BIOLOGICALLY ACTIVE EXTRACTS FROM STEPHANIA BRACHYANDRA

TECHNICAL FIELD

The invention relates to an extract, or a fraction thereof, from the plant Stephania brachyandra, which extract has multiple biological activities. The extract, or a fraction, exhibits potent anti-cancer activity, particularly anti-melanoma activity. It is also useful for treating or preventing conditions associated with angiogenesis, inflammation and immune system modulation. Further, the extract, or a fraction, has been found to be useful for treating or preventing Alzheimer's disease and for modulating glucose uptake (which means that it has potential for treating or preventing diabetes).

BACKGROUND

In Asian traditional medicine there are many preparations that are derived from plants, shrubs and herbs. There is a vast array of these, some of which are unique to certain climates or particular ethnic groups. Plants from the genus Stephania are frequently used in Asian medicinal preparations. There are numerous species of this genus found in use throughout Asia, especially in China. In particular, extracts from the root Stephania ietrandra have found extensive application. The compound tetradrine has been identified as being responsible for a number of the therapeutic activities demonstrated by Stephania tetrandra. Tetradrine has been reported to have application in treating circulatory disorders, silicosis and arthritis, and to have anti-inflammatory, immunosuppressive, aπti-fibrogenetic and antioxidant activities. Tetradrine has been reported to arrest cell cycle for lung cancer cells in culture (Lee et a/., Inter. J. Oncology, (2002) 21 : 1239-1244), indicating that the compound has anti-turrjour activity.

The isoquinoline alkaloids that are found in Stephania venosa, namely crebanine and palmatine, are reported to have cytotoxic activity, particularly on breast cancer cells (Keawpradub et a/., J. Sci. & Technol., (2001) 23: 225 - 234). Fangchinoline, a plant alkaloid isolated from Stephania venosa has been reported to inhibit vascular smooth cell proliferation (Zhang et a/., Biochem. Pharmacol., (2003) 66: 1853-1860) and also to have an anti-glycemic effect (Hou, L ₁ US 5,908, 628 (1999)).

Stephania hemandifolia extracts have testis-inhibitory substances (Jana et a/., Asian J. Andrology (2003) 5: 125-129) while preparations from other Stephania species have been reported to have analgesic, antipyretic and anti-inflammatory activity (Tsutsumi et al., Biol & Pharmaceut Bull., (2003) 26: 313-317).

Although a number of Stephania species have been investigated for potential biological activities, there is negligible published evidence for biological activities associated with Stephania brachyandra. Further, the species that comprise the Stephania genus have widely disparate physical and biological characteristics. There is considerable variability of the compounds, including alkaloids, present in plant materia! across the genus. In particular, the constituents of extracts of these Stephania species are not predictive of the constituents of extracts from Stephania brachyandra.

in the ongoing search for new biologically active extracts from plants, the applicant has investigated Stephania brachyandra and surprisingly found that extracts of this species exhibit multiple biological activities, in particular anti-cancer, anti-angiogenesis, anti- inflammation, anti-diabetes, immune system modulation, and activity in assays for Alzheimer's disease. While extracts from certain Stephania species do exhibit biological activities, as outlined above, the multiple activities of Stephania brachyandra extracts is a surprising finding.

It is therefore an object of the invention to provide a preparation containing an extract from Stephania brachyandra having anti-cancer, anti-angiogenic, anti-inflammatory, anti-diabetic, or immune system modulation activity, or activity against Alzheimer's disease, or to at least provide an alternative to existing preparations having any one of these activities.

STATEMENTS OF INVENTION

In a first aspect, the invention provides a method of treating or preventing cancer by administering to a subject an extract, or a fraction thereof, from Stephania brachyandra.

Preferably the cancer is melanoma, lung cancer, breast cancer, brain cancer, or prostate cancer, especially melanoma.

In a second aspect, the invention provides a method of treating or preventing a disease or disorder in which it is desirable to inhibit angiogenesis by administering to a subject an extract, or a fraction thereof, from Stephania brachyandra.

Preferably the disease or disorder in which it is desirable to inhibit angiogenesis is cancer, rheumatoid arthritis, retinopathy, macular degeneration, or psoriasis. The cancer may be any cancer, but is preferably melanoma, lung cancer, breast cancer, brain cancer, or prostate cancer.

In a third aspect, the invention provides a method of treating or preventing a disease or disorder in which it is desirable to inhibit inflammation by administering to a subject an extract, or a fraction thereof, from Stephania brachyandra.

Preferably the disease or disorder in which it is desirable to inhibit inflammation is joint inflammation including arthritis, lung inflammation including asthma, emphysema, and obstructive pulmonary disease, or gastrointestinal tract inflammation including inflammatory bowel disease and Crohn's disease.

In a fourth aspect, the invention provides a method of treating or preventing diabetes by administering to a subject an extract, or a fraction thereof, from Stephania brachyandra.

In a fifth aspect, the invention provides a method of treating or preventing a disease or disorder in which it is desirable to modulate the immune system by administering to a subject an extract, or a fraction thereof, from Stephania brachyandra.

Preferably the disease or disorder in which it is desirable to modulate the immune system is a disease or disorder where it is desirable to stimulate the immune system such as cancer, microbial infection, or toxicity. Alternatively, the disease or disorder in which it is desirable to modulate the immune system is a disease or disorder where it is desirable to suppress the immune system such as an autoimmune disease or the prevention of rejection following organ transplantation.

In a sixth aspect, the invention provides a method of treating or preventing Alzheimer's disease by administering to a subject an extract, or a fraction thereof, from Stephania brachyandra.

The invention also provides the use of an extract, or a fraction thereof, from Stephania brachyandra for the preparation of a medicament for treating or preventing:

1. cancer;

2. a disease or disorder associated with angiogenesis;

3. a disease or disorder associated with inflammation;

4. diabetes; 5. a disease or disorder associated with immune system modulation; or

6. Alzheimer's disease.

- A -

It is preferred that the extract from Stθphania brachyandra is an extract obtained by solvent extraction. The solvent is typically ethanol or an aqueous ethanol mix, or alternatively may be a supercritical fluid such as supercritical CO ₂ . The solvent is preferably 50-100 % ethanol in water, typically 90 or 95 % ethanol in water.

The extract, or a fraction thereof, may be administered directly or may be combined with a suitable carrier or excipient. The extract, or a fraction thereof, may be administered in the form of a solution or suspension in water or some other suitable solvent or mixture of solvents.

DETAILED DESCRIPTION

The invention provides an extract, or a fraction thereof, that has multiple biological activities and is therefore useful for treating or preventing a variety of conditions. The extract is typically an aqueous ethanol extract, although it is to be appreciated that extracts obtained using other solvents form part of this invention.

The applicant has found that an extract from Stephania brachyandra has potent anti-cancer activity, particularly anti-melanoma activity. The mode of action of this activity is, at least in part, considered not to be via inhibition of angiogenesis. However, the extract does exhibit anti-angiogenesis activity. The extract is therefore both anti-angiogenic and has anti-cancer activity. In addition to activity against cancer and angiogenesis, the extract has antiinflammatory properties and modulates the immune system.

There is often a benefit in controlling the three biological processes of angiogenesis, inflammation and immune system modulation at the same time. For example, cancer growths typically rely on angiogenesis for their survival and spread, inflammation is often a consequence of the growth of cancers, and a patient's immune system is often compromised by cancers. Rather than provide a cocktail of drugs, an extract from Stephania brachyandra may be a useful therapeutic alternative as it has the ability to attack more than one aspect of the biology of cancer.

The extract has also been shown to increase giucose uptake. The extract therefore has potential for the treatment of diabetes, or may be useful for the enhancement of physical performance such as is required in many sports and recreational activities. The extract has

additioπally found to be effective in an Alzheimer's assay and so has potential for the treatment or prevention of this disease.

In summary, the range of biological activities exhibited by an extract from Stephania brachyandra means the extract, or a fraction of the extract, is potentially useful for the treatment or prevention of a surprisingly wide range of diseases and disorders.

Cancer

A crude extract of Stephania hrachyandra strongly inhibited the growth and proliferation of melanoma cells in culture in a dose dependent manner. Complete inhibition was achieved at- 100 μg/ml. The extract aiso inhibited the growth of prostate cancer cells in a dose- dependent manner. At 100 μg/ml, the extract showed nearly 50% inhibition.

Considerable evidence has been obtained that a number of extracts from Stephania brachyandra are strong inhibitors of the proliferation of melanoma cells in vitro. There are no reports of extracts from other Stephania species showing anti-melanoma activity. Activity appears to be higher for extracts obtained using extraction solvents having higher proportions of ethanol to water. Because the extracts show other activities, such as antiinflammatory, anti-angiogenic and immune stimulatory effects, they are likely to be particularly beneficial for the treatment of melanoma in vivo. As well, there is evidence that certain fractionations of ethanolic extracts can produce more potent anti-tumour activities.

A crude extract was used for an in vivo melanoma study in mice. A 0.5% solution of a crude extract reduced the growth rate of melanoma in mice, inhibiting growth by 29%. The same extract was also effective in reducing the metastases of the tumorous lesions, and the survival rate of the mice was improved. Certain more potent fractions of the crude extract are expected to show even greater benefits for the treatment or prevention of various cancers, particularly melanoma.

Angiogenesis

The extract prepared by stirring the dried mature Stephania brachyandra (root) with 50% ethanol has significant anti-angiogenic activity. At 200μg/ml, the anti-angiogenic activity of this crude extract is approximately 3 times that of the reference compound, fumagillin (20μg/m!). Increasing the pH of the extract temporarily to 11.0 had the effect of increasing the anti-angiogenic activity by a further 28%. An extract from a 1 year old Stephania brachyandra vine had similar activity to that from a 5 year old root. If the pH of the extract is

lowered temporarily to 3.0 the activity is increased by 91%, and if the extract is passed through diatomaceous earth, the extract showed 93% higher anti-angiogenic activity.

Inflammation Extracts were shown to inhibit the production of superoxide by activated neutrophils. A solution of 200μg/ml caused greater than 50% inhibition, whereas a 200μg/ml solution of aspirin was able to produce approximately 30% inhibition under the same conditions. The effect on neutrophil activity was similar for the extracts from young and mature roots. Activity was retained when the extracts were subjected to pH changes or passed through absorptive matrices such as charcoal or diatomaceous earth. At 400μg/ml, the extract inhibited the conversion of monocytes to macrophages by up to 72% as judged by NO production. As with the neutrophils, the monocyte inhibitory effects were retained when the extracts were fractionated.

Diabetes

Extracts markedly stimulated the ability of skeletal muscle to absorb and take up glucose. A 100μg/ml solution stimulated the uptake by 4.44 times compared with a 1μM solution of insulin which stimulated uptake by 2.47 times. The ability of liver cells to take up and absorb glucose was increased by 59% by a 100μg/ml extract. These effects on glucose metabolism are strong indicators that an extract from Stephania brachyandra may be useful for treating or preventing diabetes.

Immune System

Extracts from both the mature and the young roots are inhibitory of the proliferation of T cells isolated from spleen. This was shown for both naϊve T cells and for cells that have been stimulated with the mitogen, Concanavalin A. The extract from the young vine had no effect on naϊve cells - only cells that had been stimulated were inhibited. Fractionation of the extract from the mature roots by pH adjustment to 11.0 or by passage through absorptive matrices did not cause any loss of this activity when tested with both normal cells and activated cells. Adjusting the pH to 3.0 reduced the inhibitory effect.

Alzheimer's Disease

The effect of amyloid peptide on PC-12 cell proliferation rate was measured, but showed negligible effect. However, apoptosis of these cells, which had been stimulated by the amyloid, were inhibited by the extract at 1000μg/mi. Cell apoptosis indicates that an extract from Stephania brachyandra may be useful for treating or preventing Alzheimer's disease.

Extract

While any suitable solvent may be used to obtain the extract from the plant material, the solvent is typically ethanol or an aqueous ethanol mixture. In the examples described below, the extract is prepared using 50% ethanol/water or 95% ethanol/water. The extract may be subject to any purification or modification steps, such as altering the pH, filtration or chromatography.

Wherever in this specification an extract from Stephania hrachyandra is referred to, it is to be appreciated that the term includes reference to a fraction of an extract from Stephania brachyandra. The fraction may be obtained by any suitable method such as chromatography or pH adjustment. The fraction of the extract will typically contain several compounds, although a fraction may also be a single isolated substantially pure compound.

Various parts of the Stephania hrachyandra plant have been investigated, including root and vine of the plant. The extract of the invention may be obtained from any part of the plant that provides the requisite activities. The investigations also show that the age of the plant is not critical as significant levels of many of the activities were detected in extracts from both young and mature specimens.

It will be appreciated that the extract may be administered in any suitable manner including, but not limited to, oral administration, topical administration, and administration by injection intravenously, subcutaneously, intradermally or intraperitoneally. Suitable formulations of the composition of the invention include capsules, tablets, granules, powders, creams, ointments, suspensions and solutions. A solution for oral administration may typically be an aqueous solution, although other suitable solvents may be used whether or not as co- solvents with water.

The invention is further described with reference to the following examples. It is to be appreciated that the invention is not limited to these examples.

EXAMPLES

Example 1 : Preparation of extracts

Lyophilised root from Stephania brachyandra was extracted by stirring with 50% ethanol:water for 24 hours at 20 ⁰ C. After filtration, the extract was dried and the yield determined before being re-dissolved in 50% ethanol.

Extracts of Stephania brachyandra using 95% ethanolrwater were also prepared and tested. The extract was subjected to a number of fractionation procedures, including: a. Lowering the pH of the extract to 3.0 for 2 hours with hydrochloric acid and then returning it to neutral by adding sodium hydroxide; b. Raising the pH to 11.0 for 2 hours with sodium hydroxide and then returning it to neutral with hydrochloric acid; c. Passing the extract through a bed of charcoal and using the non-absorbing material in assays; and d. Passing the extract through a diatomaceous earth and using the non-absorbing material in assays.

Anqiogertesis

Example 2: Rat Aorta Assay The aorta was removed from a rat and cleaned of adhering fatty and connective tissues before being cut into rings of approximately 3mm size. Fibrinogen was layered in the bottoms of wells of multi-well culture plates and allowed to gel by thrombin action. A ring was then layered on the top of each gel and a further layer of fibrin placed on this. The fibrinogen was prepared in MCDB131 medium supplemented with antibiotics. The double layer of fibrin was then overlaid with MCDB131 containing the test material. The final concentration of solvent in all sample wells was 2% ethanol, which is known to have negligible effect on the growth rate of the microvessels. As a control, fumagillin (20μg/m!) was assayed in triplicate on each plate. The gels were incubated at 37 ⁰ C in an atmosphere of 3%CO ₂ /97% air. The rings were examined using an inverted microscope and the growth of microvessels from their perimeters was observed. Digital pictures were taken every 2 days and the extent of microvessel growth relative to the size of the ring was determined using NIH Image software. From this the rate of growth of microvessels was determined for each well.

A 50% ethanol extract from Stephania brachyandra was assayed in triplicate and the mean growth rate (± SD) was calculated. Angiogenesis for the control was measured at 10.159 ±

1.128. Fumagiliin (20μg/ml) was measured at 7.193 ± 1.209, representing 29.20% inhibition. Stephania brachyandra (root extract) (200μg/mI) was measured at 0.690 ± 0.26, representing 93.21% inhibition.

In a separate experiment, the aπti-angiogenic activity of 95% ethanol extracts (100μg/ml) from 1 year old Stephania brachyandra root and vine was found to be slightly less than that for the extract from mature root. However, both exhibited at least 50% inhibition (Table 1).

Table 1 : Effect on Angiogenesis by Extracts and Fractions from S. brachyandra (% inhibition)

Decreasing the pH to 3.0 was detrimental to the activity of the extract from the mature root. But raising of the pH to 11.0 increased the activity markedly. Charcoal fractionation of the extract from the mature root resulted in a loss of activity. However, the diatomaceous earth fractionation produced an extract that had similar activity to that of the ethanol extract. By comparison, the pH 3.0 extract from the young vine was highly inhibitory. This is to be contrasted with the pH 3.0 extract from the mature root. The pH 11.0 extract from the young vine also shows a rather different effect to that for the mature root. Passage of the extract from the young vine through charcoal had a slight stimulatory effect, but the fraction not absorbed by the diatomaceous earth was almost completely inhibitory.

inflammation

Example 3: Neutrophil Assay

Neutrophils were prepared from fresh rat blood and diluted with phosphate-buffered saline (PBS) and adjusted to a concentration of 10 ⁷ cells per ml. The purity of the preparation was

95% or higher. The test sample was pre-incubated with a cell suspension at 37°C for 15 minutes in wells of a 96 well culture plate. Catalase and WST-1 dye reagent were then added to each incubate (including controls). Each incubate was activated with phorbol myristate acetate (100ng/mI). After incubation at 37°C for 1 hr the reaction was stopped and the level of superoxide produced in each well of the plate from oxidation of the dye WST was

determined colorimetrically at 450nm. As a positive control, triplicate wells for each of 3 concentrations of aspirin were assayed. The 50% ethanol extract was assayed at three different concentrations. Each concentration was assessed in triplicate. The extract was assessed for its ability to inhibit the production of superoxide by activated neutrophils. Activity was compared with the inhibition produced by aspirin (Table 2).

Table 2: Effect of Extracts on Superoxide Production by Activated Neutrophils (50% extract)

Absorbance % Inhibition

Cells 0.706

Cells (+Aspirin)

(200μg/ml) 0.500 29.18

(100μg/mi) 0.594 15.85

(50μg/mI) 0.670 5.08

Stephania brachyandra (root)

(400μg/ml) 0.208 70.55

(200μg/m!) 0.314 55.45

(100μg/ml) 0.472 33.10

The anti-inflammatory activity exhibited appears to be dose dependent. A 100μg/ml solution of the extract has a similar effect to a 200μg/ml solution of aspirin.

The 95% ethanol extract from mature root was subjected to various fractionation procedures and the effect on superoxide production by activated neutrophils investigated. The extracts exhibited significant anti-inflammatory activity (Table 3). Extracts having a concentration of 10Oμg/ml were found to be significantly more inhibitory than a 10Oμg/ml solution of aspirin. The extracts from the young and mature root were slightly more inhibitory. The extract from the young vines was noticeably less inhibitory than the extract from the roots. When the ethanol extract from the mature root was subjected to a decrease in pH, there was virtually no change in the anti-inflammatory activity (Table 3). But when the pH was increased to about 11.0, activity was reduced, indicating that the activity was susceptible to higher pH values. When the extract was passed through charcoal there was a slight loss of antiinflammatory activity. However, when passed through diatomaceous earth, the activity of the non-absorbed fraction was increased and was as strong as for the 1 year old root.

Table 3: Effect of Extracts on Superoxide Production by Activated Neutrophils (95% extract)

Sample % Inhibition

Aspirin 48.09 ¹ pH 3.0 Fraction from mature root 69.65 ⁴ pH 11.0 Fraction from mature root 53.57 ²

Fraction from mature root after absorption by charcoal 63.27 ⁴

Fraction from mature root after absorption by diatomaceous earth 85.74 ²

Ethanol extract of mature root 75.46 ⁴

Ethanol extract of 1 year old root 83.50 ^s

Ethano! extract of 1 year old vine 49.67 ¹

¹ p<0.05 ² p<0.02 ³ p<0.01 ⁴ pθ.005 ⁵ p<0.002 ⁶ p<0.001

(All extracts at 100μg/ml)

Example 4: Monocyte Assay

A density gradient barrier was prepared by mixing a working solution [Optiprep: diluent, 2:1 , v/v] with further diluent in a ratio of 2.3 to 5.0. A density of 1.076g/ml is required for isolating rat monocytes. Freshly isolated rat blood was cooled to 4°C. 5ml of the density gradient was layered over 5ml of the blood and 5ml of diluent was then layered gently on top. After centrifuging at 70Og for 30min at 4°C, the monocytes were collected from the top of the 1.076g/ml layer and diluted with 2 volumes of diluent. The pellet was gently resuspended in Hanks Balanced Salt Solution (HBSS). The cell concentration and the relative purity were determined. 500μl Aliquots of cell suspension were used. Aliquots (10μ!) of either the test solution or indomethacin were added to appropriate tubes and the cells incubated at 37°C for 1 hour. 10μl of LPS (5mg/ml) was then added to all tubes, except the control, to give a final concentration of LPS of approximately 20μg/ml. The cells were incubated overnight at 37°C and then centrifuged at 12,00Og (4°C) for 5min. The supernatants were collected and aliquots of these were then assayed in duplicate for the concentration of nitric oxide (NO) by the Griess reagent procedure using a kit (Sigma, St Louis, USA).

The effect of the Stephania brachyandra extract (50% ethanol) on the conversion and activation of monocytes to macrophages was assessed. The results are shown in Table 4.

Table 4: Effect of Extracts on NO Production by Activated Monocytes

NO Cone (μM) % Inhibition

Cells in the presence of LPS 9.610

Cells (No LPS) 0.044 99.5

Cells (LPS + L-NMMA) 0.566 94.1

Cells (L-NMMA) 0.091 99.1

Stephania brachyandra (root)

(400μg/ml) 2.679 72.1

(200μg/mI) 6.529 32.1

(100μg/ml) 6.791 29.3

•The Stephania brachyandra root extract shows moderate activity in this assay in a dose response manner.

In a separate experiment, the 95% ethanol extract (100μg/ml) of mature Stephania brachyandra root was shown to inhibit monocyte activation by 40% (Table 5). However, at the same concentration, the extract from the 1 year old Stephania brachyandra root inhibited this conversion by nearly 33%. The extract from the 1 year old vine was considerably more inhibitory reducing the activation by 80%. When the pH of the ethanol extract of the mature Stephania brachyandra root was lowered to 3.0, the anti-inflammatory activity increased from 40% to 65%. However, treating the extract with alkali temporarily or passing the extract through charcoal or through diatomaceous earth had little effect.

Table 5: Inhibition of Monocyte Activation by S. brachyandra Extracts

Sample % Inhibition lndomethacin 66.27 ⁵ pH 3.0 Fraction from mature root 65.44 ³ pH 11.0 Fraction from mature root 38.55 ^Z

Fraction from mature tuber after absorption by charcoal 39.52 ²

Fraction from mature tuber after absorption by diatomaceous earth 37.72 ¹

Ethanol extract of mature root 40.40 ²

Ethanol extract of 1 year old root 32.82 ¹

Ethanol extract of 1 year old vine 80.26 ⁶ p<0.05 ^: p<0.02 pθ.01 ⁴ p<0.005 p<0.002 ¹ pθ.001

Cancer

Example 5: Culturing of Tumour Cells

The cancer cells utilised in this study are:

Colon Cancer (Human adenocarcinoma) Cells (DLD-1 cells) Melanoma (Mouse) Cells (B16/F10 cells)

Breast Cancer (Human) Cells (MDA-MB-231 cells)

Prostate Cancer (Human) Cells (PC-3 cells)

Brain Cancer (Human glioma) Cells (Mo-59 cells)

Lung Cancer (Human) Cells (NCI-H 1299 cells)

For each tumour cell culture, the solutions to be tested were added to triplicate wells of a 96 well plate. DMEM culture medium containing 10% foetal bovine serum (FBS) was added to each of the wells to make the volume up to 200μI. The cells were suspended in DMEM containing 10% FBS. 100μl of the mixture was added to each well. The cells were incubated for 48h at 37°C in 5%CO ₂ /95% air. 10μl of MTT solution (5mg/ml) was added to each well and the incubation continued for a further 4 hours. Lysis buffer (100μl) was added and the incubation continued for another 4 h at 37°C. The absorbance of each well was then read at 570nm. The mean value was determined for each triplicate. Statistical significance was assessed using the Student t-test.

Example 6: Melanoma - in vitro

A Stephania brachyandra root extract (50% ethanol) was tested in duplicate at 25, 50 and 100μg/ml for its effect on the proliferation rate of B16 melanoma cells. The results are summarised in Table 6.

Table 6: Effect of Extracts on Proliferation of Melanoma Cells (O. D. + SD)

Absorbance ± SD % Inhibition

Control 0.40 ± 0.02

Stephania brachyandra (root)

25μg/ml 0.17 ± 0.06 56.5 (p<0.04)

50μg/ml 0.07 ± 0.00 83.7 (p<0.001)

100μg/mi 0.01 ± 0.00 98.0 (p<0.001)

The extract was found to be strongly inhibitory of the growth of the B16 melanoma cells. At a concentration of 100μg/ml, inhibition was about 100% while even a 25μg/mi solution caused more than 50% inhibition.

Example 7: Melanoma - in vivo

Equal numbers of male and female CB57BL mice (aged between 7 and 8 weeks at the commencement of the study) were used. The investigation involved five different dose levels of Stephania brachyandra extract, and was conducted as two separate experiments, in both experiments, the extract was added as a supplement to the drinking water.

Experiment 1:

Group A - Control group (5% ethanol final concentration).

Group B - Diet supplemented with ethanol extract of Stephania brachyandra root at 0.005% (5% ethanol final concentration).

Group C - Diet supplemented with ethanol extract from Stephania brachyandra root at

0.015% (5% ethanol final concentration). Group D - Diet supplemented with ethanolic extract from Stephania brachyandra root at

0.05% (5% ethanol final concentration).

Experiment 2:

Group A - Control group (5% Ethanol final concentration).

Group B - Diet supplemented with ethanol extract from Stephania brachyandra root at 0.15%

(5% Ethanol final concentration). Group C - Diet supplemented with ethanol extract from Stephania brachyandra root at 0.5%

(5% Ethanol final concentration).

Feeding of supplemented diets was commenced 7 days before 1 x 10 ⁵ melanoma B16 cells were injected into the left flank of each mouse. The consumption of water was measured twice weekly when the supplemented water was replaced with fresh supplemented water. From this, the dosage was determined. The water consumed, and hence the dose level of the supplements was measured on days 1, 5, 9, 12, 16, 19 and 25. Additionally, each mouse was weighed when the supplementation began, immediately prior to the injection of the melanoma cells and then after another 7 days (Day 14) and daily from Day 16 until the conclusion of the study. From Day 9 after the injection of the cells, the dimensions of each tumour growth were measured using digital callipers and the length (X), width (Y) and height (Z) determined so that the volume could be calculated daily according to the formula:

Volume = (π * X ^* Y * Z)/6 Survival rates using this data were determined. Following the sacrifice of each mouse, the tumour was excised, weighed and preserved in 10% formalin.

Methods of Melanoma growth determination: Animals were regarded as non-responders if

(1) their tumour size was less than or equal to 30mm ³ one day before the end of the trial and

(2) if tumours ulcerated at any time during the trial. Non-responders were removed completely from the group at all timepoints. Tumours that exceeded the 200mm ³ endpoint were capped using a formula of the mean plus one standard deviation above 200mm ³ on the final day. The capped value was entered in all individual timepoints where the animal's tumour exceeded the capped value for tumour size. This was calculated at the end of the experiment in order to avoid skewing the data. At the conclusion of the 21 day experimental period, each tumour was excised and weighed.

After euthanasia, each animal carcass was autopsied and the organs assessed for secondary tumours. Each tumour was also photographed in situ at the time of autopsy to provide visual reference and as backup for vascularisation scores.

Experimental design: Because there were five different doses of the Stephania brachyandra extract to be used, the study was conducted in two separate experiments. In the first experiment there were four groups comprising 5 males and 5 females each, with one control and three experimental groups. In the second experiment there were three groups comprising 5 males and 5 females each, with one control and two experimental groups. Univariate 2-way nested ANOVAs were used to examine the difference of tumour sizes between treatments and controls, samples nested within treatment and sexes (see diagrams below).

Experiment 1

Experiment 2

TREATMENTS CONTROL TREATMENT (fixed, orthogonal) / \

/ \

SAMPLES ( 2A ) (2B) (2C)

(random, nested) _{/ \} λ λ

/ \

SEX M F M λ F M F

(fixed, orthogonal)

Statistical Methods: Average tumour sizes were calculated for each sex for each day of measurement. Assumptions for normality were tested using q-q plots of studentized residual values and assumptions for heterogeneity of variances were tested using Levene's test and Residual plots. A model for the Univariate nested ANOVAs was custom made. SPSS 11 for Mac OSX was used for assumption testing and statistical analysis.

Results:

Water Consumption: The mean daily water consumption was calculated for each group. From this the mean daily dose of Stephanie brachyandra extract was calculated for each group. The data is summarised in Table 7.

Table 7: Mean Daily Consumption of Water and S. brachyandra Extract (Experiment 1)

Table 8: Mean Daily Consumption of Water and S. brachyandra Extract (Experiment 2)

The water consumption for the control and the experimental groups was fairly similar. In Experiment 1 , all three experimental groups displayed a reduced daily consumption of water. This was between 10 and 15%. However, all three extracts showed similar levels of consumption and there was no relationship to the dose level of the Sfephania brachyandra extract. The average daily water consumption by the mice receiving the high doses was similar to or less than for the unsupplemented animals (Experiment 2). The Control group in Experiment 2 consumed less water per day than the same group in Experiment 1 , and in fact was very similar to the consumption to that for the three supplemented groups in Experiment 1. However, the average daily water consumption for the group that received 0.5% Stephania extract in their water was reduced by 22%.

Tumour Size: The size of each tumour was measured throughout the experiment and the daily mean value calculated for each group overall and for each sex within each group. In both experiments, there was no significant difference between treatments, sexes and the individual groups nested within treatments. In Experiment 2, the tumours from Group C mice were obviously, though not statistically significantly, smaller than Groups A and B (Table 9). They were 29% lighter. Although there was no significant difference between sexes, females generally had smaller tumours than males. The tumours excised from the males that received 0.5% extract in their drinking water were 27% lighter than those that received no Stephania brachyandra extract. The tumours from the female mice were 35% lighter.

Table 9: Average Tumour Size (mm ³ ± SENI)

Tumour Growth Rate: Tumour growth rate was calculated from daily changes in average tumour sizes. In Experiment 2, growth rates were more variable between the groups (Table 10). The tumour growth rate of the highest concentration of extract (Group C) was 37% lower than Group B and 30% lower than the control group. The control group (Group A) had 10% lower tumour growth rates than the 0.15% S brachyandra extract (Group B).

Table 10: Tumour Growth Rate

Survival Rates: Survival rates were assessed as percentage of mice that survived each day of the experiment. In Experiment 2, all animals in the control group (Group A) had died or were sacrificed by Day 23, whereas 57% of Group C mice were still surviving on the last day of the experiment (Table 11).

Table 11 : Survival Rates (Experiment 2)

Metastases: Within the control group in Experiment 1 , metastatic growths were observed on Day 21 in the spleens of two mice that had to be sacrificed. On Day 23 there were two distinct growths in the spleen of a male mouse receiving 0.05% extract. This occupied about 1/3 of the organ. There were possibly small growths throughout the gastrointestinal (Gl) tract of a female animal from the 0.015% supplemented mice (Group C). On the day that the study ended and autopsies were performed, secondary growths were observed in the spleen of one female from the control group, from one male and one female from the 0.005% supplemented mice (Group B), in the spleen (which was also hypertrophied) from a female of Group C and in the Gl tract of a male from this group. One male in the Group D (0.05% supplement) had metastases in both stomach and intestine.

In Experiment 2, all of the mice were sacrificed by Day 23. The only metastases noted in the control group were in the liver of a male animal sacrificed on Day 23, in the spleen of a female sacrificed on Day 22 and in two females euthanased in Day 23. Among the mice receiving 0.15% extract the only signs of secondary growth were in the spleen and subcutaneously adjacent to the primary neoplasm in a female sacrificed on Day 23. This animal had a small pancreatic growth. Another female had 2 small pancreatic lesions and a similar subcutaneous one. Of the 10 animals receiving 0.5% extract, only one (a female) showed a secondary growth. This was a small lesion on its spleen.

In summary, the ethanolic extract from Stephania brachyandra root was very effective at 0.5% or approximately 12.77μl per day per mouse. It particularly i) retarded the growth rate of the tumours ii) significantly improved the survival rate of the mice iii) inhibited the spread of cancer to secondary sites.

Example 7: Prostate The same extract as for Example 6 was tested in duplicate at 25, 50 and 100μg/ml for its effect on the proliferation rate of PC-3 prostate cancer cells in culture. The results are summarised in Table 12.

Table 12: Effect of Extracts on Proliferation of Prostate Cancer Cells (O.D. ± SD)

Absorbance ± SD % inhibition

Control 0.84 ± 0.02

Stephania brachyandra (root)

25μg/ml 0.59 ± 0.05 29.6 (p<0.025)

50μg/ml 0.49 ± 0.08 42.4 (p<0.025)

100μg/ml 0.43 ± 0.01 48.7 (p<0.002)

The extract from Stephania brachyandra (root) was found to be inhibitory of the growth of PC-3 prostate cancer cells. At a concentration of 100μg/ml, inhibition was about 49%. Inhibition was also observed at lower concentrations. Further, inhibition was clearly dose dependent.

Example 8: Proliferation Rate of Cancers

The inhibitory effect of different (95% ethano!) extracts from Stephania brachyandra on the proliferation rate of cultured tumour cells is summarised in Table 13.

Table 13: Effect of Stephania brachyandra Extracts on Growth of Tumour Cells

(% of Growth of Control Cells, Mean ± SD) (100μg/ml)

A. Stephania brachyandra 5 years old (root) B. Stephania brachyandra 1 year old (root)

C. Stephania brachyandra 1 year old (vine)

The highest specificity for these extracts is for melanoma (row 1). The extract from the root of the young plant (column B) is more active than that from the older root (column A), in addition, the extract from the vine of the young plant (column C) is quite effective as an inhibitor of melanoma cell proliferation. The extract from the older root (column A) has relatively poor activity when added to the culture of breast cancer cells (row 3). But the extract from the young root (column B) and from the young vine (column C) is quite potent for this type of tumour (row 3). However, these extracts had relatively little effect on the other four tumour cells. In fact, two of the extracts stimulated the growth of the colon cancer cells.

The extract from the young root (column B) shows stronger inhibitory activity than the extract from the root of older plants (column A). For some cells, the extract from the vine of the young plant (column C) was more active than the extract from the root of the same plant (column B).

Example 9: Effect of Fractionation of Extracts on the Proliferation of Mouse Melanoma

The ethanol extract from mature root of Stephania brachyandra was subjected to a number of fractionation procedures. As shown in Table 14, both lowering and raising the pH increased anti-melanoma activity. Passing the extract through absorption matrices (charcoal and diatomaceous earth) also increased activity.

Table 14: Effect of Fractionating on the Proliferation of Mouse Melanoma Cells

(100μg/ml)

Example 10: Effect on Growth of Human Melanoma

The extracts used to investigate the growth of mouse melanoma were tested for their effects on the human melanoma cell line A-2058. Ethanol extracts and fractions were tested at seven different concentrations (Table 15).

Table 15: Inhibition of Human Melanoma Cell Proliferation (% inhibition)

p<0.05 ^' p<0.02 p<0.01 p<0.005 ¹ p<0.002 p<0.001

The extract from the mature root was significantly inhibitory (37%) only at 200μg/ml. However, the extract from the 1 year old root was much more active. It achieved significant inhibition at 50μg/ml (25%), which was approximately 4 times more active at the higher concentration. The extract from the young vine also was inhibitory. Fractionation of the extract from the mature root by exposure to acid pH or passage through charcoal or diatomaceous earth did not result in any loss of the anti-proliferative activity when added to the human melanoma cells.

Immune Modulation Example 11: Immune Modulation Assay

A spleen was isolated from a rat and a T-cell rich fraction (free of red blood cells) was prepared by density gradient centrifugation. The cell preparation was diluted to about 1.2 x 10 ⁶ cells per ml with RPMI culture medium and approximately 2.4 x 10 ⁵ cells were added to each well of a 96 well culture plate. For each concentration of the extract to be tested, 12 wells (a row) were used. Three were for the sample added at the start of the culture, three had the sample added 24 hr after culturing commences, three had the sample and the mitogen, Concanavalin A (Con A) (1μg/ml), added at the start of the culture, and three had the Con A added at the start of the culture and the test sample added after 24 hr incubation. The plate was incubated at 37°C in 5% CO ₂ /95% air for 72 hrs. The incubation was terminated by adding 15μl of MTT solution (5mg/ml) to each well and the cells were lysed 2 hrs later with 100μl of 10% Sodium Dodecyl Sulphate/45% dimethylformamide, pH4.7. The formazan crystals were dissolved by incubating overnight at 37°C before reading the absorbance at 570nm. The mean absorbance of each triplicate was determined.

The 50% ethanol extract from Stephania hrachyandra (root) was assessed for its ability to modulate the proliferation rate of spleen T-cells under 4 different culture conditions. The results are summarised in Table 16.

Table 16: Modulation by 50% Ethanol Extracts of Proliferation Rate of Rat Spleen Cells (% inhibition)

Column A Column B Column C Column D

Stephania brachyandra (root)

100μg/mI 35.21% 40.46% 30.79% 53.02%(stim)

200μg/mi 55.96% 7.80% 15.25%(stim) 53.02%(stim

Column A is the effect of adding the sample at the same time as the culture is started. Column B is the effect produced by adding the sample 24 hours after the culture is commenced. Column C is the effect when the sample and the mitogen (Concanavalin A) are added simultaneously

at the start of the culture. This effect is that produced in relation to the effect produced by Con A alone.

Column D is the effect noted when the sample is added 24 hours after the Con A has been added to the culture.

The root extract inhibits spleen cell proliferation especially when added at the commencement of the culture (Column A). When the extract was added after the cells have settled into the growth pattern, the extract was quite inhibitory at the lower concentration but much of this had disappeared at 200μg/ml (Column B). When the extract was added in conjunction with the mitogen, ConA, the extract was quite inhibitory at the lower concentration (Column C), but at 200μg/ml it produced a further 15% stimulation above that achieved by the ConA. However, if added after the cells have been stimulated and are proliferating in their activated state, the extract can eiicit a further 50% increase in the growth rate. This was the same at both concentrations suggesting that the effect is maximal and that lower concentrations of extracts would have a significant effect also.

Further investigations on immune modulation were made using 95% ethanol extracts and fractions derived from them. The results are summarised in Table 17.

Table 17: Modulation by 95% Extracts of Proliferation Rate of Rat Spleen Cells

(% inhibition)

A B C D pH 3.0 Fractionation of the Ethanol Extract (mature root)

200μg/ml 18.59% 29.67% ⁶ 70.21 % ¹ 0.89% pH 11.0 Fractionation of the Ethanol Extract (mature root)

20Qμg/ml 29.03% ¹ 50.03% ⁶ 73.44% ³ 42.83% ¹

Fractionation of Ethanol Extract by Charcoal (mature root

200μg/ml 32.12% ¹ 53.85% ⁵ 71.06% ⁴ 69.41 % ⁶

Fractionation of Ethanol Extract by Diatomaceous Earth (mature root)

200μg/m! 30.54% ¹ 50.56% 63.55% ³ 66.20% ⁵

Ethanol Extract (mature root)

200μg/ml 28.80% 46.46% ⁶ 72.55% ³ 58.61 % ⁵

Ethanol Extract (1 year old root)

200μg/ml 27.77% 47.16% ⁶ 71.73% ³ 62.36% ⁵

Ethanol Extract 1 year old vine)

200μg/ml 0.47% 1.54%(stim) 57.05% 40.93% ³

¹ p<0.05 ² p<0.02 ³ p<0.01 ⁴ p<0.005 ⁵ p<0.002 ⁶ p<0.001

A is the effect of adding the sample at the same time as the culture is started. B is the effect produced by adding the sample 24 hours after the culture is commenced.

C is the effect when the sample and the mitogen (Concaπavaiin A) are added simultaneously at the start of the culture. This effect is that produced in relation to the effect produced by Con A alone.

D is the effect noted when the sample is added 24 hours after the Con A has been added to the culture.

Alzheimer's Disease

Example 12: Alzheimer's Assay

Wells of a 96 well plate were coated with 40μg of rat tail tendon collagen and 6 x 10 ⁴ PC-12 cells (usually in 200μl) were placed in each well in Hams F-12/K media containing 10% horse serum and 5% FBS and supplemented with NGFβ at 100ng/ml. Half the media was changed every 2 days, until the cells had fully differentiated in 7 days, when they were ready for the assay. Amyloid β was made up three days previously, by suspending the peptide in PBS and incubating at 37°C, to facilitate aggregation. The peptide was suspended at 100μM. All but 45μl of media was removed and 5μl of stock Amyloid β was added, to a final concentration of 10μM. Three wells remained untreated, having 5μl of PBS added. The plates were incubated for 16 hours for the caspase assay, at 37°C in 95%air/5%CO ₂ . The cells were lysed, and spun at 10,000g for 20 min. The activity was measured by the ability of the aliquots of the supernatants to release p-nitroaniline from the peptide acetyl-Asp-Glu-Val- Asp-p-nitroanilide. The absorbance was measured at 405nm.

Example 13: Effect on Apoptosis

The extract was tested for its effect on apoptosis by assaying caspase-3 activity. The extracts were evaluated in the PC-12 cell cultures that had received β-Amyloid peptide (25- 35). The results are summarised in Table 18.

Table 18: Effect on Inhibitory Action of β-Amyloid (25-35) on the Apoptosis of PC-12 Cells by Assaying Caspase-3 Activity

Average Activity ± SD % increase

Control (2% Ethanol) 25.4 ± 7.6

Cells + β-Amyloid

(2% Ethanol) 36.2 + 15.6 42.5

Stephania brachyandra (root)

1000μg/ml 28.1 ± 4.6 10.6

The addition of the amyloid peptide increased apoptosis of the cells. This was manifested by an increase in caspase-3 activity. In this experiment, the increase was 42.5%. When a high concentration of the Stephania brachyandra (Root) extract was added with the peptide the increase was only about 11 % and so it would appear to inhibit much of the apoptotic effect.

Diabetes

Example 14: Soleus Muscle Assay

A male rat was killed and the soleus muscles were dissected in carboxygenated Krebs- Heinsleit Buffer (KHB). Each muscle was then teased longitudinally into two halves and washed in the KHB. The muscle strips were each incubated in 5ml of DMEM with 0.1% bovine serum albumin for 30 minutes at 37°C. The sample to be tested or the insulin

(positive control) was then added to the tubes containing the tissue. Each sample was assayed in duplicate. This was followed by the addition of 0.5μC| of D-[U- ¹⁴ C] glucose to each tube, except for the control tubes, which do not contain any test sample or insulin.

Incubation was continued for 2 hours at 37°C in the presence of 95%0 ₂ /5%C0 ₂ in a shaking water bath. After removing and washing, the tissue was dried in pre-weighed containers.

The tendons were excised from the dried muscle, which was then weighed. The muscle was solubiiised in a known volume of NCS solubiliser and aliquots taken and counted in a liquid scintillation counter using ACS as scintillant. The effect of the extract on the uptake of glucose by isolated rat soleus muscle is shown in Table 19.

Table 19: Uptake of Glucose by Rat Soleus Muscle

(cpm per g dry weight) % of Control

Control 800.93

Insulin (1μM) 1980.12 247.23

Stephania brachyandra (root) (100μg/ml) 3561.57 444.68

The extract at 100μg/ml produced a 4.5 fold increase in the glucose uptake. This is considerably more that achieved by a 1 μM solution of insulin.

Example 15: Hepatocytes

The perfused liver was isolated from a rat that had been starved for 24 hours. After disrupting the liver, the cells were isolated by centrifugation, washed twice and resuspended at 10 ^e cells per ml in Krebs-Ringer bicarbonate buffer containing 1 % bovine serum albumin.

The cells were incubated for 2 hours at 5%C0 ₂ /95%0 ₂ at 37°C. The cells were washed with

PBS, resuspended in 0.9ml of PBS/1 %BSA without glucose and incubated for 15 minutes. 9μl of media was removed and 9μl of sample was added to the well. Each test material was assayed in triplicate. ¹⁴ C-deoxy-giucose (0.4μCj/well) (final concentration of deoxy-glucose is 5mM) was then added. This radioactive glucose was prepared as follows: 2.5ml of PBS/(1%)BSA contains 5mM glucose + 50μl of 200μCj/mI of ¹⁴ C- 2-deoxy-glucose. After 2 hours incubation at 5%CO ₂ /95%O ₂ at 37°C, the cells were washed 3 times with ice-cold PBS containing 1OmM glucose. The cells were then solubilised with 0.5ml of NaOH. The 0.5ml of 0.5M HCI was added and 100μl of solubilised cells were added to 3.9ml of ACS counting fluid and counted. The results are expressed as the mean of the radioactive count for each triplicate + SEM in Table 20.

Table 20: Mean radioactive count per 100μl aliquot + SEM

Mean cpm/100μl ± SEM % of Control

Control (π=3) 1392.33 + 90.31

Stephanie brachyandra (root) 2218.33 + 133.93 159%

(100μg/ml)

The Stephania hrachyandra (root) extract displayed considerable ability to stimulate the ability of hepatocytes to bind and incorporate glucose. At 100μg/rπ[ it caused a 60% increase in the absorption.

Stability of Extracts Example 16: Storage The ethanol extracts were stored at 20 ⁰ C, 4 ⁰ C and -80 ⁰ C for 3 weeks. The relative activities of these at inhibiting the growth rate of melanoma B16/F10 cells were measured (Table 21).

Table 21 : Inhibition of Melanoma Cell Proliferation Following 3 Weeks Storage (% inhibition)

¹ p<0.05 ^' p<0.02 p<0.01 p<0.005 ¹ p<0.002 ¹ p<0.001

There was an obvious temperature dependence for all three extracts. Those stored at -80 ⁰ C retained almost all of their antagonistic activity while those stored at 20 ⁰ C were the least stable. The inhibition was almost identical for all three extracts that had been stored at - 80 ⁰ C. For those stored at 20 ⁰ C, the extract from the older tuber was the more stable and the extract from the young vine being the most labile. This stability behaviour is to be compared with the activity found when the extracts were fresh as described below.

The anti-melanoma activity of extracts from the mature root, young root and young vine was compared before and after 2 months in the cryo-freezer (-80 ⁰ C) (Table 22). None of the three extracts lost any of their activity on cryo-storage.

Table 22: Inhibition of Melanoma Cell Proliferation Following 2 Months Storage at - 80 ⁰ C (% inhibition)

Cone (μg/ml) Before Storage After 2 Months Storage

Mature root 50 48.49 47.71

1 year old root 50 66.47 66.53

1 year old vine 50 35.86 34.57

Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

INDUSTRIAL APPLICABILITY

The extract, or a fraction thereof, obtained from the plant Stephania hrachyandra, has multiple biological activities and is therefore potentially useful in the prevention or treatment of a variety of diseases. These include cancer (particularly melanoma), angiogenesis, inflammation, immune system modulation, Alzheimer's disease, and diabetes.