AU2013100366A4 - Therapeutic and/or Prophylactic Extract of Mahogany species - Google Patents

Abstract

The invention relates to herbal compositions comprising an enriched Mahogany extract and at least one pharmaceutically acceptable additive, wherein the extract comprises an effective amount of saponins and tannins. In particular, the present invention relates to a 5 pharmaceutical or dietary supplement composition comprising: an effective amount of an enriched Mahogany species extract and at least one pharmaceutically acceptable additive said extract comprising at least 0.0001% w/w saponins and tannins and wherein said extract has the ability to inhibit at least one activity selected from the group consisting of COX-2 activity, Caspase 3 activity, 2,2'-azino-bis(3 10 ethylbenzothiazoline-6-sulphonic acid) (ABTS) activity, AKTI1 activity and ERKI activity (GH Matters) P92696.AU 4194325_1 (GHMafe.s) P92696.AJ -n 0 o o0 N tim oC ( --I -- O 'ou n N timo OO OO :, Q Q Q N 0 S o@o 0 1 'tC A SCO ;C> C CO C Q Q fl fl T N)l ~ ~ C x F zF e Fom ~~~ ~ ~ F F )0 tQ to ~mm coao U U d <U < .1p.

Description

- 1 Therapeutic and/or Prophylactic Extract of Mahogany species FIELD The invention relates to herbal compositions comprising an enriched Mahogany extract and at least one pharmaceutically acceptable additive, wherein the extract comprises an effective amount of saponins and tannins. In particular, the present invention relates to dietary supplements or pharmaceutical formulations containing herbal compositions for the treatment or prevention of cancer and/or associated inflammation. BACKGROUND Cancer is a well known, well studied, multi-factorial disease, which is traditionally treated using chemotherapeutic drugs whose role is to slow and hopefully halt the growth and spread of the cancer. Unfortunately, the majority of drugs currently on the market are not specific, which leads to the many common side effects associated with cancer chemotherapy. Consequently, in recent times, consideration has turned towards the use of other agents such as non-steroidal anti-inflammatory drugs (NSAIDs), which have been observed to delay the development of pre-malignant tumours and reduce the incidence of different forms of neoplasia in patients that taken these drugs over prolong periods. Much has been written in the lay and scientific press about the potential of one known class of NSAID; the selective cyclooxygenase (COX)-2 inhibitors which are anti-inflammatory and analgesic agents that lack the gastrointestinal side-effects of traditional NSAIDs. Although research on COX-2 inhibitors has focussed mainly on inflammation and pain, experimental and epidemiological data suggest that COX-2 inhibitors could be used in the treatment or prevention of a broader range of diseases including cancer. Recent studies have shown that small tumours of the sympathetic nervous system (neuroblastoma) have abnormal levels of COX-2 expressed (Johnsen et al., 2004, Cancer Res. 64 (20): 7210-5). These studies report that over-expression of the COX-2 enzyme has an adverse effect on the tumour suppressor, p53. p53 is an apoptosis transcription factor normally found in the cytosol. When cellular DNA is damaged beyond repair, p53 is transported to the nucleus where it promotes p53 mediated apoptosis (Lau et al., 2007, Oncogene, 26 (13): 1920-31). Two of the metabolites of COX-2, prostaglandin A2 (PGA2) and Al (PGA1), when present in high quantities bind to p53 in the cytosol and inhibit its ability to cross into the nucleus. This essentially sequesters p53 in the cytosol and prevents apoptosis (Lau et al., 2007, supra). Coxibs such as CELEBREX (celecoxib), by selectively inhibiting the over-expressed COX-2, allow p53 to work properly. Functional p53 allows -2 DNA damaged neuroblastoma cells to commit suicide through apoptosis, halting tumour growth. COX-2 up-regulation has also been linked to the phosphorylation and activation of the E3 ubiquitin ligase HDM2, a protein that mediates p53 ligation and tagged destruction, through ubiquitination (Lau et al., 2007, surpa). The mechanism for this neuroblastoma HDM2 hyperactivity is unknown. Studies have shown that COX-2 inhibitors block the phosphorylation of HDM2 preventing its activation. In vitro, the use of COX-2 inhibitors such as CELEBREX (celecoxib) lowers the level of active HDM2 found in neuroblastoma cells. The exact process of how COX-2 inhibitors block HDM2 phosphorylation is unknown, but this mediated reduction in active HDM2 concentration level restores the cellular p53 levels. After treatment with CELEBREX (celecoxib), the restored p53 function allows DNA damaged neuroblastoma cells to commit suicide through apoptosis reducing the size of growth of the tumour (Lau et al., 2007, surpa). Recently, the FDA has approved CELEBREX for treatment of familial adenomatous polyposis (FAP) (Kujubu et al., 1991, J. Biol. Chem. 266 (20): 12866-72). COX-2 inhibitors are currently being studied in breast cancer (Chow et al., 2005, Biomed. Pharmacother. 59 Suppl 2: S281-414) and appear to be beneficial (Farooqui et al., 2007, Br. J. Cancer, 97 (11): 1523-3115. However, while NSAIDs like the COX-2 inhibitor CELEBREX are being studied for their anti-cancer properties there is still a continuing need to identify new sources of agents that can be used as a replacement for chemotherapeutic agents. Mahogany has been valued for its high-quality durable timber since the 16th century. There are three species of "true mahogany", all of which are indigenous to the Americas, including Swietenia mahagoni (L.) Jacq., S. macrophylla King and S. humilis Zucc; however, the term "Mahogany" often refer to the largest group of all Meliaceae, which includes about fifteen related species of Swietenia, Khaya and Entandrophragma. The term "genuine mahogany" applies to only the Swietenia mahoganies, wherever grown. The term "true mahogany" applies to any timber commercially called mahogany with or without qualification that it derives from the Meliaceae family. In addition to Swietenia mahoganies this especially applies to Khaya senegalensis (African Mahogany), which is from the Meliacae (Mahogany) family. Medicinally, Mahogany has been used by various aboriginal tribes in South America as well as in Malaysia, India and Indonesia. For example, Mesetemo Indians use a decoction of the crushed seeds to bring about abortion. They also use the crushed seeds with the oil from Attaleaphalerata for skin problems and children's skin allergies. In Malaysia the seeds are chewed, or swallowed in powder form to treat high blood pressure, while in India the seeds are also used to treat this as well as diabetes. In India the seeds are also used for diarrhoea. In Indonesia a decoction of the seeds is given against malaria. The leaves of Mahogany have -3 been found to have anti-diabetic properties as well as perhaps having the potential to help against hepatitis C. However, to date there is no scientific evidence to support any of these uses. SUMMARY The inventor has surprisingly discovered that extracts of Mahogany have inhibitor activity against cancer as well as other activities that are of benefit to the treatment and/or prevention of cancer and the inflammation associated with cancer. Thus, the present application discloses medicinal compositions comprising an effective amount of an enriched extract from members of Meliacae (Mahogany) family and at least one pharmaceutically acceptable additive, wherein the extract comprises an effective amount of saponins and tannins. Accordingly, in a first aspect, the present invention provides a pharmaceutical or dietary supplement composition comprising: an effective amount of an enriched extract from members of Meliacae (Mahogany) family and at least one pharmaceutically acceptable additive, wherein the extract comprises at least 0.0001% w/w saponins and tannins and wherein said extract has the ability to inhibit at least one activity selected from the group consisting of COX-2 activity, Caspase 3 activity, 2,2'-azino-bis(3-ethylbenzothiazoline-6 sulphonic acid) (ABTS) activity, AKT1 activity and ERKI activity. Any species of the Meliacae (Mahogany) family can be used as the source material for the extract including the Swietenia mahagoni (L.) Jacq., S. macrophylla King, S. humilis Zucc. and Khaya senegalensis, which is widely accepted as the only "true" mahogany besides that of the mahogany of the genus Swietenia. Preferably, the source material is Khaya senegalensis. Persons skilled in the art will appreciate that the extract can be produced from any of the plant material of the Mahogany plant including the bark, leaves, roots and seeds. In some embodiments, the compositions of the invention are admixed with at least one pharmaceutically acceptable additive selected from the group consisting of anti-oxidants, adaptogens, bio-protectants, bioavailability enhancers, trace metals, or mixtures thereof.

-4 The compounds of the present invention may be administered orally or parenterally, in a pharmacological effective amount. The term parenteral used herein includes intravenous, intramuscular, subcutaneous, intra-dermal and intra-articular. In order to use the compositions of the present invention in therapy, they will normally be formulated into a dosage form in accordance with conventional methods of pharmacy and current guidelines and relevant good laboratory and manufacturing practices. Thus, it will be appreciated that the compositions can be made into suitable dosage forms, wherein a required dosage form might be a tablet, a soft capsule, a hard capsule, a pill, a granule, a powder, an emulsion, a suspension, a spray, a syrup or a pellet. Suitable carriers can also be used in the compositions of the present invention including known diluents, flavouring agents, solubilisers, lubricants, suspending agents and binders. Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, methylcellulose, sodium carboxymethyl cellulose, cocoa butter and the like. Techniques used to prepare oral formulations are the conventional mixing, granulation and compression or capsules filling. Other forms suitable for oral administration include emulsions, syrups and aqueous solutions. Emulsions can be prepared using emulsifying agents for example lecithin, propylene glycol or sorbitan monooleate. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colourings, flavours, stabilising agents. In some embodiments, the composition of the present invention is provided as suitable nutritional supplement in foods or beverages. In a second aspect, the present invention provides a method of treating or preventing cancer comprising the step of administering an effective amount of a composition according to the first aspect. It will be appreciated by those skilled in the art that a number of cancers can be treated and/or prevented using the compositions of the present invention including, but not limited to, brain tumours, in particular glioblastoma, stomach cancer, colon cancer, multiple myeloma, breast, cervical, prostate and lung cancer. In a third aspect, the present invention provides a method of producing a pharmaceutical or dietary supplement composition according to the first aspect comprising producing an extract of a species of the Meliacae (Mahogany) family and concentrating same and admixing same with at least one pharmaceutically acceptable additive.

-5 Extraction from a species of the Meliacae (Mahogany) family can be performed by any of the conventional methods such as maceration, infusion and heat extraction by using a proper solvent. Indeed, extraction is one of the most common methods for producing efficacious substances from herbal sources. High polarity extractants include water, methanol, ethanol, and acetone. Low polarity extractants include, but are not limited to, ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, cyclohexane, normal hexane, normal butyl alcohol, benzene, or the mixture thereof. Following extraction, the Mahogany species extract can be further purified using standard techniques. Methods of purification include chromatography, crystallization, filtration, and sedimentation. DESCRIPTION OF THE FIGURES Figure 1 shows the inhibitor activity of extracts of Khaya senegalensis. Four different extraction methods were used on leaves and bark. The inhibitor activities shown are anti COX-2, anti-Caspase 3, Anti-Free Radical Scavenger ABTS, anti-CB2, anti-B2, anti-AKT1, anti-ERKI and anti-EGF Receptor. DETAILED DESCRIPTION It is to be understood that this disclosure is not limited to particularly exemplified methods and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting which will be limited only by the appended claims. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols and reagents which are reported in the publications and which might be used in connection with the disclosed methods. Nothing herein is to be construed as an admission that what is disclosed herein is not entitled to antedate such disclosure by virtue of prior invention. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings: -6 The term "comprising" is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of'. Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, and the like. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. A "medicinal composition" refers to composition that has biological activity. The medicinal composition can be used to treat, cure, mitigate, prevent (i.e., prophylactically), ameliorate, modulate, or have an otherwise favourable effect on a cancer or its associated inflammation.

-7 In the broadest aspect of the present invention there is provided an extract from a species of Mahogany which extract comprises an effective amount of saponins and tannins that are able to inhibit at least one activity selected from the group consisting of COX-2 activity, Caspase 3 activity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) activity, AKTI activity and ERKI activity. The inventor has conducted several cell based in vitro studies on extracts or enriched extracts of Mahogany species. These studies have un-expectedly indicated potent anti-cancer and anti-inflammatory activity. The term "extract" as used herein is intended to mean a concentrate of the extract components derived from the leaves, bark or other plant material of the Meliacae (Mahogany) family. Any species of the Meliacae (Mahogany) family can be used as the source material for the extract including the Swietenia mahagoni (L.) Jacq., S. macrophylla King, S. humilis Zucc. and Khaya senegalensis, which is widely accepted as the only "true" mahogany besides that of the mahogany of the genus Swietenia. Preferably, the source material is Khaya senegalensis. Also, the terms "extract of the plant", "the Mahogany extract" and "the extract" are used herein interchangeably. All known species of "true mahogany" i.e. Swietenia mahagoni (L.) Jacq., S. macrophylla King and S. humilis Zucc as well as Khaya senegalensis can be used in the present invention. The plant extract of the present invention can be obtained by any extraction method known to one skilled in the art. For extracts which may also be used as directly applicable liquid pharmaceuticals, the following extraction agents are suitable: cold water, table salt solution, diluted acetic acid, sweet wine, ethanol-water mixtures, ethanol, other low-molecular-weight alcohols, acetone, esters, ethers, and mixtures thereof. Methanol, organic solvents such as acetone, ether, dichloromethane, and supercritical gases, vacuum extraction, and freeze drying are typical and known to one skilled in the art for obtaining dry extracts. For this purpose, one may select between simple extraction methods selected from the group comprising resting maceration, moving maceration, digestion, percolation, re-percolation, evacolation and diacolation, and special extraction methods selected from the group comprising the combination of maceration and percolation, ultrasonic extraction, counter flow extraction, and extraction using separators, centrifuges, and decanters. These methods are known to one skilled in the art and reference is made, for example, to Hagers Handbuch der Pharmazeutischen Praxis (Hager's Handbook of Pharmaceutical Practice) ( 5 th edition, volume 2; pages 1026-1030, Springer Verlag, Berlin-Heidelberg-New York (1991)). Fresh plants or plant parts may be used as the starting materials, however, one typically starts with dried plants and/or plant parts, which may be mechanically pulverized before the extraction. All pulverization methods known to one skilled in the art are suitable for this purpose, crushing using a mortar is cited as an example -8 All solvents having a specific polarity, preferably organic solvents, water (distilled or non distilled) or mixtures of organic solvents and water, in particular low-molecular-weight alcohols, esters, hydrocarbons, ketones, or halogenated hydrocarbons having greater or lesser water content may be used as the solvent for performing the extractions. Examples are protic (water, alcohols, acids, primary and secondary amines) and aprotic (acetonitrile, dimethyl formamide, dimethyl sulfoxide, hexamethyl phosphoric acid triamine, nitromethane, tert amine) solvents. Extraction using water, butanol, methanol, ethanol, propanol, isopropanol, pentane, hexane, heptane, acetone, chloroform, propylene glycols, polyethylene glycols, ethyl acetate, dichloromethane, trichloromethane, and mixtures thereof is particularly preferred. The extraction is typically performed at 15 to 25'C (aqueous extracts) or at 20 to 35'C (low molecular-weight alcohols), fundamentally preferably at room temperature, in order to reliably protect temperature-sensitive extract components. After the extraction, the obtained raw extracts may optionally be subjected to further typical steps, such as purification, concentration, and/or de-colouring. If desired, the extracts thus produced may be subjected to a selective separation of individual undesired components, for example. The extracts may subsequently also be subjected to a spray or freeze-drying, for example. Once the Mahogany extract has been obtained then the exemplary embodiments of the invention providing pharmaceutical or dietary supplement compositions can be produced. In some embodiments, the pharmaceutical composition can also be described as a herbal composition having potent anti-cancer and anti-inflammatory activity. These compositions may further contain pharmaceutically or dietetically acceptable additives or excipients. Various exemplary embodiments of the invention provide a method of treatment using the composition in humans or animals in need thereof, wherein the method comprises supplementing the said humans or animals with an effective amount of a composition comprising: i) an effective amount of an enriched Mahogany species extract; ii) optionally combined with one or more known anti-inflammatory or anti cancer or immune modulating agents; and iii) further optionally combined with one or more pharmaceutically or dietetically acceptable additives or excipients. In yet another embodiment the invention provides compositions further comprising optionally effective amounts of pharmaceutically or dietetically acceptable anti-oxidants, adaptogens, anti-diabetic agents, bio-protectants, bioavailability enhancers and trace metals or mixtures thereof to form a formulation.

-9 In various exemplary embodiments of the invention, the administration of the composition can be administered orally, parenterally, nasally, rectally, vaginally, transdermally, occularly or through any other suitable route. In various exemplary embodiments of the invention, the composition is provided as a pharmaceutical or dietary supplement dosage form suitable for oral administration. Dosage forms suitable for oral administration include tablets, soft capsules, hard capsules, pills, granules, powders, emulsions, suspensions, sprays, syrups and pellets. In various other embodiments of the invention, the composition is provided as a pharmaceutical dosage form suitable for parenteral administration such as liquid formulations for administration as drops or by injection, or as solid or semisolid dosage forms for suppositories. In another embodiment, the invention provides the nutritional or dietary compositions in the form of foods or beverages. These foods or beverages comprise various exemplary embodiments of the inventive compositions containing an enriched Mahogany species extract. These foods or beverages can be prepared or provided as cereals, baby foods, healthy foods, or food for specified health uses such as solid food like chocolate or nutritional bars, semisolid food like cream or jam, or gel; and also as beverages. Specific and non-limiting examples of such food or beverage items include refreshing beverages, lactic acid bacteria beverages, drops, candies, chewing gum, chocolate, gummy candy, yoghurts, ice creams, puddings, soft adzuki bean jellies, jellies, cookies and the like. The amount of composition that will be effective in the treatment or prevention of a particular cancer will depend on the nature of the cancer, which can be determined by standard clinical techniques. In addition, the in vitro and in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will depend on the route of administration, and the seriousness or advancement of the cancer, and should be decided according to the practitioner and each patient's circumstances. Effective dosages may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, an effective amount of compositions according to various embodiments of the invention is readily determined by administering graded doses of the composition and observing the desired effect. The following examples, which include preferred embodiments, will serve to illustrate the practice of various embodiments of this invention, using appropriate doses/units of the selected individual ingredients for preparing the compositions. It is being understood that the particulars shown are by way of example and for purpose of illustrative discussion of - 10 preferred embodiments of the invention. These illustrations are not to limit the scope of the invention. EXAMPLE 1 Preparation of Extract From the Leaves of Mahogany species by Percolation Method The leaves of Khaya senegalensis were pulverized to coarse powder and about 1 Kg each of powdered material placed in different flasks and extracted with n-hexane, butanol, ethanol and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. EXAMPLE 2 Preparation of Extract From the Leaves of Khaya senegalensis by Hot-Soxlation Method The coarse powdered material of leaves of Khaya senegalensis was subjected to hot soxhalation using solvents n-hexane, butanol, ethanol and water at optimum temperature and recycled until extraction was completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature. All the above described respective solvent extracts manufactured by percolation and hot-soxhalation methods are qualitatively similar to each other. EXAMPLE 3 Preparation of Extract From the Bark of Khaya senegalensis by Percolation Method The shade dried material of bark of Khaya senegalensis was pulverized to coarse powder and about 1 Kg each of powdered material placed in different flasks and extracted with n-hexane, butanol, ethanol and water at room temperature for 24 h to 48 h., then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. EXAMPLE 4 Preparation of Extract From the Bark of Khaya senegalensis by Hot-Soxlation Method - 11 The coarse powdered material of bark of Khaya senegalensis was subjected to hot soxhalation using solvents n-hexane, butanol, ethanol and water at optimum temperature and recycled until extraction is completed, then plant extracts were filtered and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature. All the above described respective solvent extracts manufactured by percolation and hot-soxhalation methods are qualitatively similar to each other. EXAMPLE 5 Standardization of Methanolic Extract of Khaya senegalensis by HPLC Sample preparation: 100 mg of methanolic extract was weighed in 100 ml of volumetric flask and dissolved in Water:Methanol (1:1) solvent mixture and made up to the volume of 100 ml to get 1 mg/ml concentration. HPLC conditions: 20 pL of the sample was injected over reverse phase column and run with mobile phase of 0.1% of Phosphoric acid and water in the ratio of 90:10 at flow rate of 1 ml/min. and detected at 210 nm. EXAMPLE 6 Fractionation and Column Chromatography of Water Extract of Khaya senegalensis About 400 g of the water extract was fractionated into methanol soluble fraction and methanol insoluble fractions. The yield of the methanol soluble fraction is 48%. The methanol soluble fraction was subjected to column chromatography using silica gel as adsorbent and eluted in the column with n-hexane, ethyl acetate and methanol. EXAMPLE 7 Standardization of Khaya senegalensis Water Extract by HPLC Sample preparation: The water extract was accurately weighed for 100 mg in volumetric flask, dissolved in water and sonicated for 5 minutes to make up the volume to 100 ml to get the concentration of 1 mg/ml sample solution and filtered through 0.2 micron syringe filter. Mobile Phase Preparation: Accurately 1.88 g of Hexane Sulfonic Acid Sodium salt was added to 10 ml of glacial Acetic acid and 1.3 ml of Triethylamine in 1 L volumetric flask and - 12 volume is made up to the mark with double distilled water. The mobile phase is sonicated for 10 minutes and filtered through 0.45 pm filter paper. HPLC conditions: Accurately 20 [L of samples is injected over reverse phase column and ran with the mobile phase at 1 ml/min and detected at 254 nm. EXAMPLE 8 Sample Preparation Seven samples were received from Bioactive Solutions for extraction and screening for the presence of phytochemicals. The seven samples consisted of bark, seeds, seed coat and a sample of timber sawdust. The bark, seed pod and timber samples were milled using a Christy and Norris mill which uses a cross beater blade to give a fine powder to pass through a one millimetre screen. The fine powder allows for a representative sample to be taken for the extraction work. The seed was milled in a special mill and the milled material was directly used for the extractions. The powdered materials were extracted with a mixture of dichloromethane and methanol (1:1), and then extracted with 80% methanol in water twice. The extract solution was combined and concentrated under vacuum to afford a crude extract. The individual crude extracts were dissolved in 10% aqueous methanol and then extracted with hexane, three times; the combined hexane extracts were concentrated under vacuum to afford a hexane fraction. The remaining aqueous solutions were further extracted with ethyl acetate, three times and the above procedure was repeated to obtain an ethyl acetate fraction. Finally, the remaining aqueous solutions were extracted with n-butanol three times and the above procedure was repeated to afford an n-butanol fraction. The remaining aqueous solutions were concentrated under vacuum to give the aqueous fractions. In total, seven crude extracts and 28 fractions were obtained. The seed material was also extracted with hot hexane in soxhlet extraction equipment. This procedure allowed for the determination of the crude fat/oil present in the seed material. A sub sample of the crude fat/oil was taken and hydrolysed and esterified and the fatty acid profile of the oil was run on a gas chromatograph using a flame ionisation detector to identify the individual fatty acids against standard fatty acids.

- 13 Table 1 shows the weight of sample and the weight of the crude extract, the number in brackets indicates the percentage of the total extract from the sample of the material extracted. The figure in the columns headed by the solvent used in the extraction solvent indicates the percentage of the crude extract that is soluble in that solvent. Sample 001 showed that only a low percentage (7.9) was extracted using methylene chloride/methanol (1:1). The major fraction from the sample was the n-butanol extract which represented 50.8% of the crude extract. The next highest was the water soluble component (25.4%) and the Ethyl Acetate fraction at 20.8 percent. The remainder of the crude extract was soluble in hexane. Sample 002 had a much more even spread of fractions as shown in Table 1. However, the hexane fraction was still the lowest. Sample 003 had a similar fraction pattern as sample 002. The water fraction of the sample 004 had the highest amount of the crude extract. The N-butanol fraction was the second highest at 39.4% followed closely by the ethyl acetate fraction (14.7%), the hexane fraction (2.4%) was the lowest. This sample had the lowest hexane fraction of all of the samples. This sample (004) had the highest crude extract percent at 30.4%. The fraction for sample 005 was completely different to the others samples where the hexane fraction was the major fraction component of the crude extract. This sample was the seed and this result reflects that there is probably a large oil component in the crude fraction. The aqueous fraction was only 17% of the crude extract. The seed coat sample 006 had a high aqueous fraction (60.2%). The other three fractions ranged from 7.4 to 19.5% the high percent fractions were in the polar solvent range. The timber sample (007) had its highest fraction of the crude extract in the ethyl acetate fraction, followed by the aqueous fraction and then the N-butanol fraction, with the hexane fraction having the lowest percentage of the crude extract.

- 14 Table 1 Weight of crude extracts and fractions Sample Sample Crude extract Hexane Ethyl acetate N-butanol fr. Residue aqueous Number Wt (g) Wt (g) (%*) fr. (%) fr (%) (%) fr. (%) 001 19.42 1.54 (7.9%) 3.0 20.8 50.8 25.4 002 26.53 1.48 (5.6%) 12.7 30.2 24.5 32.6 003 23.93 3.14 (13.1%) 9.0 26.1 26.0 28.9 004 18.28 5.55 (30.4%) 2.4 14.7 39.4 43.5 005 8.09 2.42 (29.9%) 68.7 9.8 4.5 17.0 006 18.53 1.31 (7.1%) 7.4 11.6 19.5 60.2 007 25.55 2.10 (8.2%) 8.8 50.2 17.4 23.6 Each individual extract was tested using the following method for the possible presence of metabolite groups. The fraction were analysed on a Thin Layer Chromatography (TLC) plates. After development with a solvent mixture the TLC plates were sprayed with the metabolite testing reagents as shown in Table 2 and observing the corresponding colour changes. For observing the presence of flavonoids, the TLC plate was observed under long wavelength UV light (360nm) which produced a yellow fluorescence spot under UV light (360nm). The presence of saponins was indicated in the fraction when a foam was formed while shaking an aqueous solution of the fractions. The observation colours for the various metabolite compounds are indicated in Table 2.

- 15 Table 2 The methods used for metabolite testing Metabolites Test Observation Alkaloids Dragendorff's reagent Reddish brown colour Flavonoids Aluminium chloride Yellow fluorescence in long wavelength UV light(360nm) Tannins Vanillin/HCI Red colour Terpenoids Liebermann reaction Pink to red colour Antharaquinone Bontrager's test Pink colour Saponins Water Foam Table 3 shows the reaction of each fraction to Dragendorrff reagent thus indicating the presence of alkaloids. The N-butanol and aqueous fractions showed no presence of alkaloids. However, in the ethyl acetate fractions there was a strong alkaloid response in samples 002, 003 and 005. There were only weak responses to the presence of alkaloid in three of the samples 001, 004 and 007) and with no response with sample 006. The hexane fraction showed very strong alkaloid response in sample 005. There was only weak alkaloid response for all of the other samples and no alkaloid response from sample 007.

- 16 Table 3 Alkaloid screening using Dragendorff reagent Sample No. Hexane fraction Ethyl acetate fraction N-butyl alcohol fraction Aqueous Fraction 001 - Bark + + 002 - Bark + 003 - Bark + 004 - Bark + + 005 - seed +++

++

006 - hull + 007 - timber + Legend: - No response, + Weak response, ++Strong response, +++ Very strong response, ++++ Extremely Strong response. Table 4 shows the reaction of each fraction with aluminium chloride reagent thus indicating the presence of flavonoids. There was no flavonoid response in the N-butanol and aqueous fraction for all of the samples. The ethyl acetate fraction showed a flavonoid response for all samples. Sample 003 gave an extremely strong response for flavonoids. Sample 002 also showed a very strong response for flavonoids in the ethyl acetate fraction. All of the other samples showed a strong response except for sample 004 which showed a weak response. The hexane fraction showed a extremely strong response for flavonoids in sample 003. Sample 005 gave a very strong response to flavonoids in the hexane fraction. Samples 001 and 007 showed a weak to no response for flavonoids in the hexane fraction. While samples 004 and 006 showed a weak response for flavonoids. Sample 002 showed a strong response for flavonoids in the hexane fraction.

- 17 Table 4 Flavonoids screening using aluminium chloride Sample No. Hexane fraction Ethyl acetate fraction N-butyl alcohol fraction Aqueous Fraction 001 - Bark +/- ++ 002 - Bark ++ 003 - Bark ++++

++-

004 - Bark + + 005 - seed +++ ++ 006 - hull + ++ 007 - timber +/- ++ Legend: - No response, + Weak response, ++Strong response, +++ Very strong response, ++++ Extremely Strong response. Table 5 shows the reaction of each fraction with vanillin/hydrochloric acid reagent thus indicating the presence of tannins. Samples 005 and 006 showed no response for tannins. An extremely strong response for tannins was in the ethyl acetate fraction for samples 004 and 007. A very strong response for tannins was given in the ethyl acetate fraction for sample 003 and N-butanol fraction for sample 004. The aqueous fraction for sample 003 and 004 showed a strong response to the presence of tannins. The other sample and fractions gave no response to a weak response for tannin screening.

- 18 Table 5 Tannin screening using vanillin hydrochloride Sample No. Hexane fraction Ethyl acetate fraction N-butyl alcohol fraction Aqueous Fraction 001 - Bark + ++ ++ 002 - Bark + + + + 003 - Bark ++ + ++ 004 - Bark + ++ 005 - seed 006 - hull 007 - timber + + Legend: - No response, + Weak response, ++Strong response, +++ Very strong response, ++++ Extremely Strong response Table 6 shows the reaction of each fraction with Liebermann reagent thus indicating the presence of triterpenoids and phytosterols. The hexane fraction showed a extremely strong response for triterpenoids and phytosterols in samples 003, 005 and 007 and had very strong response for samples 002 for triterpenoids and phytosterols. Sample 004 and 006 had a strong response but sample 001 gave a weak response. In the ethyl acetate fraction two samples gave very strong response (005 and 007). Three samples had a strong response in the ethyl acetate fraction (001, 003 and 004) and sample 002 gave a weak response with sample 006 giving no response. In the N-butanol fraction two samples gave very strong responses (001 and 004). Three samples 002, 003 and sample 007 showed strong responses to this test. Sample 005 gave a weak response and 006 gave no response. In the aqueous fraction only two samples 003 and 004 showed strong responses and two samples gave a weak response (002 and 007) samples 001, 005 and 006 gave no response. Sample 006 gave no response for triterpenoids and phytosterols for the ethyl acetate, N-butanol and aqueous fractions, with a strong response in the hexane fraction.

- 19 Table 6 Triterpenoids and phytosterol screening using vanillin-phosphoric acid Sample No. Hexane fraction Ethyl acetate fraction N-butyl alcohol fraction Aqueous Fraction 001 - Bark + ++ 002 - Bark +++ + ++ + 003 - Bark ++++ ++ ++ ++ 004 - Bark ++ ++ ++ 005 - seed .. .+ + 006 - hull ++ 007 - timber .++ + Legend: - No response, + Weak response, ++Strong response, +++ Very strong response, ++++ Extremely Strong response Table 7 shows the foam production of the aqueous faction thus indicating the presence of saponins. The saponin test was only performed on the aqueous fraction as it is a foam generated test. Sample 004 showed no foaming which is negative for saponins. Sample 005 only showed weak response to saponins. Very strong response for saponins was shown by samples 001, 002 and 006. An extremely strong response was shown for sample 007.

- 20 Table 7 Saponin screening Sample 001 002 003 004 005 006 007 Saponins .+ +.. - + .+ .. Legend: - No response, + Weak response, ++Strong response, +++ Very strong response, ++++ Extremely Strong response Table 8 is a summary of the results that were found for all of the components of the Khaya senegalensis tree.

-21 Table 8 Sample Alkaloids Flavonoids Tannins Triterpenoids Antharaquinone Saponins & Phytosterols 001 - Bark + ++ ++ 002 - Bark +++ -. ++ 003 - Bark +++ - ++ 004-Bark + + 005 - seed ++- ++- + 006-hull + ++ - ++ 007 - timber + ++ ++++ ++-+ Legend: - No response, + Weak response, ++Strong response, +++ Very strong response, ++++ Extremely Strong response. The crude fat/oil determination showed that there was 46.2% as received crude fat/oil present in the seed of Khaya senegalensis. The main fatty acid present in the oil is Oleic acid. The crude fat/oil determination showed that there was 46.2% as received crude fat/oil present in the seed component (005) of the Khaya senegalensis tree. EXAMPLE 9 Estimation of Caspase 3 inhibitor Activity Caspases are the executioners of programmed cell death (apoptosis), a normal process used physiologically to eliminate specific cells or cell types without damaging surrounding cells or causing an inflammatory response. All caspases belong to a family of proteases characterized by a cysteine residue in the active site and display specificity for Asp residues. Caspases are present in all cells as a pro-caspase, which becomes activated through proteolytic cleavage by other proteases including other caspases. Caspase-3 (CPP32, Yama, and apopain) is constitutively expressed in cells as a 32 kD pro-caspase. Activation of caspase 3 occurs by cleavage of the pro-caspase into 12 and 17 kD mature fragments by caspase 8, 6 and Granzyme B. Mice deficient in caspase 3 die within three weeks of birth due to failure of neuronal apoptosis. These mice display hyperplasia, duplicated brain structures and ectopic cell masses. However, the defects are restricted to the neural tissues, other organs are normal; indicating that caspase 3 has a non-redundant role in neural apoptosis. Caspase inhibitors are currently sought as therapeutics for such diseases as Alzheimer's, Parkinson's and cerebral/myocardial ischemia. Human recombinant caspase 3 expressed in . coli was used as described in Mittl et al., (1997) (JBiol Chem. 272:6539). Caspase 3 in the presence of the substrate Ac-DEVD-AMC produces Ac-DEVD + AMC, wherein the amount of AMC formed can be read spectrofluorimetrically at 360 nm/465 nm.

- 22 Each extract (hexane, N-butyl alcohol, water and ethyl acetate) from Khaya senegalensis as described in the above Examples, were pre-incubated with 150 U/ml caspase 3 enzyme in modified HEPES buffer pH 7.4 for 15 minutes at 37'C. The reaction was initiated by addition of 50 mM Ac-DEVD-AMC (substrate) for another 60 minute incubation period. The amount of AMC formed was read spectrofluorimetrically and compared to a reference standard* as well as other known caspase 3 inhibitors: Reference Compound IC 50 (ptM) Ac-DEVD-CHO 0.0049 Ac-IETD-CHO 0.18 Ac-LEVD-CHO 0.057 Ac-VEID-CHO 0.48 Ac-YVAD-CHO >10 Z-LEHD-FMK 1.4 Z-VDVAD-FMK 0.46 Z-WEHD-FMK 5.4 As shown in Figure 1, the activity of the various extracts compared favourably with known caspase 3 inhibitors. Sample Source Conc. % Inhibition. N-butyl alcohol Extract 2 30 ig/mL 90 Ethyl acetate Extract 2 30 jtg/mL 86 Hexane Extract 2 30 ig/mL 55 Water Extract 2 30 jtg/mL 95 EXAMPLE 10 Estimation of Protein Serine/Threonine Kinase inhibitor Activity PKB (also known as c-Akt or Rac-PK) was originally identified as the transforming oncogene in a retrovirus from a spontaneous thymoma in an AKR mouse. PKB appears to be activated as a consequence of increased phosphoinositide 3-kinase (P13K) activity in cells stimulated with mitogens. Potential targets of PKB include glycogen synthase kinase-3 and p70 ribosomal protein S6 kinase. PKB is also crucial in mediating cell survival. Activation forms of both P13 kinase and PKB have been shown to protect from apoptosis induced by withdrawal of growth and survival factors, expression of c-myc, and detachment from - 23 extracellular matrix. Inhibition of AKTI (protein kinase B, alpha) may be useful in the treatment of various cancers Human protein kinase PKBa/Aktl expressed in Sf21 insect cells was used as described in Meier et al. (1998) (EMBO J. 17(24): 7294). Protein kinase PKBa/Aktl in the presence of the substrate crosstide KK and [ 3 2 P]ADPATP produces [ 3 2 P]ADPCrosstide-P and ADP. Quantitation of the activity is by scintillation counting of [ 3 2 P]Crosstide-KK. Each extract (hexane, N-butyl alcohol, water and ethyl acetate) from Khaya senegalensis as described in the above Examples, were pre-incubated with 628 mU/ml protein kinase PKBa/Aktl in modified MOPS buffer pH 7.2 at 37'C for 15 minutes. The reaction was initiated by addition of 10 tM crosstide KK, 10 tM ATP and 0.25 tCi [y- 3 2 P]ATP for another 30 minute incubation period and terminated by further addition of 3% H 3

PO

4 . An aliquot was removed to a 96 well multiscreen tray, washed 3 times with 3 % H 3

PO

4 solution, dried and counted to determine the amount of 32 P-crossitde KK formed. Compounds are screened at 10 tM. The amount of was compared to the reference compound staurosporine which has an IC 50 (ptM) of 0.051 ptM. As shown in Figure 1, the activity of the various extracts compared favourably with known protein kinase PKBa/Aktl inhibitor. Sample Source Conc. % Inhibition. N-butyl alcohol Extract 2 30 ig/mL 99 Ethyl acetate Extract 2 30 jtg/mL 99 Hexane Extract 2 30 ig/mL 84 Water Extract 2 30 jtg/mL 97 EXAMPLE 10 Estimation of Protein Tyrosine Kinase, EGF Receptor inhibitor Activity Binding of EGF or TGFa (transforming growth factor a) to the EGF receptor results in activation of intrinsic tryrosine kinase activity. Several cytosolic proteins are phosphorylated by the EGF receptor leading to activation of signalling pathways that induce mitogenesis and, in some instances, associated cell transformation. Inhibitors of EGF receptor tyrosine kinase - 24 activity may be useful for chemotherapeutic intervention in cases of malignant cellular proliferation. Human recombinant protein kinase EGFR expressed in insect cells was used as described in Xiong X et al. (2009) (Invest New Drugs. 27(1): 1). Protein kinase EGFR in the presence of the substrate poly(Glu:Tyr) and [y- 32 P]ATP produces [ 3 2 P]Poly(Glu:Tyr) and ADP. Quantitation of the activity is by scintillation counting of [ 32 P]Poly(Glu:Tyr). Each extract (hexane, N-butyl alcohol, water and ethyl acetate) from Khaya senegalensis as described in the above Examples, were pre-incubated with 0.134 Lg/ml protein kinase EGFR in modified HEPES buffer pH 7.4 for 15 minutes at 37'C. The reaction was initiated by addition of 0.2 mg/ml Poly(Glu:Tyr), 10 tM ATP and 0.25 LCi [ 7 32 P]ATP for another 30 minute incubation period and terminated by further addition of 3% H 3

PO

4 . An aliquot was removed and counted to determine the amount of [ 32 P]Poly(Glu:Tyr) formed. Extracts were screened at 10 tM. The amount of [ 32 P]Poly(Glu:Tyr) formed was compared to a reference standard*: Reference Compound IC 50 (ptM) EGFR inhibitor 0.046 As shown in Figure 1, the activity of the various extracts compared favourably with known EGFR inhibitor. Sample Source Conc. % Inhibition. N-butyl alcohol Extract 2 30 ig/mL 100 Ethyl acetate Extract 2 30 jtg/mL 100 Hexane Extract 2 30 ig/mL 82 Water Extract 2 30 ig/mL 100