Pharmaceutical Biology 2005, Vol. 43, No. 8, pp. 672–682 Muscarinic Receptor Activity of Some Malaysian Plant Species L.Y. Chung1, K.F. Yap1, M.R. Mustafa2, S.H. Goh3, and Z. Imiyabir4 Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 3Forest Research Institute Malaysia, Kuala Lumpur, Malaysia; 4Forest Research Center, Sepilok, Sandakan, Sabah, Malaysia Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. 1 Abstract Muscarinic receptor binding activity was tested on 224 plant extracts obtained from more than 50 plant families found in Malaysia. The plant extracts were evaluated by a 96-well microplate filtration–based radioligand competitive assay, centered on the ability of the plant extracts to competitively displace the radioligand, [3H]N-methylscopolamine, from binding to the muscarinic membrane receptors. The screening assay was initially carried out at 50 mg=assay point, and those showing inhibition at and above 61% were retested at 10 mg=assay point. The extracts of Ficus septica Burm. f. (Moraceae) [65.85
3.75% inhibition; mean (n ¼ 3)
SD], Polyalthia microtus Miq. (Annonaceae) (32.63
1.38% inhibition), and Popowia odoardoi Diels (Annonaceae) (35.79
7.11% inhibition) at 10 mg=assay point exhibited muscarinic properties, which are worthy of further investigation. Keywords: Ficus septica, Malaysian plants, muscarinic receptor, Polyalthia microtus, Popowia odoardoi, radioligand binding assay. Introduction Since ancient times, human being have used natural products, including plants, as remedies to treat illnesses. Today, about one-third of the top 25 prescription drugs in the world are natural products or their derivatives (Manly et al., 2002). The sources of natural products include plants, marine and microorganisms (Zhu, 1996; Cragg et al., 1997; Shu, 1997; Manly et al., 2002), and historically, plants have been an important source of pharmacologically active compounds providing for a number of muscarinic receptor antagonists. For example, atropine from the plant Atropa belladonna L. and scopolamine from Datura sp. are the most commonly used anticholinergic drugs (Evans & Evans, 2002). Anticholinergic drugs act on two distinct classes of receptors, muscarinic and nicotinic. The muscarinic class of acetylcholine receptors are widely distributed throughout the body and subserve numerous vital functions in both the brain and autonomic nervous system (Lefkowitz et al., 1990). Heterogeneity of muscarinic receptor was shown in the late 1980s when five subtypes (M1–M5) were identified using molecular biological techniques (Kubo et al., 1986; Bonner et al., 1987; Liao et al., 1989; Sokolovsky, 1989; Kashihara et al., 1992). Activation of muscarinic receptors in the periphery causes a decrease in heart rate, relaxation of blood vessels, constriction in the airways of the lung, an increase in secretions, and motility of various organs of the gastrointestinal tract, increase in the secretions of lacrimal and sweat glands, constriction in the iris sphincter and ciliary muscles of the eye (Lefkowitz et al., 1990). In the brain, muscarinic receptors influence many important functions such as learning, memory, and the control of posture (Lefkowitz et al., 1990). Classic muscarinic receptor antagonists such as atropine do not distinguish between muscarinic receptor subtypes. These nonselective compounds cover therapeutic indications such as antispasmodic, antitussive, and antibronchospastic, but their therapeutic utility is limited by the presence of side effects including mydriasis, CNS disturbances, tachycardia, and constipation. In recent studies, it has been shown that selective M1 antagonists are useful in reducing gastric acid secretion. Selective M2 antagonists may be useful in the treatment of Accepted: September 12, 2005 Address correspondence to: L.Y. Chung, Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. E-mail: chungly@hotmail.com DOI: 10.1080/13880200500383439 # 2005 Taylor & Francis Ltd. Muscarinic receptor binding of Malaysian plants 673 Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Table 1. Percent inhibition of extracts (50 mg=well) on specific binding to muscarinic receptor. No. Voucher no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 43264 143521 143521 43303 43304 143516 143516 143370 143374 143374 143523 143523 143377 143377 143358 143362 143362 133834 145372 145372 143506 143506 143509 143509 143511 143511 143502 143502 145376 145376 143524 143524 143395 145365 143368 143368 143507 143507 43125 145362 145362 145361 145361 143522 143522 43302 43306 43127 43127 43350 43309 43320 143381 143381 43344 43344 43345 Family Species Alangiaceae Alangium ebenaceum Alangium griffithii Anacardiaceae Bouea macrophylla Bouea oppositifolia Buchanania insignis Annonaceae Semicarpus leneatus Artabotrys roseus Desmos chinensis Eniscosanthum grandiflorum Goniothalamus woodii Goniothalamus gigantifolius Neouvaria acuminatiisima Orophea corymbosa Polyalthia insignis Polyalthia longipes Polyalthia microtus Polyalthia rumphii Popowia odoardoi Uvaria rufa Xylopia malayana Apocynaceae Dyera costulata Kopsia dasyrachis Burseraceae Canarium denticulatum Canarium hirsutum Dacryodes rugosa Santiria griffithii Santiria laevigata Caesalpiniaceae Chrysobalanaceae Combretaceae Cornaceae Peltophorum pterocarpum Maranthes corymbosa Terminalia superba Chionanthus laxiflorus Cupressaceae Dacrydium becarii Dacrydium elatum Parta Inhibitionb L B L L L B L L B L B L B L L B L L L S B L B L B L B L B L L R B S B L B L L L S B L B F L L L S L S L B L L S L þ þ

3þ

þ

2þ


þ


þ þ 3þ þ

3þ
3þ þ 4þ þ 3þ
4þ 3þ

þ þ
þ 2þ 2þ þ þ
þ þ 3þ
2þ þ


þ 2þ þ (Continued) 674 L.Y. Chung et al. Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Table 1. Continued. No. Voucher no. 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 43345 143503 143503 43308 43124 43124 43112 43134 43134 43324 43324 143504 143504 143519 143519 43273 145393 145393 145400 145400 145371 43108 43108 43338 43137 43323 145388 145388 43123 145398 145377 145377 145385 145385 133842 133842 133846 142698 142674 43279 145368 145380 43339 143388 143388 43129 43138 43138 143360 143360 43144 143392 143392 143393 143393 143366 143366 Family Species Dichapetalaceae Dichapetalum gelonioides Dipterocarpaceae Anisoptera costata Hopea dryobalanoides Neobalanocarpus heimii Shorea parvifolia Upuna borneensis Ebenaceae Diospyros cauliflora Diospyros tuberculata Elaeocarpaceae Euphorbiaceae Elaeocarpus petiolaris Borneodendron aenigmaticum Mallotus griffithianus Mallotus wrayi Phyllanthus emblica Fagaceae Flacourtiaceae Gittiferae Guttiferae Phyllanthus pectinata Castanopsis inermis Flacourtia rukam Homalium panayanum Mesua ferra Calophyllum blancoi Calophyllum gracilipes Calophyllum nodosum Garcinia brianii Ixonanthaceae Lauraceae Leguminosae Garcinia cuspidate Garcinia parvifolia Garcinia rostrata Ixonanthes reticulata Litsea garciae Litsea sessilis Persea Americana Dalbergia pseudo-sissoo Millettia atropurpurea Sindora echinocalyx Spatholobus macropterus Magnoliaceae Meliaceae Aromadendron elegans Aglaia affinis Aglaia argentea Aglaia shawiana Parta Inhibitionb S B L L L T L L S L S B L B L L B L B L L L T L L L B L L B B L B L B L B L L L B B L B L L L T B L L B L B L B L


þ 2þ 2þ 2þ þ 2þ 2þ 2þ

þ





2þ þ þ þ þ þ

2þ
þ

 2þ þ
2þ
2þ 2þ 2þ 2þ


þ þ

2þ þ 



(Continued) Muscarinic receptor binding of Malaysian plants 675 Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Table 1. Continued. No. Voucher no. 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 43105 143361 143361 143376 143376 143512 143512 143512 145367 145367 145367 143513 143513 142663 143355 143355 143364 143364 43104 43104 43143 145378 145378 143518 145397 145397 145392 145392 43111 145396 145396 143359 143359 43307 43329 145373 143525 43348 145363 145395 145395 145395 145364 145364 145382 145384 145384 145366 145366 143391 143391 143375 143375 143367 143369 143369 143389 Family Species Aglaia korthalsii Aglaia rivularis Chisocheton polyandra Chisocheton erythrocarpus Chisocheton macranthus Chisocheton pentandrus Chisocheton polyandrous Dysoxylum ramiflorum Dysoxylum rugulosum Sandoricum koetjape Menispermaceae Walsura chrysogene Fibraurea chloroleuca Moraceae Myrtaceae Ficus septica Decaspermum fruticosum Ochnaceae Gomphia serrata Olaceae Oleaceae Ochanostachys amentacea Chionanthus crispus Piperaceae Piper officinarum Podocarpaceae Rhamnaceae Xanthophyllum stipitatum Maesopsis eminii Vantilago dichotoma Carallia borneensis Carallia suffruticosa Gardenia tubifera Morinda rigida Rhizophoraceae Rubiaceae Praravinia suberosa Psychotria sarmentosa Timonius flavescens Rutaceae Clausena excavata Melicope accedens Melicope incana Melicope luna-akenda Melicope subunifoliata Micromelum minutum Parta Inhibitionb L B L B L B F L B F L B L L B L B L L T L F L L B L B L L B L L S L L S L L L B L S B L L B L B L B L B L L B L B






þ
þ 2þ 2þ þ

þ þ
2þ 3þ 2þ
þ 3þ 2þ 2þ þ





þ
þ
2þ þ












3þ 2þ
(Continued) 676 L.Y. Chung et al. Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Table 1. Continued. No. Voucher no. 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 143389 43325 43325 43337 43337 43327 43327 43106 43310 43341 43341 43110 43110 143514 143514 43334 43334 43347 43347 143520 143520 43145 133822 43311 145381 145381 143385 143385 145383 145383 43275 145391 145375 145375 145387 145387 143378 143378 106702 133850 133849 145389 145389 145374 106701 106701 143372 143372 145386 43322 145370 145370 145379 Family Sapindaceae Species Amesiodendron chinense Dimocarpus longan Lepisanthes alata Nephelium lappaceum Nephelium maingayi Nephelium rambutanake Pometia pinnata Walsura pinnata Sapotaceae Mimusops elengi Palaquim maingayi Scyphostegiaceae Scyphostegia borneensis Simaroubaceae Irvingia malayana Quassia indica Heritiera simplicifolia Sterculia stipulata Sterculiaceae Theaceae Schima wallichi Schima wallichii Tiliaceae Ulmaceae Urticaceae Pentace triptera Gironniera subaequaelis Dendrocnide elliptica Leucosyke winklerii Verbenaceae Callicarpa havillandii Callicarpa Callicarpa Callicarpa Callicarpa erioclona farinose fulvohirsuta havilandii Callicarpa longifolia Callicarpa stapfii Sphenodesme triflora Vitaceae Stachytarpeta jamaicencis Vitex pubescens Leea indica Zingerberaceae Alpinia fraseriana Parta Inhibitionb L L S L S F S T L L S L T B L L S L S B L L B S B L B L B L L L B L B L B L B B B B L L B L B L S L B L S
þ 2þ 2þ 2þ
2þ
2þ þ 2þ 2þ 2þ þ
2þ
þ þ

2þ
2þ

þ 





þ

þ
þ þ
þ 2þ 2þ 

2þ

Extracts were dissolved in 50% DMSO to give a concentration of 2 mg=ml for testing; 25-ml aliquots tested. B, bark; F, fruit; L, leaf; R, root; S, stem; T, twigs. b 4þ, inhibition of 81–100%; 3þ , inhibition of 61–80%; 2þ , inhibition of 41–60%; þ , inhibition of 21–40%;
, inhibition of 1–20%; , no inhibition. a Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Muscarinic receptor binding of Malaysian plants bradycardia and Alzheimer disease, whereas selective M3 antagonists affect smooth muscles (chronic obstructive airway disease, irritable bowel syndrome, and incontinence) (Eglen, 1998). To reduce side effects and to improve therapeutic activities, selective muscarinic receptor antagonists have been developed for antiulcer activity (telenzepine; M1=M4 selective; Byk), bradycardia (otenzepad; M2=M4 selective; Boehringer Ingelheim), irritable bowel syndrome (darifenacin; M3=M1 selective; Pfizer), and antibronchospastic activity (rispenzepine; M3=M1 selective; Dompe) (Eglen, 1998). As part of an ongoing screening program for potential receptor agonists and antagonists Malaysian plants that showed significant muscarinic receptor competitive binding activity were identified. The active crude extracts are subjected to further bioassay-guided isolation of active constituents. Upon successful isolation, the active constituents will be evaluated for their muscarinic receptor subtype selectivity. 677 analytical grade and obtained from standard commercial sources (Little Chalfont, Buckinghamshire, UK). Plant materials Plant samples were collected from the Forest Research Institute Malaysia, Kuala Lumpur, Malaysia (voucher no.: 5-digits series), and the state of Sabah, Malaysia (voucher no.: 6-digits series). The voucher specimens were kept at the herbaria of Forest Research Institute Malaysia (5-digits series) and Forest Research Center, Sepilok, Sandakan, Malaysia (6-digits series). Different parts of the plants were dried separately at 40C. The dried materials (100–200 g) were powdered and soaked with sufficient methanol in conical flasks for 7 days with sonication (5  sufficient volume of methanol to cover the plant materials). Methanol extracts were collected and filtered at 48, 96, and 168 h, and the conical flasks with plant materials were replaced with fresh methanol to continue extraction. The pooled extracts were evaporated at 50C in vacuo and the residues freeze-dried and kept in sample bottles at 20C until use. Materials and Methods Chemicals and reagents Crude extract dilution [3H]N-Methylscopolamine was supplied by Amersham Pharmacia Biotech UK Ltd. All other reagents were of DMSO (1 ml) was added to 4 mg of crude plant extracts and vortexed vigorously giving an initial concentration Table 2. Percent inhibition of extracts (10 mg=well) on specific binding to muscarinic receptor. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Voucher no. 43304 143506 143511 143502 145376 143524 145365 43125 43123 43127 43124 43112 145368 43104 43104 143518 43111 43325 43110 43110 106701 Family Anacardiaceae Annonaceae Apocynaceae Burseraceae Dipterocarpaceae Lauraceae Meliaceae Species Parta Inhibitionb Bouea oppositifolia Neouvaria acuminatiisima Polyalthia insignis Polyalthia longipes Polyalthia microtus Polyalthia rumphii Popowia odoardoi Dyera costulata Mesua ferra Santiria laevigata Hopea dryobalanoides Neobalanocarpus heimii Litsea garciae Sandoricum koetjape L B B B B L S L L L L L B T L L L S L T B
þ

þ þ þ þ

þ þ


3þ þ
þ
þ Moraceae Olaceae Sapindaceae Ficus septica Ochanostachys amentacea Amesiodendron chinense Pometia pinnata Verbenaceae Callicarpa stapfii Extracts were dissolved in 50% DMSO to give a concentration of 0.4 mg=ml for testing; 25-ml aliquots tested. B, bark; F, fruit; L, leaf; R, root; S, stem; T, twigs. b 4þ , inhibition of 81–100%; 3þ , inhibition of 61–80%; 2þ , inhibition of 41–60%; þ , inhibition of 21–40%;
, inhibition of 1–20%; , no inhibition. a L.Y. Chung et al. Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. 678 Figure 1. Some alkaloids isolated from Ficus species (Baumgartner et al., 1990; Buckingham, 1994; Peraza-Sanchez et al., 2002; Wu et al., 2002). Muscarinic receptor binding of Malaysian plants of 4 mg=ml. The extracts were tested at 50 and 10 mg=assay points. Membrane preparation in 10 volumes of ice-cold 50 mM Tris-HCl, pH 7.4, buffer using Ultra-Turax (2  10 s) and followed by glassTeflon pestle homogenization at 800 rpm for 20 strokes. The homogenate was centrifuged at 40,000  g using Beckman type 28 rotor at 4C for 15 min. The pellet was retained and washed twice by resuspending in icecold 50 mM Tris-HCl, pH 7.4, buffer (centrifuged at 40,000  g for 15 min). The final pellet was suspended in 5 ml ice-cold 50 mM Tris-HCl, pH 7.4, buffer, aliquoted and kept at 80C until use. Protein was determined using the Sigma Total Protein Reagent using Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Total rat brain (minus cerebellum) membrane was prepared according to the protocol described by Gattu et al. (1995) with minor modifications. Male SpragueDawley rats (250–300 g) were decapitated and the brains removed. The cerebellum was dissected out and the rest of the brain finely chopped with scissors, homogenized 679 Figure 2. Some alkaloids isolated from Popowia species (Jossang et al., 1986). 680 L.Y. Chung et al. bovine serum albumin as the standard, and the protein content corresponded to about 20 mg protein=ml. Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Muscarinic receptor binding assay The muscarinic receptor binding was assayed by a modification of the method of Bockman et al. (2001). Briefly, the membranes were thawed on ice and diluted to 0.75 mg=ml protein using 50 mM Tris-HCl, pH 7.4, buffer. The reference ligand (atropine) and radioligand ([3H]N-methylscopolamine) were diluted to 100 mM and 5 nM final concentration using 50% DMSO in deionized water and binding buffer, respectively. With its cover removed, the Multiscreen plate (GF-B, Millipore Billerica, MA, USA) was placed on vacuum manifold and the filter of each well prewetted with 200 ml of 50 mM Tris-HCl, pH 7.4, buffer. Radioligand (25 ml) was added to each well of the Multiscreen plate, followed by addition of 25 ml of 50% DMSO (total binding, 3 wells), reference compound (nonspecific binding, 3 wells), or crude extracts to the corresponding well in the plate. The reaction was initiated by adding 200 ml of diluted membranes to each well. The plate was then covered, vortexed gently, and incubated at 22C for 90 min. The reaction mixture was filtered on the vacuum manifold and washed four-times with 200 ml of ice-cold 50 mM Tris-HCl, pH 7.4. The plate was cleaned with tissues to remove excess buffer and air-dried. The filters were punched out and transferred into 5-ml scintillation vials and 4 ml of scintillation cocktail added. The vials were capped, the content shaken for a few minutes, and radioactivity counted for 3 min per vial, using a scintillation counter (Packard, Meriden, CT, USA). Data analysis The percentage inhibitory specific binding in the presence of the test compounds was calculated using a standard Figure 3. Some alkaloids isolated from Polyalthia species (Jossang et al., 1977; Marsaioli et al., 1977; Connolly et al., 1996; Said et al., 2003). Muscarinic receptor binding of Malaysian plants data reduction algorithm, and it is as follows: ½T  NSP  ½B  NSP  100 ½T  NSP where B ¼ binding in the presence of test extract, NSP ¼ nonspecific binding in the presence of excess inhibitor (reference ligand), and T ¼ total binding. Pharmaceutical Biology Downloaded from informahealthcare.com by Universiti Malaya on 12/19/11 For personal use only. Results and Discussion The crude extracts were initially screened at 100 mg=assay point and above, and under this condition, some of the extracts were not soluble in the assay buffers, which prevented proper filtration of the plates. Consequently, the extracts were screened at 50 mg=assay point (Table 1), and extracts that showed inhibition at and above 61% were retested at 10 mg=assay point. Under this condition, the samples remained soluble, and proper filtration was achieved. The extracts were screened in triplicate and the percentage inhibition averaged (Table 2). Of these 224 extracts tested at 50 mg=assay point, 42 (19%) exhibited 41–60% inhibition and 12 (5%) exhibited 61% or higher inhibition. To reduce false-positive results, extracts exhibiting above 61% inhibition and comparators were then tested at 10 mg=well. Of these, only Ficus septica gave more than 60% inhibition (65.85
3.75%). Other extracts such as Polyalthia microtus (Annonaceae) and Popowia odoardoi (Annonaceae) showed 32.63
1.38% and 35.79
7.11% inhibition, respectively. Our preliminary chemical studies and the literature indicate these plants, Ficus septica Burm. f., Polyalthia microtus Miq., and Popowia odoardoi Diels, and related species contain alkaloidal compounds, and some of the reported structures are as shown in Figures 1, 2, and 3. It is conceivable that the active constituents, which inhibited binding of the radioligand, [3H]N-methylscopolamine to muscarinic receptors, are probably alkaloids, as known muscarinic receptor agonists and antagonists possess nitrogen moieties. We have, therefore, selected Ficus septica Burm. f., Polyalthia microtus Miq., and Popowia odoardoi Diels for bioassay-guided fractionation to identify the muscarinic active constituents and to ascertain their activities. Acknowledgments The project was funded by the Ministry of Science, Technology and the Environment, Malaysia (IRPA 26-02-060127). K.F. Yap is supported by the National Science Fellowship, Malaysia. 681 References Baumgartner B, Erdermeier CAJ, Wright AD, Rali T, Sticher O (1990): An antimicrobial alkaloid from Ficus septica. Phytochemistry 29: 3327–3330. Bockman CS, Bradley ME, Dang HK, Zeng W, Scofield MA, Dowd FJ (2001): Molecular and pharmacological characterization of muscarinic receptor subtypes in a rat parotid gland cell line: Comparison with native parotid gland. J Pharmacol Exp Ther 297: 718–726. Bonner TI, Buckley NJ, Young AC, Brann MR (1987): Identification of a family of muscarinic acetycholine receptor genes. Science 237: 527–532. Buckingham J, ed. (1994): Dictionary of Natural Products. New York, Chapman & Hall, p. 769. Connolly JD, Haque MDE, Kadir AA (1996): Two 7,70 bisdehydroaporphine alkaloids from Polyalthia bullata. Phytochemistry 43: 295–297. Cragg GM, Newman DJ, Snader KM (1997): Natural products in drug discovery and development. J Nat Prod 60: 52–60. 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Liao C-F, Themmen APN, Joho R, Barberis C, Birnbaumer M, Birnbaumer L (1989): Molecular cloning and expression of the fifth muscarinic acetylcholine receptor. J Biol Chem 264: 7328–7337. Manly SP, Padmanabha R, Lowe SE (2002): Natural products or not? How to screen for natural products in the emerging HTS paradigm. In: Janzen WP, ed., High Throughput Screening. Methods and Protocols. New Jersey, Humana Press, pp. 153–168. Marsaioli AJ, Magalhães AF, Rúveda EA, Francisco de AM Reis (1980): 13C NMR analysis of some oxoaporphine alkaloids. Phytochemistry 19: 995–997. Peraza-Sanchez SR, Chai HB, Shin YG, Santisuk T, Reutrakul V, Farnsworth NR, Cordell GA, Pezzuto JM, Kinghorn AD (2002): Constituents of the leaves and twigs of Ficus hispida. Planta Med 68: 186–188. Said M, Hadi AA, Awang K (2003): Alkaloids from Polylthia sclerophylla. In: Hamzah AS, Ismail NH, Ariffin ZZ, Hamzah Z, eds., Fine Chemicals from Natural Resources. Malaysia, Pusat Penerbitan Universiti (UPENA) UiTM, pp. 275–279. Shu YZ (1998): Review: Recent natural products based drug development: A pharmaceutical industry perspective. J Nat Prod 61: 1053–1071. Sokolovsky M (1989): Muscarinic cholinergic receptors and their interactions with drugs. In: Testa B, ed., Advances in Drug Research, Vol. 18. London, Academic Press, pp. 431–509. Wu PL, Rao KV, Su CH, Kuoh CS, Wu TS (2002): Phenanthroindolizidine alkaloids and their cytotoxicity from the leaves of Ficus septica. Heterocycles 57: 2401–2408. Zhu M, Bowery NG, Greengrass PM, Phillipson JD (1996): Application of radioligand receptor binding assays in the search for CNS active principles from Chinese medicinal plants. J Ethnopharmcol 54: 153–164.