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Antithrombin activity of medicinal plants from central Florida
Journal of Ethnopharmacology 81 (2002) 277 /280 www.elsevier.com/locate/jethpharm
Antithrombin activity of medicinal plants from central Florida Natalya Chistokhodova, Chi Nguyen, Tony Calvino, Ioulia Kachirskaia, Glenn Cunningham, D. Howard Miles * Department of Chemistry, University of Central Florida, Orlando, FL 32816, USA Accepted 5 April 2002
Abstract A chromogenic bioassay was utilized to determine the antithrombin activity of methylene chloride and methanol extracts prepared from 30 plants of central Florida. Extracts of Ardisia crenata , Tetrapanax papyriferus , Lagerstroemia indica , Callistemon lanceolatus , Antigonon Leptopus , Magnolia virginiana , and Myrica cerifera demonstrated activity of 80% or higher in this bioassay system. # 2002 Published by Elsevier Science Ireland Ltd. Keywords: Ardisia crenata ; Tetrapanax papyriferus ; Lagerstroemia indica ; Callistemon lanceolatus ; Antigonon Leptopus ; Magnolia virginiana ; Myrica cerifera ; Antithrombin activity
1. Introduction The inhibition of thrombin is very important in fighting many blood coagulation and platelet disorders because thrombin is an important enzyme in the blood coagulation process. Blood coagulation is caused by a series of zymogen activations. A zymogen is an enzyme that is inactive until it acquires full enzymatic activity upon specific proteolitic cleavage of one or more of its peptide bonds. Physiology of coagulation (Collen et al., 1979) can be described by intrinsic and extrinsic pathways. Blood in vivo will not clot until a vessel wall is damaged. Vessel wall injury will lead to exposure of a layer in the vessel wall, which is full of Tissue Factor (TF). TF (thromboplastin) is present in phospholipidprotein extracts of human and animal tissues. This TF binds with factor VII (a) in plasma to form the TF- VII (a) complex. Factor VII (a) is generated by minor proteolysis of the Arg-Ile peptide bond. The complex TF- VII (a) converts factor X /X (a). Factor X circulates in plasma as a two-chain glycoprotein with molecular weight of about 67 000. Activation of factor X by a complex of tissue factor VII (a) involves at least two peptide cleavages. Factor X (a) will then convert
* Corresponding author. Fax: /1-407-823-2252. E-mail address: [email protected] (D. Howard Miles).
prothrombin to thrombin. Prothrombin or human factor II is a glycoprotein composed of a single chain and three oligosaccharide side chains giving a molecular weight of 71 600. Thrombin activates factors V and VIII/V (a) and VIII (a). The X (a) formed will combine with factor V (a) to form the prothrombinase complex. This complex is orders of magnitude more effective in the conversion of prothrombin than X (a) alone, so at this point thrombin formation really starts. In this instance factor X (a) is the enzyme that activates prothrombin and factor V (a) is the cofactor. Thrombin converts soluble fibrinogen into stabilized insoluble fibrin. The resulting fibrin monomers polymerize, undergo further cleavage by thrombin and finally form stabilized fibrin of high tensile strength by the action of factor XIII (a). This leads to the stabilization of the formed clots and thus to hemostasis or thrombosis. Alternatively, coagulation can start by intrinsic pathway after blood comes into contact with glass surfaces. Kallikrein-induced hydrolysis of XII in plasma produces factor XII (a). Factor XII is a single chain glycoprotein containing about 17% carbohydrate. Factor XII (a) activates XI, XI (a) activates IX, and IX (a) activates X. The first activated X will activate prothrombin and the first traces of thrombin then activate factors VIII and V. The extrinsic and intrinsic pathways now merge: the first X (a) will activate the first prothrombin that will lead to
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activation of more VIII and more V (Giddings and Bloom, 1994). Why target thrombin for inhibition? Thrombin is the only coagulant enzyme that directly clots fibrinogen, thereby forming a thrombus. More importantly, thrombin is the only enzyme capable of cleaving fibrinogen to form fibrin, which then polymerizes to form a matrix for the blood clot (Buchanan et al., 1995). Factor XIII (a), which covalently cross-links fibrin, is also activated by thrombin (Buchanan et al., 1995; Giddings and Bloom, 1994). Effective inhibition of thrombus formation can be achieved by using thrombin inhibitors, which have no effect on any other coagulant enzyme. This paper presents an evaluation for antithrombin activity of 30 extracts from plants of central Florida. Magnolia virginiana and Myrica cerifera are the plants that have had medicinal uses amongst Native American Indian tribes. Louisiana Choctaws boiled the leaves and stems of bayberry (M. cerifera ) in water for a fever decoction, according to Bushnell et al. (1909). Speck (1941) states that Houma Indians boiled the leaves of wax myrtle (M. cerifera ) to use the tea as a vermifuge (antihelmintic agent). The Creeks, according to Swanton (1928), used bayberry as a charm medicine, to exorcise spirits of the dead and to prevent disease. Some Indians used bayberry as a mild emetic (Buchan (1816)). The Koasati gave the decoction of the plant to children with stomachaches (Taylor, 1940). The Micmac people used the roots of bayberry as an analgesic, against headaches and inflammations; berries, bark and leaves were used as a stimulant and beverage (Chandler et al., 1979). Hot poultice of crushed, water soaked roots of wax myrtle was used by the Micmac against external inflammations (Wallis, 1922). Magnolia species were used extensively by Indian tribes and colonial settlers. Magnolia virginiana australis , for instance, has been used by Mississippi Choctaws for chills and cramps, and for the same purposes by the Houma Indians (Speck, 1904, 1941). Magnolia glauca (an old name for M. virginiana ) was widely used by the Western Indians against fevers and rheumatism (Barton, 1810). M. virginiana was reported to ease pectoral diseases, internal pains and fever, have been used as a remedy against coughs and colds, and it was suggested that its decoction was also used to stop dysentery (Kalm, 1937).
2. Materials and methods 2.1. Plant material The list of plants studied is given in Table 1. Plant samples were collected in central Florida on December 16, 1997, and identified by Tony Calvino. Voucher specimens are deposited in the reference collection of the
Laboratory of Natural Products Chemistry in the Department of Chemistry at the University of Central Florida. For each voucher specimen a number was given corresponding to the species collected.
2.2. Extract preparation The extracts were prepared by extraction with methylene chloride and methanol as described by Miles et al. (1991). The plants (50 g dry weight) were extracted in sequence with methylene chloride (8 h) and ethanol (8 h) in a soxhlet apparatus. The solvent was removed in vacuo to yield the methylene chloride extract (fraction A) and the methanol extract (fraction B).
2.3. Antithrombin activity Ependorf tubes (calibrated to deliver 1 ml) were weighed. A Finn pipette (digital microdispenser) was utilized to remove 50 ml of solution from the bottle. This solution was then placed in the weighed Ependorf tube and the solvent was allowed to evaporate. The tubes were reweighed in order to calculate the exact amount of extract in each tube (usually in the range of 30 /60 mg). The extract was then diluted to 1 ml with methanol. A calculation was then made as to how many ml should be taken from this solution to deliver 1 mg in another Ependorf tube. After performing the delivery using a micropipette, the solution was then diluted to the 1 ml mark using a Tris buffer solution that had been made up by dissolving one packet of Tri-NaCl buffer powder from Sigma Chemical Company in 1000 ml of water. The Ependorf tubes were then capped and shaken in order to thoroughly mix the solution. A Corning 96 well flat bottom plate (well diameter 6.4 mm) was utilized. Into the first well, pure methanol (50 ml) was placed for the control. Fourteen samples (50 ml each) of plant material were in first two rows (14 wells) of the plate. Then 50 ml of the thrombin solution (which has been prepared by reconstituting 500 units of Bovine Plasma Lyophilized powder from Sigma Chemical Company) was placed in each well. The plate was then incubated at 37 8C for 5 min. Subsequently, 50 ml of the chromogenic reagent (D-Phe-L-Pipecoyl-Arg p-nitroaniline) from Sigma Chemical Company which was prepared by dissolving 25 mg of this reagent in 20 ml in dH2O to yield a solution of 2 mmol/l) was placed in each well. The 96 well plate was then placed in a Molecular Devices kinetic microplate reader. The absorbance at 45 nm was measured continuously over a 5 min period. A plot of this continuous measurement was obtained as well as the absorbance at the end of the 5 min. The percent activity was calculated using the following formula and the resulting data is presented in Table 1:
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Table 1 Results of thrombin bioassay
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
Species family
Voucher specimen num- Fraction A% inhibiber tion
Fraction B% inhibition
Chenopodium ambrosioides Bert.ex Steud. (Chenopodiaceae) Ardisia crenata Roxb. (Myrsinaceae) Eupatorium capillifolium Small (Asteraceae) Baccharis halimifolia L. (Asteraceae) Phytolacca americana Linn. (Phytolaccaceae) Melia azedarach Blanco. (Meliaceae) Trachelospermum jasminoides Lem. (Apocynaceae) Myrica cerifera Bigelow (Myricaceae) Cyperus globulosus Aubl. (Cyperaceae) Lagerstroemia indica Linn. (Lythraceae) Schefflera arboricola Hayata (Araliaceae) Nerium oleander Linn. (Apocynaceae) Brassaia actinophylla F. Muell (Araliaceae) Cinnamomum camphora (L.) T.Nees & C.H.Eberm. (Lauraceae) Abrus precatorius L. (Fabaceae) Dioscorea bulbifera R. Br. (Dioscoreaceae) Poinsettia heterophylla Small (Euphorbiaceae) Malvaviscus arboreus Cav. (Malvaceae) Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) Chamaesyce maculata Small (Euphorbiaceae) Citrus aurantifolia Swingle (Rutaceae) Antigonon leptopus Hook. & Arn. (Polygoneceae) Tetrapanax papyriferum C. Koch (Araliaceae) Bidens alba (Sch.Bip.) Ballard (Asteraceae) Passiflora incarnata Ker.-Gawl. (Passifloraceae) Clerodendron thomsonae Balf. (Verbenaceae) Callistemon lanceolatus (Sm.) Sweet (Myrtaceae) Ilex Crenata (Aquifoliaceae) Magnolia virginiana Linn. (Magnoliaceae) Ambrosia artemisiifolia f. gracilissima (L.) D. Cirtu & M. Cirtu (Compositae)
1 /00197 4 /00497 5 /00597 6 /00697 11 /01197 12 /01297 13 /01397 15 /01597 16 /01697 19 /01997 20 /02097 21-02197 22 /02297 23 /02397 24 /02497 25 /02597 26 /02697 27 /02797 29 /02897 30 /03097 31 /03197 32 /03297 33 /03397 40 /04097 41 /04197 42 /04297 43 /04397 35-03597 37 /03797 18 /01897
16 20 4 49 35 42 54 81 42 85 3 27 60 42 31 61 50 51 32 54 21 90 52 48 38 8 82 46 95 41
30 80 12 30 5 63 41 71 30 79 20 4 54 71 53 30 11 10 55 76 58 2 83 79 63 56 32 77 65 30
Note: Fraction A /methylene chloride fraction. Fraction B /methanol fraction.
(Vmax)sample 100% A% 1 (Vmax)blank
3. Results and discussion Thirty plants collected in central Florida have been extracted. Crude plant extracts have been tested for the antithrombin activity. The results of the antithrombin bioassay are given in Table 1. In the antithrombin bioassay plant extracts which showed an 80% activity or higher were classified as highly active and therefore merit further study relative to drug discovery. The following plants demonstrated activity of 80% or higher: Ardisia crenata (80% CH2Cl2 fraction), Tetrapanax papyriferus (83% CH2Cl2 fraction), Lagerstroemia indica (85% CH3OH fraction), Callistemon lanceolatus (82 % CH3OH fraction), Antigonon leptopus (89% CH3OH fraction), M. virginiana (95% CH3OH fraction), M. cerifera (81% CH3OH fraction).
A. crenata is a plant for which no biological activity has been reported. Two novel triterpenoid pentasaccharides ardisicrenosides E and F were isolated from roots of A. crenata by Jia et al. (1999). Fujioka et al. (1988) isolated a novel cyclic depsipeptide FR900359 that shows inhibition of platelet aggregation and decrease of blood pressure. Asada et al. (1980) isolated several new triterpenoids (paryriogenin D, E, F, G) from the leaves of T. papyriferus. Nagasawa et al. (1978) isolated two phytotoxic fungal metabolites (/)-epiepoxydon, and (/)-deoxyepiepoxydon from a culture of an unidentified fungus on diseased L. indica leaves. These two compounds markedly inhibited germination of lettuce seed. Castillo et al. (1980) showed that crude extracts of roots of L. indica yielded alkaloids, saponins, terpenoids, steroids, tannins, polyphenols, mucins, proteins, and reducing substances. Saleh, 1973 isolated three anthocyanins from L. indica flowers and identified them as delphinidin 3-arabinoside, petunidin 3-arabinoside, and malvidin 3-arabinoside. In addition, gallic acid, methyl gallate, ellagic acid, and traces of 3-Cglycosides were found. Varma and Parthasarathy, 1975
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isolated sitosterol, erythrodiol, betulin, betulinic acid, ursolic acid, and 2-hydroxyursolic acid from leaves of C. lanceolatus. It has been previously reported by Nitao et al. (1991), that three compounds isolated from leaves of M. virginiana (4,4?-diallyl-2, 3?-dihydroxybiphenyl ether, 3,5?-diallyl-2?-hydroxy-4-methoxybiphenyl and 5,5?-diallyl-2,2?-dihydroxybiphenyl) were toxic to brine shrimp and mosquito larvae and showed strong antifungal and antibacterial activities. Isolation and identification of three triterpenes (myricadiol, taraxerol, and taraxerone) and a flavonoidglycoside (myricitrin) from the root bark of M. cerifera was reported by Paul et al. (1974). Fujimoto et al. (1992), isolated myriceron caffeoyl ester from bayberry of M. cerifera. Myriceron caffeoyl ester is an ETA receptor antagonist. In summary, seven plant extracts from central Florida have demonstrated activity of 80% or higher in the inhibition of thrombin. In addition these plants are known to contain a wide array of compounds from different structural classes. Thus these plants have excellent potential to provide new anticoagulants that may be utilized in preventing blood clotting in cancer patients.
References Asada, M., Amagaya, S., Takai, M., Olihara, Y. 1980. New triterpenoids from the leaves of Tetrapanax papyriferum . Journal of the Chemical Society Perkin transactions 1, 325 /329. Buchan, W. 1816. Every Man His Own Doctor, or a Treatise on the Prevention and Cure of Diseases. Nathan Whiting, New Heaven, p. 445. Buchanan, M.R., Brister, S.J., Ofosu, F.A. 1995. Thrombin-catalyzed amplification and inhibitory reactions of blood coagulation. In: Buchanan, M.R., Brister, S.J., Ofosu, F.A. (Eds.), Thrombin: its Key role in Thrombogenesis: Implications for its Inhibition Clinically. CRC Press, Boca Raton, FL, pp. 1 /17. Bushnell, David I., Jr. (1909) The Choctaw of Bayou Lacomb, St. Tammany Parish, Louisiana. Bureau of American Ethnology Bulletin No. 48. Washington, D.C., Government Printing Office, p.23.
Castillo, A.V., Topacio, M.R., Balasbas, M.M. 1980. Physicochemical investigation of Lagerstroemia indica Linne . NRCP Research Bulletin 35, 201 /207. Chandler, R.F., Lois, F., Shirley, N.H. 1979. Herbal remedies of the maritime indians. Journal of Ethnopharmacology 1 (58), 49 /68. Collen, D., Wiman, B., Verstracle, M. 1979. The Physiological Inhibition of Blood Coagulation and Fibrinolysis. Elsever/NorthHolland Biomedical Press, North-Holland, p. 27. Fujimoto, M., Mihara, S., Nakajima, S., Ueda, M., Nakamura, M., Sakurai, K. 1992. A novel nonpeptide endothelin antagonist isolated from bayberry Myrica cerifera . FEBT Lett. 305, 41 /44. Fujioka, M., Koda, S., Morimoto, Y., Biemann, K. 1988. Structure of FR900359, a cyclic depsipeptide from Ardisia crenata sims . The Journal of Organic Chemistry 53, 2820 /2825. Giddings, C.J., Bloom, A.L. 1994. Blood Coagulation and Fibrinolytic Mechanisms. In: Giddings, J.C. (Ed.), Thrombin, Thrombomodulin and the Control of Hemostasis. R.G. Landes Company, Austin, TX, pp. 5 /73. Kalm, P. 1937. In: Benson, A.B. (Ed.), Peter Kalm Travels in North America (English version of 1770, 2 volumes, revised from original Swedish). Wilson-Erickson, New York, pp. 109 /110. Miles, D.H., Medeiros, J., Chittawong, V., Hedin, P.A., Swithenbank, C., Lidert, Z. 1991. 3?-formyl-2?,4?,6?-trihydroxychalcone from Psidium acutangulum, Phytochemistry 30, 1131 /1132. Nagasawa, H., Suzuki, A., Tamura, S. 1978. Isolation and structure of (/)-deoxyepiepoxydon and (/)-epiepoxydon, phytotoxic fungal metabolites. Agricultural and Biological Chemistry 42, 1303 /1304. Nitao, J.K., Nair, M.G., Thorogood, D.L., Johnson, K.S., Scriber, J.M. 1991. Bioactive neolignans from the leaves of Magnolia virginiana. . Phytochemistry 30, 2193 /2195. Paul, B.D., Rao, G.S., Kapadia, G. 1974. Journal of Pharmaceutical Sciences 63, 958 /959. Saleh, N.A. 1973. Anthocyanins of Lagerstroemia indica flowers. Phytochemistry 12, 2304. Speck, F.G. 1904. Choctaw-Creek Medicines . MS, American Philosophical Society. Speck, F.G. 1941. A list of plant curatives obtained from the houma indians of lousiana. Primitive Man 14 (4), 1956. Swanton, J.R. 1928. Religious beliefs and medicinal practices of the creek indians. In: Forty-second Annual Report of the Bureau of American Ethnology, 1924 /25. Government Printing Office, Washington, D.C, pp. 472 /672. Taylor, L.A. 1940. Plants Used As Curatives by Certain Southeastern Tribes. Botanical Museum of Harvard University, Cambridge, MA. Varma, R.S., Parthasarathy, M.R. 1975. Triterpenoids of Callistemon lanceolatus leaves. Phytochemistry 14 (7), 1675 /1676. Wallis, W.D. 1922. Medicines Used by the Micmac Indians. American Anthropologist 24, 24 /30.
Antithrombin activity of medicinal plants from central Florida Natalya Chistokhodova, Chi Nguyen, Tony Calvino, Ioulia Kachirskaia, Glenn Cunningham, D. Howard Miles * Department of Chemistry, University of Central Florida, Orlando, FL 32816, USA Accepted 5 April 2002
Abstract A chromogenic bioassay was utilized to determine the antithrombin activity of methylene chloride and methanol extracts prepared from 30 plants of central Florida. Extracts of Ardisia crenata , Tetrapanax papyriferus , Lagerstroemia indica , Callistemon lanceolatus , Antigonon Leptopus , Magnolia virginiana , and Myrica cerifera demonstrated activity of 80% or higher in this bioassay system. # 2002 Published by Elsevier Science Ireland Ltd. Keywords: Ardisia crenata ; Tetrapanax papyriferus ; Lagerstroemia indica ; Callistemon lanceolatus ; Antigonon Leptopus ; Magnolia virginiana ; Myrica cerifera ; Antithrombin activity
1. Introduction The inhibition of thrombin is very important in fighting many blood coagulation and platelet disorders because thrombin is an important enzyme in the blood coagulation process. Blood coagulation is caused by a series of zymogen activations. A zymogen is an enzyme that is inactive until it acquires full enzymatic activity upon specific proteolitic cleavage of one or more of its peptide bonds. Physiology of coagulation (Collen et al., 1979) can be described by intrinsic and extrinsic pathways. Blood in vivo will not clot until a vessel wall is damaged. Vessel wall injury will lead to exposure of a layer in the vessel wall, which is full of Tissue Factor (TF). TF (thromboplastin) is present in phospholipidprotein extracts of human and animal tissues. This TF binds with factor VII (a) in plasma to form the TF- VII (a) complex. Factor VII (a) is generated by minor proteolysis of the Arg-Ile peptide bond. The complex TF- VII (a) converts factor X /X (a). Factor X circulates in plasma as a two-chain glycoprotein with molecular weight of about 67 000. Activation of factor X by a complex of tissue factor VII (a) involves at least two peptide cleavages. Factor X (a) will then convert
* Corresponding author. Fax: /1-407-823-2252. E-mail address: [email protected] (D. Howard Miles).
prothrombin to thrombin. Prothrombin or human factor II is a glycoprotein composed of a single chain and three oligosaccharide side chains giving a molecular weight of 71 600. Thrombin activates factors V and VIII/V (a) and VIII (a). The X (a) formed will combine with factor V (a) to form the prothrombinase complex. This complex is orders of magnitude more effective in the conversion of prothrombin than X (a) alone, so at this point thrombin formation really starts. In this instance factor X (a) is the enzyme that activates prothrombin and factor V (a) is the cofactor. Thrombin converts soluble fibrinogen into stabilized insoluble fibrin. The resulting fibrin monomers polymerize, undergo further cleavage by thrombin and finally form stabilized fibrin of high tensile strength by the action of factor XIII (a). This leads to the stabilization of the formed clots and thus to hemostasis or thrombosis. Alternatively, coagulation can start by intrinsic pathway after blood comes into contact with glass surfaces. Kallikrein-induced hydrolysis of XII in plasma produces factor XII (a). Factor XII is a single chain glycoprotein containing about 17% carbohydrate. Factor XII (a) activates XI, XI (a) activates IX, and IX (a) activates X. The first activated X will activate prothrombin and the first traces of thrombin then activate factors VIII and V. The extrinsic and intrinsic pathways now merge: the first X (a) will activate the first prothrombin that will lead to
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activation of more VIII and more V (Giddings and Bloom, 1994). Why target thrombin for inhibition? Thrombin is the only coagulant enzyme that directly clots fibrinogen, thereby forming a thrombus. More importantly, thrombin is the only enzyme capable of cleaving fibrinogen to form fibrin, which then polymerizes to form a matrix for the blood clot (Buchanan et al., 1995). Factor XIII (a), which covalently cross-links fibrin, is also activated by thrombin (Buchanan et al., 1995; Giddings and Bloom, 1994). Effective inhibition of thrombus formation can be achieved by using thrombin inhibitors, which have no effect on any other coagulant enzyme. This paper presents an evaluation for antithrombin activity of 30 extracts from plants of central Florida. Magnolia virginiana and Myrica cerifera are the plants that have had medicinal uses amongst Native American Indian tribes. Louisiana Choctaws boiled the leaves and stems of bayberry (M. cerifera ) in water for a fever decoction, according to Bushnell et al. (1909). Speck (1941) states that Houma Indians boiled the leaves of wax myrtle (M. cerifera ) to use the tea as a vermifuge (antihelmintic agent). The Creeks, according to Swanton (1928), used bayberry as a charm medicine, to exorcise spirits of the dead and to prevent disease. Some Indians used bayberry as a mild emetic (Buchan (1816)). The Koasati gave the decoction of the plant to children with stomachaches (Taylor, 1940). The Micmac people used the roots of bayberry as an analgesic, against headaches and inflammations; berries, bark and leaves were used as a stimulant and beverage (Chandler et al., 1979). Hot poultice of crushed, water soaked roots of wax myrtle was used by the Micmac against external inflammations (Wallis, 1922). Magnolia species were used extensively by Indian tribes and colonial settlers. Magnolia virginiana australis , for instance, has been used by Mississippi Choctaws for chills and cramps, and for the same purposes by the Houma Indians (Speck, 1904, 1941). Magnolia glauca (an old name for M. virginiana ) was widely used by the Western Indians against fevers and rheumatism (Barton, 1810). M. virginiana was reported to ease pectoral diseases, internal pains and fever, have been used as a remedy against coughs and colds, and it was suggested that its decoction was also used to stop dysentery (Kalm, 1937).
2. Materials and methods 2.1. Plant material The list of plants studied is given in Table 1. Plant samples were collected in central Florida on December 16, 1997, and identified by Tony Calvino. Voucher specimens are deposited in the reference collection of the
Laboratory of Natural Products Chemistry in the Department of Chemistry at the University of Central Florida. For each voucher specimen a number was given corresponding to the species collected.
2.2. Extract preparation The extracts were prepared by extraction with methylene chloride and methanol as described by Miles et al. (1991). The plants (50 g dry weight) were extracted in sequence with methylene chloride (8 h) and ethanol (8 h) in a soxhlet apparatus. The solvent was removed in vacuo to yield the methylene chloride extract (fraction A) and the methanol extract (fraction B).
2.3. Antithrombin activity Ependorf tubes (calibrated to deliver 1 ml) were weighed. A Finn pipette (digital microdispenser) was utilized to remove 50 ml of solution from the bottle. This solution was then placed in the weighed Ependorf tube and the solvent was allowed to evaporate. The tubes were reweighed in order to calculate the exact amount of extract in each tube (usually in the range of 30 /60 mg). The extract was then diluted to 1 ml with methanol. A calculation was then made as to how many ml should be taken from this solution to deliver 1 mg in another Ependorf tube. After performing the delivery using a micropipette, the solution was then diluted to the 1 ml mark using a Tris buffer solution that had been made up by dissolving one packet of Tri-NaCl buffer powder from Sigma Chemical Company in 1000 ml of water. The Ependorf tubes were then capped and shaken in order to thoroughly mix the solution. A Corning 96 well flat bottom plate (well diameter 6.4 mm) was utilized. Into the first well, pure methanol (50 ml) was placed for the control. Fourteen samples (50 ml each) of plant material were in first two rows (14 wells) of the plate. Then 50 ml of the thrombin solution (which has been prepared by reconstituting 500 units of Bovine Plasma Lyophilized powder from Sigma Chemical Company) was placed in each well. The plate was then incubated at 37 8C for 5 min. Subsequently, 50 ml of the chromogenic reagent (D-Phe-L-Pipecoyl-Arg p-nitroaniline) from Sigma Chemical Company which was prepared by dissolving 25 mg of this reagent in 20 ml in dH2O to yield a solution of 2 mmol/l) was placed in each well. The 96 well plate was then placed in a Molecular Devices kinetic microplate reader. The absorbance at 45 nm was measured continuously over a 5 min period. A plot of this continuous measurement was obtained as well as the absorbance at the end of the 5 min. The percent activity was calculated using the following formula and the resulting data is presented in Table 1:
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Table 1 Results of thrombin bioassay
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
Species family
Voucher specimen num- Fraction A% inhibiber tion
Fraction B% inhibition
Chenopodium ambrosioides Bert.ex Steud. (Chenopodiaceae) Ardisia crenata Roxb. (Myrsinaceae) Eupatorium capillifolium Small (Asteraceae) Baccharis halimifolia L. (Asteraceae) Phytolacca americana Linn. (Phytolaccaceae) Melia azedarach Blanco. (Meliaceae) Trachelospermum jasminoides Lem. (Apocynaceae) Myrica cerifera Bigelow (Myricaceae) Cyperus globulosus Aubl. (Cyperaceae) Lagerstroemia indica Linn. (Lythraceae) Schefflera arboricola Hayata (Araliaceae) Nerium oleander Linn. (Apocynaceae) Brassaia actinophylla F. Muell (Araliaceae) Cinnamomum camphora (L.) T.Nees & C.H.Eberm. (Lauraceae) Abrus precatorius L. (Fabaceae) Dioscorea bulbifera R. Br. (Dioscoreaceae) Poinsettia heterophylla Small (Euphorbiaceae) Malvaviscus arboreus Cav. (Malvaceae) Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) Chamaesyce maculata Small (Euphorbiaceae) Citrus aurantifolia Swingle (Rutaceae) Antigonon leptopus Hook. & Arn. (Polygoneceae) Tetrapanax papyriferum C. Koch (Araliaceae) Bidens alba (Sch.Bip.) Ballard (Asteraceae) Passiflora incarnata Ker.-Gawl. (Passifloraceae) Clerodendron thomsonae Balf. (Verbenaceae) Callistemon lanceolatus (Sm.) Sweet (Myrtaceae) Ilex Crenata (Aquifoliaceae) Magnolia virginiana Linn. (Magnoliaceae) Ambrosia artemisiifolia f. gracilissima (L.) D. Cirtu & M. Cirtu (Compositae)
1 /00197 4 /00497 5 /00597 6 /00697 11 /01197 12 /01297 13 /01397 15 /01597 16 /01697 19 /01997 20 /02097 21-02197 22 /02297 23 /02397 24 /02497 25 /02597 26 /02697 27 /02797 29 /02897 30 /03097 31 /03197 32 /03297 33 /03397 40 /04097 41 /04197 42 /04297 43 /04397 35-03597 37 /03797 18 /01897
16 20 4 49 35 42 54 81 42 85 3 27 60 42 31 61 50 51 32 54 21 90 52 48 38 8 82 46 95 41
30 80 12 30 5 63 41 71 30 79 20 4 54 71 53 30 11 10 55 76 58 2 83 79 63 56 32 77 65 30
Note: Fraction A /methylene chloride fraction. Fraction B /methanol fraction.
(Vmax)sample 100% A% 1 (Vmax)blank
3. Results and discussion Thirty plants collected in central Florida have been extracted. Crude plant extracts have been tested for the antithrombin activity. The results of the antithrombin bioassay are given in Table 1. In the antithrombin bioassay plant extracts which showed an 80% activity or higher were classified as highly active and therefore merit further study relative to drug discovery. The following plants demonstrated activity of 80% or higher: Ardisia crenata (80% CH2Cl2 fraction), Tetrapanax papyriferus (83% CH2Cl2 fraction), Lagerstroemia indica (85% CH3OH fraction), Callistemon lanceolatus (82 % CH3OH fraction), Antigonon leptopus (89% CH3OH fraction), M. virginiana (95% CH3OH fraction), M. cerifera (81% CH3OH fraction).
A. crenata is a plant for which no biological activity has been reported. Two novel triterpenoid pentasaccharides ardisicrenosides E and F were isolated from roots of A. crenata by Jia et al. (1999). Fujioka et al. (1988) isolated a novel cyclic depsipeptide FR900359 that shows inhibition of platelet aggregation and decrease of blood pressure. Asada et al. (1980) isolated several new triterpenoids (paryriogenin D, E, F, G) from the leaves of T. papyriferus. Nagasawa et al. (1978) isolated two phytotoxic fungal metabolites (/)-epiepoxydon, and (/)-deoxyepiepoxydon from a culture of an unidentified fungus on diseased L. indica leaves. These two compounds markedly inhibited germination of lettuce seed. Castillo et al. (1980) showed that crude extracts of roots of L. indica yielded alkaloids, saponins, terpenoids, steroids, tannins, polyphenols, mucins, proteins, and reducing substances. Saleh, 1973 isolated three anthocyanins from L. indica flowers and identified them as delphinidin 3-arabinoside, petunidin 3-arabinoside, and malvidin 3-arabinoside. In addition, gallic acid, methyl gallate, ellagic acid, and traces of 3-Cglycosides were found. Varma and Parthasarathy, 1975
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isolated sitosterol, erythrodiol, betulin, betulinic acid, ursolic acid, and 2-hydroxyursolic acid from leaves of C. lanceolatus. It has been previously reported by Nitao et al. (1991), that three compounds isolated from leaves of M. virginiana (4,4?-diallyl-2, 3?-dihydroxybiphenyl ether, 3,5?-diallyl-2?-hydroxy-4-methoxybiphenyl and 5,5?-diallyl-2,2?-dihydroxybiphenyl) were toxic to brine shrimp and mosquito larvae and showed strong antifungal and antibacterial activities. Isolation and identification of three triterpenes (myricadiol, taraxerol, and taraxerone) and a flavonoidglycoside (myricitrin) from the root bark of M. cerifera was reported by Paul et al. (1974). Fujimoto et al. (1992), isolated myriceron caffeoyl ester from bayberry of M. cerifera. Myriceron caffeoyl ester is an ETA receptor antagonist. In summary, seven plant extracts from central Florida have demonstrated activity of 80% or higher in the inhibition of thrombin. In addition these plants are known to contain a wide array of compounds from different structural classes. Thus these plants have excellent potential to provide new anticoagulants that may be utilized in preventing blood clotting in cancer patients.
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