METHOD FOR DELIPIDATING PLANT EXTRACTS

Abstract

The present invention is directed to a method for preparing plant extracts comprising the steps of (i) preparing a soluble extract from (a) plants or parts thereof and (b) an aqueous, alcoholic or aqueous alcoholic mixed extractant; and removal of insoluble components, (ii) concentrating the extract to a dry substance content at which the contained lipids and lipoids form suspended and/or emulgated aggregates, (iii) separating the suspended and/or emulgated aggregates by microfiltration from the concentrated extract, and (iv) optionally removing the extractant. The invention further pertains to an extract and composition comprising said extract as prepared by this method.

Claims

1. A method for preparing plant extracts, the method comprising the steps of: (i) preparing a soluble extract from (a) plants or parts thereof and (b) an aqueous, alcoholic or aqueous alcoholic mixed extractant; and removal of insoluble components; (ii) concentrating the extract to a dry substance content at which the contained lipids and lipoids form suspended or emulgated aggregates; and (iii) separating the suspended or emulgated aggregates by microfiltration from the concentrated extract.

2. The method of claim 1, wherein the membrane for the microfiltration of step (iii) has a pore size of about 0.01 to about 50 .Math.m, or a pore size of about 0.02 to about 10 .Math.m.

3. The method of claim 2, wherein the microfiltration of step (iii) is a crossflow filtration.

4. The method of claim 1, wherein the dry substance content in step (ii) is about 5 to about 50 wt-%.

5. The method of claim 1, wherein the suspended or emulgated aggregates formed in step (ii) have a diameter of about 100 nm to about 1000 .Math.m.

6. The method of claim 1, wherein the suspended or emulgated aggregates of step (ii) comprise one or more lipids or lipoids, optionally.

7. The method of claim 1, wherein the extractant in step (i) is selected from the group consisting of water, alcohols selected from the group consisting of C.sub.1-5alcohols including structural isomers thereof, methanol and ethanol; and aqueous alcoholic mixtures of C.sub.1-5alcohols including structural isomers thereof.

8. The method of claim 1, wherein the plants or plant parts are selected from the group consisting of Hypericum sp., Hedera sp., Olea europaea, Allium sp., optionally A. cepa L. , A. sativum L. , Arctostaphylos uva-ursi Spreng, Armoracia sp., Artemisia absinthium L , Artemisia annua L , Betula sp., Brassica sp., Camellia sinensis (L.) O. Kuntze, Cassia angustifolia Vahl, Cassia senna L. , Cestrum-diumum; Cucurbita sp.. Echinacea sp., Ginkgo biloba L. , Humulus lupulus L. , Hypericum perforatum L. , Ilex paraguariensis St. Hil., Laurus nobilis L. , Marrubium vulgare L. , Matricaria recutita L. , Melissa officinalis L. , Mentha species, Moringa oleifera (Syn.: Guilandina moringa), Orthosiphon stamineus Benth., Passiflora incarnata L. , Petasistis hybridus L. , Piper sp., Salvia sp., Solanum glaucophyllum, Thymus vulgaris L. , Tropaeolum sp, and Vitex agnus castus L .

9. An extract prepared by a method of claim 1.

10. A composition comprising an extract according to claim 9.

11. A method for the treatment of a disease in a subject in need thereof comprising the step of administering a composition according to claim 10 to said subject.

12. The method of claim 1, further comprising step (iv) and removing the extractant.

13. The method of claim 3, wherein the microfiltration of step (iii) is a two-step microfiltration.

14. The method of claim 13, wherein the microfiltration of step (iii) is a first microfiltration with a pore size of about 0.8 to about 10 .Math.m, followed by a second microfiltration with a pore size of about 0.01 to about 0.8 .Math.m.

15. The method of claim 13, wherein the microfiltration of step (iii) is a first microfiltration with a pore size of about 0.5 to about 2 .Math.m or about 0.5 to about 1.5 .Math.m, followed by a second microfiltration with a pore size of about 0.1 to about 0.6 or a pore size of about 0.2 .Math.m.

16. The method of claim 1, wherein the dry substance content in step (ii) is about 3 to about 30 wt% or about 6 to about 18 wt%.

17. The method of claim 1, wherein the suspended or emulgated aggregates formed in step (ii) have a diameter of about 200 nm to about 50 .Math.m or about 500 nm to about 10 .Math.m.

18. The method of claim 6, wherein the suspended or emulgated aggregates of step (ii) comprise one or more epicuticular lipids or lipoids selected from the group consisting of waxes, C.sub.16-36 fatty acid esters, C.sub.24-28 wax alcohols, C.sub.28,30,33,36 paraffins, C.sub.16-36 carbohydrates, hydrophobic aliphatic compounds, phytosterines, and phytosteroles.

19. The method of claim 7, wherein the extractant is an aqueous alcoholic mixture of C.sub.1-5 alcohols including structural isomers thereof with the alcohol present in concentrations of 5 to 90, 10 to 80 or 30 to 70 wt% alcohol content.

Description

[0032] In the following the invention will be illustrated by representative examples, none of which are to be interpreted as limiting the scope of the invention beyond the appended claims.

EXAMPLE 1

Process Step 1

[0033] 2505 kg dried and cut (cutting size 80% < 2.5 mm) St John’s wort (PhEur) were introduced into 8 parts by weight 50 wt-% ethanol. This mixture was stirred in a flask with stirring, heating and cooling apparatus at a temperature of 50±10° C. for 50±15 min. Then it was filtered over a band filter. The obtained liquid extract was concentrated at 100 mbar (max. 200 mbar) and 50° C.±15° C. in a vacuum evaporator to a dry substance content of 20±10 wt-%t. 3947 kg of turby, thin liquid extract with a dry substance weight of 14.97 wt-% resulted. The extract was left at room temperature for 24 h to settle slurry-like sediments. Subsequently, the fluid was decanted and the sediments discarded. An opaque, emulsion-like liquid with remaining suspended components resulted.

Process Step 2

[0034] Of the above material 2000 kg (±10 wt-%) extract were subjected to a crossflow filtration via a polypropylene membrane with a pore size of 1 .Math.m (Seprodyn®) at 20±10° C. and 1±1 bar. An opaque, emulsion-like liquid resulted.

Process Step 3

[0035] The obtained permeate of 1850 kg (±10 wt-%) was then microfiltered by crossflow filtration via a polyethylene membrane with a pore size of 0.2 .Math.m (Microdyn®). A clear fluorescing (in transmitted light) liquid resulted.

Process Step 4

[0036] The obtained permeate was then concentrated in an evaporator at increased temperature and reduced pressure to a dry substance content of 50 wt-% (±10%). The obtained thick extract was converted into a dry extract by spray band drying.

Analytical Data

[0037] 273 kg dry extract with a hypericin content (PhEur) of 0.22 wt-% and a flavonoid content (PhEur) of 10.15 wt-% resulted. In order to verify if the two microfiltrations completely or essentially completely preserved the desired ingredients samples were repeatedly taken from the permeates and retentates and analyzed for their content of hypericins and flavonoids.

Filtration Step Seprodyn

[0038] In the permeate of the Seprodyn filtration the content of hypericins averaged 0.25 wt-%, and for flavonoids 9.87 wt-% relative to the dry substance. In the retentate of the Seprodyn filtration 0.25 wt-% hypericins and 9.87 wt-% flavonoids were detected.

Filtration Step Microdyn

[0039] In the permeate of the Microdyn filtration the content of hypericins averaged 0.25 wt-% and for flavonoids 10.17 wt-% relative to the dry substance. In the retentate of the Microdyn filtration 0.23-0.26 wt-% hypericins and 9.99-10.17 wt-% flavonoids were detected.

[0040] These contents demonstrate that the filtrations do not reduce the desired ingredients. Consequently, a 100% or almost 100% yield of the desired ingredients resulted.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0041] A sample of 10.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magnetic stir bar and intensely stirred with 10 parts petroleum ether for 2 hours at 500 rpm and room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (300-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask did not show any added weight and the sample did not contain any petroleum ether soluble lipoids/lipids.

EXAMPLE 2 (COMPARISON)

Process Step 1

[0042] 9362 kg dried and cut (cutting size 80% < 2.5 mm) St John’s wort (PhEur) were introduced into 8 parts by weight 50 wt-% ethanol. This mixture was stirred in a flask with stirring, heating and cooling apparatus at 50±10° C. for 50±15 min. Then it was filtered over a band filter. The obtained liquid extract was concentrated at 100 mbar (max. 200 mbar) and 50±15° C. in a vacuum evaporator to a dry substance content of 20±10 wt-%. 7798 kg of turby, thin liquid extract with a dry substance content of 25.0 wt-% resulted. The extract was left at room temperature for 24 h to settle slurry-like sediments. Subsequently, the fluid was decanted and sediments discarded. An opaque, emulsion-like liquid with suspended components resulted.

Process Step 2

[0043] Then the extract was concentrated in an evaporator at elevated temperature and reduced pressure to a dry substance content of 50±10 wt-%. The obtained thick extract was transformed into a dry extract by spray band drying.

Analytical Data

[0044] 1873 kg dry extract with a hypericin content (PhEur) of 0.12 wt-% and a flavonoid content (PhEur) of 8.4 wt-% resulted.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0045] A sample of 10.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magentic stir bar and intensly stirred with 10 parts petroleum ether for 2 hours at 500 rpm and room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (300-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask showed an added weight of 0.43 g and the sample contained 4.3 wt-% petroleum ether soluble lipoids/lipids. The example 2 contained 4.3 fold the amount of lipoids/lipids compared to example 1.

EXAMPLE 3

[0046] 273.9 kg dry extract obtained according to example 1 were mixed with 1.5 kg highly dispersed silicon dioxide (Aerosil®), 22.2 kg polyoxyethylenglycol (PEG 6000) and 2.4 kg magnesium stearate resulting in 300 kg of a pelleting mixture. 300 g of this mixture were pressed to tablet cores with a weight of 539.9 mg. These tablet cores were tested for hardness, disintegration and release time (based on the release of total hypericins in a dissolution test model: required: >75% release within 60 min). The tablets had a hardness of 76 Newton, disintegrated completely in 9.49 min and released within 20 min over 75% of the total hypericin active agents.

EXAMPLE 4 (COMPARISON)

[0047] 273.9 kg dry extract obtained according to example 2 (comparison) were mixed with 1.5 kg highly dispersed silicion dioxide (Aerosil®), 22.2 kg polyoxyethylenglycol (PEG 6000) and 2.4 kg magnesium stearate resulting in 300 kg of a pelleting mixture. 300 g of this mixture were pressed to tablet cores with a weight of 543.3 mg. These tablet cores were tested for hardness, disintegration and release time (based on the release of total hypericins in a dissolution test model: required: >75 % release within 60 min). The tablets had a hardness of 46 Newton, disintegrated completely in 11.32 min and released 34% of the total hypericin active agents within 60 min.

EXAMPLE 5

Process Step 1

[0048] 2 kg liquid extract of St John’s wort with a dry substance content of 15 wt-% obtained according to example 2 (comparison, process step 1) were diluted with drinking water (aqua fontana, PhHelv) to a dry substance content of 10 wt-%. An opaque, emulsion-like liquid with suspended components resulted.

Process Step 2

[0049] Of the above material 3 kg (±10 wt-%) liquid extract were subjected to a crossflow filtration via a polypropylene membrane with a pore size of 1 .Math.m (Seprodyn®) at 20±10° C. and 1±1 bar. An opaque, emulsion-like liquid resulted.

Process Step 3

[0050] The obtained permeate was then microfiltered by crossflow filtration via a polyethylene membrane with a pore size of 0.2 .Math.m (Microdyn®). A clear fluorescing (in transmitted light) liquid resulted.

Analytical Data

[0051] The employed liquid extract and both permeates were analyzed for their contents of total hypericins and flavonoids. The liquid extract contained 0.227 wt-% total hypericins and 7.87 wt-% flavonoids relative to the dry substance content. The Seprodyn permeate comprised 0.23 wt-% total hypericins and 7.86 wt-% total flavonoids, and the Microdyn permeate comprised 0.22 wt-% total hypericins and 8.00 wt-% flavonoids relative to the dry substance content. Consequently, a 100 % or almost 100% yield of the desired ingredients resulted.

EXAMPLE 6

Process Step 1

[0052] 1500 g dried and cut (cutting size 80% < 5 mm) olive leaves (PhEur) were introduced into 10 parts by weight 70 wt-% ethanol. This mixture was stirred in a flask with stirring, heating and cooling apparatus at 50±10° C. for 50±15 min. Then it was filtered over a layer filter. The obtained liquid extract was concentrated under reduced pressure (300-100 mbar) and 50±15° C. in a vacuum evaporator to a dry substance content of 25 (20-35) wt-%. 2128 g of turby, thin liquid extract with a dry substance weight of 20.4 wt-% resulted. An opaque, emulsion-like liquid with suspended components resulted.

Process Step 2

[0053] This liquid emulsion was divided in two portions of 1064 g. For example 6 the liquid was diluted with the equal amount of tap water (aqua fontana) to a dry substance content of 10±5 wt-% and the resulting turby, thin liquid extract was subjected to a crossflow filtration via a polypropylene membrane with a pore size of 1 .Math.m (Seprodyn®) at 20±10° C. and 1±1 bar. The death volume of the equipment was roughly 500 ml. An opaque, emulsion-like liquid of 1571 g was obtained.

Process Step 3

[0054] The obtained permeate of 1571 g (±10 wt-%) was then microfiltered by crossflow filtration via a polyethylene membrane with a pore size of 0.2 .Math.m (Microdyn®) at 20±10° C. and 1±1 bar. The death volume of the equipment was roughly 500 ml. A clear brown shining (in transmitted light) liquid resulted.

Process Step 4

[0055] The obtained permeate was mixed with 20 wt-% maltodextrin (PhEur) and then concentrated in an evaporator at increased temperature and reduced pressure to dryness and the obtainned dry extract was milled.

Analytical Data

[0056] The obtained beige-brown powder extract of 111.82 g dry extract had an oleuropein content (PhEur) of 19.31 wt-%. In order to verify if the two microfiltrations completely or essentially completely preserved the desired ingredient dry extract samples were taken and the content compared with the comparison sample described in example 7.

[0057] These contents demonstrate that the filtrations do not reduce the desired ingredient. Consequently, a 100 % or almost 100% yield of the desired ingredient resulted.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0058] A sample of 3.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magnetic stir bar and intensely stirred with 100 ml petroleum ether for 30 min at room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (900-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask did show an added weight of 0.006 g, corresponding to a minimal content of petroleum ether soluble lipoids/lipids of 0.2% wt.

EXAMPLE 7 (COMPARISON)

Process Step 1

[0059] 1064 g of the opaque, emulsion-like liquid with suspended components from the end of process step 1 of example 6 were taken for comparison example 7, to allow a direct comparison.

Process Step 2

[0060] The obtained liquid extract was mixed with 20 wt-% maltodextrin (PhEur) relative to the dry substance content and then concentrated in an evaporator at increased temperature and reduced pressure to dryness and the obtained dry extract was milled.

Analytical Data

[0061] 208.6 g of a greenish-brown dry extract had an oleuropein content (PhEur) of 17.52 wt-%.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0062] A sample of 3.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magnetic stir bar and intensely stirred with 100 ml petroleum ether for 30 min at room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (900-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask showed a weight increase of 0.043 g, corresponding to a content of petroleum ether soluble lipoids/lipids of 1.43 wt-% or a 7-fold higher content compared to example 6.

EXAMPLE 8

Process Step 1

[0063] 1700 g dried and cut (cutting size 80% < 2 mm) ivy leaves (PhEur) were introduced to 10 parts by weight 40 wt-% ethanol. This mixture was stirred in a flask with stirring, heating and cooling apparatus at 50+10° C. for 50+15 min. Then it was filtered over a layer filter. The obtained liquid extract was concentrated under reduced pressure (300-100 mbar) and 50+15° C. in a vacuum evaporator to a dry substance content of 10 (5-15) wt-%.

[0064] 4970 g of turby, thin liquid extract with a dry substance weight of 6.85 wt-% resulted. An opaque, emulsion-like liquid with suspended components resulted.

Process Step 2

[0065] This liquid emulsion was divided into two equal portions (1 portion for comparison example 9) , and one portion of the thin liquid extract was subjected to a crossflow filtration via a polypropylene membrane with a pore size of 1 .Math.m (Seprodyn®) at 20+10° C. and 1+1 bar. An opaque, emulsion-like liquid resulted.

Process Step 3

[0066] The obtained permeate of 1600 g (± wt-%) was then microfiltered by crossflow filtration via a polyethylene membrane with a pore size of 0.2 .Math.m (Microdyn®) at 20±10° C. and 1±1 bar. A clear brown shining (intransmitted light) liquid resulted.

Process Step 4

[0067] The obtained brilliant clear permeate was mixed with 20 wt-% maltodextrin (PhEur) relative to the dry substance content and then spray dried. Subsequently the obtained dry extract was milled.

Analytical Data

[0068] 57.8 g dry extract had a hederacoside C content (PhEur) of 12.29 wt-%.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0069] A sample of 3.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magnetic stir bar and intensely stirred with 100 ml petroleum ether for 30 min at 500 rpm and room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (900-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask did show an added weight of 0.006 g corresponding to a minimal content of 0.2 wt% petroleum ether soluble lipoids/lipids.

EXAMPLE 9 (COMPARISON)

Process Step 1

[0070] 2485 g of the opaque, emulsion-like liquid with suspended components from end of process step 1 of example 8 were taken for the comparison example 9, to allow a direct comparison.

Process Step 2

[0071] The obtained cloudy liquid extract was mixed with 20 wt-% maltodextrin (PhEur) relative to the dry substance content and spray dried. The obtained dry extract was milled.

Analytical Data

[0072] 104.3 g dry extract with a hederacoside C content (PhEur) of 13.77 wt-% resulted.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0073] A sample of 3.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magnetic stir bar and intensely stirred with 100 ml petroleum ether for 30 min at 500 rpm and room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (900-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask had an added weight of 0.012 g and did contain 0.4 wt-% petroleum ether soluble lipoids/lipids calculated on dry extract. This is a 2-fold content compared to example 8.

EXAMPLE 10

Process Step 1

[0074] 2000 g dried and cut (cutting size 80% < 2.5 mm) St. Johns wort (PhEur) were introduced to 10 parts by weight 50 wt-% ethanol. This mixture was stirred in a flask with stirring, heating and cooling apparatus at 50±10° C. for 40±15 min. Then it was filtered over a layer filter. The obtained liquid extract was concentrated under reduced pressure (300-100 mbar) and 50+15° C. in a vacuum evaporator to a dry substance content of 13.39 (5-15) wt-%.

[0075] 2560 g of turby, thin liquid extract with a dry substance weight of 6.85 wt-% resulted-The extract was left at room temperature for 24 h to settle slurry-like sediments. Subsequently the fluid was decanted and the sediments discarded. An opaque, emulsion-like liquid with suspended components resulted.

Process Step 2

[0076] This liquid emulsion was divided into two equal portions (1 portion for comparison example 11) and one portion of the thin liquid extract was subjected to a crossflow filtration via a polypropylene membrane with a pore size of 1 .Math.m (Seprodyn®) at 20+10° C. and 1±1 bar. An opaque, emulsion-like liquid resulted.

Process Step 3

[0077] The obtained permeate was then microfiltered by crossflow filtration via a polyethylene membrane with a pore size of 0.2 .Math.m (Microdyn®) at 20±10° C. and 1±1 bar. A clear brown shining (in transmitted light) liquid resulted.

Process Step 4

[0078] The obtained brilliant clear permeate was mixed with 20 wt-% maltodextrin (PhEur) relative to the dry substance content and then vacuum oven dried. Subsequently the obtained dry extract was milled.

Analytical Data

[0079] The resulting dry extract had a Hypericin content (PhEur) of 0.196 wt-% and a flavonoid content (PhEur) of 7.49 wt%.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0080] A sample of 3.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magnetic stir bar and intensely stirred with 100 ml petroleum ether for 30 min at 500 rpm and room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (900-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask did show an added weight of 0.003 g corresponding to a minimal content of 0.1 wt% petroleum ether soluble lipoids/lipids.

EXAMPLE 11 (COMPARISON)

Process Step 1

[0081] The second portion of the opaque, emulsion-like liquid with suspended components from end of process step 1 of example 10 were taken for the comparison example 11, to allow a direct comparison.

Process Step 2

[0082] The obtained liquid extract was mixed with 20 wt-% maltodextrin (PhEur) relative to the dry substance content and vacuum oven dried. The resulting dry extract was milled.

Analytical Data

[0083] A dry extract with a Hypericin content (PhEur) of 0.197 wt % and a Flavonoid content (PhEur) of 7.366 wt % resulted.

Test for Petroleum Ether Soluble Lipoids/Lipids

[0084] A sample of 3.0 g of the dry extract was transferred to an Erlenmeyer flask with ground-in stopper and magnetic stir bar and intensely stirred with 100 ml petroleum ether for 30 min at 500 rpm and room temperature. Filtration by means of a cellulose folded filter followed and the obtained petroleum ether phase was evaporated in a tared flask to dryness (900-50 mbar, 40° C.). The tared flask was then weighed on an analytical scale. The flask had an added weight of 0.027 g and did contain 0.9 wt-% petroleum ether soluble lipoids/lipids calculated on dry extract. This is a 9-fold higher content compared to example 10.

Summary of Results

[0085] The above presented examples demonstrate that the method of the present invention allows for the quantitative or almost quantitative separation of suspended or emulsified aggregates of lipids and/or lipoids from aqueous, alcoholic or aqueous alcoholic plant extracts. The separation of those lipids and/or lipoids is specific and does not lead to a significant decrease of extracted components. The effective separation of the undesired lipids or lipoids leads to signifycantly improved release kinetics of the final drug form, for example, because lipids or lipoids cause auto-retardation.