Dihydroisocoumarins from Radix Glycyrrhizae

Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s13065-018-0427-0) contains supplementary material, which is available to authorized users.


Background
Radix Glycyrrhizae is the rhizome of Glycyrrhiza inflata Bat., Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L. They are widely distributed in the northwest and northeast of China [1]. The pharmacological activities of Radix Glycyrrhizae are mainly represented by the main triterpene saponins, glycyrrhizin, glycyrrhizic acid, glycyrrhizinic acid and its aglycone, glycyrrhetinic acid [2,3]. Its root possesses wide broad pharmacological actions. According to literature reports, its pharmacological activities include the following aspects: effects on central nerve system; cardiovascular system and endocrine system; liver, renal and pancreas functions, anti-ulcer action, anticancer action, anti-allergic and anti-inflammatory effects, anti-virus and antibacteria activities, and effect on immune function and so on [4,5]. In this paper, we describe the isolation and the structural elucidation of three new dihydroisocoumarins obtained from the 70% ethyl alcohol (EtOH) extract of Radix Glycyrrhizae. Their structures ( Fig. 1) were established by extensive spectroscopic data analysis and comparison with those of literature values. Heteronuclear Multiple-Bond Correlation (HMBC) spectrum ( Fig. 2) showed the long-rang correlations between H-7 at δ H 6.99and C-8a at δ C 108.2, C-5 at δ C 124.7, C-8 at δ C 161.1; between H-6 at δ H 7.64 and C-10 at δ C 62.2, C-8a at δ C 108.2, C-4a at δ C 141.3; between H-10 at δ H 5.09/5.17 and C-5 at δ C 124.7, C-4a at δ C 141.3, C-11 at δ C 170.8, between H-9 at δ H 1.45 and C-4 at δ C 62.3, between H-4 at δ H 4.66 and C-3 at δ C 78.5, C-4a at δ C 141.3, C-5 at δ C 124.7, between H-4 at δ H 4.66 and C-5 at δ C 124.7, which led to a conclusion that the planar structure of compound 1 was similar to that of (3R, 4R)-4,8-dihydroxy-5-(hydroxymethyl)-3-methylisochroman-1-one [6],   except for the acetylated of hydroxyl groups at C-10. Thus, the planar structure of 1 was determined as shown in Fig. 1.

Results and discussion
Compound 2 was also obtained as yellow crystal. The molecular formula was determined to be C 13 H 14 O 6 by HR-ESI-MS at m/z 289.0670 [M + Na] + . The 1 H and 13 C NMR signals of 2 were almost identical to those of 1 with slight difference at C-1, C-3, C-4, C-5, and C-4a. The CD spectrum of 2 gave an exactly opposite absorption band at 250 nm compared with that of 1, and thus 2 was suggested to be the epimer of 1 at C-3. HMBC correlations of 2 shown in Fig. 3 verified the planar structure of 2, which was the same as that of 1. The relative configurations of 1 and 2 were established by NOESY analysis (Fig. 3). For compound 2, NOESY cross-peak between active proton of C-4 and H-3 was given while for compound 1, NOESY cross-peak between active proton of C-4 and 9-CH 3 was observed, indicating the axial orientation of the active proton of C-4 as C-4 active proton could only give one NOESY cross-peak with either H-3 or 9-CH 3 .
The ECD (Electronic Circular Dichroism Spectroscopy) calculating study of 1 and 2 was performed based on the relative configuration of 1 and 2. Having two chiral centers, there are four possible stereo-isomers for 4,8-dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate as shown in Fig. 4. The ECD results of each possible isomer and the experimental CD (Circular Dichroism Spectroscopy) curves of 1 and 2 were also expressed in Fig. 4a, b. The ECD results were represented in shot dashed line in Fig. 4a, d that both gave negative cotton effect at 250 nm, and so did Fig. 4b, c that both exhibited positive cotton effect at 250 nm, indicating that C-4 orientation dominated the cotton effect around 250 nm. Thus, via comparing the ECD results with those of the experimental CD curves of 1 and 2, the absolute configurations of C-3 and C-4 were determined to be (R), (S) and (R), (R) for 1 and 2, respectively.
Compound 3 was obtained as yellow crystal. The molecular formula was determined to be C 11 H 12 O 4 by HR-ESI-MS at m/z 231.0637 [M + Na] + . The 1 H and 13 C NMR spectral data of 3 were similar to those of 2, expect for the disappearance of an acetoxy group at C-10. The absolute configuration of 3 was established by the analysis of its CD spectrum. A positive Cotton effect at 250 nm was shown in the CD spectrum (Fig. 5) of 3, indicating the (3R,4R)-configuration same as 2.
The cytotoxic activities of compounds 1-3 were evaluated using the HepG2, A549, LoVo and Hela cell lines, respectively. The IC 50 values of these compounds were shown in Table 2. As a result, all the compounds exhibited moderate or less cytotoxic activity in vitro.

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

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 H 2 O and successively partitioned with petroleum ether, dichloromethane (CH 2 Cl 2 ), 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).

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 × 10 4 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 (IC 50 ) values were confirmed. 5-Fluorouracil and dimethyl sulfoxide (DMSO, 0.1%, v/v) were used as positive control and negative control, respectively.

Conclusion
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. IC 50 value of isoliquiritigenin, licochalcone A and E on A549 cell lines were 18.5, 14.3 and 17.3 μM, respectively. Licochalcone