WO2010034971A2

WO2010034971A2 - Sub-critical water extraction of medicinal plants

Info

Publication number: WO2010034971A2
Application number: PCT/GB2009/002229
Authority: WO
Inventors: Gary William Wheatley; Kenneth Davison
Original assignee: Gary William Wheatley; Kenneth Davison
Priority date: 2008-09-23
Filing date: 2009-09-17
Publication date: 2010-04-01
Also published as: WO2010034971A3

Abstract

The patent describes the application of Sub-Critical Water Extraction (SWE) to a range of medicinal plants traditionally used in Europe or Asia to produce pharmaceutical extracts. The extraction of the following plants is covered by the scope of this patent: Rosmarinus officinalis (rosemary) and related species Matricaria recutita (German chamomile) and related species Cassia angustifolia (senna) and related species Valeriana officinalis (valerian) and related species Scutellaria baicalensis (Baikal skullcap) and related species Schisandra chinensis (Wuweizu) and related species. Zingiber officinale (ginger) and related species. Astragalus Membranaceus and related species. It is demonstrated that SWE can produce materials essentially similar, as indicated by chromatographic fingerprint, to extracts produced using conventional solvents with polarity in the range approximately represented by 50% aqueous ethanol through to methanol. Thus as the subcritical water extracts of the herbs as described in this invention are demonstrated to exhibit a composition essentially similar to that of the corresponding methanol or aqueous alcohol extract, then it can be expected that these extracts will exhibit comparable pharmacological activities to these and to the compounds contained therein. In addition to the above claims relating to the extraction of the plant material, as the water solubility of many of the active compounds is low, both oral formulations and topical formulations which improve the bioavailability and hence efficacy of the extracts are included.

Description

Sub-critical Water Extraction of Medicinal Plants.

FIELD OF THE INVENTION

The present invention relates to a method of producing an extract from medicinal plants without the use of an organic solvent. In particular the present invention relates to a novel method of producing extracts comparable in composition to those produced using a lower alcohol or water alcohol mixtures by using extraction with sub-critical water.

BACKGROUND TO THE INVENTION.

Higher plants are unsurpassed in their ability to produce biologically active molecules often of great complexity. These plant "secondary metabolites" have proven a fruitful source of new pharmaceuticals. Often the first use of these compounds has been in the form of traditional herbal medicines. However the structure of the active compounds is frequently so complex that synthetic chemistry can provide no economically viable route to the molecules and hence these have to be extracted from the plant material.

Conventional aqueous extraction of the biomass almost invariably results in a complex extract in which the target compound is diluted by a range of other unwanted phytochemicals derived from the plant, including proteins, sugars and other carbohydrates.

Extraction with a lower alcohol, typically ethanol or methanol, is traditionally a very popular means of obtaining a pharmacologically active preparation from a medicinal plant. The use of an alcohol has the advantage over an aqueous extraction that the highly polar compounds such as proteins, carbohydrates et cetera, which generally are inactive and serve only to add bulk and dilute the concentration of the active compounds, are not extracted. However the obvious disadvantages of the use of an alcohol in extraction are mat it is flammable, necessitating expensive precautions during processing, and also the recovery of the organic solvent for either disposal or for re-use after further purification is required. Methanol in addition is highly toxic, and residual levels must be rigorously limited in a pharmaceutical preparation. Indeed due to the high toxicity of methanol, it is frequently substituted by a mixture of the more polar water and the less polar ethanol to produce an extraction solvent of comparable polarity.

A more recent approach that has also been successfully applied to the production of a high potency extract is extraction with a sub-critical fluid (in effect a gas liquefied by the application of high pressure) most commonly carbon dioxide. One drawback of liquid carbon dioxide (LCO₂) extraction is that the technique is limited to less polar compounds. It will not extract the compounds most readily soluble in methanol or aqueous ethanol, including the important classes of flavonoids, lignans and other related polyphenolic compounds. The importance of polyphenolic compounds as potential new drugs is increasingly being recognised; with anti-inflammatory activity, anti-microbial activity, estrogenic activity and a wide range of activities related to the suppression of tumour growth (including inhibition of angiogenesis and reversal of multidrug resistance) being well established. A more recent example of a high pressure liquefied gas which is being evaluated as an extraction solvent and which promises capabilities not exhibited by LCO₂ is high pressure superheated water at >150°C, termed "sub-critical water". Importantly, it is capable of solubilising more polar compounds than LCO₂ extraction, including flavonoids and other polyphenolics. Sub-critical water has the unique property that the polarity of the solvent decreases in a predicable manner as temperature is increased; thus at ISO⁰C the polarity of sub-critical water is equivalent to that of a 50:50 mixture of ethanol and water, and the polarity further decreases as the temperature is increased to 230⁰C to a value equivalent to that of pure methanol. A further advantage of sub-critical water extraction is that the high temperature and pressure produce high diffusion rates which promote very efficient extraction of the raw material

The traditionally used medicinal plants for which extraction with sub-critical water is described in this invention are all reported in the scientific literature to produce a methanol or aqueous alcohol extract exhibiting important pharmacological activities.

The methanolic or aqueous alcohol extracts of the aerial parts of Rosmarinus officinalis (rosemary) contain significant levels of rosmarinic acid and also diterpenes including carnosic acid, carnosol and rosmanol along with a range of fiavonoids. These compounds have all been demonstrated to exhibit anti-inflammatory actions and various activities inhibiting the growth of cancer cells. The diterpenes and fiavonoids have also been reported to inhibit P- glycoprotein and other cellular drug efflux pumps responsible for multi-drug resistance in cancer chemotherapy.

Both methanol and aqueous ethanol extracts of German chamomile (Matricaria recutita) exhibit significant anti-inflammatory properties which are believed to be related to the fiavonoids, typified by apigenin, and related glycosides. Fiavonoids such as apigenin are also known to exhibit useful properties in the adjunctive treatment of various cancers. The methanolic extract of Cassia angustifolia (senna) leaves contains high levels of the anthraquinone glycosides, typified by sennoside A, which are known to be responsible of the established laxative action of the plant.

The methanolic extract of Valerian officinalis root contains valerenic acid and related compounds that are now established as the principal active substances responsible for the sedative and anxiolytic action of the traditionally used extracts. Such extracts also contain the valepotriates and baldrinals which may also play a role in the pharmacology. Scutellaria baicalensis (baikal skullcap) is a rich source of flavonoids, the most important of these being wogonin and baicalein, and the glycosides derived from these. Both the methanolic extract containing high levels of these flavonoids and also the purified compounds themselves exhibit strong anti-cancer activities and anti-inflammatory action. The methanolic and aqueous alcohol extracts oϊSchisandra chinensis (Wuweizu) berries contain a complex range of dibenzocyclo-octadiene lignans, including schisandrin A and gomosin A, which are thought to be largely responsible for the anti-inflammatory and anticancer activities of extracts.

The gingerol and shoagoal families of phenolic compound present in methanol and aqueous alcohol extracts of Zingiber officinale (ginger) root exhibit both anti-inflammatory and anticancer activity.

Both methanol and aqueous ethanol extracts of Astragalus membranaceus root contain the polyphenolic compounds, especially isoflavones and tanshinones, which are thought to be responsible for the observed anti-cancer and anti-microbial activities.

Thus as the subcritical water extracts of the herbs as described in this invention are demonstrated to exhibit a composition essentially similar to that of the corresponding methanolic or aqueous alcohol extract, and in most cases also to contain the actual known active compounds, then it can be expected that these extracts will exhibit comparable pharmacological activities to these and to the compounds contained therein.

A problem limiting the use of flavonoids and related polyphenolic compounds in therapeutic situations in which systemic treatment rather than local application is required is poor water solubility. This is largely responsible for the low circulating plasma levels after a single oral dose and results in the majority of the dose being excreted without absorption.

Hence this invention also describes self emulsifying formulations which increase the water solubility of these compounds, thus increasing their oral bioavailability and therapeutic efficacy.

A further enteric release formulation is described which permits the generation of active compounds locally in the intestine, and which is exemplified by the case of the flavonoid rich skullcap precipitate which shows promise as an adjunctive treatment in multi-drug resistant colon cancer.

SUMMARY OF THE INVENTION.

The present invention was made in view of the prior art described above, and the object of the present invention is to provide a means of efficiently obtaining extracts with useful pharmacological properties from certain medicinal plants without the use of an organic solvent.

To solve this problem the present invention provides a method for the extraction using sub- critical water of selected medicinal plants to produce extracts of comparable composition to those obtained with methanol or aqueous alcohol mixtures.

Further, examples of both oral and topical formulations to improve the bioavailability and hence efficacy of the therapeutic components of the extracts are included in the invention.

The scope of the invention is illustrated by the following series of examples, although not in any limitative sense.

Example 1:

The extraction apparatus consists of :

1 Two suitable stainless steel vessels capable of resisting high temperature and pressure connected by stainless steel tubing, the first to act as a reservoir in which sub-critical water is produced prior to introduction to the second (extraction) vessel. These are contained in a thermostatted oven.

2 The first vessel is connected via an inlet valve to a high pressure pump outside the thermostatted oven.

3 The extraction vessel is connected via an outlet valve to a stainless steel receiver vessel outside the thermostatted oven, but which is maintained at approximately 90⁰C.

4 A valve from the receiver vessel allows the solution that accumulates to be transferred to a suitable storage vessel.

A schematic representation of a suitable arrangement of the extraction apparatus is provided as Figure 1.

Coarsely ground rosemary botanical raw material is packed into the stainless steel extraction vessel. The system is then filled with deionised water and the temperature and pressure of the extraction vessel raised gradually to a fixed temperature in the range 150-230⁰C, most preferably 150-200⁰C, and a pressure of 85 bar respectively. The extraction system is then held at these conditions for up to 15 minutes before the resulting solution is forced from the extraction vessel into the receiver by passing a quantity of sub-critical water into the system to continue the extraction. Typically a mass of sub-critical water equivalent to twenty times the mass of botanical raw material is passed through this over the course of up to two hours. Alternatively the same extraction may be achieved in a dynamic mode by passing the same quantity of sub-critical water continuously through the botanical raw material over the course of up to two hours.

The extract is isolated from the solution resulting from the extraction by removing the water by evaporation, preferably carried out under reduced pressure to reduce the temperature required.

Alternatively the extract may be isolated from the solution by removing the water by the process of either freeze drying or spray drying.

A TLC fingerprint representative of the extract (also termed a botanical drug substance) produced in this example is given in Figure 2. Example 2.

A method as described in example 1 wherein the botanical raw material is ground German chamomile flowers.

A TLC fingerprint representative of the extract produced in this example is given in Figure 3.

Example 3.

A method as described in example 1 wherein the botanical raw material is ground senna leaves.

A TLC fingerprint representative of the extract produced in this example is given in Figure 4.

Example 4.

A method as described in example 1 wherein the botanical raw material is milled valerian root.

A TLC fingerprint representative of the extract produced in this example is given in Figure 5.

Example 5.

A method as described in example 1 wherein the botanical raw material is milled Baikal skullcap root. Unexpectedly, when the solution from the extraction is allowed to slowly cool to ambient temperature a precipitate forms. This precipitate can be collected by filtration as a light brown powder. This powder is found to be the active flavonoid fraction of the plant extract, and the remaining dissolved portion is found to contain essentially no flavonoids. A TLC fingerprint representative of the precipitate and extract produced in this example is given in Figure 6a. HPLC analysis of the precipitate, as illustrated in Figure 6b, proves it to be a mixture of main three components, with 24.0% w/w baicalein and 13.8% w/w wogonin.

Example 6.

A method as described in example 1 wherein the botanical raw material is milled Schiscmdra chinensis berries.

A TLC fingerprint representative of the extract produced in this example is given in Figure 7.

Example 7.

A method as described in example 1 wherein the botanical raw material is milled ginger root. A TLC fingerprint representative of the extract produced in this example is given in Figure 8.

Example 8.

A method as described in example 1 wherein the botanical raw material is milled Astragalus root.

A TLC fingerprint representative of the extract produced in this example is given in Figure 9. Example 9.

A Self Emulsifying Drug Delivery System (SEDDS) formulation for oral administration of the extracts containing the poorly water soluble active compounds can be prepared as follows:

Extract (selected from the extracts produced in Examples 1-8) 15% w/w Lauroyl macrogolglycerides EP (e.g Gelucire 44/14) 65-85% w/w

Surfactant (e.g. Cremophor RH40 or Labrafac are suitable) 0-20% w/w.

The extract is dispersed with stirring in the molten lauroyl macrogolglycerides at 70-80⁰C. The surfactant is then added and stirring continued for a further 5 minutes. Using suitable automatic or manual equipment the molten mixture is then dispensed into two piece hard shell gel capsules which are then sealed.

Example 10.

A cream formulation for topical application of the extracts can be prepared as follows:

Extract (selected from the extracts produced in Examples 1-8) 2% w/w

Cetostearyl alcohol EP 7% w/w

Macrogol cetostearyl ether (e.g. Cremophor A6 or A25) 3% w/w

Liquid paraffin EP 12% w/w

Parabens (e.g. Nipastat) 0.2% w/w

Deionised water 67.8% w/w

Propylene glycol EP 8% w/w

The extract is dispersed in the propylene glycol at 70-80⁰C with stirring. All other ingredients except the water are mixed at 80⁰C and then added with stirring to the water that was heated separately to 80⁰C. The dispersion of extract in propylene glycol is then added to this mixture maintained at 70-80⁰C with stirring. The formulation is then filled into tubes.

Example 11.

A hydroalcoholic gel formulation for topical application of the extracts can be prepared as follows:

Extract (selected from the extracts produced in Examples 1-8) 2% w/w

Ethanol EP 44% w/w

Carbomer (e.g. carbopol 980 NF) 3% w/w

De-ionised water 51 % w/w

Sodium hydroxide (aq) qs to neutralise

The extract is dispersed in the ethanol at 50-60⁰C with stirring. The carbomer is then added slowly to the water with rapid stirring. The extract dispersion is then added to the aqueous carbomer whilst stirring. The resulting mixture is neutralised by slowly adding aqueous sodium hydroxide to produce a smooth semi-solid. The formulation is then filled into tubes. Example 12.

An enteric coated capsule suitable for the oral administration to give a high local concentration in the colon of the botanical drug substance can be prepared as follows:

Flavonoid enriched skullcap precipitate 20% w/w

StarCap 1500™ (Colorcon) 80% w/w

The flavonoid enriched precipitate and excipient are thoroughly mixed and then using suitable automatic or manual equipment is dispensed into two piece hard shell gel capsules that are then sealed. For a size 0 capsule a target fill weight of 400mg gives a dose of 80mg of botanical drug substance. Smaller capsules may be used to produce a lower unit dose. The capsules are then enteric coated by applying an approximately 8% w/w solution of a methacrylic acid based pharmaceutical coating, Opadry Enteric 95 (Colorcon) is suitable, in an organic solvent (typically 60% propan-2-ol + 40% dichloromethane). Standard techniques (e.g. spray coating combined with coating pan) are used to give an approximate weight gain of 10% w/w for the capsules.

If desired the proprietary StarCap 1500™ excipient may be replaced by a suitable mixture of maize starch, pre-gelatinised starch and other excipients as will be recognised by those skilled in the art.

While the preferred embodiments of the invention have been described above, it will be recognised and understood by those skilled in the art that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

Claims

1. A method of preparing an extract containing pharmacologically active constituents from certain traditionally used medicinal plants, comprising reduction of the particle size of the raw material as appropriate, extracting the botanical raw material with sub-critical water, and then removing the water from the resulting solution to produce a pharmaceutically acceptable extract.

2. The method according to claims 1-4 wherein the extraction is carried out at a temperature in the range 150-230⁰C, and most preferably in the range in the range 150-200⁰C.

3. The method according to claim 1-4 wherein the extraction is carried out at a pressure in the range 15-100 bar sufficient to maintain the water in the liquid phase, and most preferably at 70-85 bar.

4. The method according to claims 1-4 wherein the medicinal plant is selected from the group consisting of the traditionally used parts of Rosmarinus officinalis (rosemary), Matricaria recutita (German chamomile), Cassia angustifolia (senna), Valeriana officinalis (valerian), Scutellaria baicalensis (Baikal skullcap), Schisandra chinensis (Wuweizu), Zingiber officinale (ginger), Astragalus membranaceus and also including species closely related to any of the foregoing.

5. The method according to claims 1-4 wherein the water is removed to yield the extract by evaporation.

6. The method according to claims 1-4 wherein the water is removed to yield the extract by freeze drying or spray drying.

7. The method according to any preceding claim wherein the sub-critical water extraction of Rosmarinus officinalis (rosemary) botanical raw material or a closely related species produces an extract with a TLC fingerprint substantially as illustrated in Figure 2, which is essentially similar to the corresponding methanol extract and exhibits a significant spot due to rosmarinic acid.

8. The method according to any preceding claim wherein the sub-critical water extraction of Matricaria recutita (German chamomile) or a closely related species produces an extract with a TLC fingerprint substantially as illustrated in Figure 3, which is essentially similar to the corresponding methanol extract.

9. The method according to any preceding claim wherein the sub-critical water extraction of Cassia angustifolia (senna) or a closely related species produces an extract with a TLC fingerprint substantially as illustrated in Figure 4, which is essentially similar to the corresponding methanol extract.

10. The method according to any preceding claim wherein the sub-critical water extraction of Valeriana officinalis (valerian) or a closely related species produces an extract with a TLC fingerprint substantially as illustrated in Figure 5, which is essentially similar to the corresponding methanol extract and exhibits a significant spot due to valerenic acid.

11. The method according to any preceding claim wherein the sub-critical water extraction of Scutellaria baicalensis (Baikal skullcap) or a closely related species produces a product with a TLC fingerprint substantially as illustrated in Figure 6a, which is essentially similar to the corresponding methanol extract and exhibits a significant spot due to baicalein. The HPLC chromatogram of the precipitate as illustrated in Figure 6b exhibits a principal peak due to baicalein and a significant secondary peak due to wogonin.

12. The method according to any preceding claim wherein the sub-critical water extraction of Schisandra chinensis (Wuweizu) or a closely related species produces an extract with a TLC fingerprint substantially as illustrated in Figure 7, which is essentially similar to the corresponding methanol extract and exhibits a significant spot due to schisandrin A

13. The method according to any preceding claim wherein the sub-critical water extraction of Zingiber officinale (ginger) or a closely related species produces an extract with a TLC fingerprint substantially as illustrated in Figure 8, which is essentially similar to the corresponding methanol extract and exhibits a significant spot due to 6-gingerol & 6-shoagaol.

14. The method according to any preceding claim wherein the sub-critical water extraction of Astragalus membranaceus or a closely related species produces an extract with a TLC fingerprint substantially as illustrated in Figure 9, which is essentially similar to the corresponding methanol and 50% aqueous ethanol extracts.

15. The use of a botanical drug substance as claimed in any of the preceding claims that consist essentially of botanical drug substances.

16. The use of a botanical drug as claimed in claim 1 S further comprising excipients.

17. The use of a botanical drug as claimed in claim 15 wherein the botanical drug substances comprise total extracts derived from the botanical raw materials.

18. The use of a botanical drug as claimed in claim 15 wherein the botanical drug substances comprise more refined fractions derived from the total extracts of the botanical raw materials.

19. The use of a botanical drug as claimed in claim 15 wherein the botanical drug substances are standardised extracts.

20. The method according to any preceding claim wherein the pharmacologically active extract is formulated in a self emulsifying drug delivery system to improve the oral bioavailability of the active constituents.

21. The method according to claim 20 wherein the self emulsifying drug delivery system is based on the formulation described in Example 9.

22. The method according to claims 20-21 wherein the pharmacologically active extract as described in any preceding claim is selected from the list Rosmarinus officinalis (rosemary), Matricaria recutita (German chamomile), Cassia angustifolia (senna), Valeriana officinalis (valerian), Scutellaria baicalensis (Baikal skullcap), Schisandra chinensis (Wuweizu), Zingiber officinale (ginger), Astragalus membranaceus and also species closely related to any of the foregoing.

23. The method according to any preceding claim wherein the pharmacologically active extract is formulated in a topical vehicle formulated to increase efficacy of the active constituents.

24. The method according to claim 23 wherein the topical vehicle is based on the formulation described in Examples 10 & 11

25. The method according to claim 23-24 wherein the pharmacologically active extract as described in any preceding claim is selected from the list Rosmarinus officinalis (rosemary), Matricaria recutita (German chamomile), Cassia angustifolia (senna), Valeriana officinalis (valerian), Scutellaria baicalensis (Baikal skullcap), Schisandra chinensis (Wuweizu), Zingiber officinale (ginger), Astragalus membranaceus and also species closely related to any of the foregoing.

26. The method according to any preceding claim wherein the pharmacologically active extract is formulated into an enteric release capsule to produce a high local concentration of the active constituents in the colon.

27. The method according to claim 26 wherein the enteric release capsule is based on the formulation described in Examples 12.

28. The method according to claim 26-27 wherein the pharmacologically active extract as described in any preceding claim is selected from the list Rosmarinus officinalis (rosemary), Matricaria recutita (German chamomile), Cassia angustifolia (senna), Valeriana officinalis (valerian), Scutellaria baicalensis (Baikal skullcap), Schisandra chinensis (Wuweizu), Zingiber officinale (ginger), Astragalus membranaceus and also species closely related to any of the foregoing.