WO2005105124A1

WO2005105124A1 - Pharmaceutical composition comprising the plant coleonema

Info

Publication number: WO2005105124A1
Application number: PCT/IB2005/001217
Authority: WO
Inventors: Johannes Gerhardus Groenewald; Riaan Meyer; Jacobus Cornelis Johannes Van Schalkwyk; Ian Augustus Dubery
Original assignee: Phyto Med Herbal Products (Proprietary) Limited
Priority date: 2004-05-04
Filing date: 2005-05-04
Publication date: 2005-11-10

Abstract

The plant material or extract of the plant may be from C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum. The pharmaceutical composition is particularly suitable for use as an immune stimulant, an antibacterial agent, an antifungal agent, an antiviral agent (in particular for treatment of HIV), an anti-inflammatory agent or an anti-oxidant. The composition may include at least one of the following compounds: 1, 2', 3'-dihydroxydihydrosuberosin (2H-1-benzopyran-2-one, 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy, 2H-1-benzopyran-2-one, 6-(3,3-dimethyloxiranyl)-7-methoxy); 5,5'-(tetrahydro-1H,3H-furo[3,4c]-furan-1,4-diyl)bis-[1S-(1 α.,3a.α,4.β.,6a)-1,3 benzodioxole; 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one; 6-(3,3-dihydroxy-3-methylbutyl)-7-methoxy-2H1-benzopyran-2-one; 7-methoxy-6-(3,3-methyl-2butenyl)- 2H-1-benzopyran-2-one; 6-[(3,3-dimethyloxiranyl)methyl]-7-metoxy-2H-1-benzopyran-2-one; 6-(2 ,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one; 6-(3,3-dimethyloxiranyl) methyl]-7-metoxy-2H-1-benzopyran-2-one; Esculetin; 3,4-dihydro-5-hydroxy-2,2,8-trimethyl-2H,6H-benzo[1,2-b:5,4-b']dipyran-6-one; 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuro[3,2-g] chromen-7-one; 7-methoxy-6-(3-methyl-2-oxobutyl)- 2H-1-benzopyran-2-one; 2,2-dimethyl-pyrano(3,2-c)(1)benzopyran-5-one; 9,10-dihydro-9-hydroxy-8,8-dimethyl-2H,8H-Benzo[1,2-b:3,4-b']dipyran-2-one; n-hexadecanoic acid; 4-(4-hydroxyphenyl)- 2-butanone; 8-[ß-D-glucopyranosyloxy)-1-methylethyl]-8,9-dihydro-2H-furo[2,3-h]-1-benzopyran-2-one; 2-isopropenyl-2,3-dihydrofuro[3,2-g] chromen-7-one; 2-(3,4-dihydroxyphenyl)-3-(ß.-D-galactopyranosyloxy)-5,7-dihydroxy-4H-1-benzopyran-4-one; Vitamin E; and pharmaceutically acceptable derivatives or salts thereof. A method of treating a patient using plant material or an extract as described above is also described, as is the compound 1, 2', 3'-dihydroxydihydrosuberosin and pharmaceutically acceptable derivatives or salts thereof for treating a patient.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE PLANT COLEONEMA

BACKGROUND OF THE INVENTION

This invention relates to a pharmaceutical composition comprising as an active ingredient plant material or an extract of a plant of the genus Coleonema, particularly Coleonema album or a hybrid thereof, to the use of such pharmaceutical compositions and to compounds extracted from the plant material for use as medicaments.

Members of the Coleonema genus, of which there are at least eight species, were originally classified as species of the genus Diosma, but have since been reclassified as a separate genus. Nevertheless, in some parts of the world, they are still commonly referred to as members of the Diosma genus. Coleonema album, the type species of the genus, is a fragrant shrub which is a dominant plant of the mountain vegetation of the southern coastal region of the Western Cape of South Africa. C. album has been domesticated and planted in many South African gardens for its show of flowers. During the flowering season the small white petals cover the ground like confetti after a wedding, hence the colloquial name "confetti bush" or "pink breath of heaven". "Sunset gold" (C. pulchrum) is another well-known species of Coleonema.

The plant is currently known for use as an insect repellant, as a deodorizer and as an aromatherapy oil.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a pharmaceutical composition for oral or topical administration comprising as an active ingredient plant material or an extract of a plant of the genus Coleonema, for example C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum, particularly C. album or a hybrid thereof.

The pharmaceutical composition for oral administration may be a tablet or a capsule containing the plant material or an extract of the plant material, a tincture or a tonic containing an extract of the plant material, or a powder comprising the plant material contained in a sachet or the like. The pharmaceutical composition for topical administration may be a salve, an ointment, a lotion, a composition for spray or aerosol application, or the like.

The plant material or the extract is preferably derived from the aerial parts of the plant, which parts may be fresh or dried.

The pharmaceutical composition is particularly for use as an immune stimulant, as an antibacterial agent, an antifungal agent, an antiviral agent, an anti-inflammatory agent or an anti-oxidant.

The pharmaceutical composition may contain from about 10 g/l to about 100 g/l plant material or extract, more particularly from about 25 g/l to about 75 g/l plant material or extract, and even more particularly about 50 g/l plant material or extract.

The pharmaceutical composition may contain at least one of the following compounds:

1, 2', 3'-dihydroxydihydrosuberosiπ (2H-1-benzopyran-2-one, 6-(2,3-dihydroxy-3-methylbutyl)-7- methoxy, 2H-1-benzopyran-2-one, 6-(3,3-dimethyloxiranyl)-7-methoxy);

5,5'-(tetrahydro-1H,3H-furo[3,4c]-furan-1,4-diyl)bis-[1S-(1a.,3a.a,4.p.,6a)-1,3 benzodioxole;

6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one; 6-(3,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one;

7-methoxy-6-(3,3-methyl-2butenyl)- 2H-1-benzopyran-2-one;

6-[(3,3-dimethyloxiranyl)methyl]-7-metoxy-2H-1-benzopyran-2-one;

6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one;

6-(3,3-dimethyloxiranyl) methyl]-7-metoxy-2H-1-benzopyran-2-one; Esculetin;

3,4-dihydro-5-hydroxy-2,2,8-trimethyl-2H,6H-benzo[1,2-b:5,4-b']dipyran-6-one;

2-(1 -hydroxy-1 -methylethyl)-2,3-dihydrofuro[3,2-g] chromen-7-one;

7-methoxy-6-(3-methyl-2-oxobutyl)- 2H-1-benzopyran-2-one;

2,2-dimethyl-pyrano(3,2-c)(1)benzopyran-5-one; 9,10-dihydro-9-hydroxy-8,8-dimethyl-2H,8H-Benzo[1,2-b:3,4-b']dipyran-2-one; n-hexadecanoic acid;

4-(4-hydroxyphenyl)- 2-butanone;

8-[β-D-glucopyranosyloxy)-1-methylethyl]-8,9-dihydro-2H-furo[2,3-h]-1-benzopyran-2-one;

2-isopropenyl-2,3-dihydrofuro[3,2-g] chromen-7-one; 2-(3,4-dihydroxyphenyl)-3~(β.-D~galactopyranosyloxy)-5J-dihydroxy-4H-1-benzopyran-4-one;

Vitamin E; and pharmaceutically acceptable derivatives or salts thereof. According to a second aspect of the invention there is provided a method of treating a patient suffering from a disease, the method comprising orally or topically administering to the patient a composition comprising as an active ingredient plant material or an extract of a plant of the genus Coleonema, for example C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum, particularly C. album or a hybrid thereof, wherein the disease is selected from the group consisting of a disease associated with a low or compromised immune system, a bacterial infection, a fungal infection, a viral infection and an inflammatory condition. For example, the disease may be HIV or AIDS, arthritis, sinusitis, larungitis or pharyngitis.

The plant material or extract may be administered to the patient in an amount of from about 500 mg to about 10 000 mg per day, more particularly in an amount of from about 1 000 mg to about 5 000 mg per day, and even more particularly in an amount of about 2 000 mg plant material or extract per day per 80 kg of body mass of the patient.

According to a third aspect of the invention there is provided the use of plant material or an extract of a plant of the genus Coleonema in the manufacture of a medicament for the treatment of a disease selected from a disease associated with a low or compromised immune system, a bacterial infection, a fungal infection, a viral infection and an inflammatory condition

According to a fourth aspect of the invention there is provided the compound 1, 2', 3'- dihydroxydihydrosuberosin (and derivatives thereof), for use in a method of treatment of the human or animal body by therapy. In addition, the pharmaceutical composition may include cyclooxygenase-1 and 2 (COX-1 and 2) inhibitor(s) and/or other suitable compounds with anti- oxidant activity.

According to a fifth aspect of the invention there is provided the use of the compound 1, 2', 3'- dihydroxydihydrosuberosin, (and derivatives thereof) in the manufacture of a medicament for the treatment of a disease selected from a disease associated with a low or compromised immune system, an bacterial infection, a fungal infection, a viral infection and an inflammatory condition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical depiction of the cytotoxity effects of a Coleonema album plant extract on cells using an XTT-based cell proliferation assay to determine the toxicity of the extract (100% ethanol extract); Figure 2 is a graphical depiction of a hybrid Coleonema album plant extract using the XTT-based cell proliferation assay (100% ethanol extract); Figure 3 is a graphical depiction of the antiviral effect of a Coleonema album plant extract on cells using the XTT-based cell proliferation assay (100% ethanol extract);

Figure 4 is a graphical depiction of the antiviral effect of a hybrid Coleonema album plant extract using the XTT-based cell proliferation assay (100% ethanol extract); Figure 5 is a graphical depiction of p24 antigen values of cells treated with various concentrations of Coleonema album plant extract (100% ethanol extract); Figure 6 is a graphical depiction is a graphical depiction of p24 antigen values of cells treated with various concentrations of hybrid Coleonema album plant extract (100% ethanol extract); Figure 7 is a graphical depiction of the HIV-1 reverse transcriptase activity of various concentrations of Coleonema album plant extract (100% ethanol extract); Figure 8 is a graphical depiction the HIV-1 reverse transcriptase activity of various concentrations of hybrid Coleonema album plant extract (100% ethanol extract);

Figure 9 is a graphical depiction of p24 antigen values of HIV-1 subtype C infected CEM/J/ ACCR5 cells treated with 50 μg/ml concentrations of Coleonema album and hybrid plant extract in acetone or ethanol;

Figure 10 is a graphical depiction of cyclooxygenase inhibition by Coleonema album plant extracts ((a): COX 2 inhibition; (b): COX 1 inhibition); ^{" "}

Figure 11 is a graphical comparison of the antioxidant activity of the different extracts with the two standard antioxidants, Trolox and Vitamin C;

Figure 12 is a graphical depiction of the linear correlation between the sample concentrations and oxygen radical scavenging capability (ORAC) for each of the Coleonema album extracts tested; and Figure 13 ia a graphical depiction of the reductive (anti-oxidant) potential of different concentrations of the different C. album extracts.

DESCRIPTION OF EMBODIMENTS

The crux of the invention is the use of plant material or an extract of a plant of the genus Coleonema, for example C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum, particularly C. album or a hybrid thereof, for medicinal purposes. The first aspect of the invention is a pharmaceutical composition for oral or topical administration comprising as an active ingredient plant material or an extract of the plant of the genus Coleonema, for example C. album, C. nubigenum, C. junipennum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum, particularly C. album or a hybrid thereof.

The natural plant material, consisting of 100% fresh or dried Coleonema, preferably the aerial parts thereof, is either extracted with a suitable extractant such as ethanol, or is dried and milled with a hammer mill.

Examples of pharmaceutical compositions of the invention are the following: A tonic or tincture containing from about 0.05 to about 1.6 g/ml of the plant extract in about 30% to 80% ethanol. A capsule containing about 50 mg to about 800 mg of the milled plant material. > An ointment containing about 0.05 g to about 1.6 g/ml of the plant extract, incorporated into an ointment, e.g. a cream base.

The second aspect of the invention is a method of treating a patient suffering from a disease, the method comprising orally or topically administering to the patient a composition comprising as an active ingredient plant material or an extract of a plant of the genus Coleonema, for example C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum, particularly C. album or a hybrid thereof, where the disease is selected from a disease associated with a low or compromised immune system (such as HIV/AIDS), a bacterial infection including dental plaque, a fungal infection, a viral infection and an inflammatory condition (such as sinusitis, laryngitis, arthritis and pharingitis).

The experimental work that follows illustrates the uses of the plant material or plant-derived extract of the invention.

The third aspect of the invention is the compound 1, 2', 3'-dihydroxydihydrosuberosin (2H-1- benzopyran-2-one, 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy, 2H-1-benzopyran-2-one, 6-(3,3- dimethyloxiranyl)-7-methoxy) and derivatives thereof, which are prenylated coumarins, for use in a method of treatment of the human or animal body by therapy, specifically for use in the treatment of those diseases listed above.

Experimental work has illustrated that at least one of the active compounds in C. album is 1 , 2', 3'-dihydroxydihydrosuberosin. This compound and derivatives thereof were shown to have antimicrobial and anti-fungal activities. This compound may be used in combination with other chemical compounds found in the Coleonema plant material, such as monoterpenes, sesquiterpenes, phenylpropanoids, phenylpropenes and prenylated phenylpropenes, coumarins, and prenylated coumarins, to give a synergistic effect.

Other experimental work has shown that the plant extract contains: 1) cyclooxygenase-1 and 2 (COX- 1 and 2) inhibiting activity; 2) several compounds with anti-oxidant activity; including: 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 6-(3,3~dimethyloxiranyl) methyl]-7-metoxy-2H-1-benzopyran-2-one, 7-methoxy-6-(3-methyl-2-butenyl)- 2H-1 -benzopyran-2-one, vitamin E, 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 6-[(3,3-dimethyloxiranyl)methyl]-7-metoxy-2H-1-benzopyran-2-one, 7-methoxy-6-(3~methyl-2-oxobutyl)- 2H-1 -benzopyran-2-one, 4-(4-hydroxyphenyl)- 2-butanone, 8-[β-D-glucopyranosyloxy)-1-methylethyl]-8,9-dihydro-2H-furo[2,3-h]-1-benzopyran-2- one, 2-isopropenyl-2,3-dihydrofuro[3,2-g] chromen-7-one, 2-(3,4-dihydroxyphenyl)-3-(β.-D-galactopyranosyloxy)-5,7-dihydroxy-4H-1-benzopyran-4- one; 3) several compounds with anti-microbial activity (antibiotic action against Gram positive and Gram negative bacteria, including Pseudomonas, Streptococcus, Staphylococcus, Enterococcus, Escherichia coli, Candida albicans, Mycobacterium tuberculosis, Mycobacterium smegmatis; and fungi, including Cladosporum cucumerinum). These compounds include: 5,5'-(tetrahydro-1 H,3H-furo[3,4c]-furan-1,4-diyl)bis-[1S-(1α.,3a.α,4.β.,6a)-1,3 benzodioxole, 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 6-(3,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 7-methoxy-6-(3,3-methyl-2butenyl)- 2H-1 -benzopyran-2-one, 6-[(3,3-dimethyloxiranyl)methyl]-7-metoxy-2H-1-benzopyran-2-one, 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 6-(3,3-dimethyloxiranyl) methyl]-7-metoxy-2H-1-benzopyran-2-one, esculetin, 3,4-dihydro-5-hydroxy-2,2,8-trimethyl-2H,6H-benzo[1,2-b:5,4-b']dipyran-6-one, 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuro[3,2-g] chromen-7-one. 7-methoxy-6-(3-methyl-2-oxobutyl)- 2H-1 -benzopyran-2-one, 2,2-dimethyl-pyrano(3,2-c)(1)benzopyran-5-one, 9,10-dihydro-9-hydroxy-8,8-dimethyi-2H,8H-Benzo[1,2-b:3,4-b']dipyran-2-one, n-hexadecanoic acid.

The identified chemical compounds may be responsible for the identified biological activities on an individual basis or may act/ contribute thereto on a synergistic basis.

The present invention is further described by the following examples. Such examples, however, are not to be construed as limiting in any way either the spirit or scope of the invention.

EXAMPLES

1. Toxicitv Testing:

1.1 Whole animal feeding studies

The objective was to determine the minimal lethal dose or alternatively, the no observed adverse effect level (NOAEL) of a 50 g/l plant extract of C. album in rats.

Test conditions: 100 ml of a plant extract in 70% ethanol and 30% water, containing 50 g/l of the extracted plant material, was received in an amber glass bottle and was kept in a refrigerator at 7°C. The mass of 10 ml of the 50 g/l plant extract (PE) was determined to be 8,75g or 875 mg per 1 ml. -

The prescribed daily therapeutic dose for a human of 80 kg body mass was given as 10 drops PE three times per day. This amounts to a total daily dose of 30 drops per day. The mass of 1 drop, according to the apothecary system, is equal to 1 grain and 15 grains are equal in mass to 1 gram, therefore, 30 drops will have a mass of 2 grams.

For this test white female rats, with body masses varying between 213g and 265g were received. It was decided, for practical reasons, to calculate the dose to be used for a 300g rat and use the same dose for all of them.

If the total daily dose for an 80kg individual is 30 drops or 2g, it is equal to 2000mg per 80kg body mass and for 1kg (1000g) body mass it will be 25mg. A rat (body mass 300g) will then receive 7,5mg as a total therapeutic daily dose. A dose thirty times more will then be 225mg for a 300g rat.

The actual mass of the 50g/l plant extract (PE) was determined to be 875mg per ml. To determine the volume in millilitres of 225mg, the percentage of 225mg in 875mg was calculated. It was 25,7% which was rounded off to 26% and 26% of 1 ml is 0,26 ml. This indicated that the 30 times therapeutic daily dose of PE for a 300g rat will be 0,26ml. Six rats in the treatment group received a daily dose of 0,26ml PE per os for 7 consecutive days while 5 rats in the control group received 0,26ml of 70% ethanol plus 30% water at the same time. Body masses of all rats were determined prior to the first doses and at termination of the experiment. One day after the last doses were given, the rats were euthanazed with pentobarbitone sodium intra peritoneally and autopsied.

Results None of both groups of rats showed any clinical signs of a disease, illness, toxicity or untoward reactions throughout the experimental period.

A decrease in body mass was seen in a few rats in both groups with the worst decrease being recorded for rat 3, control group, which started at 265g and ended at 197g. See Table 1.

Post mortem examination revealed a liver abscess in 4 of 6 treated rats and 3 of 5 control rats. No other lesions were seen in any of the rats of both groups. See Table 1.

Discussion and Conclusion Abscesses occurred in both groups of rats, which indicate a possible infection contracted in their brooder colony.

Both groups showed little or no gain in mass and many of them lost mass during this 8 day period. As there were no abscesses in control rat 3 (lost 68g), it clearly did not have an effect on the loss of mass seen in many rats in this experiment.

Conditions and the feed cubes were similar to that in the brooder colony and with previous experiments rats increased in mass indicating that something else may have resulted in these rats losing mass. The only remaining factor common to both groups was the 70% ethanol they received, which may have resulted in decreased feed intake.

These slight negative findings do not distract from the fact that the 6 rats receiving PE at 30 times the therapeutic dose, ended in a slightly better condition than the 5 in the control group.

Thirty times the therapeutic dose or 0,26ml of the 50g/l plant extract per rat per day for 7 days can therefore be regarded as the oral No Observed Adverse Effect Level (NOAEL) for this plant extract in the rat. Table 1: Rat body mass, clinical signs and necropsy lesions determination in 7 day plant extract dosing experiment.

Tr = Treatment group, C = Control group, N/a = Nothing abnormal

1.2 Brine Shrimp Assay

A further toxicity test, the brine shrimp assay, was performed to investigate potential toxic effects.

Test conditions

Brine shrimp eggs were hatched in artificial sea water and the larvae incubated with various concentrations of plant extract. As 70% ethanol alone is toxic to the brine shrimps, the plant extract was dried to a residue and redissolved in dimethyl sulfoxide (DMSO, a solubilizing agent) and water to a known concentration. Following published protocols, this was tested against the brine shrimps at 1000, 100 and 10μg ml^"1. In an alternative procedure, the ethanol was evaporated off the plant extract, which was then reconstituted to the original volume using DMSO and water. This extract was tested in decreasing concentrations from 50% (equivalent to 36,5 mg ml^"1). Barberine chloride was used as a positive control and appropriate negative controls (solvent blanks and water alone) were included in the assay.

Results and Discussion

This extract and the various dilutions thereof did not appear to be toxic to the brine shrimp larvae. Taking into account the generally accepted relationship between activity in the brine shrimp assay and possible human acute toxicity to the tested plant, these results suggest a probable lack of toxicity in the extract under investigation.

1.3 Human cell culture toxicity assay

As it was demonstrated that Coleonema plant extracts exhibit significant potency against bacterial and fungal pathogens (see below), and therefore has potential medicinal applications, the potential cytotoxicity of this plant was investigated.

Test conditions

The acetone and ethanol extracts of C. album and the hybrid species were investigated for cytotoxic properties in an in vitro assay using a human T lymphocyte cell line, CEWΛ-nkr -CCR5. This natural killer resistant sub-line of T lymphocyte CEM cells was induced to express the major HIV-1 CCR5 coreceptor (by a retroviral gene transduction technique) for use in HIV-1 neutralization assays.

The cytotoxicity effects of the extracts were determined by a colorimetric XTT-based cell proliferation assay. This assay is based on the metabolic reduction of XTT by mitochondrial enzyme activity of viable cells into a coloured formazan product that can be measured spectrophotometrically.

The dry crude extracts were redissolved in DMSO to give a final concentration of 100 mg/ml. 100 μl cells were seeded in flat bottom microtitre plates to give in a final concentration of 1 x 10⁵ cells/ml per well. Extract was added in three replicates to give a final concentration of 500.0, 50.0, 5.0, 0.5, 0.05 μg/ml. Cells were incubated for 5 days in a 5% C0₂ atmosphere at 37 °C. To evaluate cell survival, after the incubation period, 20 % XTT-solution was added and the cells incubated for a further 2 hours. Absorbance was then determined at 450 and 690 nm by a microplate reader. The percentage of cytotoxicity for each sample was calculated in reference to the untreated control cells.

Results

Table 2: Cytotoxicity analysis of C. album extracts

Conclusion

The Coleonema extracts, prepared in either acetone or ethanol, exhibited no significant level of cytotoxicity when tested up to a concentation of 50 μg/ml. A ten-fold higher concentration of 500 μg/ml however, exhibited toxic effects against the cell line used in the assay.

Antimicrobial Screening:

2.1 Antibacterial screening

The aim was to evaluate the potency of Coleonema extracts for activity against selected Gram- positive and Gram-negative bacteria.

Preparation of sample

An aliquot of the plant extract suspended in acetone was dried down to a residue and resuspended to known concentration (50 mg ml^"1) in acetone.

Testing conditions

The test organisms in the investigation of antibacterial activity included two Gram-positive bacteria, Enterococcus faecalis and Staphylococcus aureus, and two Gram-negative species, Pseudomonas aeruginosa and Escherichia coli. The microplate method was used to evaluate the antibacterial activity of the extract. This method allows the calculation of minimal inhibitory concentration (MIC) values for active plant extracts against each bacterial species. A two-fold serial dilution of plant extract was prepared in 96-well microtitre plates, and bacterial culture was added to each well. The presence of bacterial growth was detected by the addition of a tetrazolium salt which is reduced to a red-coloured formazan by biologically active organisms, in this case the dividing bacteria. When the solution in the well remains clear after addition of the compound, bacterial growth is inhibited by that concentration of the plant extract.

Neomycin was used as a positive antibiotic control and appropriate negative solvent controls were included. The assays were repeated to confirm the results. Table 3: The minimum inhibitory concentrations in mg/ml, of the acetone and ethanol plant extracts from C. album with antibacterial activity against the bacterial isolates tested

MIC results given in mg/ml + SD

A second antibacterial screen, using the above organisms, was performed using a technique known as 'bioautography' that allows the identification of individual components in a crude extract.

Test conditions:

The ability of the extract to inhibit bacterial growth was shown by bioautography. Compounds present in the extracts were separated by thin layer chromatography and the indicator organisms were used to ascertain antibacterial activity. The area/size of the zones of growth inhibition due to chemical compounds present on the plate was calculated and used as a qualitative measure of antifungal activity.

Results The Coleonema extracts (C. album as well as the hybrid) contained prominent inhibition zones which were found to be associated with different chemical compounds. The extract exhibited differential activities against the different bacteria. Besides 1, 2', 3'-dihydroxydihydrosuberosin, the structures of some of these antibacterial compounds have been determined as: 5,5'-(tetrahydro-1H,3H-furo [3,4c]-furan-1,4-diyl)bis-[1S-(1α.,3a.α,4.β.,6a)-1,3 benzodioxole, 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 6-(3,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 7-methoxy-6-(3,3-methyl-2butenyl)- 2H-1-benzopyran-2-one, 6-[(3,3-dimethyloxiranyl)methyl]-7-metoxy-2H-1-benzopyran-2-one,

6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzόpyran-2-one,

6-(3,3-dimethyloxiranyl) methyl]-7-metoxy-2H-1-benzopyran-2-one, esculetin,

3,4-dihydro-5-hydroxy-2,2,8-trimethyl-2H,6H-benzo[1,2-b:5,4-b']dipyran-6-one,

2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuro[3,2-g] chromen-7-one.

7-methoxy-6-(3-methyl-2-oxobutyl)- 2H-1 -benzopyran-2-one,

2,2-dimethyl-pyrano(3,2-c)(1)benzopyran-5-one,

9,10-dihydro-9-hydroxy-8,8-dimethyl-2H,8H-Benzo[1,2-b:3,4-b']dipyran-2-one, n-hexadecanoic acid.

2.2 Anti myco-bacteria screening

The anti-mycobacteria (TB) bacterial screening were carried out to evaluate the potency of Coleonema extracts for activity against Mycobacterium tuberculosis.

Test conditions:

The testing was performed according to the 'agar plate' method that allows the determination of the proportion of the bacterial {Mycobacterium tuberculosis) population that is resistant to the Coleonema extract. When 1 % or more of microorganisms tested are resistant to the extract, the population is considered resistant to the extract. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug that inhibited more than 9% of the bacterial population.

Results

Table 4: Anti-tuberculosis bacterial screening for Coleonema extract

Positive controls Conclusion

Extracts from Coleonema album exhibited moderate anti-TB activity while extracts from

Coleonema album hybrid exhibited good activity (< 0.5 mg/ml) (Table 4).

A second anti-mycobacteria screen, using Mycobacterium smegmatis, was performed that allows the identification of individual components in a mixture with antibiotic activity.

Test conditions

The ability of the extract to inhibit bacterial growth was demonstrated by bioautography. Compounds present in the extracts were separated by thin layer chromatography and the, indicator organism, Mycobacterium smegmatis, was used to ascertain antibacterial activity. The area/size of the zones of growth inhibition due to chemical compounds present on the plate was calculated and used as a qualitative measure of antifungal activity.

Results

The Coleonema extracts (C. album as well as the hybrid) contained five prominent inhibition zones which were found to be associated with at least five different chemical compounds.

Except for 1, 2', 3'-dihydroxydihydrosuberosin and derivatives thereof, the structures of these antibacterial compounds have yet to be determined.

3. Antiviral (HIV) testing:

XTT Cell Viability Assay: Measuring cell viability via the XTT assay (where XTT is reduced by viable cells) is a good marker for establishing the effect of different treatments on the cells.

HIV-1 infected cells have reduced viability in comparison to healthy uninfected cells. When treating HIV-1 infected cells with compounds that aid the virus in the infection process, an even greater reduction in cell viability will be observed. Treatment on HIV-1 infected cells with immuno-supportive compounds that inhibit viral replication will lead to an increase in the cell viability of the infected cells. Therefore, viability tests can be used to assess the toxic side effects of plant extracts on cells in culture, as well as the replication of HIV-1 in these cells. Peripheral blood mononuclear cells (PBMC) were used in the cell viability assay. These cells have all the necessary receptors required for HIV-1 infection. An aliquot of these cells was used to be infected with HIV-1. HIV-1 isolated from a HIV-1 seropositive donor and cultured in PBMCs was used for the cell viability assay. This viral strain has the ability to infect cells bearing either the -XCR4 or -CCR5 co-receptor; this viral strain would therefore be able to successfully infect PBMCs. After preparation of the uninfected cells and HIV-1 infected cells, 96-well plates were prepared. For each extract, eight serial dilutions were prepared in 96-well plates. Uninfected and infected PBMCs were then plated in the wells. Media and DMSO controls were included. The plates were then incubated for 4 days at 37°C 5% C0₂ in a humidified atmosphere.

On day 4 the 'XTT viability assay' was performed on the samples in the plates. Aliquots of supernatants from the wells bearing the HIV-1 infected cells were removed from all columns, to be used in the p24 antigen ELISA assay. Appropriate volumes were removed from all other wells not bearing infected cells, so as to keep the volumes in the wells the same throughout the plate. XTT reagent was then added to the wells to a final concentration of 10% (v/v). The plates were then incubated at 37° for 4 hours to allow for the reduction of the XTT dye by the viable cells and the optical density was measured. Mean drug-blank values were subtracted from the corresponding sample values. Cell toxicity values were calculated by dividing the mean OD- value of uninfected treated cells by the mean OD-value of the uninfected control cell sample. Antiviral effect values were calculated by dividing the mean OD-value of infected treated cells by the mean OD-value of the infected control cell sample (Figures 1 and 2).

P24 Antigen ELISA: The HIV-1 p24 Antigen ELISA Assay (murine monoclonal antibody) kit was purchased from Beckmann Coulter (Catalog number PN 4238020-F R 6-01). The protocol for "Procedure for quantification of HIV-1 p24 antigen in blood and tissue culture supernatants" was followed as instructed by the manufacturer. Supernatant removed from the infected treated wells from the 96-well plates was used for the p24 antigen ELISA. Positive and negative controls were included. A calibration curve of p24 antigen concentrations was constructed.

HIV-1 Reverse Transcriptase Assay:

The HIV-1 Reverse Transcriptase Assay (colourimetric) kit was purchased from Roche (Catalog number 1 468 120). The protocol for "Quantification of the inhibitory effect of reverse transcriptase inhibitors" was followed. For this assay, no cell culture supernatant was required as a source for HIV-1 reverse transcriptase. Commercially available HIV-1 reverse transcriptase was used in this assay. Eight dilutions of each extract (in duplicate) were prepared in lysis buffer supplied with the kit. After addition of HIV-1 RT and reaction mixture, the assay proceeded as stipulated by the manufacturer.

Results and Conclusion

The results for each extract tested are presented in graphic format (XTT viability: cell toxicity (CT) (Figures 1 and 2); XTT viability: antiviral effect (AV) (Figures 3 and 4); p24 values (Figures 5 and 6); and percentage reverse transcriptase (RT) activity and inhibition (Figures 7 and 8)). In Figures 1 to 4, values on each graph have to be related to the "1.0" value, which is the value for the control samples.

Therefore, values in Figures 1 and 2 above 1.0 indicate a positive effect (non-toxic), and values below 1.0 indicate a negative effect (toxic). Cell toxicity values for Figures 1 and 2 were calculated by dividing mean OD-values of uninfected treated cells by the mean OD-values of the uninfected control cell sample.

Similarly, values in Figures 3 and 4 above 1.0 indicate a positive effect (antiviral effect), and values below 1.0 indicate a negative effect (non antiviral effect). Antiviral effect values were calculated by dividing mean OD-value of infected treated cells by the mean OD-value of the infected control cell sample.

Figures 5 and 6 show the p24 antigen values at each concentration of the plant extract that cells where treated with. The p24 values can be read directly from the graph. The lower the p24 value, the lower the replication of HIV-1.

Values can also be read directly from the graphs of Figures 7 and 8, where the direct RT (reverse transcriptase enzyme) inhibition of the commercial enzyme is indicated over a concentration range.

The best conclusions can be made by referring to the p24 values, since these are a direct indication of HIV-1 replication within the cells: for each extract, the concentration by which the lowest p24 value was obtained (and therefore the lowest level of HIV-1 replication), can then be assessed for its effects on cell viability (relating it to the toxicity and antiviral effects of the extracts on the cells).

For some of the extracts, the different assays that were performed reached consensus as to which concentration of extract gave the best results, i.e. the lowest level of HIV replication with the lowest amount of toxic effects on the cells.

Table 5 shows a comparison of the different extracts concerning optimal concentration, cell toxicity, antiviral effect, p24 value, and reverse transcriptase inhibition. Table 5: A comparison between the effects of extracts on cell viability, HIV-1 replication and HIV-1 reverse transcriptase inhibition: the table shows the optimal concentration at which each extract shows low level HIV-1 replication (by referring to p24 levels).

^ffCT : Cell Toxicity **AV : Antiviral Effect

The evaluation of Coleonema extracts was repeated as described above, but with a different viral strain and human cell line. Because of its relevance in South Africa, HIV-1 subtype C virus was chosen (the subtype C epidemic has now become the most predominant subtype in Southern African countries). HIV-1 subtype C is believed to be a macrophage-tropic or NSI virus. The cell line used was CEMn /CCR5 cells, which expresses both CCR5 and CXCR4 coreceptors on the cell surface, and therefore supports the replication of the subtype C viruses.

The cells in the presence of all four extracts (C album and hybrid, extracted either with acetone or ethanol; AA, AE, HA, HE) had a higher viability compared to the infected cells without extract. Therefore, the tested extracts had the ability to decrease viral induced cytopathic effects. To confirm the results obtained by the viability assays, the p24 core protein level was again determined for each sample. This is an indication of HIV-1 replication fitness in the presence and absence of extract. This assay, for measurement of core protein, is considered as one of the most sensitive assays besides PCR, for HIV detection in vitro.

Figure 9 shows the effect of the AE, HE, AA and HA extracts (50 μg/ml)) and AZT (10μM), on the viral core protein (p24) level of acutely HIV-1 infected CEM/7/crCCR5 cell over a period of 7 days (AE, HE: ethanol extracts from C. album and the hybrid, respectively; AA, HA: acetone extracts from C. album and the hybrid, respectively).

Samples with low detectable p24 content compared to the reference inhibitors (AZT, 10 μM) and infected negative controls were regarded as being protected from infection. The cells in the presence of all four extracts had a lower p24 level compared to the infected cells (with no extract), with HE, AA and HA showing a significant decrease in p24 antigen level (p-values < 0.005), with the decrease of 50 μg/ml AA comparable to that of 10 μM AZT. The effectiveness of the various extracts was found to be as follows: AA>HE=HA>AE.

4. Immunostimulatory activity:

It was observed that human patients taking the Coleonema tincture as a tonic/immune stimulant exhibited significant improvements in their general health condition. To monitor this phenomenom under controlled conditions, the plant extract of the invention was tested on 3 male patients and one female patient over a time period of three years. Their health condition was evaluated by a general practitioner and haematological and chemical analyses of blood samples were performed by an accredited clinical pathology practice/laboratory.

Patient 1 was diagnosed HIV positive during the early part of 2000. His CD4 count before receiving the plant extract was 227 cells/μl. After starting to take the extract in August 2000, his CD4 count improved over the next month and his general health improved. By May 2002 his CD4 cell count had increased to 387 cells/μl. To June 2002 the patient has suffered no acute infections and is currently in good health.

Patient 2 was diagnosed HIV positive and with TB in September 2001. Patient 2 started to take the plant extract in November 2001. His weight has increased, and he is now able to work a full day. His general health has improved.

In addition to being HIV-positive, patient 3 was diagnosed with TB in September 2001. Patient 3 started taking the plant extract in April 2002. Since starting to take the plant extract, patient 3 has experienced an increase in energy levels and has increased in weight.

Patient 4 was diagnosed HIV positive and began taking the plant extract (15 drops twice daily) in October 2003. By the end of October, tests showed that her viral load was 1 307 copies/ml and her CD4 count was 680. Almost a year later, in September 2004, the patient's viral load had been reduced to 91 copies/ml and her CD4 count had increased to 891. At the same time, the full blood and white cell counts were found to be within the normal parameters. The patient did not take any other medication which could explain the decrease in viral load or increase in CD4 count.

In two additional studies groups of twenty and ten HIV^*" human volunteers, respectively, the members of each group were supplied with the herbal tincture over a period of three months. In the majority of cases, a general improvement in health was observed with no adverse effects. 5. Antifungal screening:

An antifungal assay determination was performed using the 'bioautography' procedure that allows detemination of individual components present in a mixture, described above.

Test conditions

The ability of the extract to inhibit fungal growth was shown by bioautography. Compounds present in the extracts were separated by thin layer chromatography and the indicator organisms, Cladosporium cucumerinum and Candida albicans, were used to ascertain antifungal activity. The area/size of the zones of growth inhibition due to chemical compounds present on the plate was calculated and used as a qualitative measure of antifungal activity.

The microplate method as described under section 2.1 was used to evaluate the anti-Candida activity of the extract. This method allows the calculation of minimal inhibitory concentration (MIC) values for active plant extracts against the test organism. Fluconazole was used as a positive antibiotic control and appropriate negative solvent controls were included. The assays were repeated to confirm the results.

Results

Table 6: The minimum inhibitory concentrations in mg/ml, of the acetone and ethanol plant extracts from C. album with anti-Candida activity (MIC values given in mg/ml ± SD. AE, HE: ethanol extracts from C. album and the hybrid, respectively. AA, HA: acetone extracts from C. album and the hybrid, respectively.

The determined average MIC value for anti-Candida effects was 0.25 mg/ml.

The Coleonema extracts (C. album as well as the hybrid) contained two prominent inhibition zones which were found to be associated with at least two different chemical compounds. The structures of these antifungal compounds were determined to be prenylated coumarins, i.e. 1 , 2', 3'-dihydroxydihydrosuberosin and derivatives thereof. Additional compounds found in the anti-Candida inhibition zones were: 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 6-(3,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuro[3,2-g]chromen-7-one, 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuro[3,2-g]chromen-7-one, 7-methoxy-6-(3-methyl-2-oxobutyl)- 2H-1-benzopyran-2-one, 2,2-dimethyl-pyrano(3,2-c)(1)benzopyran-5-one.

Anti-inflammatory screening:

Cyclooxygenase (COX, also called Prostaglandin H Synthase or PGHS) enzymes contain both cyclooxygenase and peroxidase activities. COX catalyzes the first step in the biosynthesis of prostaglandins (PGs), thromboxanes, and prostacyclins; the conversion of arachidonic acid to PGH2. Cyclooxygenase-1 (COX-1) is constitutively expressed in a variety of cell types and is involved in normal cellular homeostasis. COX-2 is responsible for the biosynthesis of PGs under acute inflammatory conditions. This inducible COX-2 is believed to be the target enzyme for the anti-inflammatory activity of nonsteroidal anti-inflammatory drugs.

6.1 Test system 1

The COX inhibitor screening assay kit (obtained from Cayman) directly measures PGF2α produced by SnCI2 reduction of COX-derived PGH2. The prostanoid product is quantified via enzyme immunoassay (EIA) using a broadly specific antibody that binds to all the major prostaglandin compounds. Thus, the Cayman COX assay is more accurate and reliable than an assay based on peroxidase inhibition. The Cayman COX Inhibitor Screening Assay includes both ovine COX-1 and human recombinant COX-2 enzymes in order to screen isozyme-specific inhibitors. The COX Inhibitor Screening Assay was performed according to the manufacturer's instructions (Cayman, 2003/4).

Traditional screening methodologies for detecting anti-inflammatory/pain potential in plants were mainly directed at the detection of the presence of a COX-1 inhibitor in the plant or herbal tincture. However, in recent years the importance of COX-2 inhibition in anti-inflammatory research has moved the emphasis from COX-1 to COX-2, and the indication of a potential medical application for anti-inflammatory use is now generally evaluated on the degree of COX- 2 inhibition. COX-1 inhibition has been associated with gastrointestinal complications and thus has a limited clinical usefulness. The current view is that a COX-2 specific inhibitor is the preferred way of treating inflammatory associated diseases. However, there are still several medications available that have both COX-1 and COX-2 inhibitor characteristics with clinical applications.

Results The results are summarized in Figures 10(a)(percentage COX 2 inhibition) and 10(b)(percentage COX 1 inhibition). These results were obtained by dilution of the samples tested. In the case of analyses 1, 3, 5 and 7, a 2000 times dilution was made and for analyses 2, 4, 6 and 8, a 4000 times dilution was used. This corresponds to 50 μg/ml and 25 μg/ml, respectively, of the original product.

Discussion

The analyzed samples show a remarkable selectivity for the inhibition of COX 2 (up to 85%) (Figure 10(a)), with very little COX 1 inhibition (10 % or less) (Figure 10(b)). The calculated IC_S0 for the C. album extract was less than (<) 15 μg/ml, which compares extremely well with other plants with reported anti-inflammatory activity in the literature.

6.2 Test system 2

This assay system involves testing for the inhibition of the COX-1 or COX-2 enzymes in an in vitro enzyme radiochemical assay. The substrate is radiolabelled arachidonic acid. This is converted by COX-1 /COX-2 to radiolabelled prostaglandins. Any inhibition of this activity (by a non-steroidal anti-inflammatory drug, or an 'active' plant extract) would result in low levels of prostaglandins being detected (by a measure of the radioactivity in the sample).

Results

The results from the radiolabelled assay indicated that the C. album extracts contained compounds that inhibited the sheep COX-1 and COX-2 iso-enzymes approximately equally well, at a level of 86% and 74%, respectively. Indomethacin was used as a positive control. Table 7: COX-1 and COX-2 inhibition by herbal tincture

Table 8: COX-1 and COX-2 inhibition of C. album and C. puichelum extracts (final test concentration = 250 μg/ml)

The results show that the active compounds were primarily found in extracts made with low- polarity solvents such as dichloromethane. Activity was significantly reduced when the extracts were evaporated to dryness with accompanying loss of volatile compounds. Discussion

The products and plants tested with system 1 show an exceptional and rare ability in their capability to inhibit human COX-2 almost exclusively, with relatively low inhibition of porcine COX-1. When tested with system 2, the extract exhibited approximately equal inhibitory activities towards COX-1 and 2 derived from sheep.

Evaluation of the herbal tincture on human patients suffering from arthritis indicated beneficial effects on their conditions for the duration of the treatment. According to current literature this plant/extract/product must be regarded as an excellent candidate for the use in inflammatory- associated diseases.

7. Anti-oxidant screening

The anti-oxidant activity determination was performed on the crude extract as well as individual components separated by thin layer chromatography.

Test conditions:

A model antioxidant system was used to assess the free radical scavenging activity of Coleonema extracts. This method is typically based on inhibition of the accumulation of oxidized products, since the generation of free radicals is inhibited by the addition of an antioxidant. - —

For the qualitative detection of the RSC of various plant extracts, a simple thin layer chromatography test was performed where the developed chromatogram is sprayed with a free radical solution. After incubation, a purple background is revealed with yellow spots at the location of the active antioxidant compound. The intensity of the yellow colour depends on the quantity and nature of the compound present at that location.

A reliable method to quantitatively determine the RSC involves the measurement of the disappearance of a coloured free radical, such as DPPH^", by a spectrophotometric assay. Specific compounds or extracts react with the stable radical by hydrogen or electron donation. DPPH^" has a purple colour that is reduced to the yellow coloured diphenylpicrylhydrazine by antioxidants. The reduction of DPPH^" is monitored by a decrease in absorbance at 515nm.

Results

Total antioxidant activity can be expressed as vitamin C equivalents calculated on a weight basis. The tests performed confirmed the ability of the Coleonema extracts to scavenge DPPH^" radicals. The RSC was quantitatively determined as the concentration of antioxidant needed to decrease the initial DPPH^" concentration by 50%.

An improved oxygen radical absorbance capacity (ORAC) assay using fluorescein as a fluorescent probe using a 96-well plate format coupled with a microplate fluorescence reader was also used to evaluate the anti-oxidant capabilities of the C. album extracts. A fluorescence decay curve was obtained by plotting the fluorescence values against the incubation times, indicating the loss of fluorescence intensity induced by AAPH over the reaction period. The ORAC value refers to the net protective area under the quenching curve of fluorescein in the presence of an antioxidant. The net area under the curve (AUC) of the standard and samples were calculated. The ORAC values were calculated using a regression equation between the Trolox concentration and the net areas under the fluorescein decay curve and were expressed as μmole Trolox equivalents / μg dry plant extract. The net area under the curve was calculated using the following equation: AUC = 0.5 + f,/f₀ + ...f fo where f, is the initial fluorescence reading at t=0 min and f,- is the fluorescence reading at time /^'.

The data were analysed by a Microsoft Excel macro program to apply the equation to calculate the AUC. The net AUC for each sample was obtained by subtracting the AUC of the corresponding blank from that of the sample.

The TLC-DPPH^" assay revealed several compounds with various radical scavenging activities, inter alia 2,3-dihydroxydihydrosuberosin. Other compounds include:

6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one, 6-(3,3-dimethyloxiranyl) methyl]-7-metoxy-2H-1-benzopyran-2-one,

7-methoxy-6-(3-methyl-2-butenyl)- 2H-1-benzopyran-2-one, vitamin E,

6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one,

6-[(3,3-dimethyloxiranyl)methyl]-7-metoxy-2H-1-benzopyran-2-one, 7-methoxy-6-(3-methyl-2-oxobutyl)- 2H-1-benzopyran-2-one,

4-(4-hydroxyphenyl)- 2-butanone,

8-[β-D-glucopyranosyloxy)-1-methylethyl]-8,9-dihydro-2H-furo[2,3-h]-1-benzopyran-2-one,

2-isopropenyl-2,3-dihydrofuro[3,2-g] chromen-7-one,

2-(3,4-dihydroxyphenyl)-3-(β.-D-galactopyranosyloxy)-5,7-dihydroxy-4H-1-benzopyran-4-one.

The antioxidant activity of plant extracts can often be correlated with the extract's total polyphenol content. This can be determined by a colourimetric oxidation/reduction assay and is expressed as gallic acid equivalents, a standard phenolic compound with potent antioxidant activity. Table 9: Summary of the phenolic content and antioxidant activity (DPPH and ORAC) of the C. album extracts (AE, HE: ethanol extracts from C. album and the hybrid, respectively. AA, HA: acetone extracts from C. album and the hybrid, respectively)

EC50 values expressed as mg/ml Vitamin C equivalence expressed as mg vitamin C equivalents per g of dry weight ORAC value expressed as μmole Trolox equivalent /μg dry plant weight Total phenolic concentration expressed as mg gallic acid equivalence/g dry extract Lower EC50 values indicate a higher antioxidant capacity. Positive controls included: Vitamin C (3.4 μg/ml) and Trolox (4.1 μg/ml)

Figure 11 shows a comparison of the antioxidant activity of the different extracts with the two standard antioxidants, Trolox and Vitamin C. This comparison indicated that HA, followed by AA, had the highest RSC of the extracts tested (AE, HE: ethanol extracts from C. album and the hybrid, respectively; AA, HA: acetone extracts from C. album and the hybrid, respectively).

Figure 12 shows the linear correlation between the sample concentrations and ORAC representing the concentration-dependent data obtained from each of the extracts tested. A linear correlation was found between the antioxidant activity and the extract concentration with the following correlation coefficient values: AE (0.958); HE (0.964); AA (0.975) and HA (0.9189) (AE, HE: ethanol extracts from C. album and the hybrid, respectively; AA, HA: acetone extracts from C. album and the hybrid, respectively).

Figure 13 shows the reductive (anti-oxidant) potential of different concentrations of the different C. album extracts, determined by the spectrophotometric detection of Fe₃ ⁺ - Fe₂ ⁺ transformations (AE, HE: ethanol extracts from C. album and the hybrid, respectively; AA, HA: acetone extracts from C. album and the hybrid, respectively).

The results indicate that all the Coleonema extracts were effective and efficient in inhibiting / scavenging free radicals. Accordingly, it is proposed that some of these compounds may lead to the development of potential drugs of considerable therapeutic value against many diseases that involve free radicals.

8. Chemical Analysis and Structural Elucidation

The chemical analyses of the active components present in the Coleonema extracts were performed by means of thin layer chromatography, high performance liquid chromatography and mass spectrometry.

Extracts were made in various organic solvents with varying polarity in order to perform a selective extract or to enrich the extract with regard to the presence of a specific compound. Extracts were fractionated by standard chromatographic techniques, in particular thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Identification of compounds was by means of HPLC coupled to spectrophotometric detection as well as mass spectrometric detection. Compounds were positively identified by comparison of ultraviolet/visible absorbance spectra and mass fragmentation patterns with chemical databases. A chemical analysis of the plant extract showed that the active ingredients included 1 , 2', 3'- dihydroxydihydrosuberosin, a prenylated coumarin, and derivatives thereof. Other bio-active agents found in the extract include monoterpenes, sesquiterpenes, phenylpropanoids, phenylpropenes and prenylated phenylpropenes, coumarins and prenylated coumarins. A detailed list is supplied in sections 2 and 7 above.

While the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated by those skilled in the art that various alterations, modifications and other changes may be made to the invention without departing from the spirit and scope of the present invention. It is therefore intended that the claims cover or encompass all such modifications, alterations and/or changes.

Claims

CLAIMS:

1. A pharmaceutical composition comprising as an active ingredient plant material or an extract of a plant of the genus Coleonema.

2. A pharmaceutical composition according to claim 1 , wherein the plant material or extract of the plant is selected from the group consisting of C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum.

3. A pharmaceutical composition according to either of claims 1 or 2, wherein the plant material or extract of the plant is from C. album or a hybrid thereof.

4. A pharmaceutical composition according to any one of claims 1 to 3, which is formulated for oral or topical administration.

5. A pharmaceutical composition according to claim 4, which is a tablet or a capsule containing the plant material or an extract of the plant material, a tincture or a tonic containing an extract of the plant material, or a powder comprising the plant material contained in a sachet.

6. A pharmaceutical composition according to claim 4, which is a salve, an ointment, a lotion, a composition for spray or aerosol application.

7. A pharmaceutical composition according to any one of claims 1 to 6, wherein the plant material or the extract is derived from aerial parts of the plant, which parts may be fresh or dried.

8. A pharmaceutical composition according to any one of claims 1 to 7, for use as an immune stimulant, an antibacterial agent, an antifungal agent, an antiviral agent, an anti- inflammatory agent or an anti-oxidant.

9. A pharmaceutical composition according to any one of claims 1 to 8, for use as an antiviral agent for the treatment of HIV infection.

10. A pharmaceutical composition according to any one of claims 1 to 8, for use as an anti- inflammatory agent for COX-1 and/or COX-2 inhibition.

11. A pharmaceutical composition according to any one of claims 1 to 10, which includes from about 10 g/l plant material or plant extract to about 100 g/l plant material or plant extract.

12. A pharmaceutical composition according to any one of claims 1 to 11, which includes from about 25 g/l plant material or plant extract to about 75 g/l plant material or plant extract.

13. A pharmaceutical composition according to any one of claims 1 to 12, which includes about 50 g/l plant material or plant extract.

14. A pharmaceutical composition according to any one of claims 1 to 13, which includes at least one of the following compounds: 1 , 2', 3'-dihydroxydihydrosuberosin (2H-1-benzopyran-2-one, 6-(2,3-dihydroxy-3- methylbutyl)-7-methoxy, 2H-1-benzopyran-2-one, 6-(3,3-dimethyloxiranyl)-7-methoxy); 5,5'-(tetrahydro-1H,3H-furo[3,4c]-furan-1,4-diyl)bis-[1S-(1α.,3a.α,4.β.,6a)-1,3 benzodioxole; 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one; 6-(3,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one; 7-methoxy-6-(3,3-methyl-2butenyl)- 2H-1-benzopyran-2-one; 6-[(3,3-dimethyloxiranyl)methyl]-7-metoxy-2H-1-benzopyran-2-one; 6-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-2H-1-benzopyran-2-one; 6-(3,3-dimethyloxiranyl) methyl]-7-metoxy-2H-1-benzopyran-2-one; Esculetiπ; 3,4-dihydro-5-hydroxy-2,2,8-trimethyl-2H,6H-benzo[1 ,2-b:5,4-b']dipyran-6-one; 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuro[3,2-g] chromen-7-one; 7-methoxy-6-(3-methyl-2-oxobutyl)- 2H-1 -benzopyran-2-one; 2,2-dimethyl-pyrano(3,2-c)(1)benzopyran-5-one; 9,10-dihydro-9-hydroxy-8,8-dimethyl-2H,8H-Benzo[1,2-b:3,4-b']dipyran-2-one; n-hexadecanoic acid; 4-(4-hydroxyphenyl)- 2-butanone; 8-[β-D-glucopyranosyloxy)-1-methylethyl]-8,9-dihydro-2H-furo[2,3-h]-1-benzopyran-2- one; 2-isopropenyl-2,3-dihydrofuro[3,2-g] chromen-7-one; 2-(3,4-dihydroxyphenyl)-3-(β.-D-galactopyranosyIoxy)-5,7-dihydroxy-4H-1-benzopyran-4- one; Vitamin E; and pharmaceutically acceptable derivatives or salts thereof.

15. A method of treating a patient suffering from a disease, the method comprising orally or topically administering to the patient a composition comprising as an active ingredient plant material or an extract of a plant of the genus Coleonema.

16. A method according to claim 15, wherein the plant material or extract is selected from the group consisting of C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum.

17. A method according to either of claims 15 or 16, wherein the disease is selected from the group consisting of a disease associated with a low or compromised immune system, a bacterial infection, a fungal infection, a viral infection and an inflammatory condition.

18. A method according to any one of claims 15 to 17, wherein the disease is HIV/AIDS.

19. A method according to any one of claims 15 to 17, wherein the inflammatory condition is selected from arthritis, sinusitis, laryngitis and pharyngitis.

20. A method according to any one of claims 15 to 19, wherein from about 500 mg to about 10 000 mg plant material or extract is administered to the patient per day.

21. A method according to any one of claims 15 to 20, wherein from about 1 000 mg to about 5 000 mg plant material or extract is administered to the patient per day.

22. A method according to any one of claims 15 to 21 , wherein about 2 000 mg plant material or extract per 80 kg body mass is administered to the patient per day.

23. A composition containing plant material or an extract of a plant from the Coleonema genus for use in a method of treatment of the human or animal body by therapy.

24. A composition according to claim 23, wherein the plant material or extract is is selected from the group consisting of C. album, C. nubigenum, C. juniperinum, C. calycinum, C. aspalathoides, C. pulchellum, C. virgatum and C. pulchrum.

25. The use of plant material or an extract of a plant of the genus Coleonema in the manufacture of a medicament for the treatment of a disease selected from a disease associated with a low or compromised immune system, a bacterial infection, a fungal infection, a viral infection and an inflammatory condition.

26. The compound 1, 2', 3'-dihydroxydihydrosuberosin and pharmaceutically acceptable derivatives or salts thereof for use in a method of treatment of the human or animal body by therapy.

27. The use of the compound 1, 2', 3'-dihydroxydihydrosuberosin and pharmaceutically acceptable derivatives or salts thereof in the manufacture of a medicament for the treatment of a disease selected from a disease associated with a low or compromised immune system, a bacterial infection, a fungal infection, a viral infection and an inflammatory condition.

28. A pharmaceutical composition according to claim 1, substantially as herein described with reference to any one of the illustrative examples.

29. A composition according to claim 23, substantially as herein described with reference to any one of the illustrative examples.

30. The use according to claim 25, substantially as herein described with reference to any one of the illustrative examples.

31. The compound for use according to claim 26, substantially as herein described with reference to any one of the illustrative examples.

32. The use according to claim 27, substantially as herein described with reference to any one of the illustrative examples.