- Research article
- Open Access
Dihydroisocoumarins from Radix Glycyrrhizae
© The Author(s) 2018
- Received: 13 November 2017
- Accepted: 30 April 2018
- Published: 11 May 2018
Radix Glycyrrhizae is the rhizome of Glycyrrhiza inflata Bat., Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L. The present paper describes the isolation and the structural elucidation of three new dihydroisocoumarins obtained from the 70% EtOH extract of Radix Glycyrrhizae. And the cytotoxic activities of these new compounds were also evaluated using four cell lines, subsequently.
A pair of new dihydroisocoumarin epimers ((3R,4S)-4,8-dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (1) and ((3R,4R)-4,8-dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (2) along with a new dihydroisocoumarin (3R,4R)-4,8-dihydroxy-3,5-dimethylisochroman-1-one (3) were isolated from Radix Glycyrrhizae. Their structures were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses, HR–ESI–MSand ECD calculation comparing with those of experimental CD spectra. Cytotoxic activities of the three compounds were evaluated using the HepG2, A549, LoVo and Hela cell lines, respectively. IC50 values indicated compounds 1–3 exhibited moderate or less cytotoxic activity in vitro.
Dihydroisocoumarin is not the common components in Radix Glycyrrhizae, a series of dihydroisocoumarin were obtained in this plant could be a supplement to the chemical study of this plant.
- Radix Glycyrrhizae
- ECD investigation
- NMR spectrum
1H NMR (600 MHz) and 13C NMR (150 MHz) spectral data of compounds 1–3 in DMSO-d 6
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
4.72 (1H, qd, 6.4, 1.6)
4.88 (1H, qd, 6.8, 1.6)
4.87 (1H, qd, 6.8, 1.2)
4.66 (1H, dd, 6.8, 1.6)
4.77 (1H, dd, 5.2, 1.6)
4.67 (1H, dd, 5.6, 1.2)
7.64 (1H, d, 8.8)
7.66 (1H, d, 8.8)
7.45 (1H, d, 8.4)
6.99 (1H, d, 8.8)
7.03 (1H, d, 8.8)
6.92 (1H, d, 8.4)
1.45 (3H, d, 6.4)
1.16 (3H, d, 6.8)
1.16 (3H, d, 6.8)
5.09 (1H, d, 12.4)
5.07 (1H, d, 12.4)
2.29 (3H, s)
5.17 (1H, d, 12.4)
5.20 (1H, d, 12.4)
2.04 (3H, s)
2.03 (3H, s)
5.70 (1H, d, 7.2)
5.96 (1H, d, 5.2)
5.82 (1H, d, 5.2)
11.20 (1H, s)
11.19 (1H, s)
10.9 (1H, s)
IC50 value of the compounds 1–3 against four cell lines (μM)
General experimental procedures
The UV spectrum was recorded on a Shimadzu UV-2201 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The IR spectrum was obtained from a Bruker IFS-55 spectrophotometer using a KBr pellet (Bruker Optik BmbH, Ettlingen, Germany). The HR-ESI–MS data were obtained on a microTOF-Q Bruker mass instrument (Bruker Daltonics, Billerica, MA, USA). CD spectra were recorded with a Biologic MOS-450 spectrometer using MeOH as solvent. 1D and 2D NMR spectra were run on a Bruker AVANCE 600 spectrometer (Bruker BioSpin, Rheinstetten, Germany). 1H chemical shifts (δH) were measured in ppm, relative to TMS, and 13C chemical shifts (δC) were measured relative to DMSO-d6 and converted to TMS scale. Column chromatography (CC) was performed on Silica gel (200–300 mesh; Qingdao Marine Chemical Co., Qingdao, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden) columns. HPLC was performed on a Shimadzu LC-10AVP liquid chromatograph with a YMC-pack C18 (ODS) column (10 × 250 mm, 5 μm, apan) and a Shimadzu LC-8AVP liquid chromatograph with a Diamonsil C18 (ODS) column (4.6 × 250 mm, 5 μm, China). All reagents for isolation were HPLC or analytical grade and were purchased from Tianjin Damao Chemical Company (Tianjin, China). Fetal bovine serum and Dulbecco’s modified eagle medium (DMEM) were from Thermo Fisher Scientific, 96-well flat bottom plate were purchased from Corning Inc. (NY, USA), 3-[4,5-dimethyl-2-thiazolyl]-2,5 diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Corporation (MA, USA).
Radix Glycyrrhizae was purchased from Anhui Yishengyuan Traditional Chinese Medicine Pellets Co., Ltd., P. R. China, and all the materials were identified by Dr. Xiao Fu, Department of Traditional Chinese Medicine, First Affiliated Hospital of Jinzhou Medical University. The voucher specimen (20150610) has been deposited at First Affiliated Hospital of Jinzhou Medical University.
Extraction and isolation
Radix Glycyrrhizae (25 kg) was cut and extracted with 70% EtOH for two times. The combined extracts were concentrated in vacuo to yield a residue, and the residue was then suspended in H2O and successively partitioned with petroleum ether, dichloromethane (CH2Cl2), ethyl acetate (EtOAc). The EtOAc crude extracts (2.3 kg) were applied on a silica gel column and eluted with petroleum ether-acetone gradient (from 500:0 to 0:100) to afford nine fractions. Fr. 6 was subjected to Sephadex LH-20, semi-preparative HPLC to yield compound 1 (12.0 mg) and 2 (9.2 mg). Fr. 7 was subjected to Sephadex LH-20, semi-preparative HPLC to yield compound 3 (15 mg).
((3R,4S)-4,8-Dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (1)
Yellow needle crystal (CH3OH); UV (MeOH) λmax(log ε) 214, 315 nm; IR (KBr) νmax 3408.9, 2920.2, 2850.0, 1674.3, 1446.1,1384.2, 1207.2, 1138.8 cm−1; CD (mdeg): Δε212 nm + 25.0,Δε250 nm − 14.6, Δε290 nm + 5.6; 1H and 13C-NMR spectral data, see Table 1. HR-ESI–MS: m/z 289.0681 [M + Na]+ (calcd. for C13H14O6Na, 289.0683).
((3R,4R)-4,8-Dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (2)
Yellow needle crystal (CH3OH); UV (MeOH) λmax(log ε) 218, 316 nm; IR (KBr) νmax 3418.5, 2920.1, 2850.8, 1675.2, 1477.9,1383.7, 1208.1, 1171.9 cm−1; CD (mdeg): Δε198 nm − 12.5,Δε212 nm + 5.0,Δε250 nm + 25.0, Δε316 nm − 3.7;1H and 13C-NMR spectral data, see Table 1. HR–ESI–MS: m/z 289.0670 [M + Na]+ (calcd. for C13H14O6Na, 289.0683).
Yellow needle crystal (CH3OH); CD (mdeg): Δε198 nm − 6.5,Δε212 nm + 7.0,Δε250 nm + 13.8, Δε316 nm − 1.1;1H and 13C-NMR spectral data, see Table 1. HR-ESI-MS: m/z 231.0637 [M + Na]+ (calcd. for C11H12O4Na, 231.0628).
Cytotoxic activity assay
Four cell lines including HepG2, A549, LoVo and Hela cell lines were purchased from the American Type Culture Collection. All the cell lines were used to evaluate the cytotoxic activities of compounds 1-3 in vitro by the method of MTT. Briefly, HepG2, A549, LoVo and Hela cells were seeded in 96-well flat bottom plates at a density of about 1 × 104 cells/well, respectively. After incubating 12–18 h, 20 μL of compounds 1–3 were added into each well at a final concentration of 1, 5, 10, 25, 50, 100 and 200 μΜ. All the cells in the plates were incubated for another 48 h respectively. Subsequently, cell lines were incubated with MTT at the concentration of 0.5 mg/mL for 4 h, and then the cells were re-suspended in 150 μL of Dimethyl sulfoxide (DMSO). Inhibitory concentrations of compounds were calculated and half maximal inhibitory concentrations (IC50) values were confirmed. 5-Fluorouracil and dimethyl sulfoxide (DMSO, 0.1%, v/v) were used as positive control and negative control, respectively.
A mount of chemical constituents have been isolated and identified from Radix Glycyrrhizae. Triterpenoids including glycyrrhizic acid, glycyrrhetinic acid and flavonoids including isoliquiritigenin, liquiritigenin, isoliquiritin, Licochalcone A, B and E are considered as the main characteristic constituents of the herb. And the anticancer bioactivities of these characteristic compounds were assayed frequently. Compared to triterpenoids, flavonoids possessed stronger anticancer bioactivities. In this study, three dihydroisocoumarins (1–3) showed the less toxicities on A549 and HepG2 cell lines than that of flavonoids constituents reported previously. IC50 value of isoliquiritigenin, licochalcone A and E on A549 cell lines were 18.5, 14.3 and 17.3 μM, respectively. Licochalcone A possessed almost the same toxicity on HepG2 cell lines (IC50, 10 μM). But the dihydroisocoumarins (1–3) showed more toxicity on A549 cell lines than that of Glycyrrhizic acid . Besides anticancer activity, dihydroisocoumarin and its derivatives also exhibited anti-inflammatory and anti-bacterial effects [8, 9]. And these bioactivities assay were also the aim of our research in the future project.
ZY and LHW conceived and designed the experiments; ZSS and WJ performed the experiments; ZSS, YXJ and ZZZ analyzed the data; ZSS and YXJ wrote the paper; ZY and LHWmodified the paper. All authors read and approved the final manuscript.
This work was financially supported by Young innovative talent training plan of College in Heilongjiang Province (UNPYSCT-2016182) and Beijing CSCO clinical cancer research foundation (201510784).
The authors declare that they have no competing interests.
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- Liu YY, Liu CS, Zeng BF, Fan BD, Li PS, Xu TH et al (2013) Research progress on germplasm resources of Glycyrrhizae Radix et Rhizoma. Zhongcaoyao 44:3593–3598Google Scholar
- Rauchensteiner F, Matsumura Y, Ya MY, Yamaji S, Tani T (2005) Analysis and comparison of Radix Glycyrrhizae(licorice) from Europe and China by capillary-zoneelectrophoresis (CZE). J Pharm Biomed Anal 38:594–600View ArticleGoogle Scholar
- Lin ZJ, Qiu SX, Wufuer A, Shum L (2005) Simultaneous determination of glycyrrhizin, a marker component in Radix Glycyrrhizae, and its major metabolite glycyrrhetic acid in human plasma by LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 814:201–207View ArticleGoogle Scholar
- Shon YH, Lee HS, Kim CH, Lim JK, Jeon BH, Nam KS (2004) Effect of herbal medicines on cytokine-induced cytotoxicity and major histo compatibility complex (MHC) class II antigen expression in rat thyroid cells. Bio Pharm Bull 27:371–374View ArticleGoogle Scholar
- Kim SC, Byun SH, Yang CH, Kim CY, Kim JW, Kim SG (2004) Cytoprotective effects of Glycyrrhizae radix extract and its active component liquiritigenin against cadmium-induced toxicity (effects on bad translocation and cytochrome c-mediated PARP cleavage). Toxicology 197:239–251View ArticleGoogle Scholar
- Zheng W, Ji YB, Li WL, Dong J, Chen N, Yu XF et al (2017) A pair of new isocoumarin epimersfrom soil fungus Hypoxylon sp. J Asian Nat Prod Res 19:993–999View ArticleGoogle Scholar
- Tang ZH, Li T, Tong YG, Chen XJ, Chen XP, Wang YT et al (2015) A systematic review of the anticancer properties of compounds isolated from Licorice (Gancao). Planta Med 81:1670–1687View ArticleGoogle Scholar
- Li YF, Plitzko I, Zaugg J, Hering S, Hamburger M (2010) HPLC-based activity profiling for GABAA receptor modulators: a new dihydroisocoumarin from haloxylon scoparium. J Nat Prod 73:768–770View ArticleGoogle Scholar
- Kim DC, Quang TH, Ngan NTT, Yoon CS, Sohn JH, Yim JH et al (2015) Dihydroisocoumarin derivatives from marine-derived fungal isolates and their anti-inflammatory effects in lipopolysaccharide-induced BV2 microglia. J Nat Prod 78:2948–2955View ArticleGoogle Scholar