GB2473069A - Methods for the Production of Sub-critical Water Extracts of Certain Plants with Healthcare Applications. - Google Patents

Abstract

A methods of preparing an extract from medicinal plants of the Skullcap family comprises the grinding (or other tissue size reduction means) of the plants, extraction in sub-critical water preferably at 150-250 and 25-100 bar and cooling of the extract, to precipitate desirable flavonoids, followed by isolation by centrifugation or filtration. The sub-critical water extract may be similar in composition to those extracts obtained by means of methanol extractions. The resultant extract may be used to prepare medicinal capsules and drugs.

Description

The Production of a Flavonoid Enriched Fraction Prepared from Certain Skullcap Species Using Sub-critical Water Extraction.

FIELD OF THE INVENTION

This invention relates to a method of producing a flavonoid rich fraction from the root of Baikal skullcap and related species using sub-critical water extraction.

BACKGROUND TO THE INVENTION.

The characteristic flavonoids produced by Baikal skullcap (Scutellaria baicalensis) and other related species are known to exhibit very important pharmacological characteristics.

Probably the most important of these are related to the treatment of cancer: * Induction of apoptosis of various cancer cell lines by increasing cellular levels of reactive oxygen species [1(0 et al 2004, CHOW et al 2007].

* Suppression of cancer cell proliferation by disruption of cellular signalling mechanisms [ZHANG et al 2008, HUANG et al 1994, IKEZOE et al 2001].

* Inhibition of angiogenesis, and hence tumour growth & metastasis [SAGAR et al 2006].

* Inhibition of ABC transporters, including P-glycoprotein, which are the "drug efflux proteins" responsible for the development of Multi-Drug Resistance (MDR) to cancer chemotherapy [EFFERTH et al 2002, KITEGAWA et al 2005].

This anti-cancer activity of skullcap flavonoids has been confirmed in a wide range of in-vivo models [TONG et al 2002, ZHANG et al 2003, BONHAM et al 2005].

Skullcap extracts inhibiting proliferation of cancer cells and containing baicalein and wogonin, have been found to exert minimal toxicity against normal human cell lines thus suggesting treatments based on these are likely to exhibit minimal undesirable side effects {HIIvIEJI et al 2007, PARAJULI et al 2009].

The ability to inhibit the drug efflux proteins which would otherwise result in MDR is particularly useful, and renders the flavonoids promising as an adjunct to standard chemotherapy in the treatment of such conditions as colon cancer in which MDR is prevalent [LINN & GIACCONE 1995].

In addition to the above anti-cancer activity, skullcap flavonoids have been demonstrated to exhibit a range of anti-inflammatory properties: * Blocking release of pro-inflammatory mediators from human mast cells which are crucial to allergic response [KEMPIJRAJ et al 2005, HSIEH et al 2007].

* Inhibition of both the COX and LOX enzymes involved in synthesis of pro-inflammatory eicosanoids [BURNETT et al 2007] Consequently skullcap flavonoids also have potential as a treatment of inflammatory diseases, particularly those with an allergic component such as atopic eczema.

Further, it is now recognised that inflammation processes are critical to the progression of most cancers and that suppressing such processes is therefore beneficial [GESCHER et al 1998, SCALBERT & WILLIAMSON 2000, LAMBERT et a! 2005].

The structure of baicalein and wogonin are illustrated in Figure 1.

The skullcap flavonoids are normally extracted using an organic solvent such as methanol and then further purified by chromatography or re-crystallisation.

This, and the lack of inherent intellectual property in such a process, has limited the use of skullcap flavonoids as pharmaceuticals to date despite the attractive pharmacological properties described previously.

In an earlier patent filed by Advanced Extraction Technology Ltd (UK Patent Application GBO8 17354 4) a process was described in which a flavonoid containing extract could be prepared from the root of IBaikal skullcap using Sub-critical Water Extraction (SWE).

Surprisingly, it was found that when the solution resulting from SWE was allowed to stand overnight then a pale yellow precipitate formed.

Subsequently it was discovered that if this were isolated, rather than allowed to remain as part of the total extract after water was removed, then it proved to contain the majority of the flavonoids extracted from the skullcap root by the SWE process.

This phenomenon is not commonly observed in the extraction of other medicinal plants by SWE, and the application of this to produce in a facile manner a highly enriched flavonoid fraction for use as a botanical drug substance forms in part the invention described in this patent.

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 and hence bioavailability. 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 [MANACH et al 2005]. This problem has been experimentally verified for the skullcap flavonoids [LAT et al 2003, DAI et al 2008].

Hence this invention describes both an enteric coated capsule to maximise local concentrations of flavonoid in the colon, and also self emulsifying formulations which increase the water solubility of these compounds, thus increasing their oral bioavailability and therapeutic efficacy.

In addition this patent also describes topical formulations of the flavonoid rich precipitate of skullcap suitable for the treatment of skin conditions involving inflammation, and also with potential as an adjunct treatment combined with chemotherapy for melanoma.

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

LITERATURE CITED: M.Bonham et al, Clin.Cancer Res., 11(10), 3905-13 (2005).

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S.Kitegawa et a!, Bio!.Pharm.Bu1L. (12), 2274-2278 (2005) C.H.Ko et al, Free Radic.Biol.Med., 36 (7), 897-9 10 (2004).

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J.D.Lambert et a!, Am.J.Clin.Nutr., 81, 284S-291S (2005), S. C.Linn & G.Giaccone, Eur.J.Cancer, 31A, 1291-94 (1995).

C.Manach et a!, Am.J.Clin.Nutr., 81, 230S-242S (2005).

P.Parajuli et a!, Planta Med, 75 (1) 41-48 (2009).

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SUMMARY OF THE INVENTION.

Example 1:

The extraction apparatus consists of: 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 thermostatically controlled oven.

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

* The extraction vessel is connected via an outlet valve to a stainless steel receiver vessel outside the oven but which is maintained at approximately 90°C.

* 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 2.

Coarsely ground skullcap root 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 150-250°C, most preferably 200-230°C 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.

After leaving the receiver vessel and being transferred to a suitable container the solution is then allowed to cool slowly over a period of several hours, most preferably in the range 12-24 hours, and during this period a precipitate forms.

The precipitate is then isolated from the solution by filtration. Alternatively the precipitate may also be isolated from the solution by centrifugation. Residual moisture is then removed from the precipitate by either drying under reduced pressure or by freeze drying.

The precipitate is found to contain high levels of the pharmacologically active skullcap flavonoids, in particular baicalein, as illustrated in the TLC and HPLC profiles given in Figures 3 and 4 respectively.

In a typical SWE run on Baikal skullcap root the following yield and composition of precipitate is obtained: 39g of precipitate per lOOg of BRM extracted Precipitate contains 24.0 w/w baicalein and 13.8% w/w wogonin.

If the remaining soluble portion (11. 8g per 1 OOg of BRM) of the extract of skullcap is recovered by removal of the water then this found to contain only trace levels of the flavonoids. This is illustrated by the HPLC profile presented in Figure 5. Typically the ratio of the content of baicalein in the precipitate and the soluble ration is >35:1. In this case it was calculated to be approximately 280:1 The general applicability and reproducibility of the procedure is good. A repeat of the procedure using a second and separate batch of botanical raw material yielded the following results: 4.3g of precipitate per lOOg of BRM extracted Precipitate contains 24.1 w/w baicalein and 8.1% w/w wogonin.

The difference between the relative proportions of the flavonoids in the precipitate from the two extractions is a consequence of the natural variability in the composition of the botanical raw material.

Example 2.

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 1 500 (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 entenc 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 I 500w 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.

Example 3.

A Self Emulsif'ing Drug Delivery System (SEDDS) formulation for oral administration of the botanical drug substance containing the poorly water soluble active compounds can be prepared as follows: Flavonoid enriched skullcap precipitate 15% w/w Lauroyl macrogolglycerides EP (e.g Gelucire 44/14) 65-85% w/w Surfactant (e.g. Cremophor RH4O or Labrafac are suitable) 0-20% w/w.

The flavonoid enriched skullcap precipitate is dispersed with stirring in the molten lauroyl macrogoiglycerides at 70-80°C. The surfactant is then added and stirring continued for a furtherS 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 4.

A cream formulation for topical application of the flavonoid enriched skullcap precipitate can be prepared as follows: Flavonoid enriched skullcap precipitate 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 Deiomsed water 67.8% w/w Propylene glycol EP 8% w/w The flavonoid enriched skullcap precipitate is dispersed in the propylene glycol at 70- 80°C with stirring. All other ingredients except the water are mixed at 80°C and then added with stirring to the water that was heated separately to 80°C. The dispersion of extract in propylene glycol is then added to this mixture maintained at 70-80°C with stirring. The formulation is then filled into tubes.

Example 5.

A hydroalcoholic gel formulation for topical application of the flavonoid enriched skullcap precipitate can be prepared as follows: Flavonoid enriched skullcap precipitate 2% w/w Ethanol EP 44% w/w Carbomer (e.g. carbopol 980 NF) 3% wfw De-ionised water 51% w/w Sodium hydroxide (a qs to neutralise The extract is dispersed in the ethanol at 50-60°C with stirring. The carbomer is then added slowly to the water with rapid stirring. The flavonoid enriched skullcap precipitate 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.

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 (18)

1. CLAIMS1. A method of preparing an extract containing pharmacologically active flavonoids from certain medicinal plants of the skullcap family, comprising reduction of the particle size of the raw material as appropriate, extracting the botanical raw material with sub-critical water, and then allowing the resulting solution to cool which results in the formation of a flavonoid enriched precipitate which is isolated by filtration or centrifugation.
2. 2. The method according to claims 1-4 wherein the extraction is carried out at a temperature in the range 150-250°C, and most preferably in the range in the range 200-230°C.
3. 3. The method according to claim 1-4 wherein the extraction is carried out at a pressure in the range 25-100 bar sufficient to maintain the water in the liquid phase, and most preferably at 70-85 bar.
4. 4. The method according to claims 1-4 wherein the medicinal plant is Baikal skullcap (Scutellaria baicalensis) and also including species closely related to the foregoing.
5. 5. The method according to claims 1-4 wherein the flavonoid enriched precipitate is isolated by filtration.
6. 6. The method according to claims 1-4 wherein the flavonoid enriched precipitate is isolated by centrifugation.
7. 7. The method according to any preceding claim wherein the sub-critical extraction of the root of Baikal skullcap or a closely related species produces a precipitate with a TLC fingerprint and an HPLC profile substantially as illustrated in Figures 4 & 5 respectively, and which for the HPLC profile exhibits as the major component a peak with a retention time corresponding to that of a baicalein standard.
8. 8. The use of a botanical drug as claimed in any of the preceding claims that consist essentially of botanical drug substances.
9. 9. The use of a botanical drug as claimed in claim 8 further comprising excipients.
10. 10. The use of a botanical drug as claimed in claim 8 wherein the botanical drug substances comprises the flavonoid enriched precipitate derived from the botanical raw material.
11. 11. The use of a botanical drug as claimed in claim 8 wherein the botanical drug substance comprises more refined fractions derived from the flavonoid enriched precipitate.
12. 12. The use of a botanical drug as claimed in claim 8 wherein the botanical drug substances are standardised extracts.
13. 13. The method according to any preceding claim wherein the botanical drug substance is formulated as an enteric coated capsule.
14. 14. The method according to claim 13 wherein the enteric coated capsule is based on the formulation described in Example 2.
15. 15. The method according to any preceding claim wherein the botanical drug substance is formulated in a self emulsifying drug delivery system to improve the oral bioavailability of the active constituents.
16. 16. The method according to claim 15 wherein the self emulsifying drug delivery system is based on the formulation described in Example 3.
17. 17. The method according to any preceding claim wherein the botanical drug substance is contained in a topical vehicle formulated to increase efficacy of the active constituents.
18. 18. The method according to claim 17 wherein the topical vehicle is based on the formulation described in Examples 4 or 5.