from daisies (Bellis perennis), since it has surprisingly been found that extracts from daisies

represent promising drug mixtures that effectively lower blood sugar levels. This is most likely done by inducing the GLUT4 translocation, which leads to increased glucose uptake in muscle and fat tissue from the bloodstream.

Another object of the invention is to provide the extract obtainable by the method and a composition comprising the extract. It is also an object of the invention to use the composition as

Dietary supplements and / or pharmaceuticals and a method for

Preparation of the composition, especially one

Dietary supplement.

According to the invention, this object is achieved by a method according to claim 1. With a view to the extract, obtainable by the invention

Process, and the composition comprising the extract according to the invention, this object is achieved by claim 9. Regarding the

Use of the composition according to the invention achieves this object by claim 12. Regarding the process for producing the

composition according to the invention, in particular one

Dietary supplement, this object is achieved by claim 15.

Preferred embodiments of the invention are specified in the subclaims.

In the following, the term “comprise” can also mean “consisting of”.

The process according to the invention for producing an extract from

Daisy (Bellis perennis) includes the steps: a) providing a material to be extracted, made from daisies;

b) extraction of the material to be extracted with an aqueous solvent to obtain an extract.

Preferably, the material to be extracted, made from daisies, is the flowers, optionally including the stems, and / or the leaves of daisies.

Particularly preferably, the material to be extracted, made from daisies, comprises the flowers of daisies.

Most preferably, the material to be extracted, made from daisies, consists of the flowers of daisies.

It is further preferred if the material to be extracted, made from daisies, is dried before the extraction.

This drying can be carried out by merely drying at room temperature (about 20 ° C.), preferably drying at room temperature for 2 to 7 days. However, it is also possible to dry the material to be extracted, made from daisies, at a slightly elevated temperature of 30 to 50 ° C for a period of 10 to 24 hours.

Regardless of the drying method, it is preferred that the material to be extracted, made from daisies, is preferably dried to a residual water content of 8 to 10% by weight before the extraction.

Preferably, the material to be extracted, made from daisies, is extracted with a suitable mill, for example one, before extraction

commercial coffee grinder, ground.

The method according to the invention is further characterized in that the solvent with which the material to be extracted is produced from

Daisy, which is extracted, comprises an aqueous solvent. Mixtures, in particular, are included as the aqueous solvent

at least water and an alcohol. The at least one alcohol preferably comprises ethanol.

The aqueous solvent preferably comprises a mixture of water and at least one alcohol, preferably ethanol, in a volume ratio of 10 to 1 to 1 to 1.

Pure water or pure alcohol can also be used as solvents in the process according to the invention.

The weight ratio of material to be extracted, produced from daisies, to aqueous solvent is preferably from 1: 1 to 1:20, particularly preferably 1: 9.

The method according to the invention is also preferably thereby

characterized in that the extraction of the material to be extracted, made from daisies, is carried out for about 5 to 120 minutes, preferably for 15 to 45 minutes.

The method according to the invention is also preferably thereby

characterized in that the method further comprises step c) treatment of the

Mixture of the material to be extracted, made from daisies, and the aqueous solvent from step b) with ultrasound.

The treatment with ultrasound is preferably carried out over a period of 5 to 120 minutes, preferably about 20 minutes.

The method according to the invention is also preferably thereby

characterized in that after the extraction, the solid and insoluble constituents are removed by centrifugation, preferably at 2500 to 3000 g, and / or filtration, preferably with a filter with a mesh size / pore size of 4 to 7.

The method according to the invention is also preferably thereby

characterized in that the aqueous solvent is removed after extraction of the material to be extracted, made from daisies to get dry extract. The aqueous solvent can preferably be removed by blowing in nitrogen.

The method according to the invention is also preferably thereby

characterized in that the extract obtained contains the compounds neochlorogenic acid, chloric acid, caffeic acid, rutin, hyperoside, isoquercitrin, guaijaverine,

Avicularin, quercitrin, kaempferol, luteolinapigenin-7-glucoside, apigenin-7-glucuronide and / or apigenin, preferably in a concentration of 0.001 to 5 mg / ml.

The present invention further relates to an extract obtainable by the method described above.

The present invention also relates to a composition comprising the extract obtainable by the process according to the invention.

It is further preferred if the composition according to the invention, containing the extract of daisies obtainable by the method according to the invention, also contains an extract from the fruits of the real guava

(Psidium Guajava).

This extract from the fruits of the real guava is preferably obtainable by a process comprising the steps

al) providing a material to be extracted, made from real guava fruits;

bl) extraction of the material to be extracted with carbon dioxide as the extraction agent in a high-pressure extraction system in order to obtain an extract.

Preferred embodiments of the method for

Preparation of an extract from the fruits of the real guava described.

In preferred embodiments, the term fruit or fruit denotes pulp, optionally with skin or skin.

The term fruit or fruits preferably does not include the seeds, kernels and / or leaves or parts thereof. Carbon dioxide is preferably used as the distraction agent for the process according to the invention. Instead of carbon dioxide, in a method according to the invention, for example, ethene, propane, ammonia, nitrous oxide, nitrous oxide and / or can also be used as the removal agent

Chlorotrifluoromethane can be used.

In a preferred embodiment, the high-pressure extraction system comprises at least one extractor. The carbon dioxide is present in the supercritical state in the extractor and extracts the material to be extracted.

In a preferred embodiment, the high-pressure extraction system comprises at least one separator. There is a lower one in the separator

Temperature and a lower pressure than in the extractor, whereby an extract separates from carbon dioxide, which is transferred from the extractor to the separator.

Destraction is preferably carried out by feeding carbon dioxide to an extractor and guiding carbon dioxide loaded with extract from the extractor into a separator in which extract from the carbon dioxide separates.

The extraction is preferably carried out in a continuous mode in which pure carbon dioxide is continuously fed to the extractor and carbon dioxide loaded with extract is continuously fed from the extractor to the separator in which extract from the carbon dioxide separates.

In a preferred embodiment, the material to be extracted comprises a puree from guava fruits or a solid obtained from

Drying of mus from fruits of the real guava.

In a preferred embodiment, the material to be extracted comprises at least 60% by weight of puree from guava fruit or a solid obtained by drying puree from fruit of real guava.

In a preferred embodiment, the material to be extracted is a pulp from real guava fruit or a solid obtained by drying a pulp from real guava fruit. The material to be extracted preferably contains no seeds, kernels and / or leaves of the real guava or parts thereof.

Preferably, the musts from the real guava fruit or the solid obtained by drying a muse from the fruits of the real guava contains no seeds, kernels and / or leaves of the real guava or parts thereof.

Before extraction, the material to be extracted is preferably dried to a water content of from 3% by weight to 50% by weight, preferably from 5% by weight to 35% by weight, particularly preferably from 7% by weight to 30% by weight.

In a preferred embodiment, the material to be extracted is dried before removal to a solid with a water content of 7% by weight to 30% by weight.

In a preferred embodiment, the material to be extracted is dried by means of a drying cabinet or vacuum chamber before the extraction.

In one possible embodiment, the material to be extracted is dried at 30 ° C. to 75 ° C., preferably at 35 ° C. to 65 ° C., for a period of 20 hours to 120 hours, preferably from 40 hours to 80 hours, before the extraction ,

In a preferred embodiment, the material to be extracted is dried in a vacuum chamber at 30 ° C. to 50 ° C. before the extraction.

If the material to be extracted is a solid, obtained by drying a musts from the real guava fruit, or comprises this solid, the solid is preferably comminuted before the extraction. The solid is preferably comminuted to a powder, preferably at least 80% by weight of the

Powder components have a diameter between 0.05 mm and 30 mm, preferably between 0.1 mm and 10 mm.

Water is preferably added as a cosolvent to the extraction material for the extraction, preferably 1% by weight to 30% by weight of water, particularly preferably 8% by weight to 20% by weight of water, based on the dry mass of the extract material. With water as the cosolvent, the yield of the extraction can be increased. The pressure in an extractor is preferably

High pressure extraction system during the extraction 100 bar to 500 bar, preferably 200 bar to 400 bar, very particularly preferably 250 bar to 350 bar.

The temperature in an extractor is preferably

High pressure extraction system during the extraction 31 ° C to 80 ° C, preferably 31 ° C to 50 ° C.

In one possible embodiment, the extraction takes place over 1 h to 8 h, preferably 2 h to 4 h.

In a preferred embodiment, the high-pressure extraction system comprises an extractor and a separator and the extractor is flowed through during the extraction by a continuous flow of carbon dioxide of 2 to 20, preferably 5 to 12, kg of carbon dioxide per hour per kg of material to be extracted, the continuous flow of carbon dioxide is fed to the separator. The amount of carbon dioxide relates to the dry mass of the material to be extracted.

A temperature of from 10 ° C. to 40 ° C., preferably from 15 ° C. to 35 ° C., is preferably present in a separator of the high-pressure extraction system during the extraction.

A pressure of 15 bar to 55 bar, preferably of 30 bar to 50 bar, is preferably present in a separator of the high-pressure extraction system during the extraction.

In a preferred embodiment, the water content of the is

extracting material at the beginning of the extraction to 3 wt.% to 50 wt.

%, preferably set to 7% by weight to 30% by weight.

In a preferred embodiment, an extract obtained is dried after the extraction, the drying preferably taking place by means of a drying cabinet or vacuum chamber, and the water content after drying preferably being <10% by weight. In a preferred embodiment, the composition of the extract obtained, in particular with regard to polyphenols, differs greatly from the composition of extracts of the real guava, obtainable by processes not according to the invention.

In a preferred embodiment, none of those polyphenols which are identified in real guava extracts obtainable by methods not according to the invention are identified in the extract obtained, the

Polyphenols are preferably identified using an HPLC method mentioned in the examples.

Polyphenols are preferably identified using an HPLC method mentioned in the examples, and the polyphenols phloridzin, phloretin, quercetin, quercitrin, isoquercitrin, hyperoside, avicularin, guaijaverin, procyanidin B1 and B2, (+) - catechin, (-) - catechin , (-) - epicatechin, gallocatechin, epicatechin gallate, gallic acid and ellagic acid include or are.

In a preferred embodiment, the extract obtained from the compounds phloridzin, phloretin, quercetin, quercitrin, isoquercitrin,

Hyperoside, avicularin, guaijaverin, procyanidin B1 and B2, (+) -catechin, (-) - catechin, (-) - epicatechin, gallocatechin, epicatechin gallate, gallic acid and ellagic acid each less than 0.8% by weight, preferably less than 0.3% by weight, based on the dry weight of the extract.

In a preferred embodiment, the composition of the extract obtained, in particular with regard to polyphenols, differs greatly from the composition of extracts of the real guava, obtainable by processes not according to the invention.

In a preferred embodiment, none of the polyphenols which are identified in extracts of the real guava, obtainable by alternative methods, is identified in the extract obtained, the polyphenols preferably being identified using an HPLC method mentioned in the examples. In a preferred embodiment, none of the polyphenols which are identified in extracts of the real guava, obtainable by alternative methods, is identified in the extract obtained, the polyphenols preferably being identified by an HPLC method mentioned in the examples, and the polyphenols being phloridzin , Phloretin, Quercetin, Quercitrin, Isoquercitrin, Hyperosid, Avicularin, Guaijaverin, Procyanidin Bl and B2, (+) - Catechin, (-) - Catechin, (-) - Epicatechin, Gallocatechin, Epicatechingallat,

Gallic acid and ellagic acid include or are.

Hyperoside, avicularin, guaijaverin, procyanidin B1 and B2, (+) - catechin, (-) - catechin, (-) - epicatechin, gallocatechin, epicatechin gallate, gallic acid and ellagic acid each less than 0.8% by weight, preferably less than 0.3% by weight, based on the dry weight of the extract.

Surprisingly, were available in the extract according to the

none of the polyphenols or flavonoids which occur in conventional extracts of real guava, for example an ethanolic extract from fruits of real guava or an extract from leaves of real guava, have been detected above. This is particularly surprising since the extract described has a much stronger inhibition of glucose transport, a more pronounced reduction in blood sugar peaks and a stronger one

Inhibition of intestinal absorption than conventional comparative extracts.

The present invention further relates to compositions as described above for use as a dietary supplement and / or

Drug.

In particular, a composition for use to reduce blood sugar peaks and / or to inhibit intestinal glucose absorption and / or to reduce weight is to be specified.

In preferred embodiments, it is a composition for use in the treatment or prevention of diabetes mellitus and / or hyperglycemia and / or obesity. The invention is also based on the idea of a method for producing a composition according to the invention, in particular one

Dietary supplement containing the extract according to the invention, the extract being obtainable by the method according to the invention. The process for producing a composition according to the invention comprises:

a) providing a daisy extract obtainable by the method according to the method described above and optionally an extract from fruits of the real guava (Psidium guajava), preferably obtainable by the method described above;

b) applying the extract or extracts to a carrier substance,

in particular by spraying a solution of the extract or the extracts onto a carrier substance in order to obtain a carrier substance containing the extract;

c) Mixing the extract-containing carrier substance with further constituents of the composition, in particular the nutritional supplement.

The extract is preferably applied in the form of a solution of the extract in aqueous ethanol or propanol.

The extract is preferably applied by spraying a solution of the extract onto a carrier substance, such as, for example, gum arabic.

The extract-containing carrier substance is preferred after the application of the

Extract dried to remove water and alcohol.

The carrier substance is preferably mixed during application.

The process ensures a particularly even distribution of

Extract in the composition.

Unless otherwise stated, the water content in the

Patent application calculated according to the following formula:

Water content (% by weight) = 100 x mass (water) / mass (water +

Dry mass).

Description of the figures 1 Effects of extracts from Bell is perennis on the GLUT4 translocation (error bars based on the SEM, n> 30, measured on 3 different days)

Fig. 2 Influence of extracts from Bel lis perennis on the blood sugar level in ovo (error bars are based on the SEM. P <0.05, ** P <0.01, *** P <0.001 and **** P <0 , 0001).

3 HPLC-DAD chromatogram of Bellis Perennis extract,

taken at 260 nm. The compounds that correspond to the respective peaks are listed in the table below.

12 14 34 Apigenin 269 0444 0 0055

12a

The invention is described below on the basis of non-limiting

Embodiments with reference to the accompanying schematic

Drawings explained in more detail.

Examples

1. Material and methods

1.1 Materials

Human insulin, CaCl ₂ , NaCl, KCl, MgS0 ₄ , KH ₂ P0 ₄ phosphate buffered

Saline (PBS), Hank's balanced salt solution (HBSS), Quercetin,

Chlorogenic acid, neochlorogenic acid and caffeic acid were obtained from Sigma-Aldrich (Schnelldorf, Germany). Guaijaverin and Avicularin came from Glentham Life Sciences (Corsham, United Kingdom). Rutin, hyperoside, isoquercitrin and quercitrin were obtained from extra synthesis (Genay Cedex, France). For the production of stock solutions, the vegetable

Compounds dissolved in Krebs Ringer phosphate HEPES buffer (KRPH; 20 mM HEPES, 1 mM CaCl ₂ , 136 mM NaCI, 4.7 mM KCl, 1 mM MgSO ₄ and 5 mM KH ₂ PO ₄ ).

A library with 2,300 water-soluble herbal extracts (PECKISH) was provided by Frank Döring (Christian-Albrechts-University Kiel, Germany). NovoRapid from Novo Nordisk was provided by Daniel Weghuber

(Paracelsus Medical University, Salzburg, Austria).

1.2 Cell culture and transfection

CHO-Kl cells that stably express the human insulin receptor (hIR) and GLUT4-myc-GFP were developed by Manoj K. Bhat (National Center for Cell Science,

13

University of Pune, India). The cells were in Ham's F12 culture medium supplemented with 100 pg / ml penicillin, 100 pg / ml streptomycin, 1%

G418 and 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA) were maintained and cultured in a humidified atmosphere at 37 ° C and 5% C0 ₂ .

1.3 Preparation of the extracts

Fresh daisy plants were collected and dried at room temperature for 1 week. The dried material was in a

Coffee grinder ground for 30 seconds. For the extract preparation, 3 g of the powder were dissolved with 30 mL EtOH (50%). The solution was with 20 min

Ultrasound treated, centrifuged and filtered with a household filter. The EtOH was blown off with N ₂ and the residues were diluted again with water. The samples were stored at -20 ° C.

1.4 TIRF microscopy

CHO-Kl hIR / GLUT4-myc-GFP cells were placed in 96-well plates (35,000

Cells / well; Mobitec, Göttingen, Germany) as in La n destroyed, P. et al. British journal of pharmacology (2014), or Stadlbauer, V. et al. PloS one (2016) described, cultivated overnight. Cell culture medium was aspirated and, after washing the cells with PBS (VWR, Vienna, Austria), replaced by HBSS (Thermo Fisher, Waltham, MA), supplemented with 0.1% BSA (Sigma-Aldrich, Schnelldorf, Germany) for 3 hours.

The cells were treated with insulin or in KRPFI buffer

Daisy extracts incubated and on an inverted Olympus IX-81

Microscope in objective TIR configuration using an Olympus 60x NA = 1.49 plan apochromatic objective. 96-well plates were placed on an x-y table (CMR-STG-MHIX2 motorized table; Märzhäuser, Wetzlar, Germany).

The scanning of larger areas was supported by a laser-controlled automatic focus hold system (ZDC2). The 488 nm emission of the diode laser (Toptica Photonics, Munich, Germany) was used to image the GFP fluorescence. After appropriate filtering that was

Fluorescence signal via an Orca-R2 CCD camera (Hamamatsu Photonics,

Herrsching, Germany).

1.5 Chicken egg test fHET 14

The HET-CAM test was carried out, as has already been done by Haselgrubler, R. et al. PloS one (2017) and Haselgrubler, R. et al. Journal of visualized experiments (2018).

In short, the eggs were at 38 ° C with an average

Humidity of 40 ± 60% incubated for 11 days. The eggs were turned over automatically and continuously, checked for fertilization by fluoroscopy and the area of the air bubble marked. The eggshell was pointed with a

Pick the tweezers in this area and add 300 pL of a buffer solution (HBSS or water) with the suspected hypoglycemic substance. 3 different plant extracts of daisies (bellis perennis) were tested.

Two extracts were used by the PECKISH extract library

(Extracts 4404 and 4407). These extracts are pure

ethanolic extracts. In addition, an extract was prepared according to the

method according to the invention ("homemade") was used.

The extracts were diluted with HBSS or water to a final concentration of ~ 300 mg / L, which was finally introduced into the air compartment of the egg using a syringe. After the incubation, the eggshell above the air bubble was carefully removed and the eggshell membrane was leveled with PBS. In the next step, the eggshell membrane was removed and the

Carefully cut the chorioallantoic membrane with microscissors. A suitable blood vessel was carefully placed on a plastic pH strip, which was taped dry with filter paper before cutting the vessel and the escaping blood was collected. The blood sugar values were checked with a blood glucose meter (Accu-Check Performa, Roche Diabetes Care GmbH,

Mannheim, Germany). At least 10 fertilized eggs were used at each time. Each experiment was repeated at least three times.

1.6 HPLC analysis

The extracts were analyzed using reverse phase chromatography with a Thermo Scientific Dionex Ultimate 3000, consisting of an LPG-3400SD pump with built-in degasser, a WPS-3000 U (T) SL cooled

Autosampler, a temperature controlled column compartment and a FLD-34000RS diode array detector (DAD) with Chromeleon software as in Müller, U. et al. 15

Molecular nutrition & food research (2018). The analyte was separated on an Accucore C18 column (150 mm x 3.0 mm inner diameter, 2.6 μm particle size; Thermo Scientific). The column temperature was set to 40 ° C and the injection volume was 1 ml. The analytes were separated by gradient elution with mobile phase A with 0.1% formic acid (FA) in water and mobile phase B with 0.1% FA in acetonitrile at a flow rate of 0.5 mL / min. The starting conditions of the elution gradient were 95% A and 5% B. After an equilibration time of 5 min, the proportion of B was increased to 20% at 8 min and to 40% at 12 min, followed by 60% B at 15 min and 80% B at 17 min for 3 min. B was reduced to 5% at 20 min to 25 min.

High resolution mass spectra were negative using a Thermo Fisher Scientific LTQ Orbitrap XL with an Ion Max API electrospray source

Ionization mode won. Capillary temperature 350 ° C, jacket gas stream 45, auxiliary gas stream 15, source voltage 3.5 kV, capillary voltage -25 V, tube lens - 90 V. The separation was carried out with an Accucore C18 column (150 mm x 3.0 mm inner diameter, 2.6 pm Particle size; Thermo Scientific under the conditions described above.

Polyphenols have been quantified against known standards, provided they are available at concentrations in a linear range of 1-1,000 mg / L.

16

1.7 Data analysis

The first image recordings were supported by the Olympus Xcellence RT software. For the calculation of the fluorescence intensity in individual cells and a quick comparison of the fluorescence signal in numerous cells in

The Spotty framework was used at different intervals

carried out in-depth analysis. Spotty is available online at

http://bioinformatics.fh-hagenberg.at/projects/microprot/. The statistical

Evaluation was carried out with the 2-way Annova in Graphpad Prism (version 6.02).

2 results

2.1 Induction of GLUT4 translocation by daisy extracts

The following is the efficacy of various daisy extracts in inducing the translocation of GLUT4 from cytosolic

Storage compartments in the plasma membrane are described. First, two purely ethanolic extracts from the PECKISH extract library were tested.

One extract was a mixture of flowers and leaves (4404), the second was only made from flowers (4407).

Third, an extract of daisy flowers according to the

Process according to the invention produced (homemade).

For the measurements, starving CHO-Kl cells, which stably express the human insulin receptor and a GLUT4-myc-GFP fusion protein, were incubated with the selected extract in low concentrations (1 mg / L).

The increase in the GFP signal in the evanescent field, which correlates with the concentration of the GLUT4 proteins in the plasma membrane, was then determined.

Fig. 1A shows the effect of the three extracts tested after 10 min incubation. The ethanolic extracts 4404 and 4407 only led to a moderate increase in the GFP signal of ~ 8% and ~ 5%, respectively. 17

However, the (homemade) extract according to the invention led to a sharp increase in the GFP signal (~ 35%).

To ensure the test was carried out, the cells were also treated with 100 nM human insulin. This led to an increase of ~ 26%, which is the result of the studies published previously by Stadlbauer, V. et al. PloS one (2016).

Based on these results, the effectiveness of the two PECKISH library extracts 4404 and 4407 was quantified by testing different concentrations from 0.1 mg / L to 10 mg / L.

As shown in Fig. 1B and C, we found a clear dose-response relationship between both extracts, with 4404 being somewhat more efficient than 4407: While 4407 had no effect at 0.25 mg / L, the addition of 4404 resulted in the same

Concentration already increased by ~ 4%. Taken together, extracts from Be / iis perennis are effective inducers of GLUT4 translocation in the absence of insulin.

2.2 Lowering the blood sugar level through daisy extract in ovo

Based on the results of the CHO-Kl-based in p / iro system, the effectiveness of Bellis-Perennis extracts was tested in ovo. For this purpose, the recently established and in Haselgrubler, R. et al. PloS one (2017), or Haselgrubler, R. et al. The Chicken of Egg Test (HET-CAM) described in the Journal of visualized experiments (2018).

With this approach it is possible to test whether the application of a

selected compound or an extract reduces the blood sugar level in a living organism. Therefore, the homemade extract and the two PECKISH extracts were dissolved in HBSS buffer (300 mg / L). NovoRapid, a fast-acting human insulin analog, was used as a positive control (3.3 U / ml) to demonstrate insulin sensitivity. After incubation with the extracts for 1 or 2 h, the blood sugar values were checked over a week

Blood glucose meter measured.

As shown in Fig. 3A, all three extracts led to a comparable drop in blood sugar levels (~ 20% after 1 h and 30% after 2 h) 18

2 h incubation time was statistically significant, with the homemade extract showing the best results.

Treatment with NovoRapid resulted in a reduction of ~ 16% after 1 h and a significant effect of ~ 33% after 2 h.

It was recently published by Haselgrubler, R. et al. PloS one (2017) showed that HBSS buffer alone also leads to a certain decrease in blood sugar levels and that ddH ₂ 0 has proven to be a better solvent for carrying out the test.

Therefore, the experiments described above were repeated under these conditions.

As shown in Fig. 2B, ddH ₂ 0 alone led to an insignificant reduction of ~ 3%, which confirms its preferred applicability. In addition, the extracts were able to significantly lower the blood sugar level at both times (~ 12% after 1 or 2 h), whereby the homemade extract showed the best results in the long term.

In summary, it can be said that the inventive

Daisy extracts lower blood sugar levels both in vitro and in vivo.

2.3 Identification and quantification of polyphenols in

Daisy extract

The polyphenol content of the homemade according to the invention

Daisy extract was applied by HPLC-MS. First, compounds were identified using mass spectrometry and UV spectra and then quantified using calibration curves of the relevant standards. 13 polyphenolic compounds were identified and quantified: rutin, hyperoside, isoquercitrin, guaijaverin, avicularin, quercitrin, quercetin (flavonols), apigenin-7-glucoside (also known as apegetrin), apigenin-7-glucuronide, apigenin (flavones), neochlorogenic acid Chlorogenic acid and

Caffeic acid (hydroxycinnamic acids). In addition, kaempferol and luteolin were identified by HPLC-MS analysis, but due to the lack thereof

Standards and overlapping retention times not quantified. 19

A representative HPLC-DAD chromatogram giving retention times and maximum wavelengths of each compound is shown in Fig. 3. Table 1 summarizes the content of identified polyphenols. The best known hydroxycinnamic acid was chlorogenic acid (2.69 mg / L), apigenin -7-glucoside and apigenin-7-glucuronide (overlapping retention times) were found to be very common flavones (0.42 mg / L), while quercitrin was found as

Main flavonol (0.1 mg / L) was detected. All other polyphenols were only detected in lower concentrations. In summary, it can be said that polyphenolic compounds are abundant in daisy extract and can help to lower blood sugar levels.

Claims

20 claims

1. Process for producing an extract from daisies (Bellis

perennis), comprising:

a) Providing a material to be extracted, made from

Daisy;

b) extraction of the material to be extracted with an aqueous

Solvent to get an extract.

2. The method of claim 1, wherein the material to be extracted comprises the flowers and / or leaves of daisies.

3. The method according to any one of the preceding claims, wherein the material to be extracted comprises the flowers of daisies.

4. The method according to any one of the preceding claims, wherein the material to be extracted is dried prior to extraction to a solid having a water content of 8% to 10% by weight.

5. The method according to any one of the preceding claims, wherein the aqueous solvent comprises a mixture of water and at least one alcohol, preferably ethanol, in a volume ratio of 10 to 1 to 1 to 1.

6. The method according to any one of the preceding claims, the method further comprising the step

c) ultrasound treatment of the mixture of the material to be extracted, made from daisies, and the aqueous solvent from step b),

includes.

7. The method according to any one of the preceding claims, wherein the

extract obtained the compounds neochlorogenic acid, chloric acid, caffeic acid, rutin, hyperoside, isoquercitrin, guaijaverin, avicularin, quercitrin, kaempferol, luteolinapigenin-7-glucoside, apigenin-7-glucuronide and / or apigenin, preferably in a concentration of 0.001 to 5 mg / mL, includes. 21

8. Extract obtainable by the method according to any one of claims 1 to 7.

9. A composition comprising the extract of claim 8.

10. The composition of claim 9, further comprising an extract from the fruits of the real guava (Psidium guajava).

11. The composition of claim 10, wherein the one extract

Real guava fruit (Psidium guava) is obtainable through a process comprising the steps

a l) providing a material to be extracted, made from real guava fruits;

12. A composition according to any one of claims 9 to 11 for

Use as a dietary supplement and / or medicine.

13. A composition according to any one of claims 9 to 11 for

Use to reduce blood sugar spikes and / or

Inhibition of intestinal glucose absorption and / or for weight loss.

14. A composition according to any one of claims 9 to 11 for

Use in the treatment or prevention of diabetes mellitus and / or hyperglycemia and / or obesity.

15. A process for the preparation of a composition according to any one of claims 9 to 11, in particular a nutritional supplement, comprising:

a) providing a daisy extract obtainable by the method according to any one of claims 1 to 7 and optionally an extract from real guava fruit (Psidium guava), preferably obtainable by the method according to claim 11;

b) applying the extract or extracts to a carrier substance, in particular by spraying a solution of the extract or 22

Extracts on a carrier substance to obtain an extract-containing carrier substance;