EP1858536A2

EP1858536A2 - Medicament consisting of plant extracts as a lipase inhibitor

Info

Publication number: EP1858536A2
Application number: EP06722585A
Authority: EP
Inventors: Jürgen SCHREZENMEIR
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-03-17
Filing date: 2006-03-09
Publication date: 2007-11-28
Also published as: AU2006224909A1; RU2007138393A; WO2006097074A3; JP2008533058A; BRPI0608007A2; DE112006001253A5; CN101151042A; WO2006097074A2; DE102005012832A1; US20080299234A1

Abstract

The invention relates to a lipase inhibitor consisting of: cardamom and/or marjoram and/or thyme and/or chilli (Capsicum frutescens) and/or evening primrose and/or oregano leaves (oreganum vugare) and/or camomile leaves (Chamomilla recutica) and/or nutmeg (Myristica fragrans) and/or apple pomace and/or corn germs and/or almond and/or hazelnut and/or walnut and/or pumpkin germs and/or Cuphea wrightii and/or evening primrose and/or common flax (Linum usitatissimum) and/or cotton burdock (Arctium lappa L) and/or flax and/or Helianthus annuus germs (sunflower) and/or marigold (Calendula officinalis) and/or Echinops banaticus and/or Mallotus philippinensis and/or rape and/or sesame and/or black cumin and/or Cynoglossum officinale and/or Lapachotea and/or Arctostaphylos uva-ursi and/or stone flower (Flor de piedra) and/or Propolis and/or Primula Veris and/or Magnolia officinalis and/or celery (Apium graveolens).

Description

Drugs from plant extracts as Lioaseinhibitor

The excessive intake of fat can cause numerous diseases, such as. Obesity (obesity), high blood lipids and type 2 diabetes, as central manifestations of the metabolic syndrome. Increased blood triglyceride and glucose levels after ingestion are associated with insulin resistance, which is central to the development of the metabolic syndrome. One way to reduce the postprandial triglyceride level is to lower the activity of the enzymes that hydrolyze the dietary fat. The pancreatic lipase (e.c. 3.1.1.3) is essential for the degradation and absorption of food-borne fats in the intestinal tract. For this reason, their reduction leads to a prevention or at least a delay of the intestinal digestion of dietary fats and consequently to a reduction of blood triglycerides. Obesity is an important risk factor for type 2 diabetes. It is generally accepted that weight reduction improves blood sugar control and helps to reduce the cardiovascular risk factor. In general, it is considered difficult to achieve weight reduction and maintain the reduced weight. Tetrahydrolipstatin (THL, Orlistat, Xenical) is a commercially available lipase inhibitor obtained from Streptomyces toxystricini. Following oral administration, orlistat prevents fat loss by blocking the active portions of the pancreatic lipase (pancreatic lipase). In clinical trials, orlistat has been shown to reduce weight and improve postprandial (after food intake)

Fat profiles (reduced postprandial triglycerides as well as fasting LDL cholesterol and total cholesterol as well as the ratio of LDL to HDL). Also, the occurrence of type 2 di- betes be reduced by orlistat. The use of orlistat leads to undesirable side effects such as oily and fatty stools. Gentler lipase inhibition is achieved by using herbal extracts and their active ingredients, which result in a delay in time rather than complete suppression of fat absorption, thus avoiding oily or fatty stools.

This can be achieved systematically by active ingredients which are self-absorbed or inactivated during digestion and thus removed from the reaction weight with the lipase-coiipase-fat complex.

The administration of lipase inhibitors is considered to be a useful medication for patients with symptoms of metabolic syndrome. In addition, a lipase-inhibiting plant extract or components isolated therefrom can be used as nutritional supplements.

Several patents cite the use of plant extracts as potential inhibitors of lipase in the digestive tract. These are Japanese applications with the following numbers: JP 2003 026 585, JP 2002 275 077, JP 2002 047 194.

In several publications, the positive effects of plant extracts on the postprandial lipid profile have been described, namely the following substances such as Zylokaria Paliurus, grape seeds, sage, Cassia mimosoides, L. var. Nomame Makine and Alpinia officinarum.

In addition, the invention has the determination of more

Plants set to work, which are useful as lipase inhibitors. To solve this problem, extracts or essences of the following substances and their mixture thereof are mentioned.

Cardamom

marjoram

thyme

Chili {Capsicum frutescens) Evening primrose

Oregano leaves (oreganum vυgare)

Chamomile leaves (Chamomilla recutica)

Nutmeg (Myristica fragrans)

Applesauce (pomace) grain germ

almond

hazelnut

walnut

Pumpkin seed Cuphea wrightii (e)

evening primrose

Oil flax (Linum usitatissimum) large burdock {Arctium lappa L) (e)

Flax (s) germs of Helianthus annuus (sunflower) (e)

Marigold (Calendula officinalis) (e, ti)

Echinops banaticus

Mallotus philippinensis (e)

Rapeseed, (e) sesame (s) black cumin (m)

Cynoglossum officinale

Lapachoteae

Arctostaphylos uva-ursi stone flower (Flor de piedra)

propolis

Primula Veris

Magnolia officinalis

Celery (Apium graveolens)

Description of the preparation of the extracts

a) CO ₂ extracts: High pressure extraction with supercritical carbon dioxide. Contains no water, no sugar and no proteins.

Cardamom:

Used part of the plant: seeds with capsules from Elettaria cardamomum.

The raw material was sourced from Guatemala. Extract consistency: oily liquid extract contains 72% essential oils

Other ingredients: α-pinene, β-pinene, β-myrcene, 1,8-cineole, linalool, α-terpineol, linalyl acetate, terpinyl acetate

Marjoram:

Used part of the plant: Leaves of the oregano Majorana

Extract consistency: oily liquid

Extract contains 80% essential oils

Other Ingredients: Sabines, Eis and Trans Sabinenhydrate, Terpinene-4-ol, Sabinenhy- dratacetat

thyme

Used part of the plant: leaves of the thymus vulgaris. The raw material was sourced from Germany. See also Cardamom above.

Chili (Capsicum frutescens): Used part of the plant: pod The raw material was sourced from India

The extract was standardized with sunflower oil Extract consistency: Otherwise, as above evening primrose:

Used part of the plant: seeds Extract stabilization with tocopherol Extract consistency: oily liquid

oregano leaves

Extract consistency: oily liquid

Ingredients 0.18% terpinene, 2.6% p-cymene, 0.07% limonene, 62% carvacrol, 1.5% caryophyllene, 0.33% thymol, 0.49% 1.8 cineole,

0.17% γ-terpinene, 0.2% β-terpinene, 1.5% 4-terpineol, 0.58% α-terpineol, 23.2% thymoquinones

Chamomile leaves Extract consistency: oily liquid

20-25% Bisabolol 5-20% Bisabololoxides 1.0-3.0% Natricin

nutmeg

Raw material sourced from Indonesia

Extract consistency: oily liquid

Ingredients: 19-24% myristicin

1-3% safrole

b) The following extracts were prepared as follows:

10 g of powder of the raw material were dissolved in e) 50 ml of pure ethanol at 37 ⁰ C. w) In water of 37 ⁰ C h) In water of 95 ⁰ C m) In 60% ethanol at 37 ⁰ C. and then incubated in a shaking water bath for 2 hours. Thereafter, the extract was stirred in a magnetic stirrer for 30 minutes and centrifuged at 6000 rpm for 10 minutes. The solvent was then removed by a Speedrac concentrator.

Applesauce (pomace) (e): Extract consistency: viscous liquid

Grain germ (s):

Extraction consistency: viscous liquid

Almond (s):

Extraction consistency: viscous liquid

Hazelnut (s):

Extraction consistency: viscous liquid

Walnut, sort S (e): Extraction consistency: viscous liquid

Pumpkin seed (s):

Extraction consistency: viscous liquid

Cuphea wrightii (e):

Extraction consistency: viscous liquid

Evening primrose (s): Used part of the plant: seeds

Extraction consistency: viscous liquid Off salmon (165Ue) Extraction consistency: viscous liquid

Off salmon (162) (e) Extraction consistency: viscous liquid

big burdock

Used part of the plant: root extraction consistency: viscous liquid

Flax (s)

Extraction consistency: viscous liquid

Seeds of Helianthus annuus (sunflower) (e): Extraction consistency: viscous liquid

Marigold (Calendula officinalis) (e, h): Extraction consistency: viscous liquid

Echinops banaticus (154: e, 152: e):

Extraction consistency: viscous liquid

Mallotus philippinensis (e): Extraction consistency: viscous liquid

Rapeseed

Extraction consistency: viscous liquid

Sesame (s) Extraction consistency: viscous liquid

black cumin (m): Cynoqlossum officinale (157, Θ):

Lapachoteae

Used part of the plant: Inner bark of the Taheebo tree (Tebubia impetiginosa) Extract consistency: viscous liquid

c) Further extractions:

Arctostaphylos uva-ursi:

Used part of the plant: Leaves of the Arctostaphylos uva-ursi Extraction liquid: water

Extraction consistency: powder, ingredient arbutin

Stone flower (Flor de Piedra): Used part of the plant:

Extraction liquid: 69% ethanol (v / v) Extract consistency: liquid Drying residue: 1.0%

propolis:

Propolis is a mixed product of various materials which collects bees from pollen and tree bark, as well as secretions of the bees themselves. Extraction liquid: propylene glycol Extraction consistency: liquid

Primula Veris: Used part: Leaves and root of Primula veris Extraction liquid: 50% ethanol (v / v) Extract consistency: Powder

Maqnolia officinalis:

Used part of the plant: bark

Extraction liquid: alcohol and water

Extraction consistency: Powder

Celery (Apium qraveolens):

Used part of the plant: tuber

Extraction liquid: unknown

Extraction consistency: oily liquid Ingredient 9% celery oil

The lipase-inhibiting substances in the plant extracts have not yet been identified.

Description of the preparation and the methods for the determination of enzyme activities.

Preparation of the test solution

To prepare the test solution from extracted powder and liquid material, 200 mg of the extracted material is dissolved in 1 ml of a suitable solvent and stirred vigorously. After centrifuging at 3400 revolutions for 5 minutes, the obtained liquid constituent is used as test solution. To customize the

The ratio between the concentration of solid components and the enzyme mass in the last experiment became the test solution in Ratio 1: 200 diluted to determine the alkaline phosphatase.

To prepare the test solution from CO ₂ extracts and oil resins, 100 μl of oily liquid extract are dissolved in 900 μl of a suitable enzyme buffer and treated with ultrasound at 100 watts for 5 minutes.

Pancreatic lipase (pancreatic lipase)

Two lipase determinations were performed to determine the activity of pancreatic lipase in vitro:

1. The assay for the determination of lipase activity consists of

- 200 μl of a 50 mM / L BICIN buffer (pH 8.0) containing 1% technical gum arabic.

- 50 μl of plant extract solution and - 25 μl of a 0.35 mM / L pancreatic lipase solution

This mixture was incubated at 37 ° for 20 minutes. The final measurement of pancreatic lipase activity is performed on a clinical chemistry autoanalyser (Konelab, Kone) using a lipase colourimeter assay with a diacylglycerol with methylresorufin third position as substrate and colipase and bile salts as essential cofactors. This method is specific for the prancreatic lipase and is based on the cleavage of the methylresorufin from the substrate by the enzymes.

For the determination of the pancreatic lipase activity in the autoanalyzer the following pipetting scheme was chosen: 1. 60 μl of reagent 1: colipase and cholate

2. 2 μl test solution + 10 μl wash solution

3. 180 seconds incubation

4. 40 μl reagent 2: colorimetric substrate and cholate 5. 120 seconds incubation

The lipase activity was determined from 12 measurement points after 83 seconds. The methylresorufin was measured photometrically at 75 nanometers.

2. Lipase inhibition under the influence of plant extracts was determined by a second lipase activity measurement method by measuring the release of the free fatty acid during the hydrolysis from triolein by the enzyme. To prepare the incubation mixture becomes a ready to use

Solution (reagent R1) used. R1 consists of:

26 mmol / L Tris buffer, pH 9.2 19 mmol / L sodium desoxycholate 0.1 mmol / L calcium chloride

0.3 mmol / L triolein 300 KU / L colipase

The incubation mixture is composed as follows:

50 μL 26 mmol / L Tris buffer, pH 9.2

50 μL 9.7 mmol / L triolein emulsion in R1

50 μL 0.134 mol / L pancreatic lipase in 26 mmol / L Tris buffer pH

9.2 50 μL test solution 1% methyl cellulose in 26 mmol / L Tris buffer (pH 9.2) was used in the study of CO ₂ extracts.

After an incubation period of 30 minutes at 37 ° C. in a water bath, the reaction was carried out by adding 3 ml of a chloroform

Heptane-methanol mixture (50: 49: 1) stopped. The liberated free fatty acids were extracted from the reaction mixture by vigorous stirring for 10 minutes. After centrifugation (3400 revolutions, 10 minutes), a 2 ml residue is recovered from the chloroformate and 1 ml of a copper reagent (94 ml of pure water, 6 ml of 1 N NaOH, 6.45 mmol / L per Cu (NO ₃₎ , 0.1 mol / L triethanolamine, 33% (w / v) NaCl) is added, this mixture is stirred vigorously for 10 minutes and centrifuged at 3400 rpm for 10 minutes, 1 ml of the supernatant fluid is removed and 1 ml Chloroform at 0.1% bathocuproin and

0.05 (w / v) of 3-tert-butyl-4-hydroxyanisole was added. The fatty acid-copper-bathocuproine complex is finally determined photometrically at 480 nm in a standard photometer.

To ensure that the decrease in lipase activity is not due to a non-specific enzyme inhibitor or denaturing of the enzyme complex, the activity of the α-amylase and the alkaline phosphatase were additionally determined. Inhibition of α-amylase may act as a reduction in postprandial blood glucose levels and is considered to be an additional beneficial effect for diabetic patients.

α-Amylase Amylase activity measurement under the influence of the plant extracts was carried out as follows: The incubation mix for the determination of the amylase activity consists of a total of 250 μL:

-200 μL 50 mM / L MOPSO buffer (pH 8.0) with 1% gum arabic -25 μL plant extract solution and

-25 μL of a 0.35 mM / L α-amylase solution

This mixture was incubated at 38 ^° C. for 20 minutes. The final measurement of pancreatic lipase activity is performed in an autoanalyzer for chemical chemistry (Konelab,

Kone).

Principle of the chemical reaction:

1. ethylene-p-nitrophenol (glucose) ₇ → Ethyien- (glucose) 3-4 ⁺ p-nitrophenol (glucose) _2- 4

2. p-nitrophenol (glucose) residues → p-nitrophenol (λ = 405 nm) + glucose

The pipette method in the autoanalyzer for the determination of α-amylase activities was carried out as follows:

1. 120 μL reagent: substrate + α-glucosidase

2. 300 seconds incubation

3. 3 μL test solution 4. 180 second incubation

The α-amylase activity is determined by measuring 5 points in 120 seconds. Alkaline phosphatase

The alkaline phosphatase activity measurement under the influence of plant extracts was carried out as follows:

The incubation mix for the determination of lipase activity consisted of

Containing 200 μL of a 50 mM / L HEDTA buffer (pH 8.0),

- 25 μL plant extract test solution and

- 25 μL of a 0.014 mM / L alkaline phosphatase solution

The mixture was incubated for 20 minutes at 22 ° C. The final measurement of pancreatic lipase activity was performed in a clinical chemistry autoanalyzer (Kone lab, Kone).

reaction equation

2-amino-2-methyl-1-propanol + 4-nitrophenyl phosphate ester → (2-amino-2-methyl-1-propanool) phosphate + 4-nitrophenoxide (λ = 409 nm)

The pipette method in the autoanalyzer for determining the alkaline phosphatase activity was as follows:

1. 150 μL reagent

2. 300 sec. Incubation

3. 3μL test solution

4. 120 sec. Incubation

The activity of the alkaline phosphatase was determined by means of 5

Measuring points in 22 seconds. Verification in animal experiments

For further verification and verification of the results found in vitro, the triglyceride-lowering effect was tested in animal experiments.

For this, a lipid challenge test in rats with Triton WR-1339 was performed to inhibit triglyceride elimination from the blood, and the postprandial increase in plasma triglyceride levels over four hours was measured. The following test substances were used:

Proteins and peptides

Protamine: strongly basic nuclear protein (of 32 AS 21 x Arg), derived from fish sperm, triglyceride lowering (Tsujita et al., 1996)

- Peptide (D4 globin digest): enzyme. Bovine erythrocyte globin hydrolyzate, triglyceride lowering (Kagawa et al., 1996, 1998)

WYP: f32-35 of the β chain of bovine hemoglobin, triglyceride decreasing (Kagawa et al., 1996, 1998)

- LVYP: f73-76 bovine β-caseins

extracts

- Ethanolic extracts - Leaves of Tabebuia impetiginosa (Lapacho)

- Arctium lappa L (burdock)

- seeds of Linum usitatissimum (flax)

- seeds of helianthus annuus (sunflower)

- Calendula officinalis (Marigold) - oleoresin

- seeds of Carum carvi (caraway) - CO ₂ extracts

- fruit with peel of Elettaria cardamomum (cardamom)

- leaves of Thymus vulgaris (thyme)

- Propolis (beeskin gum) orlistat as a positive control

As a result, the increase in plasma postprandial triglyceride levels in rats after administration of the test substances was detected. It was found that neither the protamine, peptide D4 nor the tetrapeptides a triglycerides lowering effect was found. Likewise, no desired effect was found even in some extracts in this experiment. These include flaxseed, sunflower, calendula and propolis.

With other extracts, however, a partially very clear

Reduction of triglycerides are observed; these include cumin with a reduction of 3% ± 9%, thyme with a reduction of 7% ± 7%, burdock with a reduction of 12% ± 6%, cardamom with a reduction of 13% ± 7% and in particular Outstanding the Lapachotee extract with a significant decrease of 16% ± 7%. The experiment did not differentiate which ingredients of the extracts mediate the overall observed effect. There is still a thematic area for carrying out further experiments in the animal sector, and there is a reference to the requirements of human studies.

In general, it was possible to demonstrate with this animal experiment that some of the active ingredients characterized in vitro as being active according to the invention actually also showed a similar effect in an animal experiment. It also became clear that this effect is by no means linearly transferable and in some cases even overlaid by other influences, which in extreme cases tion of the effect. However, it can be deduced from this experiment that a further review of the substances listed here as worthy of protection in the sense of a specific lipase inhibition makes sense and can be expected to yield very concrete results. In the following, further details and features of the invention will be explained in more detail with reference to two examples. However, these are not intended to limit the invention but merely to explain. It shows in a schematic representation:

Figure 1 Increase in plasma postprandial triglyceride levels in rats after administration of the test substances

Figure 2 Postprandial triglyceride levels in rat plasma after a lipid performance test.

The figures show in detail:

Figure 1 tabulates the effect of the substances administered to the rats about 0.05 hours after ingestion in relation to the triglyceride level. A distinction is made between the proteins and peptides as well as the extracts of natural active ingredients. Significantly, the proteins and peptides in this experimental configuration do not lead to the desired reduction but in part to a marked increase in

Triglyceride values resulted. This also applies to some natural active ingredients. However, other natural substances are effective in the desired direction. Significant here is the effect of the lapachote from the inner bark of the Taheebo tree (Tabe buia impetiginosa). In FIG. 2, the curves of the triglyceride values are plotted as a function of the contact time after ingestion. As expected, triglyceride levels are lowest when administering orlistat. However, in the case of the positive-action extracts already listed in FIG

Hours a desired reduction of the triglycerides can be achieved. The burdock, lapachotee and cardamom have been shown to be effective. The comparatively small increase in triglyceride levels when administering orlistat is in line with expectations and proves that the animal experiment was carried out in the past

Specification is relevant to the invention.

Claims

claims

1. Use of the following substances, substance extracts or substances as a single component or as a mixture for the preparation of an orally administered medicament which acts as a lipase inhibitor:

Cardamom and / or

Marjoram and / or thyme and / or

Chili (Capsicum frutescens) and / or

Evening Primrose and / or

Oregano leaves (oreganum vugare) and / or

Chamomile leaves (Chamomilla recutica) and / or nutmeg (Myristica fragrans) and / or

Applesauce (pomace) and / or

Grain germ and / or

Almond and / or

Hazelnut and / or walnut and / or

Pumpkin germ and / or

Cuphea wrightii and / or

Evening Primrose and / or

Oil flax (Linum usitatissimum) and / or large burdock (Arctium lappa L) and / or

Flax and / or

Germs of Helianthus annuus (sunflower) and / or

Marigold (Calendula officinalis) and / or

Echinops banaticus and / or Mallotus philippinensis and / or

Rapeseed, and / or

Sesame and / or black caraway and / or

Cynoglossum officinale and / or Lapachotea and / or

Arctostaphylos uva-ursi and / or

Stone flower (Flor de piedra) and / or

Propolis and / or

Primula veris and / or Magnolia officinalis and / or

Celery (Apium graveolens)

2. Use of the following substances, substance extracts or substance essences as a single component or mixture

Cardamom and / or

Marjoram and / or thyme and / or

Chili (Capsicum frutescens) and / or

Evening Primrose and / or

Oregano leaves (oreganum vugare) and / or

Applesauce (pomace) and / or

Grain germ and / or

Almond and / or

Hazelnut and / or walnut and / or

Pumpkin germ and / or

Cuphea wrightii and / or

Evening Primrose and / or

Flax and / or

Germs of Helianthus annuus (sunflower) and / or

Marigold (Calendula officinalis) and / or

Echinops banaticus and / or Mallotus philippinensis and / or

Rapeseed, and / or

Sesame and / or black caraway and / or

Cynoglossum officinale and / or Lapachotea and / or

Arctostaphylos uva-ursi and / or

Stone flower (Flor de piedra) and / or

Propolis and / or

Primula veris and / or Magnolia officinalis and / or

Celery (Apium graveolens) as a lipase inhibitor.