Chemistry and pharmacology of a tertiary alkaloid from Strychnos trinervis root bark

Journal of Ethnopharmacology, 19 (1987) Etsevier Scientific Publishers Ireland Ltd.

319-325

319

CHEMISTRY AND PHARMACOLOGY OF A TERTIARY FROM STR YCHNOS TRZNER VZS ROOT BARK*

MARGARETH F. de MELLO

de F.F. MELO, CID A. de M. SANTOS, and RABINDRANATH MUKHERJEE

.Cnborat&io de Tecnologia Farmace^utica, Universidade Joiio Pessoa, PB, Brazil and Instituto de Antibibticos Pernam buco, 50.000 Recife PE (Brazil) (Accepted

ALKALOID

ALDA de A. CHIAPPETA, Federal da Paraiba, Universidade Fedeml

JOSE

58.059 de

March 13, 1987)

Summary Bisnordihydrotoxiferine has been isolated as the major alkaloid from the root bark of Strychnos trineruis. This compound shows a wide antimicrobial spectrum against gram-positive, gram-negative and acid-fast microorganisms, filamentous and yeast-like fungi and also phytopathogenic microorganisms. Preliminary studies also reveal some cytotoxic activity in KB cells which was confirmed by tests against sarcoma 180 tumors.

Introduction Strychnos trineruis (Vell.) Mart. (Loganiaceae), popularly known as Quina Cruzeiro and Raiz Amargosu in the south and Cupitiio Preto in the northeast of Brazil is a creeper type plant that grows sparsely in the eastern states and somewhat more abundantly in the tropical northeast. Besides the curare-like properties of the quatemary alkaloids of this plant (Adank et al., 1953; Bovet et al., 1954), nothing more is known about S. trineruis, although the use of the roots and wood bark of the plant in traditional medicine to combat intermittent fever, to use as a diuretic and to treat nervous diseases, has been supported by some clinical observations (Peckolt, 1916). However, in a recent review (Krukoff et al., 1972) on the Strychnos species of the Americas, S. trineruis was not mentioned as a curare-type plant. In a prelimi-

*Part of this work was submitted to the Universidade PB, Brazil, in partial fulfillment of C.A.M.S.‘s MS. cology of Natural Products. Correspondence to: Dr. Rabindranath Mukherjee. 0378-8741/87/$02.80 Published and Printed

0 1987 Elsevier in Ireland

Scientific

Federal da Paraiba, degree in Chemistry

Publishers

Ireland

Ltd.

Jo&o Pessoa, and Pharma-

320

nary ph~acolo~c~ screening of S. ty~neru~ root bark, the fraction containing the tertiary alkaloids was found to have significant antimicrobial and cytotoxic activities. Therefore, a pharmacologically monitored chemical study of the tertiary fraction was initiated which resulted in the isolation of a number of bases of which bisnord~ydrotoxiferine (1) was the main alkaloid.

This paper reports the isolation and identification of the dimeric indole alkaloid, bisnordihydrotoxiferine, from the root bark of S. trineruis and its ~timicrobi~ and ctytotoxic activities. Experimental Chemistry, isolation and identification of the alkaloid Dry powdered root bark (1.5 kg) of S. tr~~e~uis,collected at Recife in December 1984, was macerated with NH,OH (50 ml, 5%) for 4-5 h and then percolated at room temperature with EtOH (95%) till the extract became very light maroon and gave a faint Mayer’s test. The total extract was then concentrated in a rotary evaporator in vacua at 40°C to a deep chocolate-colored syrup (300 g) which was then vigorously agitated with aqueous HOAc (4%, 1.7 1) until the aqueous extract responded negative to the Mayer’s test. After filtering over Celite, the gummy mass over the filter paper was discarded. The aqueous extract was then exhaustively extracted with CHCL (‘7 1) until the CHCIJ extract failed to respond to Mayer’s reagent. The CHCIJ part was then washed with ice-cold water to free it from acid, dried over anhy drous Na2S04, concentrated in vacua at 40°C to a deep brown-colored syrup (20 g) which was then diluted with chloroform (35 ml) and chromatographed over neutral Brockmann alumina, grade II-III (400 g), using C&H,, C6H6-CHC13 mixtures, CHC13and CHCl,-MeOH mixtures (19: 1 and 9 : 1) as eluents. The C,H,-CHCl, mixture (1: 1) and CHC13eluted parts following

321

evaporation furnished a slightly yellowish alkaloid (0.33 g) which crystallized from MeOH in colorless needles, m.p. above 340°C (yield, 0.02%), C&HadNd;m/z 552 (M”); [(r]g -822” (c 0.15, CHC13); Rf 0.80(CH&l,/ MeOH/NH40H; 94.5: 5.5: 0.5) and 0.64 (CHC13/MeOH; 9: 1); purple with CeIv sulphate and bright orange with Dragendorff; UV (EtOH), 293 and 323 nm (log E, 4.39 and 4.29); IR (KBr), 1640 (enamine) and 1600 cm-’ (indolme moiety); ‘H-NMR (CDC13), 7.12 (2II, t J 7.60 Hz, 9 and ll), 6.79 (lH, t J 7.60 Hz, lo), 6.40 (lII, d J 7.60 Hz, 12), 6.29 (lH[, s, 17),5.37 (2H, q J 6.50 Hz, 2 and 19), 3.72 (l&I, s, 3), 3.32 (lI-I, d J 11.60 Hz, 21), 3.12(2~,tmJ11~60Hz,5~d21’),2.98(2~,m,5’and 15), 2.47 (lH,m, 6), 2.28 (XI, m, 14), 2.08 (lH, bs, 6’), 1.81(3H, d J 6.50 Hz, 18) and 1.69 (lII, d J 12.60 Hz, 14’) ppm and m/x (5%) 552 (M”, loo), 276 (M+‘, 24), 144 (18), 143 (5), 130 (4), 122 (22) and 121(24). P~armaco~o~ica~ studies Antimicrobial activities The minimum inhibitory concentrations against 51 representative microorganisms were determined according to the agar streak method (Waksman et al., 1945). In order to determine the antimicrobial effects, a solution of bisnordihydrotoxiferine at 1000 Mg/ml was used in a series of dilutions of 1: 10 in Petri dishes using the appropriate medium for the specific development of the test microorganisms. According to the microorganism’s requirements, nutrient media of NA, GL, SA and PDA were used (components: NA = nutrient-agar - peptone 10 g, meat extract 3 g, NaC15 g, distilled water 1000 ml, agar 20 g (pH 6.90-7.10); GL = glucose-yeast extract - peptone 10 g, meat extract 3 g, NaCl 5 g, yeast extract 10 g, glucose 10 g, distilled water 1000 ml, agsr 15 g (pH 6.90-7.10); SA = Sabouraud-agar - - peptone 10 g, glucose 40 g, NaCf 7.50 g, agar 20 g, meat extract 3.50 g, distilled water 1000 ml (pH 7.00); PDA = potato-dextrose-agar - potato extract 1000 ml, glucose 20 g, agar 20 g (pH 6.80-7.00)). Each medium was melted and preserved at 45°C to homogenize with the test solution. Uniform streaks of microo~~~ms/spores were applied on the surface of the solid medium and growth or no growth of the microorganisms was observed in relation to the control. Antitumor and cytotoxic activities Preliminary testing for cytotoxic activity was performed using KB cells (human epidermal carcinoma) according to the NC1 protocol 1600 (Geran et al., 1972). KB cells were cultivated in Eagle’s basic medium with 10% serum, inoculated the day before, in monoextract, removed with 0.25% trypsin and then diluted in the completed medium 20,000-30,000 cells/ml. The test substance was diluted in the completed medium at a concentration of 100, 10, 1, and 0.10 pg/ml, using four doses (in duplicate) and a positive control

322

(6-mercaptopurine,EDSo= 0.06-0.50 @g/ml). The materialwas incubated at 37°C in presence of 5% CO2for 72 h. The cytotoxic activity was evaluated by means of dose-response. In order to test the acute toxicity, subsequent tests in vivo were performed using inbred Swiss albino mice (25-30 g). The doses were calculated in mg/kg of the body weight according to the method of Weill (1952). A control was tested which received only the vehicle of ~m~tration (10% Tween 80 and distilled water) by the intraperitonealroute. All animals were observed for 7 days after injection. Chemotherapeutictests with bisnordihydrotoxie were made with two experimental tumors, Ehrlich’s carcinoma and sarcoma 180, using six animalsin each group (control and treated) following the implant technique of Larionov (1970). The experimentwas performed 24 h after the tumors were implanted with the intraperitonealadministrationof calculated doses based on l/l0 of the LD,,. The results were calculated according to the averageresponse observed for each one of the tumors. Resultsand discussion Quantitativedeterminationof the antimicrobialeffects of bisnordihydrotoxiferine againsteight microorganismshas been reported by Verpoorte et al. (1978); however, no attempt was made to accuratelyestablishthe critical concen~ations. In the present study, qu~ti~tive tests usingthe continuous dilution technique in Petridishes were set up to determinethe minimum inhibitory concentration against 51 representativemicroorganisms.It has been found that bisnordihydrotoxiferinepresentsa wide antibacterialspectrum, being specially active againstgram-positiveorganisms.The data also show activity againsta largenumber of gram-negativeand acid-fastbacteria and againstfilamentous and yeast-likefungi, includingcertainphytopathogenie microorganisms(Table 1). TABLE 1 ANTIMICROBIAL

SPECTRUM

OF BISNORDIHIDROTOXIFERINE

Microorganisms

Species

Culture medium

MIC (rg/mVb

Bacteria (~~-positive)

Baciltur rubtilis 9 IA-16 Bacillus s~&fi~~ 27 IA-17 Bacillus mycoides IA-14 Bacillus anthracis IA-9 Bacillus cereus IA-11 ~icrococe~ citreus IA-4 Staphylococcus aureus W IA-l Sarcina lutea IA-6 Streptococcus faaealis ATCC 6067 IA-138 Streptococcus pyogenes IA-7

NA NA NA NA NA NA NA NA GL GL

l-2 10-20 40-50 40-60 30-40 30-40 20-30 5-10 40-50 50-60

323 TABLE

1 (Continued)

Microorganisms

Species

Culture medium

MIC (flg/ml)b

Bacteria (gram-negative)

Shigella paradysenteriae IA-29 Escherichia coli R IA-26 Escherichia coli S IA-27 Salmonella typhosa IA-51 Erwinia carotovora IA-106 Enterobacter (aerogenes) cloacae IA-55 Pseudomonas aeruginosa IA-39 Brucella suis ATCC 9843 IA-24 Brucella abortus ATCC IA-21 Brucella melitensis IA-22 Neisseria catarrhalis IA-31 Proteus morganii IA-3 5 Proteus mirabilis IA-33 Proteus vulgaris IA-38

NA NA NA NA NA NA NA GL GL GL GL GL GL GL

80-100 30-40 60-80 60-80 80-100 80-100 20-30 50-60 70-80 80-100 50-60 >lOO 40-50 >lOO

Mycobacterium tuberculosis 607 IA-82 Mycobacterium smegmatis IA-71 Mycobacterium phlei IA-70 Nocardia asteroides IA-3503

GL GL GL Sab

30-40 30-40 30-40 >lOO

Candida albicans IBB-60 IA-1007 Candida krusei IA-1002 Candida tropicalis IA-1004 Crytococcus neoformans IHM IA-1011 Cryptococcus neoformans ENCB IA-1010 Candida parakrusei IA-1006 Candida pseudotropicalis IA-1006 Candida utilis IA-1052 Saccharomyces cerevisiae IA-1012

Sab Sab Sab Sab Sab Sab Sab Sab Sab

80-100 100 80-100 100 80-100 100 80-100 80-100 80-100

Neurospora crassa IA-2083 Aspergillus niger ATCC 1025 IA-2003 Penicillium lilacinum IA-2023 Penicillium notatum IA-2248 Microsporum canis IA-2256 Aspergillus tlavus IA-23 16 Geotrichum candidum IA-2260

Sab Sab Sab Sab Sab Sab Sab

80-100 100 80-100 80-100 80-100 100 100

Macrophomina phaseolina IA-2322 Penicillium esclerotigenum IA-2323 Alternaria tenuissima IA-2307 Fusarium oxysporum f. vasinfectum IA-2324 Colletotrichum gloesporium IA-2325 Fusarium solani f. sp. phase& IA-2300 Agrobacterium tumefaciens IA-211

PDA PDA PDA PDA PDA PDA PDA

60-80 100 60-80 100 40-60 100 40-60

Bacteria (acid-fast)

Fungi (yeast-like)

Fungi (filamentous)

Fungi (phytopathogenic)

y Culture media: NA, nutrient-agar; tiL, glucose-yeast extract-agar; PDA, potato-dextrose-agar. b MIC, minimum inhibitory concentration.

Sab, Sabouraud-agar;

324 TABLE 2 ANTITUMOR ACTIVITY

OF BISNORDIHYDROTOXIFERINE

Tumor

Dose (mg/kg)

Average weight difference in animal T/C (g)’

Average weight of tumor + S.E.M. T/C (g)

Sarcoma 180 Carcinoma of Ehrlich

18 21

+ l.OO/+ 1.25 +0.30/-4.00

0.39 + 0.10/0.95 0.97’? 0.2711.42

Inhibition (%)

? 0.05 f 0.30

60.0 32.9

p T/C = test/control.

Bisnordihydrotoxiferine may be considered rather inactive against Ehrlich’s tumor (33%) but a distinct action (60%) against sarcoma 180 (Table 2) was observed. Conclusion From the results obtained, it may be concluded that the antimicrobial activity of bisnordihydrotoxiferine is broad but relatively weak. However, the promise of bisnordihydrotoxiferine as a cytotoxic agent is distinct, and this observation warrants further work using a broader array of animal tumors. It is also known that bisnordihidrotoxiferine is devoid of effects such as skeletal muscle relaxation (Stromborn et al., 1982). Acknowledgements The authors wish to express their sincere thanks to Professors Bhupesh C. Das, Institut de Chimie des Substances Naturelles, C.N.R.S., 91190 Gif-surYvette, France, and Norman R. Farnsworth, College of Pharmacy, University of Illinois, Chicago, U.S.A., for the mass and NMR spectra. Thanks are also accorded to Professors Alvaro A. da Silva, Laboratory of Microbiology, Silene C. do Nascimento, Division of’ Tissue Culture, and Rosalia M.O.C. Lima, Cancerology Experimental Laboratory, Instituto de Antibioticos, UFPE, Brazil, for some of the laboratory experiments. Both UFPB and UFPE groups thank CNPq and CAPES, Brazil,. for financial assistance. References Adank, K., Bovet, D., Ducke, A. and Marini-Bettolo, G.B. (1953) Ricerche sugli alcaloidi curarizzanti di varie specie di Strychnos de1 Brasile. Gli alcaloidi della Strychnos trineruis (Vell.) Mart. - Nota I. Gazzetta Chimica Italiann 83, 966-984. Bovet, D., Ducke, A., Adank, K. and Marini-Bettolo, G.B. (1954) Ricerche sugli alcaloidi curarizzanti di varie specie di Strychnos de1 Brasile. - Nota II. Studi preliminari su sette nuove specie di Strychnos. Gazzetta Chimica Italiana 84, 1141-1146.

325 Geran, R.I., Greenberg, N.H., Macdonald, M.M., Schumacher, A.M. and Abbott, B.J. (1972) Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemotherapy Report (Part 3) 3(2), l-103. Krukoff, B.A., Marini-Bettolo, G.B. and Bisset, N.G. (1972) American species of Strychnos. Lloydia 35, 193-271. Larionov, L.F. (1970) Screening anticancer drugs in the Soviet Union. Cancer Chemotherapy Report (Part 1) 54(2), 71-78. Nascimento, S.C., Mello, J.F. and Chiappeta, A.A. (1984/5) Agentes citotoxicos. Experimentos corn celulas KB. Reuista do Znstituto de Antibioticos, Recife 22(1/2), 19-26. Oyama, V.I. and Eagle, H. (1956) Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau). Proceedings of the Society for Experimental Biology and Medicine 91, 370-373. Peckolt, W. (1916) Thesis: ContribuicZo ao Estudo das Falsas Quinas Medicinoes da America do Sul Faculdade de Medicina do Rio de Janeiro, Rio de Janeiro. Stromborn, J., Huy, 0. and Bisset, N.G. (1970) Alkaloids of Strychnos wallichiana. Acta Pharmaceutics Suecica 19, 321-326. Verpoorte, R., Kode, E.W., van Doorne, H. and Baerheim Svendsen, A. (1978) Antimicrobial effect of the alkaloids from Strychnos afzelli Gilg. Planta Medica 33, 237242. Waksman, S.A. and Reilly, H.C. (1945) Agar-streak method for assaying antibiotic substances. Industrial Engineering Chemistry: Analytical Edition 17, 556-558. Weill, C.S. (1952) Tables for gonvenient calculation of median effective dose (LD,, or ED,,) and instruction in their use. Biometrics 8, 249-263.