New Sesquiterpene Lactones from Glechoma hederacea L. and Their Cytotoxic Effects on Human Cancer Cell Lines

• Abstract
• Materials and Methods
• Supporting information
• References

Abstract

Three new sesquiterpene lactones, 1α,10β-epoxy-4-hydroxy-glechoma-5-en-olide (1), 1β,10α-epoxy-4,8-dihydroxy-glechoma-5-en-olide (2), and 1β,10α;4α,5β-diepoxy-8-methoxy-glechoman-8α,12-olide (3), were isolated from the whole plant of Glechoma hederacea, together with four known sesquiterpene lactones. The structures of the three new sesquiterpene lactones were determined by spectroscopic evidence. Cytotoxic effects of the isolated compounds were examined against MDA‐MB‐231 (breast), HCT116 (colon), SW620 (colon), and DU145 (prostate) human cancer cell lines.

Glechoma hederacea L. var. longituba Nakai (Labiatae) is a perennial vine plant that is widely distributed in Korea and has been used in traditional oriental medicine for treatment of cholelithiasis, urolithiasis, dropsy, and various ailments [1], [2]. The antiinflammatory, ulcer-protective, antiviral, and cytotoxic activities of this plant have been demonstrated by in vitro and animal studies [3], [4]. G. hederacea, commonly known as “ground ivy,” “creeping Charlie,” or “grill-over-the-ground”, is a hairy herb with a creeping stem [3]. In the present study, seven sesquiterpene lactones, including three new (1–3) and four previously identified compounds (4–7), were isolated ([Fig. 1]). Four known compounds were identified as 1β,10α;4α,5β-diepoxy-glechoman-8α,12-olide (4) [5], [6], 1β,10α;4α,5β-diepoxy-8β-hydroxy-glechoman-8α,12-olide (5) [7], 1,8-epoxy-7(11)-germacren-5-one-12,8-olide (6) [8], and 1β,10α;4α,5β-diepoxy-6β-hydroxy-glechoman-8α,12-olide (7) [9] by comparing their observed and published data. The compounds 1–7 were evaluated by cytotoxicity assay against four human cancer cell lines.

Compound 1 was obtained as a white powder. HRESIMS showed a molecular ion peak [M]⁺ at m/z 264.1253 that corresponded to the molecular formula C₁₅H₂₀O₄. The IR spectrum showed absorption bands around 3469 cm⁻¹ for the hydroxyl, and 1732 and 1650 cm⁻¹ for the γ-lactone group. The signals at δ 5.29 (H-8), δ 157.0 (C-7), 78.1 (C-8), 125.4 (C-11), and 173.6 (C-12) in the ¹H and ¹³C NMR spectra of 1 ([Table 1]) further supported a sesquiterpene lactone skeleton [11], [12]. Comparison of the ¹H NMR data of 1 with those of 4 showed the presence of only one oxygenated methine proton at δ 2.86 (d, J = 9.6 Hz, H-1). The HMQC data showed two methine protons at δ 6.02 (d, J = 16.8 Hz, H-5) and 6.40 (d, J = 16.8 Hz, H-6) that corresponded to two olefinic carbons at δ 145.3 and 118.3. The HMBC spectra (Fig. 1S, Supporting Information) showed correlations between H-5 and C-3 at δ 41.9, C-4 at δ 72.9, C-7 at δ 157.0; H-6 and C-4, C-5 at δ 145.3, C-7, C-8, respectively. Based on the indicated 1D and 2D NMR data, the olefinic carbons were located at C-5 and C-6 as a trans-configured olefinic bond. The relative stereochemistry of 1 was supported by NOESY data (Fig. 2S, Supporting Information). The NOESY spectra showed cross-correlations between β-oriented H-8 and H-1, H-9α; H-1 and H-9α, whereas the correlation of H-6/H₃-14, H₃-15; H-9β/H₃-14 suggested α-orientation [13]. These NOESY correlations confirmed a 1α,10β epoxide moiety. Based on the above spectroscopic data, compound 1 was, therefore, elucidated as 1α,10β-epoxy-4-hydroxy-glechoma-5-en-olide.

Compound 2 was obtained as a white powder. HRESIMS showed a molecular ion peak [M]⁺ at m/z 280.1203, which was consistent with the molecular formula C₁₅H₂₀O₅. In the IR spectrum, 2 showed absorption bands around 3432 cm⁻¹ for the hydroxyl groups, and 1743 and 1666 cm⁻¹ for the γ-lactone moiety. The ¹H and ¹³C NMR spectra ([Table 1]) were consistent with a sesquiterpene lactone backbone [12]. The spectroscopic data were similar to those of 1. In comparison with 1, the NMR data showed the presence of an oxygenated methine proton at δ 2.68 (br d, J = 8.7 Hz) and a quaternary lactone carbon at δ 108.2 (C-8). In the HMQC data, two methine carbons at δ 151.9 and 119.4 were correlated with an olefinic proton signal at δ 6.67, which was an overlapped singlet 2H proton. The location of two olefinic methine groups was confirmed by observed correlations between H-5 at δ 6.67 and C-4 at δ 73.7, and C-6 at δ 119.4; between H-6 and C-5 at δ 151.9, C-7 at δ 156.9, and C-8 at δ 108.2 in the HMBC spectra (Fig. 1S, Supporting Information). The absence of a lactone proton at C-8 (δ 108.2), which was shifted downfield more than that of 1, and the presence of an isolated doublet methylene group at C-9 (δ 50.8) indicated that a hydroxyl group was situated at C-8 [13]. These data suggested that 2 had 8-hydroxy lactone and 5-en-olide moieties. The stereochemistry of 2 was determined by comparison with NOESY data of 1β,10α;4α,5β-diepoxy-8β-hydroxy-glechoman-8α,12-olide (5). In the NOESY spectra (Fig. 2S, Supporting Information), a correlation was observed for H₃-14 at δ 1.53 (s) and β-oriented H-9α at δ 2.87 (d, J = 14.7). NOESY correlation between an oxygenated proton H-1 at δ 2.68 and α-oriented H-9β at δ 1.27 confirmed 1β,10α-epoxide. Based on the above spectroscopic data, the structure of compound 2 was assigned as the new sesquiterpene lactone, 1β,10α-epoxy-4,8-dihydroxy-glechoma-5-en-olide.

Compound 3 was obtained as a white powder. HRESIMS data determined the molecular formula to be C₁₆H₂₂O₅, which showed a molecular ion peak at m/z 294.1356 [M]⁺ (calcd. for 294.1467). The IR band at 1729 cm⁻¹ and the UV absorption at 254 nm also indicated the presence of a γ-lactone moiety in 3. Comparison of the NMR data of 3 and 1 showed two oxygenated methine protons at δ 2.94 (dd, J = 10.5, 1.5 Hz, H-1) and δ 3.37 (dd, J = 8.7, 5.7 Hz, H-5), with no hydroxyl group in the structure of 3. These spectroscopic data ([Table 2]) were similar to those of 1β,10α;4α,5β-diepoxy-glechoman-8α,12-olide (4). The presence of isolated doublet methylene protons at C-9 and the strong correlation of the methoxy group at δ 2.97 with C-8 at δ 93.3 in HMBC spectra indicated that 3 had a 8-methoxy lactone moiety (Fig. 1S, Supporting Information). The relative configurations of the stereocenters of 3 were assigned on the basis of 2D NOESY experiments and compared to those in the literature [7]. Correlations were observed between β-oriented OCH₃ and H-9α; H-9α and H₃-14; and H₃-14 and H₃-15, which suggested the β-orientation of the methyl singlets (CH₃-14 and 15). The α-oriented correlations between H-1 and H-9β; H-1 and H-5 were observed, which confirmed 1β,10α;4α,5β diepoxide (Fig. 2S, Supporting Information). Based on the above spectroscopic data, the structure of compound 3 was therefore identified as the new sesquiterpene lactone, 1β,10α;4α,5β-diepoxy-8-methoxy-glechoman-8α,12-olide.

Seven sesquiterpene lactones were isolated and evaluated for cytotoxicity against human cancer cell lines ([Table 3]). Human cancer cells were treated with the compounds for 24 h, and the viability of the cells was determined by MTT assay. Several sesquiterpene lactones from natural sources have been reported to show cytotoxic activity [6], [9]. Due to their enzyme-alkylating ability, although the α-methylene-γ-lactone moiety has been believed to be the active moiety [10], [14], [15], sesquiterpenes including α,β-unsaturated-γ-lactone moiety showed cytotoxicity also [6], [16]. Among the seven compounds including the same lactone moiety, 2, 4, and 5 exhibited stronger cytotoxic effects than the other compounds against MDA‐MB‐231, IC₅₀ value 7.7, 6.3, and 14.1 µM, against SW620, IC₅₀ value 8.6, 5.9, and 9.8, µM, respectively. It was supposed that the cytotoxicity of the compounds on the human cancer cells is affected not only by α,β-unsaturated-γ-lactone, but also by its substituents. Further study is needed to identify this relation for the different substituents of sesquiterpene lactones.

Materials and Methods

Glechoma hederecea was collected in June 2009 in the herbal garden of the Chungnam National University and identified by one of the authors (Prof. K. H. Bae). A voucher specimen (CNU-2699) has been deposited in the herbarium at the College of Pharmacy, Chungnam National University, Daejeon, Korea. G. hederecea (7.4 kg) was extracted with ethanol. The extract was concentrated in vacuo and then suspended in distilled water. The aqueous residue was partitioned and concentrated successively with hexane, ethyl acetate, and butanol to obtain three fractions. Since the ethyl acetate soluble fraction exhibited the strongest inhibition activity on human cancer cell lines, it was subjected to silica gel column chromatography and isolation of compounds.

The extraction and isolation protocols are detailed in Supporting Information.

1α,10β-epoxy-4-hydroxy-glechoma-5-en-olide (1): white powder; mp 190–191 °C; [α]_D ²⁵ = + 78.9 (c 0.1, CHCl₃); IR (KBr) ν _max 3469 (OH), 1732 (C=O), 1650, 1100, 1004 cm⁻¹; ESIMS m/z 264 [M]⁺; HR‐ESI‐MS: m/z 264.1253 [M]⁺ (calcd. for C₁₅H₂₀O₄, 264.1362); UV (CHCl₃): λ _max (log ε) = 262 (4.2) nm; ¹H NMR (CDCl₃, 300 MHz) and ¹³C NMR (CDCl₃, 75 MHz), see [Table 1].

1β,10α-epoxy-4,8-dihydroxy-glechoma-5-en-olide (2): white powder; mp 161–162 °C; [α]_D ²⁵ = − 6.2 (c 0.13, MeOH); IR (KBr) ν _max 3432 (OH), 1743 (C=O), 1666, 1134, 944 cm⁻¹; ESIMS m/z 280 [M]⁺; HR‐ESI‐MS m/z 280.1203 [M]⁺ (calcd. for C₁₅H₂₀O₅, 280.1311); UV (MeOH): λ _max (log ε) = 272 (4.1) nm; ¹H NMR (methanol-d₄ , 300 MHz) and ¹³C NMR (methanol-d₄ , 75 MHz), see [Table 1].

1β,10α;4α,5β-diepoxy-8-methoxy-glechoman-8α,12-olide (3): white powder; mp 179–180 °C; [α]_D ²⁵ = + 15.1 (c 0.1, MeOH); IR (KBr) ν _max 2917 (CH), 1729 (C=O), 1703, 1462, 1410, 1295 cm⁻¹; ESIMS m/z 294 [M]⁺; HR‐ESI‐MS m/z 294.1356 [M]⁺ (calcd. for C₁₆H₂₂O₅, 294.1467); UV (MeOH): λ _max (log ε) = 252 (3.5) nm; ¹H NMR (methanol-d₄ , 300 MHz) and ¹³C NMR (methanol-d₄ , 75 MHz), see [Table 2].

Supporting information

The general experimental procedures, detailed protocols for extraction and isolation, protocols for the biological assays, Fig. 1S, Fig. 2S, and NMR spectra of compounds 1–3 are available as Supporting Information.

References

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  Search in Google Scholar
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  Thieme ConnectPubMedSearch in Google Scholar

Prof. Dr. KiHwan Bae

College of Pharmacy
Chungnam National University

Gung-dong 220, Yuseong-gu

Daejeon 305-764

Korea

Phone: +82 4 28 21 59 25

Fax: +82 4 28 23 65 66

Email: baekh@cnu.ac.kr

References

• 1 Jo D, Lee J, Noh J, Kim O, Kwon J. Chemical composition and electron donating and nitrite scavenging activities of Glechoma hederacea var. longituba Nakai.  J Food Sci Nutr. 2001;  6 142-146
  PubMedSearch in Google Scholar
• 2 Yamauchi H, Kakuda R, Yaoita Y, Machida K, Kikuchi M. Two new glycosides from the whole plants of Glechoma hederacea L.  Chem Pharm Bull. 2007;  55 346-347
  CrossrefPubMedSearch in Google Scholar
• 3 Kumarasamy Y, Cox P J, Jaspars M, Nahar L, Sarker S D. Isolation, structure elucidation and biological activity of hederacine A and B, two unique alkaloids from Glechoma hederaceae.  Tetrahedron. 2003;  59 6403-6407
  CrossrefPubMedSearch in Google Scholar
• 4 An H J, Jeong H J, Um J Y, Kim H M, Hong S H. Glechoma hederacea inhibits inflammatory mediator release in IFN-[gamma] and LPS-stimulated mouse peritoneal macrophages.  J Ethnopharmcol. 2006;  106 418-424
  CrossrefPubMedSearch in Google Scholar
• 5 Stahl E, Datta S N. Neue sesquiterpenoide Inhaltsstoffe der Gundelrebe (Glechoma hederacea L.).  Justus Liebigs Ann Chem. 1972;  757 23-32
  CrossrefPubMedSearch in Google Scholar
• 6 Chaturvedula V S P, Schilling J K, Miller J S, Andriantsiferana R, Rasamison V E, Kingston D G I. New cytotoxic terpenoids from the wood of Vepris punctata from the Madagascar rainforest.  J Nat Prod. 2004;  67 895-898
  CrossrefPubMedSearch in Google Scholar
• 7 Goren N, Ulubelen A. Glechomanolides and eudesmanolides from Smyrnium perfoliatum.  Phytochemistry. 1987;  26 2585-2587
  CrossrefPubMedSearch in Google Scholar
• 8 Zhang Q J, Yang X S, Zhu H Y, Wang Y, Hao X J, Song B A. A novel sesquiterpenoid from Glechoma longituba.  Chin Chem Lett. 2006;  17 355-357
  PubMedSearch in Google Scholar
• 9 Ali A, El-Gamal A A. Sesquiterpene lactones from Smyrnium olusatrum.  Phytochemistry. 2001;  57 1197-1200
  CrossrefPubMedSearch in Google Scholar
• 10 Bai N, Lai C S, He K, Zhou Z, Zhang L, Quan Z, Zhu N, Zheng Q Y, Pan M H, Ho C T. Sesquiterpene lactones from Inula britannica and their cytotoxic and apoptotic effects on human cancer cell lines.  J Nat Prod. 2006;  69 531-535
  CrossrefPubMedSearch in Google Scholar
• 11 Sanz J F, Marco J A. A germacrane derivative from Pallenis spinosa.  Phytochemistry. 1991;  30 2788-2790
  CrossrefPubMedSearch in Google Scholar
• 12 Ulubelen A, Goren N, Jakupovic J. Germacrane derivatives from the fruits of Smyrnium creticum.  Phytochemistry. 1986;  26 312-313
  CrossrefPubMedSearch in Google Scholar
• 13 Ulubelen A, Goren N, Bohlmann F, Jakupovic J, Grenz M, Tanker N. Sesquiterpene lactones from Smyrnium cordifolium.  Phytochemistry. 1985;  24 1305-1308
  CrossrefPubMedSearch in Google Scholar
• 14 Barnes J, Anderson L A, Phillipson J D. Herbal medicines. London; Pharmaceutical Press 2002: 280-281
  Search in Google Scholar
• 15 Hall I H, Lee K H, Starnes C O, Eigebaly S A, Ibuka T, Wu Y S, Kimura T, Haruna M. Antitumor agents XXX: Evaluation of alpha-methylene-gamma-lactone-containing agents for inhibition of tumor growth, respiration, and nucleic acid synthesis.  J Pharm Sci. 1978;  67 1235-1239
  CrossrefPubMedSearch in Google Scholar
• 16 Aponte J C, Yang H, Vaisberg A J, Castillo D, Málaga E, Verástegui M, Casson L K, Stivers N, Beates P J, Rojas R, Fernandez I, Lewis W H, Sarasara C, Sauvain M, Gilman R H, Hammond G B. Cytotoxic and anti-infective sesquiterpenes present in Plagiochila disticha (Plagiochilaceae) and Ambrosia peruviana (Asteraceae).  Planta Med. 2010;  76 705-707
  Thieme ConnectPubMedSearch in Google Scholar

Prof. Dr. KiHwan Bae

College of Pharmacy
Chungnam National University

Gung-dong 220, Yuseong-gu

Daejeon 305-764

Korea

Phone: +82 4 28 21 59 25

Fax: +82 4 28 23 65 66

Email: baekh@cnu.ac.kr

• Supplementary Material