Abstract
To study the photosensitivity of furocoumarins to proteins, we choose imperatorin (Imp) and Cysteine (Cys), the most active amino acid in proteins, as the experimental subjects. We used UV light to irradiate Imp, Cys and their mixture separately and dedicated from the MS that Cys was weakly oxidized to Cystine, while strongly oxidized to dithiiodipropionic acid hydrate, di-(α-hydroxy-β-dithio sulfoxide) propionic acid, di-(α-keto-β-thithian sulfone) propionic acid and Cystine sulfone disodium salt. The characteristic absorption wavelength of Imp and the mixture were red shifted from 303 nm to 312 nm. This is due to the leaving of isopentenyl moiety at Imp and the lactone bond cleavage under UV light. HPLC analysis showed that two new substances were generated under UV irradiation, and the retention time was 6.3 and 23.4 min respectively. High-resolution MS analysis showed that UV light firstly induced hydrolysis of the lactone, removal of the isopentenyl group in Imp and deamination, dehydrogenation, oxidation of the Cys, and then Imp and its photochemical products chemically reacted with Cys. The product at 6.3 min was purified by a semi-preparative HPLC and successively analyzed by MS and NMR spectroscopy, identified that it was 8-hydroxypsoralen.
Introduction
Imperatorin (Imp), which naturally exists in the plants of Angelica dahurica, Cnidium monnieri (L.) Cuss. and Glehnia littoralis, belongs to the furancoumarins [1], which has antiallergic, [2–5] anti-tumor [6–8] and other effects, [9–11] is sensitive to light and is used for the treatment of vitiligo and other skin diseases, has been widely concerned [12, 13].
Recent studies on the Imp, besides extraction, separation and analysis, are mainly focused on its metabolism and interaction. Lv et al. [14] found that penicillium janthinellum AS 3.510 exhibited good capability to metabolise Imp to its new derivatives. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and the lactone ring-opening reaction of furocoumarin skeleton. Ten transformed products were isolated and purified, with their structures identified accurately, of which two were found anti-osteoporosis activity. A new method to track coumarin metabolites in vivo was developed. [15] And Song group [16] qualitatively and quantitatively investigated Imp metabolites in rat bile by using ultra high performance liquid chromatography coupled with quadrupole time flight spectrometry (UPLC-QTOF-MS) and 32 metabolites were identified. The results demonstrated that C5H8 could be easily eliminated from Imp forming the metabolite M1. It also indicated that Imp and M1 underwent extensive metabolic reactions including oxidation, hydrolysis, methylation, glucuronide conjugation, C2H5NO2S conjugation and C3H5NO2S conjugation. The analgesic effect of coumarins from Radix angelicae pubescentis on a spared nerve injury model of neuropathy was investigated, [17] proved that osthole and columbianadin has a significant activity that can be used for a variety of pain. The antilisterial potential of Imp was investigated [18] and it was found that Imp possessed potential antilisterial activity against L. monocytogenes American Type Culture Collection (ATCC 19116, 19111, 19166, 19118 and 15313) with minimum inhibitory concentration values ranging from 15.62 to 62.5μg/mL. Mao et al. [19] studied the expression of ICAM-1 mRNA and protein of parsnip webworm treated with Imp and found a significant reduction of 74% and 79.96%, respectively, because A cDNA encoding CYP6AB3 initially epoxidizes the carbon–carbon double bond on the isoprenyl side chain of Imp. A LC-MS/MS method was developed for the simultaneous determination of Imp and its metabolite, xanthotoxol, in rat plasma and urine samples. [20] The method was successfully validated and applied to determine the pharmacokinetics of Imp in rat plasma. The cytotoxicity of coumarin was also investigated [21]. The results were inconsistent in different animal species. Not all coumarins produce cell toxicity through epoxidation.
The recent studies have not yet explored the condition, products and mechanism of the UV-catalytic reaction of Imp and protein. All these questions greatly limit our understanding to this compound. Herein, in this paper, we chose cysteine (Cys) as the representative of protein for its high activity, [22] then radiated Imp, Cys, and their mixture by UV light separately to explore the photocatalytic reaction. And then separate, purify and characterize the products to provide basic data to explain the peeling phenomenon of Angelica -processing labors in daytime.
Experimental
Chemicals
Imp was purchased from National Institutes for Food and Drug Control (Beijing, China) (batch number: 110826-200511), Cys from Shanghai Kangda Amino Acid Factory(China), methyl alcohol(AR) from Chongqing Chuandong Chemical(group)Co., Ltd(China), methyl alcohol(HPLC) from Hubei Duke Chemical Technology Co., Ltd(China). Other reagents used in this study were of AR grade.
UV irradiation
Imp solution (0.05 mg/mL) was prepared by solving Imp in water-methanol solvent (1/1, vol/vol). Cys was prepared as 0.5 mmol/L aqueous solution. 10 mL Imp solution and 10 mL Cys solution were mixed together to get the mixture. Then, Imp solution, Cys solution and the mixture were placed in a sealed quartz colorimetric cuvette with a thickness of 1 cm and a distance of 50 cm to a UV light (Philips UV germicidal lamp, TUV30W, Guangzhou Baiminghui Electric Light Source Technology Co., Ltd. China) for irradiation for 12 h. After irradiation, the absorption spectrum was scanned with a T1901 UV-Vis spectrophotometer (Persee, Beijing, China). Comparison is 25W incandescent light bulb. The operations were all in dark.
Preparing the lyophilized powder of irradiation products of Imp and the mixture
After UV irradiation, Imp and the mixture were dried by rotary evaporator at 40°C, then resolved in methanol and filtered through a 0.22μm polyvinylidene difluoride (PVDF) membrane filter for semi-preparative HPLC. A Daisogel C18 column (250 mm×3 cm, 10μm) was used for chromatographic separation. The eluting procedure was performed as follows: linear change from MeOH:H2O (55:45, v/v) to (75:25, v/v) for 0–25 min. The flow rate was 20.0 mL/min, column temperature was 25 °C and detection wavelength was 300 nm.
The separations were collected and evaporated by rotary evaporator at 40°C, then lyophilized by freeze dryer at 40-50 Pa for 24 h to get powder for later use.
Preparing the irradiation product of Cys
After UV irradiation, Cys solution was dried by rotary evaporator and resolved in methanol, then manually spotted onto a thin layer chromatographic (TLC) plate (activated GF254 nm) using a glass capillary. Plate was developed with diethyl ether/petroleum ether (3:2, v/v) to 1 cm from the top in a normal chromatographic chamber previously saturated with eluent. After development, plate was dried, and the components were visualized under 245 nm UV and marked with pencil. Then retention factor was calculated. The bands of products were scratched and products were extracted three times with 50% methanol aqueous. Extract was dried by rotary evaporator.
HPLC analysis
The lyophilized powder of the mixture irradiation products was resolved in methanol. This solution and Imp solution with or without UV-irradiation were detected by HPLC, respectively. A Marchal C18 column (250 mm×4.6 mm, 10μm) was used for analysis. The flow rate was 1.0 ml/min and other conditions were the same as those used in semi-preparative HPLC.
UV-vis analysis
The lyophilized powder of the mixture irradiation products was resolved in methanol. This solution and Imp solution were scanned from 200 to 400 nm by a UV-vis spectrophotometer.
NMR analysis
The lyophilized powder of Imp-Cys irradiation products was resolved in deuterated chloroform and then detected by a 600 DD2 NMR spectrometer (Agilent, USA).
HRMS analysis
A Solari X Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (Bruker, Germany) was used for analysis.
Results and discussion
Imp irradiation
Imp is a kind of photosensitive furanocoumarins that can undergo photo-addition, photo-dimerization and photo-oxidation. Its structure is shown in Fig. 1.
Chemical structure of Imp.
It can be seen from the figure that the furancoumarin-O9 structure is composed of 6 double-bond conjugate system, which is the electron withdrawing group of C10, and the other electron absorber of C10 is 1’-isobutylene group, their common role rendered the electronic cloud density of O9-C10 low, become a nucleophilic bond.
Figure 2 is the UV-vis spectrum of Imp before and after UV irradiation. We can see that the characteristic absorption wavelength of Imp is 303 nm while red-shifting to 312 nm after UV irradiation, indicating that the Imp under UV irradiation underwent a chemical change, perhaps due to the loss of electron-withdrawing isopentenyl group of parent nucleus.
UV-vis spectrum of Imp.
As shown in Fig. 3, before UV irradiation, only one peak showed up at 21.74 min, which corresponded to that of Imp. While two new peaks showed up at 6.36 min and 23.45 min, respectively after irradiation. Their peak area accounted for 43.12% and 1.63% of the total peak area respectively. This proved that Imp can change into at least three new substances under UV light.
UV spectrogram of Imp before (a) and after (b) UV irradiation.
Figure 4 is the HRMS of Imp before UV irradiation. Imp gave [M+H]+ ion at m/z 271.10 (C16H15O4). It produced fragment ion at m/z 203.03 (C11H7O4) by losing 68 Da which was considered as a loss of isopentenyl moiety and ion at m/z 413.27 by adding isopentenyl dimer at its double bond in isopentenyl group. Ions at m/z 563.17, 612.26 and 683.55 could be tentatively identified as Imp dimer sodium salt, additional product of two Imp and one isopentenyl and dimer of Imp-isopentenyl respectively. This proved that the double bond of isopentenyl group in Imp is active.
HRMS of Imp before UV irradiation.
Figure 5 is the HRMS of Imp after UV irradiation. Ion at m/z 113.08, produced by hydrolyzing and hydrogen-addition, could be deduced as 4-hydroxy-2,4-diene-1-valeric acid or 1-hydroxymethyl-2-methyl-3-ene dihydrofuran. Ion at m/z 263.02 could be tentatively identified as the dihydrate polymer of the first two. Ion at m/z 339.23 could be deduced as the addition product of Imp and isopentenyl group. Ion at m/z 402.23 could be deduced as the dimer of 8-hydroxypsoralen formed by hydrogen bond. It can be seen that O9-C10 bond and benzene ring could be cleaved by high-energy bombardment, indicating that they all have higher chemical activity and can generate substances above. These substances may further generate new ones through chemical reactions.
HRMS of Imp after UV irradiation.
In mammals, Cys is supplied as a component of dietary proteins and is formed through the transsulfuration pathway from methionine [23, 24]. And as a protein amino acid and biothiol, it is a very important structural and functional moiety of various proteins. Cys plays an essential role in cellular balance [25].
Figure 6a is the HRMS of Cys before UV irradiation. It can be seen that Cys is mainly in the form of polymeric hydrates before UV irradiation. After 6 h UV irradiation (Fig. 6b), sulfhydryl hydrogen left and then Cys was oxidized to Cystine and its polymers. Figure 6c is the HRMS of Cys after 12 h UV irradiation, we inferred that Cys was deaminated, dehydrogenated and oxidized under UV irradiation, then further turned into dithiopropionate hydrate, bis-(α-hydroxy-β-dithionulfone) propionic acid, bis-(α-ketono-β-dithio sulfone) propionic acid and Cystine sulfone disodium salt.
HRMS of Cys after 0h (a), 6h (b) and 12h (c) UV irradiation.
Qualitative experimental results showed that after UV irradiation of Imp-Cys mixed solution, the color changed from colorless to transparent yellow, accompanied with pungent odor. This showed that the mixture had undergone chemical changes under UV irradiation.
Figure 7 shows the UV-vis spectrum of Imp-Cys mixture before and after UV irradiation. Before the UV irradiation, the mixture had absorption peaks at 250 nm and 303 nm. The former belonged to the conjugative system of coumarin while the latter belonged to the moiety at C8. After irradiation, the spectrum changed significantly, in which the peak at 250 nm weakened while the peak at 303 nm red-shifted to 312 nm. This was due to the change of lactone and isopentenyloxy moiety.
UV-vis spectrum of Imp- Cys mixturen before and after UV irradiation.
Figure 8 shows a HRMS of Imp - Cys mixture after UV irradiation. Ion at m/z 218.27 could be tentatively deduced as 8-methoxypsoralen due to the elimination of C4H6 at isopentenyloxy moiety. Cys could be oxidized to a sulphoxide (274.27) or hydrate (274.27) and sulphone (318.30). Ion at m/z 384.29 could be tentatively identified as a dihydrate. Compared with Fig. 7, there is a clear difference that two new substances at m/z 274.27 and 384.29 emerged. Obviously, this is due to deprotonation of Cys under UV irradiation, increases the activity of thiol to give electrons, attacking isopentenyloxy moiety at Imp.
HRMS of Imp and Cys after UV irradiation.
Figure 9 shows the HPLC spectrogram of Imp-Cys mixture before and after UV irradiation. After irradiation, two new peaks emerged at 6.37 and 23.47 min, and the peak area accounted for 45.53% and 2.34% respectively. The retention time of each substance was consistent with Fig. 3, and the proportion was also close. We hypothesized that Imp changed to three substances under UV catalysis, and further reaction occured.
HPLC spectrogram of Imp and Cys before (a) and after (b) UV irradiation.
Successively, we purified the substance at 6.37 min by semi-preparative HPLC, then MS and NMR were performed. The substance gave [M–H]–ions at m/z 201.134 (C11H5O4) and its NMR data were as follows,
1H-NMR(CDCl3, 600 MHz) δ: 7.80 (1H, d, J = 9.6 Hz, H-4), 7.72 (1H, d, J = 2.4 Hz, H-2’), 7.28 (1H, d, J = 2.4 Hz, H-3’), 6.82(1H, d, J = 9.6 Hz, H-5),556.39(1H, d, J = 9.6 Hz, H-3); 13C NMR (CDCl3, 151 MHz) δ: 160.0 (s, C-2), 147.1 (s, C-2’), 144.8 (s, C-7), 144.4 (s, C-4), 139.0 (s, C-9), 129.3 (s, C-8), 126.0 (s, C-5), 115.7 (s, C-10), 114.3 (s, C-6), 110.6 (s, C-3), 106.8 (s, C-3’), So we deduced the substance at 6.3 min was 8-hydroxypsoralen.
In this study, Cys, the most active amino acid in proteins, was chose to explore the mechanism of photocatalytic reaction of Imp and proteins. The results indicated that Imp and Cys could have series of reaction under UV, the most important of which was the break of isopentenyl moiety from Imp and as a consequence, a few kinds of Imp derivatives were produced. We also found that Imp and Imp, Imp and isopentenyl fragment, Imp derivative and Cys may have addition reactions respectively. Finally, we isolated the major product of photocatalytic reaction and identified it as 8-hydroxypsoralen.