PESTICIDE

Biochemistry & Physiology

Pesticide Biochemistry and Physiology 87 (2007) 109114 www.elsevier.com/locate/ypest

Uncoupling and inhibition properties of 3,4-seco-friedelan-3-oic acid isolated from Maytenus imbricata
Silvia Ribeiro de Souza e Silva a, Gracia Divina de Fatima Silva a, b a Luiz Claudio de Almeida Barbosa , Lucienir Pains Duarte , Beatriz King-Diaz c, Francisco Archundia-Camacho c, Blas Lotina-Hennsen c,*
a

NEPLAM Departamento de Qumica, ICEx, Universidade Federal de Minas Gerais, Avenida Presidente Antonio Carlos, 6627, Pampulha, CEP 31270-901, Belo Horizonte, Minas Gerais, Brazil b LASA, Departamento de Qumica, CCE, Universidade Federal de Vicosa, Avenida PH Rolfs, s/n, Campus UFV, CEP 36570-000, Vicosa,Minas Gerais, Brazil c Departamento de Bioqumica, Facultad de Qumica, Universidad Nacional Autonoma de Mexico (UNAM), Ciudad Universitaria, Coyoacan, Mexico DF 04510, Mexico Received 9 March 2006; accepted 26 June 2006 Available online 5 July 2006

Abstract 3,4-Seco-friedelan-3-oic acid was isolated from Maytenus imbricata (Celastraceae). At low concentrations it inhibited non-cyclic electron transport and ATP synthesis in spinach chloroplasts, i.e., it behaved as a Hill reaction inhibitor, and at high concentrations it acts as an uncoupler by enhancing uncoupled electron transport and Mg2+ATPase activity. 3,4-Seco-friedelan-3-oic acid did not inhibit PSII electron transport from DPC to DCPIPox and photosystem I activity, but it enhanced from TMQH2 to MV, corroborating its action as uncoupler. It inhibits electron ow through PSII from water to sodium silicomolybdate. The whole results indicate that the 3,4seco-friedelan-3-oic acid target is at the OEC complex enzyme, the donor side of PSII. The uorescence decay data shows the formation of the K-band, which match this result, acting as inhibitor at the donor side of PSII and it as an uncoupler. 2006 Elsevier Inc. All rights reserved.
Keywords: Maytenus imbricata; Triterpenes; Photosystem II; Uncoupler-inhibitor; 3,4-Seco-friedelan-3-oic acid

1. Introduction Specimen of the genus Maytenus, are widely used in Brazilian folk medicine to treat of ulcer and inammatory problems. Maytenus imbricata (Celastraceae) is distributed throughout cerrado regions in the states of Minas Gerais and Bahia, Brazil. To our knowledge, there are no reports of M. imbricata used in traditional medicine. This is the rst report regarding phytochemical study with leaves of this specie. 3,4-Seco-friedelan-3-oic acid, a triterpenoid, has been isolated from others species of family Celastraceae [1],
*

Corresponding author. Fax: +52 56 22 53 29. E-mail address: blas@servidor.unam.mx (B. Lotina-Hennsen).

and from the leaves of M. imbricata plant 3,4-seco-friedelan-3-oic acid was isolated in addition to other compounds such as friedelin, friedelinol, galactitol, fatty esters and lupane pentacyclic triterpene. From the hexane extract of stems and branches of M. imbricata plant most of these compounds was also isolated including 3,4-seco-friedelan3-oic acid [2]. The seco-3,4-triterpenoids class are responsible for the cytotoxic activity of the crude chloroform leaf extract of Alchornea latifolia and they inhibit the enzyme topoisomerase [3]. This is the rst report regarding the activity for 3,4-seco-friedelan-3-oic acid compound that has activity on photosynthesis. In our search for natural products from Brazilian plants that aect photosynthesis, we found that 3,4-seco-friedelan-3-oic acid inhibits several photosynthetic activities including ATP synthesis and

0048-3575/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.06.008

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phosphorylating and uncoupled electron ow, as well as, PSII partial reactions and it enhances H+ATPase and photosystem I electron ow. In this research, our aim was to employ this triterpenoid as natural photosynthetic inhibitor and/or uncoupler that could led in the development of green herbicides. The genus Maytenus consists of woody and shrubby species [4] employed in the traditional medicine and investigated for phytochemical and pharmacological purposes, such Maytenus ilicifolia Mart. ex Reiss [5,6], M. blefarodes, M. macrocarpa, M. amazonica [7], M. obtusifolia [8], and M. truncata [9]. From chemical studies the Maytenus plants showed the presence of sesquiterpenes, pentacyclic triterpenes, alkaloids, avonoids and others polyphenols [10]. 2. Materials and methods 2.1. Plant material Leaves of M. imbricata were collected in the Ouro Preto city, Minas Gerais, Brazil. A voucher specimen of this plant has been deposited in the Herbarium of the Botany Departament of Universidade Federal de Vicosa, Vicosa, Minas Gerais, Brazil, collection No. 27780. 2.2. Isolation of seco-friedelan-3-oic acid Air-dried leaves (127.6 g) were ground and extracted with hexane (1000 mL) at Soxhlet apparatus. The extract was ltered and evaporated under vacuum to get solid residue (5,2 g, 4,1%). This extract was found to be active in a screening bioassay inhibiting ATP synthesis in illuminated spinach chloroplasts. The extract was subjected to column chromatography over silica gel (40 mm, 250 g) and the elution was carried out with hexane, AcOEt and MeOH gradients to give 26 fractions (120 mL) grouped in 11 sets. The set G1 were identied as esters of fatty acids (168 mgG1), G3 as friedelin (107 mg), friedelin plus friedelinol (147 mg), G5 and G6 as 3,4 seco-friedelan-3-oic acid (152 mg). The bioactive sets G5 and G6 (152 mg) was dissolved in CHCl3 and heated with coal to remove and latter re-crystallized with hexane AcOEt and CHCl3 drops obtaining 152 mg of seco-friedelan-3-oic acid (Fig. 1). This compound was identied with spectroscopic and spectrometric techniques: 1H and

C NMR techniques, including 2D experiments (HSQC, HMBC, and NOESY) as previously reported [2]. 2.3. Chloroplast isolation and chlorophyll determination Intact chloroplasts were obtained from spinach leaves (Spinacea oleraceae L.) [11,12] and chlorophyll concentration was measured as previously described [13]. 2.4. ATP synthesis and electron ow determination ATP synthesis was determined titrimetrically using a microelectrode (Orion model 8103; Ross, Beverly, MA, USA) connected to a Corning potentiometer model 12 (Corning Medical, Acton, MA, USA), with expanded scale and Gilson recorder (Kipp & Zonen, Bohemia, NY, USA) as reported by Dilley [14]. The following determinations were carried out as published by [11,12]. ATP synthesis assays were measured employing a reaction medium containing 100 mM sorbitol, 5 mM MgCl2, 10 mM KCl, 0.5 mM KCN, 1 mM sodium tricine pH 8.0, 20 lg of chlorophyll per ml as 50 lM MV (methyl viologen) as an electron acceptor in the presence of 1 mM ADP and 3 mM Pi. Light-induced non-cyclic electron transport activity from water to MV using a Clark type electrode was measured. Basal electron transport was determined by illuminating chloroplasts (20 lg chlorophyll per mL) during 1 min in the electron transport medium as previously published [11,12]. Photophosphorylating non-cyclic electron transport was measured as basal non-cyclic electron transport except that 1 mM ADP and 3 mM Pi were added [11,12]. Uncoupled electron transport was tested in the basal non-cyclic electron transport medium and 6 mM NH4Cl was added as uncoupler [11,12]. 2.5. Partial reactions determination To determine the target where the seco-friedelan-3-oic acid inhibits the chloroplasts electron transport chain, the activity on uncoupled PSII (photosystem II) and PSI (photosystem I) electron ow and their partial reactions was monitored in a similar form as the uncoupled non-cyclic electron transport medium. For uncoupled PSII from water to DCPIP (2,6-dichlorophenol indophenol), 1 lM DBMIB (2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone) and 50 lM DCPIP/300 lM K3[Fe(CN)6] were added to the medium without MV. PSII was measured by the reduction of DCPIP-supported O2 evolution, and the oxygen was measured by polarography. The partial reaction of uncoupled electron transport from water to SiMo (sodium silicomolybdate) was determined with the same reaction mixture as uncoupled electron ow without MV, except that 200 lM SiMo and 10 lM DCMU ([3-(3,4-dichlorophenyl)-1,1-dimethylurea]) were added [15]. The uncoupled PSII partial electron transport rate from DPC (diphenyl carbazide) to DCPIP reduction rate with a zspectrophotometer Beckman DU 650 and determined in

13

HOOC

Fig. 1. Chemical structure of 3,4-seco-friedelan-3-oic acid.

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thylakoids previously treated with 0.8 M Tris ((hydroxymethyl) aminomethane) (pH 8.0) and incubated for 30 min at 4 C [16]. To measure PSI electron ow from cyt b6f to MV, the uncoupled electron ow medium was used 100 lM DCPIP reduced with 300 lM sodium ascorbate was added as electron donor and 10 lM DCMU was added to inhibit PSII electron ow. To determine the uncoupled PSI electron ow from tetramethyl-p-benzohydroquinone (TMQH2) to MV, 100 lM TMQH2/300 lM ascorbate, 10 lM DCMU and 6 mM NH4Cl were added to the basal electron medium and determined polarographically [17,18]. All reaction mixtures were illuminated with actinic light from a projector lamp (GAF 2660) and were passed through a 5 cm lter of CuSO4 solution. The temperature was 20 C, and for each reaction a blank experiment was performed with the chloroplasts alone in the reaction medium. The I50 value for each activity was determined from plots of the activity in the presence at dierent concentrations of each compound. I50 is the concentration producing 50% inhibition. 2.6. Chlorophyll a uorescence measurements Chlorophyll a uorescence induction curves were measured at room temperature with a Handy PEA (Plant Ecient Analyzer) Hansatech in 5 min dark-adapted chloroplasts (20 lg Chl mL1) [11]. The maximum uorescence yield from the sample was generated using three light-emitting diodes (broad band 650 nm). The light (2500 lmol m2 s1) provided by an operating halogen saturation lamp. The pulse duration was 2 s. The reaction medium used was as the one employed in basal non-cyclic electron transport measurements. 2.7. Mg2+ATPase activity assays Mg2+ATPase activity bound to thylakoid membranes was measured according to Mills [19]. The amount of released inorganic phosphate was measured by colorimetric determination as previously described by Sumner [20]. 3. Results and discussion 3.1. ATP synthesis inhibition by 3,4-seco-friedelan-3-oic acid To know which compound inhibits ATP synthesis, all of them were assayed on photophosphorylation activity in isolated spinach chloroplasts. Fig. 2 shows that 3,4-secofriedelan-3-oic acid inhibits ATP synthesis coupled to electron transport from water to MV on freshly lysed spinach chloroplasts. The I50 value was 148 lM. The following compounds were also isolated from M. imbricata: 3,30dihydroxi-Lup-(20)-ene, 30-hydroxi-Lup (20) 29-en-3-one, dulcitol, and friedelin. They were tested on the screening bio-assay, ATP synthesis. All of them were less active than 3,4-seco-friedelan-3-oic acid. Friedelin and dulcitol did not show any inhibitory activity on ATP synthesis (data not

100

ATP - Synthesis (%)

80 60 40 20 0 0 50 100 150 200 250 300 350

I50 = 148 M

3,4,-Seco-friedelan-3-oic-acid [M]
Fig. 2. Inhibitory eect of 3,4-seco-friedelan-3-oic acid on ATP synthesis from water to MV measured as the pH rise between 8.0 and 8.01 using a combination of microelectrode Orion model 8103 Ross, connected to a Corning potentiometer Model 12 with expanded scale. Control value was 1100 lM ATP mg1 Chl h1.

shown), however, 3,30-dihydroxi-Lup-(20)-ene, 30hydroxi-Lup (20) 29-en-3-one inhibited 31% and 8%, respectively, at 300 lM and both compounds were not further studied. 3.2. Eect of 3,4-seco-friedelan-3-oic acid on chloroplasts electron transport rate In order to know the mechanism of inhibition of 3,4seco-friedelan-3-oic acid on ATP synthesis when is coupled to electron transport, its eect on basal, phosphorylating, and uncoupled electron transport from water to MV was studied on freshly lysed spinach chloroplasts. Fig. 3 shows that phosphorylating and uncoupled electron ow was inhibited up to 300 lM of the triterpenoid, after this concentration both activities are enhanced. The I50 value for uncoupled electron transport rate from water to MV is

120 100

Electron flow (%)

80 60 40 20 0 0 100 200 300 400 500 600

3,4-seco-friedelan-3-oic acid [M]

Fig. 3. Eect of 3,4-seco-friedelan-3-oic acid on photosynthetic non-cyclic electron transport activity basal (j), phosphorylating (d), and uncoupled (m) rate was measured from water to MV with a YSI (Yellow Spring Instrument) Model 5300 oxygen monitor. Control value rates were 480, 700 and 1060 lequiv e/h1 mg1 Chl, respectively.

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Table 1 Eect of the 3,4-seco-friedelan-3-oic acid on uncoupled PSII electron transport from water to DCPIP, from water to SiMo and from DPC to DCPIP Concn. (lM) PSII H2O to DCPIP a 0 100 200 300 400 333 400 333 267 200 b 100 120 100 80 60 PSII H2O to SiMo a 120 80 40 20 0 b 100 67 33 18 0 PSII DPC to DCPIP c 737 759 725 735 b 100 103 98 100 PSI DCPIPred to MV a 400 400 400 400 400 b 100 100 100 100 100 PSI TMQH2 to MV a 300 300 500 500 500 b 100 100 167 167 167

Eect of 3,4-seco-friedelan-3-oic acid on PSI from DCPIP to MV and from TMQH2 to MV. These assays were measured as indicated in Section 2. a values in lequiv e mg1 Chl h1. b values in percent. c values in lM DCPIPred mg1 Ch h1.

169 lM. The results of the eect on electron ow and ATP synthesis indicate that 3,4-seco-friedelan-3-oic acid acts as Hill reaction inhibitor at low concentrations and then as uncoupler at higher concentrations of the natural product. Fig. 3 also shows that the basal electron ow was not aected with the natural product; this last result suggests that conformational changes of the thylakoid membrane target occur when it is in an energetic state and the target is not exposed. Therefore, the triterpenoid may acts as energy transfer inhibitor or as an uncoupler; thus, further experiments will be done to clarify. To localize the action target of 3,4-seco-friedelan-3-oic acid on non-cyclic electron transport chain of chloroplasts, its eect on partial reactions of photosystem PSII and PSI was tested. Table 1 shows that this triterpenoid partially inhibits uncoupled PSII electron transport from water to DCPIPox (from H2O to QB) and from water to SiMo (from H2O to QA) as concentrations increases. The partial reaction of PSII from DPC to DCPIPox was not aected (from P680 to QB). Therefore, these results indicate that 3,4-secofriedelan-3-oic acid inhibits at the donor side of PSII, the water splitting enzyme. In the other hand the uncoupled PSI electron transport from DCPIPred to MV and from TMQH2 to MV were not aected in the presence of 3,4-seco-friedelan-3-oic acid, since the rate of uncoupled PSI electron ow were 400 and 300 lequiv e/h1 mg1 Chl, respectively, in the presence or absence of 300 lM of 3,4-seco-friedelan-3-oic acid. As well as, the electron transport from TMQH2 to MV is enhanced as concentrations of the triterpenoid increases indicated an uncoupling behavior of the compound. Thus the whole results indicate that the natural product only inhibits the non-cyclic electron transport chain at the oxygen evolving complex site. 3.3. Chlorophyll a uorescence measurements Chlorophyll a uorescence transient in the presence of 3,4-seco-friedelan-3-oic acid was measured to corroborate the interaction site of the triterpenoid at donor side of PSII. Isolated thylakoids were incubated for 5 min in the dark at

1400 1200

Fluorescence yield (Relative)

1000 800 600 400 200 0,01 0,1 1 10 100 1000

Time (ms)
Fig. 4. Fluorescence rise kinetics on thylakoids inltrated with 500 lM (dashed line) 4-seco-friedelan-3-oic acid. DCMU, 10 lM (open squares), Tris treated thylakoids (open circles) and control (continuous line). Details are in Section 2. Data are average of three replicates.

0,15

Relative Variable Fluorescence (W)

0,10

0,05

0,00 0,0 0,5 1,0 1,5 2,0

Time (ms)
Fig. 5. Comparative appearance of the K-band at about 300 ls between thylakoids incubated with 0.8 M Tris (s) and thylakoids treated with 500 lM 3,4-seco-friedelan-3-oic acid (j). The graph shows the dierence of each curve from the control with normalized relative variable uorescence on the amplitude Fj F0.

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room temperature with 500 lM of 3,4-seco-friedelan-3-oic acid, 10 lM of DCMU, and 0.8 M of Tris, which were used as positive controls [21], their uorescence kinetic behavior are well known in the literature. Fig. 4 shows the classical uorescence induction kinetics of herbicide DCMU, that induces a fast rise of the uorescence yield during the rst 2 ms. Tris, a well-known donor side inhibitor of PSII [22]; as well as the triterpenoid 3,4-seco-friedelan-3-oic acid. Fig. 5 shows the K-band calculated as the dierence of Tris and compound curve from control curve on relative variable uorescence when the O-J phase was normalized between Fj F0, Wt = Fv,t/ (Fj F0) = (Ft F0)/(Fj F0). The creation of K-band appears at 300 ls from an imbalance in the electron ow between the donor and the acceptor side of PSII [23]. When OEC is inhibited, it cannot donate electrons to YZ, which in turn cannot reduce P680 ; in this way, a maximal accumulation of P680 , Pheo+, and QA occurs [24]. Fig. 5 shows a comparative K-band between thylakoids incubated with 0.8 M Tris and thylakoids inltrated with 500 lM 3,4-seco-friedelan-3-oic acid, this compound exhibits an slightly K-band respect to Tris. This last result corroborates the polarographic results indicating that the target of the natural product is on the water splitting enzyme located at the donor side of PSII. 3.4. Mg2+ATPase activity Table 2 shows that NH4Cl enhanced the Mg2+ATPase activity as most uncoupler act, here it was used as control. The same Table 2 shows that 3,4-seco-friedelan-3-oic acid also behave as uncoupler activating the Mg2+ATPase, thus behaving as an uncoupler. These results indicate that the ammonium chloride enhance 100% the Mg2+ATPase activity, however the triterpenoid is stronger by 4.7 times more potent than the uncoupler control and this result explain why ATP synthesis is strongly inhibited (Fig. 2) than uncoupled electron transport from water to MV (Fig. 3). This triterpenoid is also more potent than tricolorin A, another natural product that acts also as Hill reaction inhibitor and as an uncoupler at the same time [25]. 3,4-Seco-friedelanTable 2 Eect of increasing concentrations of 3,4-seco-friedelan-3-oic acid and ammonium chloride on Mg2+ATPase activity Compound lM P mg1 Chl h1 Activity (%) 100 201 271 466

3-oic acid, the major Hill reaction inhibitor and an uncoupler to photophosphorylation in the hexanic extract of M. imbricata leaves, may be it is the mechanism of interaction with other plants and is probably a step forward to led green herbicides by modifying its structure and correlating the activity by quantitative structure activity relationship. Acknowledgment This work was supported by DGAPA IN205806-3. References
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3,4-Seco-friedelan-3-oic acid (lM) 0 290 100 583 300 785 500 1350 NH4Cl (mM) 0 1 3 6

220 488 549 445

100 222 250 202

Others conditions were described in Section 2.

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