Abstract
Angelica dahurica is a medicinal herb of the Umbelliferae family. The dried root of A. dahurica, also known as Angelicae dahuricae Radix, is widely used in clinical treatment. However, the aboveground part of A. dahurica which accounted for over 70% of the total plant was abandoned in the field. In order to develop the value of the aboveground part of A. dahurica, the chemical constituents and arginine kinase (AK) inhibitory activity of A. dahurica leaves were studied. 85 volatile components were identified from A. dahurica leaves by GC-MS; 39 non-volatile components including sugars, amino acids and organic acids were identified by pre-column derivatization GC-MS analysis; and 7 coumarins were qualitatively and quantitatively analyzed by HPLC. Then, an inhibitory enzyme-linked immunosorbent assay (iEIA) was applied for evaluation of AK inhibitory activity. The extracts of A. dahurica leaves exhibited well inhibitory effects on AK. Further, potential AK inhibitors were screened by grey relational analysis and their inhibitory activities were validated by iEIA. l-aspartic acid exhibited strongest inhibitory effect on AK with its IC50 value was 0.558 mM, which was much lower than that of chlorpheniramine (6.644 mM). The obtained chemical profiles displayed chemical diversity of A. dahurica leaves and will provide data support for the future development and utilization of A. dahurica leaves. The screened potential AK inhibitors from A. dahurica leaves could be candidates for development of antiallergic substances or insecticides.
Keywords: Angelica dahurica leaves, Chemical composition, Arginine kinase, Correlation analysis
1. Introduction
Angelica dahurica is a perennial herb of the Umbelliferae family. The dried root of A. dahurica which is known as Angelicae dahuricae Radix or “baizhi”, is a commonly used Traditional Chinese Medicine for expelling wind and cold, removing dampness, arresting leukorrhea, eliminating swelling, draining pus and relieving pain [1]. Angelicae dahuricae Radix could also be used as spice to give dishes a unique flavor. The clinical and industry demand of Angelicae dahuricae Radix is great, especially in China, Japan, Korea and other Asian counties and regions. However, the aboveground part of A. dahurica which accounts for over 70% of the total plant is discarded in the field. Value exploration of the aboveground part of A. dahurica will be friendly to the environment and resource utilization.
As early as about 1500 years ago, it was recorded in Annotation of Materia Medica (Ben Cao Jing Ji Zhu, 本草经集注) that A. dahurica leaves could be used in medicated bath for treatment of erysipelas and urticaria [2]. The records about application of A. dahurica leaves in bath continued until Ming Dynasty but rarely mentioned since the Qing Dynasty. Though A. dahurica leaves has not been collected in pharmacopoeia, it was reported that mashed A. dahurica leaves were effective for treatment of bee stings [3]. The historic and modern records about applications of A. dahurica leaves indicated its potential developmental values.
Arginine kinase (AK) catalyzes the reversible transphosphorylation between ATP and arginine. The generated phosphoarginine containing high-energy phosphate could serve as energy reserve. AK plays critical role in energy metabolism in invertebrates [4]. The expression of AK was reported to be closely related with flight activities, host identification, growth and development in insects. Evidences indicated that knocking down AK led to retarded development and increased mortality in insects [[5], [6], [7], [8]]. AK was also reported to be one of the major allergens of shrimps and crabs [9,10]. Furthermore, AK exists only in invertebrates and shared low homology with creatine kinase, which plays the same role in vertebrates, indicating it is a suitable potential target for development of insecticides [11] and anti-allergic medicines.
To develop potential values of A. dahurica leaves, an integrated chemical and bioactivity research strategy was applied. Firstly, GC-MS and HPLC were applied for chemical profiling. Then, the inhibitory capacities of A. dahurica leaves extracts on AK were evaluated by an inhibitory enzyme-linked immunosorbent assay (iEIA). Grey relational analysis was further applied for the screening of potential AK inhibitors. The inhibitory activities of the selected potential AK inhibitors were validated by the iEIA. The systematic chemical profiling of A. dahurica leaves provides foundation for its further value development. The screened potential AK inhibitors could be candidates for development of pesticide and antiallergic drugs which would be safer to mammals.
2. Materials
2.1. Chemicals and reagents
HPLC-grade methanol, acetonitrile and formic acid were from Fisher Scientific (Pittsburgh, PA, USA). n-Hexane (purity ≥98%) and anhydrous pyridine (purity ≥99.8%) were purchased from Aladdin biochemical Technology Co. Ltd (Shanghai, China). Salicylic acid (purity ≥99.5%) was from Ron Reagent Co. Ltd (Shanghai, China). Methoxyamine hydrochloride (purity ≥97.5%) and N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, purity ≥98.5%) were obtained from Sigma Aldrich Company (USA). Xanthotoxol, oxypeucedanin hydrate, byakangelicin, oxypeucedanin, imperatorin, phellopterin and isoimperatorin with their purities over 98% were purchased from Chengdu Push Bio-Technology Co. Ltd (Chengdu, China). l-aspartic acid, lactic acid and citric acid with their purities over 98% were purchased from Beijing Solarbio Science & Technology Co.,Ltd. (Beijing, China). Malic acid (purity ≥99.5%), shikimic acid and galactose (purity ≥98%) were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). AK enzyme-linked immunosorbent assay kit was purchased from Jiangsu Enzyme-linked Biotechnology Co., Ltd. (Jiangsu, China).
2.2. Plant materials
Five batches of fresh mature A. dahurica leaves (A、B、C、D、E) were collected from Qizhou Campus of Hebei University of Chinese Medicine and dried in shade. The voucher specimens were identified by Professor Yuguang Zheng and deposited in the Traditional Chinese Medicine Processing Technology Innovation Centre of Hebei Province with their specimen numbers were from 202210AD01 to 202210AD05.
3. Methods
3.1. Analysis of the volatile metabolites by GC-MS
3.1.1. Sample pretreatment
1 g of fully ground A. dahurica leaves powder was mixed with 1 mL n-hexane. After ultrasonication (300 W, 40 kHz) 15 min at room temperature, the extraction mixture was centrifuged at 13000 rpm at room temperature for 10 min. 100 μL of the supernatants was transferred to clean vials for GC-MS analysis.
3.1.2. Instrument parameters
The headspace injection conditions were as follows: heater temperature was set at 150 °C; quantitative ring temperature was 160 °C; transmission line temperature was 170 °C; sample bottle equilibrium time was 15 min; continuous injection time was 0.5 min.
The GC-MS analysis was performed with an Agilent 7890B5977B GC-MS (Agilent, CA, USA) coupled with a HP-5MS capillary column (30 m × 0.25 mM, 0.25 μm, Agilent, CA, USA). Helium (≥99.999%) was used as carrier gas. Evaporated samples were injected in split-mode with the split ratio set to 5:1 at a temperature of 250 °C. The oven temperature program was initially set at 45 °C, then increased to 170 °C at a rate of 4 °C/min. The electronic ionization voltage of electron-impact (EI) ion source was 70 eV. The interface temperature was 250 °C, the ion source temperature was 230 °C and the quadrupole temperature was 150 °C. The mass spectrometer was operated in full scan mode with a scanning range of 50–500 m/z. Solvent delay time was set as 2 min.
3.2. Analysis of non-volatile metabolites by pre-column derivatization GC-MS
3.2.1. Sample pretreatment
Extraction and derivatization of non-volatile metabolites were performed according to a reported protocol [12].
3.2.2. Instrument parameters
The headspace injection conditions are as follows: heater temperature was 110 °C; quantitative ring temperature was 120 °C; transmission line temperature was 130 °C; sample bottle equilibrium time was 10 min; continuous injection time was 1 min.
Aforementioned GC-MS instrument and column (see 2.1.2) was also applied for analysis of derivatized samples. 5 μL of the derivatized sample was evaporated in a 20 mL vial and injected using 5:1 split-mode at a temperature of 250 °C. The temperature gradient program and other parameters were set the same as in Ref. [12].
3.3. Analysis of the coumarins by HPLC
3.3.1. Preparation of samples and standard solutions
Standard solutions of xanthotoxol (0.05 μg/mL, 0.17 μg/mL, 0.425 μg/mL, 0.85 μg/mL, 4.25 μg/mL, 8.5 μg/mL, 17 μg/mL, 85 μg/mL, 0.85 mg/mL), oxypeucedanin hydrate (0.05 μg/mL, 0.10 μg/mL, 1.4375 μg/mL, 2.875 μg/mL, 14.375 μg/mL, 28.75 μg/mL, 57.5 μg/mL, 115 μg/mL, 1.15 mg/mL), byakangelicin (0.08 μg/mL, 0.24 μg/mL, 0.6 μg/mL, 3 μg/mL, 6 μg/mL, 30 μg/mL, 60 μg/mL, 120 μg/mL, 1.2 mg/mL), oxypeucedanin (0.06 μg/mL, 0.18 μg/mL, 2 μg/mL, 4 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, 400 μg/mL, 4 mg/mL), imperatorin (0.09 μg/mL, 0.28 μg/mL, 0.55 μg/mL, 1.1 μg/mL, 5.5 μg/mL, 11 μg/mL, 22 μg/mL, 220 μg/mL, 2.20 mg/mL), phellopterin (0.13 μg/mL, 0.32 μg/mL, 1.275 μg/mL, 2.55 μg/mL, 12.75 μg/mL, 25.5 μg/mL, 51 μg/mL, 255 μg/mL, 2.55 mg/mL) and isoimperatorin (0.07 μg/mL, 0.16 μg/mL, 0.325 μg/mL, 0.65 μg/mL, 3.25 μg/mL, 6.5 μg/mL, 13 μg/mL, 260 μg/mL, 2.6 mg/mL) were prepared by gradient dilution with 80% (v/v) methanol.
50 mg of each pulverized samples were extracted exhaustively with 1 mL of 80% (v/v) methanol by ultrasonication (40 kHz, 300 W) 50 min at room temperature. After extraction, the mixed suspension was centrifuged 10 min. The supernatant was filtered through a 0.22 μm nylon filter membrane for HPLC analysis.
3.3.2. Instrument parameters
Chromatographic separation was conducted on an Agilent ZORBAX SB C18 column (4.6 × 50 mm, 1.8 μm). The mobile phases were consisted of ultrapure water (A) and acetonitrile (B). The HPLC chromatographic condition was as follows: 0–1 min, 18% B; 1–15 min, 18%–35% B; 15–18 min, 35%–60% B; 18–25 min, 60%–85% B. The flow rate was maintained at 0.5 mL/min. 5 μL of the prepared samples was injected into the column. The column temperature was maintained at 25 °C and the ultraviolet detection wavelength was set at 310 nm.
3.4. Inhibition ELISA for evaluating AK inhibition activity
200 μL extracts of A. dahurica leaves that obtained under the condition of “3.2.1” were dried using a SpeedVac (Thermo Scientific, Inc., Bremen, Germany) at 5000 rpm and 40 °C for 180 min and then redissolved in 100 μL dimethyl sulfoxide (DMSO).
50 μL AK (20 U/L) was mixed with 5 μL the redissolved A. dahurica leaves extracts or hexane extracts from 3.1.1 or candidate compounds at various concentrations. After incubation at 37 °C for 10 min, 50 μL of the mixture was added to a 96-well immunoplate (Jianglanchun, Jiangsu, China) which was coated with purified crab AK antibody. The plate was then incubated at 37 °C for 30 min. After incubation, the mixtures were discarded and washed 5 times. Subsequently, HRP-labeled AK antibody was added and the mixtures were incubated for 30 min at 37 °C. After washing completely, the tetramethyl benzidine (TMB) substrate solution was added and the plate was incubated for another 15 min at room temperature in dark. Color development was stopped by adding 50 μL stopping solution. The luminescence intensity of each well was measured within 15 min using a microplate reader (Victor Nivo multiplate reader, PerkinElmer, USA) at 450 nm wavelength. In the blank groups, neither extract-AK mixture nor the HRP-labeled AK antibody was added. In the negative and positive groups, 5 μL of solvents (DMSO or hexane) or chlorpheniramine (2 mM) instead of A. dahurica leaves extracts or candidate compounds were applied and measured under the same condition. The inhibition rate was calculated as follows:
| Inhibition rate (%) = (AS-AB)/(AN-AB) × 100% |
AS, AB, AN are the absorbances of sample wells, blank wells and negative control wells, respectively.
3.5. Data processing and multivariate statistical analysis
For qualitative analysis, the metabolites detected by GC-MS with a similarity more than 80% to the NIST17 standard library were identified using the Agilent MassHunter analysis program (Agilent, Santa Clara, CA, USA). The coumarin components of A. dahurica leaves were qualitatively and quantitatively analyzed by comparing the HPLC chromatograms of A. dahurica leaves with reference substances mixture. SPSS 24.0 statistical software was used for grey relational analysis.
4. Results
4.1. Characterization of volatile metabolites by GC-MS
The extracted volatile components of A. dahurica leaves were analyzed by GC-MS. A typical total ion chromatogram (TIC) shows in Fig. 1. Qualitative Navigator (B.08.00) software and the NIST17 standard mass spectrometry database were applied for compound qualification and relative quantification. A total of 85 volatile components were identified including alkanes, alkenes, aldehydes, alcohols, esters and others (Table 1). The identified volatile components accounted for 92.09% of the total peak area. Alkane accounted for 47.34% of the identified components; followed by alkenes, accounted for 15.39%; aldehyde, alcohols and esters accounted for 11.43%, 10.94% and 3.35%, respectively. The compounds with the highest relative contents were 2-hexenal, 2-cyclohexene-1-ol, farnesane, saffron, β-thujene and so on.
Fig. 1.
TIC of volatile metabolites in A. dahurica leaves by GC-MS; The number of peaks was consistent with those of compounds in Table 1.
Table 1.
Identification of volatile compounds analyzed by GC-MS.
| No. | t/min | Compounds | Molecular formula | Relative content/% | Class |
|---|---|---|---|---|---|
| 1 | 2.709 | 2,5-Dimethylhexane | C8H18 | 0.13 | Alkane |
| 2 | 3.104 | 3,5-Dimethylheptane | C9H20 | 0.36 | Alkane |
| 3 | 3.582 | 2-Cyclohexen-1-ol | C6H10O | 6.52 | Alcohol |
| 4 | 3.84 | 3,4,5-Trimethylheptane | C10H22 | 0.16 | Alkane |
| 5 | 3.962 | 2,3,4-Trimethylhexane | C9H20 | 2.38 | Alkane |
| 6 | 4.329 | 2,6-Dimethylhept-3-ene | C9H18 | 0.22 | Alkene |
| 7 | 4.447 | 2-Cyclohexen-1-ol | C6H10O | 0.74 | Alcohol |
| 8 | 4.58 | 2-Hexenal | C6H10O | 10.87 | Aldehyde |
| 9 | 4.667 | Trans-3-hexen-1-ol | C6H12O | 1.18 | Alcohol |
| 10 | 4.782 | 4-Methyloctane | C9H20 | 0.76 | Alkane |
| 11 | 5.218 | 3-Ethoxy-2-methylacrylaldehyde | C6H10O2 | 0.31 | Aldehyde |
| 12 | 5.951 | 2-Ethylfuran | C6H8O | 0.52 | Furan |
| 13 | 6.517 | β-Terpinene | C10H16 | 0.21 | Alkene |
| 14 | 7.369 | 4-Methylnonane | C10H22 | 0.55 | Alkane |
| 15 | 7.655 | Sabinene | C10H16 | 0.95 | Alkene |
| 16 | 8.196 | Myrcene | C10H16 | 0.47 | Alkene |
| 17 | 8.587 | β-Phellandrene | C10H16 | 0.38 | Alkene |
| 18 | 8.895 | 4,5-Dimethylnonane | C11H24 | 0.89 | Alkane |
| 19 | 9.072 | 2,5-Dimethylnonane | C11H24 | 0.48 | Alkane |
| 20 | 9.163 | 3,6-Dimethyloctane | C10H22 | 1.65 | Alkane |
| 21 | 9.371 | β-Thujene | C10H16 | 2.86 | Alkene |
| 22 | 9.694 | Ocimene | C10H16 | 1.13 | Alkene |
| 23 | 10.03 | 3-Carene | C10H16 | 3.26 | Alkene |
| 24 | 10.31 | 5-Propylnonane | C15H32 | 3.64 | Alkane |
| 25 | 10.406 | Carbonic acid, Bis(2-ethylhexyl) ester | C17H34O3 | 0.48 | Ester |
| 26 | 10.49 | 5-Propylnonane | C12H26 | 1.22 | Alkane |
| 27 | 11.04 | Trans-1,4-dimethylcyclooctane | C10H20 | 0.3 | Alkane |
| 28 | 11.724 | 1,2-Epoxy-9-decene | C10H18O | 1.09 | Alkene |
| 29 | 11.817 | 4,6-Dimethyldodecane | C14H30 | 1.21 | Alkane |
| 30 | 12.004 | 3-Ethyl-3-methylheptane | C10H22 | 0.41 | Alkane |
| 31 | 12.899 | (+)-Trans-limonene oxide | C10H16O | 0.13 | Alkene |
| 32 | 13.058 | (−)-Trans-limonene oxide | C10H16O | 0.2 | Alkene |
| 33 | 13.832 | Cyclohexyl-dimethoxy-methylsilane | C9H20O2Si | 2.28 | Alkane |
| 34 | 13.928 | 3,4-Dimethylundecane | C13H28 | 0.41 | Alkane |
| 35 | 14.217 | α-Pinene | C10H16 | 0.36 | Alkene |
| 36 | 14.326 | Isopulegol | C10H18O | 1.24 | Alcohol |
| 37 | 15.169 | 3,8-Dimethyldecane | C12H26 | 0.21 | Alkane |
| 38 | 15.324 | 3-Methyl-5-propylnonane | C13H28 | 0.56 | Alkane |
| 39 | 15.526 | 5-Methyl-5-propylnonane | C13H28 | 0.27 | Alkane |
| 40 | 15.644 | 3,6-Dimethylundecane | C13H28 | 0.91 | Alkane |
| 41 | 15.772 | 2,4-Dimethylbenzaldehyde | C9H10O | 0.25 | Aldehyde |
| 42 | 15.915 | 4-Methyldodecane | C13H28 | 0.72 | Alkane |
| 43 | 16.658 | 3,8-Dimethyldecane | C12H26 | 0.57 | Alkane |
| 44 | 16.841 | 3,8-Dimethylundecane | C13H28 | 0.47 | Alkane |
| 45 | 16.956 | Decyl heptyl ether | C17H36O | 0.33 | Ether |
| 46 | 17.024 | 4,6-Dimethyldodecane | C14H30 | 0.68 | Alkane |
| 47 | 17.161 | 5-Propyldecane | C13H28 | 0.25 | Alkane |
| 48 | 17.326 | 2,6,10-Trimethyltridecane | C16H34 | 1.79 | Alkane |
| 49 | 17.649 | 2-Bromododecane | C12H25Br | 0.19 | Alkane |
| 50 | 17.864 | 4,6-Dimethyldodecane | C14H30 | 3.32 | Alkane |
| 51 | 18.137 | 2,6,11-Trimethyldodecane | C15H32 | 0.79 | Alkane |
| 52 | 18.33 | 2,7,10-Trimethyldodecane | C15H32 | 0.7 | Alkane |
| 53 | 18.585 | 2-Ethyl-2-methyl-tridecanol | C16H34O | 0.75 | Alcohol |
| 54 | 18.728 | 2-Isopropyl-5-methyl-1-heptanol | C11H24O | 0.23 | Alcohol |
| 55 | 18.812 | 4-Ethylundecane | C13H28 | 0.46 | Alkane |
| 56 | 19.387 | 2,6,11-Trimethyldodecane | C15H32 | 1.21 | Alkane |
| 57 | 19.999 | 2,3,5-Trimethyldecane | C13H28 | 0.17 | Alkane |
| 58 | 20.612 | 2,9-Dimethylundecane | C13H28 | 0.23 | Alkane |
| 59 | 20.997 | α-Cubebene | C15H24 | 0.24 | Alkene |
| 60 | 21.526 | Helminthogermacrene | C15H24 | 0.26 | Alkene |
| 61 | 22.378 | Tetradecane | C14H30 | 3.15 | Alkane |
| 62 | 23.391 | Isocaryophyllene | C15H24 | 0.84 | Alkene |
| 63 | 23.68 | Pristane | C19H40 | 1.31 | Alkane |
| 64 | 23.761 | 5,5-Dibutylnonane | C17H36 | 0.5 | Alkane |
| 65 | 23.864 | 2,6,11-Trimethyldodecane | C15H32 | 0.44 | Alkane |
| 66 | 24.059 | 3,8-Dimethylundecane | C13H28 | 0.3 | Alkane |
| 67 | 24.286 | β-Cubebene | C15H24 | 2.19 | Alkene |
| 68 | 24.46 | Phytane | C20H42 | 0.71 | Alkane |
| 69 | 24.7 | Crocetane | C20H42 | 3.1 | Alkane |
| 70 | 24.986 | 7-Methylhexadecane | C17H36 | 0.18 | Alkane |
| 71 | 25.318 | 2,4-Di-tert-butylphenol | C14H22O | 2.79 | Phenol |
| 72 | 25.555 | β-Cedrene | C15H24 | 0.6 | Alkene |
| 73 | 26.04 | Hexadecane | C16H34 | 1.01 | Alkane |
| 74 | 26.338 | Sulfurous acid, 2-ethylhexyl undecyl ester | C19H40O3S | 0.34 | Ester |
| 75 | 26.705 | 5,5-Dibutylnonane | C17H36 | 0.17 | Alkane |
| 76 | 26.919 | 3-Isopropyl-6,10-dimethylundecane-2-ol | C16H34O | 0.28 | Alcohol |
| 77 | 27.973 | Sulfurous acid, decyl 2-ethylhexyl ester | C18H38O3 | 0.89 | Ester |
| 78 | 28.738 | 3-Ethyl-2,6,10-trimethylundecane | C16H34 | 0.8 | Alkane |
| 79 | 29.139 | Sulfurous acid, 2-ethylhexyl octadecyl ester | C26H54O3S | 0.85 | Ester |
| 80 | 29.419 | 9-Methylheptadecane | C18H38 | 0.68 | Alkane |
| 81 | 29.82 | Sulfurous acid, butyl heptadecyl ester | C21H44O3S | 0.19 | Ester |
| 82 | 29.972 | 8-Methylheptadecane | C18H38 | 0.37 | Alkane |
| 83 | 30.165 | Sulfurous acid, 2-ethylhexyl Tetradecyl ester | C22H46O3S | 0.6 | Ester |
| 84 | 30.466 | 3-Methyltetradecane | C15H32 | 1.29 | Alkane |
| 85 | 30.793 | Heptadecane | C17H36 | 3 | Alkane |
4.2. Characterization of non-volatile metabolites by pre-column derivatization GC-MS
The A. dahurica leaf extracts were derivatized according to the experimental conditions of "2.2.1″ and then analyzed by GC-MS. A TIC shows in Fig. 2. The data were analyzed by Qualitative Navigator (B.08.00) software and compared with the NIST17 standard mass spectrometry database.
Fig. 2.
TIC of nonvolatile metabolites in A. dahurica leaves by GC-MS after derivatization. The number of peaks was consistent with those of compounds in Table 2.
A total of 39 derivative components were identified including sugars, organic acids, amino acids and alcohols (Table 2). The identified components accounted for 97.18% of the total peak area. Sugars accounted for 44.75% of the total peak area of all the identified components. Organic acids, alcohols, and amino acids accounted for 32.23%, 9.00%, and 1.83%, respectively. The relative contents of malic acid, d-glucose, d-fructose, d-galactose, myo-inositol were highest in A. dahurica leaves.
Table 2.
Identification of non-volatile metabolites analyzed by pre-column derivatization combining with GC-MS.
| No. | t/min | Compounds | Molecular Formula | Relative content/% | Class |
|---|---|---|---|---|---|
| 1 | 3.691 | Lactic acid, 2TMS | C9H22O3Si2 | 2.21% | Organic acid |
| 2 | 4.155 | l-Alanine, 2TMS | C9H23NO2Si2 | 0.29% | Amino acid |
| 3 | 4.593 | Oxalic acid, 2TMS | C8H18O4Si2 | 0.46% | Organic acid |
| 4 | 4.658 | l-Proline, 2TMS | C11H25NO2Si2 | 0.14% | Amino acid |
| 5 | 5.512 | l-Valine, 2TMS | C11H27NO2Si2 | 0.08% | Amino acid |
| 6 | 5.642 | Pinacol, 2TMS | C12H30O2Si2 | 0.01% | Alcohol |
| 7 | 6.154 | Ethanolamine, 3TMS | C11H31NOSi3 | 0.20% | Amine |
| 8 | 6.219 | l-Leucine, 2TMS | C12H29NO2Si2 | 0.07% | Amino acid |
| 9 | 6.258 | Glycerol, 3TMS | C12H32O3Si3 | 0.66% | Alcohol |
| 10 | 6.288 | 1-Hexadecanol, TMS | C19H42OSi | 1.21% | Alcohol |
| 11 | 6.613 | 2-Butenedioic acid, (Z)-, 2TMS | C10H20O4Si2 | 1.10% | Organic acid |
| 12 | 6.704 | Glycine, 3TMS | C11H29NO2Si3 | 0.50% | Amino acid |
| 13 | 7.003 | Glyceric acid, 3TMS | C12H30O4Si3 | 0.26% | Organic acid |
| 14 | 7.107 | 2-Butenedioic acid, (E)-, 2TMS | C10H20O4Si2 | 0.14% | Organic acid |
| 15 | 7.368 | l-Serine, 3TMS | C12H31NO3Si3 | 0.04% | Amino acid |
| 16 | 7.45 | Lavandulol, TMS | C13H26OSi | 0.36% | Alcohol |
| 17 | 7.623 | Arachidonic acid, TMS | C23H40O2Si | 0.13% | Organic acid |
| 18 | 7.714 | l-Threonine, 3TMS | C13H33NO3Si3 | 0.14% | Amino acid |
| 19 | 7.814 | (−)-Myrtenol, TMS | C13H24OSi | 0.05% | Alcohol |
| 20 | 8.976 | Malic acid, 3TMS | C13H30O5Si3 | 24.46% | Organic acid |
| 21 | 9.214 | Salicylic acid, 2TMS | C13H22O3Si2 | 4.26% | Internal standard |
| 22 | 9.353 | l-Aspartic acid, 3TMS | C13H31NO4Si3 | 0.17% | Amino acid |
| 23 | 9.427 | 4-Aminobutanoic acid, 3TMS | C13H33NO2Si3 | 0.41% | Amino acid |
| 24 | 11.373 | Xylose, 4TMS | C17H42O5Si4 | 0.12% | Sugar |
| 25 | 11.499 | Arabinitol, 5TMS | C20H52O5Si5 | 0.06% | Sugar Alcohol |
| 26 | 11.759 | D-Threitol, 4TMS | C16H42O4Si4 | 0.04% | Sugar Alcohol |
| 27 | 11.816 | Ribitol, 5TMS | C20H52O5Si5 | 0.08% | Sugar Alcohol |
| 28 | 12.006 | Erythritol, 4TMS | C16H42O4Si4 | 0.21% | Sugar Alcohol |
| 29 | 12.128 | d-Gluconic acid, 6TMS | C24H60O7Si6 | 0.05% | Organic acid |
| 30 | 12.631 | Shikimic acid, 4TMS | C19H42O5Si4 | 0.06% | Organic acid |
| 31 | 12.791 | Citric acid, 4TMS | C18H40O7Si4 | 1.20% | Organic acid |
| 32 | 13.333 | Quininic acid,5TMS | C22H52O6Si5 | 2.17% | Organic acid |
| 33 | 13.498 | d-Fructose, 5TMS | C21H52O6Si5 | 13.35% | Sugar |
| 34 | 13.615 | d-Galactose,5TMS | C21H52O6Si5 | 10.84% | Sugar |
| 35 | 13.806 | d-Glucose, 5TMS | C24H62O6Si6 | 20.35% | Sugar |
| 36 | 14.014 | Galactose oxime, 6TMS | C24H61NO6Si6 | 4.43% | Oxime |
| 37 | 14.742 | l-Rhamnose, 4TMS | C18H44O5Si4 | 0.10% | Sugar |
| 38 | 16.09 | Myo-Inositol, 6TMS | C24H60O6Si6 | 6.72% | Alcohol |
| 39 | 16.424 | d-Allose, oxime, 6TMS | C24H61NO6Si6 | 0.09% | Oxime |
4.3. Characterization of coumarins by HPLC
Seven coumarins, including xanthotoxol, oxypeucedanin hydrate, byakangelicin, oxypeucedanin, imperatorin, phellopterin and isoimperatorin, were identified by HPLC. The chromatograms of A. dahurica leaves and reference substances are shown in Fig. 3.
Fig. 3.
HPLC chromatograms of A. dahurica leaves (A) and reference substances mixture (B).
For the quantification of coumarins, the mixed reference solution that containing 7 coumarins was firstly diluted with 80% methanol into a series of gradient concentration solutions. Then they were analyzed under the same condition as A. dahurica leaves. Calibration curves were established between compound concentrations and the corresponding peak areas (Table 3). All the calibration curves showed good linearity (R2＞0.997). The contents of the 7 coumarins in A. dahurica leaves were then calculated according to the regression equations. Among the seven quantified coumarins, the content of oxypeucedanin (1475.58 ± 701.24 μg/g) and oxypeucedanin hydrate (498.38 ± 165.81 μg/g) were highest in A. dahurica leaves.
Table 3.
The regression equations, correlation coefficient (R2), linear ranges, limit of detection (LOD) and limit of quantification (LOQ) of the seven quantified coumarins.
| Compounds | Regression equation | R2 | Linear ranges (μg/mL) | LOD (μg/mL) | LOQ (μg/mL) | Contents (μg/g) (mean ± SD, n = 5) |
|---|---|---|---|---|---|---|
| Xanthotoxol | y = 2.42 × 107x-6557.70 | 0.9979 | 0.43–85 | 0.05 | 0.17 | 39.70 ± 28.34 |
| Oxypeucedanin hydrate | y = 2.82 × 107x+4533.68 | 0.9985 | 1.44–155 | 0.05 | 0.10 | 498.38 ± 165.81 |
| Byakangelicin | y = 1.21 × 107x-3750.76 | 0.9993 | 0.60–120 | 0.08 | 0.24 | 3.54 ± 3.27 |
| Oxypeucedanin | y = 9.06 × 106x+17829.67 | 0.9996 | 2.00–400 | 0.06 | 0.18 | 1475.58 ± 701.24 |
| Imperatorin | y = 2.33 × 107x-2060.24 | 0.9997 | 0.55–220 | 0.09 | 0.28 | 15.90 ± 8.01 |
| Phellopterin | y = 2.42 × 107x-8632.49 | 0.9995 | 1.28–255 | 0.13 | 0.32 | 27.58 ± 9.29 |
| Isoimperatorin | y = 4.76 × 107x-2168.48 | 0.9998 | 0.33–260 | 0.07 | 0.16 | 9.68 ± 10.64 |
4.4. Inhibitory effects of A. dahurica leaves extracts on AK
iEIA was applied for evaluation the inhibitory effects of A. dahurica leaves extracts on AK. The inhibition rates of the 5 batches A. dahurica leaves extracts on AK were 34.78%–62.61% which were comparable with chlorpheniramine at 2 mM (30.12%) (Fig. 4).
Fig. 4.
AK inhibition rates of A. dahurica leaves and chlorpheniramine (n = 3).
4.5. Grey relation analysis for screening of potential AK inhibitors
Grey relational analysis was applied for investigating the spectrum-effect relationships between peak areas (chemical components) and AK inhibitory activity of A. dahurica leaves extracts. The grey relational coefficients of all the identified non-volatile metabolites were ranged from 0.605 to 0.92.34 non-volatile metabolites with their coefficients more than 0.8 are listed in Table 4 which could be possible AK inhibition candidates. The higher the coeffieiects were, the more possibilities of the corresponding candidates to show AK inhibitory effects.
Table 4.
List of components with the grey relational coefficients more than 0.8.
| Evaluation items | Coefficients |
|---|---|
| l-Aspartic acid | 0.92 |
| Arabinitol | 0.912 |
| Phellopterin | 0.911 |
| l-Alanine | 0.901 |
| Glycine | 0.898 |
| Xylose | 0.884 |
| Ethanolamine | 0.881 |
| Galactose oxime | 0.88 |
| d-Glucose | 0.878 |
| l-Threonine | 0.878 |
| d-Galactose | 0.875 |
| l-Serine | 0.875 |
| d-Gluconic acid | 0.875 |
| Galactose oxime | 0.873 |
| d-Fructose | 0.872 |
| 1-Hexadecanol | 0.868 |
| D-Threitol | 0.866 |
| l-Proline | 0.864 |
| l-Valine | 0.863 |
| Myo-Inositol | 0.858 |
| 4-Aminobutanoic acid | 0.855 |
| Malic acid | 0.854 |
| 2-Butenedioic acid, (Z)- | 0.848 |
| Oxalic acid | 0.848 |
| Erythritol | 0.842 |
| Ribitol | 0.841 |
| Xanthotoxol | 0.838 |
| Citric acid | 0.828 |
| Oxypeucedanin | 0.827 |
| 2-Butenedioic acid, (E)- | 0.826 |
| Glyceric acid | 0.81 |
| Lactic acid | 0.807 |
| Byakangelicin | 0.803 |
| Shikimic acid | 0.802 |
4.6. Verification of the inhibitory effects on AK
To further validate the results of grey relational analysis, the inhibitory effects of 10 compounds, including aspartic acid, lactic acid, citric acid, malic acid, shikimic acid, phellopterin, xanthotoxol, byakangelicin, galactose and oxypeucedanin at 2 mM were tested. The inhibition rates of aspartic acid, lactic acid, citric acid, malic acid, shikimic acid were higher than that of chlorpheniramine at 2 mM, while the inhibition rates of phellopterin, xanthotoxol, byakangelicin, galactose and oxypeucedanin were lower than that of chlorpheniramine at 2 mM (Fig. 5).
Fig. 5.
AK inhibition rates of 10 compounds comparing with chlorpheniramine at 2 mM (n = 3).
Further, the IC50 values of aspartic acid, lactic acid, citric acid, malic acid, shikimic acid and chlorpheniramine were determined by evaluating their inhibitory effects on AK at 5 concentrations. As shown in Fig. 6, all of the 6 compounds exhibited dose-dependent inhibitory effects on AK. It could be noted that aspartic acid, with the highest coefficient in grey relational analysis, exhibited strongest inhibitory effects on AK. The IC50 value of aspartic acid was 0.558 mM which was much lower than that of chlorpheniramine (6.644 mM). Lactic acid, citric acid, malic acid and shikimic acid also showed strong inhibitory effects on AK with their IC50 values were 0.823 mM, 1.008 mM, 1.755 mM and 1.881 mM, respectively.
Fig. 6.
Inhibition rates of aspartic acid, lactic acid, citric acid, malic acid, shikimic acid and chlorpheniramine on AK.
5. Discussion
5.1. Volatile metabolites by GC-MS
The volatile oils of A. dahurica roots showed bioactivities such as anti-inflammation [13,14] and antioxidation [15,16]. According to literatures, the volatile oil extracted from the root of A. dahurica were mainly alkenes [17]. Present study about the volatile components of A. dahurica leaves indicated that they also contained variety of alkenes and alcohols. For instance, α-pinene, β-phellandrene, β-terpinene, 3-carene, β-cubebene and ocimene and so on existed both in A. dahurica leaves and roots but with their relative contents differed in these two parts. Gao et al. [18] applied GC-MS for comparing the volatile profiles of A. dahurica roots, leaves and stems during different growth periods. However, methanol instead of hexane was used for chemical extraction that making the results incomparable with the present study. They identified more alcohols, aldehydes and ketones.
5.2. Non-volatile metabolites by GC-MS
Malic acid was the most abundant primary metabolite identified in A. dahurica leaves. It participates in the energy metabolism of organisms and has the effects of anti-fatigue [19] and antioxidation [20]. Malic acid can improve the ability of oxidizing phosphate and energy metabolism in the processes of cell metabolism [21]. A. dahurica leaves contained 9 kinds of common amino acids, which are roughly the same as those detected in the root of A. dahurica [22]. Among them, leucine, threonine and valine are essential amino acids.
5.3. Coumarins in A. dahurica leaves
Coumarins were considered to be the main bioactive components of A. dahurica roots. They showed anti-inflammation, anti-cancer effects [23,24]. In the present study, seven coumarins, namely xanthotoxol, oxypeucedanin hydrate, byakangelicin, oxypeucedanin, imperatorin, phellopterin and isoimperatorin were detected in A. dahurica leaves. In the 2020 edition of Chinese Pharmacopoeia, imperatorin and isoimperatorin were important quality indexes of A. dahurica root [1]. A. dahurica leaves also contained these two components. Oxypeucedanin, the highest coumarin component in A. dahurica leaves, was reported to be able to enhance the anti-breast cancer effect of doxorubicin by inhibiting P-Sugar protein [25,26]. It could also reverse the drug resistance of doxorubicin in breast cancer treatment [27].
5.4. Characterization of potential AK inhibitors in A. dahurica leaves
AK has become a hot target for developing new highly selective insecticides because it exists only in invertebrates [28,29]. AK also reported to be a main allergen in seafood [9,10]. Thus, the characterization of AK inhibitors would be helpful for mammal-friendly insecticide or anti-allergy drug development. Cu2+ and some flavonoids such as myricetin, baicalin, quercetin, kaempferol and berberine showed inhibitory effects on AK [30,31]. Molecular interaction studies were applied for evaluation of the mechanisms of inhibition on AK. Some molecules might interact with AK which leading to its exposure of hydrophobic groups [31,32]. Such spacial structure alteration deactivated AK. Some arginine analogues could inhibit the activity of AK by competitively binding to the enzyme or interfering with intermediates [33]. In the present study, correlation analysis between chemical composition and AK inhibition activity was applied for screening potential AK inhibitors. Comparing with chlorpheniramine, which is a commercial anti-allergic medicine, some components from A. dahurica leaves such as aspartic acid, lactic acid, citric acid, malic acid, shikimic acid showed better inhibitory effects on AK. These compounds could be candidates for further developing of pesticides or anti-allergic drugs.
6. Conclusion
A. dahurica is a medicinal plant with its roots widely used in clinical treatment. The large aboveground part of A. dahurica is normally dumped in the field. To avoid resource waste, the chemical composition of A. dahurica leaves was profiled by GC-MS and HPLC. In total, 131 compounds were identified and quantified including alkanes, alkenes, aldehydes, alcohols, esters, sugars, organic acids, amino acids and coumarins. The chemical diversity of A. dahurica leaves implied its potential development values. The extracts and components of A. dahurica leaves exhibited strong inhibitory effects on AK. The characterized potential AK inhibitors would have a broad application prospect for the development of new insecticides or anti-allergic drugs, especially for treatment of seafood allergy.
Fundings
This work was supported by Research Foundation of Hebei Provincial Administration of Traditional Chinese Medicine (2024090), College Students’ Innovation and Entrepreneurship Training Program (202314432032) and Innovation Team of Hebei Province Modern Agricultural Industry Technology System (HBCT2023080201, HBCT2023080205).
CRediT authorship contribution statement
Aitong Yang: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation. Junyan Zhang: Writing – review & editing, Validation, Investigation. Guangying Lv: Investigation. Jiabao Chen: Data curation. Long Guo: Writing – review & editing, Resources. Yan Liu: Writing – review & editing, Supervision. Yuguang Zheng: Writing – review & editing, Supervision, Project administration, Funding acquisition. Lei Wang: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank Zi-Jing Xue for well maintenance of the plants. The authors are also grateful to all the members in Traditional Chinese Medicine Processing Technology Innovation Center of Hebei Province for fruitful discussions.
Contributor Information
Yuguang Zheng, Email: zyg314@163.com.
Lei Wang, Email: wanglei1031@126.com.
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