Isolation of Phorbol Esters from Euphorbia grandicornis and

Article pubs.acs.org/jnp

Isolation of Phorbol Esters from Euphorbia grandicornis and Evaluation of Protein Kinase C- and Human Platelet-Activating Effects of Euphorbiaceae Diterpenes Ju-Ying Tsai,†,# Dóra Rédei,§,# Peter Forgo,§ Yu Li,† Andrea Vasas,§ Judit Hohmann,*,§,⊥ and Chin-Chung Wu*,†,‡ †

Graduate Institute of Natural Products and ‡Research Center for Natural Products and Drug Development, Kaohsiung Medical University, Kaohsiung 807, Taiwan § Department of Pharmacognosy and ⊥Interdisciplinary Centre for Natural Products, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary S Supporting Information *

ABSTRACT: Human platelets contain conventional (α and β) and novel isoforms of PKC (δ and θ), and PKC activation can result in platelet aggregation and secretion reaction that are important for thrombus formation. Several tumorpromoting Euphorbiaceae diterpenes are known to act as direct activators of PKC, but many types of such diterpenes have not been studied as platelet stimulators. In the present study, two new and five known phorbol esters were isolated from Euphorbia grandicornis. Two of the isolated phorbol esters together with compounds representing ingenane, jatrophane, and myrsinane structural types were studied on PKC activation and platelet stimulation. The investigated phorbol esters and ingenane esters induced blood platelet aggregation and ATP secretion. PKC activation was demonstrated by inducing membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. The PKCactivating effect of the compounds correlated well with their efficacy to cause platelet stimulation. Moreover, by using an isoformspecific PKC inhibitor, it was found that besides conventional PKCs novel PKCs also play a positive role in platelet activation caused by phorbol/ingenane esters, especially in regulating platelet aggregation. The present results suggest that platelets afford a useful model for studying PKC activators of natural origin or their chemical derivatives. finding novel diterpenes in herbaceous plants seems to be an important task in drug discovery. Platelets are the smallest blood cells and are essential for normal hemostasis, but they also play an important role in thrombotic diseases. Platelet stimulation by physiological agonists result in activation of phospholipase C and subsequent formation of diacylglycerol (DAG), which then activates PKC. Activated PKC phosphorylates and regulates downstream substrates that can lead to platelet activation, secretion, and aggregation.8 There are three groups of PKC: the calciumdependent conventional isoforms (α, β, and γ), the calciumindependent novel isoforms (δ, θ, η, and ε), and the atypical isoforms (ζ and ι/λ). Human platelets express relatively high levels of the PKC-α, -β, -δ, and -θ isoforms and low or undetectable levels of the other isoforms.9 Moreover, platelet activation occurs rapidly (within a few minutes) and is easy to assess. These features make platelets an ideal model for studying PKC activators.

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iterpenes occurring in plants of the family Euphorbiaceae represent a large group of natural compounds with a diverse range of biological activities, including anti-inflammatory, antitumor, antiviral, cocarcinogenic, cytotoxic, and skinirritating effects.1 One of the cellular targets of Euphorbiaceae diterpenes that mediates their bioactivities is protein kinase C (PKC). Phorbol 12-myristate 13-acetate (TPA), the prototype of phorbol esters from Croton tiglium (Euphorbiaceae), is known as a tumor promoter, and the many additional biological activities of the compound are mediated through direct activation of PKC.2 In contrast to TPA, prostratin, a 12deoxyphorbol monoester from Homalanthus nutans (Euphorbiaceae), is a PKC activator without any tumor-promoting effects and exhibits promising therapeutic potential against HIV.3 In addition to phorbol esters, a number of Euphorbiaceae diterpenes were also reported as PKC activators, including ingenol esters,4 jatrophane diterpenes, 5 and daphnane diterpenes.6 Ingenol 3-angelate is a non-tumor-promoting PKC activator and is able to induce primary necrosis in dysplastic keratinocytes.7 Recently, ingenol 3-angelate has been approved for the topical treatment of actinic keratosis, precancerous lesions that can turn into skin cancers. Therefore, © 2016 American Chemical Society and American Society of Pharmacognosy

Received: June 30, 2016 Published: October 12, 2016 2658

DOI: 10.1021/acs.jnatprod.6b00603 J. Nat. Prod. 2016, 79, 2658−2666

Journal of Natural Products

Article

Figure 1. Structures of 1−11.

Table 1. 1H NMR Spectroscopic Data of Compounds 1, 2, and 5 [δ in ppm, J in Hz, CDCl3] 1a

position 1 5β 5α 7 8 10 11 12β 12α 14 16a 16b 17 18 19 20 isobutanoyl 2′ 3′ 4′ angeloyl 3″ 4″ 5″ acetyl OH-4 OH-9 OH-20 a

7.61 2.48 2.25 5.36 2.99 3.34 1.98 2.07 1.56 1.09 4.26 4.04 1.20 0.89 1.78 1.73

2b

s d (19.0) d (19.0) brs m s m dd (14.8, 7.5) dd (14.8, 12.5) d (5.3) d (11.3) d (11.3) s d (7.0) s s

7.60 2.53 2.39 5.70 3.07 3.30 2.00 2.12 1.56 1.02 3.80 3.44 1.22 0.90 1.79 4.45

2.52 sept (7.0) 1.12 d (7.0) 1.12 d (7.0)

s d (19.2) d (19.2) brs m s m dd (14.7, 6.8) m d (5.3) d (11.4) d (11.4) s d (6.5) s s (2H)

2.57 sept (7.0) 1.18 d (7.0) 1.18 d (7.0)

5a 7.58 2.53 2.49 5.68 3.01 3.27 1.99 2.05 1.54 0.81 1.07

s m m d (5.0) t (4.8) s m dd (14.2, 5.9) dd (14.2, 11.3) d (4.9) s

1.20 0.89 1.77 4.03 3.97

s d (5.9) s d (11.9) d (11.9)

2.54 sept (7.0) 1.17 d (7.0) 1.17 d (7.0)

6.09 q (6.0) 2.00 d (6.0) 1.92 s 2.06 s n.d.cc n.d.

2.09 s 5.55 s

1.92 s 5.64 brs n.d.

500 MHz. b600 MHz. cn.d., not detected.

ornamental plant in many countries. The isolation and structure determination of two phorbol analogues (16angeloyloxy-13α-isobutanoyloxy-4β,9α,20-trihydroxytiglia-1,5dien-3,7-dione and 16-angeloyloxy-13α-isobutanoyloxy4β,9α,7β-trihydroxytiglia-1,5-dien- 3-one) with unusual 5-en6-one and 5-en-6-ol functionalities was described recently from this plant.13

TPA has been studied extensively as a DAG mimetic and conventional/novel PKC activator in platelets,10−12 while the non-tumor-promoting phorbol esters or diterpenoids are less investigated. Euphorbia grandicornis Goebel (syn. E. breviarticulata Pax and E. breviarticulata var. breviarticulata Pax) is a succulent cactiform plant native to southern Africa and is used as an 2659



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Table 2. 13C NMR Spectroscopic Data of Compounds 1, 2, and 5 (δ in ppm, 125 MHz, CDCl3)

In continuation of previous investigations, reported herein are the isolation and structural characterization of seven further diterpenes (1−7) (Figure 1), which were identified as polyesters of 12-deoxyphorbol, 12-deoxy-16-hydroxyphorbol, and 12,20-dideoxy-16-hydroxyphorbol, including two new natural products (1 and 2). In the present work, two metabolites of E. grandicornis [3 and prostratin (4)], two ingenane derivatives [9 and ingenol 3-angelate (10)], two jatrophanes [esulatins B (11) and I (12)], and a myrsinanetype diterpene (13) were studied for their effects on PKC activation and platelet stimulation. The investigated ingenanes (9 and 10) were isolated from Euphorbia peplus,14 while esulatin B (11) and esulatin I (12) were obtained from Euphorbia esula.15,16 The tetracyclic diterpene 13 was isolated from Euphorbia falcata.17 It was found that the PKC-activating activity of compounds 3, 4, 8, 9, and 10 correlated well with their efficacy to cause platelet aggregation and secretion. Furthermore, the PKC isoforms involved in the platelet responses to the phorbol esters or diterpenoids were also determined.

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RESULTS AND DISCUSSION Isolation and Structure Elucidation of Diterpenes. The aerial parts of E. grandicornis were extracted with methanol at room temperature, and after concentration the extract was partitioned between chloroform and water. The chloroform phase was subjected to polyamide column chromatography using mixtures of methanol and water as eluents. The diterpene-rich fraction, obtained with methanol−water (3:2), was fractionated on silica gel using vacuum-liquid chromatography, followed by normal-phase and reversed-phase HPLC, to afford pure compounds 1−7. Compound 1 was isolated as a colorless oil, with [α]26D +16 (c 0.05, CHCl3). The positive-ion HRESIMS revealed the sodiated molecular ion peak at m/z 523.2678 [M + Na]+ (calcd for 523.2672, C29H40O7Na), indicative of a molecular formula of C29H40O7. The 1H and JMOD spectra of 1 revealed the presence of an isobutanoyl (δH 2.52 sept and 2 × 1.12 d; δC 178,9, 34.2, and 2 × 18.6) and an angeloyl group (δH 6.09 q, 2.00 d, 1.92 s; δC 168.0, 128.0, 137.4, 15.7, and 20.6) (Tables 1 and 2). Additionally, the JMOD and HSQC spectra suggested a carbon skeleton consisting of 20 carbons: four methyls (δH 1.20 s, 0.89 d, 1.78 d, 1.73 s; δC 11.6, 18.5, 10.1, and 25.6), three methylenes (δH 2.48 d, 2.25 d, 2.07 dd, 1.56 dd, 4.26 d, 4.04 d; δC 42.9, 31.7, and 69.5), six methines (δH 7.61 s, 5.36 s, 2.99 m, 3.34 s, 1.98 m, 1.09 d; δC 161.5, 127.9, 38.7, 55.8, 36.3, and 31.3), and seven quaternary carbons (δC 132.8, 209.3, 73.9, 136.6, 76.0, 63.0, and 26.4) (Tables 1 and 2). The 1H−1H COSY spectrum demonstrated three structural fragments, with the correlated protons − CH−CH (A) (δH 3.34 s, 7.61 s), CH−CH−CH− (B) (δH 5.36 brs, 2.99 m and 1.09 d), and −CH2−CH−CH3 (C) (δH 2.07 dd, 1.56 dd, 1.98 m and 0.89 d) besides two isolated methylenes [δH 2.48 and 2.25 d (J = 19.0 Hz), 4.26 and 4.04 d (J = 11.3 Hz)] (Figure 2). Fragment A (containing the typical H-1 signal of a phorbol ester at δH 7.61 s), together with a methyl (C-19), a keto group (C-3), and an O-substituted quaternary carbon (C-4), composed a methylsubstituted five-membered ring, characteristic of a 4-hydroxyphorbol ester. This was supported by the HMBC correlations detected between H-19/C-1, H-19/C-2, and H-19/C-3. One of the isolated methylenes (δH 2.25 and 2.48 d) was assigned as C5 with regard to the heteronuclear long-range correlations between C-3 (δC 209.3) and H-5α (δH 2.25 d). In addition,

a

position

1a

2b

5a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isobutanoyl 1′ 2′ 3′ 4′ angeloyl 1″ 2″ 3″ 4″ 5″ acetyl CO Me

161.5 132.8 209.3 73.9 42.9 136.6 127.9 38.7 76.0 55.8 36.3 31.7 63.0 31.3 26.4 69.5 11.6 18.5 10.1 25.6

160.4 132.8 208.6 73.5 38.6 134.7 132.4 38.5 75.9 54.9 36.5 31.7 63.6 31.0 29.5 69.3 23.3 18.0 9.4 69.3

161.3 132.8 209.3 73.8 38.5 139.9 130.3 39.2 76.1 55.8 36.3 31.7 63.2 32.6 22.9 15.4 23.3 18.5 10.1 68.3

178.9 34.2 18.6 18.6

178.3 34.0 18.5 18.5

179.0 34.3 18.6 18.6

168.0 128.0 137.4 15.7 20.6 170.4 20.8

125 MHz. b150 MHz.

Figure 2. Selected 1H−1H COSY () and HMBC (H → C) correlations for 1.

HMBC cross-peaks between H-5α/C-6 and H-5α/C-7 suggested that unit B represents the C-7−C-8−C-14 part of a phorbol-type diterpene. The HMBC correlations of the methyl 2660



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sept and 2 × 1.18 d; δC 178.3, 34.0, and 2 × 18.5) and an acetate (δH 2.06 s; δC 170.4 and 20.8) were identified (Tables 1 and 2). The methylene signals at δH 4.45 (H2-20) showed correlations in the HMBC spectrum with C-5 (δC 38.6), C-6 (δC 134.7), C-7 (δC 132.4), and the acetyl carbonyl (δC 170.4) signals, demonstrating a 20-acetate group in the molecule. On the other hand, in compound 2, a 16-hydroxy group was identified with regard to the upfield shifted H-16 signals (δH 3.80 and 3.44 d). A careful comparison of the NOESY spectra of 1 and 2 enabled the same configuration for 1 and 2 to be assumed. The structure of compound 2 was therefore elucidated as 20-acetoxy-13α-isobutanoyloxy-4β,9α,16-trihydroxytiglia-1,6-dien-3-one. Compound 4 was found to be identical in its characteristics with prostratin (Euphorbia factor T4) isolated earlier from Pimela prostrata, Euphorbia triangularis, E. cornigera, and Homolanthus nutans.21−23 Compounds 5−7 were described earlier from E. ledienii and E. triangularis as the mouse-ear irritant Euphorbia factors Le1, Le1′, and Le3′, respectively.24 Recently, compounds 6 and 7, together with 3, were isolated also from E. bothae.20 As a result of the detailed NMR study performed, the 1H and 13C NMR chemical shift assignments were determined for the first time for compound 5 (Tables 1 and 2). Effect of Euphorbiaceae Diterpenes on Platelet Aggregation. In the present study, four types of Euphorbiaceae diterpenes were investigated: phorbol esters [3, prostratin (4), and TPA (8)], ingenane esters [9 and ingenol 3-angelate (10)], jatrophane esters [esulatins B (11) and I (12)], and a myrsinane-type diterpene ester (13). In human platelets, TPA (8), ingenol 3-angelate (10), prostratin (4), 3, and 9 caused platelet aggregation in a concentration-dependent manner with EC50 values of 9.5 nM, 72.5 nM, 0.91 μM, 6.08 μM, and 5.04 μM, respectively (Figure 4). In contrast, 11−13 did not induce platelet aggregation at a concentration as high as 10 μM (Figure 4).

group at δH 1.73 with C-5, C-6, and C-7 indicated the presence of a 20-methyl group in the molecule. The structural fragment C was assigned as the C-12−C-11−C-18 part of the molecule, as confirmed by the HMBC correlations between H-12β and C9, H-12 and C-13, and H-18 and C-9. Finally, the 13,16functionalized tigliane skeleton was concluded on the basis of the H-16/C-13, H-17/C-13, H-14/C-13, H-16a,b/C-15, H-17/ C-15, H-14/C-15, H-14/C-16, and H-17/C-16 long-range correlations. Accordingly, compound 1 was elucidated as 12,20dideoxy-16-hydroxyphorbol esterified with both an isobutanoic and an angeloic acid moiety. The position of the angeloyl group at C-16 was evident from the three-bond correlation between the ester carbonyl carbon (δC 168.0) and the H2-16 protons (δH 4.26 and 4.04 d). Hydroxy groups at C-9 and C-4 were deduced from the NOESY correlation between the OH-9 group (δH 5.55) and H-14 and H-12α and between the OH-4 group (δH 2.09) and H-5β. The location of the isobutanoyl group at C-13 was determined on the basis of the chemical shift value of C-13 (δC 63.0 ppm), which was in the usual range for 13-acyl-substituted phorbol esters (δC 62.7−63.6 ppm).18−20 The configuration of 1 was elucidated by analyzing the correlations detected in a NOESY spectrum (Figure 3). NOE

Figure 3. Key NOESY correlations for compound 1.

effects between H-8 and H-11, H-8 and H3-17, and H-11 and H-12β indicated the β-position of all these protons and the methyl group. On the other hand, NOESY cross-peaks between H-12α and OH-9, H-12α and H-18, OH-9 and H-10, OH-9 and H-14, H-10 and H-1, H-1 and H-18, and H-16b and H-14 supported an α-arrangement of H-10, OH-9, H-14, and the C16-methylene and C-18-methyl groups. The trans A/B ring junction with H-10α and OH-4β was proved by the NOESY correlations between H-10 and H-5α and H-5β and OH-4. The α-position of the 13-acyl group was concluded on the basis of biogenetic considerations, since all phorbol-type diterpenes isolated so far have such functionality. All of the above data were compatible with the structure of 1 being proposed as 16angeloyloxy-13α-isobutanoyloxy-4β,9α-dihydroxytiglia-1,6dien-3-one. Compound 2 was isolated as a colorless oil, with [α]26D −18 (c 0.04, CHCl3). A molecular formula of C32H40O6 was assigned from the pseudomolecular ion peak at m/z 499.2312 [M + Na]+ (calcd for 499.2308, C26H36O8Na) in the HRESIMS. The 1H NMR and J-modulated 13C NMR spectra of 2, analyzed with the aid of 1H,1H−COSY, HSQC, and HMBC experiments, revealed close similarities to those of compound 1, with a difference in the esterification profile. From the 1H and 13C NMR data of 2, an isobutanoate (δH 2.57

Figure 4. Stimulatory effect of the Euphorbiaceae diterpenes investigated on platelet aggregation. Washed human platelets were prewarmed at 37 °C for 3 min, and then the Euphorbiaceae diterpenes were added to induce platelet aggregation. Platelet aggregation was determined by using the turbidimetric method in 96-well plates. Results are presented as means ± SEM (n = 3).

Effect of Euphorbiaceae Diterpenes on Platelet Secretion. Upon activation, platelets release granules that contain ADP, ATP, serotonin, and a variety of bioactive proteins. PKC plays an essential role in such cellular processes. In the present study, ATP released from platelets was used as a marker of platelet secretion and was determined by using a 2661



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luciferin−luciferase reaction assay. As shown in Figure 5, all phorbol esters (3, 4, 8) and ingenane esters (9, 10) that

Figure 5. Stimulatory effect of the Euphorbiaceae diterpenes investigated on platelet ATP secretion. Washed human platelets were prewarmed at 37 °C for 3 min, and then DMSO (vehicle control) or a Euphorbiaceae diterpene was added to induce platelet ATP secretion. Released ATP was determined by using a luciferin− luciferase reaction assay. Results are presented as means ± SEM (n = 3) (*p < 0.05, **p < 0.01 as compared with the control).

induced platelet aggregation were also able to induce ATP release from platelets. Moreover, the order of potencies of phorbol/ingenane esters in the ATP-release assay paralleled their potencies for induction of platelet aggregation. In contrast, 11−13, which were inactive in the platelet aggregation assay, also failed to induce platelet secretion. The Platelet-Activating Effects of Euphorbiaceae Diterpenes Are Correlated with Their PKC-Activating Activity. Next, the PKC-activating activity of phorbol/ ingenane esters was investigated. PKC activation in platelets was measured by phosphorylation of PKC substrates with a phospho-specific PKC substrate antibody. Figure 6A shows that platelet-activating phorbol/ingenane esters (3, 4, 8, 9, and 10) caused phosphorylation of a panel of PKC substrates, whereas the inactive diterpenes 11−13 did not. The rank order of potency of phorbol/ingenane esters to activate PKC paralleled their potencies for the induction of platelet activation. In addition, pretreatment of platelets with the pan-PKC inhibitor GF109203X abolished platelet aggregation and ATP secretion caused by the phorbol/ingenane esters (Figure 6B and C). These results suggest that the phorbol/ingenane ester-induced platelet activation is due exclusively to their PKC-activating effect. Effect of Euphorbiaceae Diterpenes on Downstream Signaling of PKC. In human platelets, PKC activation can lead to increased phosphorylation and activation of several signaling molecules, including MARCKS, PKD, Akt, VASP, and MAP kinases (ERK, p38), which mediate platelet aggregation or secretion. As shown in Figure 7, TPA (8), ingenol 3-angelate (10), prostratin (4), 3, and 9 all induced phosphorylation of the signaling molecules that lie downstream of PKC, and the rank order of potency was similar to that for PKC activation and platelet responses. Effect of Euphorbiaceae Diterpenes on Membrane Translocation of PKC Isoforms. The translocation of a PKC isoform from the cytoplasm to the membrane is the hallmark of PKC activation. By using subcellular fractionation and immunoblot analysis, the membrane translocation of different

Figure 6. Stimulatory effect of the Euphorbiaceae diterpenes investigated on phosphorylation of PKC substrates. (A) Washed human platelets were prewarmed at 37 °C for 3 min, and then the Euphorbiaceae diterpenes were added for another 10 min. After that, sample buffer was directly added to lyse platelets and dissolve proteins. Platelet lysates were subjected to Western blot analysis for phosphoPKC substrates. Washed human platelets were preincubated with GF109203X (1 μM) for 3 min, and then the Euphorbiaceae diterpenes were added to induce (B) platelet aggregation and (C) ATP secretion. Results are presented as means ± SEM (n = 3) (***p < 0.001 as compared with the respective controls).

PKC isoforms can be determined individually. Figure 8 shows that treatment of platelets with TPA (8) or ingenol-3-angelate (10) caused an almost complete translocation of PKC-α, -β, -δ, and -θ from cytosolic fractions to membrane fractions. Similarly, prostratin (4), 3, and 9 also elicited translocation of all four PKC isoforms, but to a lesser extent. Platelet PKC 2662



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isoforms did not show distinct selectivity of translocation in response to different phorbol/ingenane esters. This could explain the similarity in platelet responses, i.e., aggregation, secretion, and signal transduction, induced by these compounds. In other types of cells, TPA and ingenol-3-angelate may induce different patterns of PKC translocation that could lead to different biological responses. For example, in Chinese hamster ovary cells, TPA induced plasma membrane translocation of PKC-δ followed by slower nuclear translocation, while ingenol-3-angelate induced PKC-δ nuclear translocation at first (Kedei et al., 2004). Because platelets lack nuclei, it should be noticed that the effect of the phorboids on PKC nuclear translocation cannot be determined in this model. Both Conventional and Novel PKCs Mediate Euphorbiaceae Diterpene-Induced Platelet Activation. The conventional PKC-α and PKC-β isoforms have been proposed as key enzymes that positively regulate platelet aggregation, dense granule secretion, and thrombus formation in response to physiological platelet agonists.25,26 However, studies on the roles of the novel PKC-δ and PKC-θ in platelets report conflicting results.27−31 One of the reasons for the discrepancy reported is that physiological platelet agonists activate PKC via receptor-mediated complex processes and can be regulated differently. In contrast, phorbol or ingenane derivatives bypass receptor activation and directly activate PKC and may provide important insights into the functions of PKC isoforms. To explore the function of PKC isoforms, a conventional PKCspecific inhibitor, Go6976, was used in the present work. At a concentration of 1 μM, Go6976 has been shown to selectively and completely inhibit the activity of PKC-α and -β isoforms in platelets.32,33 As shown in Figure 9, Go6976 (1 μM) pretreatment only partially inhibited phorbol/ingenane esterinduced platelet aggregation. In contrast, platelet secretion caused by these compounds was more significantly inhibited by Go6976. These results indicate that, besides conventional PKCs, novel PKCs also significantly contribute to the processes of platelet activation, especially in regulating platelet aggregation. The different functions of PKC isoforms likely result from isoform-specific substrates.34 Phorbol/ingenane ester-induced phosphorylation of PKC substrates was partially reduced by Go6976 treatment, while some substrates were more susceptible than others (Figure 9C). With respect to signaling molecules downstream of PKC, phosphorylations of MARCKS, Akt, VASP, and p38 were reduced significantly by Go6976, while phosphorylations of both PKD and ERK were more resistant to this inhibitor, indicating that the former are dependent on conventional PKC isoforms, but the latter rely more on novel PKCs (Figure 9D). The dramatic reduction of phospho-MARCKS by Go6976 is of interest, as phosphoMARCKS has been reported to mediate phorbol ester-induced platelet secretion,35 and this is consistent with the present result that Go6976 largely prevented ATP secretion of phorbol/ ingenane ester-treated platelets. In the present study, all the investigated phorbol/ingenane esters exhibited PKC-activating and platelet-stimulating effects, although with different potencies. Previous X-ray crystallography and SAR studies have revealed that phorbol derivatives bind and activate PKC through hydrogen bonding, mainly through C-3O, C-9−OH, and C-20−OH.36,37 In addition, the hydrophobic ester side chains at C-12 and C-13 help to retain the phorbol ester−PKC complex in cellular membranes and thus also contribute to phorbol ester’s activity. Our results

Figure 7. Activation of downstream signaling of PKC by the Euphorbiaceae diterpenes investigated. Washed human platelets were treated as described in Figure 6, and the platelet lysates were subjected to Western blot analysis for various signaling molecules downstream of PKC.

Figure 8. Translocation of PKC isoforms caused by the Euphorbiaceae diterpenes investigated. After treatment of platelets with the Euphorbiaceae diterpenes for 1 min, the cytosolic fraction and the membrane fraction were prepared as described in the Experimental Section. Both fractions were subjected to Western blotting for determining the isoforms of PKC. 2663



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Figure 9. Effect of an inhibitor of conventional PKCs on Euphorbiaceae diterpene-induced platelet responses. Washed human platelets were pretreated with DMSO (vehicle control) or Go6976 (1 μM) for 1 min and then stimulated by Euphorbiaceae diterpenes. (A) Platelet aggregation, (B) ATP secretion, and (C) phosphorylation of PKC substrates as well as (D) signaling molecules downstream of PKC were determined as described above. Results in A and B are presented as means ± SEM (n = 3), with *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the respective controls.

are consistent with the SAR model; TPA is the most potent PKC-activating and platelet-stimulating phorbol ester (EC50 for platelet aggregation is 9.5 nM), while the 12-deoxyphorbol 13esters prostratin (4) and 3 are much less potent (EC50’s are 0.91 and 6.08 μM, respectively). Compound 3 is even weaker than prostratin (4); this may be due to lack of C-20−OH. In the case of ingenane esters, C-3−O−CO, C-9O, and C20−OH have been recognized as three essential pharmacophores.38,39 It was shown here that ingenol 3-angelate (10) exhibits potent PKC-activating and platelet-stimulating activity (EC50 for platelet aggregation is 72.5 nM), but compound 9 is 70-fold less potent (EC50 = 5.04 μM). Because the only structural difference between 10 and 9 is C-20−OH, our data support the important role of C-20−OH in the PKC-activating effect of ingenane esters. Previous studies have suggested that the ability of phorbol esters to induce platelet aggregation and secretion is linked to their tumor-promoting activity.40,41 In these studies, phorbol esters that are active as tumor promoters are also capable of activating platelet PKC and eliciting platelet aggregation, while phorbol esters without any tumor-promoting effect are also inactive against platelets. However, it was shown here that ingenol 3-angelate (10) and prostratin (4), both not tumor promoters, still potently caused platelet aggregation and

secretion, indicating that the platelet-stimulating effect of phorbol or ingenol derivatives is correlated with their PKCactivating effects rather than the ability to promote tumor. To the best of our knowledge, this is the first report that has investigated the platelet-stimulating effects of ingenol 3angelate (10) and prostratin (4). The present data also raise concerns about the potential thrombotic complications using the PKC activators in clinical therapy, especially when they are administered systemically and at high doses. In conclusion, in the present work, PKC activation in human platelets by Euphorbiaceae diterpenes has been demonstrated by inducing the membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. Moreover, platelet aggregation and secretion in response to Euphorbiaceae diterpenes correlated very well with their PKC-activating ability. Since the platelet responses occurred within a few minutes and could be detected easily and quantitatively measured, the present results suggest that platelets appear to be a useful model for screening PKC activators of natural origin or their chemical derivatives.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined in chloroform using a PerkinElmer 341 polarimeter.

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13α-Acetoxy-4β,9α,20-trihydroxytiglia-1,6-dien-3-one (prostratin) (4): colorless oil; 1H and 13C NMR data are in good agreement with literature data.42 13α-Isobutanoyloxy-4β,9α,20-trihydroxytiglia-1,6-dien-3-one (5): colorless oil; for 1H NMR and 13C NMR spectroscopic data, see Table 1. 20-Acetoxy-13α-isobutanoyloxy-4β,9α-dihydroxytiglia-1,6-dien3-one (6): colorless oil; 1H and 13C NMR data were identical with literature values.20 20-Acetoxy-13α-(2-methylbutanoyloxy)-4β,9α-dihydroxytiglia1,6-dien-3-one (7): colorless oil; 1H and 13C NMR data were identical with literature values.20 Biological Assays. Materials. TPA and prostratin were purchased from Santa Cruz Biotechnology, and ingenol-3-angelate was from Cayman Chemical. Antibodies against p-(Ser) PKC substrate, p-AKT Ser 308, p-VASP, p-p-38, and p-PKD were purchased from Cell Signaling, and the antibody against p-ERK was from Santa Cruz Biotechnology. Preparation of Washed Human Platelets. Human blood anticoagulated with acid citrate dextrose was obtained from healthy human volunteers who had not taken any drugs within the prior 2 weeks. The study was approved by the Institutional Review Board of Kaohsiung Medical University (KMUH-IRB-20110285), and informed consent was obtained from every volunteer. The platelet suspension was then prepared according to the method described previously.43 Platelets were finally suspended in Tyrode’s solution containing Ca2+ (2 mM), glucose (11.1 mM), and bovine serum albumin (3.5 mg/mL) at a concentration of 3 × 108 or 6 × 108 platelets/mL. Measurement of Platelet Aggregation. Platelet aggregation was determined by the turbidimetric method in a 96-well plate using a Synergy HT multi-detection microplate reader (BioTek, USA). Platelet suspensions (3 × 108 platelets/mL) were incubated with dimethyl sulfoxide (DMSO, vehicle) or the test compounds at 37 °C under a fast shake condition (20 Hz). The extent of platelet aggregation was measured as the maximal increase of light transmission at 450 nm within 15 min after the addition of a test compound. Measurement of Platelet Secretion. Platelet secretion of ATP was determined by using a luciferin−luciferase reaction assay.44 Platelet suspensions (3 × 108 platelets/mL) were incubated with DMSO or test compounds at 37 °C for 5 min and centrifuged at 14 000 rpm at 4 °C for 1 min. The content of ATP in the supernatants was determined using an ATP bioluminescent assay kit (Sigma, USA) according to the manufacturer’s protocol. Western Blot Analysis. After treatment with a test compound, platelet suspensions were lysed by addition of 2× sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min. Lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 h and incubated with primary antibody overnight. The membrane was then incubated with HRP-conjugated goat anti-rabbit IgG (GE Healthcare) for 1 h. Finally, the membrane was detected by a chemiluminescence detection kit (Millipore, MA, USA). Equal loading was confirmed by detection of GAPDH (GT239, Gentex). PKC Translocation. For preparation of cytosolic fractions and membrane-rich fractions, a previous method45 was used with minor modification. After treatment with the test compounds, platelet suspensions were lysed by addition of 0.1% digitonin at 4 °C for 3 min. The total lysates were centrifuged at 14 000 rpm at 4 °C for 10 min. The supernatant was defined as the cytosolic fraction. The pellet was lysed by 1% Triton X-100 buffer at 4 °C for 10 min and was centrifuged at 14 000 rpm at 4 °C for 10 min. The supernatant was defined as the membrane fraction. Both fractions were dissolved in SDS sample buffer, boiled, and subjected to SDS-PAGE and Western blot for determining PKC-α, -β, -δ, and -θ. Statistical Analysis. Results are expressed as the means ± standard error of the mean (SEM). Statistical significance was calculated by one-way analysis of variance (ANOVA); p < 0.05 was considered statistically significant.

NMR spectra were recorded on a Bruker Avance DRX 500 spectrometer at 500 MHz (1H) and 125 MHz (13C) (compounds 1, 3−6) and on a Bruker Ultrashield Plus 600 spectrometer at 600 MHz (1H) and 150 MHz (13C) (compound 2) with TMS as internal standard. Two-dimensional NMR data were acquired and processed with standard Bruker software. For 1H−1H COSY, HSQC, and HMBC experiments, gradient-enhanced versions were used. Lowresolution APCI-MS was performed on an API 2000 Applied Biosystems instrument in the positive mode. The samples were injected into an acetonitrile flow. High-resolution MS data were recorded on a Waters-Micromass Q-TOF Premier mass spectrometer equipped with an electrospray source. The resolution was over 1 ppm. The data were acquired and processed with MassLynx software. Column chromatography was carried out on polyamide (50−160 μm, MP Biomedicals), and vacuum-liquid chromatography (VLC) on silica gel G (15 μm, Merck). Separations were monitored by TLC on Merck 60 F254 (0.25 mm) plates and visualized by staining with concentrated sulfuric acid. HPLC was performed on a Waters instrument with detection at 254 nm on LiChrospher RP-18 (5 μm, 250 × 4 mm, Merck) and LiChrospher Si 100 (5 μm, 250 × 4 mm, Merck) columns. Plant Material. The aerial parts of E. grandicornis were collected in March 2008 from cultivated stock in Szeged-Kecskés, Hungary. The plant was identified by Zsanett Hajdú (Department of Pharmacognosy, University of Szeged). A voucher specimen (No. 769) has been preserved in the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary. Extraction and Isolation. The fresh plant material (10 kg) was crushed in a blender and then extracted with 38 L of MeOH at room temperature. The methanol extract was concentrated in vacuo and extracted with 5 × 500 mL of chloroform. The organic phase (29.4 g) was subjected to polyamide column chromatography (180 g) with mixtures of methanol and water (3:2 and 4:1, each 2000 mL) as eluents. The fraction (0.6 g) obtained with methanol−water (3:2) was fractionated by VLC on silica gel using a gradient system of n-hexane− acetone (9:1, 8:2, 7:3, 6:4, 1:1, and 3:7). Fractions with similar compositions according to TLC monitoring were combined, affording fractions I−XI. Fraction III, obtained with n-hexane−acetone (8:2), was fractionated by NP-HPLC, using cyclohexane−ethyl acetate− ethanol (140:40:1) as eluent (flow rate 0.5 mL/min). Peaks eluted at retention times 10.0, 18.4, and 23.0 min afforded pure compounds 1 (1.7 mg), 6 (1.0 mg), and 3 (1.6 mg), respectively. Fraction IV, obtained with n-hexane−acetone (8:2) was separated by the same manner, yielding 7 (tR = 17.1 min, 2 mg) besides compounds 1 (1.0 mg), 6 (10.3 mg), and 3 (21.4 mg). Fraction VI, eluted with nhexane−acetone (7:3), was subjected to HPLC separation using cyclohexane−ethyl acetate−ethanol (70:30:3) as eluent (flow rate 0.4 mL/min) to isolate compounds 3 (8.8 mg), 5 (tR = 18.1 min; 14 mg), and 2 (tR = 21.0 min, 4.7 mg), respectively. Fraction VII, obtained with n-hexane−acetone (6:4), was fractionated first by NP-HPLC with the eluent cyclohexane−ethyl acetate−ethanol (70:30:3) and then by RPHPLC using methanol−water (8:2) (flow rate 0.5 mL/min), affording compound 4 (2.0 mg). 16-Angeloyloxy-13α-isobutanoyloxy-4β,9α-dihydroxytiglia-1,6dien-3-one (1): colorless oil; [α]26D +16 (c 0.05, CHCl3); for 1H NMR and 13C NMR spectroscopic data, see Table 1; APCIMS positive mode m/z 501 [M + H]+, 483 [(M + H) − H2O]+, 395 [(M + H) − H2O − (CH3)2CHCOOH]+, 295 [395 − CH3CHC(CH3)COOH]+, 277 [295 − H2O]+, 249 [277 − CO]; HRESIMS m/z 523.2678 [M + Na]+ (calcd for 523.2672, C29H40O7Na). 20-Acetoxy-13α-isobutanoyloxy-4β,9α,16-trihydroxytiglia-1,6dien-3-one (2): colorless oil; [α]26D −18 (c 0.04, CHCl3); for 1H NMR and 13C NMR spectroscopic data, see Table 1; APCIMS positive mode m/z 477 [M + H]+, 459 [(M + H) − H2O]+, 371 [(M + H) − H2O − (CH3)2CHCOOH]+, 311 [371 − CH3COOH]+; HRESIMS m/z 499.2312 [M + Na] + (calcd for 499.2308, C26H36O8Na). 16-Angeloyloxy-13α-isobutanoyloxy-20-acetoxy-4β,9α-dihydroxytiglia-1,6-dien-3-one (3): colorless oil; 1H NMR and 13C NMR spectroscopic data were identical with those of published data.20 2665



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00603. Copies of 1H, 13C, 1H,1H COSY, HSQC, HMBC, and NOESY spectra of 1 and 2 and 1H NMR spectra of 3−7 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +36 62 546453. Fax: +36 62 547404. E-mail: [email protected] (J. Hohmann). *Phone: +886 7 3121101. E-mail: [email protected] (C.-C. Wu). Author Contributions #

J.-Y. Tsai and D. Rédei contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Ministry of Science and Technology, Taiwan (MOST 103-2320-B-037-013-MY3), and the Hungarian Scientific Research Fund (OTKA K109846) is gratefully acknowledged. D.R. is a grantee of the János Bolyai Research Fellowship of the Hungarian Academy of Sciences.

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REFERENCES

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