Gh.aplerl 0!/momedicine and:7/nliprolozoai:Pfanl :Producls

ANCIENT ETHNOMEDICINE

Nature is still humankind's greatest chemist and pharmacognosy of many

compounds that remain undiscovered in plants are beyond the imagination of even our

best researchers (Seiber et. al. 1983, Soejarto and Farnsworth 1989). In many

countries of the world there is a well-established system of traditional medicine, whose

remedies are being compiled with renewed interest. There are written documents of

the early civilizations in China (2735 BC), India (2500-3000 BC), the near East and

Egypt (1900 BC), Mesopotamia, ancient Greece and Rome etc. (Hamburger and

Hostettman 1991, Holland 1994, Siddiqui and Hakim 1991) and by others such as the

ancient Egyptians with the first people who gathered roots ("rhizotomi"). For what were

once termed "women's concoctions" for want of proper documentation, these

documents trace the use of medicinal plants as a source for relief from illness back

over five millennia. The Greek compendium of Dioscorides, Materia Medica included

all plants used in the first century AD in the Mediterranean area and Egypt. TheShang

Nung of China contained the first list of medicinal plants in that part of the world and

this knowledge was incorporated into the 141h century Pen-tsao (Bannerman 1983).

We also have the first descriptions of medicinal herbs by Hippocrates, Theophrastus

and Galen (Capasso 1985). Such documentations of medicinal plants have

foreshadowed the present day developments of newer pharmaceutical technologies for

mass production. Traditional plant remedies are shown to have the potential to cure

malaria both clinically and parasitologically, particularly since modern treatment of

malaria are not accessible to rural communities wh.ich account for 80 % of populations

of African countries e.g. Uganda (Bitawha et. al. 1997), Nigeria (Etkin 1997) and in

most Asian countries.

DEVELOPMENT OF ETHNOMEDICINE IN INDIA

India inherits a very precious herbal heritage preserved by the dwellers in its

varied regions in their vernacular forms. Sushruta Samhita deals with surgery and

Charak samhita with medicine in which a comprehensive account of therapeutic use of

drugs, a remarkable description of the properties and therapeutic use of about 2,000

remedies derived mostly from vegetable sources is available (Kapoor 1968). The

herbal basis of in Hausa medicine (Africa), Buddhist, Yunani and Chinese systems of

medicine too contributed to an important acceptance in traditional medical practice in

the overall global nature of effective cures which now exist side by side with newer

4

Ghaplerl 0!/momeclicine ancl.7lnliprolozoai:P/anl :Procfucls

uses of pharmaceutically prepared drugs in contemporary medicine in the Asian

region.

The starting point in the recording of medicinal plants was done in Sir William

Jone's memoir on "Botanical Observations of Select Plants" (Chakravarty 1975). Table

1 is compilation of such historical records.

s. No. Name of text/ Publication Year of Author of text publication

1 Rigveda 4500 B C N.a

2 Atharvaveda 2000-900 B C N.a

3 Sushruta Samhita 1000 B.C N.a

4 Charak samhita 1000 B.C N.a

5 Botanical Observations of Select Plants Early 191h century Sir William Jone

6 Catalogue of Indian Medicinal Plants and Drugs 1810 John Fleming

7 Materia Medica of Hindustan 1813 Ainslie

8 The Bengal Dispensatory and Pharmacopoeia 1841 0' Shaughnessy

9 Pharmacopoeia of India 1868 Waring

10 Flora Indica 1874 Roxburgh

11 Materia Medica 1877 U.C. Dutta

12 Pharmacographia of India 1879 Fluckiger and Hand bury

13 Vegetable Materia Medica of western India 1883 Dymock

14 Supplement to the Pharmacopoeia 1891 Mohideen Sherif

15 Materia Medica of Madras 1891 Hooper

16 Pharmacographica Indica 1890-93 Dymock, Warden and Hooper

17 Economic Products of India (6 volumes) 1889~1896 Sir George Watt

18 Indigenous Drugs of India 1896 Kanai LaiDey

19 Indian Medicinal Plants (4 volumes) 1923 Kirtikar and Basu

20 Indian Materia Medica 1927 Nadkarni

21 Indigenous drugs of India 1958 Sir Ram Nath Chopra

22 Wealth of India (11 volumes) 1948-till date C.S.I.R

23 Compact Disc's (C.D.) & Web Sites 1998 onwards C.S.I.R

N.a -Not available

Table 1: Some historical written records of Indian medicinal plants based on

ethnomedicinal collections and medicinal value.

5

Ghaplerl 0!/momedicine and:Jlnliprolozoaf:Pfanl :Producls

Detailed ethnobotanical contributions are being tabulated from field surveys in

almost all regions of the sub-continent. Recent integrated multi-disciplinary research

on indigenous medicinal plants is sponsored under the composite Drug Research

Scheme by the I.C.M.R. and by C.C.R.A.S. at Regional Science and medical

institutions besides being conducted in C.S.I. R. laboratories (Gupta 1994 ). It is

important to understand the challenges of medical pluralism upon ethnomedicine and

the ways in which this system manages to survive in a period of general social change

which is occurring as a result of various tribal welfare measures of the government and

also with increased interaction with the non tribals.

Web sites like NAPRALERT (Natural Products Alert) and many others like

INMEDPLAN (The Indian Medicinal Plant Distributed Database Network) are now

available. The Foundation for Revitalization of Local Health Traditions was formed to

document, save and encourage the continued usage of India's rich ethno-botanical

tradition. The Foundation also acts as the Network Secretariat for the database,

INMEDPLAN, which is a network on nine main agencies spread across India,

collecting information in botany, ecology, phytochemistry, pharmacology,

pharmacognosy, ethnomedicine, bibliography and abstracts, agrotechnology and

traditional systems of medicine.

SELECTIVITY OF ANTIPROTOZOAL PLANT PRODUCTS

Compounds found to possess high activity in screening tests using one

particular organism should be evaluated further for 'broad spectrum' activity as activity

against other organisms can be found. Many natural products from plants have been

shown to possess activity against one or more species of protozoa but few have been

shown to be highly selective antiprotozoal agents (Wright and Phillipson 1990). Often

in vivo activity can be shown without in vitro effects and vice-versa partly because of

metabolic activation, transformation and therefore the development of methods, which

allow the detection of some pro drugs in vitro screening, has been undertaken. Natural

products isolated from plants contain a multiplicity of constituents, which from time to

time are grouped under diversified categories of compounds. The prominent ones are:

Alkaloids, Terpenes and quassinoids, and miscellaneous compounds (Siddiqui et. al.

1998).

6

G.hapler! 0!/momedicine and!7Jnliprolozoai:P/anl :Producls

Alkaloids are again classified into different groups such as Quinoline,

Quinazoline, Acridone, lndolizidine, Quinolizidine etc. (Michael 1994, Michael 1995).

Attention has been focused on the quassinoids in recent years because several of

them have shown promising antitumour, antiviral, antimalarial and amoebicidal

activities. The quassinoids are a group of degraded triterpenes found in various

species belonging to the family Simaroubaceae. They are heavily oxygenated,

fundamentally C-20 compounds and bitter in nature, isolated from theSimaroubaceous

plants (Gupta and Ali 1996).

To determine preferential selectivity towards one or more species of protozoa,

comparisons of in vitro activities have shown that a number of quassinoids are highly

effective against both Entamoeba histolytica and Plasmodium falciparum. Although

quassinoids have been found to be generally cytotoxic they exhibit a high degree of

selective toxicity against P. falciparum in vitro. (Wright et. al. 1988). Quassinoids

appear to display marked selectivity towards P. fa/ciparum as compared to E.

histolytica, Giardia intestinalis and Toxoplasma gondii when tested in vitro (Wright et.

al. 1993).

On a preliminary screening, alkaloidal extracts of the stem bark of Unonopsis

buchtienii, the alkaloids and sterols isolated from these were studied. Of the alkaloids,

liriodenine exhibited the highest activity against Leishmania major and L. donovani

(IC10o = 3.12 f.lg/ml). On the other hand, 0-methylmoschatoline and the petroleum

ether extract without alkaloids showed an interesting in vitro activity against

Trypanosoma brucei with an IC100 of 6.25 f.lg/ml (Waechter et. al. 1999).

1.1.1 ANTIPLASMODIAL VIS A VIS ANTIAMOEBIC ACTIVITY

Simultaneous testing of large number of plant samples for antiprotozoal activity

followed by direct effects of any given plant product like emetine formulations derived

from Ipecacuanha, quinines from Cinchona, terpenes, quassinoids from Brucea sps.

etc are difficult to separate because of:

i. Common properties of plant products (alkaloids, terpenes, quinones and

miscellaneous compounds)

ii. Selectivity and sensitivity to specific antiamoebic agents

iii. Varied antiprotozoal cultures and pathogenesis (Clark 1998)

7

Ghaplerl 0!/momechcine and:Jlnliprolozoal:?fanl :?roducls

iv. The two or more biological components involved both in vivo and in vitro.

Qinghaosu is also active in vitro against the free-living amoeba Naegleria

fow/eria, and it shows antitrematode activity, notably against Schistosoma japonicum,

S. mansoni and Clonorchis sinensis (Hien and White 1993).

At the beginning of the nineteenth century, chemical techniques were

developed that made possible the isolation of vegetable alkaloids. After making crude

extracts sophisticated chemical works are required for identification of individual

compounds using techniques such as chromatography, Nuclear Magnetic Resonance

and Mass spectroscopy. Better biological work is required for screening and

elucidation of the mechanism of action, metabolism of plant products for example the

unique artemisinin endoperoxide, stable ozonoides, trioxanes etc., to synthesize

molecules modeled on these moieties. There is an ever-growing need for creating

newer biopharmaceuticals with unique and reproducible properties, for clinical trials.

Such a modern approach which prefers the use of individual compounds has emerged

from the wealth of data of medicinal plants which belong to the oriental systems of

medicine which are considered to be more holistic (Kirby 1997). Metronidazole and

emetine have been successfully used in the treatment of malaria, Giardiasis,

Trichomoniasis and can be considered to be a broad-spectrum antiprotozoal drug

(James 1985, Raju 1979).

Table 2 shows some common effects of antiprotozoal plant products and drugs

with comparable effects of antiamoebic activity, particularly by some antimalarials.

t:metme Nl u.ur 2,3-dehydroemetine NT 0.16 metronidazole NT 0.22 5-chloro-8-hydroxyquinoline NT 0.19 amodiaquine NT 0.07 mepacrine NT 0.58 Primaquine NT 23.2 Chloroquine NT 24.8 Quinine NT 14.8 Quinidine NT 16.6 Quinidinone NT 7.4 Cinchoamine NT 14.8 1 0-methoxycinchonamine NT 4.5 3-epiquinamin NT 12.9

Cinchona ledgeriana Alkaloids ICso (l!g/ml) Keene et.al. 3-lsocorynantheol NT 4.1 1987 3p,17p-18,19- NT 1.7 dehydroochrolifuanine 3p,17a-18,19- NT 2.0 dehydroochrolifuanine 3a, 17p-Cinchophylline NT 0.96 3a, 17a-Cinchophylline NT 2.2

ECso (ng/ml) Druilhe et.al. Cinchona sps. Alkaloids Dihydroquinidine 74.1 NT 1988 (Rubiaceae) 9-Epiquinine 1179 NT

Quinine 252.2 NT Quinidine 67.7 NT Cinchonine 67.5 NT

Contd.

9

Plant species & Extract/ Class-of Name of compound Dose efficiency Dose efficiency Reference (Family) compound and in P. falciparum in E. hfstolytica

Part used

MIC (1-lg/ml) Tona et.al. Cryptolepis Maceration in ND ~ 7.81 1998 sanguinolenta water (Asclepideaceae) ICso (J.lg/ml) Kirby et. al

lndolequinoline Cryptolepine 0.031 1995 alkaloid

MIC (1-lg/ml) Mbela et.al. Gardenia jovis Aq. Ethanol ND 3.13 1992 ton antis n-butanol ND 1.56 (Rubiaceae) Formal HCI ND 1.56

Ether ND 6.25 ICso (1-lg/ml) Weenen

Petroleum ether ND >499 et.al. 1990 Dichloromethane ND 50-99 Methanol ND >499

MIG (1-lg/ml) Tona et.al. Harungana Decoction ND 62.5 1998 madagascariensis ICso (1-lg/ml) (Hypericaceae) Ethanol ND 29 Gessler

Petroleum ether ND 10 et.al. 1994 Ethylacetate ND 10 water ND 88

MIC (J.lglml) Tona et.al. Nauc/ea latifolia Decoction ND >500 1998 (Rubiaceae) Decoction ND >125

Water ND 15 1-1g< 1Cso<22 Gbeassor et.al. 1989b

MIC(1-1 g/ml) Sharma & Parthenium Sesquiterpene Parthenin 10-12.5 Bhutani hysterophorous lactone 1988 (Asteraceae) ICso (1-lg/ml) Hooperet. al

1.289 1990 ICso (J.lg/ml) ICso (1-lg/ml) Wright et. al

Simarouba amara Quassinoids Glaucarubinone 0.004 0.168 1988 (Simaroubaceae) 2'-Acetylglaucarubinone 0.008 0.155

Ailanthinone 0.015 0.063 Holacanthone 0.007 0.162 Metronidazole NT 0.320

ICso(J.lM) ICso (l-IM) Wright et. al Simarouba amara Quassinoids Ailanthinone 0.019 0.132 1993 (Simaroubaceae) Glaucarubinone 0.008 0.323

ICso (J.IM) ICso (1-1M) Wrightet. al Srtychnos henningsii Alkaloids Holstiline 31.5 Inactive at 63 1994 (Loganiaceae) Holstiine 32.7 Inactive at 65

CQ diphosphate 0.156 NT Emetine dihydrochloride 0.13 3.07 Metronidazol NT 1.87

ICso (1-lg/ml) ICso (1-lg/ml) Wrightet. al Strychnos Alkaloids Usambarensine 0.38 0.49 1991 usambarensis 3'4'-Dihydrousambarensine 0.01 2.18 (Loganiaceae) Nb- methlusambarensine 2.39 4.12

Chloride Usambarine "'1.85 0.46 18,19-Dihydrousambarine 1.07 0.65 oxallate Strychnopentamine 0.09 7.70 Strychnopentamine 0.09 10.10 methanesulphonate lsostrychnopentamine base NT 10.70 Akagerine 6.98 17.1 Tubulosine 0.020 NT

Contd.

10

Plant species & Extract/ Class of Name of compound Dose efficiency Dose efficiency Reference (Family) compound and in P. falciparum in E. histolytica

Part used

ICso (!!M) ICso (!!M) Wrightet. al Strychnos Alkaloids 5,6-Dihydroflavopereirine 3.02 24.31 1994 usambarensis lsostrychnopentamine 0.765 19.5 (Loganiaceae) Strychnofoline 13.8 inactive at 52

Strychnopentamine 0.164 14.0 Strychnopentamine 0.140 15.7 methane sulphonate Usambarensine 0.880 1.13 3',4'- 0.023 5.02 Dihydrousambarensine Nb- methylusambarensine 5.345 9.22 chloride Usambarine -4.11 1.02 18, 19-Dihydrousambarine 1.98 1.20 oxalate

ICso(!!M) ICso (!!M) Wrightet. al Strychnos variabilis Alkaloids o- Acetlisoreticuline 22.1 Inactive at 66 1994 (Loganiaceae) lsoreticuline -92 Inactive at 74

Retulinal/isoretulinal 36 Inactive at 75 Retuline -92 Inactive at 74 lsostrychnobiline 2.07 Inactive at 41 Didehydroisostrychnobiline 1.67 Inactive at 35 monomethanesulphonate 12'- 1.58 Inactive at 34 Hydroxyisostrychnobiiine monomethanesulphonate 1.03 Inactive at 35 Strychnobiline

ICso(!!M) ICso(!!M) Marshall et. N.a Bisbenzylisoquinol Funiferine 0.63 108 al 1994 (Mennispermaceae) ine alkaloids Tiliagene 6.32 >411

Daphnoline 0.96 46.4 Aromoline 1.36 105 Homoaromoline 3.46 >82.2 Oxyacanthine HCI 1.06 74.4 Thalisopidine 0.09 41.2 Phaenthine 1.46 43.6 Tetrandrine 0.57 NT lsotetrandrine 0.16 NT Tetrandrine methiodide >65.4 >32.7 Pycnamine 0.83 31.9 Fangchinoline 1.43 104 Berbamine 0.45 17.8 Obamegine 0.74 55.4 Dinklacorine 3.92 54.8 lsochondodendrine 22.0 >421 Trigilletimine 42.1 >448 Cocsoline 1.16 NT Cocsulinine z88.9 >222 lsotrilobine 2.06 18.8 Cocsuline methiodide >17.8 >355 Gilletine 1.81 38.3 lnsularine picrate 2.07 >294

4- aminoquinoline CQ diphosphate 0.20 NT Emetine hydrochloride NT 2.23

5- nitroimidazole Metronidazole NT 1.87 ICso (!!g/ml) ICso (!!g/ml) Ohigashi

Vernonia amygdalina Steroid giucosides Vemonioside A1 139.7 <12.5 et.al. 1994 (Asteraceae) Vemonioside A2_ 94.1 <12.5

Vemonioside AJ 245.1 -Vemonioside A.. 81.8 <12.5 Vemonioside 81 46.1 <12.5 3-0xovemoniol 81 50.7 <12.5 lsovemoniol ~ 156.4 <12.5

ICso (!!g/ml) Wright et. al Standard drugs 5- nitroimidazole Metronidazole NT 0.320 1987

Emetine hydrochloride NT 1.230 4- aminoquinoline CQ diphosphate NT 15.1 Cinchona alkaloid Quinine NT 24.0

MIC (!!g/ml) Mahajan et. Standard drugs 5- nitroimidazole Metronidazole NT 0.312- 0.625 al 1974

nitroimidazole Tinidazole NT 0.625-1.25

ND-Not detected, NT- Not Tested, N.a- Not available

Table 2: Plants possessing common activities against both P. falciparum and E. histolytica are shown along with their dose efficacies.

11

Gb.aplerl 0!lmomedicine and!7/nliprolozoai:P/anl :Producls

1.1.2 ANTIMALARIAL PLANTS

Herbal remedies have led to global imports of millions of dollar by various

pharmaceutical agencies in Europe and the Americas for cost effective symptomatic

relief from, the diagnosis of malaria and the objective assessment of the impact of the

treatment on circulating parasitaemia (Warell 1997). Prior to 1820, ague was treated

with the suspension of finely powdered Cinchona bark in wine (Meshnick 1997).

Among the first to be studied were Opium and Cinchona because of their medicinal

value (Smith 1976). The Cinchona alkaloids were used for the treatment of malaria

initially as crude preparations, powders and extracts, and later in the 191h century as

purified salts especially so, quinine (Were 1987). The second most important plant

from ethnobotanical origin, emerged 150 years later, when the plant Artemisia annua

was rediscovered as a Chinese traditional remedy, along with its chemical

characterizations, studied to isolate Quinghaosu (Quinghaosu Antimalaria

Coordinating Research Group 1979). Ethnobotanical records afforded the discovery of

a succession of synthetic drugs that exhibited potent antimalarial activity in a 30-year

period between 1925 and 1955 (Greenwood 1995).

A historical review and extensive compilation of plants showing anti plasmodial

activity has been published recently (Sharma and Sharma 1998) which is a prelude as

Part I to the following tables of Part II. (Tables 3a and 3b).

12

Plant species & Part used Extract/fraction Dose efficiency Plasmodium sp. & Reference (Family) Strain

ICso (J.Ig/ml) Ailanthus excelsa Leaves, 80 % methanol 16 & 180 P. falciparum 307 & Od2 Tahiret. al. (Simaroubaceae) Stem bark 80% methanol 27 & 50 1999 Annona muricata ICso (J.Ig/ml) P. falciparum W-2 & 0-6 Antoun et. al. (Annonaceae) Leaf 95% ethanol 20 & > 63 1993

ICso (J.Ig/ml) Annona squamosa Leaves, 80 % methanol 2 & 30 P. falciparum 307 & Od2 Tahiret. al. (Annonaceae) Stem bark 80 % methanol 8.5 & 120 1999 Andrographis 1 00% inhibition P. falciparum FCR-3 Rahman et. paniculata Whole plant Methanol 2.5 mg/ml (after 48 h.) al. 1999 (Acanthaceae) Chloroform 0.025 mg/ml Aphanamixis ICso (J.Ig/ml) P. falciparum 06&W2 Mackinnon et. po/ystachya Wood Ethanol 16.8 & >20 al. 1997 (Meliaceae) Artemisia indica Stem Methanol ECso(glm~ P. falciparum K1 Chanphen et. (Asteraceae) 6.60 X 10 al. 1998 Ayush- 64 %cured P. falciparum Chari et. al. Containing N.a N.a 72.4 1985 Alstonia scholaris (Apocyanaceae) Swertia chirata (Gentianaceae) Caesalpinia bonducella (Caesalpineaeceae) Picrorrhiza kurroa (Scophulariaceae)

ICso (J.Iglml) Azadirachta indica Leaves, 80 % methanol 5.8 & 1.7 P. falciparum 307 & Od2 Tahiret. al. (Meliaceae) Stem bark 80 % methanol 8.5 & 40 1999 Azadirachta indica ICso(J.Ig/ml) P. falciparum D6&W2 Mackinnon (Meliaceae) Leaves Ethanol 2.5 & 2.45 et. al. 1997

Azadirachta indica Seeds Meythl B IOso (J.Iglml) P. falciparum Perrow et. al. (Meliaceae) cyclodextrin 5-50 1996 Bourreria ICso (J.Iglml) P. falciparum W-2 & 0-6 Antoun et. al. succulenta Stem 95% ethanol > 63 & 21 1993 (Boraginaceae) Castilla elastica ICso (J.Ig/ml) P. falciparum W-2 & 0-6 Antoun et. al. (Moraceae) Stem 95% ethanol 13 & > 63 1993

ICso (J.I glml) P. falciparum 06 & W2 Mackinnon et. Cedre/a Bark Ethanol >20 & 15.0 al. 1997 salvadorensis Fruit Ethanol 18.7 & 15.3 (Meliaceae) Leaves Ethanol 8.3 & >20

Wood Ethanol ·13.8 7 13.2 Cedre/a odorata ICso (J.I g/ml) P. falciparum 06 & W2 Mackinnon et. (Meliaceae) wood Ethanol 1.20 & 1.11 al. 1997

ICso (J.Ig/ml) P. falciparum W-2 & 0-6 Antoun et. al. Ceiba pentandra Stem 95% ethanol 13 & > 63 1993 (Bombaceae)

ICso (J.I glml) P. falciparum 06 & W2 Mackinnon et. Chukrasia tabularis Leaves Ethanol 6.36 & 3.39 al. 1997 (Meliaceae)

ICso (ng/ml) P. falciparum 0-6, K-1, Cimanga et. Crypt ole pis Root bark Aqueous 122, 93 & 168 W-2 al. 1997 sanguinolenta 80% Ethanol 72,56 & 142 (Asclepiadaceae) Aqueous Average P. berghei yoelii &

80% Ethanol parasitaemia P. berghei berghei 19.4 & 39.8 11.9& 18.9

Dysoxylum ICso(Jl g/ml) P. falciparum 06 & W2 Mackinnon et. fraseranum Leaf Ethanol 13.1 & >20 al. 1997 (Meliaceae) Wood Ethanol 10.2 & 16.4

ICso (J.I g/ml) P. falciparum 06 & W2 Mackinnon et. Guarea g/abra Bark Ethanol 19.7 & >20 al. 1997 (Meliaceae)

ICso (J.I g/ml) P. falciparum W-2 & 0-6 Antoun et. al. Guarea guidonia Leaf/ 95% ethanol 17 & > 63 1993 (Meliaceae) Stem Guarea pryriformis ICso (J.Ig!ml) P. falciparum 06 & 'f'/2 Mackinnon et. (Meliaceae) Wood Ethanol 13.9 & 13.3 al. 1997

Contd.

13

Plant species & Part used Extract/fraction Dose efficiency Plasmodium sp. & Reference (Family) Strain

ICso (It g/ml) P. falciparum Francois et. Hannoa klaineana Stem bark Dichloromethane 2.400 NF 54, clone A1A9 al.1998 (Simaroubaceae) Petroleum ether 8.833

Methanol 0.672 Water- 1.658 decoction

ICso (~tgfml) Harrisonia Leaves, 80 % methanol 60 & 50 P. falciparum 3D7 & Dd2 Tahiret. al. abyssinica Stem bark 80 % methanol 10 & 4.7 1999 (Simaroubaceae)

% suppression at P. berghei berghei Aweet. al. Kaya grandifolia 50 mg/kg/day 1991 K. ivorensis Stem bark Aqueous 55.1 K. senegalensis Stem bark Aqueous 62.2 (Meliaceae) Stem bark Aqueous 44.7

ICso (~tg/ml) Kaya senegalensis Leaves, 80 % methanol 5.5 & 4.3 P. falciparum 3D7 & Dd2 Tahir et. al. (Meliaceae) Stem bark 80 % methanol 150 & 25 1999

Seeds 80 % methanol 4.3 & 38 Seeds water 30 & 31.5

% suppression at P. yoelii nigeriensis Awe et. al. 50, 100, 200 and 1998 400 mg/kg/day

Mangifera indica Stem bark Methanol 32.6 (Anacardiaceae) 51.8

62.2 78.2 ICso (~tg/ml)

Maytenus Leaves, 80 % methanol 65 & 5.1 P. falciparum 3D7 & Dd2 Tahiret. al. senegalensis Stem bark 3.9 & 10 1999 (Celastraceae)

ICso (~tg/ml) P. falciparum W-2 & D-6 Antoun et. al. Miconia racemosa Stem 95% ethanol 25 & 39 1993 (Melastomataceae)

ICso (~tg/ml) P. falciparum W-2 & D-6 Antoun et. al. Myrica cerifera Leaf 95% ethanol 12 & > 42 1993 (Myriaceae)

ICso (~tg/ml) P. falciparum Nigerian & Benoit-Vical Nauc/ea /atifolia Stem Infusion 6.9.& 7.5 FcB1 et. al. 1998 (Rubiaceae) Decoction 7.3 & 7.0

Root Infusion 5.9 & 6.0 Decoction 4.0 & 3.8

1 00% inhibition P. falciparum FCR-3 Rahman et. Piper sarmentosum Leaves Methanol 0.8 mg/ml (after 48 h.) al. 1999 (Piperaceae) chloroform 0.05 mg/ml

ICso (~tg/ml) Pseudocederla Leaves, 80 % methanol 15 & 50 P. falciparum 3D7 & Dd2 Tahiret. al. kotosifyi Stem bark 80 % methanol 40 &45 1999 (Meliaceae)

ICso (~tg/ml) P. falciparum K1 Marshall et. Rhazya stricta N.a Aqueous 11 al. 1995 (Apocyanaceae) Methanol

%complete P. berghei Das and recovery & NK65 Beuria 1991

Streb/us asper survival of mice (Moraceae) Stem bark Aqueous 30 Tinospora 100% inhibition P. falciparum FCR-3 Rahman et. paniculata Stem Methanol 2.5 mg/ml (after 72 h.) al. 1999 (Menispermaceae)

ICso (~tg/ml) Trichilia emetica Leaves, 80 % methanol 17.5 & 2.5 P. falciparum 3D7 & Dd2 Tahiret. al. (Meliaceae) Stem bark 80 % methanol 8.5 & 200 1999 Trichilia glabra ICso (~tglml) P. falciparum D6 & W2 Mackinnon et. (Meliaceae) Bark Ethanol 8.75 & 19.8 al. 1997 Trichilia hirta ICso(lt g/ml) P. falciparum D6 & W2 Mackinnon et. (Meliaceae) Leaves Ethanol 16.2 & >20 al. 1997 Triclisia trifolia ICso (It g/ml) P. falciparum D6 & W2 Mackinnon et. (Meliaceae) Leaves Ethanol 10.3 & 15.2 al. 1997

N.a- Not available

Table 3a: A composite table of different crude extracts derived from various parts of plants showing their in vitro and in vivo antiplasmodial activities against different species and strains of Plasmodia. Three plants with the most effective crude extracts/fractions have been highlighted.

14

Plant species Part used Class of compound Compound Dose efficiency Plasmodium Reference &(Family) sp. &strain

Ancistrocladus heyneanus ICso (llg/ml) P. falciparum Bring mann (Ancistrocladaceae) Roots Triterpene Betulic acid 10.46 NF, clone et. al. 1997 Triphyllum peltatum A1A2 (Oioncophyllaceae)

ICso (llg/ml) P. falciparum & Hallock et. Ancistrocladus korupensis Leaves & Naphtyltetrahydro Korupensamine A 0.31 & 0.56 P. berghei al. 1994 (Ancistrocladaceae) Twigs isoquinoline alkaloids Korupensamine B 0.18 & 0.41

ICso (llg/ml) P. falciparum Hallock et. Ancistrocladus korupensis Leaves Naphtyltetrahydro Korupensamine E 2 al. 1997 (Ancistrocladaceae) isoquinoline alkaloids

ECso (llg/ml) Chanphen Artemisia indica Stem Hydroxy Exiguaflavanone A 4.6 X 1006 P. falciparum et. al.1998 (Asteraceae) flavanones Exiguaflavanone B 7.05 X 10-6 K1

Maackiain 4.70 X 10'5

2-{2,4-dihydroxyphenyl)-5,6- 2.70 X 10'5

methylenedioxybenzofuran ICso (llg/ml) P. falciparum Milhau et.

Artemisia vulgaris Leaves & Essential oil NO 527 & 125 FcB1 & al.1997 (Asteraceae) Twigs Nigerian

ICso(ll M) P. falciparum Oketch-Asparagus africanus Roots Steroid Sapogenin Muzanzagenin 61, 163,23 & 16 K39, VI/Sd, Rabah et. (Liliaceae) Colourless oil (+)-nyasol -.-. 12 & 12 307 & Od2 al. 1997 Azadirachta indica ICso (%) P. falciparum Ohar et. al. (Meliaceae) Seeds Oil NO 0.0016 NF54 1998

ICso (ll g/ml) P. falciparum Campbell Brunsvigia littoralis Bulb Alkaloids Lycorine 0.62 & 0.7 010 & FAC8 et. al. 1998 (Amaryllidaceae) 1,2-0i-0-acetyllycorine 1.0 & 1.0

Ambelline >20 & >20 11-0-Acetylambelline >20 & >20 Crinine >20 & >20 3-0-Acetylcrinine >20 & >20

ICso (llg/ml) P. falclparum Dou et. al. Caste/a texana Aerial Quassinoids 11-0-trans-p- 0.92 & 0.75 D6&W2 1996 (Simaroubaceae) parts coumaroylamarolide

(Twigs & amarolide Not active at 1 01! Leaves) g/ml

chaparrinone 0.25 & 0.20 glaucarubolone 0.125 & 0.20 chaparrin 0.25 & 0.35 holacanthone 0.010 & 0.012 15-0-glucopyranosyl Not active at 10 glaucaroubol 1-19/ml

ICso (n g/ml) Cryptolepis sanguinolenta Root bark Alkaloids Cryptolepine 27,33&41 P. falciparum Cimanga et. (Asclepiadaceae) Cryptolepine HCI 41,62&52 0-6, K-1 & W-2 al. 1997

11-hydroxy cryptolepine 31, 45& 59 Quindoline 63, 87 & 108 Neo cryptolepine 35,51 & 65

Cryptolepine 13.7 & 45.4 P. berhei yoelii Cryptolepine HCI 10.8 & 15.6 & P. berghei 11-hydroxy cryptolepine Not done & 43.2 berghei Quindoline 42.1 &48.2 Neo cryptolepine Not done

ICso (ll g/ml) P. falciparum Milhau et. Eucalyptus globulus Leaves & Essential oil NO >1000 & 1000 FcB1 & al. 1997 (Myrtaceae) Twigs Nigerian

ICso (ng/ml) P. falciparum Kardono et. Eurycoma longifolia Roots Quassinoid Eurycomanone 47.7&48.1 D6&W2 al. 1991 (Simaroubaceae) [}-carboline alkaloid 7- methoxy-13 carboline-1- 3144 &2978

propionoic acid Fagara rhetza ICso(ll M) P. falciparum Shibuya et. (Rutaceae) Bark Acid amide Hazaleamide 43 al. 1992

Contd.

15

Plant species Part used Class of compound Compound Dose efficiency Plasmodium Reference &(Family) sp. & strain

ICso (~ g/ml) P. falciparum Likhitwitaya Garcinia cowa Bark Xanthones 7-0- Methylgarcinone 2.50 wid et. al. (Guttiferae) Cowan in 3.00 1998a

Cowanol 1.60 Cowaxanthone 1.50 13-Mangostin 3.00

ICso (fl g/ml) P. falclparum Francois Hannoa chlorantha Seeds & Quassinoids Chaparrinone 0.037 NF 54, clone et. al. 1998 (Simaroubaceae) Roots 14- Hydroxychaparrinone 0.188 A1A9

15- Desacetylundulatone 0.047 6-a-Tigloyloxyglaucarubol 0.257

Chaparrinone 90% P. berghei 14- Hydroxychaparrinone 84% Anka 15- Desacetylundulatone 98% 6-a-Tigloyloxyglaucarubol not active

ICso (fl g/ml) P. falciparum Milhau et. Juniperus communis Leaves & Essential oil ND 508&408 FcB1 & al. 1997 (Cupressaceae) Twigs Nigerian

ICso (fl g/ml) P. falciparum Milhau et. Lavendula augustifolia Leaves & Essential oil ND 267 &471 FcB1 & al. 1997 (Lamiaceae) Twigs Nigerian

ICso (~ g/ml) P. falciparum Bringmann Murraya euchrestifolia Branch. Carbazole alkaloids Murrayafoline A >50 NF, clone et. al. 1998 (Rutaceae) leaves & Bismurrayaquinone A >50 A1A2

Root bark 1-hydroxy-3-methylcarbazole 7.62 Murrayamine 31.62 2-(2'-Hydroxy-9'- 11.27 methylcarbazozyi0-3-methylcarbazol-1 A-quinone 2-(2'-Bis-(diacetoxy-3- >50 methylcarbazole) 1 A-Diacetoxy-3- 1.79 methylcarbazole

ICso (fl g/ml) P. falciparum Milhau et. Myrtus communis Leaves & Essential oil ND >1000 & 1000 FcB1 & al. 1997 (Myrtaceae) Twigs Nigerian

ICso (~ M) P. falciparum Likhitwitaya Nepenthes thorelii Roots Naphthaquinones Plumbagin 0.27 T9194 widet. al. (Nepenthaceae) 2-methylnaphthazarin 5.79 1998b

octadecyl caffeate 11.76 isoshinanolone 20.83 droserone 22.06 5-Methoxy-2-methyl-1 ,4- 0.89 naphthoquinone 5-Methoxy-2-methyl-1 ,4- 2.75 naphthaquinone-2,3-epoxide 3-Hydroxy-5-methoxy-2- 32.11 methyl-1 A-naphthaquinone 3-Acetyloxy-5-methoxy-2- 123.07 methyl-1 A-naphthoquinone 3,5-Dimethoxy-2-methyl-1,4- '3.88 naphthaquinone 2,2-Dimethyl-3-hydroxy-3- 56.82 methoxycarbonyl-4-methoxy-1-inden-1-one 3-Chloro-5-methoxy-2-methyl- 168.07 1 A-naphthaquinone

ICso (~ g/ml) P. falciparum Milhau et. Origanum vulgare Leaves & Essential oil ND 516 &488 FcB1 & al. 1997 (Lamiaceae) Twigs Nigerian

ICso(~ M) P. falciparum Sittie et. al. Phyllanthus fratemus Whole Alkamides E,E-2,4-octadienamide 192.8& 200 307 & Dd2 1998 (Euphorbiaceae) plant E,Z-2,4-decadienamide 87.1 & 91.3

ICso (fl g/ml) P. berghei Yao et. al. Polyalthia nemoralis Roots N.a Zinpolyanemine 2.5 1994 (Annonaceae) Cupric bis ( pyridine-N-oxide- 10

2-thiolate

Contd.

16

Plant species Part used Class of compound Compound Dose efficiency Plasmodium Reference I &(Family) sp. &strain

ICso (I! g/ml) P. falciparum Milhau et. Rosmarinus officina/is Leaves & Essential oil NO 267 & 220 FcB1 & al. 1997 (Lamiaceae) Twigs Nigerian

ICso (n g/ml) P. falciparum Figueiredo Salacia kraussii Root Quinone methides 28-nor-isoiguesterin-17- 94.0 & 79.9 K1 & P. et. al. 1998 (Celastraceae) carbaldehyde falciparum NF

17 -(methoxycarbonyl)-28-nor- 27.6 & 37.1 54 isoiguesterin 28-hydroxyisoiguesterin 114.4 & 140.2

Celastroloids Celastrol 180.9 & 254.2 Pristimerin 190.4 & 270.9 lsoguesterol 22.9 & 54.1

ICso (I! g/ml) P. falciparum Milhau et. Salvia officina/is Leaves & Essential oil NO >1000 & 1000 FcB1 & al. 1997 (Scrophulariaceae) Twigs Nigerian

ICso (I! g/ml) P. falciparum Castaneda Simira mexicana Stem bark Alkaloid Harman 0.97 et. al. 1991 (Rubiaceae)

ICso (!! M) P. falciparum Valsaraj et. Terminalia bellerica Fruit rind Lignans Termilignan 9.6 307 al. 1997 (Combretaceae) Thannilignan >50

7-hydroxy-3',4'- >50 9methelenedioxy flavan Anolignan 20.5

ICso (I! g/ml) P. falciparum Ohigashi et. Vernonia amygdalina Whole Sesquiterpene Vemodalin 4.0 K1 al.1994 (Asteraceae) plant lactone Vemolide 8.4

Hydroxyvemolide 11.4 Vemodalol 4.2

ICso(!l M) P. falciparum Oketch-Vernonia brachyca/yx Roots Coumarins 2'-epicycloisobrachycou- 160 & 54 307 & Dd2 Rabah et. (Asteraceae) marinone epoxide al. 1997

Cycloisobrachycoumarinone 111 & 54 epoxide

ICso (ng/ml) P. falciparum Mackinnon N.a. N.a Limnoids (Gedunin Gedunin 39&20 D6&w2 et. al. 1997 (Meliaceae) derivatives) 1 ,2-Dihydrogedunin > 1000 & 840

1 ,2-Epoxygedunin 2580 & 980 1 ,2-Dihydro-313-gedunol 4210 & 2440 7 -Deacetylgedunin 2610 & 1280 7 -ketogedunin > 1000 & > 1000 Tetrahydrogedunin 2500 & 900 21-Acetylgedunin 133 & 39 23-Acetylgedunin 832 & 156 Hexahydrogedunin 10,000 & 2130 Hirtin 173 & 96

N.a-Not available, N.D.- Not detected

Table 3b: A composite table of purified compounds derived from antimalarial plants showing their dose related effects against different species and strains of Plasmodia. Three plants with the most effective compounds have been highlighted.

17

Gliapler! Gllinomedicine and :7/nliprolozoaiYYanl Yhducls

1.1.3 ANTIAMOEBIC PLANTS

Amoebiasis is a major cause of morbidity and mortality in tropical areas, It is

more closely related to sanitation and socioeconomic status than to the climate

(Stanley 1996) and is known to be a major health problem in China, Mexico, the

eastern portion of South America, South-East and West Africa, and the whole of

South-East Asia including the Indian subcontinent (WHO 1985, Adams and MaCleod

1977). An estimated 480 million people, or 12% of the world's population are infected

with Entamoeba histolytica, amoebiasis causes an estimated 40,000-1,10,000 deaths

per year worldwide.

It is therefore not surprising that life style, the environmental conditions in the

tropics and the nonavailability of guaranteed conventional medical cure that people

have looked for natural symptomatic cures and resorted to self medication. A number

of traditionally found natural products are believed to be of medicinal value and have

been further studied for scientific validation.

Metronidazole was originally described by Casar and John in 1959 (Tanowitz

et. al. 1975), and is still the drug of choice in amoebiasis much as Chloroquine is in

malaria. Losch 1875 recorded the use of quinine enemas to treat patients with

amoebiasis.

Table 4 shows a compilation of research work of in vitro studies with various

plants against different strains of E. histolytica.

18

Plant species Part used Extract/ Name of Dose E. histolytfca · Reference (Family) Class of compound efficiency (in vitro)

compound

Acacia auriculiformis Legumes Ethanol ND Low activity at E. histolytica Uniyal et. (Mimosaceae) 1000 ~g/ml STA al. 1990

MIG (~g/ml) E. histolytica Tona et. al. Afchornea cordifolia Leaves Decoction ND 125 1998 (Euphorbiaceae)

100% killing E. histolytica Mirelman Allium sativum Cloves Crude garlic- at SAW et. al1987 (Liliaceae) extract oil Allicin 50 ~g/ml 1734Ric1AR

30 ~g/ml MIG (~g/ml) E. histolytica Bories et.

Annona cherimolia Fruits Methanol ND >100 Rehman al. 1991 Annona muricata Fruits Methanol >100 (Annonaceae)

EC1oo(~g/ml) E. histolytica Shah et. al. Ardisia oxyphylla Roots Dichloromethane 200 BY 80 1987 (Myrisnaceae) Petroleum ether 200

insoluble portion Rapanone 200 MIG (~g/ml) E. histolytica Sohni et.

Berberis asiatica N.a N.a ND 100 SFL3 al. 1995 (Berberidaceae)

MIG (~g/ml) E. histolytica Tona et. al. Bridelia ferruginea Root bark Decoction ND 62.5 1998 (Euphorbiaceae) Stem bark Decoction ND 250

ICso (~g/ml) E. histolytica Gillinet. al. Bruce a Fruits Quassinoids Bruceantin 0.018 HM-1 1982 antidysenterica Simalilactone D 0.047 (Simaruobaceae) Ailanthinone 0.068

Glaucarubolone 0.12 Glaucarubinone 0.14 Ailanthone 0.14 Glaucarubin 1.57

MIG (~g/ml) > E. histolytica Tona et. al. Cajanus cajan Leaves Decoction ND 500 1998 (Fabaceae)

MIG (~g/ml) E. histolytica Tona et. al. Carica papaya Mature Decoction ND ~ 7.81 1998 (Caricaceae) seeds

Immature Maceration in ND 62.5 seeds water

ICso (~g/ml) E. histolytica Anturlikar Cassia fistula Seeds Ethanol ND 109.65 364 in vitro et. al. 1993 (Caesalpineaceae)

ICso(~g/ml) E. histolytica Heinrich et. Caste/a texana Aerial parts Ethanol ND 31-63 NIH 200 al. 1992 (Simaroubaceae) Dichloromethane ND 4-8

Water ND 63 MIG (~g/ml) E. histolytica Tona et. al.

Ceiba pentandra Stem bark Decoction ND 125 in vitro 1998 (Bombaceae)

ICso (~ M) E. histolytica Yu et. al. Centipeda minima whole plant Sesquiterpene Brevilin A 4.5-9 in vitro 1994 (Asteraceae) lactone

ICso(~g/ml) E. histolytica Heinrich et. Chenopodium Aerial parts Ethanol ND 125-250 NIH 200 al. 1992 graveolens Dichloromethane ND 31-63 (Chenopodiaceae) Water ND >250

MIC(~g/ml) E. histolytica Shah et. al. Chonemorpha Roots N.a Chonemorphine 25 BY eo & SFL3 1987 fragnans dihydrochloride 100 (Apocyanaceae)

ICso (~g/ml) E. histolytica Keene et. Cinchona ledgeriana Leaves Fraction A ND 0.21 NIH 200 al. (Rubiaceae) Fraction B ND 3.0 1986

Fraction C ND 0.52 MIG (~g/ml) E. histolytica Tona et. al.

Cissius areloides Leaves Decoction ND > 500 1998 (Vitaceae)

Contd.

19

Plant species Part used Extract/ Name of Dose E. histolytica Reference (Family) Class of compound efficiency (in vitro)

compound

Clematis dioica ICso (llg/ml) E. histolytica Calzada et. (Ranunculaceae) Leaves Methanol ND 1294.19 HM 1-1 MSS al 1998

ICso ()lg/ml) E. histolytica Sharma Commiphora wightii Gum -oleo Chloroform NO 0.22 NIH 200 and (Burseraceae) resin Aqueous NO 0.24 Sharma

Petroleum ether NO 0.88 1996 Costus afer MIC (llg/ml) E. histolytica Tona et. al. (Zingiberaceae) Juice Decoction NO 125 1998 Crossopteryx MIC (llg/ml) E. histolytica Tona et. al. febrifuga Leaves Decoction NO 125 1998 (Rubiaceae)

ICso (llg/ml) E. histolytica Calzada et. Cuphea pinetorum Root Methanol 73.23 HM 1-1 MSS al 1998 (Lythraceae) Flavonoid Kaempferol 7.93

Quercetin 114.30 Datura arborea MIC (llg/ml) E. histolytica Tona .et. al. (Solanaceae) Leaves Decoction NO 125 1998 Dialum englerianum MIC (llg/ml) E. histolytica Tona et. al. · (Caesalpineaeceae) Stem bark Decoction NO 62.5 1998 Draceana reflexa MIG (llg/ml) E. histolytica Tona et. al. (Agavaceae) Leaves Decoction NO 62.5 1998

MIC (llg/ml) E. histolytica Tona et. al. Euphorbia hirta Leaves Maceration in NO 250 1998 (Euphorbiaceae) Whole water 31.25

plant Maceration in water

Fuchsia microphylla Stem/ ICso ()lg/ml) E. histolytica Calzada et. (Onagraceae) Leaves Methanol NO 759.87 HM 1-1 MSS al 1998 Garcinia kola MIC (llg/ml) E. histolytica Tona et. al. (Ciusiaceae) Stem bark Decoction NO 125 1998

ICso(llM) E. histolytica Gonzlez-Gossypium Seed oil N.a Gossypol 0.015 HM-1:1MSS Garza nad herbacium Said-(Malvaceae) Fernandez

1988 ICso(!lM) E. histolytica Gonzalez-

Gossypium sp. Seed oil N.a (-)-Gossypol 0.010 HM-1 Garza et. al (Malvaceae) 0.016 HK-9 1993

0.038 HM-3 0.026 HM-2 0.029 HM-38 ICso()lg/ml) E. histolytica Heinrich et.

Gouania polygama Leaves Ethanol NO 125-250 NIH 200 al. 1992 (Rhamnaceae) Dichloromethane NO 125-250

Water NO >250 Heinsia pulchella MIC (llg/ml) E. histolytica Tona et. al. (Rubiaceae) Root bark Decoction NO 15.62 1998 Helianthemum ICso()lg/ml) E. histolytica Calzada et. glomeratum Stem/ Methanol NO 158.22 HM 1-1 MSS al 1998 (Cistaceae) Leaves

dilution E. histolytica White 1933 Holarrhena Bark & Alkaloid Norconessine 1: 5000 integerrima Seeds Conessine 1:20,000 (Apocyanaceae)

MIC (llg/ml) E. histolytica Tona et. al. Hymenocardia acida Stem bark Decoction NO 31.25 1998 (Euphorbiaceae) Root bark Decoction NO 250

Significant E. histolytica Villaescosa Jasonia glutinosa Aerial parts Acetone NO inhibition at Rahman et. al. 1996 (Asteraceae) 100)lg/ml

MIC ()lg/ml) E. histolytica Tona et. al. Jatropha curcas Leaves Decoction NO 31.25 1998 (Euphorbiaceae) Justicia insularis MIC (llg/ml) E. histolytica Tona et. al. (Acanthaceae) Leaves Decoction NO > 500 1998 Mangifera indica MIC (llg/ml) E. histolytica Tona et. al. (Anacardeaceae) Stem bar1< Decoction NO $7.81 1998

Contd.

20

Plant species Part used Extract/ Name of Dose E. hlstolytica Reference (Family) Class of compound efficiency (/n vitro)

compound

MIC (~g/ml) E. histolytica Tona et. al. Maprounea africana Leaves Decoction ND 62.5 1998 (Euphorbiaceae) Root bark Decoction ND 31.25 Morinda morindoides MIC (~g/ml) E. histolytica Tona et. al. (Rubiaceae) Leaves Decoction ND 15.62 1998 Myrtanthus arboreus MIC(~g/ml) E. histolytica Tona et. al. (Moraceae) Leaves Decoction ND > 500 1998 Ongokea gore MIC (~g/ml) E. histolytica Tona et. al. (Oiacaceae) Stem bark Decoction ND > 500 1998 Parathesis ICso(~g/ml) E. histolytica Calzada et. chiapensis Stem/ Methanol ND 1170.68 HM 1-1 MSS al 1998 (Myristicaceae) Leaves Paropsia brazzeana MIC (~g/ml) E. histolytica Tona et. al. (Fiacourtiaceae) Root bark Decoction ND !> 7.81 1998 Pentac/etra MIC (~g/ml) E. histolytica Tona et. al. macrophylla Stem bark Decoction ND 250 1998 (Mimosaceae) Phytollaca MIC (~g/ml) E. histolytica Tona et. al. dodecandra Leaves Decoction ND > 500 1998 (Phytollacaceae)

ECso (~g/ml) E. histolytica Harries et. Picrasma excelsa Wood Quassinoid Quassin 0.5 NIH 200 al. 1982 (Simaroubaceae) Indole alkaloid Canthin-6-one 23

ICso (~g/ml) E. histolytica Ghosal et. Piper longum Fruits Ethanol 1000 NIH 200 al. 1996 (Piperaceae) Hexane 1000

Chloroform 500 n-Butanol 100 (Soluble) n-Butanol Not active (insoluble)

I

f Piperine Not active

98% E. histolytica Joshi et. al. Piper schmidtii Whole Hexane ND trophozoites 1990 (Piperaceae) plant killed at1000

~g/ml

MIC (~g/ml) E. histolytica Ahmed et. Prosopis juliflora N.a. Alkaloids Juliflorine 10 al. 1996 (Leguminosae) Julifloricine 10

Benzene insoluble 7.5 Alkaloidal portion

MIC (~g/ml) E. histolytica Tona et. al. Psidium guajava Leaves Decoction ND 62.5 1998 (Myrtaceae) Stem bark Decoction N.D. < 7.81 Pleridium aquilinum MIC (~g/ml) E. histolytica Tona et. al. (Pteridaceae) Twigs Decoction ND > 500 1998 Quassia africana MIC (~g/ml) E. histolytica Tona et. al. (Simaroubaceae) Root bark Decoction ND 31.5 1998

ICso(~g/ml) E. histolytica Heinrich et. Quercus oleoides Bark Ethanol ND 125-250 NIH 200 al. 1992 (Fagaceae) Dichloromethane ND 125-250

Water ND >250 Rauwolfia obstrata MIC (~g/ml) E. histolytica Tona et. al. (Apocyanaceae) Root bark Decoction ND 31.5 1998 Rubus corlifolius ICso(~g/ml) E. histolytica Calzada et. (Rosaceae) Stem/ Methanol ND 72.42 HM1-1 MSS al 1998

Leaves Sida rhombifolia MIC (~g/ml) E. histolytica Tona et. al. (Malvaceae) Leaves Decoction ND 62.5 1998

/ Contd.

21

Plant species Part used Extract/ Name of Dose E. histolytica Reference (Family) Class of compound efficiency (in vitro)

compound

Strychnos 100% E. histolytica Gasquetet. gossweileri Root bark Quaternary Diploceline inhibition at Rehman al. 1992 (Loganiaceae) alkaloid 50 J.lg/ml

At 1.5 E. histolytica VanBeek mg/ml Du et. al. 1984

Taebernaemontana Leaf+ twig Ethanol NO 20-80% aurantiaca T. chippii Leaf 20-80%

Root bark 80-100% T. contortarta Stem bark 0-20%

Leaf +Twig 80-100% T. crassa Stem bark 20-80%

Leaf 0-20% T. dichotoma Leaf 20-80%

Stem bark 20-80% T. eglandulosa Leaf 20-80%

Stem bark 20-80% T. g/andu/osa Leaf +Twig 20-80% T. heterophylla Leaf+ twig 0-20% T. longiflora Leaf+ twig 20-80% T. orienta/is Root bark 20-80% T. pachysiphon Stem bark 20-80% VanBeek T. penduliflora Leaf 20-80% et. al. 1984

Stem bark 80-100% T. psorocarpa Leaf 100%

Root bark 100% Stem bark 20-80%

T. undulata Stem bark 100% T. ventricosa Leaf 80-100% (Apocyanaceae) Stem bark 0-20% Tetracera poggei MIC (J.lg/ml) E. histolytica Tona et. al. (Dilleneaceae) Leaves Decoction NO > 500 1998

ICso(J.tM) E. histolytica Gonzalez-Thespepsia Seed oil N.a. (+)-Gossypol 0.235 HM-1 Garza et. al pupulana 2.086 HK-9 1993 (Malvaceae) 0.225 HM-3

0.455 HM-2 1.085 HM-38

Tithonia diversifolia MIC (J.lg/ml) E. histolytica Tona et. al. (Asteraceae) Leaves Maceration in NO 62.5 1998

water MIC (J.lg/ml) E. histolytica Bhutani et.

Ty/ophora indica Aerial parts Ethanol 150.0 & 75.0 NIH 200 al. 1987 axenic &

Tylophorine 6.25 &6.25 Polyaxenic hydrochloride

Phenanthroindolizi T ylophorine 400 & 400 dine alkaloids T y1ophorinine 25 & 50

Dernethyltybphorine 3.12 & 12.5 4-methoxy-14- 50 & 25 hydroxylylophorine Aoetyltylophorine 400 & 400 cl-septicine 300 &150

Tylophora hirsuta Aerial parts Ethanol ( Asclepiadaceae)

Phenanthroindolizi T ylohirsutinine 50 & 100 dine alkaloids 13a- 25 &12.5

rnethyttylohirsutinine 13a- 400 & N.D. rnethyttylohirsutinidine Tylohirsutinidine 200 & N.D. 13a- 50 & N.D. hydroxylylophorine 13a-hydroxysepticine 800 & 800 isotylocrebine 25 &25 14-deoxy-13a- 12.5 & 12.5 rnethyttylohirsutinidine

Contd.

22

Plant species Part used Extract/ Name of Dose E. histolytica Reference (Family) Class of compound efficiency (in vitro)

compound

Vitex madiensis MIC (f.lg/mi) > E. histolytica Tona et. al. (Verbanaceae) Leaves Decoction NO 500 1998 Voacanga africana Maceration in MIC (f.lg/ml) E. histolytica Tona et. al. {Apocyanaceae) Root bark water NO 62.5 1998

MIC (f.lg/ml) E. histolytica Sohniet. Whole formulation N.a Ethanol NO 1000 SFL3 al. 1995 containing Boerrhavia diffusa (Nyctaginaceae) Tinospora cordifolia (Menispermaceae) Berberis asiatica (Berberidaceae) Terminalia chebula (Combretaceae) Zingiber offici nate (Zingiberaceae)

N.a-Not available, NO-Not Detected

Table 4:A compilation of research work showing in-vitro studies with various strains of E. histolytica for screening with plant extracts, different fractions and purified compounds.

23

G.hapler I Cllmomedicine and:7lnh"prolozoaf'?lanl '?roducls

1.2.1 FORMULATIONS FROM CINCHONA

The first time a natural product gained wide acceptance as a treatment for

malaria was in the 161hcentury. It was then that the therapeutic action of the bark of the

Cinchona tree was disclosed by the natives of Peru to Jesuit missionaries, who, in

turn, brought the word of its utility to Europe (Klayman 1985, Smith 1976). Peruvian

bark was brought to Spain in 1636 and its ingredient, quinine was later named after the

fourth countess of Cinchona, wife of the viceroy of Peru (Gibbs 1985). Cinchona

contains mixture of more than twenty alkaloids (Amabeoku 1991 ). Quinine the alkaloid

isolated in 1834 from Cinchona bark by the French chemist Pelletier became the main

treatment for malaria until the 1930's when the synthetic antimalarials were developed

{Phillipson and Wright 1991 ). The antimalarial action of quinidine was described by the

Madras Cinchona Commision in 1866-1868 (Phillips 1984 ). For three centuries it stood

virtually alone as the sole effective treatment for a life-threatening infectious disease

(Greenwood 1992, Mukherje 1946).

Quinine A quinoline methanol and a highly active schizontocidal drug with a short

half life, is used orally and in injectable form. It affects on the intracellular transport of

membrane components & macromolecules, and on phospholipase activity (Panisko

and Keystone 1990) and inhibition of the haem polymerase leading to the

accumulation of soluble haem toxic for the parasite. CQ resistant parasites

accumulate the drug to a lesser extent than do sensitive parasites. Quinoline

methanols may exert their action by disrupting the membrane trafficking events

involved in the uptake of metabolites (Foley and Tilley 1997).

Primaquine It is an 8-aminoquinoline and reduces the likelihood of relapses by its

activity against the liver stages.

Pyrimethamine This drug is sporontocidal and an inhibitor of dihydrofolate

reductase required to inhibit the mammalian enzyme (Panisko and Keystone 1990).

Chloroquine It is the prototype of the synthetic 4-aminoquinolines and inhibits the

polymerization of haem.

Mefloquine It is a synthetic 4-quinoline methanol, developed for itsschizontocidal

activity against chloroquine-resistant P. fa/ciparum and is shown to bind to erythrocyte

24

G.haplerl 0!/momechcine and:7lnliprofozoai7Yanl Producls

membrane phospholipids (Chevli and Fitch 1982).

Proguanil This is a dihydrofolate reductase inhibitor.

Sulfonamides and Sulfones These drugs have a mild schizontocidal effect and

are recommended against CQ resistant P. falciparum only.

Sulfadoxine-pyrimethamine (Fansidar) This combination was originally developed

and promoted for its efficacy against chloroquine-resistant P. falciparum.

Halofantrine The drug has action similar to that of mefloquine. Absorption of

halofantrine is very erratic and even with therapeutic doses, serious complications can

arise.

Amodiaquine It is a 4-aminoquinoline related to chloroquine.

Atovaquone An antimalarial drug combined with proguanil in an attempt to prevent

or delay the emergence of resistance, is currently in advanced phases of international

clinical development for the treatment of uncomplicated falciparum malaria. It reduces

the parasite oxygen consumption rate in a concentration-dependent manner (Murphy

and Lang-Unnasch 1999).

Tebuquine It is a 4-aminoquinoline, significantly more active than amodiaquine and

chloroquine as compared to its analogues fluorotebuquine and dehydroxytebuquine

(0' Neill et. al. 1997). WR 194,965 phosphate, a new antimalarial agent containing a

biphenyl ring structure was found active against CO-resistant P. fa/ciparum (Karle and

Karle 1988).

1.2.2 FORMULATIONS FROM ARTEMISIA

The herb Artemisia annua called "quinghaosu" has been used for many

centuries in Chinese traditional medicine as treatment for fever & malaria and was first

recorded in an Apothecary's list, dated 168 BC titled "Prescriptions for 52 kinds of

disease" from the Mawangdui Han dynasty tomb in Chansha, Hunan province (WHO,

Geneva 1994, Hien and White 1993). In 1971, Chinese chemists isolated from the

leafy portions of the plant the substance responsible for its reputed medicinal action.

This compound called qinghaosu (QHS, artemisinin) is a sesquiterpene lactone

containing an epoxide function (Valecha and Tripathi 1997, Avery et. al. 1996). Its

25

G.haplerl 0!/momedicine and!7/nhprolozoal:?fanl :?roducls

structure includes an endoperoxide bridge (C-0-0-C), which is unique among

antimalarial drugs. Its plasmodicidal effects have been confirmed in experimental

studies in animals and in clinical trials against both Chloroquine sensitive and resistant

strains of P. falciparum (Gu et. al. 1983). Artemisinin derived drugs have been proved

particularly effective in treatment for severe malaria even for multi drug resistant

malaria (Geldre et. al. 1997, Day 1996, Bharel et. al. 1996). Artemether is as effective

as quinine in the treatment of cerebral malaria in children (Hensbroek et. al. 1996). The

drug is now cleared for marketing in India for hospital use. M/SThemis Chemical Ltd.,

Bombay is currently one of the licencees for the drug, which markets the drug in 3 x2

ml injections, a pack for Indian hospitals under the brand name EMAL (Kumar 1998).

Artemisinin It is poorly soluble in water and decomposes in other solvents.

Therefore its derivative, dihydroartemisinin was produced. Its action seems to be a

haemin-catalyzed reduction of the peroxide moiety, which results in free radicals and

reactive aldehydes that subsequently kill the malarial parasites.

Dihydroartemisinin It is a hemiacetal (lactol) and exists as a mixture of a and 13

anomers

Artemether A methyl ether of dihydroartemisinin that is synthesised by a two step

procedure.

Artesunate It is a water soluble hemisuccinate derivative.

Arteether It is the 13-anomer of the ethyl ether of dihydro artemisinin and has similar

chemical and physical properties as artemether.

Artelinic acid It is a moderately stable, second generation water-soluble compound.

1.2.3 MICELLANEOUS ANTIMALARIAL FORMULATIONS

Ayush - 64 In India, the scientists at Central Council of Research inAyurveda and

Siddha (C.C.R.A.S.) worked for more than a decade to evolve the formula of a coded

antimalarial medicine "Ayush 64", a simple formulation combining four drugs together

in definite proportions. This drug, is not only free from any harmful side effect but also

improves the functioning of the liver and doesn't give the usual after effects of malaria

like giddiness and depression. The results are best and quickest in P. vivax while in P.

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0./iapler I 01/momedicine and:7lnliprolozoal'?fanl '?roducls

fa/ciparum a slightly longer therapy is needed. M/S. Gudimani Enterprises (India) has

adopted this formula and commercialising the drug, which is named as " Malcurin"

(Sharma et. al. 1981, Gujjar 1983).

Tubulozole isomers Recently recognized as a new class of potential antimalarial

agents, are thought to inhibit malaria by interaction with tubulin or some other

component of the microtubules (Dieckman -Schuppert and Franklin 1990).

Antibiotics Like tetracycline, minocycline, doxycycline, linomycin, clindamycin,

erythromycin, spiramycin are inhibitors of protein synthesis. Pyrimethamine, proguanil,

cycloguanil and pyrimethamine plus sulfadoxine combination (Fansidar) are antifolates

which preferentially interfere with folic acid cycle of the parasite (Bhasin 1996).

lonophore antibiotics are also positive antimalarial agents causing cytocidal effect

against all parasitic stages rather than being stage specific in nature (Gumila et. al.

1997).

Xanthones Combines with haem to form soluble complexes, . thus inhibiting the

process of haem polymerization (lgnatuschenko et. al. 1997)

Antimalarial oxidant drugs Enhance the production of oxygen radicals inside

parasitized erythrocytes or act to make parasite more susceptible to attack by oxygen

radicals (Winter et. al. 1997b ).

Iron chelators {Reversed Siderophores, RSFs) Probably induce an iron deficit in

the parasites by displaying physicochemical properties that favour iron extraction

beyond membrane barriers of P. falciparum-infected RBC (Lytton et. al. 1993).

Rufigallol A recently identified active antimalarial compound showed a remarkable

synergistic antimalarial interaction with another structurally similar compound exifone

or Ad lone (Winter et. al. 1996a).

Pyronaridine A Mannich base has recently proved to be effective in both

chloroquine and multidrug-resistance areas. Benflumetol, a fluoromethanol is under

development for concurrent use with artemether, but limited efficacy data are available.

A series of chalcones and their derivatives have been synthesized and identified as

novel potential antimalarials (Li et. al. 1995).

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1.3 AMOEBICIDES

Metronidazole (1-f3 hydroxyethyl-2-methyl-5-nitroimidazole )-In vitro antiamoebic

activity was first described with metronidazole by Gordeeva 1965. Loss of majority of

ingested starch granules within 6 hours and replacement with vacuoles of different

sizes resulted in death within 12 hours (Grewal 1968).

Metronidazole was shown to possess mutagenic activity (Connor et. al. 1977).

It was also shown to induce lung tumors and increase incidence of malignant

lymphomas (Rustia et. al. 1972). In addition to the possibility of developing resistance

to the drug through widespread and unnecessary use, metronidazole is intended only

for elimination of tissue-invasive organisms and is not very effective against intestinal

lumen infections (Clark 1998).

The major Ipecacuanha alkaloids, emetine and cephaeline are used in a

number of linctus and pastille preparations in amoebiasis treatment. The general

effectiveness and safety of ipecac syrup is generally acceptable but it is also known for

its toxicity as involving primarily gastrointestinal, cardiovascular and neuromuscular

foci (Manno and Manno 1977)

Direct and indirect antiamoebic effects of a given compound in its selective

sensitivity to specific antiamoebic agents e.g. Emetine, Dehydroemetine, Chloroquine,

Conessine, 8-hydroxyquinolines, Enterovioform (5 chloro 7 iodo 8 hydroxyquinoline),

Chloroquine [7 chloro 4 (4-diethylamine-1-methylbutyl) aminoquinoline], Entobex (4,7

phenanthroline-5,6 quinone), Diloxanide furoate, lntestopan (Quina-quinaldine

derivative). etc. is found to be remarkable in E. histofytica (Youssef 1968).

Emetine, and related ipecac alkaloids correlate well with their capacity to inhibit

protein synthesis in vivo and in vitro. Puromycin and Cycloheximide are amoebicides,

which inhibit protein synthesis secondary to effects on other metabolic pathways in

animal cells by more than 50% at minimal concentration and eventually, causes death

of cent-percent amoebae (Entner 1979, Entner and Groll man 1973).

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1.4 DNA-BINDING OF DRUGS AND NATURAL COMPOUNDS

Extracts by definition are multi-substance mixtures obtained from specific parts

of the plant with active ingredients, which are qualitatively and quantitatively different.

Such phytogenerics, which deals with extract identical preparations for the

pharmaceutical and clinical applications, requires the understanding of the bioactivity,

bioavailability and bioequivalence of phyopharmaceuticals (Loew 1997). Field work

exploring the medicinal uses of plants by indigenous peoples in remote areas coupled

with the introduction of ex vivo assays are able to determine their biological effect, and

has facilitated the discovery of bioactive molecules produced by medicinal plants.

Methyl green reversibly binds polymerised DNA and the ability of known and unknown

DNA active compounds to disrupt the DNA/methyl green complex can be conveniently

and easily assessed by spectral changes resulting by its intercalation (Burres et. al.

1992).

Drugs that interact with nucleic acids are well known anticancer

chemotherapeutic agents and their cytotoxicity is reflected in detailed structural

changes in DNA. Antibiotics, antiviral and antiprotozoal agents have bioactivities in

common in complexing with DNA, a rationale that can be exploited in drug design.

Chloroquine is known to enhance the cytotoxic activity in multiple-drug resistant

leukemic cells by an increased formation of cytoplasmic vacuoles and is a modulator of

anticancer drug action (Zamora and Beck 1986). A broad range of biologically active

compounds including chloroquine form molecular associations with Doxorubicin

(adriamycin) and may have common properties in multi-drug resistance in cancer cells

and drug resistance in P. falciparum. In vitro sensitivity of P. fa/ciparum to drugs

particularly to chloroquine are based on alterations of DNA or inhibit its synthesis (Lee

and lnselburg 1993). The alterations in absorbance of DNA methyl green complex in

the presence of plant extracts of medicinal value has been studied extensively and is

related to their antioxidant and other cytotoxic activity.

29