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Study on flavonoid and bioactivity features of the pericarp of Citri Reticulatae ‘chachi' during storage
⁎Corresponding authora at: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China. wh@scau.edu.cn (Hong Wu)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
The mature pericarp of Citri Reticulatae 'Chachi' (PCRC) is one of the six traditional Chinese medicinal materials that should be used after long storage, and it was regarded that the longer the medicine was stored, the better. However, the aging mechanism of the medicine is not clear. To further investigate the effect of aging on the main active flavonoids of PCRC, ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) and metabolomics analysis were used to analyze the flavonoids of PCRC stored for different periods. In the results, 219 flavonoids were detected. 5,7,3′,4′,5′-pentamethoxy dihydroflavone and 2′-hydroxy-3,4,5,3′4′,6′-hexamethoxychalcone were found from PCRC for the first time. According to the clustering analysis of metabolites, aging times of 0 year, 1 year, and 2 year were clustered into one group, and aging times of 3 year, 4 year, and 29 year were clustered into the other group. Quantitative analysis showed that the former group contained a greater amount of 4 flavonoids than the latter group, while the latter group contained a greater amount of 15 polymethoxyflavonoids. The newly harvested PCRC was compared with the other 5 groups of PCRC (stored for 1, 2, 3, 4, and 29 years). Eight flavonoids, tectochrysin, apigenin, 2′-hydroxyisoflavone, luteolin, 6-hydroxyluteolin, gallocatechin, quercetin -O- acetylhexoside and apigenin −7-O-[β-D-glucuronide (1 → 2) -O-β-D-glucuronide], were used as marker components to discriminate newly harvested PCRC and aging PCRC. In addition, the antioxidant potency composite index (APC) indicated that the PCRC stored for three or four years had stronger antioxidant activity than the PCRC stored for other periods. By means of molecular docking, it was reviewed that the amount of antiviral components against SARS-CoV-2 in freshly harvested PCRC was significantly higher than that in aging PCRC. The results in this study supplied scientific data for quality control, evaluation, and rational utilization of PCRC and basic information for further analysis of the metabolic regulation of the active components of the PCRC.
Keywords
Pericarp of Citri Reticulatae 'Chachi'
Aging period
Flavonoids
UPLC-MS/MS
Antioxidation
SARS-CoV-2
1 Introduction
The traditional Chinese medicinal material, 'Chenpi' (Citri Reticulatae Pericarpium), is the dry mature pericarp of Citrus reticulata Blanco and its cultivars. According to the cultivation place and cultivar, Citri Reticulatae Pericarpium was divided into two types, 'Chenpi' and 'Guang Chenpi' (National Pharmacopoeia Commission, 2020). 'Guang Chenpi' refers to the Citri Reticulatae Pericarpium cultivated in Xinhui, Guangdong, China, which is the dry, mature pericarp of the cultivar Citrus reticulata Blanco, Citrus reticulata 'Chachi'. The dry, mature pericarp of Citrus reticulata 'Chachi' (PCRC) is of higher quality. Modern phytochemical studies have shown that the chemical constituents of PCRC are flavonoids, volatile oils, polysaccharides, and alkaloids (Luo et al., 2019). The main active components are flavonoids, including flavonoid glycosides and polymethoxyflavonoids (Fu et al., 2018). According to the Chinese Pharmacopoeia, PCRC regulates qi flow to strengthen the spleen and dry dampness to reduce phlegm. It is mainly used to treat abdominal distension, lack of appetite, vomiting, and diarrhea, and coughing with phlegm (National Pharmacopoeia Commission, 2020). Modern pharmacological studies also show that it has antioxidative, antibacterial, antiviral, anti-inflammatory, antitumor, and hypolipidemic effects (Liu et al., 2013; Xu et al., 2014; Gao et al., 2011; Chen et al., 2017; Yu et al., 2019; Zeng et al., 2020a, 2020b). It has a long history that PCRC was used as medicine. PCRC was recorded in a pharmaceutical monograph written approximately 2000 years ago in the Eastern Han Dynasty, “Shennong's classic of materia medica” (Gu, 2007). Currently, the number of preparations employing PCRC recorded in the Chinese Pharmacopoeia (2020 Edition) is as high as 176 (National Pharmacopoeia Commission, 2020). In addition, PCRC is also the most popular food seasoning or ingredient in China. Among them, the annual market sales of PCRC produced from Xinhui reached 10 billion RMB, ranking first for three consecutive years in influence ranking of agricultural products on development forum for the regional agricultural brand of China.
In traditional Chinese medicine, there is the theory of “aging medicine”. Aging medicine refers to the medicinal material stored and maintained by specific methods and then used after aging. The process of storage, maintenance, and aging is the process of changing the properties and efficacy of the medicinal material, making it more suitable to the clinical needs of traditional Chinese medicine (Wang et al., 2012). As early as 1500 years ago, in the Southern Dynasties·Liang Dynasty, Hongjing Tao recorded Citri Reticulatae Pericarpium as one of the six aging traditional Chinese medicinal materials, which should be used after aging (Sun, 1985). Since then, in the Tang, Song, Yuan, Ming, and Qing Dynasties, there have been records that the longer the Citri Reticulatae Pericarpium was stored, the better (Sun, 1985; Su, 1988; Wang, 1987; Du, 1993; Ni, 2005; Wang, 1998). Looking up all the ancient books recording traditional Chinese medicinal materials, the reasons for long storage treatment of aging medicine were not discussed (Wang et al., 2017). In recent years, with the help of modern instrumental analysis, researchers have rapidly promoted the development of research on the effect of aging on the active components of Citri Reticulatae Pericarpium. Wang et al (Wang et al., 2016) studied the effect of aging on the component accumulation and biological activity of Citri Reticulatae Pericarpium. The results showed that the content of combined polyphenols and flavonoids increased significantly during the aging process, and the antioxidant activity increased. By means of HPLC, Liu et al (Liu et al., 2020) showed that the contents of three flavonoids, hesperidin, nobiletin, and hesperetin, significantly increased as the storage time was prolonged. Zheng et al (Zheng et al., 2010) quantitatively studied the changes in five flavonoids, hesperidin, nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, hesperetin, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone, in ten batches of PCRC with different storage periods, and the results showed that the contents of five flavonoids tended to increase as storage time increased. Fu et al (Fu et al., 2017) determined Citri Reticulatae Pericarpium stored for 36 months by HPLC-dual wavelength detection and found that the content of five flavoids of sinensetin, 4,5,7,8-tetramethoxyflavone, nobiletin, hesperetin, and 5-O-demethylnobiletin increased, while the content of hesperidin decreased. In the above results, the characteristics of specific components in the aging process of PCRC were not consistent with each other and were even contradictory. The reason for the deviation of those results was hard to analyze. However, based on metabonomics, a study indicated that up to 92 flavonoids were determined in the pericarp of freshly harvested Citrus reticulata 'Chachi' (Zhang et al., 2020). Currently, most published works have determined only 3–5 flavonoids to study the change in flavonoids in PCRC during aging. It was hard to show the whole pattern. In this study, to comprehensively evaluate the change of flavonoids in PCRC during aging, UPLC-MS/MS and widely targeted metabolomics analyses were employed to fully investigate the quantity and composition of flavonoids, and the antioxidant potency composite index (APC) was determined to compare the activity. Moreover, the novel coronavirus found at the end of 2019 was named 2019 novel coronavirus or “2019-nCoV” by the World Health Organization (WHO) on January 12, 2020 (Wu et al., 2020). As of June 12, 2021, Beijing time, more than 176 million people had been infected with COVID-19, and more than 3.8 million people had died all over the world. Unfortunately, there is currently no specific drug for COVID-19 in the world. Wu reported that flavonoids such as neohesperidin, hesperidin, baicalin, kaempferol 3-O-rutinoside, and rutin from different sources, with antiviral, antibacterial, and anti-inflammatory activities, could effectively interact with some targets of SARS-CoV-2 (Wu et al., 2020). Therefore, to study the possible effect of the abundant flavonoids in PCRC on the prevention and treatment of COVID-19, further study by means of molecular docking was used to analyze the binding affinity of some important flavonoid glycosides and polymethoxyflavones at target proteins of SARS-CoV-2. The results in this study showed that the aging process promoted the accumulation of important pharmacologically active compounds, polymethoxyflavones, and supplied evidence of active components to the statement of “the longer the medicine was stored the better” for PCRC. However, the contents of narirutin, neohesperidin, and hesperidin with higher binding affinity with target proteins in SARS-CoV-2 were most abundant in freshly harvested PCRC. The results supplied scientific data for the quality control, evaluation, and rational utilization of PCRC.
2 Materials and methods
2.1 Plant materials
The fruits of Citrus reticulata 'Chachi' were harvested in November 1990, 2015, 2016, 2017, 2018, and 2019 Fig. 1 from Langmei Orchard, Fumei Village, Shuangshui Town, Xinhui District, Jiangmen City, Guangdong Province. Gatherer was Shu-shen Zhang. All samples were identified to be genuine by associate Professor Rong-jing Zhang. After harvest, stains on the surface of the fruits were washed off with clean water. Pericarps were peeled off the fruits, dried under sunlight, and put in sealed bags for storage. The PCRC was stored in a cool and dry place for aging. They were removed and dried under sunlight several times a year. Sample information is listed in Table 1.
Images of the PCRC samples. Bars = 2 cm.
No.
Harvest and preparation time
Aging period (year)
Common name
Origin
C0
Nov 2019
0
Freshly harvested pericarps
Langmei Orchard, Fumei Village, Shuangshui Town, Xinhui District, Jiangmen City, Guangdong Province
C1
Nov 2018
1
1-year-old pericarps
C2
Nov 2017
2
2-year-old pericarps
C3
Nov 2016
3
3-year-old pericarps
C4
Nov 2015
4
4-year-old pericarps
C29
Nov 1990
29
29-year-old pericarps
2.2 Sample preparation
2.2.1 Sample preparation and extraction for flavonoid metabolomic analysis
The freeze-dried samples were crushed using a mixer mill (MM 400, Retsch) for 1.5 min at 30 Hz. One hundred milligrams of powder from each sample was weighed and extracted with 1.0 mL 70% aqueous methanol. The resulting mixtures were stored at 4 °C overnight and vortexed three times to ensure complete extraction. Following centrifugation at 10,000 g for 10 min, the extracts were absorbed and filtered (SCAA-104, 0.22 μm pore size) before UPLC-MS/MS analysis (Chen et al., 2013).
2.2.2 Quality control (QC) samples
QC samples were prepared by blending the extracts of samples in all groups with equal amounts. Four replicate QC samples were prepared separately, named mix01 to mix04. QC samples were determined with the same method as well as the samples. In instrumental analysis, one QC sample was determined after every three samples to evaluate the repeatability of the whole analysis process.
2.3 Methods
2.3.1 Determination of total flavonoid content and total antioxidant activity
Three pieces of PCRC were randomly removed, powdered by a pulverizer, and sieved through a 40 mesh sieve. PCRC powder accurately weighing 0.5 g was transferred into a conical flask, followed by 32 mL of 70% aqueous methanol solution. The mixture was treated with an ultrasonic cleaner (ultrasonic power 100 W, water temperature 60 °C) for 1 h. Then, it was centrifuged at 6000 rpm for 10 min. Supernatant liquid was collected. The extraction was repeated three times. The supernatant liquid of three extractions was combined and diluted to 100 mL in a volumetric flask to give the sample solution. Three replicate sample solutions were prepared separately from every PCRC sample. Determination of total flavonoid content adopted from the method of Meyers et al (Meyers et al., 2003) was slightly adapted. Determination of total antioxidant activity employed a detection kit from Beyotime Biotechnology Co., Ltd. and followed the FRAP and ABTS procedures. The DPPH method referred to the method of Kong et al (Kong et al., 2017) with a small adaption.
2.3.2 HPLC conditions
The sample extracts were analyzed using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems 6500 Q TRAP, www.appliedbiosystems. com.cn/). The analytical conditions were as follows, HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm*100 mm); solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program,100:0 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 15.0 min; flow rate, 0.40 mL/min; temperature, 40 °C; injection volume: 2 μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS.
2.3.3 ESI-Q TRAP-MS/Ms
LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 6500 Q TRAP LC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 500 °C; ion spray voltage (IS) 5500 V; ion source gas I (GSI), gas II(GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions were performed with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the metabolites eluted within this period (Chen et al., 2013).
2.3.4 Molecular docking
The protein structures of the spike (PDB ID: 6 VSB, chains A, B, C), 3CLpro (PDB ID: 6 LU7, chain A), PLpro (PDB ID: 4OVZ, chain A), and RdRp (PDB ID: 6NUS, chain A), four potential antiviral targeting proteins for SARS-CoV-2, were downloaded from the Protein Data Bank (www.rcsb.org). Three-dimensional molecular structures of flavonoids and positive control drugs were downloaded from the compound database of PubChem (https://pubchem.ncbi.nlm.nih.gov/). Lopinavir, ritonavir, ribavirin, chloroquine, arbidol, and favipiravir were used as positive controls because they were recommended in the COVID-19 Diagnosis and Treatment Plan (trial version 8) (National Health Commission of the people's Republic of China, 2020).
Three-dimensional molecular structures of flavonoids and positive control drugs were ligands. The protein structures of Spike, 3CLpro, PLpro, and RdRp were used for binding. Molecular docking experiments employed Auto Dock Vina software. A grid box (126 Å × 126 Å × 126 Å) centered at 226.49, 226.728, 221.894 Å was used for the spike. A grid box (60 Å × 60 Å × 60 Å) centered at (-28.059, 9.486, 61.528) Å was used for 3CLpro. A grid box (60 Å × 60 Å × 60 Å) centered at (-13.953, 51.391, −31.750) Å was used for PLpro. A grid box (126 Å × 126 Å × 126 Å) centered at (152.179, 167.664, 166.985) Å was used for the RdRp.
2.4 Data analysis
2.4.1 PCAa
Unsupervised PCA (principal component analysis) was performed by the statistics function prcomp within R (www.r-project.org). The data were unit variance scaled before unsupervised PCA.
2.4.2 Hierarchical cluster analysis and Pearson correlation coefficients
The HCA (hierarchical cluster analysis) results of samples and metabolites are presented as heatmaps with dendrograms, while Pearson correlation coefficients (PCCs) between samples were calculated by the cor function in R and presented as only heatmaps. Both HCA and PCC were carried out by R package heatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum.
2.4.3 Differential metabolites selected
Significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute Log2FC (fold change) ≥ 1. VIP values were extracted from the OPLS-DA results, which also contained score plots and permutation plots, and were generated using the R package MetaboAnalystR. The data were log-transformed (log2) and mean-centered before OPLS-DA. To avoid overfitting, a permutation test (200 permutations) was performed.
2.4.4 KEGG annotation and enrichment analysis
Identified metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and annotated metabolites were then mapped to the KEGG pathway database. Pathways with significantly regulated metabolites mapped to were then fed into MSEA (metabolite set enrichment analysis), and their significance was determined by hypergeometric test p-values.
3 Results
3.1 Determination of total flavonoid content
Total flavonoid contents of PCRC from six groups with different aging periods were determined by spectrophotometry. The results showed that with increasing aging time, the total flavonoid contents of PCRC first decreased, then increased, and then decreased again (Fig. 2). Three groups with shorter aging periods (C0, C1, C2) had a lower total flavonoid content than three groups with longer aging periods (C3, C4, C29). The total flavonoid content of the C3 group was the highest, which was 0.83%. This result indicated that different aging periods had different impacts on the flavonoids of PCRC.
Total flavonoid content of PCRC with different aging periods. Note: Total flavonoid content (with rutin as the standard) = concentration of total flavonoid in extract solution of PCRC*volume of solution*100%/weight of PCRC sample.
3.2 Metabolism features of flavonoids
3.2.1 Qualitative and quantitative analysis of flavonoid metabolites
To evaluate the effect of aging on flavonoids of PCRC, widely targeted metabolomics analysis was employed for the six groups of PCRC. Fig. 3a, b show that the total ion current (TIC) chromatogram of quality control samples. Fig. 3c, d show multipeak chromatograms of metabolites using multiple reaction monitoring (MRM). Based on the MetWare database, the metabolites of samples were determined qualitatively and quantitatively by UPLC-MS/MS. Under MRM mode, multipeak chromatograms of metabolites show detectable components. Every color indicated one metablite. Characteristic ions were screened by triple quadrupole, and their signal strength was detected on the detector. Data files were opened by MultiaQuant software to integrate peaks and make calibrations. The peak area indicates the relative content of the component. In this study, 219 flavonoid metabolites were detected, including 82 flavones, 56 flavonols, 26 flavonoid C-glycosides, 19 dihydroflavones, 7 isoflavones, 7 tannins, 5 flavanols, 5 dihydroflavonols, 4 anthocyanins, 3 chalcones, 2 other flavonoids, 2 proanthocyanidins, and 1 dihydroisoflavone. Among them, 5,7,3′,4′,5′-pentamethoxy-dihydroflavones and 2′-hydroxy-3,4,5,3′,4′,6′-hexamethoxychalcone. were detected in PCRC for the first time. The metabolites detected are listed in Table S1, including precursor, integration value, and corresponding metabolite name.
TIC of quality control samples and multi-peaks chromatograms of metabolites using multiple reactions monitoring. a, b shows positive TIC chromatogram and negative TIC chromatogram of quality control samples. c, d shows positive and negative ion current multi-peaks chromatograms of metabolites in quality control samples using MRM. Horizontal ordinate represents retention time of detected metabolites, vertical coordinate represents the ion current strength of detected ion.
3.2.2 Multivariate analysis of the metabolites
The ion current strength data of the flavonoid metabolites were used to establish a cluster heat map, and the samples were analyzed by principal component analysis (PCA). A cluster heat map showed that three replicate samples from the same groups clustered and indicated that the data were reliable (Fig. 4a). In addition, clustering results of samples from different aging groups showed that six groups of different aging periods first clustered into two classes. C0, C1, and C2 clustered into one class, and C3, C4, and C29 clustered into the other. According to the length of the aging, they were further divided into four classes: C0 was one class by itself, C1 and C2 were clustered into a class, C3 was one class by itself, and C4 and C29 were clustered into a class. This result indicated that flavonoid metabolites of PCRC varied with the length of the aging, and flavonoid metabolites of PCRC were more similar as their lengths of the aging were more closed. In Fig. 4b, PC1 and PC2 explained 71.28% of the total difference, and three replicate samples from the same groups were combined. C1 was close to C2. C4 was close to C29. The results were consistent with the cluster heat map.
Difference of the flavonoid metabolites in groups of PCRC. a Clustering analysis of flavonoid metabolites in 6 groups of PCRC. The accumulation level of metabolites from high to low was indicated by colors from red to green. b PCA of flavonoid metabolites in 6 groups of PCRC. Horizontal ordinate shows PC1 value, and vertical coordinate shows PC2 value.
3.3 Screening and analysis of the differential metabolites between freshly harvested PCRC and aging PCRC
By means of clustering analysis and PCA of the 6 groups of PCRC samples, it was found that the flavonoid metabolites significantly varied over the aging period. Comparing the ion strength of flavonoids in PCRC samples with different aging periods, differential metabolites were screened out. The results showed that differential metabolites in five comparing pairs (C0 vs C1, C0 vs C2, C0 vs C3, C0 vs C4, C0 vs C29) were 55 (31 increased, 24 decreased), 55 (23 increased, 32 decreased), 58 (45 increased, 13 decreased), 51 (30 increased, 21 decreased), 49 (27 increased, 22 decreased) (Fig. 5), separately. Overall, the content of 45.2% metabolites (99 components) changed significantly. This result indicated that the aging process significantly affected the content of flavonoid metabolites. To identify the differential metabolites from the flavonoid metabolites between freshly harvested PCRC and the other five groups of aging PCRC (C1, C2, C3, C4, C29), a Venn diagram was employed (Fig. 6). There were 21 common flavonoid metabolites among the five pairs of PCRC groups: C0 vs C1, C0 vs C2, C0 vs C3, C0 vs C4, and C0 vs C29 (Table 2). Seven flavonoids (tectochrysin, apigenin, 2′-hydroxyisoflavone, luteolin, 6-hydroxyluteolin, gallocatechin, quercetin-O-acetylhexoside) were detected in five groups of aging PCRC samples and were not detected in freshly harvested PCRC samples. Apigenin-7-O-[β-D-glucuronide (1 → 2)-O-β-D-glucuronide] was only detected in freshly harvested PCRC samples.
Volcano diagram of differential metabolites. Note: Every point in the volcano diagram represents a metabolite. Horizontal ordinate represents the logarithmic value of quantitative fold change of a certain metabolites between two groups of samples. Vertical coordinate represents VIP value. The greater absolute value of horizontal ordinate indicates the bigger fold change of expression amount between two groups of samples. The greater absolute value of vertical coordinate indicates the expression change is more significant and the differential metabolite found by screening is more convincing. The green point indicates the expression of the differential metabolite down-regulated. The red point indicates the expression of the differential metabolite up-regulated. The black point indicates the expression of the differential metabolite is not significant.
Venn Diagram of differential flavonoid metabolites in five pair groups for comparison.
Components
Relative content
C0
C1
C2
C3
C4
C29
Methyl gallate
19785 ± 1931f
106395 ± 15635e
157937 ± 11140d
275190 ± 19796c
463257 ± 21037b
686393 ± 22563a
Tectochrysin
0c
21822 ± 888a
20924 ± 1532ab
22378 ± 898a
18803 ± 1959b
18717 ± 1338b
Apigenin
0d
69684 ± 3765c
194160 ± 23714b
176273 ± 13523b
234647 ± 15452a
191600 ± 13688b
2′-Hydroxyisoflavone
0d
5850 ± 1480c
858 ± 3963bc
11922 ± 3714b
40597 ± 3258a
13405 ± 1608b
Luteolin
0e
99007 ± 2736d
183020 ± 31610c
399967 ± 49360a
321533 ± 39569b
194553 ± 16338c
Isosakuranetin
126767 ± 10722f
888373 ± 49580e
1784533 ± 102701a
1090167 ± 75982d
1629566 ± 57415b
1266533 ± 13737c
Eriodictyol
76828 ± 2182e
533217 ± 7574d
990117 ± 103771b
1122450 ± 108947a
851797 ± 17286c
586707 ± 13189d
6-Hydroxyluteolin
0e
36007 ± 7907d
80031 ± 11845c
109570 ± 7182b
110593 ± 6913b
455953 ± 22678a
Homoeriodictyol
1007713 ± 177275e
59225333 ± 4433949c
136000000 ± 10570000a
59222667 ± 6087200c
88361667 ± 2979724b
47430333 ± 2243746d
Hesperetin
417317 ± 52441d
26183667 ± 965291c
67591333 ± 5854918a
29585667 ± 3944180c
44605333 ± 838270b
24821667 ± 657704c
Gallocatechin
0d
4142 ± 7174d
40853 ± 13747b
91122 ± 8330a
24953 ± 3451c
14235 ± 2317 cd
Galloylgallic acid
1147 ± 1020d
0d
0d
12176 ± 2365c
29879 ± 3464b
59776 ± 6372a
5,6,7,8,3′,4′-Hexamethoxyflavanone
588443 ± 30549f
3761167 ± 134203e
5750100 ± 163503d
10055433 ± 231343b
10927667 ± 297248a
8091967 ± 60152c
5,4′-Dihydroxy-6,7-dimethoxyflavone-8-C-β-D-glucoside
67129 ± 10490d
239023 ± 95537c
340777 ± 109329c
815490 ± 50100b
755750 ± 27979b
1595533 ± 113445a
Kaempferol-3-O-(6′'-acetyl)-glucoside
255677 ± 29171a
48383 ± 6765b
26931 ± 2738bc
16761 ± 1068 cd
4683 ± 8111 cd
0d
Quercetin-O-acetylhexoside
0b
50017 ± 86631b
94460 ± 84150b
324543 ± 49272a
293150 ± 12284a
256400 ± 36369a
Isorhamnetin-acetyl hexoside
937593 ± 77367a
264943 ± 11698b
160227 ± 9203c
57247 ± 9497d
16441 ± 15156d
0d
Hesperetin C-malonylhexoside
37950667 ± 2265878a
9506000 ± 120643b
8876433 ± 770626b
2485000 ± 253531c
1109267 ± 51664c
609720 ± 167100c
Tricin 7-O-(6′'-O-Malonyl) -Beta-D-Glucoside
24980667 ± 204510a
8171500 ± 260968b
6992133 ± 575853b
2804133 ± 51771c
1316667 ± 96968 cd
588470 ± 25840d
Apigenin-7-O-[β-D-glucuronosyl(1 → 2)-O-β-D-glucuronoside)
952890 ± 39778a
0b
0b
0b
0b
0b
Apigenin-O-rutinoside-O-Hexoside
1155600 ± 67073a
218540 ± 20069bc
336853 ± 35740b
151580 ± 133363 cd
37273 ± 64559de
0e
3.4 Analysis of contents change of main flavonoids
Flavonoids are a kind of main active component in PCRC, including flavonoid glycosides and polymethoxyflavones. Fifteen polymethoxyflavones were screened out from 219 detected metabolites of flavonoids. They were tangerine, nobiletin, 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (demethylnobiletin), 3,5,6,7,8,3′,4′-heptamethoxyflavone, 5-hydroxy-6,7,3′,4′-tetramethoxyflavone, 5,7,8,4′-tetramethoxyflavone, monohydroxy-hexamethoxyflavone, 7-hydroxy-3,5,6,8-tetramethoxyflavone, sinensetin (5,6,7,3′,4′-pentamethoxyflavone), 5,6,7,8,3′,4′-hexamethoxyflavone, isosinensetin (3′,4′, 5,7,8-pentamethoxyflavone), skullcapflavone II(5,6′- dihydroxy-6,7,8,2′-tetramethoxyflavone), natsudaidain(3-hydroxy-5,6,7,8,3′, 4′-hexamethoxyflavone), 5,7,3′,4′,5′-pentamethoxydihydroflavone,and 2′-hydroxy-3,4,5,3′4′,6′-hexamethoxychalcone. 5,7,3′,4′,5′-pentamethoxydihydroflavone was first detected in PCRC. To make the change of the contents of flavonoid glycosides and polymethoxyflavones in PCRCs of different aging periods more obvious, the relative content of flavonoid glycosides (Fig. 7) and polymethoxyflavones (Figs. 8 and 9) were compared in the form of histograms.
The change of contents of four flavonoid glycosides.
The change of contents of eight polymethoxyflavones.
The change of contents of seven polymethoxyflavones.
The flavonoid glycosides with higher relative contents in PCRC were hesperidin, neohesperidin, naringin, and narirutin. The polymethoxyflavones with higher relative contents in PCRC were tangerine and nobiletin. By comparison, it was found that as the aging period extended, four flavonoid glycosides, hesperidin, neohesperidin, naringin, and narirutin, decreased, while polymethoxyflavones increased.
As shown in Fig. 7, as the aging period was extended, the neohesperidin content decreased. The neohesperidin content of the C3 group was not significant; however, the trend generally decreased. The hesperidin content also decreased as the aging period extended. After three years, the change in hesperidin content was not significant; however, it was still decreasing. Narirutin content decreased at first and then increased in the first two years of storage, slowly decreased after two years, and did not change significantly after four years. The change in naringin content was in accordance with the change in narirutin content. Generally, in the aging process, the changes in the contents of the four flavonoid glycosides slightly fluctuated but eventually significantly decreased. Flavonoid glycosides in PCRC decreased as the aging period extended.
In Fig. 8, the contents of tangerine and nobiletin slightly fluctuated; however, the change was not significant. Generally, their contents in the groups with shorter aging periods (C0, C1, C2) were lower than those in the groups with longer aging periods (C3, C4, C29), and the change was not significant among the C3, C4, C29 groups. The content of 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (demethylnobiletin) fluctuated greatly, and the content in the C0, C3, and C29 groups did not change significantly and was higher than that in the other groups. The content of 3,5,6,7,8,3′,4′-heptamethoxyflavone did not change significantly from group C0 to group C1, increased gradually from group C1 to group C4, and decreased slightly in groups C4 and C29. The content of 5-hydroxy-6,7,3′,4′-tetramethoxyflavone fluctuated and was highest in group C3 and then decreased. The content of 5,7,8,4′-tetramethoxyflavone decreased from group C0 to group C1, increased gradually from group C1 to group C3, and slightly decreased from C3 to group C29. The contents of monohydroxy-hexamethoxyflavone and 7-hydroxy-3,5,6,8-tetramethoxyflavone had similar changing trends, which gradually decreased from group C0 to group C2, sharply increased from group C2 to group C3, and fluctuated from group C3 to group C29 with relatively slight changes.
In Fig. 9, the content of sinensetin (5,6,7,3′,4′-pentamethoxyflavone) first decreased and then increased to its highest level from group C0 to C3 and did not change significantly thereafter. The content of 5,6,7,8,3′,4′-hexamethoxyflavone increased to its highest level from group C0 to group C4 and slightly decreased from group C4 to group C29. The content of natsudaidain (3-hydroxy-5,6,7,8,3′,4′-hexamethoxyflavone) slightly fluctuated and was lower in the groups with shorter aging periods (C0, C1, C2) than in the groups with longer aging periods (C3, C4, C29). The content of isosinensetin (3′,4′,5,7,8-pentamethoxyflavone) was nearly unchanged from group C0 to group C2, increased to its highest level from group C2 to group C3, and gradually decreased thereafter. The content of skullcapflavone II (5,6′- dihydroxy-6,7,8,2′-tetramethoxyflavone) had a change of fluctuation, which gradually decreased from groups C0 to C2, increased from group C2 to group C3, and fluctuated from group C3 to group C29. The content of 5,7,3′,4′,5′-pentamethoxydihydroflavone remained stable from group C0 to group C2, then increased, and obtained the highest content in group C29. The content of 2′-hydroxy-3,4,5,3′4′,6′-hexamethoxychalcone steadily increased from group C0 to group C29.
In general, in 15 polymethoxyflavones, the content of most of them in PCRC did not have a certain simple trend. However, the contents of polymethoxyflavones in groups with shorter aging periods were lower than those in groups with longer aging periods.
3.5 Evaluation of total antioxidant activity
To investigate the impact of the change in flavonoid content in PCRC during the aging process on the bioactivity, FRAP, ABTS, and DPPH methods were employed to determine the total antioxidant activity of the extract solutions of PCRC in six groups with different aging periods (Table 3). The antioxidant potency composite index (APC) was used to evaluate the total antioxidant activity. The results were C3 > C4 > C2 > C29 > C0 > C1, and the APCs of group C3 and group C4 were significantly higher than those of the other four groups, indicating that they had better antioxidant potency. Wang et al(Wang et al., 2017) reported that the total phenols of freshly harvested PCRC had the best antioxidant potency, as tested by the peroxyl radical scavenging capacity (PSC) test. PCRC aged for 1 year and 13 years had the best oxygen radical absorbance capacity (ORAC) test, and PCRC aged for 6 years had the best cellular antioxidant activity (CAA) test. Note: FRAP/ABTS method use equivalent weight of FeSO4·•7H2O/Trolox, in unit of mM, the larger the stronger potency. DPPH method use 50% inhibiting concentration (IC50), in unit of mg/mL, the smaller the stronger potency.
No.
Total antioxidant activity
IC50
APC
FRAP(mM)
ABTS(mM)
DPPH(μg/mL)
C0
1.66 ± 0.04b
0.45 ± 0.01c
2.43 ± 0.43ab
38.45
C1
1.39 ± 0.02d
0.55 ± 0.02b
2.58 ± 0.10a
32.98
C2
1.65 ± 0.08bc
0.51 ± 0.07bc
2.45 ± 0.10ab
44.97
C3
2.09 ± 0.15a
0.77 ± 0.01a
1.23 ± 0.08c
140.75
C4
1.49 ± 0.05c
0.79 ± 0.03a
2.07 ± 0.41b
86.35
C29
1.78 ± 0.08b
0.44 ± 0.02c
2.48 ± 0.12ab
41.69
3.6 Molecular docking
To recognize the effect of PCRC in preventing and treating COVID-19, molecular docking was used to evaluate the binding energy of flavonoids and positive control drugs separately at the protein structures of Spike, 3CLpro, PLpro, and RdRp. A lower binding energy indicates stronger affinity and better potential efficacy. The results of the positive control drugs are shown in Table 4-1. Lopinavir had the lowest binding energy at 3CLpro, RdRp, and spike proteins, with values of −6.20 kcal/mol, −10.10 kcal/mol, and −11.60 kcal/mol, respectively. Ribavirin had the lowest binding energy at the PLpro protein, with a value of −7.30 kcal/mol.
positive control drug
Binding energy(kcal/mol)
3CLpro
RdRp
PLpro
Spike protein
Lopinavir
−6.20
−10.10
−6.23
−11.60
Ritonavir
−6.13
−8.67
−6.20
−9.37
Ribavirin
−5.90
−7.70
−7.30
−7.50
Chloroquine
−6.10
−6.97
−5.07
−7.13
Arbidol
−6.03
−8.03
−5.00
−7.00
Favipiravir
−4.73
−6.20
−6.43
−6.07
Table 4-2 lists the 32 components from PCRC that had lower binding energy at 3CLpro than lopinavir. Isoschaftoside had the lowest value of −9.40 kcal/mol. In addition, some flavonoids abundant in PCRC had lower binding energies than lopinavir, e.g., hesperidin −6.47 kcal/mol, naringin −7.80 kcal/mol, narirutin −8.87 kcal/mol, neohesperidin −8.17 kcal/mol, nobiletin −6.80 kcal/mol, and tangeretin −6.80 kcal/mol.
No.
Flavonoids name
Binding energy(kcal/mol)
No.
Flavonoids name
Binding energy(kcal/mol)
1
Isoschaftoside
−9.40
17
Ononin
−7.63
2
Vitexin
−9.00
18
Phlorizin
−7.40
3
Narirutin
−8.87
19
Tricin 7-O-(6′'-O-Malonyl) -Beta-D-Glucoside
−7.20
4
Kaempferin
−8.87
20
Gallocatechin
−7.13
5
Isorhoifolin
−8.83
21
Nicotiflorin
−7.10
6
Quercitrin
−8.73
22
Hesperetin
−7.07
7
Linarin
−8.70
23
Luteolin
−6.97
8
Astragalin
−8.47
24
6-Hydroxyluteolin
−6.90
9
Neohesperidin
−8.17
25
Tangeretin
−6.80
10
Naringenin-7-O-glucoside
−8.13
26
Nobiletin
−6.80
11
Rhoifolin
−8.00
27
5,6,7,8,3′,4′-Hexamethoxyflavanone
−6.73
12
Lonicerin
−7.90
28
Apigenin
−6.70
13
Naringin
−7.80
29
Tectochrysin
−6.50
14
Sissotrin
−7.80
30
Hesperidin
−6.47
15
Tiliroside
−7.73
31
2′-Hydroxyisoflavone
−6.37
16
Cynaroside
−7.67
32
Saponarin
−6.33
Table 4-3 lists the 13 components from PCRC that had lower binding energy at RdRp than lopinavir. Linarin had the lowest value of −11.93 kcal/mol. In addition, some flavonoids abundant in PCRC had lower binding energies than lopinavir, e.g., naringin −10.77 kcal/mol, narirutin −11.77 kcal/mol, and neohesperidin −10.20 kcal/mol.
No.
Flavonoids name
Binding energy(kcal/mol)
No.
Flavonoids name
Binding energy(kcal/mol)
1
Linarin
−11.93
8
Rhoifolin
−10.73
2
Isorhoifolin
−11.80
9
Saponarin
−10.70
3
Narirutin
−11.77
10
Hesperetin
−10.40
4
Lonicerin
−11.13
11
Neohesperidin
−10.20
5
Isoschaftoside
−11.10
12
Gallocatechin
−10.07
6
Nicotiflorin
−11.00
13
Cynaroside
−10.07
7
Naringin
−10.77
Table 4-4 lists the 12 components from PCRC that had lower binding energy at PLpro than ribavirin. Neohesperidin had the lowest value of −7.83 kcal/mol. In addition, naringin and narirutin had values of −7.80 kcal/mol and 7.50 kcal/mol, respectively.
No.
Flavonoids name
Binding energy(kcal/mol)
No.
Flavonoids name
Binding energy(kcal/mol)
1
Neohesperidin
−7.83
7
Astragalin
−7.57
2
Naringin
−7.80
8
Kaempferin
−7.53
3
Quercitrin
−7.80
9
Narirutin
−7.50
4
Isorhoifolin
−7.67
10
Cynaroside
−7.33
5
Linarin
−7.63
11
Nicotiflorin
−7.33
6
Lonicerin
−7.57
12
Gallocatechin
−7.33
Table 4-5 lists the five components from PCRC that had lower binding energy at spike than lopinavir. Isoxaphoroside had the lowest value of −13.27 kcal/mol. Naringin and hesperidin had values of −11.80 kcal/mol and 11.60 kcal/mol, respectively.
No.
Flavonoids name
Binding energy(kcal/mol)
No.
Flavonoids name
Binding energy(kcal/mol)
1
Isoschaftoside
−13.27
4
Rhoifolin
−11.80
2
Lonicerin
−11.83
5
Isorhoifolin
−11.67
3
Naringin
−11.80
6
Hesperidin
−11.60
To compare the potential antiviral activities of PCRC with different aging periods against SARS-CoV-2, the total content of flavonoids with lower binding energy than the positive control drug was added up for separate targeting proteins and separate aging periods. Fig. 10a shows that the total content of 32 flavonoids with lower binding energy than the positive control drug at 3CLpro varied with the aging period. It was found that the content of group C0 was highest. Of the rest of the groups, the content of group C3 was the highest. Fig. 10b, c, and d show the results for the binding target proteins RdRp, PLpro, and Spike, respectively. Generally, the total content of flavonoids with lower binding energy than the positive control drug was highest in group C0, decreased in the one-year aging process, increased to the second-highest level, and decreased as the aging period extended.
The total content of flavonoids with lower binding energy than positive control drug docking with the four targeting proteins in PCRC with different aging periods. a The total content of flavonoids with lower binding energy than positive control drug at 3CLpro. b The total content of flavonoids with lower binding energy than positive control drug at RdRp. c The total content of flavonoids with lower binding energy than positive control drug at PLpro. d The total content of flavonoids with lower binding energy than positive control drug at Spike.
4 Discussion
In the latest report, 92 flavonoids were detected from the methanol extract solution of PCRC (Zhang et al., 2020), and up to 56 polymethoxyflavones were identified from PCRC (Zeng et al., 2017). The marker components of PCRC recorded in the Chinese Pharmacopeia (2020 version, One Sections) were hesperidin, nobiletin, and tangeretin (National Pharmacopoeia Commission, 2020). Studying the main components, hesperidin, nobiletin, and tangeretin, in PCRC were beneficial to the quality evaluation of PCRC (Wang et al., 2016; Liu et al., 2020; Zheng et al., 2010; Fu et al., 2017; Zhang et al., 2020). However, in the aging process, the contents of polyphenols, flavonoids, and particularly polymethoxyflavones undergo a series of changes (Wang et al., 2016; Zhang et al., 2020). Therefore, investigating the overall change in flavonoids in the aging process of PCRC is a prerequisite and guarantees reasonable quality control and standard establishment for PCRC. In this study, PCRC samples from the same tree (excluding the group of aging periods of 29 year), same orchard, and stored under the same conditions were used as objects. The total flavonoid contents of PCRC from six groups with different aging periods were determined by spectrophotometry. The results showed that with increasing aging time, the total flavonoid contents of PCRC decreased at first, then increased, and then decreased again. Furthermore, UPLC-MS/MS and widely targeted metabolomics analysis were employed to intensively investigate the composition of the flavonoids in PCRC with different aging periods. Up to 219 metabolites of flavonoids were detected in all samples, including 82 flavones, 56 flavonols, 26 flavonoid C-glycosides, 19 dihydroflavones, 7 isoflavones, 7 tannins, 5 flavanols, 5 dihydroflavonols, 4 anthocyanins, 3 chalcones, 2 other flavonoids, 2 proanthocyanidins, and 1 dihydroisoflavone. Among them, two flavonoids, 5,7,3′,4′,5′-pentamethoxy- dihydroflavones and 2′-hydroxy-3,4,5,3′,4′,6′-hexamethoxychalcone, were detected in PCRC for the first time. By clustering analysis, it was found that samples of freshly harvested PCRC and PCRC with aging periods of 1 year and 2 year clustered into one class. Samples of PCRC with an aging period of 3 year and over 3 years clustered into the other class. This result indicated that qualitative and quantitative changes occurred in the component composition of PCRC with an aging period of over 3 years. Further analysis reviewed that polymethoxyflavones in groups with longer aging periods (3, 4, 29 year) were more abundant than those in groups with shorter aging periods (0, 1, 2 year). Polymethoxyflavones are one kind of important component in PCRC. Because their molecular structures are more lipophilic, they penetrate biofilms more easily and enhance bioavailabilities. Therefore, the bioactivity of polymethoxyflavones was better than that of the relevant structure without methoxylation (Hung et al., 2018; Li et al., 2006). Polymethoxyflavones not only have good antioxidative activity but also have an effect on relieving metabolic syndromes such as hypertriglyceridemia, fatty liver, and insulin resistance by regulating enteric microorganisms and amino acid metabolism (Fu et al., 2017; Ding et al., 2008; Zeng et al., 2020a, 2020b). They have antitumor effects by inhibiting the growth of tumor cells and inducing the apoptosis of tumor cells (Whitman et al., 2005; Hung et al., 2018; Li et al., 2006). The results reviewed the aging process to enhance the compound number and total content of the polymethoxyflavones in PCRC and further indicated the scientific meaning of “the longer the aging period is, the better” for PCRC.
However, regarding the antioxidative effect, the potency of PCRC in the groups aged 3 years was better than that in the other 5 groups. The group with the longest aging period of 29 years did not present a significant advantage. Its potency was only equal to that of the group with an aging period of 2 years. The results explained the record of an aging period of 2 ∼ 3 years in a medical book written by Yuhe Xu in the Qing Dynasty (Xu, 1990; He et al., 2019).
Of all the differential flavonoid metabolites in the five pairs of PCRC groups, C0 vs C1, C0 vs C2, C0 vs C3, C0 vs C4, and C0 vs C29, 21 were the same. Seven flavonoids (tectochrysin, apigenin, 2′-hydroxyisoflavone, luteolin, 6-hydroxyluteolin, gallocatechin, quercetin-O-acetylhexoside) were detected in five groups of aging PCRC samples and were not detected in newly harvested PCRC samples. Apigenin-7-O-[β-D-glucuronide (1 → 2)-O-β-D-glucuronide] was only detected in freshly harvested PCRC samples. This result indicated that the aging process might prompt the synthesis or decomposition of some flavonoids by initiating special biochemical metabolic processes. It was reported that some key microbes associated with C. reticulata 'Chachi' could be involved in the improvement of its health properties (He et al., 2019). In addition, 5,7,3′,4′,5′- pentamethoxy dihydroflavone and 2′- hydroxy-3,4,5,3′4′,6′- hexamethoxychalcone were found from PCRC for the first time. They can not be found in all other literatures. Similar substances, like Citrus polymethoxyflavone (2S)-5,6,7,3′,4′-pentamethoxyflavanone (PMF) was a novel Nrf2 (nuclear factor E2-related factor 2) activator with potential prevention against oxidative insults (Zhou et al., 2019). However, whether these two new flavonoids in PCRC have pharmacological activity and possible application deserve further study. Seven flavonoids appeared, and flavonoids disappeared in the aging process, which might be the main object components for later studies relating to flavonoid metabolism regulated by microbes in the aging process.
Currently, COVID-19 is still prevalent around the world. Although vaccines were invented, there is still much uncertainty. New mutated viruses emerged and led to an enhanced ability to spread and continue to deteriorate the epidemic situation in some local areas. Finding active compounds to prevent and/or treat COVID-19 is important and urgent. Some flavonoids were reported to interact with some targets of SARS-CoV-2 (National Health Commission of the people's Republic of China, 2020). Interfering with the spike protein of SARS-CoV-2 and the ACE2 receptor of the host or inhibiting RNA polymerase and important proteases (3CLpro and PLpro) inhibit virus reproduction and might be a potential treatment for COVID-19 (National Health Commission of the people's Republic of China, 2020). Molecular docking was performed using flavonoids from PCRC targeting the spike protein, 3CLpro, PLpro, and RdRp of SARS-CoV-2. It was found that many flavonoids from PCRC had better affinity for the target than positive control drugs. The total content of flavonoids with lower binding energy than the positive control drug was highest in newly harvested PCRC. This result indicated that newly harvested PCRC would be more appropriate for the preparation to prevent or treat COVID-19. The results are only based on molecular docking. In vivo and in vitro experiments should be further carried out to evaluate the antiviral activity of PCRC flavonoids against SARS-CoV-2.
5 Conclusions
UPLC-MS/MS and metabolomics analysis were employed to analyze the flavonoids of PCRC with different aging periods for the first time. The results showed that the aging process prompted qualitative and quantitative changes in flavonoids in PCRC, and polymethoxyflavone significantly increased in PCRC aged for 3, 4, and 29 years. However, the potential antiviral components against SARS-CoV-2 decreased as the aging period extended. The results of APC experiments showed that PCRC with aging periods of 3 and 4 years presented significantly higher antioxidative potency than the other groups, and PCRC with an aging period of 29 years did not present an antioxidative advantage. Therefore, it was recommended to use PCRC with an aging period over 3 years according to the content of bioactive polyemethoxyflavones, and for antioxidative use, PCRC with an aging period of 3 ∼ 4 years would be better. For use in preparation preventing or treating COVID-19, newly harvested PCRC would be more appropriate. In addition, 5,7,3′,4′,5′-pentamethoxy dihydroflavone and 2′-hydroxy-3,4,5,3′4′,6′- hexamethoxychalcone were found from PCRC for the first time. At the same time, seven flavonoids (tectochrysin, apigenin, 2′-hydroxyisoflavone, luteolin, 6-hydroxyluteolin, gallocatechin, quercetin-O-acetylhexoside) were detected in aging PCRC samples, and apigenin-7-O-[β-D-glucuronide (1 → 2)-O-β-D-glucuronide] disappeared after aging. The results in this study supplied scientific guidance information for fully evaluating component changes in PCRC with different aging periods and rational utilization in medicine and supplied important clues for intensively investigating the synthesis of related compounds and the regulatory mechanism of metabolism in the aging process.
Acknowledgements
This study was supported by Key realm R&D Program of Guangdong Province(2020B020221001), The National Natural Science Foundation of China (31570180), Agricultural Science and Technology Innovation and Promotion Project of Guangdong (2018LM2160), Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2019KJ125), Fund of National Modern Agricultural Industrial Park of Xinhui Tangerine Peel and Fund of Development and Application of Active Components of Medicinal Plants.
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Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103653.
Appendix A
Supplementary material
The following are the Supplementary data to this article:Supplementary data 1
Supplementary data 1
