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All issues Volume 32 (2021) BIO Web Conf., 32 (2021) 02010 Full HTML
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BIO Web Conf.
Volume 32, 2021
III International Scientific and Practical Conference “Problems and Prospects of Scientific and Innovative Support of the Agro-Industrial Complex of the Regions” 2021
Article Number 02010
Number of page(s) 10
Section Actual problems of plant protection
DOI https://doi.org/10.1051/bioconf/20213202010
Published online 13 August 2021

© The Authors, published by EDP Sciences, 2021

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1 Introduction

Blue-berried honeysuckle Lonicera caerulea L., family Caprifoliaceae is known as a natural source of food, beverages and nutraceuticals due to its rich chemical composition, enriched with nutrient and biologically active compounds. The increased focus on these berries is due to their phenolic composition, antioxidant activity, and potential health benefits. The high content of phenols in Lonicera caerulea L. is directly related to their biological activity. Popularity of phenolic compounds has grown in recent years as they are excellent antioxidants. Antioxidant intake has been shown to be effective in preventing cancer, cardiovascular disease, osteoporosis, obesity, diabetes, and other health problems [Dias et al., 2017]. The antioxidant properties of plant phenolic compounds are relevant in the field of nutrition (inhibition of lipid oxidation), physiology (protection against oxidative stress) and cosmetology. Phenolic compounds provide antioxidant activity through direct reduction of reactive oxygen species (ROS), inhibition of enzymes involved in oxidative stress, binding of metal ions responsible for ROS production, and stimulation of endogenous antioxidant defense systems [Hossain et al., 2016]. The quality and quantity of phenolic compounds in plants usually depends on the stage of growth, the parts of the plant used and the growing conditions in the environment [Bujor O.-C., 2016].

In this regard, the purpose of this work is the simultaneous assessment of phenolic compounds in the berries of Lonicera caerulea L. of various species collected in different climaticgeographical zones of Russia. This study is a complete qualitative study of phenols and other compounds, leading to the identification of a large number of phenolic secondary metabolites isolated from Lonicera caerulea L. berries of various species.

Initial LC-MS/MS screening suggested that 82 target analytes detected in EtOH-extracts of Blue-berried honeysuckle. Therefore, tandem mass spectrometry was used in this study for comparative small molecule profiling of four Lonicera varieties cultivated in the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources.

thumbnail Fig. 1.

Polymorphism of wild Lonicera caerulea L. berries presented in the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources

2 Experimental

2.1 Materials

The object of the study was the four varieties of Blue-berried honeysuckle Lonicera caerulea L. of breeding varieties obtained as a result of many years of research from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources. There were a varieties: №1043-11 (St. Petersburg); №1043-08 (St. Petersburg); №863 (Japan); №860 (Wild Lonicera from Amur river). The berries were harvested at the end of July 2020. All samples morphologically corresponded to the pharmacopoeial standards of the State Pharmacopoeia of the Russian Federation [SPh XIV, Russia, 2018].

2.2 Chemicals and Reagents

HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), MS-grade formic acid was from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was prepared from a SIEMENS ULTRA clear (SIEMENS water technologies, Germany), and all other chemicals were analytical grade.

2.3 Fractional maceration.

To obtain highly concentrated extracts, fractional maceration was applied. In this case, the total amount of the extractant (methyl alcohol of reagent grade) is divided into 3 parts and is consistently infused on potato with the first part, then with the second and third. The infusion time of each part of the extractant was 7 days.

2.4 Liquid chromatography

HPLC was performed using Shimadzu LC-20 Prominence HPLC (Shimadzu, Japan) was used, equipped with an UVsensor and a Shodex ODP-40 4E reverse phase column to perform the separation of multicomponent mixtures. The gradient elution program was as follows: 0.01-4 min, 100% CH3CN; 4-35 min, 100-25% CH3CN; 35-50 min, 25-0% CH3CN; control washing 50-60 min 0% CH3CN. The entire HPLC analysis was done with a ESI detector at wavelengths of 230 ηm and 330 ηm; the temperature corresponded to 17°C. The injection volume was 1 ml.

2.5 Mass spectrometry

MS analysis was performed on an ion trap amaZon SL (BRUKER DALTONIKS, Germany) equipped with an ESI source in negative ion mode. The optimized parameters were obtained as follows: ionization source temperature: 70 ° C, gas flow: 4 l / min, nebulizer gas (atomizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500V, fragmentary: 280 V, collision energy: 60 eV. An ion trap was used in the scan range m / z 100 -1.700 for MS and MS/MS. The capture rate was one spectrum/s for MS and two spectrum/s for MS/MS. Data collection was controlled by Windows software for BRUKER DALTONIKS. All experiments were repeated three times. A four-stage ion separation mode (MS/MS mode) was implemented.

3 Results and Discussion

Four of the most consumed extracts of Lonicera caerulea L. have been selected. All of them have a rich bioactive composition. There were four extracts from a varieties: №1043-11 (St. Petersburg); №1043-08 (St. Petersburg); №863 (Japan); №860 (Wild Lonicera, Amur river) from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources.

High accuracy mass spectrometric data were recorded on an ion trap amaZon SL BRUKER DALTONIKS equipped with an ESI source in the mode of negative-positive ions. The four-stage ion separation mode (MS/MS mode) was implemented. The combination of both ionization modes (positive and negative) in MS full scan mode gave extra certainly to the molecular mass determination (Fig. 2,3,4). The positive-negative ion mode provides the highest sensitivity and results in limited fragmentation, making it most suited to infer the molecular mass of the separated polyphenols, especially in cases where concentration is low. By comparing the m/z values, the RT and the fragmentation patterns with the MS2 spectral data taken from the literature [Abeywickrama et al., 2016; Abu-Reidah et al., 2015; Rafsanjany et al., 2015; Goufo et al., 2020; Paudel et al., 2013; Jaiswal et al., 2014; De Rosso et al., 2014; Marzouk et al., 2018; Barros et al., 2012; Pradhan & Saha, 2016; da Silva et al., 2019; Ruiz et al., 2013; Ruiz et al., 2010; Razgonova et al., 2020; Kajdzanoska et al., 2010] or to search the data bases (MS2T, MassBank, HMDB). A unifying system table was compiled of the molecular masses of the target analytes isolated from the EtOH-extract of Lonicera caerulea L. for ease of identification (Table 1). The 82 target analytes shown in Table 1 belong to different polyphenolic families: flavones, flavonols, flavan-3-ols, flavanones, anthocyanins, hydroxycinnamic acids, hydroxybenzoic acids, stilbenes, proanthocyanidins and belong to others classes of compounds.

In addition to the reported metabolites, a number of metabolites were newly annotated in Lonicera caerulea L. There were flavonols: Dihydrokaempferol, Rhamnetin I, Rhamnetin II, Taxifolin-3-O-glucoside, Mearnsetinhexoside, Horridin; flavones: Chrysoeriol, Apigenin-Opentoside, Chrysoeriol-7-O-glucoside; flavanone Naringenin; flavan-3-ols: Catechin, Epicatechin, Biochanin A-7-O-glucoside; essential amino acids: L-Pyroglutamic acid, Tyrosine; polypeptide 5-Oxo-L-propyl-L-isoleucine; sterols: Ergosterol, Fucosterol, Beta-Sitosterin; triterpenoids: Betunolic acid, Oleanoic acid; anabolic steroid Vebonol, indole sesquiterpene alkaloid Sespendole; iridoids: Monotropein, p-Coumaroyl monotropein, p-Coumaroyl monotropein hexoside; Myristoleic acid, etc.

The CID-spectrum (collision induced dissociation spectrum) in positive ion modes of Dihydrokaempferol from extracts of Lonicera caerulea L. (variety SPb 10438) is shown in Fig. 5. The [M + H]+ ion produced three fragment ions at m/z 270.99, m/z 193.01, m/z 127.03 (Fig. 5). It was identified in the bibliography in extracts from Potato [Oertel et al., 2017]; F. glaucescens [Hamed et al., 2020]; Echinops [Seukep et al., 2020]; Rhodiola rosea [Lee et al., 2016]; Rhodiola crenulata [Daikonya et al., 2011].

The CID-spectrum in positive ion modes of Dihydrokaempferol from extracts of Lonicera caerulea L. (variety Wild Lonicera from Amur river) is shown in Fig. 6. The [M + H]+ ion produced one fragment ion at m/z 448.92 (Fig. 6). The fragment ion with m/z 448.92 yields three daughter ions at m/z 376.96, m/z 344.93, and m/z. 286.95. The fragment ion with m/z 376.96 yields two daughter ions at m/z 344.92, and m/z 286.99. It was identified in the bibliography in extracts from Rubus ulmifolius [da Silva et al., 2019]; Vitis vinifera [Goufo et a., 2020]

thumbnail Fig. 2.

Chemical profiles of the Lonicera caerulea L. (variety SPb 1043-11) sample represented total ion chromatogram from MeOHextract.
thumbnail Fig. 3.

Chemical profiles of the Lonicera caerulea L. (variety SPb 1043-8) sample represented total ion chromatogram from MeOH-extract.
thumbnail Fig. 4.

Chemical profiles of the Lonicera caerulea L. (variety Wild lonicera from Amur river) sample represented total ion chromatogram from MeOH-extract.
Table 1.

Identified target analytes in MeOH extracts of berries of Lonicera caerulea L.

thumbnail Fig. 5.

CID-spectrum of dihydrokaempferol from extracts of Lonicera caerulea L. (variety SPb 1043-8), m/z 289.98.
thumbnail Fig. 6.

CID-spectrum of Taxifolin 3-O-glucoside from extracts of Lonicera caerulea L. (variety Wild Lonicera from Amur river), m/z 466.92.

4 Conclusions

Blue-berried honeysuckle Lonicera caerulea L. contains a large number of polyphenolic compounds and other biologically active substances. In this work, we first tried to conduct a comparative metabolomic study of biologically active substances of wild Blue-berried honeysuckle obtained from locations in Khabarovsk territory and from the collection of N.I. Vavilov AllRussian Institute of Plant Genetic Resources (St.Petersburg). HPLC in combination with a BRUKER DALTONIKS ion trap (tandem mass spectrometry) was used to identify target analytes in extracts.

The results showed the presence of 82 biologically active compounds corresponding to the Blue-berried honeysuckle Lonicera caerulea species. In addition to the reported metabolites, a number of metabolites were newly annotated in blue-berried honeysuckle. There were flavonols: Dihydrokaempferol, Rhamnetin I, Rhamnetin II, Taxifolin-3-O-glucoside, Mearnsetin-hexoside, Horridin; flavones: Chrysoeriol, Apigenin-O-pentoside, Chrysoeriol-7-O-glucoside; flavanone Naringenin; flavan-3-ols: Catechin, Epicatechin, Biochanin A-7-Oglucoside; essential amino acids: L-Pyroglutamic acid, Tyrosine; polypeptide 5-Oxo-L-propyl-L-isoleucine; sterols: Ergosterol, Fucosterol, Beta-Sitosterin; triterpenoids: Betunolic acid, Oleanoic acid; anabolic steroid Vebonol, indole sesquiterpene alkaloid Sespendole; iridoids: Monotropein, p-Coumaroyl monotropein, p-Coumaroyl monotropein hexoside; Myristoleic acid, etc.

The findings may support future research into the production of various pharmaceutical and dietary supplements containing blue-berried honeysuckle Lonicera caerulea L. extracts. A wide variety of biologically active compounds opens up rich opportunities for the creation of new drugs and biologically active additives based on extracts from this family Caprifoliaceae.

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All Tables

Table 1.

Identified target analytes in MeOH extracts of berries of Lonicera caerulea L.

In the text

All Figures

thumbnail Fig. 1.

Polymorphism of wild Lonicera caerulea L. berries presented in the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources
In the text
thumbnail Fig. 2.

Chemical profiles of the Lonicera caerulea L. (variety SPb 1043-11) sample represented total ion chromatogram from MeOHextract.
In the text
thumbnail Fig. 3.

Chemical profiles of the Lonicera caerulea L. (variety SPb 1043-8) sample represented total ion chromatogram from MeOH-extract.
In the text
thumbnail Fig. 4.

Chemical profiles of the Lonicera caerulea L. (variety Wild lonicera from Amur river) sample represented total ion chromatogram from MeOH-extract.
In the text
thumbnail Fig. 5.

CID-spectrum of dihydrokaempferol from extracts of Lonicera caerulea L. (variety SPb 1043-8), m/z 289.98.
In the text
thumbnail Fig. 6.

CID-spectrum of Taxifolin 3-O-glucoside from extracts of Lonicera caerulea L. (variety Wild Lonicera from Amur river), m/z 466.92.
In the text

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