Kaempferia Parviflora (Black Ginger) Constituents on Nitric Oxide (NO) Production Was Examined

日本食品化学学会誌（日食化誌）、Vol. 25(3), 2018 152

Regular article 日本食品化学学会誌、 Vol. 25(3), 152-159(2018) Japanese Journal of Food Chemistry and Safety (JJFCS)

Inhibitory effect of black ginger Kaempferia( parviflora) constituents on nitric oxide production (Received May 24, 2018) (Accepted September 21, 2018)

Hiroyuki Fuchino a), Nanami Fukui b), Osamu Iida a), Hiroshi Wada b), Nobuo Kawahara a)

a) Research Center for Medicinal Plant Resources, National Institutes of Biomedical Innovation, Health and Nutrition b) Faculty of Pharmaceutical Sciences, Tokyo University of Science

Abstract The inhibitory effect of Kaempferia parviflora (black ginger) constituents on nitric oxide (NO) production was examined. From the methanolic extract of K. parviflora rhizome, 16 flavones, 14 flavanones, 2 diarylheptanoids, 2 chalcones and a stilbene were isolated and their inhibitory activities toward NO production were examined. As a result, two methoxyflavones, 5,7,3’,4’-tetramethylluteolin and 5,7-dimethoxyflavone showed significant activities. Most of the flavanone constituents, except for 5,7,3’-trimethoxy-4’-hydroxyflavanone, showed lower activities. The most potent active constituent of Black ginger was the chalcone flavokavin B; its derivative, flavokavin A, also showed significant activity. The curcumin and its derivative, 1-(4-hydroxyphenyl)-7-phenyl-1,6-heptadiene-3,5-dione, showed signiﬁcant activity. Only one stilbene was isolated, and it showed low activity.

Keywords : inhibition of NO production, anti-inﬂammatory effect, black ginger, Kaempferia parviflora, polymethoxy ﬂavonoid

Ⅰ Introduction Polymethoxyflavonoids are known to be constituents of black ginger and may play a role in several of its Kaempferia parviflora Wall ex. Baker belongs to the pharmaceutical properties7, 8). While the anti-inflammatory Zingiberaceae and is called “black ginger” or “black turmeric” activities of the constituents of black ginger have been based on the color of its rhizome. K. parviflora grows in examined, minor products have not yet been the target of Thailand (Local name; krachai dum), and has been used in folk large-scale extraction3). medicine as a tonic1). It has also been reported to have anti- In this report, we describe the inhibitory activities of cancer2), anti-inﬂammation3) and other bioactivities. Recently, isolated constituents of black ginger including many minor its dietary properties4, 5) have received some attention, and it compounds on nitric oxide production to evaluate its anti- has become a popular health food. Since Black ginger is on the inﬂammatory activity. list of raw materials that are not regarded as pharmaceuticals unless a medical property is proclaimed, which is maintained by the Ministry of Health, Labor and Welfare of Japan6), Ⅱ Material and Methods and is thus at the border between pharmaceuticals and non- pharmaceuticals, many health food products containing black Instruments ginger can now be found in the Japanese market. However, An SCA-165DRS (Astec Co. Ltd.) CO2 incubator was imported Black ginger seems to vary considerably in quality, used for incubation. An xMark microplate reader (Bio-Rad and therefore methods for maintaining quality control are Laboratories, Inc.) was used. NMR spectra were measured on necessary. a Bruker Ascend 600 (600 MHz). HPLC was performed on

Corresponding author: Hiroyuki Fuchino, Research Center for Medicinal Plant Resources, National Institutes of Biomedical Innovation, Health and Nutrition, 1-2, Hachimandai, Tsukuba, Ibaraki 305-0843, Japan 153 Jpn. J. Food Chem. Safety, Vol. 25(3), 2018

a Shimadzu LC-10ADvp series (pump; LC-10AD VP, Diode column temperature; 35°C. Array Detector (DAD); SPD-M-10A VP, Column oven; CTO- G: Nacalai Tesque COSMOSIL Packed Column Cholester, 10 10A) and a JASCO-2000 Plus series (pump; PU-2080 Plus, mm i.d.×250 mm, gradient methanol-water, flow rate; 3.0 DAD; MD-2018 Plus, Column oven; CO-2060 Plus). Recycling mL/min, column temperature; 35°C. preparative HPLC was performed on a Japan Analytical H: Waters X Bridge prep C18, 5 µm, 10 mm i.d.×250 mm, Industry LC-908W. gradient with acetonitrile-water, flow rate; 2.5 mL/min, column temperature; 35°C. Materials I: Imtakt Unison UK-C18, 10 mm i.d.×250 mm, 3 µm, The Black ginger used in this study was purchased from gradient with methanol-water, flow rate; 3.0 mL/min, Exotic Plants Ltd. (Chiba, Japan). column temperature; 40°C. J: Nacalai Tesque COSMOSIL Packed Column PBr, 10 mm Chemicals i.d.×250 mm, 0.1%TFA/acetonitrile-0.1%TFA/water, flow F-12 Ham medium, L-glutamine, and lipopolysaccharide rate; 3.5 mL/min, column temperature; 40°C. (LPS) were purchased from Sigma-Aldrich Co. LLC., K: Waters X Bridge prep C18, 5 µm, 10 mm i.d.×250 mm, and mouse recombinant interferon (IFN)-γ, N -1- 70% A-30% B (A; 0.1% trifluoroacetic acid in acetonitrile, naphthylethylenediamine dihydrochloride, sulfanilamide and B: 0.1% trifluoroacetic acid/water), flow rate; 3.0 mL/min, phosphoric acid were obtained from Wako Pure Chemical column temperature; 40°C.) Industry, Co. Ltd., WST-1 was purchased from Roche G Diagnostics GmbH and N -monomethyl-L-arginine acetate Dried rhizome of K. parviflora (1.94 kg) was pulverized (L-NMMA) was purchased from Dojindo Laboratories, Ltd. and extracted with methanol (2.5 L) under reflux for 2 h. The RAW264.7 cells were purchased from the European Collection extract was then filtered and concentrated under reduced of Cell Cultures (ECCC). Fetal Bovine Serum (FBS) was pressure to give a residue (231 g). The residue was suspended obtained from Life Technologies Corp. An MS-8096R 96-well with water, partitioned between ethyl acetate, n-butanol plate (Sumitomo Bakelite Co. Ltd.) was used. and water, and concentrated to obtain three fractions: KP-A (153 g), KP-B (12.8 g) and KP-W (70.5 g), respectively.

Cell Culture KP-A was subjected to column chromatography on SiO2 F-12 Ham medium supplemented with 10%FBS and (internal diameter (i.d.): 14.3 cm, solvent system: chloroform- L-glutamine (2 mM) was used. Cells were incubated at 37°C in ethyl acetate) to give 36 fractions (FR1-1 - 36). FR1-10 - 11 an atmosphere of 5%CO2. were combined and crystallized from ethanol to afford 1 (44.1 mg). The filtrate was subjected to medium-pressure Isolation of the constituents of the K. parviflora liquid chromatography (MPLC) (SIL-G3-300 YAMAZEN, rhizome n-hexane/ethyl acetate (=85:15), ﬂow rate 15 mL/min) to afford (In this section, the analytical conditions for HPLC are 120 fractions (FR2-1 - 120). FR2-25 - 39 were combined and abbreviated as follows: purified by HPLC (condition: A), column chromatography

A: Waters X Bridge prep C18, 5 µm, 10 mm i.d.×250 mm, (SiO2, i.d.: 0.6 cm, solvent system: n-hexane-ethyl acetate), gradient with acetonitrile-water, flow rate; 2.5 mL/min, recycling HPLC (JAIGEL GS-310+JAIGEL GS-320, methanol) column temperature; 35°C. and crystallization (methanol) to give 2 (0.4 mg) and 3 (1.7 B: Waters X Bridge prep C18 5 µm, 10 mm i.d.×250 mm, mg). FR2-41 - 57 were combined, 1 was removed by repeated gradient with methanol-water, flow rate; 3.5 mL/min, crystallization, and the filtrate was subjected to HPLC (B) to column temperature; 40°C. give 3 (2.0 mg) and 4 (14.0 mg). FR2-59 - 79 were combined C: Kanto Chemicals, Co. Ltd., Mightysil Si60 10 mm i.d.×250 and subjected to HPLC (B) to afford 5 (3.8 mg), 6 (2.7 mg) and mm 5 µm, gradient with n-hexane-ethyl acetate, flow rate; 7 (1.9 mg). Crude crystal (CR-1) was obtained from FR1-13 - 3.0 mL/min, column temperature; 35°C. 15 by crystallization (chloroform-methanol) and a portion of D: Waters Atlantis T3, 5 µm, 10 mm i.d.×250 mm, gradient CR-1 (50 mg) was subjected to HPLC (C) to afford 1 (2.3 mg), with acetonitrile-water, flow rate; 3.0 mL/min, column 6 (2.5 mg), 8 (0.6 mg), 9 (7.2 mg) and 10 (21.5 mg). Crude temperature; 40°C. crystal (CR-2) was obtained from FR1-16 by crystallization E: Waters X Bridge prep phenyl, 5 µm, 10 mm i.d.×250 mm, (ethanol) and a portion of CR-2 (50 mg) was purified by HPLC gradient with methanol-water, flow rate; 3.0 mL/min, (D) to give 11 (1.7 mg), 8 (20.2 mg), 9 (8.3 mg) and 10 (0.8

column temperature; 40°C. mg). The ﬁltrate of CR-2 was chromatographed on SiO2 (i.d.: F: Phenomenex Kinetex 5 µ Biphenyl 100A, 10 mm i.d.×250 3.2 cm, n-hexane- ethyl acetate) to give 22 fractions (FR3-1

mm, gradient methanol-water, flow rate; 3.0 mL/min, - 22). FR3-10 was re-chromatographed on SiO2 (i.d.: 2.0 cm, 日本食品化学学会誌（日食化誌）、Vol. 25(3), 2018 154

chloroform-methanol) to obtain β-sitosterol (160.1 mg). FR3- (1.2 mg), 28 (0.7 mg) and 29 (1.2 mg). FR5-91 was puriﬁed

12 was crystallized from acetonitrile to afford 9 (36 mg) and by column chromatography on SiO2 (i.d.: 6.0 cm, n-hexane- the ﬁltrate was subjected to HPLC (1st, B; 2nd, E) to give 12 (1.0 ethyl acetate - ethyl acetate-methanol) to afford 52 fractions mg), 13 (0.6 mg) and 14 (2.3 mg). FR3-13 was fractionated (FR6-1 - 52). Fr6-12 - 17 were combined and crystallized from to give 11 fractions (FR4-1 - 11) by HPLC (1st, B, 2nd, E) and methanol to give 30 (1588.7 mg). FR6-6 - 7 were subjected FR4-5 was subjected to HPLC (F) to give 15 (1.2 mg) and 16 to HPLC (K, J) to afford 31 (2.3 mg) and FR6-24 - 29 were (2.0 mg). FR4-10 was purified by HPLC (F) to give 17 (1.3 purified by HPLC (J) to give 32 (162.0 mg) and 33 (63.4 mg). FR3-14 was purified by HPLC (1st, B, 2nd, G) to give mg). FR1-34 - 36 were puriﬁed by column chromatography on

18 (2.2 mg). FR3-15 was subjected to HPLC (H) to give 19 SiO2 (i.d.: 8.0 cm, chloroform-ethyl acetate), crystallization (13.5 mg). FR3-16 was subjected to HPLC (B) to give 20 (5.1 (methanol) and HPLC (B) to give 34 (3.0 g) and 35 (20.4 mg). mg). Compound 21 (45.7 mg) was obtained by crystallization All of the obtained compounds were elucidated by several of FR3-17 from methanol. FR3-18 was purified by HPLC two-dimensional NMR spectra (COSY, HSQC, HMBC and (B) to afford 23 (8.7 mg). FR1-17 - 22 were combined and NOESY) and finally confirmed by comparison of their NMR chromatographed on SiO2 (i.d.: 6.0 cm, n-hexane-ethyl acetate spectral data to those in the literature. Relative configurations - ethyl acetate-methanol) to give 91 fractions (FR5-1 - 91). between C-2 and 3 for flavanonols were determined by FR5-9 was subjected to HPLC (1st, B, 2nd, G) to give 22 (1.8 1H-NMR spectral data, however absolute configurations for mg). FR5-29 - 31 were purified by column chromatography flavanones and flavanonols have not been determined yet. on SiO2 (i.d.: 4.0 cm, n-hexane-ethyl acetate) and then HPLC (G) to afford 23 (8.2 mg) and 24 (4.2 mg). Fr5-54 - 59 was Assay of the ability to inhibit NO production crystallized from methanol-chloroform to give 25 (1119.3 mg) activity 9, 10) and its filtrate was combined to Fr.5-45-53 and purified by Macrophage-like cells (RAW264.7) were seeded into a 96- 5 column chromatography on SiO2 (i.d.: 4.2 cm, n-hexane-ethyl well plate (2.0×10 cells/200 μL/well) and incubated under st nd acetate) and HPLC (1 , I, 2 , J) to give 26 (16.5 mg), 27 5%CO2 at 37°C for 2 h. IFN-γ and LPS were added to each

Table 1. Flavones isolated from black ginger

Compound R1 R2 R3 R4 R5 References

1 (3,7-dimethyl galangin) OCH3 OH OCH3 H H 13

4 (izalpinin)* OH OH OCH3 H H 14

6 (techtochrysin) H OH OCH3 H H 13

8 (retusin) OCH3 OH OCH3 OCH3 OCH3 13

9 (4’,7-dimethylapigenin) H OH OCH3 OCH3 H 13

10 OCH3 OH OCH3 OCH3 H 13

11 (gonzalitosin I) H OH OCH3 OCH3 OCH3 15

18 * OH OH OCH3 OCH3 OCH3 16

19 (ayanin) OCH3 OH OCH3 OCH3 OH 17

25 OCH3 OCH3 OCH3 H H 13

30 OCH3 OCH3 OCH3 OCH3 H 13

31 * OH OCH3 OCH3 OCH3 H 16

32 OCH3 OCH3 OCH3 OCH3 OCH3 13

33 H OCH3 OCH3 H H 13

34 H OCH3 OCH3 OCH3 H 13

35 H OCH3 OCH3 OCH3 OCH3 13 * First reported isolation from K. parviflora. 155 Jpn. J. Food Chem. Safety, Vol. 25(3), 2018

Table 2. Flavanones isolated from black ginger

Compound R1 R2 R3 R4 R5 References Fig. 1. A stilbene isolated from black ginger

3 (pinostrobin) H OH OCH3 H H 18

5* H OH OCH3 OCH3 H 19 well to reach 0.3 ng/mL and 100 ng/mL, respectively, and 12* OH OH OCH3 OCH3 H 20 sample solutions dissolved in DMSO were added to reach 10 14 (pinocembrin)* H OH OH H H 21, 22 μM. The plate was incubated at 37°C under 5%CO2 for 16 15 (sakuranetin)* H OH OCH3 OH H 23 h. L-NMMA (100 μM), an inhibitor of iNOS, was used as a 16* H OH OCH3 OH OCH3 23 positive control.

20* OH OCH3 OCH3 H H 24

21 H OCH3 OCH3 H H 18 Inhibition of NO production

23* H OCH3 OCH3 OCH3 H 19 Supernatants of culture medium after incubation were

24* OH OCH3 OCH3 OCH3 H 25 recovered, and 50 μL of 1% sulfanilamide in 5% H3PO4 and

26* OH OCH3 OCH3 OCH3 OCH3 26 0.1% N-1-naphthylethylenediamine dihydrochloride were added. The mixture was allowed to stand at room temperature 27* H OCH3 OCH3 OCH3 OCH3 27, 28 under light-shielded conditions for 15 min. Absorbance at 28* H OCH3 OCH3 OH H 29 550 nm was measured (reference wavelength: 655 nm) on 29* H OCH3 OCH3 OH OCH3 30 a microplate reader. The inhibition of NO production was See the footnote for Table 1. measured as follows:

Table 3. Diarylheptanoids isolated from black ginger Inhibition of NO production (%)

= (1 － (AS －AN) / (AD －AN))×100

AN : Untreated

AS : Sample/LPS/IFN-treated

AD : DMSO/LPS/IFN-treated

Samples that exhibited inhibitory activity were dissolved

Compound R References in DMSO to give different ﬁnal concentrations (3, 5, 10, 30, 50 μM for 2, 21, 23, 29, 33, 34; 1, 3, 5, 10, 30 μM for 7, 2 H 31 17, 22, 35; 5, 10, 30, 50, 100 μM for 3, 13, 16, 27, 28) 17* OH 32 and their inhibitory effects against NO production were then See the footnote for Table 1. reexamined. From these results, the half-maximal inhibitory

concentration (IC50) was calculated. Table 4. Chalcones isolated from black ginger

Cell viability After removal of the supernatant, WST-1 reagents (10 μL)

were added and cells were incubated at 37°C under 5%CO2 for 2 h. Absorbance at 450 nm (reference wavelength: 655 nm) was measured by a microplate reader.

Compound R References Cell viability was calculated as follows:

7 (flavokavin B) H 33 Cell viability (%) = AS / AD×100

22 (flavokavin A)* OCH3 34 AS : Sample/LPS/IFN-treated

See the footnote for Table 1. AD : DMSO/LPS/IFN-treated 日本食品化学学会誌（日食化誌）、Vol. 25(3), 2018 156

Fig. 2. Inhibitory activities of isolated products on nitric oxide production (mean ± SD, n=3) Bar graph: Percent Inhibition of NO Production (%); Line graph: Cell Viability (%) Concentration of samples is 10 μM except for L-NMMA (100 μM). 157 Jpn. J. Food Chem. Safety, Vol. 25(3), 2018

Table 5. IC50 values of isolated compounds (mean ± SD, n=3) (IC50 7.53 and 6.61 μM, respectively), although curcumin is

IC50 (µM) known to strongly inhibit nitric oxide production (11). Only Diarylheptanoid one stilbene was isolated, and it showed low activity. Most 2 7.53 ± 0.30 of them showed no significant cytotoxicity, and thus were 17 6.61 ± 3.32 thought to have little, if any, inﬂuence on activity. Recently, Sae-Wong et al. reported that methoxyﬂavonoids Chalcone isolated from black ginger suppressed inducible nitric oxide 7 2.51 ± 1.17 synthase expression in RAW 264.7 cells (12), and discussed 22 7.82 ± 1.77 the mechanisms of their anti-inflammation activities. They isolated 12 constituents of the chloroform fraction Stilbenoid of black ginger and found that 5,7-dimethoxyflavone (33), 13 26.0 ± 1.71 trimethylapigenin (34) and tetramethylluteolin (35) showed

Flavone potent anti-inflammation activities, which was consistent with 33 9.03 ± 5.04 our results. However, their report did not address the activities 34 14.2 ± 4.19 of flavanones, chalcones or diarylheptanoids. 35 16.6 ± 0.50 With regard to the relationship between these structures and their inhibitory activities toward nitric oxide production, Flavanone while substitution at the C-3 position with a hydroxyl or 3 35.9 ± 8.26 methoxyl group tended to reduce the activity of the flavone 21 14.3 ± 6.04 and flavanone skeletons, methylation of a hydroxyl group at 23 20.1 ± 5.73 the C-5 position tended to enhance the activity (e.g., 3 vs. 21, 16 45.0 ± 3.96 and 16 vs. 29). Substitution on the B-ring reduced the activity 27 31.1 ± 0.52 of the flavanone skeleton (Figs. 1 and 2). As mentioned before, 28 45.0 ± 5.30 curcumin is known to exhibit significant activity, and 17, 29 16.1 ± 6.92 which has a hydroxyl group on an aromatic ring of curcumin, also showed potent activity. Since several methoxyflavones, chalcones and diarylheptanoids all showed significant activities, the anti- Ⅲ Results and Discussion inflammatory effect of Black ginger is considered to be due to these constituents. Overall, 36 compounds were isolated from the methanolic extract of black ginger rhizome. Among them, compounds 4, 5, 12, 14, 15, 16, 17, 18, 20, 22, 23, 24, 26, 27, 29, Ⅳ Acknowledgement 29 and 31 are first isolated from Black ginger in this study, and especially 29 is first isolated from natural resources. This study was financially supported by the Ministry The inhibitory activities of these compounds toward nitric of Agriculture, Forestry and Fisheries, “Plant variety oxide production were examined. The main constituents classification investigation of Kaempferia parviflora” in 2015. were methoxy flavonoids; many isomers with differently- substituted positions were found, which could be useful for discussing structure-activity relationships. Among a Ⅴ References series of polymethylflavones, 5,7-dimethylluteolin (33) showed the most significant activity (IC50 9.03 μM) and 1) Handa, S. S., Rakesh, D. D., Vasisht, K., “Compendium 5,7,4’-trimethoxyflavone (34) showed moderate activity of Medicinal and Aromatic Plants ASIA”, Volume II,

(IC50 14.2 μM). On the other hand, most of the flavanone Italy, ICS-UNIDO, 2006, p. 99. constituents, except for 5,7-dimethoxyflavanone (21) (IC50 2) Potikanond, S., Na, T. M., Nimlamool, W., Sookkhee, S., 14.3 μM) and 5,7,3’-trimethoxy-4’-hydroxyflavanone (29) Mungkornasawakul, P., Mungkornasawakul, P., Wikan,

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論文

黒ショウガ（Kaempferia parviflora）の成分の一酸化窒素産生抑制効果について（2018 年 5 月 24 日受付）（2018 年 9 月 21 日受理）

渕野裕之 a)、福井ななみ b)、飯田修 a)、和田浩志 b)、川原信夫 a)

a) 国立研究開発法人医薬基盤・健康・栄養研究所薬用植物資源研究センター b) 東京理科大学薬学部

キーワード : NO 産生抑制、抗炎症作用、黒ショウガ、Kaempferia parviflora、ポリメトキシフラボノイド

概要黒ショウガ（ Kaempferia parviflora）の成分の一酸化窒素（ NO）産生抑制活性について検討を行った。黒ショウガ根茎のメタノール抽出エキスから 16 種のフラボン、 14 種のフラバノン、 2 種のジアリルヘプタノイド、 2 種のカルコン、 1 種のスチルベン化合物を単離しそれらの NO 産生抑制活性を調べた。その結果、2 種のフラボン化合物である 5,7,3’,4’-tetramethylluteolin と 5,7-dimethoxyflavone が、顕著な抑制活性を示した。フラバノン化合物においては、5,7,3’-trimethoxy-4’-hydroxyflavanone を除いては活性は低かった。最も活性が強かったのは、カルコン化合物である flavokavin B であり、その類縁体である flavokavin A も同様に強い活性を示した。クルクミンとその類縁体である 1-(4-hydroxyphenyl)-7-phenyl-1,6-heptadiene-3,5-dioneも顕著な活性を示した。唯一のスチルベンは活性は低かった。

連絡先：〒 305-0843 茨城県つくば市八幡台 1-2 国立研究開発法人医薬基盤・健康・栄養研究所薬用植物資源研究センター渕野裕之