Medicinal plants in Suriname: screening of plant extracts for receptorbinding activity

Phytom edicine Vol. 4 (1), pp. 59- 65,1 997 © 199 7 by Gu sta v Fischer Verlag

Medicinal plants in Suriname: screening of plant extracts for receptorbinding activity J. A. HASRAT1, J.-P. DE BACKER2, G. VAUQUELlN2 and

A. J. VLlETINCK1 I

2

Department of Pharm aceut ical Sciences, University of Antwerp, Antwe rp, Belgium . Depa rt ment of Protein-chemistr y, Free University of Brussels, St. Genesius Rod e, Belgium.

Summary Ligand-b inding-studies on twelve different receptors were used for screening extracts from plants from Suriname (South-America ). The results on 5-HT j A , A j and NMDA receptor s promote furthe r investigation of Microtea debilis, Hibiscus bifurcatus, Irlbarchia purpurascens, Miconia ciliata and Mimosa myriadena. Key words: ligand -binding-stud ies, receptor s, Suriname, medicinal plants.

Introduction Plants are one of the most importa nt tools in traditional medicine from Suriname. Since Suriname is a multi-cultural society (Asians, Africans and American-Indians), not all plants used in traditional medicine are indigenous; some of th em were imported from the other continent s. Over the past fifteen years several erhno boranic investigations on medicinal plant s of Surina me have been carried out, more specifically in the fields of ar row poisons (Plotkin, 1990; 1993 ) and antimicrobial activities (Verpoorte, 1982 a; 1982 b; 1983; 1987). Th ese plant s, however, have not been documented prop erly up till now, so that much of the knowl edge might be lost soon. Mo reover, many plant s or related species that are found in Suriname and from which noth ing is known in Suriname as regard to their medicinal appli cat ions, are used elsewhere in the South-American continent in traditional medicine. According to William s (Williams, 1991 ) the use of a screening approach can greatly facilitate the discovery of intere sting leads for the developme nt of new and safer dru gs. For the screening we used ligand -binding studies (LBS), a meth od that has proven to be useful for this purpose (Williams, 1991). With LBS it is possible to investigate very rapidly the affinity of products for specific receptors so that many samples can be investigated simultaneously. Anoth er ad vantage of LBS is th at only minute amo unts of ma-

terial are requ ired to evaluate a new compound (5- 10 mg for 20-30 assays) comp ared to what was needed before radio-binding became available (50-200 mg for one assay). The expenditure decrea sed a bout 30 times for one compound per assay (Williams, 1991 ). Assuming that th ese benefits also apply to the screening an d evaluation of crude plant extracts from Suriname, we present their screening on different receptors in human and animal tissues. Since many medicinal plants are used by traditional healers for th e treatment of distu rbance s of the central nervous system, we selected th e following recept ors: a j , a 2A , a 2B and P2 adr energic receptors; M j , M 2and M J muscarinic receptors; 5-HT j A and 5-HT 2 serotonin recept ors; D. and D 2 dopaminergic recepto rs; A j adenos ine recept or and the Nmethyl-Dvaspart are (N MDA) excita tory amino acid receptor compl ex.

Material and Methods

Plants. Th e plants and plant parts used are described in Table 1. The vegetation of Suriname ist part of the Amazon ra inforest. Th e plants were collected in a region of 50-60 kilometers from the seacost. Geographically, thi s re-

60

J. A. Hasrat et al.

gion lies in the young and old costal area and partly in the Zanderij series. The plants were identified by M. Werkhove from the herbarium of the University of Suriname, where the voucher specimens have been deposited. • Plant extracts: The plant material was sun-dried, powdered and extracted with 80 %, ethanol by maceration. The macerated plant suspension was filtered and the marc was packed into a percolator and percolated with 80 % ethanol until exhaustion. The filtrate and percolate were combined and evaporated under reduced pressure at a temperature of 40°C till dryness. For LBS the residue was dissolved in DMSO at concentrations of 100 ug/ml and 10 ug/rnl. • Partition of extracts: The plant extracts which produced a pronounced decrease in radioligand binding to certain receptors were partitioned between water and chloroform. These fractions were then evaporated till dryness under reduced pressure at a temperature of 40°C. For LBS, the residue of the water layer was dissolved in a dimethylsulfoxyde (DMSO)/water mixture, whereas the residue of the chloroform layer was dissolved in DMSO only. Two concentrations, 100 ug/ml and 10 ug/ml, were prepared and the DMSO concentration did not exceed 1 % in the test tube. • Applications of the plants: There was no information about the traditional use for some plants, but information about related species could be found. The information on the medicinal use of some of the plants in Suriname has been found in Heyde (Heyde, 1985), and for some others or related species in Morton (Morton, 1981). The applications of the plants or related species are presented in Table 2.

Radioligand-binding assays • Materials: Sources of drugs were as follows: [3H]prazosin (22 Ci/mmol), [3H]SCH 23390 (80 Ci/mmol), [3H]RX 821002 (44-49 Ci/mmol) and [3H]1,3-di-propyl-8-cyclopentylxanthine ([3H]DPCPX) (107 Ci/mmol) were from Amesham, U. K.; [3H]spiperone (24 Ci/mmol), [3H]rauwolseine (80 Ci/mmol), [3H]dihydroalprenolol, [3H]DHA (48 Ci/mmol), [3H]quinuclidinyl benzylate, [3H]QNB (33 Ci/mmol) and [3H]MK-801 (15-30 Ci/mmol) were from New England Nuclear, Boston, MA; serotonin (5-hydroxytryptamine; 5-HT), yohimbine, 8-hydroxy-2-(di-npropylamino)tetralin (8-0H-DPAT), isoproteronol bitartrate were from Sigma; (+)-butaclamol was from Research Biochemicals Inc., (U.S.A.); spiperone from Janssen Pharmaceutica (Beerse, Belgium); and atropine sulfate monohydrate from Aldrich Europe (Beerse, Belgium). (R)-N6-(2phenyl-l-methylethyll-adenosine [R(-)PIA] was fromBoehringer Mannheim (Germany). Phentolamine hydrochloride was a gift from Ciba Geigy. • Membrane preparation: Brains from individuals, who died suddenly from heart attack were provided by the Department of Neurology, Academic Hospital, Free University Brussels (AZ-VUB), Belgium. Brains were removed within 5 h after death and immediately frozen at -30°C. Subsequent manipulations were performed at 0-4 0C. Small pieces of frontal cortex, hippocampus, putamen, pons and submandibular gland were dissected by a neuroanatomist. Cortex and putamen tissue were first homogenized with an ultraturax for 15s in 10vol. of 50mM Tris-HCl (pH 7.5)110 mM MgCl 2 (buffer A) and then further homogenized with a motor-driven Potter Elvehjem homogenizer (10 strokes at maximum speed). The homegenate was

Table 1. Plants and parts used for screening. Plant species with their voucher specimen number

Vernacular name

Family

Plant/Part

Averrhoa bilimbi L. (10) Bellucia grossularioides Triana (14) Commelina nudiflora L. (8) Costus niveus GFW Meyer (15) Crescentia cujete L. (9) Cucurbita moschata Duch. (7) Hibiscus bifurcatus Cav. (17) Irlbarchia purpurascens (Lisianthus uliginosus) Aublet (Maas) (16) Miconia ciliata (Rich) DC. (3) Microtea debilis Swartz (13) Mimosa myriadena Benth. (4) Momordica charantia L. (6) Peperonia pellucida HBK (18) Phyllantus amarus Schum. etThonn. (5) Physalis angulata L. (12) Pseudocalymna alliacea Sandw. (19) Scoparia dulcis L. (11)

Birambi Mispel Gado dede Sangrafoe Kalebas Pompoen Jorka okro Savanne bluebell

Oxalidaceae Melastomaceae Commelinaceae Zingiberaceae Bignoniaceae Cucurbitaceae Malvaceae Gentianaceae

leaves leaves whole plant aerial part leaves flower aerial part whole plant

Mispel Eiwit wiwiri Wacht een beetje Wilde soprpo Konsaka wiwiri Fini bita Batoto bita Knoflook liaan Sisibi wiwiri

Melastomaceae Phytolaccaceae Mimosaceae Cucurbitaceae Piperaceae Euphorbiaceae Solanaceae Bignoniaceae Scrophulariacea

leaves whole plant aerial part whole plant whole plant whole plant whole plant aerial part whole plant

Medicinal plants in Suriname centrifuged at 2000 g for 15 min and the superna tant was then further centrifuged at 29 000 g for 20 min. The resulting pellet was washed thre e times by resuspension in fresh buffer A and centrifuged at 29 000 g for 20 min, resuspended in 20 mM Tris-HCI (pH 7.5)/10 mM MgCl 2 buffer containing 10 % (v/v) glycerol and stored in liquid nitoge n. Pieces of hippo campus, pons and submandibular gland were hom ogenized in 10 mM sodium phosphate (pH 7.5)/ 145 mM NaC I (buffer B) as above. The homogenat e was cent rifuged for 15 min at 500 g and then for 20 min at 29 000 g. Th e latter pellet was washed 3 times in buffer B as abov e, suspended in buffer B conta ining 10 % (v/v) glycerol and stored in liquid nitrogen. Male Wistar rats (250-300 g) were killed by decapit ation. Lungs and kidneys were dissected, freed of connecti ve tissue, and homogenized in 145 mM NaCI as above. The homogenate was centrifug ed for 15 min at 1000 g and then for 20 min at 29 000 g. Th e pellet was washed 3 times in buffer A, suspended in buffer A conta ining 10 % (v/v) glycero l, and sto red in liquid nitrogen. Membranes from rat forebrain (witho ut striat um) for adenosine-1 receptor assays were prep ar ed by homogeni zing the tissue in 0.32 M sucrose solution as above for 30 sec at 500 g. The homogenate was first centrifuged at 1000 g for 10 min and the supernatant centrifuged at 30 000 g for 30 min. The pellet was then suspended in H 20 , kept on ice for 30 min and centrifuged once more at 30 000 g for 30 min. Th e pellet was then washed twice (by resuspension and centrifugation as above) with 50 mM Tris-H CI (pH 7.4) and the final pellet was resuspended in 50 mM Tris-H CI (pH 7.4) and incubated for 30 min with 2 D/ml adeno sine deaminase at 37°C. The membrane suspension was stored in liquid nitrogen.

61

Memb ranes from rat forebrain (witho ut striatu m) for NMDA recept or compl ex assay were prepared by homo genizing the tissue with an ultr aturax for 15 sec in 10 vol. of 5 mM Tris-H CI (pH 7.4) and further with a motor-driven Potter Elvehjem homogenizer (10 strokes at maximum speed). The homogenate was centrifuged at 500 g for 15 min and the superna tant cent rifuged at 29 000 g for 20 min. The latter pellet was washed once more by resuspension in fresh buffer and centrifuged at 29 000 g and stored in liquid nitrogen. On the day of the assay, the pellet was thawed and washed 3 times by resuspension in fresh 5 mM Tris-H CI (pH 7.4) buffer and centrifugation at 29 000 g. Protein concentrations were determined according to the Sopachem total protein assay (Sopar-Biochem) or by the method of Pierce (Smith et aI., 19 85), using bovine albumin as sta ndard. • Radioligand binding: The followin g procedure was adopted for most competition binding experiments. Me mbrane s (0.5- 2 mg protein/ml, final concentration) were incubated with a fixed concentr ation of radioligand and tw o concentra tions of the plant extracts in 200 ul of buffer under the conditio ns given in Table 3. At the end of the incubation, the samples were filtered und er reduced pressure through a glass fibre filter (Whatman GF/B), and rapidly washed four times with 4 ml of ice-cold incubation buffer. The amou nt of radioli gand remaining on the filters was subsequently determined by liquid scintillation counting. Binding of [3H]prazosin to a t adr enoceptors was performed in hum an fro nta l cortex membr anes following the method described by Bott ari et al. (1983). Binding of [3H]RX 821002 to a 2A and a 2B adr enoceptors was perform ed in hum an fronte! cortex as described by Vauque!in et al. (199 0).

Table 2. Applicat ions of th e plant s or related species. Plant name

Applicat ion s

Averrhoa bilimbi Bellucia grossularioides Commelina nudi{/ora

diabetes, inflammation, hypertension, hepat itis, diarrh ea sunstro ke, diuretic, sterility in wo men, sedative, itchi ng kidney complaints, wou nds, venerea l diseases, hypertension , menstru ation cramps, haemorrhage of the uterus, malaria fever, diuretic, tuberculosis, abor tivum flatulence, exorcism, thr oarache, typhu s, pur gative to aid delivery, cough and cold, lungcomp laint s, skin diseases jaundice, measles and small pox contusions, nervousness, fever, sprai ns, emollient, cold remedy, inflame d eyes, emetic infected wounds, dysentry, stomache, carminative and bitter tonic, emmenagogue, nervous dyspep sia, venera l diseases sunstroke, diur etic, depu rative and cooling dr ink, itching, sudurific, night sweats, seda tive protienu ria, coughs, sto mache asthma , to nic, coughs and cold, emetic, nephr itis, nervou sness appetite, to nic, colds and fever, arthritis, hypertension, diabetes, kidney sto nes, colic eyebath, constipatio n, recta l inflammation, heart complaints colic; sto mac he, fever, colds and influenza, kidney disord ers nephritis, jaundice, gonorr hea, mala ria, diuretic rheumatic pain, vermifuge, fever, colds bro nchitis; cough, diabetes, urinary burning, jaundice, rash, broncho-pulmonary complaints, tonic, febrifuge

Costus niveus Crescentia cuiete Cucurbita moschata Hibiscus bifurcatus Irlbarchia purpurascens Miconia ciliata Microtea debilis Mimosa myriadena Momordica charantia Peperomia pellucida Phyllantus amarus Physalis angulata Pseudocalymna alliacea Scoparia dulcis

62

j. A. Hasrat et al.

Several of the radioligands are able to bind to more than one receptor; binding of such radioligands to undesired receptors was prevented by masking these receptors with selective ligands. [3H]RX 82 1002 has proved to be a powerful ligand for the identification and characterization of
ser et al. (1989). [3H]Spiperone ist a Drdopamine antagonist, but it also labels 5-HT2 receptors in membranes from the human CNS (De Keyser et aI., 1985 ). The huma n putamen possesses a high concentration of D2 receptors and binding of [3H]spiperone to the 5-HT 2 receptors was masked with O.3IlM mianserin (De Keyser et aI., 1985). Binding of [3H]dihydroalpreno lol [3H]DHA to ~2 adrenoceptors was performed in rat lung membranes, as described by Severne er al. (1985). Binding of [3H]quiniclidinyl benzylare [3HlQNB to M 1-, M 2- and M r muscarinic receptors was determined in membranes from human hippo campus, pons and submandibular gland respectively, as described by Vanderheyden et al. (1990) . Binding of [3H]DPCPX to Al receptors in rat forebrain membranes was performed as described by Lohse et al. (1987) . Binding of [3H]MK-801 to NMDA receptor chan nels was determine d as described by Kornh uber et al. (1989 ). The results presented are expressed as the fraction (percentage) of the radioligand still bound to the receptor.

Table 3. Conditions for competition binding studies for different receptors. Radioligand

Receptor

Tissue

Type

(nM)

Concen- Non-specific binding tration (nM) Condition

al ·adrenergic

H uman cortex

[lH]prazosin

0.69

5

11.lM phentolamine

a2A-adrenergic

Human cortex

[3H]RX 821002

3.5 a

6

30 I.lM yohimbine

~B-adrenergic

Rat kidney

[3H]RX 821002

8.3

2

30 11M yohimbine

5-H T 1A·serotonin

Human cortex

[3H]rauwolscine

9.9 b

5

0.1 11M 8-0H DPAT

1.3/7C

2

111M (+)-butaclamol

O.13d

0.5

111M (+)-bu taclamol

Kn

[3H]SCH 23392 D]-dopamine/5HTz- H uman cortex serotonin Human putamen [3H]spiperone D 2-dopamine pz-adrenergic

Rat lung

PH]DHA

1.3

5

Mj-muscarinic " ~.

Human hippocampus Human pons

[3H]QNB

0.091 e

0.5

lOOI1M (-j isoproteronol 111M atropine

[l H]Q N B

0.0 74 e

0.5

1 11M atropine

Human submandibular gland Rat forebrain Rat forebrain

[lH ]Q NB

0.12 e

0.5

111M atropine

[3H]DPCPX [3H]MK -801

0.28

004 3

1OI.lM R-PIA

M1·muscarinic " ·

~

M j-muscarinic" **

AI-adenosine NMDA receptorchannel complex

Incubation conditions" 15 min at 3]oC, TB 50mM + 10 mM MgCl 2 + O.3I.lM 5HT 15 min at 3]oC, TB 50mM + 2 mM MgCI 2 + 0.3 1.l 5HT 15 min at 37°C, TB 50 mM + 2 mM M gClz + 0.3 11M 5HT 15 min at 37°C, TB 50mM + lOmM MgCl z + 5 1.lM (-)adrenaline 20 min at 30°C, TB 50 mM + 10 mM MgCl z + 145 mM NaCI 20 min at 30 ·C, TB 50 mM + 10mM MgCl z + 145mM NaCl + 0.3 11M mianserin 20 min at 30 ·C, TB 75 mM + 25 mM MgCl z 30 min at 30 ·C, PB 10 mM + 145 mM Na Cl 30 min at 30°C, PB 10 mM + 145 mM NaCI 30 min at 30°C, PB 10 mM + 145mM NaCl 60 min at 25 ·C, TB 50 mM ** 60 min at 20°C, TB 5 mM~~

"Incubation in Tris-HCl buffer (TB) or phosphate buffe r (PB) pH 7.5; ' f "TB pH 704. *.,*Acetylcholine receptor subtype. aFrom Vauquelin et al. (1990); bfrom Convents et al. (1989); 'from De Keyser et al. (1989); dfrom De Keyser et al. (1985 ); "from Vand erheyden et al. (1990).

Medicinal plants in Suriname

Results The results of the screening are shown in table 4. None of the plant extracts interfered significantly with the a l , D/5HT2 , D 2 , ~2' M l and M 3 receptors and even an increase in the binding was observed in certain instances. On the contrary, most plant extracts (11 from 17) decreased the binding of [3H]rauwolscine to the 5-HT 1A receptor by more than 40 %, 4 to 5 extracts interfered with the binding of the radioligands to the M 2 and A l receptors and NMDA recep-

63

tor channel, and 2 extracts inhibited the binding of [3H]RX 821002 to the a 2A and a 2B receptors. The most important effects (a decrease of the radioligand binding of more than 60 %) were shown by Microtea debilis on the Al receptor, Momordica charantia on the a 2A receptor and Scoparia dulcis, Irilbarchia purpurascens and Hibiscus bifurcatus on the 5-HT lA -receptor, The results of the activity of the partition fractions of the plant extracts, which decreased the radioligand binding to some receptors to a high extent, are given in Table 5. In gen-

Table 4. Results of the screening of 80 % ethanol extracts of plants of Suriname on several receptors using inhibition of the binding of a suitable radioligand for any of these receptors as bioassay. For more details about the experimental procedures see table 3 and text. The results are expressed as percentage of the radio ligand still bound to the receptor. Every value in the table is the average, with its standard error, of the results of three to six experiments (in all experiments every sample was measured in triplo). The inhibition of the binding of the radioligand was measured with two concentrations of the extracts, I = 10 ug/ml and II = 100 ug/ml. The inhibition of the binding of the radioligand to the NMDA receptor complex was performed with the addition of glycine (GLY) to the incubation buffer. Table4A aZA II

Plant name

Averrhoa bilimbi Belluciagrossularioides Commelina nudiflora Costus niueus Crescentia cujete Cucurbita moschata Hibiscus bifurcatus lrlbarcbiapurpurascens Miconia ciliata Microteadebilis Mimosa myriadena Momordica charantia Peperonia pellucida Phyllantusamarus Physalis angulata Pseudocalymnaalliacea Scoparia dulcis

80 ± 7 102 ± 4 83 ± 5 89 ± 10 84 ± 1 114±3 75 ± 2 66 ± 7 137 ± 9 79 ± 8 128 ± 9 68 ± 5 83 ± 4 128 ± 31 90 ± 14 83 ± 1 89 ± 5

86 ± 5 115 ± 12 87 ± 3 96 ± 42 82 ± 7 103 ± 13 77 ± 2 73 ± 5 154 ± 2 80 ± 12 135±9 59 ± 21 87 ± 6 169 ± 39 107 ± 4 82 ± 5 92 ± 8

II 96 ± 16 81 ± 19 111 ± 7 88 ± 5 114 ± 6 92 ± 18 100 ± 7 79 ± 8 93 ± 5 108" 5 109" 3 100 ± 5 83 ± 18 106" 5 98" 10 95 ± 2 93 ± 6 111 ± 26 100 ± 6 94 ± 4 94" 18 92 ± 6 32 ± 5 96" 8 90 ± 7 85 ± 6 101 " 1 100 ± 4 97 ± 4 106 ± 6 88 ± 4 98" 4 48 ± 4 74" 7

98 ± 12 110±8 87 ± 4 108 ± 10 72 ± 11 115 ± 3 88 ± 4 108 ± 5 102 ± 3 104 ± 5 104 ± 29 116 ± 5 94 ± 6 133±7 122 ± 2 93 ± 10 94 ± 38

D/5HT z I II

II

II

81 ± 1 92 ± 10 81 ± 2 97 ± 5 65 ± 7 115 ± 3 77 ± 1 98 ± 3 121 ± 2 96 ± 2 85 ± 2 43 ± 5 100 ± 3 136±1 117 ± 4 99

±

4

54

±

9

81 ± 25 71 ± 15 89" 2

55 ± 4 85 ±1 66 ± 11 93 ± 19 69 ± 5 209 ± 66 68 ± 12 138 ± 63 79 ± 12

56 ± 2 49 ± 11 51 ± 13 46 ± 6 46 ± 4 55 ± 14 35 ± 8 35 ± 4 697 ± 44 47 ± 10 443 ± 17 290 ± 96

80" 5 52" 4 94 ± 9 108 ± 12 45 ± 4 77±11 99 ± 16 104 ± 31 63 ± 10 22 ± 9

92 ± 7 92±13 90 ± 2 79 ± 33 94 ± 2 61 ± 8 97 ± 3 86 ± 1 100 ± 3 76 ± 10 136 ± 10 133±4 96 ± 15 92 ± 5 106 ± 5 86 ± 2 101 ± 4 98 ± 9 98 ± 3 74 ± 10 63 ± 5 81 ± 8 137 ± 3 177 ± 23 87 ± 14 99 ± 5 134 ± 5 132±4 112 ± 7 82 ± 9 97 ± 18 87 ± 12 76 ± 7 61 ± 6

II 102± 11 105 ± 9 94 ± 3 87 ± 4 96 ± 5 102 ± 4 93 ± 2 113 ± 3 113±3114±10 110 ± 4 110 ± 5 80 ± 1 82 ± 4 90 ± 4 86 ± 2 72 ± 2 72 ± 15 85 ± 4 84 ± 8 78 ± 2 120 ± 5 101 ± 1 132 ± 8 84 ± 5 86 ± 6 119 ± 1 109 ± 10 101 ± 2 97 ± 6 108 ± 16 121 ±4 93 ± 1 93 ± 3

Table4B. ~2 Plant name

M3 II

Averrhoabilimbi 98 ± 25 94 ± 34 Bellucia grossularioides 91 ± 15 90 ± 14 Commelina nudiflora 94 ± 33 144 ± 26 88 ± 7 Costus niueus 88 ± 1 89 ± 15 97 ± 13 Crescentia cujete Cucurbita moschata 91 ± 5 94 ± 13 85 ± 1 84 ± 3 Hibiscus bifurcatus 84 ± 29 Irlbarchia purpurascens 87 ± 2 Miconia ciliata 91 ± 16 81 ± 2 86 ± 2 86 ± 2 Microteadebilis 81 ± 2 Mimosa myriadena 86 ± 2 91 ± 2 Momordica charantia 81 ± 1 90 ± 7 84 ± 9 Peperonra pellucida 93 ± 34 91 ± 2 Phyllantusamarus 124 ± 12 97 ± 27 Physalis angulata Pseudocalymna alliacea 102 ± 19 105 ± 23 82 ± 20 96 ± 5 Scoparia dulcis

97 ± 4 112±4 108 ± 3 128 ± 2 106" 6 99" 2 111" 5 116 ± 7 107 ± 5 118 ± 5 128 ± 3 136" 1 126 ± 3 94 ± 5 110±2 108 ± 5 107" 1

99

±

2

99 ± 7 105 ± 3 125" 2 102 ± 2 99

±

4

126 ± 11 118 ± 5 112±4 117±3 123 ± 2 134 ± 3 124 ± 3 99

A,

II

II

±

2

102 ± 3 107 ± 2 102 ± 2

II

64 ± 22 71 ± 39 82 ± 18 62 ± 12 59 ± 15 72 ± 34 85 ± 23 88 ± 19 86 ± 5 73 ± 11 129 ± 8 124±4 90 ± 13 51 ± 18 72 ± 6 89 ± 1 84 ± 9 87 ± 8 99 ± 21 53 ± 18

74 ± 92 ± 72 ± 89 ± 90" 97 ±

93 ± 20 80 ± 4 44 ± 13 90 ± 4 84 ± 6 87 ± 7 118 ± 3 121 ± 5 74 ± 32 70 ± 11 61 ± 35 53 ± 13 56 ± 15 54 ± 5

93" 9 81 ±1 94 ± 1 96 ± 6 87 ± 1 78 ± 2 80 ± 5

93" 95 ± 85 ± 93 ±

11 1

14 4 10 2 2 1

2 2

72 ± 85 ± 87 ± 83 ± 88 ± 99

±

NMDA+Gly II

3 7 2

1 7 2

90 ± 1 89 ± 1 68 ± 7 87 ± 4 82 ± 2 73 ± 6 86 ± 2 96 ± 7 80 ± 1 86 ± 3 76 ± 2

101 ± 3 69 ± 3 88 ± 11 52 ± 10 78 ± 4 100 ± 3 87 ± 2 103 ± 4 78 ± 2 108±1 101 ± 6 91 ± 2 98 ± 4 73 ± 4 102 ± 1 55 ± 5 105 ± 1 80 ± 7 5±1 50 ± 01 38 ± 3 87 ± 1 117 ± 4 124 ± 1 106 ± 4 72 ± 4 101 ± 1 95 ± 3 104±2 72 ± 16 94 ± 1 110 ± 1 106 ± 23 45 ± 7

I 92,,4 97 ± 2 135 ± 1 94 ± 1 95 ± 2 127 ± 4 89 ± 3 88 ± 2 91 ± 3 99 ± 2 92 ± 10 85 ± 3 109" 6 108 ± 3 105 ± 1 98 ± 3 92 ± 1

II 105±1 66 ± 3 151 ± 4 82 ± 3 111 ± 1 166 ±10 33 ± 1 57 ± 1 49 ± 3 98 ± 1 49 ± 3 116 ± 7 118 ±11 122 ±1 120 ± 1 114 ±1 79 ± 5

64

J. A. Hasrat et al.

eral, the results with the partition fractions were in good accordance with the results of the total extract from which they were obtained (Table4). In concentrations up to l0011g/ml the chloroform extract of Scoparia dulcis blocked to a high degree the radioligand binding to respectively the Ai and 5-HT 1A receptors and the chloroform extract of Microtea debilis blocked the Ai' the 5-HT 1A and the 5-HT2 receptors. The aqueous extracts of Scoparia dulcis and Microtea debilis had a pronounced blocking effect on the radioligand binding to the a 2a, the 5-HT 1A and the Ai receptors.

Discussion There is a lack of published information about the use of LBS as screening method of crude plant extracts. A few publications described the use of phytochemicals in radioligand binding assays, but as far as we know is this the first publication of the results of the screening of crude plant extracts by LBS. Using LBS as screening method will only allow to detect active components with a mechanism of action that involves the selected receptors. Therefore, no firm conclusion can be made about the opportunity of the use in traditional medicine of plants whose extracts were negative in the present screening tests.

A few plant extracts decreased radio ligand binding to some receptors to a high extent. The results of this investigation supports the use of some plants in traditional medicine. For example: Microtea debilis is used against proteinuria. It is now established that adenosine receptors are involved in the protection of some types of proteinuria (Oswald et al., 1995). Hibiscus bifurcatus is believed to influence ,the mind'. 5HT 1A receptors are involved in the modulation of emotion (Hoyer et al., 1994). So the activity on 5HT 1A receptors could explain its use in traditional medicine. By actions on a 2A and Ai receptors the applications of Scoparia dulcis against bronchitis, broncho-pulmonary complaints and urinary burning could be explained. From lrabacbia purpurascens no applications could be found in the literature, but for related species they exist. If there is a chemotaxonomic relation, the application of the related species for nervous dyspepsia could be explained by interaction with the 5-HT 1A receptor. There is a search for compounds that could interfere with the NMDA receptor channel complex due to the benefit these substances could have in a variety of neuropathological conditions (Meldrum and Garthwaite, 1990). It is interesting to find that there are probably compounds present in the extracts of Miconia ciliata and Mimosa myriadena that interfere to a high degree with the [3H]MK-801 binding. Related speciesof Miconia ciliata are used as sedativa and relat-

Table 5. Results of the inhibition of the binding of some radio ligands to their particular receptor by partition fractions of 80 % ethanol extracts of some of the plants mentioned in table 4. For more details about the experimental procedures see table 3 and text. The results are expressed as percentage of the radioligand still bound to the receptor. Every value in the table is the average, with its standard error, of the results of three to six experiments (in all experiments every sample was measured in triplo). The inhibition of the binding of the radioligand was measured with two concentrations of each of the partiton fraction, I = 10 ug/ml and II = 100 ug/ml, C = Chloroform, W = Water. The inhibition of the binding of the radio ligand to the NMDA receptor complex was performed with or without addition of glycine (GLY) to the incubation buffer. Plant name Be/luciagrossularioides Commelina nudiflora Hibiscus bifurcatus Irlbarchia purpurascens Miconia ciliata

part. fro

C W

Mimosa myriadena

C W

Physalisangulata Pseudocalymniaal/iacea Scopariadulcis

5HT1A II

56 53 61 53

±2 ±2 ±1 ±6

20 43 30 38

±3 ±5 ±3 ±2

II

117 ± 5 80 ± 2 110 ± 14 104 ± 2 108 ± 15 64 ± 10 70 ± 2 88 ± 7 100 ± 15 75 ± 7 100 ± 7 118±17 109 ± 6 102 ± 6

17 ± 1 59 ± 10 27 66 ± 16 119 ± 14 102 150 ± 14 108 130 ± 15 87 95 ± 10 51 ± 5 88 ± 2 55

88 ± 6 81 ± 7 69 ± 7 54 ± 7 56 ± 10 54 ± 19

90 ± 6 48 ± 5

51 ± 2 22 ± 3

NMDA-Gly

Al II

25 ± 3 69 ± 9

C W C W C W C W

5HT 2 II

I

108 ± 2 112 ± 9

C W C W C W C W C W

Microtea debilis

Peperoniape/lucida

a 2A

44 ± 4 81 ± 2

NMDA+Gly

II

I

I

112 ± 19 86 ± 4 212 ± 5 190 ± 2

85 ± 5 54 ± 3 144 ± 12 119±6

82 ± 1 158 ± 2 91 ± 1 129±3 68 ± 2 61 ± 14

60 106 72 148 41 25

85 ± 2 157 ± 2

59 ± 3 151 ± 2

79 ± 1 119 ± 3

70 ± 4 44 ± 3

±4 ±8

85 ± 1 61 ± 2

20 ± 2 11 ± 2

87 ± 5 69 ± 3 230±22146±11 88 ± 3 87 ± 8 153 ± 6 207 ± 2 96 ± 3 58 ± 5 88 ± 13 43 ± 3 92 ± 5 82 ± 11 219 ± 14 237 ± 8

±5 ±3 ±6

107 ± 5 100 ± 7

62 ± 7 46 ± 5

85 ± 2 152 ± 12

90 ± 5 88 ± 5 87 ± 3

83 ± 4 55 ± 4 72 ± 7

63 ± 11 28 ± 16 110±4 71 ± 12 37 ± 13 75 ± 4

58 ± 5 75 ± 6

52 ± 5 81 ± 1

103 239 160 254 92 ± 15 33 ± 2 108 ± 2 83 ± 4

II

±9 ±5 ±8 ± 14

57 ± 14 59 ± 2

111 236 207 245

±5 ±3 ±6 ±6

79 ± 4 155 ± 1 112±5 152 ± 1

79 86 105 142

1 ±4 ±2 ±2 ± 10 ±1

±

±7 ±4 ±9 ±5

Medicinal plants in Suriname ed species of Mimosamyriadena against nervousness. At the time the [3H]MK-801 binding assays were performed no selective ligand was available for the receptors involved, so that non-specific binding could not be measured. On the basis of these results a few plants were selected for further examination, namely Microtea debilis for activity on Aradenosine receptors, Hibiscus bifurcatus and Irilbachia purpurascens for activity on 5-HT 1A receptor and Miconia ciliata and Mimosa myriadena for activity on the NMDA receptor channel complex. There are some extracts that increased the binding of the radioligand, the reasons for this is not known at present. It is possible that by changing the conformation of the receptor by some plant compounds the affinity of the radioligand for the receptor can be increased. There could also be substances present in the extract that formed insoluble products with the radioligand. It is worthwhile to investigate these results further. We also observed in a few cases that the binding of the radioligand could be inhibited to the same level by both partition fractions. This phenomenon can be explained by the presence of different compounds in both the aqueous and organic layers, that bind to the receptor or by a distribution of the same ligand-binding compounds between the two phases. LBS will be further used for the bioguided-isolation of the active components in the extracts of plants of interest. Acknowledgements Hasrat, J. A. was a recipient of a grant of Algemeen Bestuur voor Ontwikkelingssamenwerking (ABOS). G. Vauquelin is Research Director at the National Funds for Scientific Research, Belgium. References Bottari, S. P., Vauquelin, G., Lescrainier, J. P., Kaivez, E. and Vokaer, A.: Identification and characterisation of alpha I-adrenergic receptors in human myometrium by [3H]prazosin binding. Biochem. Pharmac. 32: 925-928, 1983. Convents, A., De Keyser, J., De Backer, J.-P. and Vauquelin, G.: [3H]rauwolscine labels alpha 2-adrenoceptors and 5-HT I A receptors in human cerebral cortex. Eur. J. Pharmac. 159: 307-310,1989. De Keyser, J., De Backer, J.-P., Ebinger, G. and Vauquelin, G.: Regional distribution of the dopamine D 2 receptors in the mesotelencephalic dopamine neuron system of human brain.]. Neurol. Sci. 71: 119-127, 1985. De Keyser, j., Walraevens, H., Convents, A., Ebinger, G., Vauquelin, G.: [3H]SCH 23390 labels a novel 5-hydroxytryptamine binding site in human blood platelet membranes. Eur. J. Pharmacol. 162: 437-445,1989. Galitzky, J., Senard, J. M., Lafontan, M., Stillings, M., Montastrue, J. L. and Berlan, M.: Identification of human platelet u 2 adrenoceptors with a new antagonist [3H]RX 821002, a 2methoxy derivative of idazoxan. Br. J. Pharmac. 100: 862-866, 1990. Heyde, H.: In: Surinaamse planten als volksmedicijn, 1985. Hoyer, D., Clarke, D. E., Fozard, J. R., Hartig, P.R., Martin,

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G. R., Mylecharane, E.J., Saxena, P.R., Humphrey, P.P.A.: VII. International Union of Pharmacology Classification of receptors for 5-Hydroxytryptamine (Serotonin). Pharmacological Reviews 46: 157-203, 1994. Kornhuber, J., Mack-Buckhardt, E. H. E, Kornhuber, M. E., Riederer, P.: [3H]MK-801 binding sites in post-mortem human frontal cortex. Eur. J. Pharmacol. 162: 483-490, 1989. Lohse, M.J., Lindenborn-Fotinos, J., Redington, M., Schwabe, U. and Olsson, R. A.: 8-Cyclopentyl-l,3-dipropylxanthine (DPCPX), a selective high affinity antagonist radioligand for Al adenosine receptors. Naunyn Schmiedeberg's Arch. Pharmacol. 336: 204-210, 1987. Meldrum, B. S. and Garthwaite, J.: Excitatory amino acid neurotoxicity and neurodegenerative disease. Trands Pharmacol. Sci. 11: 379,1990. Morton, J. E: In: Atlas of Medicinal Plants of Middle America, Bahamas to Yucatan (Thomas, C. C. ed.) Illinois, 1981. Oswald, H., Gleiter, Ch. and Muhlgauer, B.: Therapeutic use of theophylline to antagonize renal effects of adenosine. Clinical Nephrology 43: S33-S37, 1995. Plotkin, M.J.: "Strychnos medeola: A New Arrow Poison from Suriname". In: Ethnobiology: Implications and Applications, Brazil, 1990. Plotkin, M. J. In: Tales of a Shaman's Apprentice. Penguin Books, New York, 1993. Severne, Y., Wemers, c., Vauquelin, G.: AgonistlN-ethylmaleimide mediated inactivation of beta-adrenergic receptors. Biochem. Pharmac. 34: 1611-1617, 1985. Smith, P.K., Krohn, R. 1., Hermanson, G. T., Mallia, A. K., Gartner, E H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B.J. and Klenk, D. c.: Measurement of protein using bicinchoninic acid. Analyt. Biochem. 150: 76-85,1985. Van der Heyden, P., Gies, J.-P., Ebinger, G., De Keyser, J., Landry, Y. and Vauquelin, G.: Human Mj-, M 2- and Mj-muscarinic cholinergic receptors: binding characteristics of agonists and antagonists. J. Neurol. Sci. 97: 67-80, 1990. Vauquelin, G., De Vos, H., De Backer, J.-P. and Ebinger, G.: Identification of U 2 adrenergic receptors in human frontal cortex membranes by binding of [3H]RX 821002, the 2-methoxy analogue of [3H]idazoxan. Neurochem. Int. 17: 537-546,1990. Verpoorte, R., Tjin, A., Tsoi, A., van Doorne, H. and Baerheim Svendsen, A.: a) Medicinal plants of Suriname I: Antimicrobial activity of some medicinal plants. J. Ethnopharmacol. 5: 221-226,1982. Verpoorte, R., Ruigrok, C. L. M. and Baerheim Svendsen, A.: b) Medicinal plants of Suriname II: Antimicrobial active alkaloids from Aspidosperma marcgraviantum. Planta Med. 46: 149-152, 1982. Verpoorte, A., Kos-Kuick, E., Tsin, A., Tsoi, A., Ruigrok, C. L. M., de Jong, G. and Baerheim Svendsen, A.: Medicinal plants of Suriname III: Antimicrobially active alkaloids from Aspidosperma marcgraviantum. Planta Med. 48: 283-289,1983. Verpoorte, R. and Dihal, P.P.: Medicinal plants of Suriname IV: Antimicrobial activity of some medicinal plants. J. Ethnopharmacol. 21: 315-318,1987. Williams, M.: Receptor binding in the drug discovery process. Med. Res. Reviews 111: 147-184, 1991.

Address A. J. Vlietinck, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Antwerp, Belgium.