Introduction
Honey can be divided into different types according to the differences in nectar source plants, different honey components are different, and metabolic components are also different (Zhao et al., 2023). In addition to physical and chemical components such as sugars, enzymes, minerals, vitamins, flavonoids, hydrogen peroxide and amino acids, some honey also contains iconic ingredients, such as caffeic acid in Jingtiao honey, 4-hydroxyquinoline in Jujube honey, Acacia honey with locust glycoside and so on (Wu et al., 2022; Khallouki et al., 2020; Xing et al., 2017). Non-target metabolomics provides a reliable analytical platform for the qualitative and quantitative systematic study of endogenous metabolite components in organisms and is widely used in the study of animals, plants and microorganisms (Pankaj et al., 2023). It provides a reliable analysis platform for the qualitative and quantitative systematic study of endogenous metabolite components, and can well detect metabolite processes in organisms and obtain information about the originality of honey. It provides theoretical basis for the antibacterial effect, antioxidant activity and molecular mechanism of improving oxidative stress-related inflammation of honey, and provides new ideas for the application of multiomics technology in honey medicine (Wang et al., 2022). At present, honey metabolomics research methods are also becoming more mature and increasingly important, and differences in metabolic components and contents lead to different antibacterial activities of different honeys.
Studies have shown that the composition and bacteriostatic activity of honey have a significant correlation with the nectar of nectar from nectar-source plants and a variety of mineral elements in the soil at the place of origin (Grainger et al., 2021). The source of nectar from the Hengshan Astragalus honey plant is the Astragalus from the Hengshan Nature Reserve, Hengshan is the main Astragalus production area in Shanxi, altitude 1 612 m–2 778 m, pollution-free, original and unique geology has shaped its authentic quality. Chen found that Astragalus had a high content of selenium, barium, calcium, etc., and its flowers were rich in flavonoids, fructose, and other substances (Chen et al., 2011). It can be inferred from this that the perfect combination of regional and ecological excellence and authentic Astragalus honey source plants will surely achieve the unique ingredients and effects of Hengshan Astragalus honey. However, currently there are few studies on Astragalus honey, and research on the efficacy of Astragalus honey has rarely been reported in the Hengshan Nature Reserve.
In this study, the antibacterial properties, secondary metabolite components and bacteriostatic effects of Hengshan Astragalus honey were analyzed by taking Hengshan Astragalus honey, Shanxi Acacia honey, Xinjiang Jujube honey and Longxi Astragalus honey as control honey from four medicinal plants. It provides a theoretical basis for the study of the antibacterial mechanism of Hengshan Astragalus honey and also provides scientific support for the development and utilization of the high-quality Hengshan Astragalus honey.
Materials and Methods
Materials
Experimental materials The five types of honey are supplied directly from the source, namely Hengshan Astragalus honey (HH) (Hunyuan, China), Shanxi Acacia honey (AH) (Jinzhong, China), Shanxi Jingtiao honey (JH) (Jingzhong, China), Longxi Astragalus honey (LH) (Longxi, China) and Xinjiang Jujube honey (XH) (Akesu, China), All has identified as single nectar by palynology; The three wound infections were Escherichia coli (ATCC25922, China), Pseudomonas aeruginosa (ATCC9027, China), and Staphylococcus aureus (ATCC6538, China), all purchased from the Beijing Biological Preservation Center.
Experimental reagents Methanol, formic acid, acetonitrile, ammonia, LC-MS grade, CNW Technologies, Inc; Ethanol, oxalic acid, sulfuric acid solution, rutin standard, sodium thiosulfate standard solution, starch indicator, catalase, aluminum trichloride, potassium iodide, ammonium molybdate, activated carbon, thiourea, 2,4-dinitrophenylhydrazine.
Main instruments and equipment EXION LC System (SCIEX) Ultra Performance Liquid Chromatography Instrument, SCIEX 6500 QTRAP+ Triple Quadrupole Mass Spectrometer, Sciex brand. Digital Brix Meter LH-B55, PH Meter (Hangzhou Luheng Biotechnology Co., Ltd. Portable acidity measure PHB-4), electronic balance JH1102 (Shanghai Jingke Tianmei Scientific Instrument Co., Ltd.), SB-5200D Ultrasonic Cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.), 722E Visible Spectrophotometer (Shanghai Spectroscopic Instrument Factory), high-speed TGL-16 desktop centrifuge.
Experimental methods
Physical and chemical indicators of honey The determination of honey moisture content, 20.0 g of honey samples were accurately weighed and the volume was set to 100 mL with distilled water, and the sugar content was determined by a sugar meter in accordance with SN/T 0852-2012 “Inspection Regulations for Import and Export of Honey”, and the sugar content was converted into a percentage of water (Hao et al., 2016). The pH of honey is measured using a pH meter. The determination of the total flavonoid content was determined according to the national standard (GB/T20574-2006), The content of flavonoids was quantitatively determined by AlCl colorimetric method according to the yellow depth of Al complex. According to the national standard (GB/T5009.1-2003), the hydrogen peroxide content was measured using the iodine measurement method. Vitamin content is determined colorimetrically by 2,4-dinitrophenylhydrazine (Jiang et al., 2019).
Component determination of the Ultra High Performance Lliquid Chromatography Mass Spectrometer (UHPLC-MS). Chromatographic conditions, mass spectrometry conditions according to the Baiqu experimental protocol (Zha et al., 2018): using ultraperformance liquid chromatography of the EXION LC System (SCIEX), chromatographic separation of target compounds using a Waters UPLC liquid chromatography column. Phase A of liquid chromatography is an aqueous solution containing 0.1 % formic acid, and phase B is acetonitrile. The column oven temperature is 40 °C, the autosampler temperature is 4 °C, and the injection volume is 2 µL. The time unit is min, and the flow rate unit is mg·kg−1.
Honey bacteriostatic experiment by punching method Referring to the bacteriostatic experiment of Elizabeth et al. (2016), slightly modified. Three tubes of cryopreservation strains −4 °C were inoculated in beef paste peptone liquid medium, incubated at 37 °C for 24 h to amplify, and diluted with normal saline to a bacterial suspension at a concentration of 105–106 CFU/mL for later use. Dilute honey samples with sterilized distilled water to 10 %, 20 %, 30 %, 40 %, 50 %, 60 %. Spread 15 mL of pure nutrient agar medium in a 90 mm Petri dish with a syringe; After cooling and solidification, the beef paste peptone medium at about 50 °C is added to the bacterial capacity of 3 %, the bacterial solution is mixed with the medium evenly. Absorb 15 mL of the mixed medium with a syringe and inject it into the upper layer of the pure medium plate to make a double-layer plate. Use a hole punch to make 6 holes (diameter 6mm) evenly on the medium, then add 200µL honey diluent or blank control (sterile water) in the holes, and place the petri dish in a constant temperature incubator at 37 °C for 24 h. The minimum bactericidal concentration (MBC) of the honey samples was the minimum honey concentration corresponding to allowing less than 0.1 % bacterial growth, 3 times parallel per group.
Data processing The acquisition and quantification of raw metabolic data for target compounds are performed using SCIEX Analyst Work Station Software (Version 1.6.3). MS conventer software is used to convert the raw mass spectrum to TXT format. Then use the Biotree DB self-written R package combined with the self-built database to complete peak lifting, annotation, and other work. Multidata statistical analysis was used to perform principal component analysis (PCA), cluster analysis (CA), and orthogonal partial least squares discriminant analysis (orthogonal PLS-DA, OPLS-DA). Differential metabolites were selected by combining the variance multiple value and the variable importance in project (VIP) value of the OPLS-DA model. Volcanic maps and hierarchical clustering maps were drawn by https://www.omicshare.com/ and the enrichment of the antibacterial differential metabolite pathway was performed in the KEGG network.
Finally, the diameter (mm) of the inhibition circle was measured using the cross-method using a vernier caliper, and the sensitivity of the three bacteria to different honeys was judged according to the size of the inhibition circle. The diameter of the inhibition circle (Yao, 2002): d ≥ 25 mm is extremely sensitive (++++), 25 mm > d ≥ 20 mm is highly sensitive (+++), 20 mm > d ≥ 15 mm is moderately sensitive (++), 15 mm > d > 6 mm is low sensitivity (+), d = 6 mm without the inhibitory circle is insensitive (−).
Results and Discussion
Analysis of physical and chemical indexes of honey The moisture, pH value, total flavonoid content, hydrogen peroxide content, and vitamin C content of five honeys were determined, and the results are shown in Table 1.
Table 1. Physical and chemical indexes of five kinds of honey.
| Physical and chemical parameters |
Longxi Astragalus honey |
Shanxi Jingtiao honey |
Hengshan Astragalus honey |
Shanxi Acacia honey |
Xinjiang Jujube honey |
| Moisture content /(%) |
18.11 ± 1.21b |
19.16 ± 1.17ab |
19.96 ± 0.40a |
15.01 ± 0.94c |
20.31 ± 0.57a |
| PH value |
4.26 ± 0.09b |
3.68 ± 0.06d |
4.02 ± 0.04c |
4.14 ± 0.01bc |
4.79 ± 0.03a |
| Total flavonoid content (mg·g−1) |
0.443 ± 0.01a |
0.025 ± 0.01b |
0.349 ± 0.00c |
0.066 ± 0.00d |
0.124 ± 0.00e |
| Hydrogen peroxide content (mg·kg−1) |
8.06 ± 0.38b |
8.54 ± 0.11b |
7.74 ± 0.45b |
6.80 ± 0.36a |
8.26 ± 0.29b |
| Vitamin C content (mg·kg−1) |
94.67 ± 11.98a |
131.10 ± 2.71b |
193.71 ± 10.00c |
555.14 ± 10.79d |
113.96 ± 2.89e |
Different letters in colums indicate a different homogenous group (α = 0.05).
The level of moisture content is closely related to the growth and biochemical reaction of microorganisms, and honey with high water content is easy to be infected by bacteria and fermentation and deterioration, and it is of great significance to study and determine the moisture content of honey. Table 1 showed that the water content of different honeys was different. The water content from high to low was as follows: Xinjiang Jujube honey > Hengshan Astragalus honey > Shanxi Jingtiao honey > Longxi Astragalus honey > Shanxi Acacia honey. The results of the difference significance analysis showed that the difference between Hengshan Astragalus honey and Xinjiang Jujube honey was not significant. It was significantly different from Shanxi Jingtiao honey; it was very different from Longxi Astragalus honey and Shanxi Acacia honey. In addition to the influence of the honey source region, the climate of the honey source, the harvesting time, etc. (Sui, 2011), which may also be related to the degree of dehydration during honey processing and the different degree of crystallization of the five honeys before the test.
The pH value of microbial growth and reproduction is mostly 7.2–7.4, the pH value of organic acid in honey is between 3.2–4.5, and the amount of combined acid reaches 0.43 %, which is enough to inhibit the growth and reproduction of a variety of pathogenic bacteria (Liu et al., 2023). The acidity of five honey samples was determined and it was found that the pH value of five honeys, except Xinjiang Jujube honey, was slightly higher than this range, and the rest were in this range. The pH of the five honeys was much lower than the pH range of microbial growth and reproduction. The pH of Hengshan Astragalus Honey is 4.02 ± 0.04, which is low among the five honeys, and its acidity is speculated to be sufficient to inhibit the growth and reproduction of some pathogenic bacteria. Analysis of the differences showed that the pH values of Hengshan Astragalus honey were significantly different from those of Shanxi Jingtiao honey, Xinjiang Jujube honey, and Longxi Astragalus honey, and significantly different from Shanxi Acacia honey. The pH value of honey is related to the nectar plant species, the production environment, and the climate. In addition, the condition and detection method of honey will also affect the measured pH value. The factors that affect the pH value of honey are diverse and factors that affect the pH value of honey need to be further studied.
Flavonoids are antioxidant, eliminate free radicals, antiradiation, promote tissue regeneration, and other effects, and it is of great significance to study their bacteriostatic properties (Liu et al., 2023; Rao, et al., 2022; Mandal and Mandal et al., 2011). The total flavonoid content of Hengshan Astragalus honey was 0.349 mg·g−1, which was only lower than that of Longxi Astragalus honey (0.443 mg·g−1). The total flavonoid content of the two Astragalus honeys was significantly higher than that of the other three honeys, which was speculated to be related to the higher total flavonoids content in Astragalus. The results of this study showed that the total flavonoid content of the five honeys was significantly or very significant.
Hydrogen peroxide is an important antibacterial active substance in honey, which can achieve an antibacterial effect by releasing strong oxidizing free radicals and binding to the hydrogen sulfide groups of proteins in microbial cells, inactivating enzymes (Mandal and Mandal et al., 2011).
We found that the lowest hydrogen peroxide content of Shanxi Acacia honey was 6.80 ± 0.36 mg·kg−1, and the hydrogen peroxide content of other honeys was very significant, while the hydrogen peroxide content of other honeys was not significant. Brudzynski found that high concentrations of hydrogen peroxide had a significant effect on the bacteriostatic activity of honey (Brudzynski et al., 2006).
As one of the nutrients of honey, vitamin C has various effects, such as antibacterial, bacteriotic, antioxidant, and antitumor (Zhang et al., 2021). The findings of the study showed that the vitamin C content of honey from different sources was different and the difference between them was very significant. The vitamin C content of Hengshan Astragalus honey was (193.71 ± 10.00) mg·kg−1, second only to Shanxi Acacia honey (555.14 ± 10.79) mg·kg−1.
Different honey has different chemical composition and content, Hengshan Astragalus honey has a low pH value, a high content of total flavonoids and vitamin C. These substances are the main markers to distinguish Hengshan Astragalus honey, and also lay a material foundation for it to have good bacteriostatic properties. Total flavonoids are well-known antioxidants and important bacteriostatic components (Basualdo et al., 2007). Vitamin C is an important physiologically active substance in honey and promotes wound healing (Cheraskin et al., 1982).
UHPLC-MS analysis
Identification and analysis of secondary bacteriostatic metabolites UHPLC-QE-MS was used to identify five secondary bacteriostatic metabolites of honey, which could judge that the results of extraction and detection of metabolites were stable and the data were reliable and reproducible. A total of 334 secondary metabolites were identified by UHPLC-QE-MS and 87 key inhibitory secondary metabolites in 31 classes were screened according to the properties of the bacteriostatic components (mainly including 13 phenols, 8 alkaloids, 7 flavonoids, 6 phenylpropionins, 5 hydroxylic acids and their derivatives, 3 flavonoids, etc.). The analysis of the principal components showed that five secondary bacteriostatic metabolites of honey could be well separated (Fig. 1). Further OPLS-DA analysis was carried out on Hengshan Astragalus honey and other honeys in Shanxi Province, as shown in Fig. 2A–D, indicating that the displacement model was basically reasonable and that the differences between groups between Hengshan Astragalus honey and the other four honeys were small and the differences between the groups were large, indicating that there were differences in the types and contents of different honey components.
Screening of differential bacteriostatic metabolites and analysis of differential metabolic pathways The differential metabolites between Hengshan Astragalus honey and other honey were screened according to the filter criteria VIP > 1 and p-value < 0.05. The volcano map (Fig. 3A–D) was drawn and there were 14, 35, 5 and 13 bacteriostatic metabolites between the Hengshan Astragalus honey and the Longxi Astragalus honey, the Shanxi Jingtiao honey, the Shanxi Acacia honey and the Xinjiang Jujube honey, respectively. The highest concentration of Hengshan Astragalus Honey is maltotriose (Organic oxygen compounds, 0.0737), D-2-Aminobutyric acid (carboxylic acids and their derivatives, 0.0145), tanshinneoketone (diterpenoids, 0.013), 3α,6β-BETA-diacrotonyloxyscopola-7β-ol (alkaloids, 0.0111), artemisinin (sesquiterpenes, 0.002), and the substances with lower content are chrysin (flavonoids, 9.367E-06), Caffeic acid (phenylpropanoids 9.053E-06), N-γ-acetyl-N-2-formyl-5-methylkynurenine (amino acids and their derivatives, 8.265E-06), N-acetyl-L-glutamic acid (amino acids and their derivatives, 6.567E-06), fumarate (organic acids and their derivatives, 4.962E-06), so the author believes that the above substances are unique markers of Hengshan Astragalus honey.
The results of the clustering of Hengshan Astragalus honey and other honeys are shown in Fig. 4A–D. Among the differential bacteriostatic metabolites of Hengshan Astragalus honey and Longxi Astragalus honey, pregnenolones, phenols, steroids and their derivatives, and flavonoids are clustered into one class, and other substances are clustered into a group, the former substances have strong anti-inflammatory effects and can promote physiological properties of wound healing, among which sancin C has obvious inhibitory reactive oxygen species and NO release ability to promote the expression of inflammatory cytokines. Among the bacteriostatic metabolites of Hengshan Astragalus honey and Xinjiang Jujube honey, phenols, alkaloids, steroids and their derivatives were clustered into one class, while abscisic acid, which was a plant hormone, was clustered in a separate class, and the former 1′-acetoxypiperoxyphen acetate phenolic had a significant inhibitory effect on fungi. The latter abscisic acid is an organic acid, which has a strong acidity and can inhibit the growth and reproduction of microorganisms, thus playing a bacteriostatic role.
Antibacterial effect of honey The experiment of drilling method showed that all the five honey samples could inhibit the three bacteria (Table 2). The MBC value of Hengshan Astragalus honey to both E. coli and P. aeruginosa gramnegative bacteria was 30 %, showing high inhibition. The MBC value of the gram-negative Staphylococcus aureus was 50 %, showing moderate inhibition. Combined with the conclusion of physicochemical experiment, the analysis showed that the two kinds of Astragalus honey had high flavonoid content alone, which did not mean that the antibacterial performance was similar. Multiple physicochemical indexes are needed to evaluate the bacteriostatic ability. Antibacterial effect was affected by phenols, flavonoids, osmotic pressure, acidity, antimicrobial peptides, royal pulp main protein and other factors.
Table 2. Minimum bactericidal Concentration of five kinds of honey (MBC).
| Name |
MBC (%) |
| Escherichia coli |
Staphylococcus aureus |
Pseudomonas aeruginosa |
| Longxi Astragalus honey |
50 |
60 |
30 |
| Shanxi Jingtiao honey |
60 |
50 |
40 |
| Hengshan Astragalus honey |
30 |
50 |
30 |
| Shanxi Acacia honey |
40 |
50 |
40 |
Xinjiang
jujube
honey |
60 |
60 |
50 |
When the concentration of honey was 50 %, the overall antibacterial effect was consistent and good (Table 3). The strongest bacteriostatic effect was Shanxi Jingtiao honey, and the three kinds of bacteria were highly sensitive to it. There was no significant difference between the other four, except for the extremely strong inhibitory effect of Shanxi Jingtiao honey on Pseudomonas aeruginosa. They all showed extreme sensitivity. By comparing the antibacterial effect of Hengshan Astragalus honey with other four kinds of honey, We found that the antibacterial effect was similar to that of locus Shanxi Acacia honey.
Table 3. Size and antibacterial grade of five types of honeys inhibition circle.
| Honey sample |
Escherichia coli |
Staphylococcus aureus |
Pseudomonas aeruginosa |
| Bacteriostatic ring diameter (mm) |
Bacteriostatic grade |
Bacteriostatic ring diameter (mm) |
Bacteriostatic grade |
Bacteriostatic ring diameter (mm) |
Bacteriostatic grade |
| Longxi Astragalus honey |
21.65 ± 1.18b |
+++ |
28.10 ± 1.08a |
++++ |
27.29 ± 0.93ab |
++++ |
| Shanxi Jingtiao honey |
32.87 ± 1.41a |
++++ |
28.57 ± 0.45a |
++++ |
29.41 ± 1.18a |
++++ |
| Hengshan Astragalus honey |
28.64 ± 2.15ab |
++++ |
18.31 ± 0.15c |
++ |
25.63 ± 2.01b |
++++ |
| Shanxi Acacia honey |
28.63 ± 2.28ab |
++++ |
26.26 ± 0.95b |
++++ |
25.73 ± 0.44b |
++++ |
| Xinjiang jujube honey |
29.30 ± 8.21a |
++++ |
17.1 ± 0.12c |
++ |
22.57 ± 0.99b |
+++ |
Different lowercase letters indicate that the size of the inhibition circle formed by different honey issignificantly different (p < 0.05), and the difference between the same letters is not significant.
Combined with the experimental physical and chemical conclusions, the content of two Astragalus honey flavonoids was the highest. Antibacterial experiments showed that Longxi Astragalus honey had the best antibacterial effect against Staphylococcus aureus and Pseudomonas aeruginosa, but its antibacterial effect on Escherichia coli was weak. However, Hengshan Astragalus honey had a strong antibacterial effect on Escherichia coli and Pseudomonas aeruginosa, but its bacteriostatic ability against the Staphylococcus aureus was not excellent. Similar Shanxi Jingtiao honey flavonoids had lower content, but their hydrogen peroxide content was high, and they showed a strong bacteriostatic ability against all three bacteria. In general, although phenols, flavonoids, and other compounds are the main antibacterial components of honey, their total antibacterial effect was also affected by the complex composition of honey, and the antibacterial effect of each component and its mutual mechanism of action are not clear.
The results showed that the composition and content of secondary metabolites in different honeys were different. This may be related to the different types of honey sources, the climate of the origin, the time of honey harvest and the honey processing process (Sun, 2022). The high content of flavonoids and polysaccharides in Astragalus flowers in Longxi and Hengshan areas is the main reason for the high content of flavonoids in Astragalus honey. In this study, 5 kinds of honey mainly from Hengshan Astragalus honey were compared and analyzed. It was found that there were abundant bacteriostatic secondary metabolites in honey, and the key bacteriostatic secondary metabolites mainly included 13 phenols, 8 alkaloids and 7 flavonoids. Phenols (flavonoids are polyphenols) can produce antibacterial effects by inhibiting the activity of enzymes such as bacterial dehydrogenase and oxidase (Wang et al., 2017; Ou et al., 2023), alkaloids inhibit the proliferation of bacterial cell walls and thus the bacteria (Dian et al., 2022), while the antibacterial effect of flavonoids may be related to inhibiting the tricarboxylic acid cycling pathway of the bacteria and thus inhibiting their respiration. The secondary metabolites with relatively high content were maltose, D-2-aminobutyric acid and artemisinin. It is worth mentioning that Wen and other scientists have also shown that artemisinin has antibacterial, anti-parasitic, antipyretic and immune-promoting effects (Wen, 2009). This study also found that metabolites with the same properties and characteristics were more likely to gather in one class, such as Hengshan Astragalus honey and Longxi Astragalus honey flavonoid clusters and carboxylic acid clusters. This is consistent with the results of (Qin et al., 2022).
The inhibition process is relatively complex, honey is the result of multi-targets effects. For example, the physicochemical experiment and antibacterial experiment results of this study showed that Hengshan and Longxi Astragalus honey, both of which were source plants of Astragalus, had higher flavonoid content but different antibacterial properties. Shanxi Jingtiao honey has low flavonoid content, but high hydrogen peroxide content, which has strong antibacterial ability. The antibacterial effect was affected by osmotic pressure, acidity and other factors, phenols, flavonoids and other substances, and the antibacterial effect was different for different species. For example, In this experiment, the antibacterial effect of Hengshan Astragalus honey against gram-negative Escherichia coli and Pseudomonas aeruginosa was better than that against grampositive Staphylococcus aureus. To sum up, it can be inferred that all samples of honey have a certain antibacterial ability, different honey to play the antibacterial effect of different components, the types of antibacterial bacteria are also different. The effective components of Hengshan Astragalus honey with good antibacterial properties are flavonoids, and its low pH value also plays a role in the physical antibacterial function. However, the specific role of the effective components still needs to be further studied.
Acknowledgements This work was supported by the Program for Scientifc and Technological Innovation of Higher Education Institutions in Shanxi (2022L459, 2021L384), the special project of Industry, university and research of ShanxiDatong University (2022CXY11, 2022CXY8), Datong University college students innovation and entrepreneurship training program (XDC2022188), Yungang Studies Special Project of Shanxi Datong University (2022YGZX005). We would like to thank Shanxi Beiyue Shenqi Biotechnology Co., LTD., Shanxi Hengguang Beiqi Biotechnology Co., LTD Provide high quality experimental materials. We would like to acknowledge Shanghai Biotree Biotech Co., Ltd. for their helpful suggestions concerning the data analysis.
Author Contributions YFZ: project administration (equal); conceptualization (equal); methodology (equal); writing—review & editing (equal). JD: writing—original draft (equal); writing—review & editing (equal); data curation (equal). FZT: data curation (equal). MMW: data curation (equal). YC: data curation (equal). BYM:data curation (equal). SYL: data curation (equal). YZ: writing—review & editing (equal). HB: methodology (equal). JW: methodology (equal). HLZ: methodology (equal).
Conflict of interest There are no conflicts of interest to declare.
Ethical approval Ethics approval was not required for this research.
