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BY 4.0 license Open Access Published by De Gruyter Open Access July 12, 2022

GC-MS analysis of Vespa velutina auraria Smith and its anti-inflammatory and antioxidant activities in vitro

  • Qi Wang , Si-Tong Zhou , Xiu-Mei Wu , Xiu-Qin Pang , Lian-Li Ni , Shi-Meng Yuan , Zhi-Bin Yang , Yue-Hua Li EMAIL logo and Huai Xiao EMAIL logo
From the journal Open Chemistry


Vespa velutina auraria Smith is an edible and medicinal insect in China. This study demonstrated the in vitro antioxidant and anti-inflammatory bioactivities and the volatile composition identification determined by Gas chromatography mass spectrometry (GC-MS). The antioxidant activity screening results showed that the ethanol extracts of both the fresh and dried samples exhibited an efficient antioxidant activity for three models, 2,2′-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt) free radicals scavenging capacity, 1,1-diphenyl-2-picrylhydrazyl scavenging capacity, and ferric reducing antioxidant power. The anti-inflammatory activity screening in vitro indicated that ethanol extracts had considerable inhibitory effect on Tumor Necrosis Factor-α and Interleukin-1β (IL-1β) in macrophages, but had no influence on IL-6 expression. GC-MS analyses of volatile composition of V. auraria identified 46 components, representing 75.76% of the total peak areas from fresh sample, and 34 components, 84.70% of the total peak areas from dried ones. The volatile constituents were very different in the petroleum ether part of fresh and dried ones. The three major components are hentriacontane (7.76%), n-hexadecanoic acid (6.54%), and palmitoleic acid (4.50%) in the fresh sample, while they are benzeneacetaldehyde (13.11%), dodecanoic acid (7.08%), and oleic Acid (6.72%) in the dried sample.

Graphical abstract

1 Introduction

Insects have been widely used in traditional medicine for centuries in East Asia, Africa, and South America, and this has gradually attracted attention as sources of modern drugs. In China, more than 100 medicinal insects, including the wasp, were recorded in the “Compendium of Materia Medica,” which was also known as Ancient Chinese Encyclopedia. The natural products and extracts of insects have various pharmacological activities [1,2,3]. The wasp, vespae nidus and wasp venom have been used in the treatment of arthritis, headache, hemorrhage, analgesic, and sexual vigor [4,5,6]. We were committed to insect biomedical R&D and had developed a wasp venom plastic for treating pain, thrombus, and Ischemia reperfusion injury [7,8,9]. Although the wasp stings threaten health and wasp is a natural enemy of bees, it can catch agricultural pests such as Heliothis armigera Habner, Ascotis selenaria, and Cnaphalocrocis medinalis Guenee.

Vespa velutina auraria Smith is a species of wasp. Most of them are widely distributed in north-eastern India, southern and central China (including Yunnan, Sichuan, Tibet, and Taiwan), and Indonesia [10]. They mainly spread in farmland, river ditch, mountain, forest, orchard, and other places with suitable climate and sufficient food source [11]. V. auraria is used as a folk medicine in China for the treatment of arthritis and rheumatism since ancient times. Simultaneously, it is used to treat these diseases by making wasp wine [12,13], which was widely used in Jingpo, a Chinese national minority, and has been recorded in the Pharmacopoeia of the People’s Republic of China. In addition, the wasp pupae are fried and used as a culinary delicacy in China due to its abundant nutrient contents [14].

The research on V. auraria mainly focuses on elemental and protein analysis [14,15], melanization [16], invasion, and distribution [17]. However, the active ingredients and pheromone are not clear, and the mechanism of pharmacological action is a terra incognita. Therefore, the antioxidant, the anti-inflammatory, and the antibacterial activity attached to ethanolic extracts of fresh and dried V. auraria were tested and compared. The volatile compounds of the fresh and dried V. auraria were determined and contrasted to explore more biological activities and the possible chemical components.

2 Materials and methods

2.1 Wasp samples

The wasp used for this study were collected in a fresh condition from Yunan province, China, and identified to be V. auraria by Professor Zi-Zhong Yang at Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, Dali University. Voucher specimens (number: 201506) were deposited at the same laboratory. Prior to the experiments, the live insects were frozen at –20°C and divided into two parts after they expired. One part is dried in a drying cabinet at 45°C and the other part is not processed. Both of them are crushed into a certain size of particles. The fresh and dried wasps consist of about 65.57 and 2.99% water, respectively.

2.2 Preparation of extracts

50 g samples of fresh and dried V. auraria were ultrasonically extracted 3 times with 95% ethanol, at a solid to liquid ratio of 1:10, 30 min for each time. The extracted solution was filtrated, concentrated, and lyophilized to obtain the ethanol extracts, with a yield of 286.48 mg/g for fresh and 320.72 mg/g for dried sample.

Taking 50 mg of above ethanol extracts and is extracted again with petroleum ether to get petroleum ether part, which was used for volatile composition analysis by GC-MS.

2.3 Antioxidant activity determination of ethanol extracts

2.3.1 2,2′-Azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt) (ABTS) scavenging capacity assay

ABTS free radicals scavenging capacity was measured according to the reported method [18]. Ascorbic acid was used as positive control. ABTS radical cation (ABTS·+) was produced by reacting ABTS stock solution (7 mM) with potassium persulfate (2.45 mM) and the mixture was kept in the dark at room temperature for 12 h before use. The concentrated ABTS was diluted with ethanol to a final absorbance of 0.72 ± 0.02 at 734 nm on the UV-6000PC UV-Vis spectrophotometer (Metash, Shanghai, China). Then, 100 µL of the examined ethanol extract solution was added to 2 mL of ABTS solution and equilibrated at 30°C for 10 min, and the absorbance of the solution was measured at 734 nm. The inhibition ratio (%) was calculated as I = ([A 0A S]/A 0) × 100%, where A 0 is the absorbance of ABTS solution without sample, and A S is the absorbance after adding sample. A series of extract solutions with concentrations of 0.125, 0.25, 0.50, 1.00, and 2.00 mg/mL was tested to estimate the inhibition activity and calculate the IC50.

2.3.2 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity assay

The free radical scavenging activity of extracts on 1,1-diphenyl-2-picrylhydrazyl (DPPH) was assessed using the reported method [19]. Ascorbic acid was used as the positive control. 3 mL of 50% ethanol and 1 mL of DPPH ethanol solution (1 mM) were added to 1 mL of the examined ethanol extract solutions (with the final concentration of 0.125, 0.25, 0.50, 1.00, and 2.00 mg/mL) at room temperature for 40 min, and then the absorbance of the solution was tested at 517 nm. The inhibition ratio (%) calculated was consistent with the above.

2.3.3 Ferric reducing antioxidant power (FRAP) assay

The FRAP of ethanol extracts was measured according to the method [20]. Ascorbic acid was used as positive control. The FRAP reagent was prepared before the test by mixing 10 mL of acetate buffer (300 mM and pH 3.6) with 1 mL of TPTZ solution (10 mM in 40 mM HCl) and 1 mL of FeCl3 (20 mM). Then, the mixture was incubated at 37°C. 200 µL of the examined ethanol extract solution or different concentrations of FeSO4 solution were added to 3 mL of FRAP and equilibrated at 37°C for 10 min. The absorbance of the solution was measured at 593 nm. The results were expressed in mmol FeSO4 equivalents per gram of ethanol extracts and ascorbic acid.

2.4 Anti-inflammatory activity determination of ethanol extracts

RAW 264.7 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL). They were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged by trypsinization during logarithmic phase. They (1 × 106 cells per mL) were treated with Lipopolysaccharide (LPS, 10 μg/mL) for 4 h and then cultivated with or without ethanol extract solutions (75, 150, and 300 μg/mL) for 24 h. 50 μL of the culture supernatant was taken out to determine the level of Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Interleukin-6 (IL-6) using respective enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

2.5 Analysis of the volatile constituents by gas chromatography mass spectrometry (GC-MS)

GC-MS was performed with a gas chromatography instrument (Agilent Technologies 7890A, Agilent Technologies, Inc. Santa Clara, CA, USA) coupled to a mass spectrometer (Agilent Technologies 5975C, Agilent Technologies, Inc. Santa Clara, CA, USA).

GC/MS (electron ionization; EI) conditions: Helium was used as the carrier gas (0.9 mL/min). All analyses were performed using the following temperature ramp: the program was initiated by a column temperature set at 80°C and maintained for 1 min, increased from 80 to 240°C at the rate of 5°C/min and then kept at 240°C for 5 min. The splitless injection was conducted. The mass spectrometer was operated with EI ion source at 70 eV, and the mass range was from m/z 40 to m/z 400. The scan rate is 0.2 s. The transfer line temperature and ionization source temperature were 280 and 230°C, respectively. The components were identified by matching their recorded mass spectra with those of the reference compounds in the National Institute of Standards and Technology (NIST) mass spectral library, and the similarity was greater than or equal to 80%. The relative percentage of the constituents was expressed as percentages by peak area normalization.

2.6 Statistical analysis

The experimental data are represented as + s. A one-way analysis of variance was performed with SPSS 26.0 software. After the homogeneity of the variance test, the experimental data with uniform variance were statistically analyzed by the pairwise comparison LSD method. Besides, the data with uneven variance were analyzed by the rank sum test. Values of P < 0.05 were considered statistically significant. According to the results, GraphPad Prism 6.01 software was used for mapping.

3 Results

3.1 Antioxidant activity of V. auraria ethanol extracts

The methods of ABTS, DPPH, and FRAP are based on single-electron transfer reaction. In the present study, the ABTS assay values of the two ethanol extracts showed that the clearance rate of ABTS free radicals increases as the concentration increases in the range of 0.125–2.00 mg/mL (Figure 1a), and there was a positive correlation between the increase in concentration and the inhibition in the DPPH scavenging capacity assay in the concentration range of 0.125–1.00 mg/mL (Figure 1b). The free radical scavenging activities of DPPH and ABTS were evaluated by IC50 values, and the amount of Fe2+ equivalent per gram of ethanol extracts is also shown in Table 1. The extract of dried wasp had a better activity for clearance of ABTS free radicals and DPPH scavenging with IC50 values of 748.50 ± 22.90 and 365.33 ± 19.55 μg/mL, while the values were 957.51 ± 51.46 and 478.19 ± 28.22 μg/mL for extract of fresh wasp, respectively. Fresh and dried wasp ethanol extracts showed similar total reducing capacity for FRAP, 1 g ethanol extracts of fresh and dried wasp were equivalent to the antioxidant capacity in 5.12 ± 0.07 and 5.49 ± 0.07 mmoL Fe2+, respectively. The results obtained in this work showed that V. auraria ethanol extracts possessed antioxidant activity. Comparing the IC50 values of the free radical scavenging activities of DPPH and ABTS for dried and fresh wasps, it was determined that the ethanol extract of dried wasp had a little stronger antioxidant activity than that of the fresh one. Phenolics have favorable antioxidant activity naturally. We hypothesized that the stronger antioxidant activity of dried wasp extract must be due to the phenolic contents. Further study of the phenolic content testing by using Folin–Ciocalteu method [21] supported the above speculation, the content was 27.15 mg/g for fresh wasp extract and 38.54 mg/g for dried one.

Figure 1 
                  The effects of V. auraria extract for the inhibition of the ABTS (a) and DPPH (b).
Figure 1

The effects of V. auraria extract for the inhibition of the ABTS (a) and DPPH (b).

Table 1

Antioxidant activities of the V. auraria extracts (x ± s, n = 3)

IC50 (μg/mL) IC50 (μg/mL) Fe2+ (mmol/g)
Fresh wasp 957.51 ± 51.46 478.19 ± 28.22 5.12 ± 0.07
Dried wasp 748.50 ± 22.90 365.33 ± 19.55 5.49 ± 0.07
Ascorbic acid 30.00 ± 0.07 17.57 ± 0.02 20.58 ± 0.03

3.2 Effect of V. auraria extract on the expression of inflammatory mediators

In the research, the RAW 264.7 cells were treated with LPS to produce large amounts of inflammatory factors and they were tested after adding the ethanol extracts. Results found that LPS increased the amount of TNF-α, IL-1β, and IL-6 produced by macrophages and the extract from dried wasp reduced the production of TNF-α and IL-1β (Figure 2). TNF-α was considerably suppressed in a dose-dependent manner. Ethanol extracts had no influence on IL-6 expression. V. auraria may inhibit inflammatory responses primarily by regulating TNF-α.

Figure 2 
                  Inhibitory effects of ethanol extracts on TNF-α (a), IL-1β (b), and IL-6 (c) released in RAW 264.7 cells (note: compared with the untreated group, *P < 0.05, **P < 0.01; compared with the LPS group, Δ
                     P < 0.05, ΔΔ
                     P < 0.01).
Figure 2

Inhibitory effects of ethanol extracts on TNF-α (a), IL-1β (b), and IL-6 (c) released in RAW 264.7 cells (note: compared with the untreated group, *P < 0.05, **P < 0.01; compared with the LPS group, Δ P < 0.05, ΔΔ P < 0.01).

3.3 The volatile compositions in V. auraria

As shown in Table 2, GC-MS analyses of the petroleum ether part of fresh wasp were extracted for the identification of 46 different components, representing 75.76% of the total peak areas, and 34 components were identified from the corresponding part of dried wasp that accounted for 84.70% of the total peak areas. Carboxylic acids and carboxylic esters, representing 28.37% (15) and 55.23% (21) of the total peak areas, were identified in the fresh and dried samples, respectively. The three major components are hentriacontane (7.76%), n-hexadecanoic acid (6.54%), and palmitoleic acid (4.50%) in the fresh sample, while they are benzeneacetaldehyde (13.11%), dodecanoic acid (7.08%), and oleic acid (6.72%) in the dried sample. It can also be seen from Figure 3 that the samples are different.

Table 2

Volatile composition in the petroleum ether parts of the fresh and dried V. auraria

No. Retention time (min) Name Formula Molecular weight Fresh Dried
Content (%) Similarity (%) Content (%) Similarity (%)
1 4.987 1-Tridecene C13H26 182 1.68 95
2 6.831 Pyrazine, tetramethyl- C8H12N2 136 5.60 91
3 7.564 Benzaldehyde C7H6O 106 2.88 94
4 9.031 Hexadecane C16H34 226 0.58 95 0.46 94
5 9.964 Benzeneacetaldehyde C8H8O 120 0.39 92 13.11 91
6 13.597 Hexadecane, 2,6,10,14-tetramethyl- C20H42 282 0.19 93
7 14.352 Octadecane C18H38 254 0.47 98
8 15.719 Heptadecanoic acid, ethyl ester C19H38O2 298 0.09 91
9 16.052 Heptadecane, 2-methyl- C18H38 254 0.14 91
10 16.63 Benzyl alcohol C7H8O 108 2.11 96
11 17.541 2-Bromo dodecane C12H25Br 248 0.68 90
12 17.729 Phenylethyl alcohol C8H10O 122 2.24 93
13 17.796 Butylated hydroxytoluene C15H24O 220 0.33 98
14 18.174 Benzeneacetaldehyde, .alpha.-ethylidene- C10H10O 146 0.09 83 2.00 94
15 19.262 Heptanoic acid C7H14O2 130 0.72 91
16 20.918 Heptadecane C17H36 240 1.05 91
17 22.529 Tetradecanoic acid, ethyl ester C16H32O2 256 0.26 92 2.90 96
18 22.895 Octanoic acid C8H16O2 144 2.46 90
19 23.751 Ethyl 9-tetradecenoate C16H30O2 254 0.06 99
20 24.362 Nonadecane C19H40 268 1.86 95 0.18 95
21 26.562 Nonanoic acid C9H18O2 158 1.49 90
22 27.05 Megastigmatrienone C13H18O 190 0.12 98
23 27.795 Eicosane C20H42 282 1.38 91
24 28.228 Hexadecanoic acid, methyl ester C17H34O2 270 0.25 99
25 28.884 Cyclohexane, (1-octylnonyl)- C23H46 322 0.10 91
26 29.55 Hexadecanoic acid, ethyl ester C18H36O2 284 0.94 93 4.01 97
27 30.172 n-Decanoic acid C10H20O2 172 1.02 98
28 30.339 E-11-Hexadecenoic acid, ethyl ester C18H34O2 282 0.83 98 2.49 99
29 31.094 Heneicosane, 11-decyl- C20H42 437 0.16 94
30 31.205 Heneicosane C21H44 296 1.90 91
31 31.572 1-Docosene C22H44 308 0.37 98
32 32.383 Octadecanal C18H36O 268 0.17 95
33 32.416 Triacontyl acetate C32H64O2 480 0.25 95
34 32.872 Diethyl Phthalate C12H14O4 222 0.37 98
35 34.516 Nonadecane, 9-methyl- C20H42 282 1.90 93
36 34.916 Indole C8H7N 117 0.34 76 0.22 95
37 35.838 Triacontyl acetate C32H64O2 480 0.24 95
38 36.127 Octadecane, 2,6,10,14-tetramethyl- C22H46 310 0.36 94
39 36.305 Octadecanoic acid, ethyl ester C20H40O2 312 0.24 99
40 36.86 Ethyl oleate C20H38O2 310 2.40 99 5.75 99
41 37.127 Dodecanoic acid C12H24O2 200 0.98 91 7.08 99
42 37.749 Pentacosane C25H52 352 3.51 98 0.21 96
43 39.804 Tetracosane C24H50 338 1.15 99
44 40.36 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- C20H34O2 306 1.52 94 1.44 99
45 40.86 Heptadecane, 9-octyl- C25H52 352 2.76 90
46 41.471 Triacontane, 1-bromo- C30H61Br 500 0.79 92
47 43.104 Dibutyl phthalate C16H22O4 278 0.42 93 0.79 93
48 43.659 Tetradecanoic acid C14H28O2 228 3.66 99 5.51 99
49 43.904 Hexacosane C26H54 366 2.41 91
50 44.426 1-Hexacosene C26H52 364 0.92 94
51 44.981 Z-7-Tetradecenoic acid C14H26O2 226 0.80 98
52 46.815 Octacosane C28H58 394 4.37 99
53 47.359 Nonacosane C29H60 408 1.32 96
54 48.781 2,6,10,14-Tetramethyl-7-(3-methylpent-4-enylidene) pentadecane C25H48 348 0.71 90
55 49.748 n-Hexadecanoic acid C16H32O2 256 6.54 98 5.51 99
56 50.703 Palmitoleic acid C16H30O2 254 4.50 99 3.74 99
57 52.358 Triacontane C30H62 422 3.41 92
58 55.002 Hentriacontane C31H64 437 7.76 94
59 56.191 Oleic acid C18H34O2 282 2.66 99 6.72 99
60 57.558 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280 3.59 99
61 59.346 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- C18H30O2 278 1.39 99 1.89 99
62 60.057 Octadecane, 1-iodo- C18H37I 380 2.57 93
Total content 75.76 84.70
Figure 3 
                  Total ion current of volatile composition analyzed on V. auraria. (a) The petroleum ether part of the fresh V. auraria; (b) The petroleum ether part of the dried V. auraria.
Figure 3

Total ion current of volatile composition analyzed on V. auraria. (a) The petroleum ether part of the fresh V. auraria; (b) The petroleum ether part of the dried V. auraria.

The antibacterial research was carried out on the petroleum ether and the ethanol extracts of fresh and dried V. auraria (data not shown). Inhibition effect of Staphylococcus aureus, Candida albicans, and Escherichia coli were not seen in the ethanol extracts.

4 Discussion

In general, the body’s antioxidant defense system is dynamically balanced, and the generation and removal of free radicals in the body are also in equilibrium. They may lead to chain reactions in the body and are able to cause DNA fragmentation, cell damage, and lipid peroxidation when the generation of free radicals exceeds the system’s ability to eliminate them. Modern medical research shows that the generation and development of aging, cancer, cardiovascular disease, and inflammation can be induced by free radicals and their metabolites [19]. Determination of the antioxidant properties of samples is important for the investigation of their use in various fields such as food, medicine, and cosmetics. Insects possess antioxidant compounds which aid in curbing various pathologies [22]. There are few studies of the Vespa extracts. In previous studies, only the aqueous extract of V. affinis have been researched about the antioxidant activities. It is reported that the aqueous extract of V. affinis had significant antioxidant enzyme activities and could prevent the H2O2-induced intracellular reactive oxygen species production in monocytes [23]. This study demonstrated the bioactivities about the antioxidant and anti-inflammatory characteristics of the V. auraria ethanol extracts for the first time. Results showed that the ethanol extract of V. auraria demonstrated a dose-dependent increase in the inhibition of ABTS.

TNF-α is an important inflammatory factor in the inflammatory response and is widely found in the blood, synovial tissue, and synovial fluid of patients with rheumatoid arthritis (RA) [24]. It is involved in a variety of pathophysiological processes in RA (including the onset of anemia) and is an important driving and maintenance factor for the development of RA. In this research, the TNF-α was considerably suppressed in a dose-dependent manner.

Pheromone usually consists of two types of compounds, one is straight-chain aliphatic alcohols, aldehydes or esters, and the other is acyclic mono-, sesqui-, and diterpenes (alcohols or acetates) [25]. The major constituents of petroleum ether part were dominated by aliphatic hydrocarbons, carboxylic acids, and esters in fresh wasp, and they were the possible components of pheromones, while for dried ones, aliphatic hydrocarbons were less both in quantity and content. 23 aliphatic hydrocarbons, that is, 40.30% of the total peak areas were identified in the fresh sample, but just 4 alkanes were identified in the dried one. Phenylethyl alcohol is a very common semiochemical that is found in a broad range of insect species, and it usually as the aggregation, sex, alarm pheromone to use. [26] Phenylethyl alcohol was 2.24% in the dried sample. Fewer components have been identified in dried wasps than fresh samples, and fresh samples contain large amounts of aliphatic hydrocarbons, while the main components in dried samples are carboxylic acids and esters. This indicates that V. auraria is rich in aliphatic hydrocarbons, and most of the volatile hydrocarbons were lost in the drying process.

5 Conclusion

Insects are used for the treatment of different types of diseases all over the world. This study reported the bioactivities about antioxidant and anti-inflammatory characteristics of V. auraria ethanol extracts for the first time. The ethanol extract from dried wasp had higher antioxidant activity than the fresh ones. Meanwhile, the results of anti-inflammatory activity demonstrated that the ethanol extract of dried wasp had considerable inhibitory effect on TNF-α in a dose-dependent manner in the macrophages and it also had a significant inhibitory effect on IL-1β. The volatile constituents were very different in fresh and dried ones. The straight-chain aliphatic alcohols, aldehydes, or esters may be the possible components of wasp pheromones. In order to have a clear picture, further investigations are required to identify the active principles present and more possible pheromones in V. auraria and it is currently in progress.

# These authors contributed equally to this work.

  1. Funding information: This work was supported by The National Natural Science Foundation of China (No. 82160822 and 82060765); The Special Program of Science and Technology of Yunnan Province (202002AA100007); Medicine Pieces Industry Development Special Fund of Yunnan Province [Grant number 2019-YG-067]; The Natural Science Foundation of Yunnan Province [Grant number 2017FA050]; Key Science and Technology Support Project of Dali [Grant number D2019NA01].

  2. Author contributions: Huai Xiao and Xiu-Mei Wu conceived and designed the experiments; Si-Tong Zhou, Lian-Li Ni, Shi-Meng Yuan, Zhi-Bin Yang, and Yue-Hua Li performed the experiments; Si-Tong Zhou and Xiu-Mei Wu analyzed the data; Qi Wang, Si-Tong Zhou, Xiu-Qin Pang, and Huai Xiao wrote the manuscript; and Huai Xiao polished it. Huai Xiao and Zhi-Bin Yang acquired funding for the research. All authors reviewed and approved the final version.

  3. Conflict of interest: The authors report no conflicts of interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: All the data of this manuscript are available from the authors.


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Received: 2021-12-29
Revised: 2022-05-31
Accepted: 2022-06-02
Published Online: 2022-07-12

© 2022 Qi Wang et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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