OA16218A

OA16218A - Treatment of parasitic infections in humans and animals

Info

Publication number: OA16218A
Application number: OA1200900136
Authority: OA
Inventors: Motlalepula Gilbert Matsabisa; William Ernest Campbell; Peter Ian Folb; Peter John Smith
Original assignee: South African Medical Research Council; University Of Cape Town
Priority date: 2004-11-04
Filing date: 2005-11-02
Publication date: 2015-04-10

Abstract

The invention provides a process for the production of a substance or composition for the treatment, by therapy or prophylaxis, of parasitic infections, in particular malarial infections such as Plasmodium falciparum infections, of the human or animal body. The process comprises extracting the substance or composition from roots of the plant species Dicoma anomala, by an extraction using an organic solvent to obtain a liquid extract containing the substance or composition and removing the solvent from the liquid extract to leave a dried extract containing the substance or composition. The invention extends also to the use of the substance or composition in the manufacture of a medicament or preparation for such treatment of infections; to a substance or composition for use in such treatment of said infections; to compounds for use in such treatment of said infections; and to a method of treating said infections using such compounds.

Description

THI5 INVENTION relates to the treatment of parasitic infections in humans and animais. More partîcularly, the invention relates to a substance or composition, and to one or more compounds for use in such treatment, which may be therapeutic or prophylactic, to a method of such treatment, and to a process for the production of such substance or composition and compounds, for the treatment of parasitic infections, the substance or composition and compounds, the methods of treatment and the process of production ail being useful for, but not limited to, the prophylaxis or treatment of malarial parasitic infections by Plasmodium species, tn parttcularP. falciparum.

According to the invention there is provided a process for the production of a substance or composition for the treatment, by therapy or prophylaxis, of parasitic infections, in particular malarial infections such as Plasmodium falciparum infections, of the human or animal body, the process comprising the steps of:

extracting the substance or composition from a startîng material comprising plant roots of the plant species Dicoma anomala, by subjecting the piant roct startîng material to extraction using an organic solvent to obtain a liquid extract containing the substance or composition; and i

«

I

Ί removing the solvent from the liquid extract to leave a dried extract containing the substance or composition.

While the extraction may be a coid (room température) extraction, it may be a hot (Soxhlet) extraction. Thus the extraction may be a hot extraction carried oui at an elevated température in the range 35 - 140°C, for example 35 - 50°C. Conveniently, however, the extraction is a coid extraction carried out at a température of at most 40°C.

The plant root starting material may be a solid plant material in a fînely divided form, having a particle size not exceeding 100pm, preferably not exceeding 10pm. Thus, in other words, the plant root starting material may be in powdered form having a particle size of at most 100pm, preferably at most 10pm.

One or more organic iiquids may be used as the solvent for the extraction, either separately in sequence, or as mixtures or blends. The solvent may be a polar solvent having a polarity index in the range 0.00-5.00. Preferably each liquid oc blend of Iiquids used as the solvent thus may hâve a polarity index of at teast 0.00 and at most 5.00. in particular, the solvent may hâve a polarity index of 1.0 - 4.8 and may comprise at least one member selected from the group consisting of the solvents set forth in the following table, Table 1, in whichthe polarity indices of the solvents are also listed:

TABLE 1

Solvent

Benzene n-Butanol

Butyl Acetate

Carbon Tetrachloride

Polarity Index

2.7

3.9

4.0

1.6

Chloroform	4.1
1,2 Dichloroethane	3.5
Dichloromethane	3.1
Dioxane	4.8
Ethyl Acetate	4.4
di-Ethyl Ether	2.8
Methyi-t-Butyf Ether	2.5
Methyl Ethyl Ketone	4.7
n-Propanol	4.0
iso-Propanol	3.9
di-iso-Propanol	2.2
Tetrahydrofuran	4.0
Toluene	2.4
T richloroethylene	1.0
Xylene	2.5

A particularly preferred solvent has been found to be dichloromethane (CH₂CI₂).

The process may include subjecting the plant root starting matériel to a preliminary extraction, using a non-polar organic soivent having a polarity index of less than 3.90, preferably a polarity index ïn the range 3.10 - 3.50, followed by drying to evaporate residual non-polar solvent from the solid plant materia!, and then subjecting the dried plant root materiaI, from which the non-polar solvent has been removed, to the extraction, to obtain the liquid extract containing the substance or composition. The preliminary extraction may be carried out with the non-polar solvent at a température of 15 - 30^oC, the drying being carried out at a température not exceeding 40°C, and both extractions being carried out to exhaustion. In particular, the process may include subjecting the dried extract containing the substance or composition to removal of tannin therefrom using a polymer of an organic amide.

Tnus, în a preferred embodiment of the process of the présent invention the powdered plant root starting materia! is subjected te a preüminary extraction, which may

Si

I be substantially to exhaustîon, at room or ambient température (15 - 30°C), using a nonpolar solvent such as hexane, the hexane extract beîng discarded and residual hexane in the solid plant material beîng removed by évaporation by drying the solid plant matériel at a température not exceeding 40°C. In this embodiment of the process the dried plant material, from which the hexane has been removed, will then be subjected to a further extraction, to obtain the phamnaceutically active constîtuents of the plant material, the further extraction also preferably being to exhaustîon, using the selected polar solvent, such as dichloromethane. The dichloromethane, after the further extraction, may then be removed at a température not exceeding 40°C from the plant material to provide a dichloromethane extract of the plant material and to leave a dried solid plant resîdue, which is discarded. The dichloromethane extract may then be subjected to removal of tannin therefrom using Polyamide S, i.e. a polymer of an organic amide available from Riedel de Haen.

D.anomala is a small and low-growing herb with small, stiff and bluish-grey leaves. It has a protea-like head of flowers which opens in mid-summer. The herb grows fiat along the ground on North-facing rocky hillsides. It prefers full sun and welldrained soit and has been propagated from seed only. D.anomala is distributed from the Eastem Cape province in South Africa, through the KwaZulu-Natal-, Free State-, Gauteng-, Mpumalanga- and Northem provinces in South Africa, and into Zimbabwe and Lesotho. It is also found in Botswana, Namibia and thence to Zambia and Uganda in the North.

For the purpose of the présent invention, the Applicant has employed D.anomala roots collected from the Morifi and Mohales' hoek areas in Lesotho, from the Reitz area • >

in the Free State, and from the Sikhukhuni area in the Northern province. Roots bought from herb markets in Durban, in the KwaZulu-Natal province, hâve also been investigated. The D.anomala used has been identified by vocicher specimen No. 98142 deposîted ai the Bolus Herbarium at the Universîty of Cape Town, in the Western Cape Province of South Africa.

The dichloromethane extract obtained as described above from D.anomala was found, after the tannin remova), to hâve substantial antiplasmodial activity which outweighs ils cytotoxicity to an extent which indicates that it comprises a substance or composition suitable for the thérapeute or prophyiactic treatment of malaria in the human or animal body.

Thus, the dichloromethane extract of D.anomala was found to hâve plasmocidal effects both on the chloroquine-sensitive D10 strain and on the chloroquine-resistant FAC8 strain of P.faldparum, with IC_5[) values of 2000ng/m£ and 6000ng/mt for the D10 and FAC8 strains respectively. The applicant found the antiplasmodial activity of the dichloromethane extract to exceed the cytotoxic activity thereof with regard to a number of human cancer cell fines, by a factor of 10 (Le, by an ordsr of magnitude). In this regard the dichloromethane extract (and the sesquiterpenes displaying antiplasmodial activity discussed hereunder) were found to hâve an antiplasmodiai selectivity index of about 10 on a number of cancer cell Unes. The antiplasmodial selectivity index (SI antiplasmodial) can be expressed by:

SI antiplasmodial = IC₅₃antiplasmodial ! IC_Sûcytotoxicity.

After fractionating of the dichîoromethane extract into a number of fractions and after testîng the antiplasmodial activity of these fractions, it was found that antiplasmodia) activity is confined to thoss fractions which contain members of a sériés of asymmetrical sesquiterpene dimers whose molecular masses differed from one another by 18 atomic mass units (characteristic of severa! losses of water - H₂O), from a minimum molecular mass of 230 up to a maximum molecular mass of 539.

In particular, antiplasmodial activity was found to be présent in the fraction of the

D.anomala dichioromethane extract which contains two asymmetrical sesquiterpene dimers having molecular masses respectively of 506.23879 atomic mass units and 508.23980 atomic mass units, these masses differing from each other substantially by 2 atomic mass units. The molecular ratio of the heavier compound to the lighter compound (î.e. of the 508.23980 atomic mass unit compound to the 506.23879 atomic mass unit compound) was found to be 1:3, as determined by their Ή spectra, liquid chromatography - mass spectroscopy (LC-MS) and by atmospheric pressure chemical ionisation high resolution mass spectroscopy (APCI HRMS). The minor (heavier) of the two compounds was shown by APCI HRMS to hâve a highest molecular mass of M⁺+1 509.2545 atomic mass units, consistent with the empirical molecular formula of while the major (lighter) compound had a highest molecular mass of M*+1 = 507.2388 atomic mass units, determined by APCI ARMS, consistent with an empirical molecular formula of C_:oH_3i0₇.

The compounds of farmulae C_3DH₃₆O₇ or C,₀H_MO₇ were found to hâve antiplasmodial activity against the chloroquine-sensitive (D10) strain of P.falciparum with an IC_M value of 200ng/mt. Thîs amounted substantialiy to a 10-fold increase in activity over that of the crude dichioromethane extract.

The dichloromethane extract of D.anomala was a!so tested in vitro against Gram négative bacteria and Gram positive bacteria and was found to be bactericidally active at minimum effect concentrations (MEC) of 250pg/mt, which is an indication of bacteriocidal activity against bacteria! infections and utility in combatting bacteria! infections.

When the dichloromethane extract of D.anomala was tested for toxicity in test animais, no toxicity was found when the extract was administered at levels of up to 100mg/kg of body mass, and no morphological changes were found to the spleen, liver or kidneys of test animais. Toxicology was determined by deaih, hair-straightening as a sign of loss of weü-being, and loss of body weight as a sïgn of loss of weil-being. In respect of these parameters the test animais were found not to deviate from control animais.

The active sesquiterpene compouna's were concentrated empîoying solid phase extraction (SPE), using 10g 018 end capped (EC) 70mt Îsolute cartridges. Cartridges 20 were conditioned thrice with methanol and then thrice with water, followed by sample loading at 4mg/mt) (i.e. 200 mg/50 mi), followed in tum by percoiation to obtain Fraction 1, by washing with 100% water to obtain Fraction 2, and by sequential cartridge-elution with 50mt mixtures of MeCN and water in the proportions given in the following table, Table 2, to obtain Fractions 3-12:

l

TABLE 2

Eluant 12

Fraction 34 %MeCN în 10%20% mixture (by volume)

4 56

6 78

30% 40% 50% 60%

8 910

10 1112

70% 80% 90% 100%

Analytical high pressure liquid chromatography (HPLC) séparations and préparative scale purification were carried out on the active sesquiterpene compounds using as the mobile phase mixtures of MeCN (0.05% by volume tetrafluoroacetic acid (TFA)) and water (0.05% by volume TFA), and using a gradient of 30 - 100 % MeCN in 40 minutes. The flow rates used were 1 mf/minute for analysis and SOmi/mlnute for préparative scale purification. The column used was a Phenomenex Prodigy - using ODS 2-type siiica, C-18 and 5pm (particle size) (4.6 x 150mm); and the ultra-violet (U\7) range for data collection was 200 - 600nm. The pure compounds had a maximum absorbance (λ max) at a wavelength of 225nm. Sample strength was 10mg/t and injection volume was 50μί for analytical séparation and 500m£ for préparative séparation.

With regard to structural élucidation of the active sesquiterpene compounds, mass spectra were obtained at the same time as the HLPC run, using a Thermo Quest LCQ (Finnegan) high resolution mass spectrometer connected inline with the high pressure liquid chromatograph. The liquid chromatograph/ mass spectrometer set-up was equipped with both electraspray ionization (ESi) and atmospheric pressure chemical ionization (APCI) probes and an ion trap.

Nuclear magnetic résonance (NMR) spectra (1H, 13C, corrélation spectroscopy (COSY), distortionless enhancement by polarîzation transfer (DEPT) 135), heteronuclear multiple quantum cohérence (HMQC), and heteronuclear multiple bond quantum corrélation (HMBC) were recorded on a Bruker-DR X-400 spectrometer (11-1:400,13 MHz, 13C:100.62 MHz) and a Bruker-DRX-500 spectrometer with inverse détection and z-gradient equipment (1H:500,13 MHz, 13C:125.77MHz) ai 23°C in CDCI3, The chemical shifts (δ) were related to the signal of the residual solvent.

The asymmetrical sesquiterpene antiplasmodially active dimer compounds of the D.anomala extract of the présent conversion hâve been found to be no more than sparingly soluble and the bioavailability of the compounds will dépend on such routes of the administration as are found to be possible (orally, rectaily and întravenously). In principle formulation of the active compounds into one or more of capsules, tablets, syrups, injectable liquids and herbal tinctures (optionally stabilized) is possible, the formulation or formulations selected depending on bioavailability and pharmacokinetics of the compounds.

Furthermore, the invention extends also to the use of a substance or composition in the manufacture of a médicament or préparation for the therapeutic or prophylactic treatment of parasitic infections, in particular malarial infections such as P.falciparum infections, of the human or animal body, the substance or composition comprisîng an organic solvent root extract of a plant of the species D. anomala.

Further, the invention extends also to the use of a substance or composition in the manufacture of a médicament or préparation for the therapeutic or prophylactic treatment of parasiîic infections, in particular malaria! infections su ch as P.falciparum infections, of the human or animai body, the substance or composition comprising at least one asymmetrical sesquiterpene dimer having a molecular mass in the moiecuiar range 230 - 539 atomîc mass units.

The substance or composition may comprise a plurality of asymmetrical sesquiterpene dîmers having molecular masses in the moiecuiar range 230 - 539 atomic mass units.

The substance or composition may comprise at least one asymmetrical sesquiterpene dimer, having a structural formula as shown in any one of Figures 2 and 6 - 9 as described hereunder; or ii may comprise ai least one dérivative of an asymmetrical sesquiterpene dimer having a structural formula as sbown in any one of Figures 2 and 6-9, the dérivative being selected from the group consistîng of:

dérivatives in which one or more of any α-methylene groups forming part of ylactones and any exocyclic methylene groups in the dimer are reduced to methyl groups;

dérivatives in which one or more of any y-lactones are opened by hydrolysis to produce hydroxy-carboxylic acids;

dérivatives in which one or more of any carboxyl groups in the dimer are reduced to methylene groups;

dérivatives in which one or more of any primary alcohol groups in the dimer are oxidized to aldéhyde groups; and dérivatives in which one or more of any secondary alcohoi groups in the dimer are converted to ketone groups and acetate groups.

The invention extends aiso to the plant extracts (and to the antipiasmodially active sesquîterpene compounds contaîned therein) as substances or compositions for the treatment, by therapy or prophyiaxîs, of bacterial and in particular plasmodia! (malaria!) infections of the animal and particularly the human body.

Thus, more particularly, the invention extends further to a substance or composition for use in a method of treatment or prophylaxis of parasitic infections of the human or animal body, in particular malarial infections such as P. falctparum infections, by admînistering an effective amount of said substance or composition to the human or animal body, said substance or composition forming part of an organic solvent root extract extracted from roots of plants of the species D. anomala.

The substance or composition may comprise at least one asymmetrical sesquîterpene dimer having a molecular mass in the molecular mass range 230 - 539 atomic mass units. In particular, the substance or composition may comprise at least one asymmetrical sesquîterpene dimer having an empirical formula selected from the group consisting of C30H36O7 (with a molecular mass in the range 508.239 - 508.240 atomic mass units) and C30H34O7 (with a molecular mass in the range 506.238 506.239 atomic mass units). The substance or composition may comprise a plurality of asymmetrical sesquîterpene dimers having molecular masses in the molecular range 230 - 539 atomic mass units.

The invention extends further also to a substance or composition for use in a method of treatment or prophylaxis of parasitic infections, in particular malarial infections such as P. falciparum infections, of the human or animal body by admînistering an «

V

I effective amount of said substance or composition to the human or animal body, the substance or composition comprising at ieast one asymmetrical sesquiterpene dimer having a molecular mass in the molecular range 230 - 539 atomic mass units.

The substance or composition may comprise a plurality of asymmetrical sesquiterpene dimers having molecular masses in the molecular range 230 - 539 atomic mass units.

In particular, as indicated above, the substance or composition may comprise at least one asymmetrical sesquiterpene dimer having an empirical formula selected from the group consisting of C30H36O7 (with a molecuiar mass in the range 508.239 - 508.240 atomic mass units) and C30H34O7 (with a molecular mass in the range 508.238 - 506.239 atomic mass units).

The asymmetrical sesquiterpene dimers may be présent in the aforementioned substance or composition at a concentration of at least 0.08% by mass, e.g. at least 0.2% by mass. Preferably, the concentration of the asymmetrical sesquiterpene dimers is at least 50% by mass, more preferably at least 70% and most preferably at ieast 99% by mass.

In particular, the substance or composition may be in a form selected from the group consisting of capsules, tablets, syrups, injectable préparations, herbai tinctures and supposrtories. Thus the substance or composition may be for use in a method of treatment of prophylaxis in which the administering of the substance or composition is in unit dosage form. Preferably, the administering of the substance or composition may be such as to attain an effective sérum concentration, by mass of the dimers, in the human or animal body. More particulariy, the administering cf the substance or composition may be at an effective daiiy dosage rate, the rate being dépendent on the body mass of the subject.

Preferably the extract is one resuiting from a cold or hot extraction perfomned on the D.anomala roots using an organic solvent, in particular dichloromethane, having a polarity index of 0-5, for exampis the solvents set forth in Table 1. When the substance or composition comprises the sesquiterpene compounds from the sériés whose molecular masses differ by 18 atomic mass units from one another from the sériés, in particular the two asymmetrical sesquiterpene dimers having molecular masses of 506.23879 atomic mass units and 508.23980 atomic mass units respectiveiy, in more or less refined form, so that they are présent at concentrations greater than those présent in the extracts, in particular the dichloromethane extracts, of D.anomala, the active compounds may form at ieast 0.2 % by mass of the substance or composition, preferably at Ieast 50%, more preferably at Ieast 70% and most preferably at Ieast 99%.

The substance or composition may comprise at Ieast one asymmetrical sesquiterpene dimer having a structurai formula as foilows:

o o

o λ

Instead or în addition, the substance or composition may comprise at least one asymmetrical sesquiterpene dimer having a structural formula as follows:

Instead or in addition, the substance or composition may comprise at least one asymmetrical sesquiterpene dimer having a structural formula as follows:

The invention extends to an asymmetrical sesquiterpene dimer which has a structural formula as shown in any one of Figures 2 and 6-9.

The invention extends further to a dérivative which can be obtained from an organic solvent root extract of a plant of the species D. anomola, the dérivative being of an asymmetrical sesquiterpene dimer which can be obtained from an organic solvent root extract of a plant of the species D. anomola.

The dérivative may be a dérivative of a said dimer which has a molecular mass in the molecular mass range 230 - 539 atomic mass units.

The dérivative may be selected from the group consisting of:

dérivatives in which one or more of any α-methylene group forming part of γlactones and any exocyclic methylene groups in the dimer are reduced to methy! groups;

dérivatives in which any γ-lactones in the dimer are opened by hydrolysis to produce hydroxyl-carboxylîc acids;

dérivatives in which one or more of any primary alcohol groups in the dimer are oxidized to aldéhyde groups; and dérivatives in which one or more of any secondary alcohol groups are converted to ketone groups and acetate groups.

The dérivative may be a dérivative of a said dimer which has a structural formula as shown in any one of Figures 2 and 6-9,

The invention furthermore extends to a method of treatment or prophylaxis of parasitic infections, in particular malarial infections such as P.falciparum infections, of the human or animal body, the method comprising administering to a human or animal subject an effective amount of a substance or composition comprising an organic solvent root extract extracted from roots of plants of the spectes D. anomala.

The invention furthermore extends also to a method of treatment or prophylaxis of parasitic infections, in particular malarial infections such as P.falciparum infections, of the human or animal body, the method comprising administering to a human or animal subject an effective amount of a substance or composition comprising at least one asymmetrical sesquîterpene dimer having a molecular mass in the molecular range 230 — 539 atomic mass units.

The substance or composition may comprise a pluralîty of asymmetrical sesquîterpene dimers having molecular masses in the molecular range 230 - 539 atomic mass units.

The administering of the substance or composition may be in unit dosage form.

In particular, the administering of said substance or composition may be such as to attain an effective sérum concentration, by mass of the dimers, in the human or animal body. More particularly, the substance or composition may be adminîstered at an effective daily dosage rate, the rate being dépendent on the body mass of the subject.

The structure of the asymmetrical sesquîterpene dimers forming the active ingrédients of the présent invention will be discussed with référencé to the following schematic drawings, in which:

Figure 1 shows a piot of milli-absorbence units (mAU) against wavelength in nanometers (nm);

Figure 2 shows a general schematic structural représentation of the dimers of the présent invention;

Figures 3 and 4 respectively show schematic structura! représentations of monomers into which the dimers of Figure 2 can be split;

Figure 5 shows a schematic structural représentation of an a-methylene-y-lactone forming a substituent in the dimers and monomers of Figures 2-4; and

Figures 6-9 respectively show structural formulae of certain asymmetrical sesquiterpene dimers of the présent invention.

Figure 1 shows the UV spectrum of two of the active asymmetrical sesquiterpene dimers of the présent invention, i.e. the asymmetrical sesquiterpene dimers, in solution in dichloromethane and at a concentration in total of 0.2%, in the ratios in which the dimers in question are présent in the extracts from D.anomala, and in D.anomala itself, the dimers in question being those discussed above and having molecular masses best represented as 506.23879 atomic mass units and 508.23980 atomic mass units respect! vely.

Figure 2 shows the best représentation of a structural formula which the applicant is presently able to provide for both the above dimers.

Figures 3 and 4 show respectively monomers into which the dimers of Figure 2 can be split, and it will be noted that the dîmer and the monomers each contain the amethylene-y-lactone of Figure 5.

Figures 6-9 respectively show structural formulae of four additional asymmetrical sesquiterpene dimers, different from the two illustrated by Figure 2, also forming active ingrédients of the présent invention.

Without being bound by theory, the applicant believes that it may be possible to obtain a number of semi-synthetic dérivatives of the asymmetrical sesquiterpene dimers obtained from D.anomala. Th us, the σ-methylene groups of the y-lactones, as well as »

* any other exocyctic methylene groups în the dimers can be reduced to methyi groups as fbilows:

The y-lactones can be opened by hydrolysis to produce hydroxyroarboxylic acids as follows:

Carbonyl groups can be reduced to methylene groups as follows:

Primary alcohols can be oxidised to aldéhydes, which can be further derivatised as follows:

Finally, secondary alcohols can be converted to ketones and acétates as follows:

; and

—CH — C-C-CH II Ü

It is believed that at least some of the dérivatives of the asymmetrical sesquiterpene dimers obtainable as set forth above can hâve anti-malarial activity, possibly enhanced anti-malarial activity compared with that of the dimers derived from D.anomala.

The invention will now be described, by way of non-limiting illustration, with référencé to the following worked Examples.

EXAMPLE 1

Piant material from D.anomala was authentîcated and a voucher specimen [No. 98142] was deposited with regand to the plant material at the Bolus Herbarium of the University of Cape Town. The piant material was washed free of the fungal growth, bacterial growth and/or soil, and was dried at room température away from direct sunlight. The plant material was then ground, using a plant grinder, to a particle size of at most 10pm. The ground plant material was stored in dry cardboard boxes in a wellventilated store priorto extraction.

To carry out cold extractions on the plant material, 500g batches of ground dried plant material were weighed and extracted using 5f cold (room température) dichloromethane. The extraction was carried out over a period of 24 hours, accompanied by agîtation/shaking at 130 rpm, after with the extraction mixture was filtered, and a further 5t cold dichloromethane was added for a further extraction cycle. The extraction cycles were repeated until the material was extracted to exhaustion, after which the fïltrate obtaîned from each cycle was pooled, and the dichloromethane was removed by rotary évaporation at most 40°C, to leave the plant extract. The extract was then dried under a fume hood away from direct sunlight, after which yields were determined, and the dried extract was soreened for antimalaria! activity.

Extra cts which showed no antimalarial activity were discanded, and those with antimalarial activity had tannins and polyphenolic compounds removed therefrom by dissolving 200mg of the dried dichloromethane extract in 100 mi methanol to which 5g of Polyamide S (an organic amide polymer) powder was added, followed by thorough mixing and filtration under vacuum using a Buchner funnel. The résidus was washed with a further 100m£ of methanol, followed by a further said filtration, after which the methanol filtrâtes containing the tannin-free dissolved extract were pooled. The methanol was evaporated under vacuum and the tannin-free extract was dried under a fume hood.

The residue remaining after the methanol extraction was washed twice with 20 mf batches of Ν,Ν-toimethylformamide (DMF) to extract the tannin materîal from the residue. The DMF was then removed under vacuum and the remaining extract was dried under a fume hood.

The dichloromethane extract, the methanol extract and the DMF extract were tested for antimalarial activity, and, except for the dichloromethane extract, were discarded.

The dichloromethane extract was purified by a solid phase extraction (SPE), C-18 end capped SPE reverse phase cartridges beîng used, having a capacity of 10g and 70mL An amount of 200mg of the dried dichlonomethane extract, from which tannins and polyphenolic compounds had been removed was prepared for the SPE extraction by complété dissolution thereof in 15mE of MeCN, the solution being centrifuged for 15 minutes at 13000 rpm, after which the supematant liquid was decanted. Then 35m£ of distilled de-ionized water was added to the supematant liquid, followed by centrifuging for 15 minutes at 13000 rpm, followed by decanting and filtration of the supematant liquid for use in the SPE.

The SPE cartridges were conditioned by washing thrice with 50m£ of MeCN, followed by washing thrice with 50 mi of distilled de-ionized water. Sample loading at 4mg/m( was carried out by loading the 50mt solution obtained from the 200mg extract on each column. To each column a vacuum was gently applied after a one minute interval, to obtain a percolate which was collected as a tiret fraction. The percoiate was concentrated by freeze drying. The cartridges were each then washed five times with 50mf aliquots (250mE in total) of de-ionized water, the water then being removed by freeze drying, to leave a second or washed fraction.

Cartridge elution was carried out by eluting each cartridge five times with 50mt aliquots (250mt in total) of solutions of MeCN mixed with water, in which the concentrations of MeCN and water were varied stepwise as shown hereinabove in Table 2, in which Fractions 3 - 12 are shown, Fractions 1 and 2 being the first and second fractions referred te above derived respectively from sample loading and cartridge washing.

For testing the plant extracts, malaria parasites of the species P. falciparum were used, which had been maintained in culture flasks at 37°C in an atmosphère comprising, by volume, 3% oxygen, 4% carbon dioxide and 93% nitrogen, prior to testing of the plant extracts. The parasites were cultured in a culture medium which was changed daily, and was diluted with fresh red blood cells every 2 - 3 days so that at least 5% of the red blood cells were infected at any time. Parasitemia was observed by means of Giemsa R66 stained thin smears. The smears were fixed with methanol and exposed to a 10% by mass solution of Giemsa-R66 in phosphate-buffered saline solution forten minutes at room température. The thin smears were observed on slides which were rinsed under a stream of tap water and wiped dry with lint-free tissue priorto smearing. The slides were viewed under a light microscope with a 100X objective lens under oil having aviscosity of 1.250 centiStokes, and the parasitised red cells were counted.

The plant extracts were prepared for use by reconstitution thereof, the dichloromethane extracts being initially dissolved in 100 μ t of methanol, and then made up to 2ng/mt with distilled de-ionized water. The stock solutions formed in this way were steriüsed using 0.22pm stérile filter units followed by storage in a freezer at -1B°C until needed for screening.

Microtiter or microdilution plates were used, each consisting of 96 flat-bottomed weils arranged in eight rows (A - H) and twelve columns (1-12). A volume of 1 00 μί of the culture medium was pipetted into each well, except for the weils of column 3, into which 200μί of the reconstituted plant extract was pipetted. Two-fold serial dilutions were made of each extract from within the plate, using a multi-channel Eppendorff dispenser. Column 1 was reserved for a red blood cell blank and column 2 was reserved for a parasite control. A volume of 100μ£ of a 2% hematocrit (2% by mass of red blood cells) was pipetted into the wells of column 1, and 100 μΕ of 2% hematocrit and

100μΐ of2% hematocrit with 2% parasitemia (Le. 2% of the cells were parasitised) were added to the rest of the wells of the plate as a control. The effects of the methanol or other solvent on the parasites were tested, by placing the plates in an airtight desiccator flushed with a gas mixture, by volume, of 3% oxygen, 4% carbon dioxide and 90% nitrogen, in which the plates were incubated at 37°C for 43 hours.

One of the mîcrotiter/microdilution plates is illustrated in the following table, Table

3. Starting with column 3 in the plate and progressing to column 12, the amount of culture medium was successively halvecf (Le. 100% in column 3, 50% in column 4, 25% in column 5 and so forth down to 0.1953% in column 12). The highest concentration of extract, in column 3, was 100 OOOng/mt, and the lowest concentration, in column 12, was 195ng/mL The chloroquine was tested at concentrations of 200ng/mf - 0.39ng/m£. As indicated above, column 1 was employed for a red blood ce!! blank, and column 2 was employed for a parasite control, a first sample being tested in rows A and B, a second sample being tested in rows C and D, a third sample being tested in rows E and F and a fourth sample being tested in rows G and H.

B	C	10 0	\| 50	25	12.5	6.25	3.125	1.5625	0.78125	0.3906	0.1953
B	C
B	c	10	50	25	12.5	6.25	3.125	1.5625	0.73125	0.3906	0.1953
		0
B	c
B	c	10	50	25	12.5	6.25	3.125	1.5625	0.78125	0.3906	0.1953
		0
B	g
B	c	10	50	25	12.5	6.25	3.125	1.5625	0.78125	0.3906	0.1953
		0
B	c

For antiplasmodial screening a parasite lactate dehydrogenase assay was employed. The test plates were removed from the incubator, and their contents were resuspended. The wefls of another ciean 96 well (8 rows x 12 coiumns) microtitar piste had 10ΟμΕ of Maistat™ reagent added thereto, for reactinc with parasite lactate dehydrogenase. From each of the wells of each plate from the incubator 15μί of liquid 10 was transferred to the corresponding well in the microtiter plate contaîning the Maistat™ reagent, and to each of these welis containing the Malstat'^u reagent was added 25μ£ of a solution of 0.24mM (milliMolar) phenyl ethyl sulphate (PES) and 1,96mM nitro blue tetrazoleum (NBT). The formation of a purple colour was an indication of parasite survival. Réaction of the Malsiat’* was allowed to take place in the dark for 10 minutes, and absorbence at 620nm was read in a Model 7520 Microplate Reader manufactured by Cambridge Technology, Inc. Each plate had its well in column 1 containing only red blood cells bianked. The parasite percentage survival was calculated from the absorbence readings using the following formula:

average reading of test welis - average reading of blarik welis % parasite survival = 100 x_______________________________________________________ average reading of contro) welis - average reading of blank welis

Dose response curves were prepared by plotting the absorbence readings against the concentrations of the extracts, and from these response curves, the IC» was in each case determined, by extrapolation, as the concentration that would inhibit 50% of the parasite population. If desired, standard déviations could be calculated from the absorbence readings.

The extracts showing activity against the malaria parasites were subjected to cytotoxicity studies. Fully attached, confluent cells with a viability greater than 95% were used to assay cytotoxicity.

Microdilution plates were prepared by diluting stock cultures of the extracts with the liquid medium thereof, to a density of 50 000 cells/mi, for use in the MTT cytotoxicity assay described hereunder. A volume of 200u£ of the diluted stock culture containing 10

000 cells was added to each well of a 96 well microdilution plate having 8 rows and 12 columns, except for columns 1 and 2 which served as a blank and a parasite contrai respectiveiy. Each plate was incubated at 37°C in an incubator which was continuously flushed with a carbon dioxide atmosphère. The plates were incubated until the cells 5 became confluent.

The extracts were prepared as described above for antiplasmodial screening. In each case the effect oF the solvent used to dissolve the plant extract was tested on the cells.

To détermine cell viability the cells were trypsinîzed with a 5% by mass trypsin ethylene diamine tetracetic acid solution in phosphate-buffered saline (PBS) to detach them from the fiask in which they were contained. The cells were then spun at 1500 rpm for five minutes in a centrifuge, after which supernatant liquid was aspirated therefrom, 15 and the celis were washed twice with the liquid used as their culture medium. The cells were then re-suspended in culture medium to obtain a cell suspension, and an aliquot of the cell suspension was mixed with an equal volume of trypan blue. After thoraugh mixing a drop of the mixture was piaced in a haematocytometer and covered with a cover slip. The cells were allowed to settle for two minutes, and a count of non-viable 20 cells, nameîy those which staîned blue, were made as a percentage, i.e. the number of cells that stained blue in each hundred ceils.

Cytotoxicity was then assayed. When the cells reached confluence, the medium was aspirated therefrom, and 5mi of said 5% trypsin ethylene dismine tetracetic acid 25 was used to detach them. The cell suspension was centrifuged at 2050 rpm for two

J t

minutes, after which the trypsin ethylene diamine tetracetic acid was aspirated and the cells were washed with culture medium. The number of viable cells was counted on a haematocytometer after staining the sl/de as described above, and the celis were seeded at 10 000 cells/well in the wells of a 96 weil flat-bottomed microtiter plate. The cetls were allowed to settle and attach for 24 hours, after which the culture medium was aspirated and fresh medium and test extracts were added. The cells were exposed serially to eleven half dilutions ofthe extracts for 48 hours.

After the 48 hour incubation period 50pf of a 1mg/mf solution of [3-(4,5dimethy!ihlazol)-2-y!]-2,5-dithemyl-tetrazoiiumbramide (MTT) prepared in PBS in ali of the wells. The plates were re-incubated for a further 4 hours at 37°C in an incubator, after which the plates were centrifuged at 2050 rpm for 10 minutes. The culture medium was aspirated in a fume hood. The assay is based on the réduction of the MTT to a purpie insoluble formazan product by the reducing enzymes présent only in metabolicaliy active live organisme. Formazan crystals were obtained, and 100pt of dimethylsulphoxide was added thereto in each well, after which the absorbence at 620 nm was read on the abovementioned Micraplate Reader, from which ce!) survival was calculated.

A selectivity index was employed to détermine whether or not a given plant extract was to be regarded as cytotoxic or antiplasmodîal. To calculate the selectivity index the same units were used for cytotoxicity and antiplasmodial concentrations, according to the following formula:

Antiplasmodial IC_M

Sélectivity Index (antiplasmodial) = _________________________

Cytotoxicity ÎC_a

EXAMPLE 2

Example 1 was repeated with regard to the dichloromethane extract, except that the SPE cartrîdges were conditioned by washing once with 50 mE of methanol followed by washing once with 50 mE of distilled deionised water (înstead of washing thrice with MeCN and then thrice with water as in Example 1). As in Example 1,4mg/mE of crude extract was loaded on each column. Sound compounds were eluted at a rate of 17mt/mînute and as detaüed above in Table 2, Le. with mobile phases comprising MeCN and water in the proportions shown in Table 2.

Eluant Fraction 5, 6 and 7, comprising respectively 30% MeCN, 40% MeCN and 50% MeCN, ali by volume, contained respectively 0.58 ±0.065pg/mf of extract, 0.55 ±0.12pg/mi of extract and 0.54 +0.11pg/mE of extract. Each of Eluant Fractions 5, 6 and 7 showed more antimaiariaî activity-than the crude dichloromethane extract.

Pure compounds were obtained by collectîng individual peaks from the 40% MeCN fraction (Eiuant Fraction 6), which had a number of prominent peaks. A C18 HLPC Haisil 100 se mî-p réparai ivs cartridge was used, having dimensions of (250 x 10mm) and a particle size of 5pm. A détection waveiength of 215nm was used, with a flow rate of 2.5 mf/minute, and an injection volume of 216μί, the mobile phase being an MeCN/water mixture comprising 60% MeCN by volume. This procedure resulted in the isolation of the compounds whose structural formulae are shown respectively in Figures and 7. The cornpound of Figure 6 was found to display an ICso of 0.19pg/m£; and the cornpound of Figure 7 displayed an 1C_Sq of 0.28pg/mt.

EXAMPLE 3

Example 1 was repeated with regard to the dichloromethane extract and, in addition to the compounds of Figure 2, the compounds of Figures 8 and 9 were isolated.

The IC50 values of O.19pg/mi and Û.28pg/mf respectîveiy of the compounds of

Figures 6 and 7 were an approximately ten-fold improvement over the antimalarial activity of the crude extract, being a two-fold to five-fold improvement over the respective fractions from which they were isolated. The compounds of Figures 6 and 7 displayed an antiplasmodial selectivity index of 1/2, demonstrating twice as much cytotoxicity as antimalarial activity, but the compounds of Figures 6 and 7 were marginally more antimalarially active than the compounds of Figures 2, 8 and 9.

It is expected that, for the purpose of treating persons suffering from malaria, the plant extract, or its active ingrédients, will be capable of administration by way of one or more of capsules, tablets, syrups, injectable Iiquids, herbal tinctures (which may be 20 standardised), suppositories, or the like.

Claims

1. A process for the production of a substance or composition for the treatment, by therapy or prophylaxie, of parasitic infections, in particular malariai infections such as Plasmodium falciparum infections, of the human or animal body, the process being characterized in that it comprises the steps of:

extracting the substance or composition from a starting material comprising plant roots of the plant species Dicoma anomala, by subjecting the plant root starting material to extraction using an organic solvent to obtain a liquid extract containing the substance or composition; and removing the solvent from the liquid extract to leave a dried extract containing the substance or composition.

2. The process as claîmed in Claim 1, characterized in that the extraction is a cold extraction carried out at a température of at most 40 °C.

3. The process as claîmed in Claim 1 or Claim 2, characterized in that the plant root starting material is a solid plant material in a finely dîvided form, having a particle size not exceeding 10pm.

4. The process as claîmed in any one of the preceding claims, characterized in that the solvent is a polar solvent having a polarîty index in the range 0.00-5.00.

λ

5. The process as claimed in Claim 4, characterized in that the soi vent has a poiarity index of 1.0 - 4.8 and comprises at Ieast one member selected from the group consisting of

benzene, n-butanol. butyî acetate, carbon tetrachloride, chloroform, 1,2 dichioroethane, dichloromethane, dîcxane, ethyl acetate, di-ethy! ether, n-propanol, methyM-outyl ether, methyl ethyl ketone, iso-propanol, di-iso-propanol, tetrahydrofuran, toluene, trichloroethylene,

xylene.

6. The process as claimed in Ciaim 5, characterized in that the solvent is dichloromethane (CHoC^).

7. The process as claimed in any one of the precedîng daims, characterized in that it includes subjecting the plant root starting material to a preliminary extraction, using a non-polar organic solvent having a poiarity index of less than 3.90, foilowed by drying to evaporate residual non-poiar solvent from the solid plant material, and then subjecting the dried plant root material, from which the non-polar solvent has been removed, to the extraction, to obtain the lîquid extract containing the substance or composition.

8. The process as claimed in Claim 7, characterized in that the preliminary extraction is carried out with the non-poiar solvent at a température of 15 - 30°C, the drying beîng carried out at a température not exceeding 40°C, and both extractions beîng carried out to exhaustîon.

9. The process as claîmed in any one of the preceding daims, characterized in that it includes subjecting the dried extract containing the substance or composition to removal of tannin therefrom using a polymer of an organic amide.

10. Use of a substance or composition in the manufacture of a médicament or préparation for the therapeutic or prophylactic treatment of parasitic infections, in particular malariaf infections such as P.falciparum infections, of the human or animal body, characterized in that the substance of composition comprises an organic solvent root extract of a plant of the species D. anomale.

11. Use of a substance or composition in the manufacture of a médicament or préparation for the therapeutic or prophylactic treatment of parasitic infections, in particular malarial infections such as P.falciparum infections, of the human or animai body, characterized in that the substance of composition comprises at least one asymmetrical sesquiterpene dîmer having a molecular mass in the molecular mass range 230 - 539 atomic mass units.

12. The use as claîmed in Claim 10 or Claîm 11, characterized in that the substance or composition comprises at least one asymmetrical sesquiterpene dimer, having a structural formula as shown in any one of Figures 2 and 6-9.

13. The use as claimed in Claim 12, characterized in that the substance or composition comprises at least one dérivative of an asymmetrical sesquiterpene dimer having a structural formula as shown in any one of Figures 2 and 6-9, the dérivative being selected from the group consisting of:

dérivatives in which one or more of any α-methylene groups forming part of γ-lactones and any exocyclic methylene groups in the dimer are reduced to methyl groups;

dérivatives in which one or more of any γ-lactones are opened by hydrolysîs to produce hydroxy-carboxylic acids;

dérivatives in which one or more of any primary alcohol groups in the dimer are oxidized to aldéhyde groups; and dérivatives in which one or more of any secondary alcohol groups in the dimer are converted to ketone groups and acetate groups.

14. A substance or composition for use in a method of treatment or prophylaxis of parasitic infections of the human or animal body, in particular malarial infections such as P. falciparum infections, by adminîsterîng an effective amount of said substance or composition to the human or animal body, said substance or composition being characterized in that it forms part of an organic solvent root extract extra cted from roots of plants of the species D. anomala.

15. The substance or composition as claîmed in Claim 14, characterized in triât it comprises at least one asymmetrical sesquiterpene dimer having a molecular mass in the molecular range 230 - 539 atomic mass units.

5

16. The substance or composition as claîmed in Claim 14 or Claim 15, characterized in that it comprises at least one asymmetrical sesquiterpene dimer having an empirical formula selected from the group consisting of (with a molecular mass in the range 508.239 - 508.240 atomic mass units) and C30H34O7 (with a molecular mass in the range 506.238 - 506.239 atomic mass units).

17. A substance or composition for use in a method of treatment or prophylaxis of parasitic infections, in particular malaria! infections such as P.falciparum infections, of the human or animal body by administering an effective amount of said substance or composition to the human or animal body, the substance or composition

15 being characterized în that it comprises at least one asymmetrical sesquiterpene dimer having a molecular mass in the molecular range 230 - 539 atomic mass units.

18. The substance or composition as claîmed in Claim 17, characterized in that the substance or composition comprises at least one asymmetrical sesquiterpene

20 dimer having an empirical formula selected from the group consisting of C30H36O7 (with a molecular mass in the range 508.239 - 508.240 atomic mass units) and C30H34O7 (with a molecular mass in the range 506.238 - 506.239 atomic mass units).

19. The substance or composition as claimed in any one of Claims 14-13 inclusive, characterized in that, in the substance or composition, the asymmetricai sesquîterpene dimers are présent at a concentration of at least 0.2% by mass.

20. The substance or composition as claimed in Ciaim 19, characterized in that, in the substance or composition, said concentration is at least 99% by mass.

21. The substance or composition as claimed in any one of Claims 14 - 20 inclusive, characterized in that the substance or composition is in a form selected from the group consisting of capsules, tablets, syrups, injectable préparations, herbal tinctures and suppositories.

22. The substance or composition as claimed in any one of Claims 14 - 21 inclusive, characterized In that the substance or composition is in unit dosage form.

23. The substance or composition as claimed in any one of Claims 14-22 inclusive, characterized In that it comprises at ieast one asymmetrical sesquîterpene dimer having a structura! formula as folJows:

ü o

D

I

24. The substance of composition as claimed in an/ one of Claims 14-23 inclusive, characterized in that it comprises at least one asymmetrical sesquîterpene dimer having a structural formula as follows:

25. The substance of composition as claimed in any one of Claims 14- 24 inclusive, characterized in that it comprises at îeast one asymmetrical sesquîterpene dimer having a structura! formula as follows:

r «

26. The substance of composition as claimed in any one of Ciaims 14- 25 inclusive, characterized in that it comprises at least one asymmetrical sesquiterpene dimer having a structural formula as follows:

27. The substance of composition as claimed in any one of Ciaims 14-26 inclusive, characterized in that it comprises at least one asymmetrical sesquiterpene dimer having a structural formula as follows:

28. An asymmetrical sesquîterpene dimer characterized in that it has a structural formula as shown in any one of Figures 2 and 6-9.

29. A dérivative of an asymmetrical sesquîterpene dimer characterized in that both the dérivative and dimer can be obtained from an organic solvent root extract of a plant of the species D. anomola.

30. A dérivative as claimed in Claim 29, characterized in that said dimer has a molecular mass in the molecular mass range 230-539 atomic mass units.

31. A dérivative as claimed in Claim 29 or claim 30, characterized în that the dérivative is selected from the group consîsting of:

dérivatives in which one or more of any a-methylene group forming part of γlactones and any exocyclic methylene groups in the dimer are reduced to methyl groups;

dérivatives in which any γ-lactones in the dimer are opened by hydrolysis to produce hydroxyl-carboxylic acids;

32. A dérivative as claimed in any one of Clairns 29 - 31, characterized in that the asymmetrical sesquiterpene dimer has a structural formula as shown in any one of Figures 2 and 6-9.