An Investigation on the Antioxidant Capabilities, Chemical Analysis and Potential Green Applications of the Yellow Bedstraw (Galium Verum) Ethanol Extract

doi:10.21203/rs.3.rs-4376256/v1

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An Investigation on the Antioxidant Capabilities, Chemical Analysis and Potential Green Applications of the Yellow Bedstraw (Galium Verum) Ethanol Extract

https://doi.org/10.21203/rs.3.rs-4376256/v1

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Plants are important sources of phytochemicals with multiple uses in medicine and other technical fields. Much research attention has been focused on the high natural antioxidant activity of extracts obtained from various plants. Antioxidant activity is a promise not only for biochemical behaviour but also for other reactions involving electron transfer, such as metal protection. Antioxidant activity using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method, total polyphenol (via the Folin-Ciocalteu method), and total flavonoids measured with the aluminium chloride (AlCl₃) technique were determined for various ethanol extracts of Yellow Bedstraw or Galium verum (GV). Other methods, such as reverse-phase high-performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS), are also emerging, both to reveal the chemical composition of Galium verum extract and to support the presence of antioxidant compounds. The various chemical constituents are classified into categories based on their possible applications: metabolic activity, antioxidant, antimicrobial, antifungal, anti-inflammatory and active components in different drug, treatments or other green industrial usage.

Physical sciences/Chemistry/Analytical chemistry

Physical sciences/Chemistry/Chemistry publishing

Physical sciences/Chemistry/Environmental chemistry

Galium verum

ethanol extract

antioxidant activity

HPLC

GC-MS

In the last decade, the research, studies, and utilization of extracts from different parts of plants have practically exploded. Research has shown that plants have a complex and rich content of phytochemicals in the flowers and leaves and in the aerial parts in general (Abu-Orabi et al., 2020; Beldean-Galea et al., 2008; Bradic, et al., 2021; Bursal et al., 2019; Singleton et al., 1999; Xie and Schaich, 2014; Hodisan et al., 2022; Tava et al., 2020; Laanet et al., 2023; Schmidt et al., 2014). The difficulty in structuring the various extracts in accordance with this criterion is due to the chemical composition's complexity, which adds to the interest. Among the most important additional criteria for classifying plant extracts are the extraction medium, the extraction method, the field of use of the extract, and complementary methods of study and analysis. A very brief and up-to-date description of some natural plant extracts is given. The most used extraction mediums are hydromethanolic extract (Xie and Schaich, 2014; Lakić et al., 2010), hydroethanolic extract (Bradic, et al., 2021; Negru et al., 2021; Turcov et al., 2022; Costa et al., 2023) ethylic extract (Negru et al., 2021; Al-Rifai et al., 2017; Bursal et al., 2019), methanolic extract (Bota et al., 2021; Abu-Orabi et al., 2020; Milevic et al., 2022; Bradic et al., 2019) or others (Mocan et al., 2019; Wairata et al., 2022). These extracts have been obtained using different methods: maceration (Lakić et al., 2010; Bota et al., 2021; Turcov et al., 2022), reflux extraction (Turcov et al., 2022; Milevic et al., 2022; Bradic et al., 2019) ultrasound extraction (Bota et al., 2021; Turcov et al., 2022) decoction (Schmidt et al., 2014) or microwave extraction (Mocan et al., 2019).

Because antioxidant activity is by far the most important characteristic of plant extracts, the main determinations are focused on the identification and dosage of phenolic compounds (Abdelwahab et al., 2017; Akhtar et al., 2018; Sari et al., 2023; Singleton et al., 1999; Turcov et al., 2022; Mocan et al., 2019; Negru et al., 2021) and flavonoids (Bungau et al., 2012; Akhtar et al., 2018; Sari et al., 2023; Abdelwahab et al., 2017). The analytical methods used in this sense were mainly the Folin-Ciocalteu (Bota et al., 2021; Mocan et al., 2019; Turcov et al., 2022), HPLC (Farcas et al., 2018; Bota et al., 2021; Hodisan et al., 2022; Bursal et al., 2019) and complementary methods: EPR spectroscopy (Farcas et al., 2018), GC-MS (Abu-Orabi et al., 2020; Abdelwahab et al., 2017; Ciotlaus et al., 2020; Zaichikova et al., 2020; Tava et al., 2020; Laanet et al., 2023, microplate assay ( Sari et al., 2023) or statistical analysis (Costa et al., 2023).

Any plant extract, as does Galium verum, besides phenolic compounds and flavonoids, also contains polysaccharides, aldehydes, and alcohols (Akhtar et al., 2018; Ciotlaus et al., 2020; Bradic, et al., 2021), terpenes (Al-Snafi, 2018; Negru et al., 2021; Abu-Orabi et al., 2020; Beldean-Galea et al., 2008; Bradic et al., 2019; Ciotlaus et al., 2020) and anthraquinones (Bradic, et al., 2021; Al-Snafi, 2018; Mocan et al., 2019).

The chemical profile of a certain plant extract, harvested from a certain geographical area, is the solution for choosing the main fields where the plant could be used: for food and nutrients (Lakić et al., 2010; Bungau et al., 2012; Abdelwahab et al., 2017), in cosmetics (Lakić et al., 2010; Turcov et al., 2022), in pharmaceutical and medicinal purposes (Bradic, et al., 2021; Schmidt et al., 2014; Bungau et al., 2012; Bursal et al., 2019), and in other industries as green corrosion inhibitors because of their redox behavior (Panchal et al., 2021; Al-Otaibi et al., 2014; Al Shibli et al., 2022; Dehghani et al., 2022; Badea et al., 2023). For the use of plant extracts in these fields, the most important are the antioxidant activity (Abu-Orabi et al., 2020; Singleton et al., 1999; Akhtar et al., 2018; Schmidt et al., 2014; Wairata et al., 2022; Abdelwahab et al., 2017) and the antibacterial and antifungal activity (Bradic, et al., 2021; Al-Snafi, 2018; Mocan et al., 2019; Akhtar et al., 2018; Al-Rifai et al., 2017).

The plants, known as Yellow Bedstraw or Lady's Bedstraw (Galium verum), are part of the spontaneous flora in Southeastern Europe. These plants have long been used in traditional medicine for healing internal and external ailments, such as supporting the normal functioning of the liver and biliary tract and the electrolytic balance. Galium verum belongs to the Rubiaceae family and is an herbaceous perennial flowering plant. There are more than 650 species of Galium verum spread around the world, but only four of them are officially recognized.

Galium verum has been studied for its antioxidant activity, which can play a role in different domains: ingredients in cosmetic products (Lakić et al., 2010; Turcov et al., 2022) pharmaceuticals, and medicine (Al-Snafi, 2018; Mocan et al., 2019).

Investigations of Galium verum with different extraction techniques have been reported: maceration in hydro-alcoholic solutions (ethanol, methanol) with different concentrations, ultrasound-assisted extraction, and reflux extraction. These extraction techniques are based on several variations of parameters such as solid-liquid ratio, solvent type, and extraction time (Farcas et al., 2018, Al-Snafi, 2018; Bota et al., 2021; Mocan et al., 2019; Antoniak et al., 2023; Antoniak et al., 2023; Layali et al., 2022; Ciotlaus et al., 2020).

The aim of this study was a comparative analysis of commercial and laboratory extracts of Galium verum from the spontaneous flora of the Transylvanian Plateau, Brasov, Romania.

These extracts, because of their composition and antioxidant action, could contribute to improving the quality of human life by reducing oxidative stress. These extracts could also have other potential uses in the technique by interfering in oxidation and reduction reactions, such as their possible use as environmentally friendly corrosion inhibitors for various metals or alloys in aqueous media, especially for mild steel (Panchal et al., 2021; Al-Otaibi et al., 2014; Al Shibli et al., 2022; Dehghani et al., 2022; Badea et al., 2023). The extraction methods employed in this study are predicated upon the differential distribution of the various components within a mixture when subjected to a polar solvent. The process of maceration was conducted utilizing polar solvents, specifically ethanol and water. Extraction protocols are continuously optimized to achieve high extract quality with high yields, cost-effectiveness, and reproducibility.

Among the instrumental methods that may be used to analyze these extracts, the chromatographic methods RP-HPLC and GC-MS were chosen (Ciotlaus et al., 2020; Zaichikova et al., 2020; Tava et al., 2020; Laanet et al., 2023; Laanet et al., 2023). Besides these methods, specific methods may also be used for the analysis of biologically active compounds, such as the evaluation of antioxidant activity and both total polyphenol and total flavonoid content of Galium Verum extracts.

2.1. Materials

The chemicals employed for the assays, namely ethanol, acetic acid, sodium carbonate, aluminum chloride and sodium acetate were of analytical quality. The chemicals 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid, quercetin, caffeic acid, umbelliferon, and kaempferol standards were obtained from the Sigma-Aldrich Company. The Folin-Ciocalteu reagent was procured from Merck Co. One of the analyzed Galium verum extracts was supplied by Dacia Plant S.R.L. Romania (www.daciaplant.ro).

2.2. Extract sample preparation

The desiccated botanical specimen was acquired from the marketplace. The plant's dried aerial portions were finely chopped and afterwards underwent phytochemical extraction. The extraction procedure was conducted using a 1:1 mixture of ethanol and water employing the maceration technique. A quantity of 10 grams of plant powder was subjected to maceration with 100 ml of solvent, maintained at room temperature for a duration of 1 days. The solution was periodically agitated. The suspension was subjected to vacuum filtration using a 0.45 µm Millipore filter.

Another technique used for extraction was a reflux extraction using a Soxhlet device. In this case, the extraction was also carried out in ethanol. For reflux extraction by Soxhlet first there were weighed 35.81 g of the dried plant, then were fine-cut and introduced into the cardboard cartridge. A volume of 260 ml of ethyl alcohol was introduced into the flask. The extraction time was about 3 hours. During that period the solution was recirculated 8 times.

The extracted solutions were stored at a temperature of 4 ℃ in a refrigeration unit until they were ready for further analysis.

2.3. Assay of Total Phenolic Content

The Folin-Ciocalteu test has become known as the main method for assessing the overall polyphenolic content in various extracts. The colorimetric technique described is founded on the electron transfer process occurring in an alkaline environment, where phenolic compounds donate electrons to generate a blue chromophore. This chromophore is composed of a complex formed by phosphotungstic and phosphomolybdenum, and its absorption at maximum wavelength is directly proportional to the overall concentration of polyphenolic compounds.

The detectability of the decreased Folin-Ciocalteu reagent can be achieved using a spectrophotometer within the wavelength range of 690 to 750 nm. A reaction temperature of 37°C was chosen in order to minimize the time necessary to achieve the maximum coloration. The method employed in this study followed the guidelines outlined in the International Organization for Standardization ISO 14502-1, which utilized gallic acid as the standard reference compound. A calibration curve was initially constructed having as reference a compound with significant antioxidant properties.

Amounts of polyphenols with antioxidant properties is expressed in gallic acid equivalents, GAE (mg/ml). In the concentration range of 0.05–2.0 mg/ml (gallic acid) a straight dependence of absorbance with concentration is obtained. The line equation is y = 4.3916x + 0.1236. The correlation coefficient R₂ = 0.9975 indicated a good correlation of the results. The reaction with the Folin-Ciocalteu reagent takes place, as mentioned above, in basic medium, at pH ~ 10. In order to achieve the best possible results, several basic substances were tested. Among these Na₂CO₃ has been the most successful. The use of sodium hydroxide can lead to the formation of precipitates due to the complexation of some inorganic compounds presented in the sample.

The Folin-Ciocalteu reagent was employed with minor adjustments to assess the total phenolic content of each extract (Singleton et al., 1999). In a test tube, 8 ml of distilled water, 1 ml of a standard working solution or analytical solution, and 1 ml of a diluted Folin-Ciocalteu reagent (at a 1:10 dilution) were added. The solution was agitated and allowed to stand for a duration of 1 minute. Subsequently, 2 ml of a 20% Na₂CO₃ solution were introduced, and the temperature was maintained at 37 ℃ for a period of 1 hour. The measurement of absorbance for the resultant blue hue was conducted at a wavelength of 765 nm using a SPECORD 210 PLUS UV-VIS spectrophotometer manufactured by Analytik-Jena. The quantification of total phenolic components was performed by expressing the concentration in milligrams of gallic acid equivalent (GAE) per millliliter of extract.

2.4. Assay of Total Flavonoids Content

Total flavonoids content was determined by spectrophotometry method with aluminium chloride (AlCl₃), in which aluminium ions interact with flavonoid molecules at pH 4.8 in HAc/NaAc buffer solution forming an aluminium flavonoid chelates with an intense absorption band at 430 nm. In the mixture between 1 ml pH 4.8 HAc/NaAc buffer solution and 2 ml 2% AlCl₃ solution was added 1 ml Galium verum extract sample and was incubated for 15 min at 40 ℃, the absorbance of the reaction mixtures was measured at 430 nm. In the blank, the extract sample was replaced by distilled water. Flavonoids content was expressed in µg quercetin equivalents (QE) per g dried extract by using a standard curve of quercetin with concentration range from 0.1-1 µg/ml. All measurements were replicated three times (Lakić et al., 2010; Mocan et al., 2019).

2.5. Assay for antioxidant activity

The measurement of the hydrogen donating, or radical-scavenging ability of the extraction solution was conducted utilizing the stable radical DPPH. The DPPH scavenging analysis for each sample was conducted using a modified DPPH technique (Xie and Schaich, 2014).

Solution extracts were produced from each sample of Galium Verum, with concentrations ranging from 0.25 -15 µg/ml and were then diluted with 100% methanol. The DPPH solution was made by dissolving 2 mg of DPPH in 50 ml of methanol, resulting in a concentration of approximately 3 mM. A total volume of 0.2 ml of each pre-prepared extracted sample was combined with 2.8 ml of DPPH solution. The solution was subjected to incubation at ambient temperature, under conditions of darkness. The measurement of absorbance was conducted at a specific wavelength of 517 nm following a time period of 60 minutes, utilizing gallic acid as the positive control. The DPPH radical scavenging capacity of each extracted sample was determined by employing the subsequent equation:

DPPH radical scavenging rate (%) = (A_control-A_sample) x 100 / A_control.

where A_control is the absorbance of the DPPH solution (without extract sample), A_sample is the absorbance of the extracted sample. The antioxidant activity of standard and samples was expressed as IC₅₀ value in µg GAE/ml corresponding to the inhibition of 50% DPPH radical. The graphically method was used for IC₅₀ determination from linear variation plot of the inhibition percent with the antioxidant concentration. All experiments were performed in triplicate (Bota et al., 2021).

2.6. Analysis of phenols by reverse-phase high-performance liquid chromatography (RP-HPLC)

The determination of phenols by RP-HPLC was conducted using an HPLC ACME 3000 Young Lin Instrument, equipped with SP 930D module and UV 730D detector module, operating under the following chromatographic conditions: isocratic conditions with methanol: water: acetic acid at a ratio of 300:700:2 as a mobile phase, and an elution flow set at 1 ml/min. Chromatographic separation of sample constituents was achieved using a reverse-phase YMC-Pack ODS AQ column, 150 cm long and with an internal diameter of 4.6 mm. Each sample, with a volume of 0.2 µl, was injected into the chromatograph at room temperature.

Through RP-HPLC chromatographic analysis, the content of various polyphenolic compounds in the prepared extracts was determined. Initially, calibration curves were established for several polyphenol standards present in the analyzed extract samples, including Gallic Acid, Catechin, Vanillic Acid, Caffeic Acid, Kaempferol, Quercitin, and Umbelliferone. The equations representing the calibration curves of the external standards utilized are presented in Table 1.

Table 1

Polyphenolic content obtained by RP-HPLC analysis
Compound	Retention time (min)	Equation of the calibration line	R²
Gallic acid	2.2	Y = 28572x + 457.97	0.9943
Catechin	2.83	Y = 21015x + 8.6669	0.9972
Vanillic acid	4.36	Y = 33957x − 121.42	0.9998
Caffeic Acid	4.6	Y = 42330x -157.55	0.9960
Kaempferol	15.1	Y = 207,88x – 4.5454	0.9966
Quercetin	22.1	Y = 19277x-1233.2	0.9998
Umbelliferone	6.4	Y = 1739,9x + 2751.8	0.9988

2.7. GC-MS analysis

The GC-MS analysis of the Soxhlet extract obtained from the aerial parts of Galium verum was conducted using a Thermo GC-MS system (Model Trace 1310 ISQ7000), coupled with an HP-5MS capillary column measuring 30 m in length, 0.32 mm in internal diameter, and coated with a 0.25 µm film thickness. For GC-MS spectroscopic detection, an electron ionization system with an ionization energy of 70 eV was employed. Helium served as the carrier gas at a flow rate of 30 cm/s, and the injection volume was set at 1 µL. The mass transfer line and injector temperatures were maintained at 220 ℃ and 290 ℃, respectively.

The oven temperature was programmed as follows: starting at 45 ℃ for 1 minute, then ramped up to 250 ℃ at a rate of 5 ℃/min, and finally held at 250 ℃ for 5 minutes. Samples, diluted to 1 µL, were injected in split mode with a split ratio of 120:1. The relative percentage of chemical constituents present in the dried extracts was determined using peak area normalization.

3.1. Determination of Polyphenol Content

By analysing the experimental data, you may observe that in case of the extract obtained at reflux with the Soxhlet apparatus, when the solvent was ethanol, the polyphenol content was the lowest, 1.97 mg gallic acid/g, compared to 2.22 mg gallic acid/g for the extracts obtained by maceration with hydroalcoholic solutions. These differences are due to the higher water solubility of these compounds compared to their solubility in ethyl alcohol. Previous studies reported in the literature show a content of 2.0-4.6 mg gallic acid/g dried extract (Lakić et al., 2010). The results obtained are comparable taking into account the dilution. In Table 2 are presented the obtained results comparative with other researchers. The Galium Verum studied extracts are: ethanolic Soxhlet reflux extract (GV-SOX), prepared hydroethanolic extract (GV-L), and Dacia Plant hydroethanolic extract (GV-DP).

Table 2

Comparative data of different Galium verum extracts.
Solvent	Extraction Method	Extraction Time, h	[Ref]
100% ethanol	reflux extraction	2	GV -SOX
50% ethanol	maceration	24	GV-L
50% ethanol	maceration	-	GV-DP
80% methanol	maceration	24	(Lakić et al., 2010)
80% methanol	maceration	24	(Lakić et al., 2010)
70% ethanol	maceration	168	(Farcas et al., 2018)
100% methanol	-	-	(Al-Snafi, 2018; Layali et al., 2022)
50% ethanol	reflux extraction	1	(Turcov et al., 2022)*
50% ethanol	maceration	192	(Turcov et al., 2022)*
100% ethanol	ultrasound maceration	0.12	(Mocan et al., 2019)
96% ethanol	ultrasound maceration	0.5	(Antoniak et al., 2023)*
* Some authors report the measurements to dried plant, instead of dried extract

As it could be seen there were taken into consideration the solvent, the extraction time, the type of extraction and the corresponding GAE obtained.

In Fig. 1 are presented comparatively the GAE (mg gallic acid / g dried extract) and the total flavonoids (µQE / g dried extract) of the analyzed Galium verum extracts and confirm that the obtained results are comparable with other data from literature (Lakić et al., 2010; Mocan et al., 2019).

Some authors reported higher values, but they do not give enough information for comparison (Al-Snafi, 2018; Turcov et al., 2022; Antoniak et al., 2023; Layali et al., 2022). For example, a value of 58.47 ± 4.40 mg gallic acid / g row material (Antoniak et al., 2023). The value above was obtained for 15 g, that means about 3.89 mg GAE/1g of dried plant which is close to the lower values of phenolic content obtained in this study, and also by other authors (Lakić et al., 2010; Mocan et al., 2019). Main cause of these differences is probably due to different preparation of solvent and dried extract, which means that further studies should be done in order to find the best extraction technology and also to use a uniform unit measure.

3.2. Determination of Total Flavonoids Content

This study revealed a variation of total flavonoids content from 6.55–6.91 µQE/g dried extract in macerates to 7.37 µQE/g dried extract in Soxhlet extraction. The difference between the macerates was not significant, but an increase of the flavonoid concentration in the Soxhlet extraction could be observed. Flavonoids showed a higher solubility in 100% ethanol, than in a hydroalcoholic solvent.

From Fig. 2, the total flavonoids content resulting from these experiments is in good agreement with other literature data obtained for Galium verum extracts, if the same methods and data processing were used (Lakić et al., 2010; Mocan et al., 2019). A higher TFC was found for Galium verum sample extracted with the ultrasonic maceration technique in 100% ethanol (Mocan et al., 2019), than for the GV macerated in 80% methanol (Lakić et al., 2010), the latter being a more polar solvent. Also, in Fig. 2 it can be seen that the Total flavonoids content was not consistent with the data of other authors (Lakić et al., 2010; Turcov et al., 2022) who used different ways of expressing concentrations.

3.3. Determination of Extracts Antioxidant Activity

The beneficial effects of plant extracts on health are due to the high antioxidant activity of their polyphenols content. In order to determine the antioxidant activity of Galium verum extract, the free radical scavenging capacity was measured. In DPPH, the nitrogen atom unpaired electron is reduced through the acceptance of a hydrogen atom from the antioxidants, leading to the formation of the equivalent hydrazine compound. DPPH is a chemically stable paramagnetic radical. The unpaired electron originating from the nitrogen atom exhibits delocalization, so becoming part of the entire molecule. The solution exhibits a dark purple coloration, indicating a pronounced absorption peak at a wavelength of 517 nm. Determinations of the absorbance (A), after 60 minutes and of the antioxidant activity (%) of the three extracts, in different concentration from 0.25 to 15 µg/ml in 100% methanol were done. The antioxidant activity is due to the neutralization of free radicals. The obtained data are recorded in Table 3. IC₅₀ data from the linear plot equations for DPPH study of Galium verum extract samples were noted in Table 3.

Table 3

DPPH radical scavenging rate (%) of 0.25-15 g/ml Galium verum extracts.
µ
Extract conccentration, (µg/ml)	GV -SOX	GV-DP	GV-L
Extract conccentration, (µg/ml)	DPPH RSR %	DPPH RSR %	DPPH RSR %
IC₅₀	3.744±0.09	3.352±0.07	3.203±0.06
0.25	3.85±0.12	4.75±0.16	4.89±0.16
0.5	8.7±0.06	13.5±0.013	15.5±0.08
1	16.6±0.09	20.4±0.09	22.2±0.02
2.5	38.5±0.05	41.6±0.05	43.3±0.06
5	63.7±0.09	69.6±0.05	72.3±0.02
7.5	77.1±0.17	79.8±0.12	81.4±0.06
10	80.5±0.06	82.5±0.11	83.8±0.13
12.5	81.4±0.18	83.6±0.16	85.1±0.12
15	82.2±0.14	84.0±0.15	85.7±0.12

The determination of the antioxidant activity of Galium verum extracts led to the following results: both extracts in 50% ethanol, GV-L and GV-DP, presented a higher antioxidant activity than the extract in 100% ethanol, GV-SOX; all found IC₅₀ values were ranged from 3.203 to 3.744 µg/ml; all IC₅₀ values of the analyzed samples were higher than those obtained for the gallic acid standard, for which an IC₅₀ value of 2.607 ± 0.025 µg/ml was found. Thus, we can say that the antioxidant activity of the extracts in 100% ethanol and 50% ethanol have an antioxidant activity slightly lower than gallic acid, knowing that a low IC₅₀ value corresponding to a high antioxidant activity.

Comparison between IC₅₀ values found after determination of antioxidant activity for three Galium verum extracts in 100% ethanol and hydroalcoholic solvents studied in this paper with literature data from other researchers for Galium verum extracts in different solvents was achieved with difficulty and limited because the DPPH methods were different, the standards used were not always the same and the results are few and expressed in different systems of units. In another research (Lakić et al., 2010), a Galium verum macerate sample in 80% methanol, obtained 3.1 µg/ml for IC₅₀ was used. The result is supported by the theory that the nature of the solvent can influence the value of the antioxidant activity. The obtained results are correlated with determinations of phenolic acids content, as these compounds are known to be responsible for antioxidant activity, neutralizing free radicals. Similar results were obtained by other authors as it could be seen from Fig. 3.

3.4. RP-HPLC chromatography

In Fig. 4 are represented the chemical structures of the phenolic compounds which were identified in the Galium verum extract.

Figure 5 shows the chromatogram obtained for the extract of Galium verum analysis. After RP-HPLC chromatographic analysis of the extracts, the polyphenol content was determined and quantified using calibration curves for each compound.

In Table 4 are presented the experimental results obtained from the analysis of Galium verum extracts by the RP-HPLC method.

Table 4

Amounts of polyphenols in samples analyzed by RP-HPLC method.
	GV-DP	GV-SOX	GV-L
Gallic acid (mg/ml)	0.15	0.49	0.23
Catechin (mg/ml)	0.18	0.20	0.51
Vanillic acid (mg/ml)	0.07	0.09	0.158
Caffeic acid (mg/ml)	0.008	0.008	0.006
Umbelliferone (mg/ml)	2.03	1.83	1.72
Kaempferol (mg/ml)	2.02	1.98	2.72
Quercetin (mg/ml)	0.33	0.22	0.91

From the experimental results it can be seen that in all samples were identified the compounds: gallic acid. caffeic acid. vanillic acid. and umbelliferon. Besides these compounds. the flavanols: catechin, kaempferol and quercetin were identified.The concentration of these compounds in extracts differs. depending on the extraction technique and the used solvent.Higher amounts of flavanols were extracted in hydroalcoholic solutions and higher amounts of phenolic acids were extracted in alcoholic media by reflux extraction. The obtained results are comparable with data found in the literature. It is indicated that the obtained extract is rich in chlorogenic acid, rutin, coumarin and ferulic acids and quercetin (Farcas et al., 2018). The average quercetin content in the samples was 0.36 mg/ml.

3.5. Chemical profile of Galium verum extract

To fully characterize the extracts, the sample obtained by reflux extraction using ethyl alcohol as solvent was analyzed by GC-MS chromatography.More than 80 compounds were identified in the ethanolic extract. These include alcohols (n-propanol, isopropanol, tert-butanol, isobutanol, phenyl ethanol, benzyl alcohol, furfuryl alcohol), carboxylic acids (acetic acid, benzoic acid, isovalerianic acid, mystic acid). fatty acids (linoleic acid, linolenic acid, palmitic acid, stearic acid, arachidic acid). hydrocarbons (eicosane, heptadecatetraene, toluene, tetradecane, phenanthrene), aldehydes (benzaldehyde, furfuraldehyde), esters (dimethoxymethylacetate, butyl acetate), phytol, linalool, coumarin, D (+)-carvone. The same classes of compounds have also been identified by other authors who have studied the components of Galium verum extracts (Al-Snafi, 2018). Other compounds soluble in alcohol were also observed, such as terpenes, flavoring substances whose presence was also marked in other GC_MS spectra, such as that of the essential oil separated from GV (Ciotlaus et al., 2020; Zaichikova et al., 2020; Tava et al., 2020; Laanet et al., 2023; Laanet et al., 2023). The presence of numerous aromatic compounds such as phenols, alcohols or their condensed derivatives by dehydration as ethers, esters, with groups favoring oxidation reactions was observed. From Tables 5. to Table 9., an attempt was made to classify the compounds obtained from the GC-MS analysis according to their source (fats and carbohydrates) but also according to their role in living organisms: metabolic activity, antioxidant, antimicrobial, antifungal, anti-inflammatory and active components in different drugs or treatments.

Table 5

Components and metabolites of natural fats from GV-SOX extract.
Substance	Retention time, min	Percentage, %
Palmitic acid	29.17	6.3116
Linolenic acid	31.95	6.1761
Linoleic acid	31.84	3.0841
Butyric acid. octyl ester	23.00	1.8878
Stearic acid	32.24	0.1239
Glycerin	8.95	0.0695
1-Monoacetin	15.16	0.0450
2,3-Dihydroxypropanal	8.73	0.0387
Myristic acid	25.74	0.0321
Palmitic acid ethyl ester	29.68	0.0252
Arachidic acid	35.07	0.02306
2-Monopalmitin	37.02	0.01712
Diethyl-acetal-isovaleraldehyde	8.17	0.01496
Heptadecanoic acid 15-methyl-ethyl ester	32.72	0.01228

Table 6

Compounds with metabolic activity and health interest from GV-SOX extract.
Substance	Retention time, min	Percentage, %
2-Methyl- benzene methanol	12.86	2.2275
2,3,4-Trimethyloxetane	3.49	0.1442
2,2'-Dimethyldecane	9.04	0.0746
Tetradecane	18.76	0.0616
Benzoic acid	13.35	0.0398
5,6,7,7-Tetrahydro-4,4,7a-trimethyl-2(4H)-benzofuranone	21.63	0.0162
Hexyl-chloroformate	6.31	0.0129
phenanthrene	26.28	0.0129
1,2- Dimethylindoline	16.76	0.0114
3-methyl benzyl alcohol	12.63	0.0107
1,1,2-Triethoxyethane	7.58	0.0106
Gamma-Hydroxybutyrate	7.24	0.0090
Fluorene-9-one	25.51	0.0090
Phosphosal	16.55	0.0076

Table 7

Carbohydrates and their derivatives from GV-SOX extract.
Substance	Retention time, min	Percentage, %
1,3,6- Trideoxy − 3,6- epithio -D- Fructose	19.66	1.5557
3-O-Methyl-d-glucose	24.15	1.2028
5-Hydroxy-methyl -furfural	14.84	0.0973
D- Allose	20.5	0.0681
3-Deoxy-d-mannoic lactones	22.7	0.0154
6-Deoxygulitol	9.56	0.0071

Table 8

Flavoring and fragrance agents, terpenoids, volatile and essential oils from GV-SOX extract.
Substance	Retention time, min	Percentage, %
Phytol	31.54	1.1888
Benzyl benzoate	25.98	1.0478
Neophytadiene	27.15	0.0560
2-(Ethoxyethoxy)-3-methyl-1,4-butanediol	6.76	0.0553
n-Cetane	22.8	0.0389
n-Dodecane	14.23	0.0386
Isobutyl alcohol	3.24	0.0269
Hexahydro-farnesyl-acetone	27.26	0.0258
Trans, trans-2,6-dimethyl-2,6-octadiene-1,8-diol	21.2	0.0181
Butyl acetate	5.3	0.0164
3-Furaldehyde	5.65	0.0148
1,8,11,14- Heptadecatetraene	28.78	0.0144
Linalool	11.8	0.0141
D (+)-Carvone	15.33	0.0130
1,1-Diethoxy-2-methylpropane	1.91	0.0097
1,1-Dietoxi-2-metilbutan	8.21	0.0089
2,4-Hexadien-1-ol	7.38	0.0069
Alcohol phenyl ethyl	12.14	0.0057

Table 9

Compounds with antioxidant, antibacterial, anti-inflammatory, antifungal activity, bioinsecticides and biopesticides, including flavonoids and polyphenols from GV-SOX extract.
Substance	Retention time, min	Percentage, %
Neophytadiene	27.15	0.0560
2-(Ethoxyethoxy)-3-methyl-1,4-butanediol	6.76	0.0553
1-Isobutyl-7,7-dimethyl-octahydro-isobenzofuran-3a-ol	24.28	0.0525
Diethoxymethylacetate	5.16	0.0309
2-Methoxyvinylphenol	16.92	0.0265
Coumaran	14.65	0.0233
Phloretic acid	23.42	0.0223
4-Hydroxy-4-isopropyl-5-methyl-2-hexynylacetate	24.63	0.0206
(E)-4-(3-Hydroxyprop-1-en-1-yl)-2-methoxyphenol	25.43	0.0181
3-Deoxy-d-mannoic lactones	22.7	0.0154
1,3,5-Benzentriol	11.18	0.0151
Isovanilic acid	22.08	0.0140
Benzilic alcohol	10.14	0.0138
Benzene acetaldehyde	10.39	0.0106
Furfuryl alcohol	6.04	0.0102
Gamma-Hydroxybutyrate	7.24	0.0090
2,2,11,11- Tetramethyldodecane	17.21	0.0089
Cyclohexyl-methyl-ether	6.38	0.0084
3-Methyl-1,5-pentanediol	12.04	0.0084
3-Phenyl-1-butanol	17.56	0.0081
4-Methoxy-carbonyl-4-butanolides	14.57	0.0075
Alcohol phenyl ethyl	12.14	0.0057
N-(neopentroxy)-dimethylamine	9.26	0.0056
Ammelide	13.91	0.0032

Some compounds have multiple functions, they can be antioxidants and metabolites or essential oils at the same time. Another example is that of phytol, which is a precursor for vitamins E and K1 and also a linkage to chlorophyll. Also, a degradation product of chlorophyll, 2-Ethyl-3-methyl-maleimide was highlighted in GC-MS analysis.

Galium verum extracts have valuable biological properties, which make them suitable to be used to improve human health, but they can also have many other uses based primarily on the antioxidant activity of the constituents.

A potential application of the extracts is their use as corrosion inhibitors (Badea et al., 2023) for various metallic materials, such as soft or mild steels in aqueous, acidic, or neutral media. Plant extracts offer advantages as corrosion inhibitors due to their biodegradability and minimal toxicity. Plant extract, unlike conventional inhibitors, does not generate detrimental residues in the environment following its application. Moreover, plant extract exhibits lower toxicity towards human health and the environment in comparison to conventional inhibitors. Recent investigations have demonstrated the efficacy of Galium verum extract as an environmentally friendly corrosion inhibitor, similar with other botanical extracts (Fig. 6).

The objective is to conduct investigations on the electrochemical characterization of the inhibitory effects of polyphenols, and maybe other compounds, to determine the electrochemical mechanism of corrosion inhibition.

Different extraction procedures, as maceration and reflux extraction were applied for the establishment of the chemical profile of alcoholic extract of Galium verum (aerial parts). the multicomponent phenolic pattern and the antioxidant activity.

Because of the broad scope of Folin-Ciocalteu assay, substances that are not phenols or are rarely thought of as phenols (such as proteins) can also react under the reaction conditions. When utilized with careful consideration, taking into consideration potential confounding factors in specific samples and doing pertinent background investigation, the assay has the ability to generate highly useful outcomes. In numerous instances, the utilization of aggregate analysis proves to be more enlightening compared to extensive volumes of data that pose challenges in summarization due to the utilization of diverse procedures, such as high-performance liquid chromatography (HPLC), which facilitates the separation of a substantial quantity of individual molecules. The identification of individual reactants and the calculation of their respective contributions to the overall FC phenol content of a mixture can be achieved due to the predictable reaction exhibited by the mixture. The investigation using Folin-Ciocalteu assay demonstrates limited sensitivity towards various adsorbents and precipitants. Consequently, conducting a differential test by comparing the results before and after several treatments can prove to be advantageous.

Even Galium verum is a common plant from spontaneous flora in the Eastern Europe and it was the object of some research studies, it is chemical profile confirm that the analysed alcoholic and hydroalcoholic extracts have certain properties, based mainly on the redox activity, but which is not limited to it and brings new and original information that can be starting points for other studies with the aim of capitalizing on the substances identified in the chemical composition.

In conclusion. the following statements underline the originality of this study.

Galium verum plants from the geographical aria, namely Brasov Plateau, from Romania haven’t been studied before. More data should be achieved for a more comprehensive characterization of this specie, because of the difference produced by soil or climate. The Phenol content was determined using Folin-Ciocalteu method and expressed in GAE. equivalent mg gallic acid/mg dried plant. The extraction method showed some little variable effect on the phenolic content. which was comparative with literature data. So, this approach opens the perspective of new, more advanced studies and the creation of a database in which to identify those plants with maximum potential for a certain substance in the composition of the extracts.

The extracts also showed a remarkable antioxidant activity which was consistent with the total polyphenol content. The amounts of the most important contained polyphenols in samples (gallic acid, catechin, vanillic acid, caffeic acid, umbelliferon, kaempferol, quercetin) were determined by RP-HPLC analytical method.

The chemical profile obtained by experimental determinations demonstrated a complex composition of Gallium Verum extracts. Compounds from the class of phenolic acids. flavonoids and more than 80 compounds from various classes (hydrocarbons, alcohol ethers, carboxylic acids, esters, carbonyl compounds, linalool, coumarone, D(+)-carvone) were identified by GC-MS chromatography, also comparative with literature data. This study also conducted a classification of the compounds derived from the GC-MS analysis based on their origin (fats and carbohydrates) as well as their functions in living organisms, such as metabolic activity, antioxidant properties, antimicrobial effects, antifungal properties, anti-inflammatory properties, and their role as active components in various pharmaceuticals or medications.

Knowing the chemical composition of Gallium Verum extract is important in identifying its potential as a corrosion inhibitor. The extract contains polyphenols, which are known to be effective oxidant species that can be absorbed on the metal surface and function as cathodic inhibitors. Polyphenols are inexpensive, biodegradable, renewable, and safe for both the environment and humans. Research has shown that polyphenols have a very promising potential to be used as both green and effective corrosion inhibitors.

Competing interests

The authors declare no competing interests.

Funding

This research was funded by the University of Oradea. within the Grants Competition “Scientific Research of Excellence Related to Priority Areas with Capitalization through Technology Transfer: INO-TRANSFER-UO”. Project No.327/21/12/2021.

Author Contribution

All authors have made a substantial contribution to conducting this work, the drafting and writing of this manuscript, and all provide final approval for this version to be published and agree to be accountable for all aspects of the work. Contributions are as follows: Conception, design, analysis and interpretation, drafting of the article, statistical analysis, and final approval of the article: all authors.

Data Availability

The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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An Investigation on the Antioxidant Capabilities, Chemical Analysis and Potential Green Applications of the Yellow Bedstraw (Galium Verum) Ethanol Extract

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Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials

2.2. Extract sample preparation

2.3. Assay of Total Phenolic Content

2.4. Assay of Total Flavonoids Content

2.5. Assay for antioxidant activity

2.6. Analysis of phenols by reverse-phase high-performance liquid chromatography (RP-HPLC)

2.7. GC-MS analysis

3. Results and discussion

3.1. Determination of Polyphenol Content

3.2. Determination of Total Flavonoids Content

3.3. Determination of Extracts Antioxidant Activity

3.4. RP-HPLC chromatography

3.5. Chemical profile of Galium verum extract

4. Conclusions

Declarations

Competing interests

Funding

Author Contribution

Data Availability

References

Additional Declarations

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