Isolation and structure elucidation of antioxidant compounds from stem and root barks of Daphne giraldii

Abstract Two new compounds, aphegiractin A1/A2 (1a/1b), and seven known compounds were isolated by phytochemical work on EtOAc-soluble ingredients extracted from stem and root barks of Daphne giraldii. Their structures were established based on extensive spectroscopic methods, including HRESIMS, CD experiments, 1D and 2D NMR. All compounds were evaluated for their antioxidant activity to DPPH, ABTS radical scavenging activity and inhibitory activity on tyrosinase. Of these compounds, compound 3 exhibited significant antioxidant activities.


Introduction
Daphne giraldii, belonging to family Thymelaeaceae and genus Daphne Linn., is mainly distributed in Shanxi, Gansu, Sichuan and Qinghai provinces of China [1].In China, the root and stem bark of D. giraldii is one of the sources of traditional Chinese medicine "Zushima" [2], which has been used for a long history to treat many diseases, such as bruises, aches, traumatic injury, rheumatoid arthritis and bronchitis [3][4][5].The phytochemical studies on D. giraldii have isolated and identified various types of chemical compounds, mainly including coumarins, flavonoids, lignans, diterpenes and so on [6][7][8][9][10][11]. Coumarins are considered as the main active components of D. giraldii [12].Previous phytochemical studies have shown that 35 coumarins, including 21 mono-coumarins and 14 bis-coumarins, have been discovered from D. giraldii.Daphnetin (7,8-hydroxycoumarin) has been reported to possess antimicrobial and antioxidant activities, which is one of the main active components of Chinese patent medicine "Zushima tablet" [13].A present phytochemical investigation on the constituents of this species led to the isolation of one pair of coumarin enantiomers, daphegiractin A 1 /A 2 (Figure 1), as well as seven known compounds.The hydroxyl group at C-7 of daphegiractin A 1 /A 2 nucleus is condensed with phenyl group at C-6 to form a pyran ring linear pyranocoumarin.In this study, the antioxidant activities of those compounds were evaluated by in vitro tests including DPPH radical scavenging assay, ABTS radical scavenging assay and tyrosinase inhibition activity (Figure 1 and 2).

Results and discussion
Compound 1 was assigned with the molecular formula C 14 H 14 O 6 on the basis of its HRESIMS m/z 279.0850   ) indicated the connection of the 3 0 ,4 0 -dihydroxy-2 0 ,2 0 -dimethyldihydropyran ring with C-6, 7, thus establishing the planar structure of compound 1.The coupling constant of compound 1 (J 3 0 ,4 0 ¼ 8.5 Hz) demonstrated a trans configuration [14,15].Because of the weak CD Cotton effect, compound 1 was thought to be a racemic mixture.Compound 1 was subjected to chiral chromatography column to obtain 1a and 1b in a ratio of 1: 1.The absolute configurations of compounds 1a and 1b were established by comparing the calculated and experimental ECD curves.As illustrated in Figure 3, the absolute configurations of 1a and 1b were unambiguously assigned as 3'S, 4'R and 3'R, 4'S.Thus, the two novel compounds 1a and 1b were given the trivial names daphnegiractin A 1 and daphnegiractin A 2 , respectively.The structures of seven known compounds were identified as (-)-rutadetin (2) [16], (±)-kazinol U (3) [17], (±)-7,4 0 -dihydroxy-3 0 -methoxyflavan (4) [18], (2S)-7,3 0dihydroxy-4 0 -methoxyflavan (5) [19], (±)-7,4 0 -dihydroxyflavane (6) [20], (-)-simulanol (7) [21], (-)-lariciresinol (8) [22] by the interpretation of their NMR data and comparison with those reported in the literatures.Antioxidant activities of all compounds were evaluated by the DPPH, ABTS free radical scavenging tests tyrosinase inhibitory activity (see Figure 4).As shown in Figure 5, a positive correlation existed between the concentration of the compounds and antioxidant activities.As for the DPPH radical scavenging assay, the results showed that the IC 50 values of compounds 3, 5, and 8 (190.60,241.20 and 116.50 lM, respectively) were significantly lower than those of other tested compounds, which indicated that they showed potential antioxidant activities.The antioxidant activity of compound 1 (849.83lM) (a mixture of A1 and A2) did not give significant antioxidant activity.In the ABTS þ radical scavenging assay, compounds 3-8 showed potential ABTS þ scavenging activity but all of these compounds showed lower scavenging activity than ascorbic acid.In addition, compounds 3-5 were evaluated for their inhibitory activities against tyrosinase at the concentrations of 12.5, 25, 50, 100, and 200 lM.As shown in Table 2, compound 3 showed the most  potential activity with the IC 50 value of 15.39 lM compared with positive control arbutin (165.39 lM).According to the results, it be inferred that the antioxidant abilities of the stem and root bark of D. giraldii may be closely attributed to the contents of the flavone with orthophenolic hydroxyl group.

Plant material
The stem and root barks of Daphne giraldii were purchased from Anguo in Hebei province.The plant was identified by Professor Jiuzhi Yuan, School of traditional Chinese medicine, Shenyang Pharmaceutical University.A voucher specimen (Accession number: Dgenk-2016-8) was deposited in the herbarium of Shenyang Pharmaceutical University.

Extraction and isolation
The air-dried stem and root barks of D. giraldii (30.0 kg) were extracted with 90% EtOH cold soak for two hours and then extracted by heat reflux with 75% EtOH for two times.3.4.Anti-oxidant activity assay 3.4.1.1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH) scavenging assay This assay was carried out according to the procedure of Andonova et al. [23] with some modifications.100 ll different concentrations of sample (12.5-200 lM) and 100 ll DPPH solution were added to 96-well microplate.Instead of a sample, a 1& DMSO solution was used as a blank control.The reaction mixture was incubated at 37 C for 30 minutes, and the absorbance of the solution was detected at 517 nm.The experiment was conducted in triplicate.Ascorbic acid was used as positive control.The activity to scavenging the DPPH was calculated by the following formula: DPPH scavenging activity (%) ¼ [(A 1 -A 0 )]/A 0 Ã 100%, where A 0 is the absorbance of the blank, A 1 is the absorbance of all samples or ascorbic acid.
3.4.2.2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid) (ABTS þ ) scavenging activity The scavenging activity to ABTS þ was determined according to the method of Liu et al. [24].Working mother liquor of ABTS þ : 7 mmol L À1 ABTS solution and 2.45 mmol L À1 potassium persulfate solution was mixed in equal volume and reacted in the dark for 12-16 h at room temperature.Then, the ABTS working solution was diluted 30-40 times with anhydrous ethyl alcohol.100 ll different concentrations sample (12.5-200 lM) and 100 ll ABTS solution were added to 96-well microplate.Instead of a sample, a 1& DMSO solution was used as a blank control.Ascorbic acid was taken as positive control.The absorbance was measured at 734 nm.Tests were carried out in triplicate.The activity to scavenging the ABTS þ was calculated by the following equation: ABTS þ scavenging activity (%) ¼ [(A 1 -A 0 )]/A 0 Â100%, where A 0 is the absorbance of the blank, A 1 is the absorbance of all samples in the stable state when mixed with reaction solution.

The assay of inhibitory activity to tyrosinase
The assay of inhibitory activity to tyrosinase was conducted according the method reported by Fan et al. [25] with some modification.In the experiment, 2 mmolÁL À1 Ltyrosine was used as substrate.The total reaction system was 200 ll.In sequence, 50 ll of tyrosinase solution, 50 ll or 100 ll of phosphate-buffered saline (PBS, 2.5 mmolÁL À1 ), 50 ll of tyrosinase solution (2000 U/ml) and 50 ll of test solution were added into a 96-well plate.Instead of a sample, a 1& DMSO solution was used as a blank control.In this system, the concentration gradient of test solution (including positive control arbutin) is 12.5, 25, 50, 100 and 200 lM.The solution in the testing tubes was shaken vigorously and incubated in the dark for 30 min at 37 C.Then, the absorbance of mixture was measured at 475 nm.The activity was calculated as the formula: inhibition (%) ¼ [(A-B)-(C-D)]/(A-B) Ã 100%, where A is the absorbance after incubation with the blank solution, B is the absorbance before incubation with the blank solution, C is the absorbance after incubation with the sample solution, and D is the absorbance before incubation with the sample solution.

Figure 4 .
Figure 4. Antioxidant activity of the compounds from stem and root barks of D. giraldii: (a) Scavenging activity to DPPH; (b) Scavenging activity to ABTS þ.

Table 2 .
Samples with antioxidant activities.