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(I1) Patent Number: KE 59

(45) Date of grant: 23/10/1996

(12) PATENT
 
(51) Int.CL.5: A 61M 1/36, B 03C 1/00

(21) Application Number: 1996/000174

(22) Filing Date: 27/02/1996
 
(73) Owner: DOLLY MUTHONI NJIRU-GAKUNJU of, P.O. BOX 67687, NAIROBI, KENYA, Kenya

(72) Inventor: DOLLY MUTHONI NJIRU-GAKUNJU
 
(54) Title: A PROCESS OF EXTRACTING AN ISOQUINOLINE ALKALOID OF THE BENZO (C) PHENANTHRIDINE TYPE OF PROVEN USE IN THE TREATMENT OF MALARIA FROM A PLANT OF THE GENUS TODDALIA FOUND IN THE RIFT VALLEY PROVINCE OF KENYA

(57) Abstract: A process of extracting an Isoquinoline alkaloid of the benzo (c) phenanthridine type of proven use in the treatment of malaria from a plant of the genus Toddalia, found in the Rift Valley Province of Kenya.
 
INTRODUCTION
Traditional medicine involves the total combination of knowledge and practice, whether explicable or not, used in diagnosis, prevention and elimination of physical, mental and social diseases. It relies on past experience and observations handed down from generation to generation verbally or in writing. A traditional medicine practitioner or healer is a person recognized by the community he/she lives in as being competent to provide health care using vegetable, animal and mineral substances. The traditional healer might use certain other methods based on social, cultural and religious background as well as knowledge, attitudes and beliefs that are relevant to the community regarding physical, mental and social well-being and causes of diseases and disability. These practitioners include herbalists, hone setters, traditional birth attendants, traditional psychiatrists, spiritual healers and other specialists [Muriuki et al., 1989].

The medical and pharmaceutical sectors have always exploited plants as sources of medicine. The increased use of traditional medicinal plants must necessarily involve the transference of the knowledge of their use and potential from the traditional to the modern sector. This necessitates a change in the context in which such medicines will be used; from a cultural to a hospital context. The concept of cultural use has been.

 The primary objective of research in traditional medicines is to expand modem medical practice and to cut down the bill for modern medicines. In short, to supplement and complement modern medicine. All ethnomedicinal lore should be studied before ethnic groups or the plants they use become genetically swamped, endangered, or extinct [Waane et al, 1990].

Current Usage
Modern medical facilities in the Third World are inadequate or totally lacking, particularly in remote areas. In most cases therefore, some 60%40% of the inhabitants of rural areas rely on traditional medical practices [Mshigeni, 1990].

Traditional medicine found a place in the World Health Organization programme in the early eighties [Olawayo et al. 1990]. The practice of traditional medicine, an indispensable cultural heritage, has been legalized and integrated into primary health care in most African developing countries. Primary health care has been adopted as the appropriate strategy for developing national health systems. This system has become imperative for technologically less advanced countries given their general state of economic crisis. However, primary care still demands the use of therapeutic preparations and in the face of declining foreign exchange earnings, governments are finding it increasingly difficult to make essential drugs available to their rapidly growing populations. The use of traditional medicine thus finds natural expression, and further development, in primary health care, where, in many cases, it bridges the gap between the availability of and the demand for essential drugs.

The World Health Organization has acknowledged that the target of providing total health coverage for everybody by the year 2000 can only be met by incorporating digoxin, scopolamine, digitoxin, pilocarpine, and quinidine. These are some of the different chemical compounds of known structures derived from plants. The assumption that most, if not all of the higher plant derived drugs of known structure are now produced commercially by synthesis is wrong. Of the individual drugs, the few commercially produced by synthesis are; emetine, caffeine, theobromine, theophylline, pseudoephedrine, ephedrine and papaverine. This is not to imply that most of the naturally occurring drugs have not been synthesized, indeed they have. However, practical industrial syntheses for such important drugs as morphine, codeine, atropine, digoxin etc are not available. The alkaloid reserving for example, can be commercially extracted from natural sources for about $0.75/g, where as a multistep and difficult synthesis is available that yields reserving at about $1.251g. It should be obvious which of the two sources is used to produce this pharmaceutical [Wagner et al., 1976].

Problems
Forty per cent of all pharmaceuticals presently in use are derived from natural sources (plants, fungi and other microorganisms, animals etc) either used directly as such, or with some modifications [Nkunya at al., 1990]. However, the use of crude plant extracts without any scientific evaluation could lead to serious complications. Ineffective drugs could be used as a matter of belief or tradition; under/over-doses could be taken; highly toxic drugs with short term, long term, or cumulative effects could be prescribed. The last two effects are difficult to recognize than others, and are hence potentially very serious.

In addition to these, the preparation, handling and storage of crude drugs can lead to
decomposition or transformation of the hitherto active constituents to ineffective and/or
 
Extended biological-Evaluation e.g. Antimalarial activity tests Molluscicidal activity test Filaricidal activity test. First-level investigations provide an indication of pharmacological effect about traditional materia medic. Second-level investigations comprising bioassay-modeled tests are designed to extend the knowledge of effects suggested by the first-level procedures. Based on data from Level 1 and Level 2 findings, third-level experiments are specific tests tailored to confirm the pharmacodynamic/kinetic properties and clinical efficacy of traditional drugs. Level 2 and Level 3 tests may be combined in monitoring fractionation of crude drug constituents with potential application in conventional therapeutics [Kyerematen et al., 1987].

MALARIA
Malaria and Herbalism
Malaria due to Plasmodium falciparum is a major cause of mortality in many less developed countries, particularly in Africa [Figure 1.1]. Herbalists in Africa commonly treat the recurrent fevers typical of malaria with plant extracts. Since immunity is capable of resolving most cases of falciparum malaria in regularly exposed (semi-immune) adults without drug intervention, such extracts may simply contain antipyretic agents which can relieve the symptoms of the disease while cure is achieved immunologically. However, finding clinically useful antimalarial compounds in such extracts could provide significant medical and economic benefits [Khalid et al., 1985].

In Africa, plants continue to be used in the treatment of malaria, either for their anti-parasitic activity, or because they possess, or arc believed to possess other therapeutic value for a patient with malaria [Weenen et al., 1990]

As there are currently no scientific methods applicable for the selection of plants that can be expected to contain novel biologically active substances, the planning for plant based drug development programmes must utilize approaches that are considered to be "non-scientific" by some, e.g. selection of plants based on folkloric use. A majority of the currently useful drugs derived from plants were discovered through scientific investigation of folkloric claims of human efficacy. The discovery of reserpine and artemisinin are the most recent success stories based on a pursuit of folklore. However, quinine, digoxin, digitoxin, tubocurarine, morphine, codeine and a majority of other useful drugs were derived from plants in a similar fashion [Krogsgaard Larsen et al. 1983]

This does not apply to all the drugs in use which are derived from plants. For example, vincristine, an alkaloid isolated from Vinca rosea (Catharanthus roseus), is an antineoplastic agent which had a folkloric history of treating diabetes (Svoboda 1961], taxol, a new antileukemic and antitumour agent from the stem hark of Trams brevifolia was isolated by random screening of plants [Wani et al., 1971]
 
Natural Products

Description: I:\For Milka\patents_Raw_data\KE000059\1\current\000007.tifDescription: I:\For Milka\patents_Raw_data\KE000059\1\current\000008.tif Atovaquone, a novel hydroxynaphihoquinone broad-spectrum anti-infective synthetic drug developed by Welcome Research Laboratories is currently showing clinical promise for the treatment of malaria and the AIDS associated diseases Pneumacystis carinii pneumonia and toxoplasmosis. The drug is the end product of half a century of research by numerous groups who have investigated the antiparasitic properties of many related compounds. Atovaquone is the only member of the series to show therapeutic activity in humans when taken orally [Hudson, 1993].

Problems with Modern Resistance
Plasmodia] drug resistance has been defined as the ability of a parasite population to multiply or to survive in the presence of concentrations of a drug that normally destroy parasites of the same species or prevent their multiplication. Complete resistance denotes the parasites ability of withstanding maximum doses tolerated by the host (Wernsdorfer, 1992). This definition must now be qualified in the light of present knowledge of sulfonamide metabolism in certain individuals, with a statement to the effect that the form of the drug active against the parasite must gain access to the parasite or the infected red blood cell for the duration of time necessary for normal action. This qualification depends on certain observations;
(a) In some individuals, sulfonamides or sulfones may be metabolized abnormally, e.g. become bound to proteins and released too slowly to maintain an effective antiparasitic level.

(b) Parasites resistant to chloroquine may not encounter lethal quantities of this drug because the membrane of the host red blood cell and the parasite membrane become selectively impermeable to drug. This is due to the acidity of the metabolic products of the parasite-lactate production by chloroquine-susceptible parasites which reduces the pH of the red blood cell, thus inducing absorption of the drug. This is however only part of a complex process [Black et al., 1986].
Resistance to currently available antimalarial drugs is a major problem across the tropical world. Resistance to chloroquine now occurs in all the countries with endemic malaria apart from Central America and parts of Western Asia (Figure 1.4) [Alder 1992b].

Major and alarming changes in resistance have occurred in Africa south of the Sahara and in islands off the eastern coast. Chloroquine-resistant P. falciparum has been reported from some African countries, namely Angola, Burundi, Central African Republic, Comoros, Gabon, Kenya, Madagascar, Malawi, Cameron, Namibia, Sudan, Uganda, Tanzania, Zaire and Zambia [Black et al., 1986].

Resistance to more than one antimalarial agent known as multi-drug resistance has also been recorded in some areas, e.g. resistance of P. falciparum to sulfadoxine-pyrimethamine (fansidar) has been reported from East African countries such as Kenya, Tanzania and Zambia. Cases of resistance to quinine have been reported in Cameroon, Congo, Guinea, Nigeria, Senegal, Tanzania and Zambia [Alder 1992c].
 

 
(a) Molluscicidal activity

Of the linear furanocoumarins bergapten and isopimpinellin, obtained from Melicope stipitata (Four 1974].

(b) Piscicid al activity
Recorded for alloimperatorin and the methyl ether derivative (Feuer 1974].

(c) Antitumour activity

(i) Broad spectrum antitumour activity of acronycine, first isolated from Acronychia bauri by Svoboda in 1966 [Jewers et al. 1973].

(ii) Benzophenanthridines, although known for many years, the benzophenanthridine alkaloids have only recently become of interest for their tumour-inhibitory activity. Two alkaloids have stimulated this interest, nitidine from a number of Zanthoxylum species and fagaronine from Zanthoxylum (Fasara) xantizoxyloides [Messmer et al. 1972].

(d) Antiviral activity

(1) Recorded for sessiliflorene and the sessiliflorols obtained from Melicope sessiliflora (Chan et al. [989]

(ii) For the flavonols tematin and meliternatin [Simoes et al 1989].

Description: I:\For Milka\patents_Raw_data\KE000059\1\current\000013.tif
 
 Ethnomedicinal Uses of Toddalia asiatica

(Synonym; Toddalia aculeatus Pcrs).


Some of the ethnomedicinal uses of T. asiatica are outlined in Appendix I which is adapted from 1991 Natural Products ALERT database (Napralert) at the University of Illinois, Chicago.

Other uses include;

The leaves are boiled and the stem used as vapor both for nasal and bronchial pains. Fruits or their decoction are taken for coughs and for stomach-ache. The roots are sometimes cooked with soup for this treatment. Juice from the roots is rubbed into incisions around the wrists and ankles and some of it is taken orally at the same time as a remedy for paralysis caused by snake-bite. It is said to be a powerful emetic [Watt et al. 19621.

The fruit is used by the Masai and Chagga as a cough remedy and the root is the treatment of indigestion and influenza. They also take the leaf, coated in butter, for lung diseases and rheumatic pains. A decoction of similar components is used for pneumonia and rheumatism. A decoction of the root is a Tanzanian antidote for poisoning by bad meat. The Masai use the plant as food and the decoction of the root for stomach ache, rheumatism and as an antihelmintic [Watt et al. 1962].

In Madagascar, the root is used as a tonic in malaria and in indigestion. The root bark has been used as a stimulant and stomachic and for malaria, intermittent fever, cholera, diarrhea, rheumatism and syphilis [Torte et al. 19691. An infusion of the root bark administered in mild cases of malaria was found beneficial, but experiments carried

Description: I:\For Milka\patents_Raw_data\KE000059\1\current\000015.tifDescription: I:\For Milka\patents_Raw_data\KE000059\1\current\000016.tifDescription: I:\For Milka\patents_Raw_data\KE000059\1\current\000017.tifDescription: I:\For Milka\patents_Raw_data\KE000059\1\current\000018.tifOn the basis of the ID50s obtained using the aqueous extracts (Section 2.3.2) appropriate extract concentrations were prepared for in vitro antimalarial activity tests which were carried out as described in Section 2.2.3.

Acid/Base Alkaloidal Extraction.


Thin layer chromatography of the above methanolic extract (TA 106 DM) using plastic-backed TLC plates and the solvent system Ethyl acetate: Methanol: Water: Ammonia in the ratio 100:17:13:3 drops showed the presence of alkaloids after spraying with Dragendorfrs reagent and obtaining orange spots. Assuming that the antimalarial activity in the extracts was due to the presence of alkaloids, an attempt was made to concentrate them by an acid/base extraction method (Scheme 4.2) which takes advantage of the polarity differences in the various components. This was the first attempt in trying to identify the compounds responsible for the antiplasmodial activity.

Acid/base extraction of the extract TA 106 DM

The methanolic extract was freeze-dried and weighed. The extract (6.08g) was re-dissolved in a small amount of methanol, a little at a time until the whole extract went into solution. This was extracted with twice the volume of various solvents (Scheme 4.2). The methanolic residue was acidified with 10 % acetic acid, stirred and filtered. It was then extracted first with petroleum ether (100 ml x 3) followed by chloroform (100 ml x 3). The aqueous acidic methanolic residue was treated with 10 % ammonium hydroxide solution to a pH of 9 and extracted first with chloroform (100 ml x 3) then with ethyl acetate (100 m I x 3). On the basis of TLC, the chloroform.
 
In vitro Antibacterial and Antifungal Activity tests

The organisms tested in this project were Escherichia coli, Bacillus subtilis and Saccharomyces cereviriae. The petroleum ether, the acidic and the basic chloroform extracts were tested at three different concentrations (400, 200, 100 pg/disk) under three different lighting conditions (light, dark, UV) using the Nutrient Agar method [Clark et al. 1981, Lin et al 1990]. The test plates were incubated for 24 hrs and the zone of inhibition measured in millimeters. The procedure was repeated after 48 hrs.

In vitro Cytotoxicity Test

The brine shrimp lethality test [Meyer et al. 1982, Tyler 1988] was carried out at three different concentrations (1000, 100, 10µg/m1) on the petroleum ether, acidic and basic chloroform extracts from acid/base extraction of the methanolic extract of Toddalia asiatica.

MULTIPLE CHROMATOGRAPHIC TECHNIQUES FOR SEPARATION AND PURIFICATION


Thin Layer Chromatography
Thin Layer Chromatography (TLC) was constantly employed in this project.
Precoated silica gel (Merck 5735) plastic-hacked TLC plates were used as they are column was first washed through with n.-hexane under vacuum and the sample to be eluted was then applied as a dry powder adsorbed onto silica gel. Elution then proceeded under vacuum, with solvents of increasing polarity.

For both Sections the solvent system found appropriate for separation of components in T. sciatica’s basic chloroform extract was ethyl acetate: Methanol: Water: Ammonia in the ratio 100:17:13:3 drops. Different fractions were collected, examined by TLC and those that were similar were pooled.

Preparative Thin Layer Chromatography (Prep. TLC)
Prep. TLC was used for separation and purification of extracted compounds.

The plates were prepared by mixing 45 g of silica gel (60 PF Merck 7749) with 90 ml distilled water to produce a slurry which was shaken briefly but vigorously to ensure uniform wetting and then applied by spreading to glass plates (20 x 20cm) to produce thin layers of uniform thickness (0.5 or 0.75 mm). The prepared plates were then dried for about 30 minutes, placed in an oven at 105-120°C for 3-4 hours.

Soluble samples were applied to the plates by capillary (using a Pasteur pipette that had been drawn out under heat so that the outlet was as small as possible) as a thin band at 1.2 cm. distance from the edge of the plate. The plate was then placed in a TLC tank containing a suitable eluting solvent, previously chosen on the basis of TLC studies, and which had been allowed to equilibrate for 30 minutes prior to development. The plates were developed to a distance of 15 cm; multiple developments was sometimes used to separate mixtures of compounds with similar Rf values. After visualization, band(s) containing the required compound(s) were removed from the plate by
 
ANALYSIS OF SEMI-PURE EXTRACTS/FRACTIONS

In vitro Antimalarial Activity tests

These were carried out on accurately weighed extracts/fractions as described under Section
Spectral Studies
Instrumentation

Infra Red (IR) spectrum was recorded as KBr disc on a Perkin-Elmer 781 Infra Red Spectrophotometer. UV spectra were obtained in methanol using Perkin-Elmer 552 UVNIS spectrophotometer. FTIR spectrum was obtained on Perkin-Elmer Model 1720 PIA spectrophotometer. High resolution electron impact mass spectra (E1MS) and accurate mass measurements were obtained on an AEI-MS 902 double focusing instrument by direct probe insertion, operating at 70 eV and at elevated temperature. Nuclear Magnetic Resonance spectra were recorded on a Bruker AMX-400 (400 MHz) instrument. Deuterated solvents were used to run NMR spectrum.
 
Sequential bioassay guided extracts in vitro antimalarial study results.

Different extracts from Toddalia asiatica (dichloromethane, methanol and water) showed antiprotozoal activity (Table 2) in a dose-dependent manner (Figures 3-5). However, the methanolic extract showed the highest activity of an average value of 0.98 } lend using chloroquine-sensitive strains of P. falciparum isolates K67, K39 and M24. ENT7 did not achieve appropriate growth for the tests.

Acid/Base Extraction of TA 106 DM

Acid/base extraction of the methanolic extract (6.08g) yielded 0.1893 g (3.1%) of petroleum ether soluble extract, 0.0246 g (0.4%) of the acidic chloroform extract, and 0.99 g (16.3%) of the basic chloroform extract.

10% acetic acid may be used to make soluble salts of weak alkaloidal bases. Filtering ensures the removal of all the non-alkaloidal precipitating material. Extracting this acidic mixture with petroleum spirit (40-60)0C removes all the fatty material. Chloroform extraction of the acidic solution takes the non-alkalods or the weakly basic alkaloids (weaker than those that formed salts with the above weak acid). The water layer is made basic by adding 10% ammonium hydroxide solution in excess. This ensures that all the alkaloids are released from their salt form. Extracting the basic form with chloroform removes the alkaloids and the very polar alkaloids are extracted with ethyl acetate at the next stage. The above process ensures concentration of alkaloids from an alkaloidal extract.

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 Constituents are dihydronitidine and oxynitidine which could he breakdown products of nitidine.
 

ISOLATE                                 CQ(S/R)                     ID50(µg/ml)

                                        CQ      TA 106(13)                      NITIDINE

UPA               S             16.0                 56.0                    67.0
K39                S            4.43                  40.3                     45.1
SL/D6            S            6.59                  29.3                     76.0
11133             S            5.86                 40.4                      73.6
Average                        8.22                41.5                      65.4
IT12               R            65.9                9.20                       42.0
FCR3                        47.4                37.5                       165.0
FCB               R           28.4                108.0                      47.5
Average                      47.2                 51.6                        84.8

Table 8. In vitro chemosensitivityof P falciparrum to TA 106(13), nitidine and chloroquine
Footnote;
CQ-Chloroquine
S-Sensitive
R-Resistant

 
WHAT IS CLAIMED
1. The process of extracting the active compound isoquinoline alkaloid(nitidine) from the root bark extract of Toddalia asiatica which posses in-vitro anti-malarial activity against both chloroquine sensitive (ID50 5µg/ml-1) and chloroquine resistant 01350 20 pg/m1-1 culture adopted plasmodium falciparum isolates.

2. The process of extracting the active compounds as in claim 1 which comprises maceration and aseptical filtration of the extract.

3. The process of extracting the active compounds as in claim 1 using foria transforms infra-red spectroscopy.

4. The process of extracting active compounds as claimed in 3 by use of ultra-violet spectroscopy.

5. The process for extracting active compound claimed in 4 above using (NRM) Nuclear Magnetic Resonance Technique.

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