Anti-inflammatory activities of isopimara-8(14),-15-diene diterpenoids and mode of action of kaempulchraols P and Q from Kaempferia pulchra rhizomes

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Anti-inflammatory activities of isopimara-8(14),-15-diene diterpenoids and mode of action of kaempulchraols P and Q from Kaempferia pulchra rhizomes Nwet Nwet Wina, Besse Hardiantia,b, Shiori Kasaharaa, Hla Ngwec, Yoshihiro Hayakawaa, , ⁎ Hiroyuki Moritaa, ⁎

a

Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan Sekolah Tinggi Ilmu Farmasi Makassar, Perintis Kemerdekaan Street KM13.7, Makassar 90242, Indonesia c Department of Chemistry, University of Yangon, Yangon 11041, Myanmar b

ARTICLE INFO

ABSTRACT

Keywords: Inflammation Kaempferia pulchra Isopimarane diterpenoids Kaempulchraols P and Q

Inflammation is an extensively recognized link to many pathological diseases. It is a host response for protection from infections and tissue damage. Infections trigger acute inflammation; however, persistent infection will contribute to chronic inflammation and higher disease susceptibility. Deregulated inflammatory responses can cause excessive or long-lasting tissue damage, manifested as cancer, immune disorders, diabetes, etc. NF-κB is a central mediator of pro-inflammatory gene induction and functions in both innate and adaptive immune cells; therefore, the anti-inflammatory regulation of NF-κB is needed. Natural products reportedly play an important role in controlling the inflammatory response pathways. However, the anti-inflammatory activities of isopimara8-(14),15-diene diterpenoids have not yet been fully elucidated. To elucidate the anti-inflammatory activities of the isopimara-8(14),15-diene diterpenoids, we investigated 21 isopimara-8(14),15-diene diterpenoids previously isolated from Kaempferia pulchra rhizomes. Eleven compounds exhibited NO inhibitory activity against lipopolysaccharide (LPS)-induced RAW264.7 cells, with IC50 values ranging from 30 to 100 μM. Furthermore, the most potent kaempulchraols P and Q, with IC50 values of 39.88 and 36.05 μM, respectively, inhibited the NFκB-mediated transactivation of a luciferase reporter gene, IL-6 production, and COX-2 expression, with an effective dose of 25 μM. These findings provide new insights into the anti-inflammatory activities of the isopimara8(14),15-diene diterpenoids.

Inflammation is a remarkable process that requires the stringent management of the regulatory functions of the immune defense systems, to control infection and resolve inflammation. This prevents the undesirable persistence of the immune responses and reduces the risk of disease onset.1 NF-κB is a central mediator of the human immune response. The activation of NF-κB by multiple inflammatory stimuli, including cytokines and pathogen-derived molecules such as lipopolysaccharide (LPS),2 which is frequently detected in tumors,3 results in the expression of numerous target genes encoding cytokines, cell adhesion molecules, and inflammatory enzymes, including nitric oxide (NO)-producing nitric oxide synthase (iNOS) and prostanoid-producing cyclooxygenase (COX-2) across many cellular processes.4–7 The constitutive activation of the NF-κB pathways is often associated with inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and asthma. Therefore, NF-κB-targeted therapeutics might be effective in inflammatory diseases.8 Some natural products reportedly play an important role in

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controlling the inflammatory response pathways.9,10 Among them, the isopimarane diterpenoids are structurally diverse and functionally noteworthy natural products. They are abundantly found in plants11,12 and fungi13 and possess various biological activities, such as cytotoxic,14 anti-microbial,15 anti-malarial,16 anti-inflammatory,17 and anti-viral18 properties, as well as inhibitory activities against acetylcholinesterase,19 α-glucosidase,13 and nitric oxide production.20 However, the anti-inflammatory activities of the isopimara-8(14),15diene diterpenoids have not been fully elucidated. The NO-inhibitory activities of some highly oxygenated isopimarane diterpenoids from Orthosiphon stamineus, from Okinawa, Taiwan, Indonesia, Vietnam, and Myanmar, have been explored against LPS-activated macrophage-like J774.1 cells, and 2-O-deacetylorthosiphonone A (IC50 35.0 μM), orthosiphols A (IC50 11.5 μM), B (IC50 20.5 μM), D (IC50 14.4 μM), and X (IC50 6.4 μM) have been identified as potent NO inhibitors.21–23 In a previous study, we identified 21 isopimara-8(14),15-diene diterpenoids, including kaempulchraols E (1), F (2), H − M (3–8), P − R

Corresponding authors. E-mail addresses: [email protected] (Y. Hayakawa), [email protected] (H. Morita).

https://doi.org/10.1016/j.bmcl.2019.126841 Received 10 September 2019; Received in revised form 6 November 2019; Accepted 15 November 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Nwet Nwet Win, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.126841

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Fig. 1. Structures of isopimara-8(14),15-dienes isolated from K. pulchra rhizomes. Table 1 Inhibitory effects of isopimara-8(14),15-dienes isolated from K. pulchra rhizomes on NO production in LPS-induced RAW264.7 cells. Compounds

% Inhibition

IC50 (μM)

5 μM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 L-NMMA monoacetate

17.44 5.57 9.01 27.21 16.48 17.71 4.09 18.12 9.97 15.23 20.72 −0.32 21.36 5.03 33.26 18.38 13.38 4.21 3.43 17.25 17.45 22.79

10 μM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.40 1.50 0.42 1.56 0.20 0.35 0.23 0.23 0.70 2.02 4.28 0.31 0.21 0.49 0.06 0.20 0.21 0.53 0.47 0.21 0.17 0.12

17.72 13.35 15.16 14.01 7.80 9.03 10.81 7.80 3.82 9.55 14.90 9.88 16.67 16.88 32.46 14.81 6.28 9.37 7.09 11.11 14.38 27.02

50 μM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.46 0.50 0.95 0.25 0.60 0.23 0.32 0.35 2.72 3.50 0.06 0.20 0.61 0.15 0.15 0.21 0.23 0.12 0.44 0.10 0.15

35.21 33.41 34.32 47.51 25.12 7.90 46.58 20.99 65.64 71.56 59.48 80.27 24.94 51.39 40.07 5.89 46.60 37.12 50.79 37.12 59.34 46.34

(9–11), T (12), V (13), W (14), 9α-hydroxyisopimara-8(14),15-dien-7one (15), 7β,9α-dihydroxypimara-8(14),15-diene (16), (1S,5S,9S,10S,11R,13R)-1,11-dihydroxypimara-8(14),15-diene (17), sandaracopimaradien-1α,2α-diol (18), (2R)-ent-2-hydroxyisopimara8(14),15-diene (19), (1R,2S,5S,9S,10S,11R,13R)-1,2,11-trihydroxypimara-8(14),15-diene (20), and 7α-hydroxyisopimara-8(14),15-diene (21), from a CHCl3-soluble extract of Kaempferia pulchra rhizomes (Fig. 1) and reported that some are potent HIV-1 Vpr inhibitors.11,12,24,25 We recently found that the CHCl3-soluble extract

100 μM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.67 0.12 0.32 0.85 0.50 0.61 0.26 0.12 0.55 1.85 3.00 0.10 0.32 0.35 0.20 0.15 0.20 0.40 0.15 0.15 0.29 0.38

26.34 33.75 24.07 80.48 35.50 −3.85 74.25 20.82 81.70 81.82 80.48 82.13 47.55 60.40 41.59 −5.96 50.35 49.21 81.79 22.37 67.59 58.16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.59 0.61 0.38 0.15 0.31 0.21 0.55 0.12 0.12 0.17 0.12 0.06 0.21 0.10 0.15 0.12 0.31 0.46 0.06 0.53 0.15 0.21

> 100 > 100 > 100 53.77 > 100 > 100 56.17 > 100 39.88 36.05 41.49 32.79 > 100 48.39 > 100 > 100 95.34 100.00 48.71 > 100 41.69 65.50

exhibits NO inhibitory activity, with an IC50 value of 31.43 μg/mL, without showing any cytotoxicity in LPS-induced RAW264.7 cells. Thus, some of the isolated 21 isopimara-8(14),15-diene diterpenoids could be the anti-inflammatory phytoconstituents in this plant. To test this hypothesis, we investigated the anti-inflammatory activities of the 21 compounds and the more detailed inhibitory modes of action of two highly potent compounds. The anti-inflammatory activities and cytotoxic effects of compounds 1–21 were evaluated by monitoring the NO inhibition and cell viability 2

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Table 2 Cell viability of RAW264.7 cells treated with isopimara-8(14),15-dienes isolated from K. pulchra rhizomes during LPS-induced NO production. Compounds

% Cell survival 5 μM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 L-NMMA monoacetate

72.93 103.85 104.27 57.73 86.61 76.94 121.21 85.66 98.66 106.23 71.48 100.32 89.27 140.37 93.30 113.97 78.37 113.23 97.48 65.71 76.94 111.80

10 μM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

15.13 0.14 2.69 4.31 3.32 9.69 6.51 9.40 0.49 25.17 23.62 10.96 2.19 9.97 0.92 2.05 6.79 16.40 1.41 7.35 17.54 2.62

65.70 85.13 113.35 77.40 92.44 82.05 104.47 85.27 149.15 111.27 98.26 107.03 84.74 81.17 83.29 98.80 86.73 70.41 115.02 50.85 65.82 92.64

50 μM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.27 11.03 6.36 2.76 34.86 1.06 14.42 10.47 3.68 27.08 21.43 14.92 20.72 1.27 3.89 5.80 17.25 10.61 3.75 4.17 0.07 3.89

52.17 50.05 89.40 67.61 86.82 68.75 109.61 27.46 108.55 60.28 88.69 6.17 85.70 60.19 50.70 73.32 35.86 70.61 114.07 60.89 58.76 107.45

100 μM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.21 0.42 1.34 19.73 6.08 0.78 10.25 0.14 27.58 1.00 18.95 0.21 3.96 0.21 5.37 10.54 0.57 18.88 8.20 5.09 10.18 6.29

66.99 47.73 101.05 3.78 77.78 76.01 37.60 27.59 66.77 3.39 4.72 3.73 70.31 22.06 45.75 71.32 27.84 73.52 4.35 66.53 34.23 103.12

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

22.42 8.13 10.11 0.85 3.82 0.78 11.38 2.97 8.70 0.64 0.78 0.07 1.34 0.07 6.29 0.42 0.35 5.87 0.28 4.74 3.25 7.50

Fig. 2. Effects of 9 and 10 on NF-κB activation by a reporter gene assay (A), and on cell viability (B) in RAW264.7-NF-κB-luc cells. The data points and bars represent mean ± SD (n = 3).

of LPS-induced RAW264.7 cells, respectively. Compounds 4, 7, 9–12, 14, 17–19, and 21 showed anti-inflammatory activities, with IC50 values ranging from 30 − 100 μM (Table 1). In particular, kaempulchraols P (9), Q (10), and T (12) inhibited the NO production with IC50 values of 39.88, 36.05, and 32.79 μM, respectively, which were levels comparable to that of the positive control, L-NMMA monoacetate (IC50 65.50 μM). All of the tested compounds did not show any cytotoxicities at 5 μM and 10 μM, whereas the active compounds 4, 10, 11, 12, and 19 exhibited cytotoxicities at 100 μM. Furthermore, the active compound 12 was cytotoxic at 50 μM (Table 2). Thus, the lowest IC50 dose of compound 12 (32.79 μM) for the NO production is probably caused by its cytotoxicity. Considering the preliminary results from the NO inhibition and cytotoxicity assays, we selected compounds 9 and 10 and investigated their dose-dependent effects on the activation of NF-κB, a central mediator of the human immune response in inflammation, by a reporter

gene assay. As shown in Fig. 2A and B, both compounds inhibited the activation of NF-κB in RAW264.7-NF-κB-luc cells at doses of 5, 10, and 25 μM, without showing any cytotoxicity. Interestingly, 5–25 μM of compounds 9 and 10 inhibited the NF-κB activation (Fig. 3A and B) in LPS-induced RAW264.7-NF-κB-luc cells, which mimic the inflammation caused by infection, when the inflammation response was induced by 100 ng/mL of LPS. The LPS molecule, a component from the cell walls of Gram-negative bacteria, is one of the most powerful activators of macrophages and is involved in the production of pro-inflammatory cytokines by activating the NF-κB pathway. These results suggested that both compounds inhibited the pro-inflammatory cytokine production. To confirm the effects of these compounds on the pro-inflammatory cytokines, we measured the level of IL-6 production in the LPS-induced macrophage RAW264.7 cells, using enzyme-linked immunosorbent assay (ELISA) kits. As shown in Fig 4., 25 μM of compounds 9 and 10 significantly inhibited the production of IL-6. Therefore, the vascular 3

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Fig. 3. Effects of 9 and 10 on NF-κB activation by a reporter gene assay (A) and on cell viability (B) against LPS-induced RAW264.7-NF-κB-luc cells. The data points and bars represent mean ± SD (n = 3).

inflammation mediated by IL-6 can be alleviated by these compounds. The modulation of the activity of pro-inflammatory enzymes is also one of the most important mechanisms in anti-inflammatory drug discovery. Therefore, the effects of compounds 9 and 10 on the expression of a pro-inflammatory enzyme, cyclooxygenase-2 (COX-2), were examined by western blotting (See S3.4 for experimental details). The LPS treatment induced the COX-2 expression (Fig. 5). Under these conditions, 25 μM of compounds 9 and 10 diminished the expression of COX2 in RAW264.7 macrophages after 24 h treatment (Fig. 5). These results implied that compounds 9 and 10 could be applicable to the COX-2 expression-associated human diseases,26 as well as to the pain and swelling of inflammation.27 The inhibitory activities of kaempulchraols P (9) and Q (10) in the LPS-induced RAW264.7 NF-κB reporter activity were comparable to cordycepin, which was reported as potent antiinflammatory effect in RAW264.7 cells.28 A structure–activity relationship study of the active compounds revealed that the presence of the additional α-OAc group at C-1 favored the inhibition of NO when the β-OH group is present at C-6, as confirmed by the % NO inhibitions of 10 and 9 at 50 μM [10 (71.56) > 9 (65.64)]. In contrast, the presence of the α-OH group at C-1, instead of the α-OAc group in compounds 1 and 3, led to weaker activity as confirmed by the % NO inhibitions of compounds 10, 1, and 3 at 50 μM [10 (71.56) > 1 (35.21), 3 (34.32)]. Hence, as a potent NO inhibitor, the α–OAc group at C-1 and the β–OH group at C-6 could be important functional groups in the isopimara-8(14),15-dienes. In the case of cytotoxic effect, compound 12 was the most toxic agent against RAW264.7 cells with an IC50 value of 32.61 μM. On the other hand, the substitution of α-OAc at C-7 with either β-OAc or β–OH increased the cell viability (IC50 > 50 μM) when the β–OH group is present at C-6, as shown in compounds 13 and 14. These observations could suggest that the presence of the α-OAc group at C-7 may enhance the cytotoxicity. In summary, we investigated the anti-inflammatory activities of 21 isopimara-8(14),15-diene diterpenoids isolated from the CHCl3-soluble extract of K. pulchra rhizomes. We found that compounds 4, 7, 9–11, 14, 18, 19, and 21 exhibit obvious NO inhibitory activity in LPS-induced RAW264.7 cells. Furthermore, an investigation of the mechanisms of action of the most active kaempulchraols P (9) and Q (10) suggested that these compounds effectively inhibited NF-κB-mediated transactivation, as well as IL-6 production and COX-2 expression. Thus,

Fig. 4. Effects of 9 and 10 on IL-6 production. The data points and bars represent mean ± SD (n = 3). ***, P < 0.0001, Very significantly different from the LPS-treated control.

Fig. 5. Effects of 9 and 10 on COX-2 expression in LPS-induced RAW264.7 cells, analyzed by western blotting.

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the isopimarane diterpenoids are suggested to be potent inhibitors of the NF-κB pathways and could be further explored as potential antiinflammatory lead compounds. In addition, these findings suggest that K. pulchra is a promising source of natural medicines to manage inflammation diseases by targeting the NF-κB pathway. Notably, the rhizomes have been used for self-medication by local cancer and AIDS patients.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grants JP17H05435, 17H06398, 19K23974, and 19H04649), the JSPS RONPAKU Program (R11815), 2019 Director Leadership Expenses, Institute of Natural Medicine, University of Toyama, and the Takahashi Industrial and Economic Research Foundation (H.M.).

20. 21. 22. 23. 24. 25. 26. 27. 28.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126841.

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