WO2009068872A1

WO2009068872A1 - Antiviral

Info

Publication number: WO2009068872A1
Application number: PCT/GB2008/003947
Authority: WO
Inventors: Shouming Zhong; Hongwen Yu; Edward Blair
Original assignee: Phynova Limited
Priority date: 2007-11-27
Filing date: 2008-11-26
Publication date: 2009-06-04
Also published as: GB0723609D0; GB2455151A

Abstract

The present invention relates to a novel antiviral product and its use in the treatment of the flaviviridae family of viruses including the genus flavivirus, particularly Dengue.

Description

ANTIVIRAL TECHNICAL FIELD OF THE INVENTION

The present invention relates to an antiviral, and its use in the treatment of infections/ diseases caused by one or more genus of the flaviviridae family of viruses, including viruses from the genus: Flavivirus, Pestivirus, Unassigned Flaviviradae and Tentative Species in the Genus Hepacivirus. Inparticular, it relates to the use of an extract of Astragalus in the manufacture of a medicament for use in the treatment of Dengue Virus, West Nile Virus, Japanese Encephalitus and Yellow fever (all mosquito bourne viruses) as well as other related viruses.

The applicant has previously described in UK application number 0612025.7, which at the time of filing remains unpublished, that an Astragalus extract, and defined fractions therof, exhibit antiviral activity against Hepatitis C virus (HCV) in:

• A replicon test,

• A polymerase test (NS5B), and

• Protease tests (NS3/4A).

Based on, particularly, the polymerase test data they suggested that the Astragalus extract and defined fractions thereof might exhibit activity against other viruses within the Flaviviradae family of viruses.

Subsequently, they have demonstrated, as set out herein, that the Astragalus extract disclosed in the earlier application, together with a variant thereof, are active in reducing viral load against Dengue viruses and that as a consequence of the phylogenetic relationship of the Flaviviruses based on the NS3 helicase region (Fig 17) and E protein amino acid identity (Fig 18), together with the activity previously demonstrated against HCV, argue that this provides credible evidence that extracts of Astragalus, and defined fractions thereof, including single compounds isolated therefrom, are likely to exhibit activity against the Flaviviridae identified in Tables 1.1 to 1.4 herein. In particular the evidence supports the use of extracts of Astragalus in the manufacture of a medicament for use in the treatment of at least the following mosquito bourne Flaviviruses:

• Dengue Virus (DEN);

• West Nile (WN) and Japanese Encephalitis (JE), two very closely related viruses;

• Kunjin virus (KUN), Murray Valley encephalitis'virus (MVEV), St. Louis encephalitis virus (SLEV); and

• Yellow fever.

Additionally, there is strong phylogenetic evidence suggesting the extract will also be active against:

• Tick bourne Flavivirus including: o Kyasanur Forest disease virus (KFD) o Langat virus (LGT) o Louping ill virus (LI) o Omsk hemorrhagic fever virus (OHF) o Powassan virus (POW) o Tick-borne encephalitis virus (TBE)

• Pestivirus including: o Bovine viral diarrhea virus 2 (BVDV), and o Classical swine fever virus (CSFV)

• Unassigned viruses including o GBV-A, and o GBV-C and

• Tentative Species in the Genus Hepacivirus, including o GBV-B BACKGROUND OF THE EWENTION

In applicant's international application WO2005079823 there is disclosed a four herb combination product which in addition to providing symptomatic relief had been shown, by way of a replicon assay, to exhibit anti-HCV activity (inhibition of 41.8 %) at a dilution of 1/350.

The product is composed of four herbal extracts and is formulated as follows:

Herbal extracts:

• Milk Thistle Fruit dry extract 0.200 g

• Chinese Sage Root dry extract 0.225 g

• Schisandra Fruit dry extract 0.400 g

• Astralagus Root dry extract 0.585 g

Excipients:

• Macrogol 6000 powder 0.600 g

• Ferwogel 30.385 (molecular weight

3.5-4.O xIO⁶) 0.07Og

• Mannitol EZ 0.16Og

• Aerosil 200 0.050g

• Aspartame 0.050 g

• Caramel powder 0.100 g

• Peppermint powder aroma 0.06Og

The applicant investigated this product further and surprisingly discovered that an anti- HCV activity appeared to derive from only one plant, demonstrating both good activity and no detectable cell cytotoxicity. This discovery forms the basis of UK patent application no 0612025.7, as yet unpublished. That plant is Astragalus, and it has further been determined that a fraction purified by a factor of greater than 10, more particularly greater than 50, more particularly still greater than 75 and most particularly between 75 and 200 is particularly active.

The active fraction can be produced in good yield in effectively a three stage process comprising:

1) An alcoholic extraction (which may be repeated);

2) An ethanol- water precipitation process (which may be repeated); and

3) A systematic solvent fractionation step with a plurality of solvents of different polarity.

Preferably, the systematic solvent fractionation utilizes a number of different solvents commencing with the least polar and finishing with the most polar. The active fraction is a dichloromethane fraction or a solvent with similar polarity.

The active fraction (and sub- fractions) can be characterized with reference to one or more markers, one or a combination of which may be responsible for the anti-HCV activity and polymerase inhibition. Particular markers identified include:

• Astragaloside I,

• Astragaloside IV,

• Formononetin-7-O-β-D-glucoside; and

• 3'-hydroxyl- formononetin -7-O-β-D-glucoside.

Additionally they can be identified by way of TLC or HPLC fingerprints as set out in the detailed description.

The plant which exhibits the activity is a member of the Leguminosae family, more particularly Huang Qi:

Pharmaceutical name: Radix Astragali Membranaceous;

Botanical name: Astragalus membranaceus (Fisch) Bge. or Astragalus membranaceus

Bge var. Mongholicus Hsiao (hereafter Astragalus). The plant may be referred to as Milkvetch in Europe and it is the root which is used.

In traditional Chinese herbal medicine a dosage (based on dry raw material) of 9-3Og and occasionally up to 6Og is used. Typically it is taken as a decoction. According to Pharmacopoeia of the People's Republic of China (English Edition 2000) VoI I a cold water extraction method gives a water soluable extract of not less than 17%.

According to Chinese Herbal Medicine, Materia Medica, Revised edition, chemically the root of Astragalus membranaceus is known to contain, as major ingredients, D-β asparagine, 2'4'-dihydroxy-5, 6-dimethoxyisoflavane, calycosin, formononetin, cycloastragenol, astragalosides, choline, betaine, kumatakenin, sucrose, glucuronic acid and β-sitosterol.

Its chemical constituents are discussed further in Chinese Drugs of Plant Origin, Springer- Verlag which further discusses it's pharmacology and the symptomatic relief in the treatment of chronic hepatitis.

According to Chang HM and But PP (1987) Pharmacology and applications of Chinese Materia Medica (VoI ii) World Scientific Publishing, Astragalus membranaceus may enhance immunological function. More particularly reference is made to the use of a 100% decoction, 0.4ml daily, to "protect" the liver and a clinical trial in which Huangqi injection achieved normalization of GPT levels in 80% of patients. (This is a symptomatic treatment.)

Additional art disclosing the use of Astragalus include:

CNl 616097 which discloses a lozenge containing attenuated Newcastle disease virus vaccine or deactivated Newcastle disease virus vaccine, 10-120 hemagglutination units, astragalus root l-20000mg, liquiritigenin 1-lOOOmg, supplementary material and stabilizer. It is stated to be suitable for preventing SARS and other viral infectious respiratory tract diseases and to have auxiliary treatment effects on hepatitis B and C. CNl 607003 which discloses the use of a composition comprising Chinese caterpillar fungus (50-90 parts) with astragalus root (10-50 parts) as an auxiliary therapeutic agent for hepatitis C. It is stated to adjust immune function and improve curative effect.

US2005/0074428 which discloses an adjuvant agent for use in combination with interferon and ribivarin for treating Hepatitis C containing 50-90wt % cordyceps sinensis and 10-50wt% astragalus membrancens.

CN 1448163 which discloses a Chinese recipe comprising astragalus root among some eleven herbs to treat various diseases including hepatitis C.

CN1393255 which discloses a nineteen herb mix, including astragalus root, for treating hepatitis C.

CN1593586 which discloses a pill, for treating hepatitis C, comprising at least 15 herbs one of which is raw astragalus root.

US2005/0147699 which discloses the use of an astragalus radix and codonopsis pilosulae radix mixed extract for inhibiting carcinogenesis and metastasis. It states that Astragalus is recorded, in the traditional pharmacopea, to treat chronic nephritis, albuminuria, myositis, antihypertensive, coronary artery disease, cerebral infarction, peptic ulcer (duodenal and gastric ulcer), renal disease and diabetes mellitus. It makes no reference to its potential use as an anti viral agent.

Indeed none of the documents suggests that the herb Astragalus extract can be used alone as an antiviral to treat:

• Positive-stranded, single stranded RNA including: o The genus - Flavivirus, (Table 1.1); o The genus - Pestivirus, (Table 1.2); o The genus - Unassigned Flaviviradae (Table 1.3) or o Tentative Species in the Genus HCV (Table 1.4) which may be considered separate to the HCV viruses.

WO 2004/112720 teaches the use if GRII antagonists (glucocorticoids), including Astragaloside II, as compounds that inhibit viral replication.

US6555523 discloses flavin compounds, including formononetin compounds, to treat viral infections.

Both of the above relate to the use of single compounds rather than extracts comprising a plurality of components, several of which may exhibit activity and which together provide a beneficial effect.

On the other hand, Biol. Pharm. Bull vol 19(9) 1166-1169 discloses the oral administration of a decoction of astragali radix against Japenese Encephalitis virus infection in mice.

CN1985877 discloses the use of an astragalus extract to treat viral Hepatitis.

BMC Bioinformatics vol 7 (sup 5) 2006 provides a phylogenetic tree for the Flaviviridae.

In contrast, the Astragalus extract of the invention might be used alone as an antiviral to treat one or more of the viruses listed in the tables below:

Table 1.1 Flavivirus

Table 1.3 Unassigned viruses

Table 1.4 hepacivirus

Whilst in UK application no 0612025.7 it is stated that the data generated against HCV suggest a broader application of the extracts to treat diseases caused by the Flaviviridae, no additional test data supporting this supposition had been generated.

The applicant has since generated additional data supporting the use of an. Astragalus extract against another single-stranded RNA-containing virus, and a member of the different genus flavivirus, (particularly Dengue) which data supports the broader specific claims made herein.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided an Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament for the treatment of a disease caused by a genus of the flaviridae family selected from: o the genus - Flavivirus, (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus — Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4).

In a most preferred embodiment the Astragalus extract or an active fraction thereof or a compound isolated therefrom are used against the following viruses: • Flavivirus o Tick-borne viruses

^■ Kyasanur Forest disease virus;

^■ Langat virus

^■ Louping ill virus

^■ Omsk hemorrhagic fever virus ^■ Powassan virus

^■ Tick-borne encephalitis virus o Mosquito-borne viruses

^■ Dengue virus

^■ Japanese encephalitis virus

^■ Murray Valley encephalitis virus

^■ St. Louis encephalitis virus

^■ West Nile virus

^■ Kunjin virus

^■ Yellow fever virus

• Pestivirus o Bovine viral diarrhea virus o Classical swine fever virus

• Unassigned viruses o GB virus A o GB virus C

• Hepacivirus o GB virus B

The extract has proved capable of significantly reducing viral load in viruses from two of the four genus of flaviviridae.

Preferably the active fraction is an extract of Astragalus purified by at least a factor of 10 with reference to dried raw plant material, and which is characterized in that it comprises at least one marker selected from the group consisting of:

• Astragaloside I;

• Astragaloside IV;

• Formononetin-7-o-β-d-glucoside; and

• 3 ' -hydroxyl-formononetin-7-o-β-d-glucoside.

Preferably it comprises all four markers. In a particularly favoured embodiment, the extract is purified by at least a factor of 50, most preferably by a factor of between 75 and 200 and can be characterized by the presence of the four markers identified above. In fact, at least six defined peaks can be identified in this fraction.

In a preferred embodiment the extract is used or formulated as a botanical drug.

Most preferably the formulation is from a single herb extract.

In an alternative embodiment the extract may be used as a food, dietary supplement or food additive. The term food includes food or drink and articles used for components of such articles.

The botanical terms used herein are intended to have the meanings as used by the FDA in their directive "Guidance for Industry - Botanical Drug Products" (June 2004) which directive is incorporated by reference. It will, however, by understood by persons skilled in the art that in different countries different terminology may be used. Reference to the FDA guidance terminology is used for consistency and should not be taken to be limiting. Thus differing but equivalent terms used by, for example, the EMEA in their Guidelines on Quality of Herbal Medicinal Products / Traditional Herbal Medicinal Products (CHMP/ THMP adopted March 2006), will be understood by the skilled person to be encompassed by the terms used herein.

According to a second aspect of the present invention there is provided Astragalus raw material packaged or otherwise sold in a manner which indicates it is suitable and intended for use as an antiviral to treat infections caused by a virus from one of: o the genus - Flavivrus, (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4). Preferably the raw material is cultivated and harvested from a designated production site under managed agricultural practice.

According to a third aspect of the present invention there is provided the use of Astragalus or an extract thereof or an active fraction thereof or an isolated compound therefrom in the manufacture of a food, dietary supplement or food additive for use as an antiviral to treat infections caused by a virus from one of: o the genus - Flavivirus, (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4).

The preferred method for producing an extract of Astragalus (which exhibits antiviral activity and which is purified by at least a factor of 10 with reference to dried raw plant material) which is characterized in that it comprises at least one marker selected from the group consisting of:

• Astragaloside I;

• Astragaloside IV

• Formononetin-7-o-β-d-glucoside; and

• 3 '-hydroxyl-formononetin-7-o-β-d-glucoside comprises:

1) an alcoholic extraction (which may be repeated);

2) an ethanol-water precipitation process (which may be repeated); and either

3.1) a systematic solvent fractionation step with a plurality of solvents of different polarity or

3.2) a macro-porous resin column purification.

Preferably, the alcoholic extraction is an ethanolic extraction. The ethanolic extraction is preferably conducted under reflux. Most preferably it is repeated. Preferably high concentration ethanol is used (greater than 50% by concentration.) The alcoholic extraction is followed by an ethanol-water precipitation and the supernatant recovered. The ethanol is recovered leaving a concentrate.

The concentrate is then dissolved in water to give an aqueous solution which is subjected to systematic solvent fractionation using solvents of different polarity. Preferred solvents are: Petroleum ether, dichloromethane, and ethyl acetate.

Alternatively a macro-porous resin column purification process may be employed in which the purification comprises running the extract through the column, washing with a low concentration ethanol (between 30 and 50% ethanol concentration) and eluting using a high concentration ethanol (typically 90% ethanol).

According to a forth aspect of the present invention there is provided a method for producing an extract of Astragalus which exhibits antiviral activity against a virus from one of: o the genus - Flavivirus (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4). and which is purified by at least a factor of 10 with reference to dried raw plant material, and which is characterized in that it comprises at least one marker selected from the group consisting of: o Astragaloside I; o Astragaloside IV o Formononetin-7-o-β-d-glucoside; and o 3 '-hydroxyl-formononetin-7-o-β-d-glucoside comprising:

1) an alcoholic extraction (which may be repeated);

2) an ethanol-water precipitation process (which may be repeated); and either 3.1) a systematic solvent fractionation step with a plurality of solvents of different polarity, or

3.2) a macro-porous resin column purification.

The active fraction from each of the methods is referred to as an AS-C fraction due to its activity and the presence of common marker compounds. The benefit of the macro- porous resin separation is its scalability and the fact it avoids the use of undesirable solvents e.g. petrol ether and dichloromethane.

According to a fifth aspect of the present invention there is provided a method of treating infections caused by a virus from one of: o the genus - Flavivirus, (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4). comprising administering an effective amount of an. Astragalus extract or an active fraction thereof or an active compound isolated therefrom to a patient or animal.

The Astragalus extract or an active fraction thereof or an active compound isolated therefrom is preferably provided in unit dosage form. Most preferably it is provided in a form suitable for oral delivery e.g. a filled capsule. The skilled person will appreciate other dosage forms may alternatively be presented.

Preferably the extract is used in an amount equivalent to an amount of between 9 and 3Og of dried raw material / day.

In one embodiment the Astragalus or an active fraction thereof is administered by way of a combination treatment with at least one other immuno-modulatory or antiviral drug e.g. Interferon and/ or Ribavirin.

The drugs may be administered together simultaneously or sequentially. The botanical drug, food, dietary supplement or food additive preferably comprises an extract of Astragalus purified by at least a factor of 10 with reference to dried raw plant material, and is characterized in that it comprises at least one marker selected from the group consisting of:

• Astragaloside I;

• Astragaloside IV

• Formononetin-7-o-β-d-glucoside; and

• 3 ' -hydroxy l-formononetin-7-o-β-d-glucoside.

It may be further characterized by the presence of characteristic spots present by way of a TLC fingerprint as shown in any one of Figs 4-6 or by the characteristic peaks present by way of a HPLC chromatogram as shown in any of Figs 16a-16d.

According to a sixth aspect of the present invention there is provided an Astragalus extract fraction having an HPLC fingerprint showing a primary peak for Astragaloside I substantially as shown in any of Figs 16a to 16d.

According to a seventh aspect of the present invention there is provided an Astragalus extract having a TLC fingerprint comprising six identifiable spots substantially as illustrated in any of lanes 2 to 5 of Fig 14 or 15.

The present invention will be further illustrated, by way of Example only, with reference to the following methodology and data in which:

Fig 1 illustrates the anti-hepatitis C virus activity of both Salvia (SMR) and Astragalus (AMR) extracts together with toxicity data;

Fig 2 is a flow diagram of an alcoholic extraction process giving rise to a primary extract containing less than 15% by weight of the starting raw material;

Fig 3a is a flow diagram showing the fractionation of the primary extract (8) to give a more concentrated active fraction or secondary extract (AS-C (14)); Fig 3b is a flow diagram showing the fractionation of the primary extract to give a more concentrated alternative active fraction or secondary extract (ASC-90(30));

Fig 3 c is a flow diagram showing the fractionation of the primary extract to give a more concentrated alternative active fraction or secondary extract (ASC-90(50));

Fig 4 is a TLC fingerprint of a number of different fractions (UV254nm); Fig 5 is a TLC fingerprint of a number of different fractions (UV365nm); Fig 6 is a TLC fingerprint of a number of different fractions (daylight); Fig 7 is a graph showing the activity of various fractions in a replicon assay; Fig 8 is a graph showing the activity of ASC 705 (14) at different concentrations; Fig 9 is a flow diagram showing the further fractionation of the secondary extract;

Fig 10 is a TLC fingerprint of the five fractions AS-C-I to AS-C -V in chloroform/ methanol 9:1

Fig 11 is a TLC fingerprint of the five fractions AS-C-I to AS-C -V in chloroform/ methanol/ water 8:2:02

Fig 12 is a flow diagram showing the isolation, purification and chemical identification of the components present in different AS-C-I sub fractions

Fig 13a is a TLC chromatogram of "ASC-I-I" in chloroform: ethanol: water 85:15:1;

Fig 13b is a TLC chromatogram of "ASC-I-2" and "ASC-I-3" in chloroform: ethanol: and water 85:15:1 (UV 254nm);

Fig 13c is a TLC chromatogram of "ASC-I-2" and "ASC-I-3" in chloroform: ethanol: water 85:15:1 (iodine);

Fig 14 is a TLC chromatogram under UV 254nm of ASC 90(30) and ASC 90(50) extracts;

Fig 15 is a TLC chromatogram under sunlight of ASC 90(30) and ASC 90(50) extracts;

Figs 16 a - b are HPLC fingerprints of ASC 90(30) Figs 16 c - d are HPLC fingerprints of ASC 90(50) 8 003947

Fig 17 is a representation of the phylogenetic relationship of the flaviviridae family of viruses based on NS3 helicase region;

Fig 18 is representation of the phylogenetic relationship of the flaviviridae family of viruses based on E protein amino acid identity;

Fig 19 is a diagrammatic representation of the clinical manifestations of Dengue virus infection;

Fig 20 is a diagrammatic representation shown in factors influencing disease severity;

Fig 21 shows graphically the cytotoxicity data generated in Example 8; Fig 22 show graphically the activity data generated in Example 8; and Fig 23 shows graphically Den 2 assay data generated in Example 8.

DETAILED DESCRIPTION

Whilst the current application focuses on the non - HCV applications of the Astragalus extract, the methodology and data disclosed in UK patent no 0612025.7 are included for reference.

METHODOLOGY

EXAMPLE 1

Activity of component herbs

Following on from the discovery that the four herb combination demonstrated anti hepatitis C activity (by replicon Assay) the Applicant looked at the activity of the individual herbs. Fig 1 illustrates the activity of the PYN 17 constituents:

• AMR - Astragalus membranaceus root;

• SCF - Schisandra chinensis fruit;

• SMR - Salvia miltiorrhiza root;

• SMF - Silybum marianum fruit; and

• BCSMRO404 - A purified SMR compared to the control: • PYNl 7 - four herb combination.

Both AMR and SMR appear to demonstrate inhibitory activity in the HCV replicon assay. This is particularly evident at high concentrations, as shown in the left hand panel of Fig 1. The right hand panel of Fig 1 shows cytotoxicity data in the same replicon cells. This indicates that AMR displays no observable cell cytotoxicity whereas SMR is cytotoxic at high concentrations.

Having determined that the Astragalus membranaceus demonstrated activity and a lack of toxicity they sought to identify specific active fractions.

EXAMPLE 2.

Astragalus Root Extraction Process

Root material was dried in an oven at 6O⁰C for 3 hours, pulverised into a coarse powder and passed through a sieve (10 mesh). It was then subjected to an alcoholic extraction as set out in Fig 2.

Briefly this comprised the following steps:

1. To 10Og of coarse powdered Astragalus membranaceus (1 ) was added 70% ethanol (10 folds of raw material). This was then refluxed for 1.5 hours and the solution (2a) separated from the residue (3a);

2. To the residue was added 70% ethanol (8 folds of raw material). This was then refluxed for 1 hour and again the solution (2b) was separated from the residue (3b);

3. The solutions (2a and 2b) were combined, and the ethanol recovered under vacuum at a pressure of 0.08MPa to give 50ml of a concentrate (4);

4. To the concentrate was added 95% ethanol. The solution was allowed to stand over night and the precipitate separated from the supernatant (5) by filtration; 5. Again the ethanol was recovered under vacuum at a pressure of 0.08MPa and a concentrate (6) obtained;

6. The concentrate was dried at 60-70°C under vacuum at a pressure of 0.08MPa to give a solid extract (7).

7. The solid extract was then ground to a powder (8). This powdered primary extract had a solid yield of between 11.7 and 13% by weight compared to the dry weight of starting material, i.e. It had been purified by a factor of about 8.

The content of Astragaloside IV₅ the standard chemical marker for Astragalus was greater than 0.4 %.

EXAMPLE 3

Extract purification/ extraction / characterisation

The primary extract obtained by the method illustrated in Fig 2 was fractionated as set out in Fig 3 a.

Briefly this comprised the following steps:

1. To the primary extract (8) was added 95% ethanol to 80% and this was again allowed to stand over night. The precipitate (9) "AS-A" was separated from the supernatant (10) "AS-F" by filtration;

2. The ethanol was recovered under vacuum at a pressure of 0.08MPa and a concentrated solution (11) obtained;

3. Water was added to the concentrated solution (11) to form a water solution (12) which was successively partitioned with: a. Petroleum ether to give a fraction (13) "AS-B"; b. Dichloromethane to give a fraction (14) "AS-C"; and c. Ethyl acetate to give a fraction (15) "AS-D"; together with d. The Water faction (16) "AS-E". The fractions (9), (10), (13), (14), (15) and (16) were subjected to further activity tests as set out in Table 2 below:

Table 2.

Sample Source Total Starch (%) Fraction codes weight(g) Yield (%)

AS-A (9) precipitates 129.0 0 12.9

AS-F (10) 80% ethanol 4.0 25 12.4 soluble fraction

AS-B (13) petroleum ether 2.0 0 0.21 fraction

AS-C (14) methylene 10.5 0 1.1 dichloride fraction

AS-D (15) ethyl acetate 1.1 0 0.12 fraction

AS-E (16) aqueous fraction 100.0 0 10.5

Figs 4-6 are TLC plates showing from left to right, fractions (9), (10), (13), (14), (15), and (16). The TLC plates are silica gels and the developing system was chloroform/ methanol/ water (8:2:0.2).

Fig 4 shows detection with UV at 254nm; and

Fig 5 shows detection with UV at 365nm;

Fig 6 is observed in daylight following treatment with 10% sulphuric acid in ethanol followed by heating.

Fraction (14) shows a plurality of distinct spots. At least 6 are clearly seen at 254nm

Fraction 14 AS-C was found to be particularly active in the replicon assay (Example 4 Table 8). It had a solid yield of about 1.1% by weight compared to the dry weight of starting material. In fact it had been purified by a factor of about 90%. This fraction had a chromatographic profile as illustrated in Figs 4-6.

In alternative protocols, Figs 3b and 3 c, the powder extract (8) from Fig 2 is treated as follows:

1. Add water (two fold) and sonicate to dissolve the powder;

2. Filter and keep the supernatant;

3. Add to a column filled with a macroporus resin;

4. Elute sequentially with either: a. 3BV water, 3BV 30% ethanol, 3BV 90% ethanol and concentrate (Fig 3b) or b. 3BV water, 3BV 50% ethanol, 3BV 90% ethanol (Fig 3c) and concentrate.

More specifically the preferred extraction processes for AS-C 90 (30) and AS-C 90 (50 are as follows

Extraction:

The raw material of Astragalus was pulverized and extracted twice with 70% ethanol (10 times of the weight of the raw material for 1.5 hours and then 8 times for 1 hour). The extracted solutions were combined and the ethanol recovered under vacuum to about half of the weight of the raw material. 95% ethanol was added to the concentrated extract to give an ethanol concentration of 80%. The concentrated extract was set aside over night and filtered to obtain the solution. The solvent was recovered under vacuum from the solution to dryness with a temperature not exceeding 70°C to produce a dried extract (AS-C).

Purification through macro porous resin Production of AS-C 90 (30):

Dissolve the dried extract in water (twice the amount of the starting raw material) , sonicate and filter. Apply the solution to a column filled with pre-treated macro porous resin. The quantity of the resin in the column was about 9 times (volume in 95% ethanol/raw material weight) of the starting raw material. Elute the column with purified water (about 3 times the column volume) until sugar test became negative. Continue the elution with 30% ethanol (about 3 times the column volume). Change the eluting solvent to 90% ethanol and continue the elution until 3 times of the column volume ethanol was used. Collect the 90% ethanol elution portion and recover the solvent under vacuum to dryness to obtain AS-C 90 (30). (Fig 3b)

Production of AS-C 90 (50):

Dissolve the dried extract in water (twice the amount of the starting raw material), sonicate and filter. Apply the solution to a column filled with pre-treated macro porous resin. The quantity of the resin in the column was about 9 times (volume in 95% ethanol/raw material weight) of the starting raw material. Elute the column with purified water (about 3 times the column volume) until sugar test became negative. Continue the elution with 50% ethanol (about 3 times the column volume). Change the eluting solvent to 90% ethanol and continue the elution until 3 times of the column volume ethanol was used. Collect the 90% ethanol elution portion and recover the solvent under vacuum to dryness to obtain AS-C 90 (50). (Fig 3c)

The resulting extracts have an appearance, by TLC, as illustrated in Figs 14 and 15 when examined using the following methodology:

TLC Method and Chromatogram

1. TLC Method

Take adequate amount of the reference substances of AS-C-I-I, AS-C-I-2 and Astragaloside IV respectively, put them into a flask and dissolve with methanol as the mixed reference solution (lmg/ml of each substance). Take adequate amount of each test sample, respectively, dissolve them with methanol - chloroform mixture (6:1) solution as the sample solution with a concentration of 2.5mg/ml, respectively. Apply lOμl of each test samples solution together with the reference solution onto a TLC plate (254nm fluorescent). Develop the plate with chloroform - methanol - water (8: 2: 0.2). Dry the plate and then spray with 10% sulphuric acid-ethanol solution. Heat with hot air blow until the spots became clear.

Under UV light (254nm), the test samples showed the same spot as the reference substance AS-C-I-2. (Fig 14)

Under the sunlight, the test sample showed the same spots as the reference substances AS-C-I-I and Astragaloside IV. (Fig 15)

In each of Figs 14 and 15 the sample order is:

1. Mixed reference substances of Astragaloside I (AS-C-I-I), Formononetin-7-O-β-D- glucoside (AS-C-I-2) and Astragaloside IV

2. Test sample batch No. 0412 , AS-C-90 ( 30 ) Fraction

3. Test sample batch No. 0604 , AS-C-90 ( 30 ) Fraction

4. Test sample batch No. 0412 , AS-C-90 ( 50 ) Fraction

5. Test sample batch No. 0604 , AS-C-90 ( 50 ) Fraction

By HPLC chromatography the extracts appear as illustrated in Figs 16a- 16d.

HPLC Method & Chromatogram

1. HPLC Detection Method

Apparatus: Waters HPLC Systems: Waters 2420 ELS Detector, 717 plus Autosampler & Pump 600 Controller with Empower™ Software.

Chromatographic Conditions:

Column: Atlantis ® dC18 ( 5μm , 250^χ4.6mm )

Mobile Phase: % A acetonitrile % B water - acetic acid = 200:1 Gradient Settings: As set out in Table 3: Table 3

Flow rate: lml/min

Chromatographic column temperature : 25°C

Gain: 20 Gas pressure: 20

Nebulizer heater level: 50% Drift tube heater set-temp: 60°C

Preparation of Sample Solution

Take precisely 20mg of the sample, put it into a 10ml flask and dissolve it with 80% ethanol. Ultra-sonicate for 5 minutes and add solvent to the volume, shake it well and cool it down; filter it with 0.45μl microporous filtering film to obtain the sample solution. Load lOμl of the sample for testing.

In Figs 16 a-d the peaks can be identified as follows:

Peak Index:

1. Formononetin-7-O-β-D-glucoside (AS-C-I-2 )

2. Astragaloside IV

3. Astragaloside I (AS-C-I-I)

A quantitative analysis is given in Table 4 below: Table 4. Preferably, and standardised extract is standardised to one or more of the "mean" figures, i.e. to within plus or minus 40%, more preferably plus or minus 30%, and most preferably to within plus or minus 20%.

Most preferably standardisation is against the largest component, namely, Astragaloside

EXAMPLE 4 Activity 1

In order to test the activity of the fractions ASA to ASF lOmg of each sample was dissolved in 1 ml of DMSO and sonicated for 15 minutes. Two additional samples PA (crude Astragalus extract as WO2005079823) and INFα (standard treatment) were run as comparators. The solubility of the samples is shown in Table 5 below:

Table 5.

The DMSO solutions were then diluted 1/10 in tissue culture medium and filtered. This concentration was called NEAT. lOμl per well was used, total volume in the test well lOOμl. All concentrations were set up as 5 replicates. 3 replicates were of the same dilution as the DMSO.

The dilutions of the extract samples are shown in Table 6 below and those for IFNα are shown in Table 7.

Table 6.

Table 7.

In the first assay the replicon cells were plated out at a concentration of 5 x 10³ / well in 90μl. The following day the test samples were added in lOμl. The plates were incubated for a further 72 hours and harvested and assayed using the Dual Luciferase Assay Promega. The replicon cell line expresses Renilla luciferase.

The samples were tested for cytotoxicity by setting up a separate 96 well plate with each sample and concentration in duplicate. The cells were labeled with tritiated thymidine for 24 hours and harvested.

For the second assay a separate plate with the top 5 concentrations was set up and incubated for 2 hours with wst-1 (Roche) and the OD read at 450nm and 630nm. Wst-1 is a viable cell stain.

Results

Assay 1: Mean of reading from cells without any drug added 198906. The results are shown in Table 8.

Table 8.

% inhibition of expression of Renilla luciferase

The readings obtained with no added drug were quite high.

The result obtained with the interferon at these concentrations is typical of this assay. It seemed from these results that inhibition was obtained with some of the samples at the higher concentrations so the assay was repeated.

Assay 2.

The results are shown in Table 9

Table 9.

Mean of reading from cells with no drug added. 71676.

% inhibition of expression of Renilla lucif erase

The dilutions were tested for cytotoxicity in the wst-1 assay and this plate was also examined under the microscope. This assay will not detect cytostatic agents. Sample 4 killed the cells at the top 2 dilutions; they looked less healthy at the 3^rd concentration. AU the other samples had cells that were healthy in appearance. This is reflected in the wst-1 results.

Assay 2 wst-1 results.

The results are shown in Table 10

Table 10.

Mean OD reading at 450nm

It should be noted that the IFN alpha concentrations are pg/ml. The results are illustrated graphically in Figs 7 and 8.

Fig 7 shows the raw replicon scores of the samples against concentration. ASC-0705 is clearly the best performer and outperforms IFN alpha (standard treatment). It also demonstrates that the best activity is at a concentration above 10ug/ml.

Fig 8 shows activity against concentration. Clearly the optimum concentration is above about 1.36uM EXAMPLE 5

Further purification

The fraction (14 AS-C) was subjected to a chromatographic separation step as illustrated in Fig 9 and as set out below. Briefly this comprised the following steps:

1. 6.2g of the Dichloromethane extract (14) "AS-C" from Astragalus root was separated by silica gel column chromatography and eluted in gradient elution with chloroform, chloroform/methanol (100:5), chloroform/methanol (100:10), chloroform/methanol (100:15), and methanol and 25 fractions obtained which were initially split into six pools (17; 18; 19; 20; 21 and 22);

2. Pooled fractions 1-14 (17) contained no significant chemicals;

3. Fraction 15 (18) had a weight of 241 mg and was further purified twice with Sephadex column chromatography and eluted with methanol to get a fraction (23)

"AS-C-II" which was shown to be active BUT which also exhibited toxicity;

4. Fraction 17 (19) had a weight of 231 mg and was further separated into two fractions by way of silica gel column chromatography eluting with chloroform/methanol (94:6). The two fractions weighed 13mg, fraction (24) "AS- C-III" and a 14mg (25) "AS-C-IV";

5. The 18^th fraction (20) was left to stand and 649mg of a white powder precipitated as AS-C-I. This was found to exhibit activity with no toxicity;

6. The 22^nd fraction (21) weighing 219mg was separated by Sephadex column and Silica column to obtain an 18 mg fraction (26) "AS-C-V".

The extracts which were tested are identified in Table 11 below:

Table 11

Ref Sample Source Total % content Factorial weight with change from reference to raw material dry raw material

(14) AS-C 6.2g 0.79671%* 125

(20) AS-C -I AS-C 18^th 649mg 0.08339% 1,199 fraction eluted

(23) AS-C -II AS-C 15 th 17mg 0.00218% 45,871 fraction eluted

(24) AS-C -III AS-C 17 rth 13mg 0.00167% 59,880 fraction eluted

(25) AS-C -IV AS-C 17 .th 14mg 0.00179% 55,865 fraction eluted

(26) AS-C -V AS-C 22^: nd 18mg 0.00231% 43,290 fraction eluted

* Calculated on basis that lOOg of primary extract (8) contains 12.85% of the starting material and accordingly the equivalent weight of dry raw material is 778.2g

Each of the five referenced sub fraction samples was subjected to analysis by TLC and the results are illustrated in Figs 10 and 11.

Fig 10 shows silica gel plates developed in chloroform / methanol (9:1). The left hand plates is viewed under UV at 254nm and the right hand plate under daylight (spray with 10% sulphuric acid in ethanol and then heat the plate until the spots are clearly seen).

Fig 11 shows silica gel plates developed in chloroform / methanol/ water (8:2:0.2). The left hand plates is viewed under UV at 254nm and the right hand plate under daylight (spray with 10% sulphuric acid in ethanol and then heat the plate until the spots are clearly seen).

In both cases the samples read from left to right: AS-C-I; AS-C-II; AS-C-III; AS-C-IV;

AS-C-V. EXAMPLE 6

Activity

Each of six samples: "AS-C"; "AS-C -I"; "AS-C -II"; "AS-C -III"; "AS-C -IV"; and "AS-C -V" were dissolved in DMSO to give a 10 mg/ml stock solution. The samples were:

1. analysed for purity and identity; and

2. tested for anti HCV activity.

1. Purity and Identity

The six samples, AS-C, AS-C-I₅ AS-C-II, AS-C-III, AS-C-IV, AS-C-V were analysed with respect to purity and identity using LC-UV and LC-MS. The analysis gave the following data

• AS-C fraction contained several (>6) small peaks (m/z 269, 301, 303, etc.)

• AS-C-I two main peaks (m/z 269, 301)

• AS-C-II one peak (m/z 301)

• AS-C-III one peak (m/z 303)

• AS-C-IV one peak (m/z 269)

• AS-C-V no peaks As at this stage, the components corresponding to the peaks had not been identified so the only estimation that could be done was to compare the peak areas with the DMSO peak area:

I. "AS-C-II" and "AS-C-IV" contained a single peak which is higher than the DMSO peaks.

II. "AS-C" and "AS-C-I" are mixtures;

III. "AS-C-III" contains a single peak which is lower than the DMSO peak.

IV. "AS-C-V" does not contain any peak visible in UV.

It should be noted that the purity was analysed with UV detection and any compounds that do not absorb in UV will not be detected. Material from the peaks was analysed with MS to determine the m/z (given above).

2. Anti-HCV Activity

The samples were tested for anti-HCV activity in: a) a replicon assay (Reblikon), b) a NS5B assay and c) a NS3/NS4A full length protease assay.

To estimate the selectivity of any "hit" in the replicon assay, the samples were also tested for cytotoxicity in Huh7 cells.

The results are illustrated in Table 12 below which additionally includes purity, peak area and identity data.

Table 12

FRACTION AS-C AS-C-I AS-C-Π AS-C-m AS-C-IV AS-C-V

TEST a) REPLICON, EC50 3.7 36 17 >50 >50 >50 (μg/ml)

b) NS5B, EC50 (μg/ml) 17 19 >100

cl) NS3/NS4, %inh @100 μg/ml 45 46 c2) NS3/NS4, %inh @10 μg/ml 19 27 37

d) Huh7, CCSO (μg/ml) >100 >100 >100

no peaks

Purity (LC-UV) 97% 98% detected

2339866 11689463

PeαA area (2.67 min) (2.57 min)

Identity (LC-MS) m/z 303 m/z 269

From the table the following can be determined:

Sample, "AS-C" (mixture)

• a) Inhibits HCV replication in replicon cells showing EC₅₀ = 3.7 μg/ml;

• b) Inhibits NS5B at 9.2 μg/ml; m

• c) Gives 95 % inhibition at a concentration of 100 μg/ml in the NS3/NS4 assay.

• d) Shows no cytotoxicity in concentrations of up to 100 μg/ml.

AS-C-I and AS-C-II • a) also show some activity in the replicon assay but at higher concentrations. The determined EC₅₀ values were 36 and 17 μg/ml respectively

EXAMPLE 7

Further characterisation

In order to further characterise the "AS-C" fraction (14) which exhibited the strongest activity, reference was made to fraction "AS-C-I" (20) as this also exhibited activity and thus it was concluded it contained one or more of the actives responsible for the activity which could serve as markers.

Accordingly, and with reference to Fig 12, the following protocol was followed:

1. 6.2g of (14) AS-C was separated by silica gel column chromatography and eluted with chloroform/methanol (100:15). The precipitated white powder from the 18^th fraction (20) was referred as "AS-C-I" (649mg).

2. The ASC-I (20) was dissolved in methanol with supersonication, and then filtrated. The filtrate was separated by Sephadex LH-20 column to obtain three major compounds as showed in the TLC plates illustrated in Figs 13-15.

3. Pooled fractions 4-6 (28) showed a spot when sprayed with sulphuric acid and heated;

4. Pooled fractions 9-11 (30) showed 2 spots when the plate was placed in an iodine steam and observed at UV254 _nm-

5. Pooled fractions 4-6 (28) had trace amounts of pigment after purifying by Silica gel column and eluted with chloroform/methanol: 9:1 (32)

6. This sample (32) was dissolved in acetone, refrigerated and re-crystallized to obtain "AS-C-1-1" (33).

7. Pooled fractions 9-11 (30) showed fluorescent spots when purified by a gel column (34), and then TLC method (35). The two fluorescent bands were separated. The band with a higher Rf value was eluted to obtain "AS-C-I-2" (36); while the one with a lower Rf value was purified by Sephadex LH-20 to obtain "AS-C-I-3" (37).

The three compounds identified were analyzed by mass spectrum and nuclear magnetic resonance spectroscopy, respectively. The structures of the three compounds were identified by comparing the data obtained with the existing literature on chemical structures and are shown in Table 13.

Table 13

Sample No. Spectra tested Structural analysing results

AS-C-I-I ESI, ¹HNMR, Astragaloside I 1³CNMR, COSY, HMQC, HMBC

AS-C-l-2 ESI, ¹HNMR, Formononetin-7-O-β-D- 1³CNMR, COSY, glucoside HMQC, HMBC

AS-C-I-3 ESI, ¹HNMR, 3'-hydroxyl- HMQC, HMBC formononetin -7-0-β-D- glucoside

These compounds have the following structures:

Formula 1 Astragaloside I (AS-C-I-I)

Formula 2: Formononetin-7-O-β-D-glucoside (AS-C-I-2)

glcO

Formula 3: 3'-hydroxyl- formononetin -7-O-β-D-glucoside (AS-C-I-3)

EXAMPLE 8

Investigation of activity against Dengue Virus

The objective was to test: a) The cytotoxicity of extracts ASC, ASC 90(30) and ASC 90(5O)₅ and b) Test the effects of extracts ASC, ASC 90(30) and ASC 90(50) on dengue virus infection METHOD

Preparation of test solutions

Each test sample (ASC 0506, ASC 90 (30) and ASC 90 (50) (10 mg) was re-suspended in 1 ml of DMSO to obtain the concentration of 10 mg/ml. The completely dissolved extract was diluted by a 100-fold with growth medium and, therefore, this stock solution had the final concentration of 100 μg/ml (in the presence of 1% DMSO). To test the effects of the extract on cell viability and dengue virus infection, the stock solution was serially diluted with the maintenance medium as follows.

100 μg/ml (DMSO = 1%) a 2-fold dilution = 50 μg/ml (DMSO = 0.5%)

Serial 10-fold dilutions: 5 μg/ml (DMSO = 0.05%)

0.5 μg/ml (DMSO = 0.005%)

100 μg/ml (DMSO = 1%) a 5-fold dilution = 20 μg/ml (DMSO = 0.2%)

Serial 10-fold dilutions: 2 μg/ml (DMSO = 0.02%)

0.2 μg/ml (DMSO = 0.002%)

Determination of cytotoxicity

Vero (an African green monkey kidney cell line) or Eahy 926 (an endothelial cell line) were seeded onto 96-well plates (20,000 cells/ well) in 100 μl of growth medium (10% FBS-MEM + 2 niM L-glutamine for Vero cells and 10% FBS-DMEM F-12 for Eahy 926) and incubated at 37⁰C with 5% CO₂ for 24 hr. Thereafter, the medium was discarded and the cells were incubated with 100 μl of extracts ASC, ASC 90(30), or ASC 90(50) at the concentrations of 0.2, 0.5, 2, 5, 20, 50, and 100 μg/μl. Cells that had been incubated with 100 μl of the maintenance medium (2% FBSMEM + 2 BiM L-glutamine for Vero cells and 5% FSB-DMEM F-12 for Eahy 926) or DMSO (the diluent of the compounds) at the concentrations of 0.02%, 0.05%, 0.2%, 0.5%, and 1% served as the control. At 24, 48, and 72 hr post incubation, the cells were subjected to an MTT cell proliferation assay (ATCC) according to the manufacturer's protocol. Standard controls for cell proliferation at the indicated time points were also established by plating different numbers of cells in each well:

10⁴, 105, 10⁶ cells/well for Vero or 10³, 10⁴, 5 x 10⁴, 10⁵ cells/well for Eahy 926. The absotbance which reflects the number of cells was read out at 595 run (with the reference wavelength of 630 nm). The numbers of DMSO or compound-treated cells were obtained from the predicted value on the standard curve [OD (standard - blank) vs. the number of cells]. The percentage of cell viability was calculated based on the following formula:

% viability = The number of DMSO or compound-treated cells x 100

The number of non-treated cells, all standard controls, DMSO or extract-treated cells, and non-treated cells were tested in triplicate for each experiment. Two independent experiments were performed.

Determination of the effects of the extracts on dengue vivus infection

Vero or Eahy 926 cells were seeded onto 24-well plates (250,000 cells/well) in 1 ml of growth medium (10% FBS-MEM + 2 mM L-glutamine) and incubated at 37⁰C with 5% CO₂ for 24 hr. Thereafter, the medium was discarded and cells were incubated with dengue virus serotype 2 (strain 16681) at an MOI of 1 or 5 in the total volume of 250 μl at 37°C for 2 hr. The virus supernatant was then removed and the cells were washed twice with 1 ml of plain medium and further incubated with 1.25 ml of maintenance medium (2% FBS-MEM + 2 mM L-glutamine) which contains compounds ASC, ASC 90(30) or ASC 90(50) at the concentration of 5 or 50 μg/μl, or DMSO (the diluent of the compounds) at the concentration of 0.05% or 0.5%. At 24, 48, and 72 hr post infection, mock (negative control) and dengue virus infected cells were harvested and assayed for the viability by staining with tryptan blue or propidium iodide. The percentage of dengue virus infection was assessed by immunofluorescence staining of intracellular dengue viral E and NSl and subsequent analysis by flow cytometry. In addition, titers of infectious virus in the culture supernatants were determined at the indicated time points by a focus forming unit assay.

RESULTS Cytotoxicity

Vero and Eahy 926 cells were cultured in the presence of extracts ASC, ASC 90(30), and ASC 90(50) at the concentrations of 0.2, 0.5, 2, 5, 20, 50, and 100 μg/ml up to 3 days. The extract-treated cells were harvested daily and determined for the viability by an MTT assay based on cell proliferation. DMSO-treated cells collected at the same time points served as the control. Results from two independent experiments showed that all extracts did not cause significantly cell death in both cell lines tested but, instead, were likely to induce cell proliferation as compared with the control. In addition, cell viability appeared to be time-dependent since it became decreased on day 3 after culture establishment. Both the cell lines that had been treated with the extracts still exhibited more than 50% of cell viability even after the highest concentration (i.e., 100 μg/ml) of the extracts was utilized (except on day 3). These results suggested that the cytotoxic concentration (CC50) of each extract may be higher than 100 μg/ml. Therefore, the concentrations of 5 and 50 μg/ml were chosen as a representative of low and high doses of the compounds, respectively, for use in further study on the effects of the extracts on dengue virus infection.

The results are shown graphically in Fig 21.

Activity against Dengue virus infection

Vero and Eahy 926 cells were incubated with dengue virus serotype 2 (strain 16681) at an MOI of 1 or 5 for 2 hr and thereafter the virus supernatant was replaced with fresh medium containing extracts ASC, ASC 90(30), or ASC 90(50) at the final concentrations of 5 or 50 μg/ml. The cultures were further incubated up to 3 days. Mock and dengue virus-infected cells that had been treated or left untreated with DMSO served as controls for dengue virus infection.

In dengue virus-infected Vero cell culture (MOI=I and 5), treatment with extracts at the concentration of 5 μg/ml yielded the similar percentage of viable cells as compared with DMSO-treated control. The viability of dengue virus-infected cells tended to decrease in the presence of 50 μg/ml of the extracts, particularly ASC and ACS 90(50), as determined by tryptan blue and propidium iodide staining. Dengue virus infection resulted in the expression of viral E and NSl antigens in Vero cells in a dose-dependent and a time-dependent manner (Fig 22) and almost all of the cells (about 90%) could be infected with dengue virus by day 3 post infection. Regardless of the virus doses, treatment with the extracts at 5 μg/ml did not affect the percentage of viral antigen expression as well as the production of infectious virus as assessed by immunofluorescence staining and a focus forming unit assay, respectively. However, treatment with ASC and ASC 90(50), but not ASC 90(30), at 50 μg/ml (Fig 23) resulted in a lower level of virus production by about one log¹⁰ FFU/ml, than treatment with DMSO in the dengue virus-infected cell culture (MOI=5). When Vero cells were infected with a lower dose of virus (MOI=I), a slightly reduction of viral antigen expression was observed following treatment with 50 μg/ml of either the ASC or the ASC 90(50). More interestingly, the supernatants of these culture had a significant decrease in the virus titer by about 2-3 log¹⁰ FFU/ml as compared with that of the DMSO-treated control. Dengue virus-infected Eahy 926 cell culture (MOI=5) showed the profile of cell viability upon treatment with the extracts, similar to dengue virus-infected Vero cell culture, at both the concentrations tested. Specifically, treatment with 50 μg/ml of the ASC and ASC 90(50) as well as, to a lesser extent, the ASC 90(30), tended to induce cell death in the infected Eahy 926 culture at a higher level than DMSO-treated control. Following dengue virus infection, relatively low levels of viral E and NSl expression were detected regardless of treatment with the extracts; therefore, it would be somewhat difficult to assess the extract activity against dengue virus infection based on the viral antigen expression under the condition tested. A focus forming unit assay was utilized as an alternative approach to determine the effect of the extracts on dengue virus production. Results showed that, virus titers in dengue virus-infected Eahy 926 cell culture containing 50 μg/ml of the ASC and ASC 90(50) were about 2-3 log¹⁰ FFU/ml lower than those in the DMSO- treated or the ASC 90(30)-treated cultures. This was consistent with the results observed in dengue virus-infected Vero cell culture

Conclusion

Extracts ASC, ASC 90(50) and ASC 90(30) at all the concentrated tested were not toxic to Vero and Eahy 926 cells in the absence of dengue virus infection. The predicted CC50 of each extract may therefore be higher than 100 μg/ml. However, treatment with 50 μg/ml of the ASC and ASC 90(50) resulted in certain extents of cell death in dengue virus-infected Vero and Eahy 926 cultures. This phenomenon was coincident with a slightly reduction of viral antigen expression and a significant decrease in virus production in the same cultures following treatment with these extracts. The induction of cell death by the extracts ASC and ASC 90(50) may be one of the potential mechanisms to reduce the initial pool of infectious viruses during dengue virus infection of the susceptible cells.

The benefits of the reduction in viral load can be most effectively demonstrated with reference to Figs 19 and 20. Thus, where a patient presents with symptoms (Fig 19) early use of the extract can result in a reduction of viral load on a log scale with the benefit (Fig 20) that the patient develops a "fever" as opposed to a "hemorrhagic fever".

Claims

1. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament for the treatment of a disease caused by a genus of the flaviviridae family selected from:

• the genus - Flavivirus, (Table 1.1);

• the genus - Pestivirus, (Table 1.2);

• the genus — Unassigned Flaviviradae (Table 1.3) or

• tentative Species in the Genus HCV (Table 1.4).

2. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in claim 1 in which the Flavivirus is a mosquito bourne flavivirus.

3. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in claim 2 wherein the mosquito bourne flavivirus is selected from: o Dengue Virus (DEN); o West Nile (WN) virus; o Japanese Encephalitis (JE) virus; o Kunjin virus (KUN); o Murray Valley encephalitis virus (MVEV); o St. Louis encephalitis virus (SLEV); and o Yellow fever.

4. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in claim 1 in which the Flavivirus is a tick bourne flavivirus.

5. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in claim 4 wherein the tick bourne flavivirus is selected from: o Kyasanur Forest disease virus o Langat virus o Louping ill virus o Omsk hemorrhagic fever virus o Powassan virus o Tick-borne encephalitis virus

6. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in claim 1 in which the Pestivirus is selected from:

• Bovine viral diarrhea virus 2, and

• Classical swine fever virus.

7. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in claim 1 in which the unassigned Flaviviridae is selected from:

• GBV-A₅ and

• GBV-C.

8. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in claim 1 in which the tentative Species in the Genus Hepacivirus, is o GBV-B

9. An Astragalus extract or an active fraction thereof or an active compound isolated therefrom, for use in the manufacture of a medicament as claimed in any of the preceding claims in which the extract, active fraction thereof or compound isolated therefrom is or is derived from a single herb extract.

10. An Astragalus extract, or an active fraction thereof or compound isolated therefrom as claimed in any of the preceding claims wherein the active fraction is an extract of Astragalus purified by at least a factor of 10 with reference to dried raw plant material, and which is characterized in that it comprises at least one marker selected from the group consisting of:

• Astragaloside I;

• Astragaloside IV

• Formononetin-7-o-β-d-glucoside; and

• 3 ' -hydroxyl-formononetin-7-o-β-d-glucoside.

11. An Astragalus extract, or an active fraction thereof or compound isolated therefrom as claimed in claim 10 wherein the active fraction is an extract of Astragalus purified by a factor of 50 with reference to dried raw plant material.

12. An Astragalus extract, or an active fraction thereof or compound isolated therefrom, as claimed in claim 11 wherein the active fraction is an extract of Astragalus purified by a factor of between 75 and 200 with reference to dried raw plant material.

13. An Astragalus extract, or an active fraction thereof or compound isolated therefrom, as claimed in any of claims 9-12 which is derived from an alcoholic extract.

14. An Astragalus extract, or an active fraction thereof or compound isolated therefrom, as claimed in claim 13 in which the extract has been subject to an ethanol-water precipitation process.

15. An Astragalus extract, or an active fraction thereof or compound isolated therefrom, as claimed in claim 13 or 14 in which the extract has been subject to a systematic solvent fractionation step with a plurality of solvents of different polarity or separation on a macro-porous resin

16. Pea. Astragalus extract, or an active fraction thereof or compound isolated therefrom, as claimed in claim 15 wherein the active fraction is obtained from a dichloromethane fractionation or a solvent of similar polarity.

17. Pea. Astragalus extract, or an active fraction thereof or compound isolated therefrom, as claimed in claim 15 wherein the extract is separated on a macro- porous resin, washed with water, washed with either 30% or 50% ethanol and eluted with 90% ethanol.

18. An Astragalus extract, or an active fraction thereof or compound isolated therefrom, as claimed in any of the preceding claims wherein the Astragalus is Astragalus membranaceus (Fisch) Bge. or Astragalus membranaceus Bge var. Mongholicus Hsiao.

19. Pea. Astragalus raw material packaged or otherwise sold in a manner which indicates it is suitable and intended for use as an antiviral to treat infections caused by a virus from one of: o the genus - Flavivirus, (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4).

20. The use of Astragalus or an extract thereof or an active fraction thereof or an isolated compound therefrom in the manufacture of a food, dietary supplement or food additive for use as an antiviral to treat infections caused by a virus from one of: o the genus - Flavivirus, (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4).

21. A method of treating infections caused by a virus from one of: o the genus - Flavivirus, (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4) comprising administering an effective amount of an Astragalus extract or an active fraction thereof or an active compound isolated therefrom to a patient or animal.

22. A method as claimed in claim 21 wherein the Astragalus extract, or an active fraction thereof, is provided in a unit dosage form.

23. A method as claimed in claim 22 wherein the unit dosage form is for oral delivery.

24. A method as claimed in any of claims 21-23 wherein a daily dosage is equivalent to 9-3Og of raw material.

25. A method as claimed in any of claims 23-24 wherein administration is with at least one other immuno-modulatory or anti viral drug.

26. A method as claimed in claim 25 wherein the other drug is interferon and / or Ribavirin.

27. An Astragalus extract fraction having an HPLC fingerprint showing a primary peak for Astragaloside I substantially as shown in any of Figs 16a to 16d.

28. An Astragalus extract fraction as claimed in claim 27 showing a secondary peak for Formononetin-7-O-β-D-glucoside

29. An Astragalus extract fraction as claimed in claim 27 or 28 showing a tertiary peak for Astragaloside IV

30. An Astragalus extract fraction as claimed in any of claims 27-29 comprising no more than seven additional peaks of prominence between the peak for Astragaloside IV and the peak for Astragaloside 1.

31. An Astragalus extract having a TLC fingerprint comprising six identifiable spots substantially as illustrated in any of lanes 2 to 5 of Fig 14 or 15.

32. A method for producing an extract of Astragalus which exhibits antiviral activity against a virus from one of: o the genus - Flavivirus (Table 1.1); o the genus - Pestivirus, (Table 1.2); o the genus - Unassigned Flaviviradae (Table 1.3) or o tentative Species in the Genus HCV (Table 1.4). and which is purified by at least a factor of 10 with reference to dried raw plant material, and which is characterized in that it comprises at least one marker selected from the group consisting of: o Astragaloside I; o Astragaloside IV o Formononetin-7-o-β-d-glucoside; and o 3 ' -hydroxyl-formononetin-7-o-β-d-glucoside comprising:

1) an alcoholic extraction (which may be repeated);

2) an ethanol-water precipitation process (which may be repeated); and either

3.1) a systematic solvent fractionation step with a plurality of solvents of different polarity, or

3.2) a macro-porous resin column purification.

33. A method for producing an extract of Astragalus as claimed in claim 32 wherein the extract is separated on a macro-porous resin, washed with water, washed with a low concentration ethanol and eluted with a high concentration ethanol.

34. A method for producing an extract of Astragalus as claimed in claim 33 wherein the low concentration ethanol is either 35% or 50% ethanol and the high concentration ethanol is 90% ethanol.