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Biological activities of benzoquinones from Badula barthesia and Embelia angustifolia
Phytomedicine, Vol. 5(3), pp. 199-203 © Gustav Fischer Verlag 1998
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Biological activities of benzoquinones from Badula barthesia and Embelia angustifolia A. K. Lund, A. Adsersen and U. Nyman Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark
Summary In our screening program for antihypertensive plant constituents extracts of the leaves and bark of Badula barthesia showed strong in vitro inhibition of angiotensin converting enzyme (ACE). Rapanone (1), 2,S-dihydroxy-3-tridecyl-l,4-benzoquinone, was isolated as an active constituent of the leaves. The IC so values of rapanone and three 3-alkyl-2,S-dihydroxybenzoquinones, (2)-(4), (Z)-2,S-dihydroxy-3(pentadec-8-enyl)-I,4-benzoquinone, (Z,Z)-2,S-dihydroxy-3-(heptadeca-8,I1-dienyl}-1,4-benzoquinone and (Z)-2,S-dihydroxy-3-(heptadec-8-enyl)-1,4-benzoquinone recently isolated from Embelia angustifolia were determined. The following IC so ± S.D. values have been obtained (1) 36 ± 4.6 [AM, (2) 19 ± 6.2 [AM, (3) 19 ± 8.7 [AM and (4) 16 ± 3.0 [AM. The IC so value for the reference compound captopril was determined to 12 ± 2.6 nM. The antimicrobial activity of the four compounds was determined by thin layer chromatography agar overlay technique as minimum growth inhibitory amount in ug. One yeast, Candida albicans, and four bacteria, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were used as test organisms. Key words: angiotensin converting enzyme, ACE, antihypertensive effect, antimicrobial activity, rapanone, 2,5-dihydroxybenzoquinones, Badula barthesia, Embelia angustifolia, Myrsinaceae, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, plants Reunion Island
•
Introduction
Hypertension is considered as an important factor for several cardiovascular diseases and there is a need for development of improved antihypertensive drugs. In the renin-angiotensin system two enzymes are active, renin and angiotensin converting enzyme (ACE). Renin converts angiotensinogen to angiotensin I, then ACE converts angiotensin I into the active vasoconstrictive octapeptide angiotensin II and inactivates bradykinin. Angiotensin II also stimulates the synthesis and release of the hormone aldosteron. These factors contribute to the hypertensive effect of ACE. An inhibition of the renin-angiotensin system will lead to vasodilation, diuresis, Na-excretion and a fall in blood pressure (Wyvratt and Patchett, 1985). Badula barthesia (Lam.) A.DC. (vernacular name Bois de savon), Myrsinaceae, is endemic to Reunion Island in the Indian Ocean and Embelia angustifolia
(A.DC.) A.DC. in DC. (vernacular name Liane savon), Myrsinaceae is endemic to Reunion Island and Mauritius. Both species are used in traditional medicine. On Reunion Island leaves and bark of B. barthesia are used as diuretics, in the treatment of kidney disorders, inflammation of the bladder, gastritis, for the circulation and different skin disorders (Lavergne, 1990). E. angustifolia is used locally as a diuretic, in the treatment of kidney disorders, nephritis, inflammation of the bladder and gastritis (Lavergne, 1990). ' The use of the two species indicates that the active constituents could be compounds with antihypertensive and/or antimicrobial activity. In our screening program of plants for potential antihypertensive effect based on inhibition of ACE leaves and bark of B. barthesia and leaves of E. angustifolia showed strong inhibition (Adsersen and Adsersen, 1997).
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A. K. Lund et al.
The present paper deals with the isolation of rapanone from B. barthesia and the determination of the ACE inhibition and the antimicrobial activity of rapanone and three recently isolated 3-alkyl-2,5-dihydroxybenzoquinones from E. angustifolia (Lund et aI., 1997).
•
Materials and Methods
General Experimental Procedures
TLC was carried out on pre-coated silica gel plates, Kiselgel 60 F2S4 (Merck) impregnated with NaH 2P04 , eluent CH 2CI2 • The spots corresponding to benzoquinones being red in visible light. NMR spectra were recorded in CDCI 3/DMSO-d6 (2:1) on a Bruker AC200. lH and l3C NMR: 200 MHz and 50.3 MHz respectively using TMS as internal standard. The mass spectrum was recorded on a Jeol JMS AX505 mass spectrometer. The ion source was run in El mode, 70eV ionization energy. UV: Shimadzu UVvisible recording spectrophotometer, UV-265. IR: Perkin-Elmer 781 infrared spectrophotometer. The melting point is corrected and determined on a digital melting point apparatus. Plant material
Leaves and bark of Badula barthesia, Myrsinaceae, were collected in October 1994, at Colorado, St. Denis, Reunion Island. The plant material was air-dried immediately after collection. A voucher specimen (A. & H. Adsersen, 5503) is deposited in Herbarium C, Botanical Museum, Copenhagen, Denmark. ACE assay
In vitro ACE inhibitory activity was measured as described by Elbl and Wagner (1991) and later modified by Hansen et al. (1995) using angiotensin converting enzyme from rabbit lung (EC 3.4.15.1, Sigma) and dansyltriglycine as substrate. IC so value of 12 ± 2.6 nM for captopril. The substances for determining the dose response curves were dissolved in a 10% acetone-buffer solution. Test organisms
The test organisms used were Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538) and Candida albicans (IMI 349010). Isolation of rapanone
The dried and powdered leaves (375 g) of B. barthesia were extracted in a Soxhlet apparatus for about 6 hr with Et 20. The extract was partitioned in the system
pentane-Met.il-l-Hju (10:9:1) and the polar phase evaporated (yield 14.4 g). The polar phase was subjected to chromatography over silica gel (Kieselgel 60, 70-230 mesh Art. 7734, Merck) impregnated with 6-7% of KH 2P04 and 5% of water. 45 g KH2P04 was dissolved in 750 ml water and mixed with 400 g silica gel. The mixture was filtered, almost to dryness, on a Buchner funnel. The silica gel was dried and activated at 120°C for about 24 hours. The warm silica gel was transferred to a tared bottle. The bottle was closed and the silica gel was cooled to room temperature. The amount of silica gel was determined and 5% water was added. The filtrate was evaporated in a tared flask and the amount of residual phosphate was determined. CH 2Cl2-toluen (1:1) with increasing amounts of tert.butanol (2-5%) was used as eluent. Fractions containing compound 1 with R, = 0.45 were combined and refractionated. Compound 1 was crystallized from the fractions with MeOH or CH 2CI2 • All crystals were combined and recrystallized from CH2CI2 • In total 2.46 g of compound 1 was isolated corresponding to 0.65% yield from dry plant material. Compound 1: orange crystals, M.p. 139, 1-140, 1 °C (corrected). lH and l3C NMR, IR, UV and MS data were identical with previously published data for rapanone (Ogawa and Natori, 1968, Shah et aI., 1987, Chow et aI., 1991). Determination of ACE inhibitory activity
The IC so values are reported as the mean value ± standard deviation of at least 3 separate experiments. The IC so values were determined according to Fig. 2 by non-linear iterative curve-fitting of the data to the equation: % inhibition = (100% x [inhibitorj'u/Ijinhibitor]" + [ICSO]ll) using the program GraFit 3.01 (Erithacus Software, Stainess, UK). Statistical differences between mean IC so and between mean n were examined by a two-tailed Students t-test at a 0.05 level of significance. Determination of antimicrobial activity
A thin layer chromatographic agar overlay technique (Saxena et aI., 1995) was used to determine the activity of the four compounds against two gram negative bacteria: Escherichia coli and Pseudomonas aeruginosa and two gram positive bacteria: Bacillus subtilis and Staphylococcus aureus and the yeast Candida albicans. The minimum growth inhibitory amount (MIA) for the compounds was determined by applying the pure compounds to TLC-plates, silica gel, (Kieselgel 60, Merck) 10 x 10 ern. A seeded agar medium with 10 6 CFUlml was distributed over the TLC-plates and the plates
Biological activities of benzoquinones
201
1 R=
Fig. 1. The structures of the 3alkyl-2,S-dihydroxybenzoquinones.
H*OOH HO I o I R
were incubated overnight at 37°C. After incubation the TLC-plates were sprayed with an aqueous solution (1 mg/ml) of dimethylthiazolyldiphenyltetrazolium bromide (MTT) (Riedel de Haen 33751). After approximately half an hour inhibition zones were observed as yellow spots against a blue background. All experiments were done in duplicate.
•
Results and discussion
Identification
Rapanone (1), 2,5-dihydroxy-3-tridecyl-l,4-benzoquinone; Orange crystals from methylenchloride; M.p. (corr.) 139.1-140.1 °C, lit. 140-142°C (Shah et al., 1987, Chow et al., 1991). The UV spectrum of 1 shows the characteristic pattern for a 2,5-dihydroxy-l,4-benzoquinone with absorption at about 288 and 417 nm (Ogawa and Natori, 1968). The IR spectrum is also characteristic for a 2,5-dihydroxy-l,4-benzoquinone, with bands at 3305 (OH) and 1610 (>C = 0) em:'. IH NMR: s ca. 10.60 (2H, br, OH), 5.76 (lH, s, H6),2.35 (2H, t, J = 6.8 Hz, H-l'), 1.25 (22H, m, H-2'H-12'), 0.87 (3H, t, J = 6.3 Hz, H-13'). The EI mass spectrum of 1 shows rn/z (rel.int.): 322 [M]+ (85) and 154 [M-168]+ (100) pointing to 2,5-dihydroxy-l,4benzoquinone structure with a single side chain, -C 13H27 , at position 3. IH and 13C NMR, IR, UV and MS data were identical with previously published data for rapanone (Ogawa and Natori, 1968, Shah et al., 1987, Chow et al., 1991). Fig. 1 shows the structures of rapanone and the three 3-alkyl-2,5-dihydroxy-benzoquinones from E. angustifolia (Lund et al., 1997). ACE inhibitory activity
Fig. 2 shows the dose-response curves (in acetone) for the inhibition of ACE by the four 3-alkyl-2,5-dihydroxybenzoquinones. The IC so values were determined by non-linear curve-fitting of the data to the graph. The following IC so ± S.D. values have been obtained (1) 36 ± 4.6 [!M, (2) 19 ± 6.2 [!M, (3) 19 ± 8.7 [!M and (4) 16 ± 3.0 [!M. The IC so value for 1 is significantly different from the IC so values for compounds 2-4. The IC so
2 R= 3
R=
4
R=
value for the reference compound captopril is according to the literature 14 nM (Elbl and Wagner, 1991). In our laboratory the IC so for captopril is determined to 12 ± 2.6 nM. In Fig. 2 n was determined to (1) 2.59 ± 0.35, (2) 1.68 ± 0.35, (3) 1.47 ± 0.22 and (4) 1.34 ± 0.24. The n value for 1 is significantly different from the n values for compounds 2-4. When the value of n is unity, the inhibitor shows pure competitive inhibition. As n, for the four compounds (1)-(4), is greater than unity, the inhibition is not a pure substrate inhibition. The curves in Fig. 2 might suggest that the substrate and the inhibitors bind to different domains on the enzyme or that the inhibitors show allosteric inhibition. Studies have established that ACE has two functional catalytic sites (Soubrier et al., 1993). From the inhibition data of captopril n was determined to be about 2 (Hansen, 1995).
100 UJ
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20
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Fig. 2. Determination of ACE inhibitory activity. The ICso values were determined by non-linear iterative curve-fittingof the data to the equation: % Inhibition = (100% x [inhibitorj-l/tlinhibitor]" + [ICso]D) using the program GraFit 3.01. The following ICso values have been obtained (1) 36 ± 4.6 !!M, (2) 19 ± 6.2 !!M, (3) 19 ± 8.7!!M and (4) 16 ± 3.0 !!M. The ICso values are reported as the means ± S.D. of at least three separate experiments. o Compound 1; .A. Compound 2; 'V Compound 3; • Compound 4
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A. K. Lund et al.
The ACE inhibitory activity of the 3-alkyl-2,5-dihydroxybenzoquinones seems to some degree to be influenced by the saturation and/or the length of the side chain. Rapanone (saturated and with the shortest side chain) shows IC so = 36 u.M, while compounds 2 and 4 (monounsaturated and 2 with the shortest side chain) show IC so = 19 [.lM and IC so = 16 [.lM, respectively. Compound 3 with two double bonds has IC so = 19 u.M, It seems to be the first time 3-alkyl-2,5-dihydroxybenzoquinones have been reported as ACE inhibitors. Nicotianamine is until now the most active ACE inhibitor isolated from plant material. The compound is isolated from soy sauce and has an IC so value determined to 0.26 u.M. As soy beans is one of the raw materials in the production ofsoy sauce, the origin of nicotianamine in soy sauce might be considered to be soy bean (Kinoshita et aI., 1993).
Antimicrobial activity The results from the antimicrobial tests are shown in Table 1 together with the MIA values for the two reference compounds streptomycin and amphotericin. The most active compound was 2 being active against the two gram positive bacteria, B. subtilis (MIA = 5 ug) and S. aureus (MIA = 1.25 ug), respectively. The minimum growth inhibitory amount for compound 2 is only 5 times (B. subtilis) and 1.6 times (S. aureus) the values for Streptomycin. None of the compounds showed any activity against the yeast C. albicans in the amount of 10 [.lg or less. According to the literature rapanone (1) is biologically active as an anthelmintic/antiparasitic agent (Shah et aI., 1987) and is known to stimulate succinate oxidase activity of intact mitochondria from rat liver (Ozawa et aI., 1965). As the compounds 2-4 are new no biological activity has so far been published. Our results with respect to ACE inhibitory activity and antimicrobial activity supports the traditional use of the two plant species as diuretics and as remedies in the treatment of inflammation of the bladder, gastritis and skin disorders, which could be due to microbial infections.
Acknowledgements
A special thank to Bente Gauguin and Katrine Juhl Krydsfeldt for technical assistance. Bjarke Ebert for computer assistance. Carl Erik Olsen, The Royal Veterinary and Agricultural University,for making the mass determination. Collection of plant material was supported by a travel grant from Julius Waels and Helga Waels Foundation. The Velux Foundation and the Thorkil Steenbeck Foundation, the Ib Henriksen Foundation and the Danish Council for Technical Research are greatefully acknowledged for provision of the NMR instrument.
•
References
Adsersen, A. and Adsersen, H.: Plants from Reunion Island with alleged antihypertensive and diuretic effects - an experimental and ethnobotanical evaluation. ]. Ethnopharmacal. 58 (3): 189-206, 1997. Chow, P.W., Sim, K. Y., Lim, P. L. and Chung, V. c.: Constituents of Ardisia elliptica; BC NMR and Mass spectra of rapanone and related quinones. Bull. Singapore Natl. Inst. Chem. 19: 87-93, 1991. Elbl, G. and Wagner, H.: A new method for the in vitro screening of inhibitors of angiotensin-converting enzyme (ACE), using the chrornophore- and fluorophore-labelled substrate, dansyltriglycine. Planta Med. 57: 137-141, 1991. Hansen, K., Nyman, u., Smitt, U. W., Adsersen, A., Gudiksen, L., Rajasekharan, S. and Pushpangadan, P.: In vitro screening of traditional medicines for antihypertensive effect based on inhibition of the angiotensin converting enzyme (ACE). J. Ethnopharmacol. 48 (1): 43-51, 1995. Hansen, K.: In vitro screening for the detection of angiotensin converting enzyme (ACE) inhibitors in selected medicinal plants and identification of the active principles. Ph. D. dissertation. Department of Pharmacognosy, The Royal Danish School of Pharmacy, Copenhagen, Denmark, 1995. Kinoshita, E., Yamakoshi, J. And Kikuchi, M.: Purification and identification of an angiotensin l-converting enzyme inhibitor from soy sauce. Biotech. Biochem. 57 (7): 1107-1110,1993. Lavergne, R.: Tisaneurs et plantes medicinales indigenes de lile de La Reunion. Editions Orphie 3 et 5 Avenue d'Orleans, Livry Gargan, France, pp. 271-273 (B. barthesia), pp. 347-349 (E. angustifolia), 1990. Lund, A. K., Lemmich,]., Adsersen, A. and Olsen, C. E.: Benzoquinones from Embelia angustifolia. Phytochemistry 44 (4): 679-681, 1997.
Table 1. The minimum growth inhibitory amount (MIA) for the four 3-alkyl-2,5-dihydroxybenzoquinones. Compound
E. coli
P. aeruginosa
1 2 3 4 Streptomycin Amphotericin
10 ug
>10/tg 10 ug >10 [.lg 0.8 [.lg
10 ug >10/tg >10 [.lg >10 ug 2 [.lg
B. subtilis 10 [.lg 5 ug 10 [.lg 10 [.lg 1 [.lg
S. aureus >10 ug 1.25 ug >10 [.lg 5 ug 0.8 ug
C. albicans >10 [.lg >10 ug >10 ug >10 [.lg 1.2 ug
Biological activities of benzoquinones Ogawa, H. and Natori, S.: Hydroxybenzoquinones from Myrsinaceae plants. III. The structure of 2-hydroxy-5methoxy-3-pentadecenylbenzoquinone and ardisiaquinones A, Band C from Ardisia spp. Chem. Pharm, Bull. 16 (9): 1709-1720, 1968. Ozawa, H., Natori, S. and Mamose, K.: Biochemical studies on quinone derivatives. I. Effects of naturally occurring benzoquinone derivatives on mitochondrial preparations. Chem. Pharm. Bull. 13: 1029-1035, 1965. Saxena, G., Farmer, S., Towers, G. H. N. and Hancock, R. E. W.: Use of specific dyes in the detection of antimicrobial compounds from crude plant extracts using a thin layer chromatography agar overlay technique. Phytocbem. Anal. 6: 125-129, 1995. Shah, V., Sunder, R. and De Souza, N. ].: Chonemorphine and rapanone antiparasitic agents from plant sources. J. Nat. Prod. 50 (4): 730-731,1987. Soubrier, F., Wie, L., Hubert, c., Clauser, E., Alhenc-Gelas, F.
203
and Corvol, P.: Molecular biology of the angiotensin I converting enzyme: II. Structure-function. Gene polymorphism and clinical implications. J. Hypertens. 11: 599-604, 1993. Wyvratt, M. ]. and Patchett, A. A.: Recent developments in the design of angiotensin-converting enzyme Inhibitors. Med. Res. Rev. 5 (4): 483-531,1985.
• Address A. Adsersen, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen 0, Denmark Tel.: +4535370850 Fax: +4535370845 e-mail: [email protected]
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I~~~~!~mml
Biological activities of benzoquinones from Badula barthesia and Embelia angustifolia A. K. Lund, A. Adsersen and U. Nyman Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark
Summary In our screening program for antihypertensive plant constituents extracts of the leaves and bark of Badula barthesia showed strong in vitro inhibition of angiotensin converting enzyme (ACE). Rapanone (1), 2,S-dihydroxy-3-tridecyl-l,4-benzoquinone, was isolated as an active constituent of the leaves. The IC so values of rapanone and three 3-alkyl-2,S-dihydroxybenzoquinones, (2)-(4), (Z)-2,S-dihydroxy-3(pentadec-8-enyl)-I,4-benzoquinone, (Z,Z)-2,S-dihydroxy-3-(heptadeca-8,I1-dienyl}-1,4-benzoquinone and (Z)-2,S-dihydroxy-3-(heptadec-8-enyl)-1,4-benzoquinone recently isolated from Embelia angustifolia were determined. The following IC so ± S.D. values have been obtained (1) 36 ± 4.6 [AM, (2) 19 ± 6.2 [AM, (3) 19 ± 8.7 [AM and (4) 16 ± 3.0 [AM. The IC so value for the reference compound captopril was determined to 12 ± 2.6 nM. The antimicrobial activity of the four compounds was determined by thin layer chromatography agar overlay technique as minimum growth inhibitory amount in ug. One yeast, Candida albicans, and four bacteria, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were used as test organisms. Key words: angiotensin converting enzyme, ACE, antihypertensive effect, antimicrobial activity, rapanone, 2,5-dihydroxybenzoquinones, Badula barthesia, Embelia angustifolia, Myrsinaceae, Candida albicans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, plants Reunion Island
•
Introduction
Hypertension is considered as an important factor for several cardiovascular diseases and there is a need for development of improved antihypertensive drugs. In the renin-angiotensin system two enzymes are active, renin and angiotensin converting enzyme (ACE). Renin converts angiotensinogen to angiotensin I, then ACE converts angiotensin I into the active vasoconstrictive octapeptide angiotensin II and inactivates bradykinin. Angiotensin II also stimulates the synthesis and release of the hormone aldosteron. These factors contribute to the hypertensive effect of ACE. An inhibition of the renin-angiotensin system will lead to vasodilation, diuresis, Na-excretion and a fall in blood pressure (Wyvratt and Patchett, 1985). Badula barthesia (Lam.) A.DC. (vernacular name Bois de savon), Myrsinaceae, is endemic to Reunion Island in the Indian Ocean and Embelia angustifolia
(A.DC.) A.DC. in DC. (vernacular name Liane savon), Myrsinaceae is endemic to Reunion Island and Mauritius. Both species are used in traditional medicine. On Reunion Island leaves and bark of B. barthesia are used as diuretics, in the treatment of kidney disorders, inflammation of the bladder, gastritis, for the circulation and different skin disorders (Lavergne, 1990). E. angustifolia is used locally as a diuretic, in the treatment of kidney disorders, nephritis, inflammation of the bladder and gastritis (Lavergne, 1990). ' The use of the two species indicates that the active constituents could be compounds with antihypertensive and/or antimicrobial activity. In our screening program of plants for potential antihypertensive effect based on inhibition of ACE leaves and bark of B. barthesia and leaves of E. angustifolia showed strong inhibition (Adsersen and Adsersen, 1997).
200
A. K. Lund et al.
The present paper deals with the isolation of rapanone from B. barthesia and the determination of the ACE inhibition and the antimicrobial activity of rapanone and three recently isolated 3-alkyl-2,5-dihydroxybenzoquinones from E. angustifolia (Lund et aI., 1997).
•
Materials and Methods
General Experimental Procedures
TLC was carried out on pre-coated silica gel plates, Kiselgel 60 F2S4 (Merck) impregnated with NaH 2P04 , eluent CH 2CI2 • The spots corresponding to benzoquinones being red in visible light. NMR spectra were recorded in CDCI 3/DMSO-d6 (2:1) on a Bruker AC200. lH and l3C NMR: 200 MHz and 50.3 MHz respectively using TMS as internal standard. The mass spectrum was recorded on a Jeol JMS AX505 mass spectrometer. The ion source was run in El mode, 70eV ionization energy. UV: Shimadzu UVvisible recording spectrophotometer, UV-265. IR: Perkin-Elmer 781 infrared spectrophotometer. The melting point is corrected and determined on a digital melting point apparatus. Plant material
Leaves and bark of Badula barthesia, Myrsinaceae, were collected in October 1994, at Colorado, St. Denis, Reunion Island. The plant material was air-dried immediately after collection. A voucher specimen (A. & H. Adsersen, 5503) is deposited in Herbarium C, Botanical Museum, Copenhagen, Denmark. ACE assay
In vitro ACE inhibitory activity was measured as described by Elbl and Wagner (1991) and later modified by Hansen et al. (1995) using angiotensin converting enzyme from rabbit lung (EC 3.4.15.1, Sigma) and dansyltriglycine as substrate. IC so value of 12 ± 2.6 nM for captopril. The substances for determining the dose response curves were dissolved in a 10% acetone-buffer solution. Test organisms
The test organisms used were Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538) and Candida albicans (IMI 349010). Isolation of rapanone
The dried and powdered leaves (375 g) of B. barthesia were extracted in a Soxhlet apparatus for about 6 hr with Et 20. The extract was partitioned in the system
pentane-Met.il-l-Hju (10:9:1) and the polar phase evaporated (yield 14.4 g). The polar phase was subjected to chromatography over silica gel (Kieselgel 60, 70-230 mesh Art. 7734, Merck) impregnated with 6-7% of KH 2P04 and 5% of water. 45 g KH2P04 was dissolved in 750 ml water and mixed with 400 g silica gel. The mixture was filtered, almost to dryness, on a Buchner funnel. The silica gel was dried and activated at 120°C for about 24 hours. The warm silica gel was transferred to a tared bottle. The bottle was closed and the silica gel was cooled to room temperature. The amount of silica gel was determined and 5% water was added. The filtrate was evaporated in a tared flask and the amount of residual phosphate was determined. CH 2Cl2-toluen (1:1) with increasing amounts of tert.butanol (2-5%) was used as eluent. Fractions containing compound 1 with R, = 0.45 were combined and refractionated. Compound 1 was crystallized from the fractions with MeOH or CH 2CI2 • All crystals were combined and recrystallized from CH2CI2 • In total 2.46 g of compound 1 was isolated corresponding to 0.65% yield from dry plant material. Compound 1: orange crystals, M.p. 139, 1-140, 1 °C (corrected). lH and l3C NMR, IR, UV and MS data were identical with previously published data for rapanone (Ogawa and Natori, 1968, Shah et aI., 1987, Chow et aI., 1991). Determination of ACE inhibitory activity
The IC so values are reported as the mean value ± standard deviation of at least 3 separate experiments. The IC so values were determined according to Fig. 2 by non-linear iterative curve-fitting of the data to the equation: % inhibition = (100% x [inhibitorj'u/Ijinhibitor]" + [ICSO]ll) using the program GraFit 3.01 (Erithacus Software, Stainess, UK). Statistical differences between mean IC so and between mean n were examined by a two-tailed Students t-test at a 0.05 level of significance. Determination of antimicrobial activity
A thin layer chromatographic agar overlay technique (Saxena et aI., 1995) was used to determine the activity of the four compounds against two gram negative bacteria: Escherichia coli and Pseudomonas aeruginosa and two gram positive bacteria: Bacillus subtilis and Staphylococcus aureus and the yeast Candida albicans. The minimum growth inhibitory amount (MIA) for the compounds was determined by applying the pure compounds to TLC-plates, silica gel, (Kieselgel 60, Merck) 10 x 10 ern. A seeded agar medium with 10 6 CFUlml was distributed over the TLC-plates and the plates
Biological activities of benzoquinones
201
1 R=
Fig. 1. The structures of the 3alkyl-2,S-dihydroxybenzoquinones.
H*OOH HO I o I R
were incubated overnight at 37°C. After incubation the TLC-plates were sprayed with an aqueous solution (1 mg/ml) of dimethylthiazolyldiphenyltetrazolium bromide (MTT) (Riedel de Haen 33751). After approximately half an hour inhibition zones were observed as yellow spots against a blue background. All experiments were done in duplicate.
•
Results and discussion
Identification
Rapanone (1), 2,5-dihydroxy-3-tridecyl-l,4-benzoquinone; Orange crystals from methylenchloride; M.p. (corr.) 139.1-140.1 °C, lit. 140-142°C (Shah et al., 1987, Chow et al., 1991). The UV spectrum of 1 shows the characteristic pattern for a 2,5-dihydroxy-l,4-benzoquinone with absorption at about 288 and 417 nm (Ogawa and Natori, 1968). The IR spectrum is also characteristic for a 2,5-dihydroxy-l,4-benzoquinone, with bands at 3305 (OH) and 1610 (>C = 0) em:'. IH NMR: s ca. 10.60 (2H, br, OH), 5.76 (lH, s, H6),2.35 (2H, t, J = 6.8 Hz, H-l'), 1.25 (22H, m, H-2'H-12'), 0.87 (3H, t, J = 6.3 Hz, H-13'). The EI mass spectrum of 1 shows rn/z (rel.int.): 322 [M]+ (85) and 154 [M-168]+ (100) pointing to 2,5-dihydroxy-l,4benzoquinone structure with a single side chain, -C 13H27 , at position 3. IH and 13C NMR, IR, UV and MS data were identical with previously published data for rapanone (Ogawa and Natori, 1968, Shah et al., 1987, Chow et al., 1991). Fig. 1 shows the structures of rapanone and the three 3-alkyl-2,5-dihydroxy-benzoquinones from E. angustifolia (Lund et al., 1997). ACE inhibitory activity
Fig. 2 shows the dose-response curves (in acetone) for the inhibition of ACE by the four 3-alkyl-2,5-dihydroxybenzoquinones. The IC so values were determined by non-linear curve-fitting of the data to the graph. The following IC so ± S.D. values have been obtained (1) 36 ± 4.6 [!M, (2) 19 ± 6.2 [!M, (3) 19 ± 8.7 [!M and (4) 16 ± 3.0 [!M. The IC so value for 1 is significantly different from the IC so values for compounds 2-4. The IC so
2 R= 3
R=
4
R=
value for the reference compound captopril is according to the literature 14 nM (Elbl and Wagner, 1991). In our laboratory the IC so for captopril is determined to 12 ± 2.6 nM. In Fig. 2 n was determined to (1) 2.59 ± 0.35, (2) 1.68 ± 0.35, (3) 1.47 ± 0.22 and (4) 1.34 ± 0.24. The n value for 1 is significantly different from the n values for compounds 2-4. When the value of n is unity, the inhibitor shows pure competitive inhibition. As n, for the four compounds (1)-(4), is greater than unity, the inhibition is not a pure substrate inhibition. The curves in Fig. 2 might suggest that the substrate and the inhibitors bind to different domains on the enzyme or that the inhibitors show allosteric inhibition. Studies have established that ACE has two functional catalytic sites (Soubrier et al., 1993). From the inhibition data of captopril n was determined to be about 2 (Hansen, 1995).
100 UJ
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100
Concentration (IJM]
Fig. 2. Determination of ACE inhibitory activity. The ICso values were determined by non-linear iterative curve-fittingof the data to the equation: % Inhibition = (100% x [inhibitorj-l/tlinhibitor]" + [ICso]D) using the program GraFit 3.01. The following ICso values have been obtained (1) 36 ± 4.6 !!M, (2) 19 ± 6.2 !!M, (3) 19 ± 8.7!!M and (4) 16 ± 3.0 !!M. The ICso values are reported as the means ± S.D. of at least three separate experiments. o Compound 1; .A. Compound 2; 'V Compound 3; • Compound 4
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The ACE inhibitory activity of the 3-alkyl-2,5-dihydroxybenzoquinones seems to some degree to be influenced by the saturation and/or the length of the side chain. Rapanone (saturated and with the shortest side chain) shows IC so = 36 u.M, while compounds 2 and 4 (monounsaturated and 2 with the shortest side chain) show IC so = 19 [.lM and IC so = 16 [.lM, respectively. Compound 3 with two double bonds has IC so = 19 u.M, It seems to be the first time 3-alkyl-2,5-dihydroxybenzoquinones have been reported as ACE inhibitors. Nicotianamine is until now the most active ACE inhibitor isolated from plant material. The compound is isolated from soy sauce and has an IC so value determined to 0.26 u.M. As soy beans is one of the raw materials in the production ofsoy sauce, the origin of nicotianamine in soy sauce might be considered to be soy bean (Kinoshita et aI., 1993).
Antimicrobial activity The results from the antimicrobial tests are shown in Table 1 together with the MIA values for the two reference compounds streptomycin and amphotericin. The most active compound was 2 being active against the two gram positive bacteria, B. subtilis (MIA = 5 ug) and S. aureus (MIA = 1.25 ug), respectively. The minimum growth inhibitory amount for compound 2 is only 5 times (B. subtilis) and 1.6 times (S. aureus) the values for Streptomycin. None of the compounds showed any activity against the yeast C. albicans in the amount of 10 [.lg or less. According to the literature rapanone (1) is biologically active as an anthelmintic/antiparasitic agent (Shah et aI., 1987) and is known to stimulate succinate oxidase activity of intact mitochondria from rat liver (Ozawa et aI., 1965). As the compounds 2-4 are new no biological activity has so far been published. Our results with respect to ACE inhibitory activity and antimicrobial activity supports the traditional use of the two plant species as diuretics and as remedies in the treatment of inflammation of the bladder, gastritis and skin disorders, which could be due to microbial infections.
Acknowledgements
A special thank to Bente Gauguin and Katrine Juhl Krydsfeldt for technical assistance. Bjarke Ebert for computer assistance. Carl Erik Olsen, The Royal Veterinary and Agricultural University,for making the mass determination. Collection of plant material was supported by a travel grant from Julius Waels and Helga Waels Foundation. The Velux Foundation and the Thorkil Steenbeck Foundation, the Ib Henriksen Foundation and the Danish Council for Technical Research are greatefully acknowledged for provision of the NMR instrument.
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References
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Table 1. The minimum growth inhibitory amount (MIA) for the four 3-alkyl-2,5-dihydroxybenzoquinones. Compound
E. coli
P. aeruginosa
1 2 3 4 Streptomycin Amphotericin
10 ug
>10/tg 10 ug >10 [.lg 0.8 [.lg
10 ug >10/tg >10 [.lg >10 ug 2 [.lg
B. subtilis 10 [.lg 5 ug 10 [.lg 10 [.lg 1 [.lg
S. aureus >10 ug 1.25 ug >10 [.lg 5 ug 0.8 ug
C. albicans >10 [.lg >10 ug >10 ug >10 [.lg 1.2 ug
Biological activities of benzoquinones Ogawa, H. and Natori, S.: Hydroxybenzoquinones from Myrsinaceae plants. III. The structure of 2-hydroxy-5methoxy-3-pentadecenylbenzoquinone and ardisiaquinones A, Band C from Ardisia spp. Chem. Pharm, Bull. 16 (9): 1709-1720, 1968. Ozawa, H., Natori, S. and Mamose, K.: Biochemical studies on quinone derivatives. I. Effects of naturally occurring benzoquinone derivatives on mitochondrial preparations. Chem. Pharm. Bull. 13: 1029-1035, 1965. Saxena, G., Farmer, S., Towers, G. H. N. and Hancock, R. E. W.: Use of specific dyes in the detection of antimicrobial compounds from crude plant extracts using a thin layer chromatography agar overlay technique. Phytocbem. Anal. 6: 125-129, 1995. Shah, V., Sunder, R. and De Souza, N. ].: Chonemorphine and rapanone antiparasitic agents from plant sources. J. Nat. Prod. 50 (4): 730-731,1987. Soubrier, F., Wie, L., Hubert, c., Clauser, E., Alhenc-Gelas, F.
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and Corvol, P.: Molecular biology of the angiotensin I converting enzyme: II. Structure-function. Gene polymorphism and clinical implications. J. Hypertens. 11: 599-604, 1993. Wyvratt, M. ]. and Patchett, A. A.: Recent developments in the design of angiotensin-converting enzyme Inhibitors. Med. Res. Rev. 5 (4): 483-531,1985.
• Address A. Adsersen, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen 0, Denmark Tel.: +4535370850 Fax: +4535370845 e-mail: [email protected]