Advances in Biological Chemistry
Vol.4 No.2(2014), Article ID:44409,7 pages DOI:10.4236/abc.2014.42015

Pharmacological Prospects of Oxygenated Abietane-Type Diterpenoids from Taxodium distichum Cones

Norihisa Kusumoto1,2*, Nobuhiro Aburai2, Tatsuya Ashitani2,3, Koetsu Takahashi2,3, Ken-ichi Kimura2,4

1Institute of Wood Technology, Akita Prefectural University, Noshiro, Japan

2The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Japan

3Faculty of Agriculture, Yamagata University, Tsuruoka, Japan

4Graduate School of Agriculture, Iwate University, Morioka, Japan

Email: *kusumoto@iwt.akita-pu.ac.jp

Copyright © 2014 by authors and Scientific Research Publishing Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

Received 28 January 2014; revised 6 March 2014; accepted 15 March 2014

ABSTRACT

Eight naturally occurring diterpenoids, including 6,7-dehydroroyleanone, taxodal, taxodione, salvinolone, 14-deoxycoleon U, 5,6-dehydrosugiol, sandaracopimaric acid, and xanthoperol were isolated from Taxodium distichum cones and their biological properties evaluated in vitro against six different biological screening targets. Taxodione showed potent activity against a number of different targets, and salvinolone and 14-deoxycoleon U showed remarkable inhibitory activities against prolyl oligopeptidase (POP) and 17α-hydroxylase/C17,20-lyase (CYP17), respectively. These three compounds also showed strong cytotoxic activities against HL60 and K562 human leukemia cells. The structure-activity relationships of these compounds have also been considered. The findings in this study could lead to enhanced pharmacological prospects for the natural abietanetype diterpenoids consisting in conifer cones.

Keywords:Abietane Diterpenoid; Conifer Cone; Antibacterial Activity; 17α-Hydroxylase/C17,20-Lyase; Prolyl Oligopeptidase; Cytotoxicity

1. Introduction

Natural occurring terpenoids such as mono-, sesquiand diterpenoids are formed in large amounts as secondary metabolites in coniferous species. Monoand diterpenoid mixtures are well-known to be the major components of resins produced by plants belonging to this particular species, and their synergistic self-defense functions, including their chemical and physical defensive roles, have been reported [1] . Furthermore, the chemical defenses of conifers are strongly reliant on specific components being present in the different parts of the tree (e. g., trunk, branch, root, needle, and cone). In our previous work, we focused on the hydrophobic terpenoid constituents in the cones of Taxodium distichum, and reported the isolations and identifications of twelve oxygenated diterpenoids, including ten abietane-type diterpenoids, as well as one novel norabietane-type diterpenoid and one isopimarane-type diterpenoid, together with an evaluation of the antitermitic and antifungal properties of these compounds against wood decomposers [2] [3] . Oxygenated abietane-type diterpenes are widely distributed in the plant kingdom and have been reported to show potent cytotoxic, antifungal, and antibacterial activities [4] - [7] .

Based on our previous investigations of the natural terpenoids in conifer cones, it was envisaged that the diterpenoids isolated from T. distichum cones would not only show activities against wood decomposers but would also show activity against other organisms. Furthermore, the results of our previous studies effectively highlighted the interesting bioactivities of oxygenated abietane-type diterpenoids [8] -[11] . The aim of the current study, therefore, was to evaluate the pharmacological prospects of eight natural diterpenoids, including 6,7-dehydroroyleanone (A), taxodal (B), taxodione (C), salvinolone (D), 14-deoxycoleon U (E), 5,6-dehydrosugiol (F), sandaracopimaric acid (G) and xanthoperol (H), which were isolated from T. distichum cones against six different biological targets in vitro. These unique methods for the evaluation of natural products were previously developed by our group during the course of several screening programs [12] -[18] . We also considered the structure-activity relationships of these compounds.

2. Materials and Methods

2.1. Chemicals

The eight natural diterpenoids (Figure 1), including 6,7-dehydroroyleanone (A), taxodal (B), taxodione (C), salvinolone (D), 14-deoxycoleon U (E), 5,6-dehydrosugiol (F), sandaracopimaric acid (G) and xanthoperol (H), were isolated from the n-hexane extract of fallen Taxodium distichum cones. The isolation procedures and techniques used for the structural identification of these compounds have been described previously in the literature [2] . Liposidomycin C-(III) and propeptin were isolated from the fermentation broth of Streptomyces sp. SN- 1061M [12] and Microbispora sp. SNA-115 [14] , respectively. Pisiferdiol was isolated from Chamaecyparis pisifera [17] . Ketoconazole and camptothecin were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. Sanguinarine chloride was purchased from Funakoshi Co. Ltd., Tokyo, Japan. The immunosuppressive drug FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd (currently known as Astellas Pharma Inc., Tokyo, Japan).

Figure 1. Eight diterpenoids isolated from the cones of Taxodium distichum.

2.2. Antimicrobial Activity against Acid-Fast Bacillus

A inoculating loop of Mycobacterium phlei (IFO3158) was pre-cultured in a tube for 4 days at 37˚C and then suspended in agar medium (pH 6.8) consisting of glycerin (30 g), meat extract (5 g), peptone (10 g), NaCl (3 g) and agar (1.5 g) per liter of distilled water (pH 6.7). Each of the compounds (A-H) was soaked into an 8 mm paper disc (40 μg/disc) before being placed on the prepared agar surface. Liposidomycin C-(III) (1.2 μg/disc) was used as a positive control for the antimicrobial screening [12] . The prepared dishes were incubated for 2 days at 37˚C and the diameters of inhibition and partial inhibition zones (mm) were measured.

2.3. Inhibition of 17α-Hydroxylase/C17,20-Lyase (CYP17)

The compounds (A-H) were screened for their CYP17 inhibitory activity according to a previously reported procedure [13] . Each compound (A-H) was used at 20 μg/ml of the final concentration, and the inhibition (%) of the C17,20-lyase reaction was calculated from the area of androstenedione using 17α-hydroxyprogesterone as a substrate. An antifungal clinical drug ketoconazole was used as a positive control at a concentration of 50 μM [13] .

2.4. Inhibition of Prolyl Oligopeptidase (POP)

The POP [formerly known as prolyl endopeptidase (PEP): EC 3.4.21.26] inhibition assay was performed as previously reported [14] . Each compound (A-H) was used at a final concentration of 10 μg/ml and the IC50 values (μM) were measured when significant levels of POP inhibition (%) were observed. A new peptide inhibitor, propeptin (IC50 = 0.55 μM) was used as a positive control [14] .

2.5. Inhibition of Ca2+-Signal Transduction in the Mutant Yeast

The Ca2+-signal transduction inhibitory activities of the compounds were tested according to a previously reported procedure [15] . Each compound (A-H) was soaked into an 8 mm paper disc (40 μg/disc) before being placed on the surface of YPD ager medium containing the mutant strain of Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ) (YNS17) and 0.3 M CaCl2. After 3 days of incubation at 28˚C, the diameters of the growth and inhibitory zones around the disc were measured. FK506 (0.02 μg/disc) was used as a positive control [15] .

2.6. Inhibition or Activation of Protein Phosphatase (PP) 2C

Screening for PP2C inhibitors or activators was carried out according to a previously reported procedure [16] [17] . The phosphatase activity of PP2C was assayed using α-casein as a substrate on the malachite green detection method. BIOMOL GREEN™ (AK-111, Biomol Research Laboratories, Inc., Plymouth Meeting, PA, USA) was used for the PP2C reaction. Each compound (A-H) was used at a final concentration of 40 μg/ml and the IC50 values (μg/ml) or activation activities (%) were measured when significant levels of PP2C inhibition or activation were observed. Sanguinarine (IC50 = 2.50 μM) was used as an inhibition control [16] and pisiferdiol (130% at 100 μM) was used as an activation control [17] .

2.7. Cytotoxicity against HL60 and K562 Cells

The antitumor activities of the compounds were evaluated against human promyelocytic leukemia HL60 cells (RCB-0041, 4 × 104 cells/ml) and human chronic myelogenous leukemia K562 cells (ATCC CCL-243, 5 × 104 cells/ml) using an MTT assay, according to a previously described procedure [18] . The cells were grown in 96-well plates and treated with the compounds at a variety of different concentrations at 37˚C under a humidified atmosphere containing 5% CO2 for 4 days. The viable cell percentages were calculated from the ratio of the A570 values of the treated and control cells. Camptothecin (IC50 = 0.02 μM to HL60 cells and IC50 = 0.10 to K562 cells) was used as a positive control [18] .

3. Results and Discussion

The results for the six different in vitro biological screening experiments that were conducted for each compound are shown in Table 1. In terms of the antimicrobial activities of the compounds against Mycobacterium

Table 1. Results of the in vitro screening experiments for the eight abietane-type diterpenes isolated from the cones of Taxodium distichum.

The hyphen indicates no activity was observed. aAntibacterial activities on the growth of Mycobacterium phlei (IFO 3158) at a concentration of 40 μg/disc. The inner inhibitory diameters (mm) are shown with the outer diameter. Liposidomycin C-(III) was used as a positive control at the concentration of 1.2 μg/disc. bInhibitions of CYP17 (17α-hydroxylase/C17,20-lyase) at a final concentration of 20 μg/ml. Ketoconazole was used as a positive control at a concentration of 50 μM. cInhibitions of prolyl oligopeptidase (POP: EC 3.4.21.26) at a final concentration of 10 μg/ml. Propeptin was used as a positive control. dCa2+-signal transduction inhibitory activities using a mutant strain of Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ) (YNS17) at a final concentration of 40 μg/ml. FK506 was used as a positive control at a concentration of 0.02 μg/ml. eInhibitions of protein phosphatase (PP) 2C at a final concentration of 40 μg/ml. Sanguinarine was used as an inhibition control and pisiferdiol (130% at 100 μM) was used as an activation control. fCytotoxic values (IC50) against the HL60 (RCB-0041, 4 × 104 cells/ml) and K562 (ATCC CCL-243, 5 × 104 cells/ml) human leukemic cell lines. Camptothecin was used as a positive control.

phlei (IFO 3158), only taxodione (C), salvinolone (D), and 14-deoxycoleon U (E) showed inhibition zones at 40 μg/ml. The anti-Mycobacterium activities of compounds C and E were the highest of the eight diterpenoids tested, and partial inhibition zones were also observed for these two compounds (Figure 2(a)).

14-Deoxycoleon U (E) was the only compound of the eight compounds tested to show inhibitory activity towards CYP17 (17α-hydroxylase/C17,20-lyase), with 53% inhibition at a concentration of 20 μg/ml against C17,20- lyase. The screening results for prolyl oligopeptidase (POP: EC 3.4.21.26) activity revealed that salvinolone (D) showed 79% inhibition at 10 μg/ml. The IC50 value of salvinolone (D) was determined to be 26.0 μM (8.15 μg/ml) using serial dilution. All of the other compounds tested showed weak levels of inhibition.

The Ca2+-signal transduction inhibitory activities of the compounds were evaluated using the hypersensitive mutant yeast Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ) (YNS17), and a notable growth restored zone (20.1 mm) was observed on taxodione (C) at 40 μg/ml together with an inhibitory zone (10.5 mm) (Figure 2(b)). 7-Keto formed abietatrienes, compounds D (14.4 mm), E (15.2 mm) and F (14.2 mm), also showed restored zones around the discs. Compounds A, B, G and H did not show any signs of activity.

Of the eight compounds tested, only taxodione (C) showed any activity as a protein phosphatase (PP) 2C inhibitor, with 92% inhibition at 40 μg/ml. The IC50 value of taxodione (C) was determined to be 56.4 μM (17.7 μg/ml) by serial dilution.

In terms of their cytotoxic activities against the human leukemia cell lines, 6,7-dehydroroyleanone (A) (IC50 = 4.46 μM), taxodione (C) (IC50 = 4.14 μM) and 14-deoxycoleon U (E) (IC50 = 3.94 μM) showed notable activities against HL60 cells; whereas taxodione (C) (IC50 = 6.05 μM), salvinolone (D) (IC50 = 6.05 μM) and 14-deoxycoleon U (E) (IC50 = 5.15 μM) showed notable activities against K562 cells. The only compounds to show specific cytotoxic activity against towards one cell line were 6,7-dehydroroyleanone (A) and salvinolone (D), which showed specificity towards the HL60 and K562 cell lines, respectively.

Figure 2. The growth effects of the abietane-type diterpenes (A-H) on Mycobacterium phlei (a) and Saccharomyces cerevisiae (b). Figure Labels: (a) Antibacterial activities on the growth of M. phlei (IFO 3158) at a concentration of 40 μg/disc. Liposidomycin C-(III) (asterisk) was used as a positive control at a concentration of 1.2 μg/disc. (b) Ca2+-Signal transduction inhibitory activities using a mutant strain of S. cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ) at a concentration of 40 μg/disc. FK506 (asterisk) was used as a positive control at a concentration of 0.02 μg/disc.

From the results of the six different in vitro screening assays, quinone methide taxodione (C), 7-keto formed abietatrienes 14-deoxycoleon U (E), and salvinolone (D) showed the highest levels of activity in a number of different assays, and were the most remarkable of the eight diterpenoids tested. The biochemical activities of taxodione-type compounds containing two double bonds conjugated with a carbonyl group have previously been investigated [4] [5] [11] , and the IC50 value for the cytotoxicity of compound C against HL60 cells (4.14 μM, 4 days) from the current study was similar to a result reported in a recent study [11] (4.8 μM, 3 days). In contrast, the structures of the notable POP inhibitor salvinolone (D) and the CYP17 inhibitor 14-deoxycoleon U (E) are based on a 5,6-dehydrosugiol framework. Interestingly, however, 5,6-dehydrosugiol (F) was much less active than these two compounds in all of the screening experiments conducted in the current study. The higher activity of salvinolone (D) compared with 5,6-dehydrosugiol (F) was attributed to the presence of the C-11 hydroxy group in salvinolone (D), which represents the only difference between the two compounds (Figure 3). Furthermore, a report concerning the structure-activity relationships of oxidized abietane diterpenes against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) has already been reported [5] , where a catechol with a carbonyl function at C-7 together with an alcohol or ketone at C-6 of the 12-hydroxyabietatriene structure was required structures for potent antimicrobial activity. Although the results of this study were in agreement with these requirements, further consideration could be needed because xanthoperol (H), which is a 12-hydroxyabietatriene compound that is oxidized at C-6 and C-7, showed no activity against any of the targets tested in the current study. Thus, the hydroxyl group at C-11 could be a more important factor in determining the activities of these compounds than the presence of an alcohol or ketone at C-6 position.

4. Conclusions

Remarkable levels of biological activity have been observed in some of the eight naturally occurring diterpenoids isolated from Taxodium distichum cones. Taxodione (C) and 14-deoxycoleon U (E), in particular, were the most active of the eight compounds tested and showed good levels of in vitro activity against five of the targets investigated. The antibacterial activities of compounds C and E against M. phlei have not been reported previously elsewhere. However, further considerations will be necessary, because these compounds also appeared to exhibit antimicrobial activity against M. tuberculosis or M. avium complex (MAC) [12] . It is noteworthy that taxodione (C) inhibited Ca2+-signal transduction. The prospects of natural terpenoids that showed inhibitory activity towards Ca2+-signal transduction in the mutant strain of S. cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ) were discussed in our previous studies [19] [20] . Furthermore, taxodione (C) was the only compound to show potent inhibitory activity against PP2C with an IC50 value of 56.4 μM. Sanguinarine, which is a plant benzo (c) phenanthridine alkaloid, has been reported to show potent PP2C inhibition with an IC50 value of 2.50 μM [16] . Although taxodione (C) provided a lower level of PP2C inhibition than that of sanguinarine and the structurally

Figure 3. Comparison of pharmacological activities of the 11-hydroxylated (double line) and non-hydroxylated (broken line) 5,6-dehydrosugiol structure compounds investigated with the six different screening methods.

related compound chelerythrine (IC50 = 11.0 μM) [16] , the PP2C inhibitory activity of this compound is particularly interesting because of its unique character.

From our previous studies, 6,7-dehydroroyleanone (A) showed remarkable termicidal activity and taxodione (C), 14-deoxycoleon U (E) and xanthoperol (H) showed potent antifeedant activity against the subterranean termite Reticulitermes speratus Kolbe [2] . Furthermore, taxodione (C), salvinolone (D) and 14-deoxycoleon U (E) showed remarkable levels of mycelial growth inhibition against wood-decaying fungi [3] . Most of the previously reported antitermitic and antifungal compounds showed notable pharmacological activities during the in vitro biological screening experiments conducted in this study. It is necessary to investigate the functional mechanisms of each active compound, because the novel findings of this study could enhance the pharmacological prospects of the natural abietane-type diterpenoids found in conifer cones.

Acknowledgements

This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science Fellows (Grant no. 225181).

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NOTES

*Corresponding author.