Next Article in Journal
Unlocking Nature’s Clock: CRISPR Technology in Flowering Time Engineering
Next Article in Special Issue
Unlocking Therapeutic Potential: Comprehensive Extraction, Profiling, and Pharmacological Evaluation of Bioactive Compounds from Eclipta alba (L.) Hassk. for Dermatological Applications
Previous Article in Journal
Characterization of the F-Box Gene Family and Its Expression under Osmotic Stress in Birch
Previous Article in Special Issue
Chemical Composition and Skin-Whitening Activities of Siegesbeckia glabrescens Makino Flower Absolute in Melanocytes
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 12
Issue 23
10.3390/plants12234019
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Phytochemical Profiles and In Vitro Immunomodulatory Activities of Extracts Obtained from Limonium gmelinii Using Different Extraction Methods

by
Dariya Kassymova
1,*,
Galiya Zhusupova
1,*,
Vyacheslav Ogay
2,
Aizhan Zhussupova
3,
Kumar Katragunta
4,
Bharathi Avula
4 and
Ikhlas A. Khan
4
1
Department of Chemistry and Technology of Organic Substances, Natural Compounds and Polymers, NPJSC Al-Farabi Kazakh National University, Al-Farabi Ave. 71, Almaty 050040, Kazakhstan
2
Stem Cell Laboratory, National Center for Biotechnology, Korgalzhyn Highway 13/5, Astana 010000, Kazakhstan
3
Department of Molecular Biology and Genetics, NPJSC Al-Farabi Kazakh National University, Al-Farabi Ave. 71, Almaty 050040, Kazakhstan
4
National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
*
Authors to whom correspondence should be addressed.
Plants 2023, 12(23), 4019; https://doi.org/10.3390/plants12234019
Submission received: 22 September 2023 / Revised: 15 October 2023 / Accepted: 23 October 2023 / Published: 29 November 2023
(This article belongs to the Special Issue Structural and Functional Analysis of Extracts in Plants IV)
Browse Figures
Review Reports Versions Notes

Abstract

:
Limonium (L.) gmelinii is a valuable pharmacopoeial Kazakhstani plant. Several studies have reported on the various biological activities of the plant. The purpose of our research was to study and compare the extraction yields, immunomodulatory activities, and chemical compositions of extracts from the above-ground parts of L. gmelinii obtained via conventional extraction (CE; Extract 1) and ultrasound-assisted extraction (UAE; Extract 2). The extracts were characterized by a considerable number of polyphenols and flavonoids: 378.1 ± 4.5 and 382.2 ± 3.3 GAE mg/g, and 90.22 ± 2.8 and 94.61 ± 1.9 QE mg/g in Extract 1 and Extract 2, respectively. Extract 2 had a slightly higher extraction yield (33.5 ± 2.4%) than Extract 1 (30.2 ± 1.6%). Liquid Chromatography–Diode-Array Detection–Electrospray Ionization–Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) revealed the presence of 54 biologically active compounds in both extracts. It was shown that the studied extracts stimulate the secretion of TNF-α and IL-6 by intact mouse peritoneal macrophages and splenic lymphocytes, whilst they have an inhibitory effect on the secretion of these cytokines by activated immune cells. Both extracts demonstrated similar patterns of stimulation and inhibition in a splenocyte proliferation assay. Altogether, the L. gmelinii extracts obtained via CE and UAE might be suggested as effective immunomodulatory agents. The application of UAE for this purpose seems to be more efficient with a view of obtaining of a highly potent extract in a much shorter time.

1. Introduction

Limonium (L.) Mill is a diverse genus of perennial grasses and shrubs, composed of almost 600 species [1]. A wide range of breeding options, including mixed reproduction (sexual, vegetative, and apomixis), with the occurrence of hybridization and polyploidy, determine the wide cosmopolitan distribution (arid and saline soils, rocky areas, deserts, and steppes) of its representatives along with a diverse spectrum of biological activities [2]. Plants of the genus are broadly used in decorative [1], medicinal [3], and food purposes [4], for the decontamination of soils from heavy metals [5], etc.
The broad spectrum of the proven therapeutic activity of certain species has been the object of numerous studies on the chemical compositions of their extracts and methods of the optimal extraction of biologically active compounds (BACs). For instance, a phenolic-rich ethanolic extract of L. delicatulum, a halophyte growing in the salt marshes of the Mediterranean coast, demonstrated significant antioxidant and antimicrobial activities [6]. Its methanolic extract was found to act as an inhibitor of certain enzymes, including cholinesterase [7] and tyrosinase [8]. Another representative of halophytes common in Mediterranean salt marshes, L. densiflorum, exhibited notable antioxidant, anti-inflammatory, antimicrobial, antitumor [9], and antiviral activities [10]. Antitumor and immunomodulatory effects were attributed to the polysaccharides of L. sinense (Girard) Kuntze [11]. The inhibitory activity of the aqueous extracts of L. brasiliense has been demonstrated against xanthine oxidase upon the treatment of hyperuricemia [12], as well as their antibacterial activity against bacteria with multidrug resistance [13]. Secondary metabolites isolated from L. tubiflorum were examined as antiviral components targeting SARS-CoV-2 in in silico studies, and some myricetin derivatives were suggested as potential COVID-19 drug candidates [14].
The most well-studied species of the genus L. Mill growing in Kazakhstan are L. gmelinii (Willd.) Kuntze, L. myrianthum (Schrenk) Kuntze, L. leptophyllum (Schrenk) Kuntze, and L. caspium (Willd.) Gams. Antibacterial, antifungal, antimalarial, and antileishmanial activities were demonstrated by compounds isolated from L. caspium [4]; similar effects were shown by secondary metabolites from L. myrianthum, L. leptophyllum, and L. gmelinii [15]. As such, preclinical tests of an extract from L. gmelinii roots showed its antioxidant, hepatoprotective, antibacterial, antimutagenic, antitumor, and antiviral properties [16,17]. It has also been shown that extracts from the roots and rhizomes of L. gmelinii protect neurons, astrocytes, and cerebral endothelial cells from oxidative and inflammatory responses in vitro and improve motor functions after ischemic stroke in vivo [18]. Data on the antidiabetic, cytotoxic, and anti-inflammatory properties of lignanamides isolated from the ethyl acetate extract of L. gmelinii roots [19].
Hence, Limonium species are therapeutically important and can be used for various medicinal purposes, the anti-inflammatory and antioxidant properties of which have been better studied, and these are what possibly make them useful in both acute and chronic inflammation.
Recently, plant-sourced phytochemicals have been in the spotlight because they represent a critical part in immunotherapies to combat the spread of cancer, autoimmune diseases, and infection, and they have promising potential for use as immune-enhancing agents and regulators [20]. It is known that an abnormal immune response against individual cells could have serious consequences, such as chronic inflammation leading to the above-mentioned diseases [21].
Macrophages and lymphocytes are known as key players in the development and resolution of inflammation, as they secrete a variety of inflammatory mediators, including several cytokines, inorganic reactive radicals, and reactive oxygen and nitrogen intermediates [22]. TNF-α and IL-6 are important biomarkers of proinflammatory responses; the dysregulation of their production is associated with a variety of inflammatory disorders [22]. An uncontrolled proliferation of lymphocytes can also lead to undesirable conditions like chronic or autoimmune diseases [23]. In general, immunomodulators modify immune responses, which, in turn, may lead to increased or decreased immune reactions [20].
The principal aim of the current research was to study and compare the immunomodulatory effect of L. gmelinii extracts obtained using methods of conventional extraction (CE) and ultrasound-assisted extraction (UAE) by measuring the levels of TNF-α and IL-6 produced by murine macrophages and splenic lymphocytes, as well as the proliferation of splenocytes. To the best of the authors’ knowledge, these properties have not yet been investigated in L. gmelinii species.

2. Results

2.1. Preparation of Extracts and Their Standardization

In our study, the efficiency of the CE and UAE methods of extraction was assessed via the yield of extracts and the content of the main secondary metabolites in them (Table 1). According to the yield, the L. gmelinii extracts obtained via 45 min ultrasonication (Extract 1) and 5 h double maceration (Extract 2) amounted to 30.2 ± 1.6% and 33.5 ± 2.4%, respectively (Table 1). The total phenolic contents (TPCs) of the extracts obtained via UAE and CE were 378.1 ± 4.5 mg gallic acid equivalent (GAE)/g and 382.2 ± 3.3 mg GAE/g, respectively. In the case of the total flavonoid content (TFC), the results were slightly different, 90.22 ± 2.8 mg quercetin equivalent QE/g for UAE and 94.61 ± 1.9 mg QE/g for CE. However, there were no significant differences (p > 0.05) between the two methods.

2.2. Identification and Characterization of Phenolic Acid Derivatives, Flavonoids, and Lignanamides

Extract 1 and Extract 2 were further examined using Liquid Chromatography–Diode-Array Detection–Electrospray Ionization–Quadrupole Time-of-Flight Mass Spectrometry (LC-DAD-QToF-MS). Data were obtained using both positive and negative electrospray ionization source (ESI) modes, and it was observed that phenolic acids and their derivatives were ionized in the negative mode, whereas flavonoids were ionized in both positive and negatives modes. At the same time, lignanamides showed better ionization in the positive mode than in the negative mode. The identified compounds are summarized in Table 2, including the retention time, molecular formulas, m/z values in both positive and negative modes, and major high-resolution mass fragment ions. As displayed in Table 2, the extracts of the above-ground parts of L. gmelinii were composed of phenolic acid derivatives (sulfated) (1–10); flavonoids (11–36); lignanamides (37–46); and other compounds, such as amino acids, citric acids and their derivatives, hydroxyquinoline (quinoline derivative), and N-trans feruloyl tyramine (alkaloid) (47–54).
LC-DAD chromatograms are presented in Figure 1a at 254 nm, 280 nm, and 330 nm wavelengths, and representative LC-MS total current chromatograms in the positive and negative modes of ionization are presented in Figure 1b. No differences were observed in the chromatographic profiles between Extract 1 and Extract 2 of the above-ground parts of L. gmelinii.
  • Phenolic acids and sulfated derivatives (1–10):
A total of 10 phenolic acids, such as gallic acid, protocatechuic acid, syringic acid, glucogallin, and their sulfated derivatives, were identified and tentatively characterized in the above-ground parts of L. gmelinii. The observed fragment ions are listed in Table 2.
  • Flavonoids (11–36):
Flavonoids were one of the major classes of compounds observed in the extracts. The tentative characterization of flavonoids was performed on the basis of our previous in-house report [18]. Gallocatechin, apigenin, quercetin, and myricetin derivatives, as well as their aglycone fragment ions, were the main flavonoids, as shown in Table 2.
  • Lignanamides (37–46):
Lignanamides are nitrogen-containing compounds, with characteristic fragment ion at m/z 287.0547 observed for limoniumin derivatives (38, 40–43) [3,19].
  • Other compounds (47–54):
In addition to the above-mentioned classes, the investigated extracts contained choline-O-sulfate, leucine and isoleucine amino acids, citric acids and their derivatives, a quinoline derivative, and N-trans feruloyl tyramine alkaloid.

2.3. Study of the Immunomodulatory Activity of the Extracts

2.3.1. Effect of L. gmelinii Extracts on the Cytokine-Producing Activity of Murine Macrophages and Lymphocytes

Extracts 1 and 2 were dissolved in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fischer Scientific, MA, USA) at the following concentrations: 1 mg/mL; 500; 100; 50; 25; 10; and 5 µg/mL. Splenic lymphocytes and peritoneal macrophages were obtained from male C57BL/6 mice to serve as target cells. Lymphocytes were activated with 5 µg/mL of Concanavalin A (ConA) (Sigma-Aldrich, St. Louis, MO, USA) and further cultured in an RPMI-1640 medium supplemented with Extracts 1 and 2 for 72 h. Peritoneal macrophages were stimulated with 500 pg/mL of lipopolysaccharides (LPSs) (Sigma-Aldrich, St. Louis, MO, USA), treated with the extracts, and incubated for 24 h at 37 °C and 5% CO2. The levels of cytokine production were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits.

Effect of L. gmelinii Extracts on IL-6 Levels in Murine Macrophages and Lymphocytes

According to our results, presented in Figure 2a, Extracts 1 and 2 exerted a stimulatory effect on IL-6 production by non-activated macrophages at a concentration of 100 μg/mL to 5 μg/mL (Figure 2a). The maximum stimulatory effect on IL-6 production by non-activated macrophages was found at a concentration of 10 μg/mL of both extracts.
As expected, macrophages responded to LPS as a result of increased levels of IL-6 (747.73 pg/mL; 755.54 pg/mL), effectively suppressed by both extracts (Figure 2b). The maximum suppression of almost 3.5-fold in IL-6 production caused by macrophages was observed at the same concentration of 100 μg/mL of both extracts (Figure 2b).
Similar immunostimulatory and immunosuppressive effects of Extracts 1 and 2 were found on splenic lymphocytes. Treatment with the extracts caused an increase in IL-6 secretion in non-activated lymphocytes, as shown in Figure 3a. IL-6 production reached the maximum at a concentration of 5 μg/mL of Extract 1 and 10 μg/mL of Extract 2. On the contrary, the application of the extracts to ConA-activated splenic lymphocytes led to a significant decrease in the level of IL-6 (Figure 3b). The maximum inhibitory effect on IL-6 expression was caused by Extracts 1 and 2 at a concentration of 100 μg/mL (Figure 3b).
Thus, no significant difference in the effects of Extracts 1 and 2 on the level of IL-6 production was observed in both intact and activated macrophages and lymphocytes.

Effect of L. gmelinii Extracts on TNF-α Levels in Murine Macrophages and Lymphocytes

The effects of the L. gmelinii extract on TNF-α secretion in murine macrophages and lymphocytes were also studied. There was the same tendency towards an increase in the TNF-α level in intact cells and its reduction in LPS-stimulated macrophages and ConA-activated lymphocytes.
As shown in Figure 4a, Extracts 1 and 2 at the same concentration of 1 mg/mL significantly increased TNF-α secretion in intact macrophages to 2.41 ± 0.19 pg/mL and 2.65 ± 0.84 pg/mL, respectively. However, treatment of activated macrophages with Extracts 1 and 2 at a concentration of 1 mg/mL suppressed the level of TNF-α production (Figure 4b). No effects were observed at lower concentrations of the plant extracts.
When examining the level of TNF-α in non-activated lymphocytes, a statistically significant increase was observed at the same concentration of 1 mg/mL of both extracts. These results are shown in Figure 5a. However, when the splenic lymphocytes were stimulated with ConA, the extracts inhibited the secretion of TNF-α at all doses (Figure 5b). Treatment with Extracts 1 and 2 significantly decreased cytokine levels, with the maximum reduction at a concentration of 100 μg/mL.

2.3.2. Effect of L. gmelinii Extracts on the Proliferation of Murine Splenic Lymphocytes

The proliferation of lymphocytes is a critical event in a proper immune response leading to the initiation and development of inflammation.
In this study, we determined the immunomodulatory effects of Extracts 1 and 2 on the proliferation of splenic lymphocytes in response to stimulation with ConA. An analysis of cell proliferation showed that Extract 2 more strongly suppressed the proliferation of activated splenic lymphocytes than Extract 1 (Figure 6a). A significantly high inhibition of the number of splenic lymphocytes (Extract 1, 65,750 ± 850) and (Extract 2, 32,835 ± 465) was produced at a dose of 1 mg/mL in comparison to the control (1,789,364 ± 511 and 1,788,125 ± 650, respectively).
However, both extracts had a stimulatory effect on the proliferation of non-activated lymphocytes (Figure 6b). The maximum number of proliferating cells was observed upon the addition of 50 µg/mL of Extract 1 and 25 µg/mL of Extract 2 (Figure 6b).

3. Discussion

The currently patented method for obtaining a valuable extract from the roots of L. gmelinii is 5 h maceration with 50% ethanol [24]. The extract obtained via this method contains a rich set of biologically active compounds: flavonols and their glycosides, condensed and hydrolysable tannins, pyrogallol, gallic and ellagic acids, proanthocyanidins, polyphenolic compounds, and previously undescribed forms of flavan-3-ols [15,16,17,18,25]. As such, polyphenols of botanic origin have been proven to act as immunomodulators [26]. The aim of the current research was to study the immunomodulatory activity of extracts from the above-ground parts of L. gmelinii, as well as to examine how ultrasonic-assisted treatment affects the extraction process and chemical composition of the obtained extracts.
It is reported that UAE can serve as a more efficient and environmentally friendly method due to its short processing time, lower consumption of solvent and energy, less environmental pollution, and low maintenance cost of equipment [27]. Moreover, several studies have shown an increase in the yield of extracts and their activity when using ultrasound in comparison with other methods [28,29]. At the same time, some authors have noted that the yields of the secondary metabolites obtained when using UAE are lower than or equal to those obtained when using CE [30].
In our study, the efficiency of the CE and UAE methods of extraction was assessed via the yield of extracts and the content of the main secondary metabolites in them (Table 1), and it was found that Extracts 1 and 2 differed insignificantly. The TPC values were 378.1 ± 4.5 mg GAE/g DW in Extract 1 and 382.2 ± 3.3 mg GAE/g DW in Extract 2, and these are much higher than those reported for L. gmelinii extracts from other countries, at 54.42 mg GAE/g DW [31] and 5.60 ± 0.45 mg GAE/g DW [32]. In reports [31] and [32], the TFC values as mg of catechin equivalent (CE) were a little over 2 mg CE/g DW and 1.57 ± 0.10 mg CE/g DW, respectively, which is significantly lower than those in our results. This may be due to the impact of geographical origin, environmental and seasonal variations [33], and the methods and conditions of extraction on phytochemical composition, which may alter the amount of phenolics [27].
As stated above, the polyphenolic complex is supposed to be a predominant class in the hydroethanolic extracts of L. gmelinii, with the biological activity possibly attributed to the presence of these secondary metabolites. The chemical constituents of L. gmelinii detected using LC-QToF-MS were phenolic acid derivatives (sulfated) (1–11); flavonoids (12–43); lignanamides (44–53); and other compounds, such as amino acids, citric acids and their derivatives, hydroxyquinoline (quinoline derivative), and N-trans feruloyl tyramine (alkaloid) (54–62). No differences in the chromatographic profiles were observed among the CE and UAE extracts from the above-ground parts of L. gmelinii. The obtained extracts displayed complex chemical compositions, which led to the fair assumption that they may have immunomodulatory activity.
It was also of interest to determine whether there was a difference in the biological activities of the extracts. The in vitro effects of the L. gmelinii extracts on the induction of murine macrophages and splenic lymphocytes that produce proinflammatory cytokines (IL-6 and TNF-α) and their action on the proliferative responses were studied for the first time.
There have been numerous studies on the immunomodulatory activity of phenolic-rich plant extracts and dietary polyphenols, and their data are in good agreement with the data obtained in this study. An immunosuppressive action linked to the downregulation of the gene expression of proinflammatory cytokines associated with the presence of polyphenolic and flavonoid constituents was suggested for a number of plants [34,35,36]. On the contrary, some reports showed an immunostimulatory effect of phenolic-rich extracts on the production of proinflammatory cytokines. As such, the MeOH extract of P. campechiana boosted the production of IL-6 and TNF-α by murine macrophages in a concentration-dependent manner [37]. Another study demonstrated that a herbal composition rich in phenols and flavonoids increased TNF-α and decreased IL-10 and IL-6 levels in CP-induced immunosuppressed mice [38].
Extracts 1 and 2 isolated from L. gmelinii effectively suppressed the increased levels of TNF-α and IL-6 in LPS- and ConA-stimulated macrophages and lymphocytes, respectively. At the same time, the treatment of non-activated macrophages and lymphocytes with Extracts 1 and 2 significantly increased the secretion of cytokines. The same stimulatory or inhibitory effects of Extract 1 and Extract 2 on lymphocyte proliferation depending on their state were also verified. However, no significant difference was noted in the immunomodulatory activity of the L. gmelinii extracts, supported by the above data on chemical composition. In similar studies of the immunomodulatory effects of Limonium species, it was demonstrated that the polysaccharides of L. sinense improved the phagocytosis activity of macrophages in immunosuppressed mice and increased the proliferation and production of IFN-γ and IL-2 by splenocytes in vitro [11]. The extract of L. duriusculum and the isolated flavonoid apigenin inhibited proinflammatory NF-κB-dependent transcriptional responses in HCT116 and THP-1 cells [39]. These observations, as well as the results of the current study, raise the prospect of using the extracts of L. gmelinii and related species for the treatment of diseases associated with immune disorders.

4. Conclusions

The results of the current study demonstrate that the method of obtaining extracts (CE and UAE) from the above-ground parts of L. gmelinii does not significantly affect the yield, TPC, TFC, chemical composition, or immunomodulatory activity. Therefore, UAE could have advantages over CE, as it is significantly faster and simpler and has a lower solvent consumption and “greener” approach [27]. The LC-QToF-MS analysis clearly showed impressive compositions of both extracts, rich in biologically active compounds with remarkable immunomodulatory potential, such as apigenin [39], epigallocatechin-3-gallate [20] and quercetin [20]. This work reported, for the first time, that extracts from the above-ground parts of L. gmelinii may modulate the inflammatory mode of murine macrophages and lymphocytes via the stimulation or suppression of TNF-α and IL-6 production and splenocyte proliferation depending on the status of these immune cells. The data obtained provide possibilities for further research into the mechanisms of the immunomodulatory activity of L. gmelinii extracts, which could be recommended for further preclinical and clinical studies.

5. Materials and Methods

5.1. Chemicals and Reagents

Ethanol ≥96% was purchased from Talgarspirt. Folin–Ciocalteu reagent, gallic acid ≥96%, and quercerin ≥95% were purchased from Sigma-Aldrich. Sodium carbonate and aluminum chloride were bought from AppliChem GmbH. LPS (from Escherichia coli 0111:B4) was from Sigma-Aldrich, and DMEM high-glucose medium was from Gibco. The used acetonitrile, methanol, and formic acid were of HPLC-certified grade, and water was purified using a Milli-Q system (Millipore).

5.2. Plant Raw Materials

The above-ground parts of L. gmelinii were collected in the territory of the Almaty region during the flowering period of 2020 and identified at the Institute of Botany and Phytointroduction in Almaty, Kazakhstan, with the voucher specimen number 154,958. During the trip to the territory of the Zhanalyk village of the Talgar region, the coordinates of the place of the collection and the growth of L. gmelinii species were noted (Table 3), and raw material was collected for the study and production of extracts. The raw materials were identified for authenticity using macro- and microscopy methods upon independent assessment at the laboratories of the Institute of Botany and Phytointroduction and al-Farabi Kazakh National University (Almaty, Kazakhstan), upon which they were thoroughly cleaned and dried and subsequently milled to a particle size of 3 mm. Quality control of plant raw materials was carried out in accordance with the requirements of the State Pharmacopeia of the Republic of Kazakhstan.

5.3. Preparation of Extracts and Their Standardization

5.3.1. Maceration and Ultrasonic-Assisted Extraction

To obtain the extract via UAE, the extraction of the milled plant material was carried out once under the optimum conditions [40] in an Elmasonic S 450 ultrasonic bath (Elma Electronic UK Ltd., Bedford, UK) at a ratio of raw material to solvent of 1:5 for 45 min at 30 °C. The filtered aqueous–alcoholic extract was concentrated using a rotary evaporator, model IKA RV20 (IKA®-Werke GmbH & Co. KG, Staufen im Breisgau, Germany), at a temperature of 40–45 °C in a vacuum to dryness until a brown crystalline powder was obtained.
To obtain the extract via CE, 5 h double extraction was carried out with a six-fold excess of 50% ethanol at a temperature of 20–23 °C, and the plant material was subsequently washed with water to remove the extract residues. The first and second extracts were mixed and concentrated to dryness under mild conditions on a rotary evaporator to obtain a dry extract. The extract yield was calculated using the formula:
Y i e l d , % = W e i g h t   o f   d r i e d   e x t r a c t W e i g h t   o f   p l a n t   m a t e r i a l × 100

5.3.2. Total Phenolic Content

The total phenolic contents (TPC) in the extracts were determined using the Folin–Ciocalteu reagent in accordance with the methodology in [41] with some modifications. About 200.0 mg of the substance was transferred into a 100.0 mL volumetric flask and quantitatively dissolved in 30% ethanol. Then, a 2.0 mL aliquot was taken and diluted with distilled water in a 100.0 mL volumetric flask.
Next, 1.0 mL of this solution was mixed with 1.0 mL of the Folin–Ciocalteu reagent and 15.0 mL of distilled water and left for 6 min. Subsequently, 3.0 mL of 20.0% sodium carbonate solution was added, and the flask was placed in a dark place. After 120 min, the absorbance corresponding to the total amount of polyphenols was measured at 760 nm on a JASCO J-715 spectrophotometer (Jasco Corp., Tokyo, Japan) using distilled water as a reference solution. Gallic acid was used as a standard for plotting a calibration line.

5.3.3. Total Flavonoid Content

The assessment of the total flavonoid content was carried out according to the method described in [42] with some modifications; quercetin was used as a standard. The dry extract was dissolved in 100.0 mL of 90% ethanol to prepare a solution with a concentration of 1.0 mg/mL. Then, 2.0 mL of a sample solution and 1.0 mL of 1% aluminum chloride in 95% ethanol were mixed and diluted to 25.0 mL in a volumetric flask. After 20 min, the optical density of the solution was measured on a spectrophotometer (Jasco Corp., Tokyo, Japan) at a wavelength of 415 nm. A solution consisting of 2.0 mL of the sample solution was diluted to 25.0 mL in a volumetric flask with 95% ethanol and used as a reference.

5.4. Chemical Characterization of Extract from Above-Ground Part of L. gmelinii Using Liquid Chromatography–Diode-Array Detector–Quadrupole Time-of-Flight Mass Spectrometry (LC-DAD-QToF)

5.4.1. Sample Preparation

About 20 mg of the extract from the above-ground parts of L. gmelinii was sonicated in 1.0 mL of methanol for 5 min followed by centrifugation for 10 min at 5000× g rpm separately. The supernatant-filtered solution was used for analyses.

5.4.2. Instrumentation and Analytical Conditions

The LC-QToF-MS method was used for the analysis of the extracts from the above-ground samples of L. gmelinii using the reported method [18].

5.5. In Vivo Experiments to Determine Immunomodulatory Activity

5.5.1. Preparation of the Test Specimens

Extracts 1 and 2 were dissolved in culture medium DMEM at the following 7 concentrations: 1 mg/mL; 500 µg/mL; 100 μg/mL; 50 μg/mL; 25 µg/mL; 10 μg/mL; and 5 µg/mL. The diluted solutions were sterilized using a polyethersulfone membrane filter with a pore size of 0.2 μm (TPP).

5.5.2. Animals

Male C57BL/6 10–12-week-old mice were purchased from Masgut Aikimbayev’s National Scientific Center for Especially Dangerous Infections (Almaty, Kazakhstan). The animals were housed in a temperature-controlled environment (23 °C) with 60% relative humidity applying a 12 h light/dark cycle. The animals had ad libitum access to food and water. The experimental procedures involving animals were in full compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, National Academy Press, 1996) [43] and were approved by the Local Ethics Committee for Animal Use in the National Center for Biotechnology (Protocol No. 4 from 8 September 2020).

5.5.3. Isolation of Lymphocytes

The animal was sacrificed via cervical dislocation and treated with a 70% ethanol solution; the abdomen was carefully cut, and the spleen was removed. The spleen was immediately transferred to a Petri dish with DMEM for washing. After this procedure, the spleen was transferred to a glass homogenizer filled with DMEM (4–5 mL) and homogenized with a Teflon pestle. The homogenate containing mainly lymphocytes was carefully removed with a Pasteur pipette and transferred into a sterile centrifuge tube (15 mL). Splenocytes were pelleted via centrifugation at 1500 rpm for 5 min. To remove erythrocytes, 3 mL of BD Pharm Lyse® lysing solution (BD Biosciences, Dubai, UAE) was added to the cell pellet and kept for 5 min on ice. After the hemolysis of erythrocytes, the splenocytes were washed three times with large volumes of DMEM to completely remove the lysing solution. Then, 2 mL of culture medium RPMI-1640 containing 10% fetal bovine serum (FBS) was added to the cell pellet and thoroughly resuspended with a vortex (Vortex 1, IKA-Werke GmbH & Co. KG, Staufen im Breisgau, Germany). The cells were counted with a TC20® automatic cell counter (Bio-Rad, Hercules, CA, USA).

5.5.4. Isolation of Peritoneal Macrophages

The animal was sacrificed via cervical dislocation and treated with a 70% ethanol solution. The skin was removed from the abdomen without piercing the abdominal wall. Then, 3 mL of DMEM was injected with some air into the abdominal cavity. In this case, the abdomen of the mouse swells, tightening the injection site. Then, using surgical forceps or a spatula, the abdominal wall was massaged for 1 min to separate peritoneal macrophages into suspension. Using a syringe, fluid was collected from the lateral side of the abdominal cavity and then transferred into sterile centrifuge tubes.
Peritoneal macrophages were isolated from other cells via their high adhesivity to a plastic surface. For this, peritoneal cells were suspended at a concentration of 2 × 106 cells/mL in RPMI-1640 medium containing 10 mM HEPES and 10% FBS, placed in polystyrene 24-well plates at 1 mL per well, and left for 1 h at 37 °C and 5% CO2. Then, the non-adherent cells were carefully removed, and the monolayer of macrophages was washed once with the DMEM. Cell viability was determined using 0.1% trypan blue. Macrophages were incubated in 1 mL of RPMI-1640 culture medium for 24 h at 37 °C and 5% CO2.

5.5.5. Determination of Cytokine Levels

The measurement of cytokine levels was performed using ELISA. ELISA was performed using the following comsigmial kits: a Mouse TNF alpha SimpleStep ELISA kit and an IL-6 Mouse SimpleStep ELISA kit (all ELISA kits were from Abcam, Cambridge, UK). All procedures were performed according to the manufacturer’s instructions.

5.5.6. Lymphocyte Proliferation Assay

Extracts 1 and 2 were dissolved in a complete nutrient medium, DMEM or RPMI-1640, at the following 7 concentrations: 1 mg/mL; 500 µg/mL; 100 μg/mL; 50 μg/mL; 25 µg/mL; 10 μg/mL; and 5 µg/mL. The splenic lymphocytes of C57BL/6 mice served as target cells. Prior to the addition of the preparations, lymphocytes were activated with ConA (Sigma-Aldrich) at a concentration of 5 µg/mL, after which they were cultivated in RPMI-1640 medium with the addition of Extracts 1 and 2 for 72 h. Splenic lymphocytes were stained with anti-Ki67 antibodies and analyzed using a Muse cellular analyzer (MilliporeSigma, Darmstadt, Germany).

5.6. Statistical Processing of Data

All data are presented as mean ± standard deviation. Statistical significance was calculated using a one-way analysis of variance (ANOVA) test. p < 0.05 was considered statistically significant. The statistical analysis was conducted with software Statistica 6.0 (StatSoft, Tulsa, OK, USA).

Author Contributions

Conceptualization, D.K., G.Z. and V.O.; methodology, D.K., G.Z., V.O., and B.A.; investigation, D.K., V.O., B.A. and K.K.; formal analysis D.K., V.O., A.Z., G.Z. and K.K.; visualization, D.K., A.Z., V.O. writing—original draft preparation, D.K., A.Z., V.O., B.A. and K.K.; writing—review and editing, A.Z., V.O., G.Z., B.A. and K.K.; supervision, G.Z.; Resources, I.A.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Committee of Science, Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. AP19174425, 2023–2025).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Koutroumpa, K.; Theodoridis, S.; Warren, B.H.; Jiménez, A.; Celep, F.; Doğan, M.; Romeiras, M.M.; Santos-Guerra, A.; Fernández-Palacios, J.M.; Caujapé-Castells, J.; et al. An Expanded Molecular Phylogeny of Plumbaginaceae, with Emphasis on Limonium (Sea Lavenders): Taxonomic Implications and Biogeographic Considerations. Ecol. Evol. 2018, 8, 12397–12424. [Google Scholar] [CrossRef] [PubMed]
  2. Palacios, C.; Rosselló, J.A.; González-Candelas, F. Study of the Evolutionary Relationships among Limonium Species (Plumbaginaceae) Using Nuclear and Cytoplasmic Molecular Markers. Mol. Phylogenet Evol. 2000, 14, 232–249. [Google Scholar] [CrossRef] [PubMed]
  3. González-Orenga, S.; Grigore, M.N.; Boscaiu, M.; Vicente, O. Constitutive and Induced Salt Tolerance Mechanisms and Potential Uses of Limonium Mill. Species. Agronomy 2021, 11, 413. [Google Scholar] [CrossRef]
  4. Gancedo, N.C.; Isolani, R.; de Oliveira, N.C.; Nakamura, C.V.; de Medeiros Araújo, D.C.; Sanches, A.C.C.; Tonin, F.S.; Fernandez-Llimos, F.; Chierrito, D.; de Mello, J.C.P. Chemical Constituents, Anticancer and Anti-Proliferative Potential of Limonium Species: A Systematic Review. Pharmaceuticals 2023, 16, 293. [Google Scholar] [CrossRef] [PubMed]
  5. Gadetskaya, A.V.; Tarawneh, A.H.; Zhusupova, G.E.; Gemejiyeva, N.G.; Cantrell, C.L.; Cutler, S.J.; Ross, S.A. Sulfated Phenolic Compounds from Limonium caspium: Isolation, Structural Elucidation, and Biological Evaluation. Fitoterapia 2015, 104, 80–85. [Google Scholar] [CrossRef] [PubMed]
  6. Medini, F.; Fellah, H.; Ksouri, R.; Abdelly, C. Total Phenolic, Flavonoid and Tannin Contents and Antioxidant and Antimicrobial Activities of Organic Extracts of Shoots of the Plant Limonium delicatulum. J. Taibah Univ. Sci. 2014, 8, 216–224. [Google Scholar] [CrossRef]
  7. Bakhouche, I.; Aliat, T.; Boubellouta, T.; Gali, L.; Şen, A.; Bellik, Y. Phenolic Contents and In Vitro Antioxidant, Anti-Tyrosinase, and Anti-Inflammatory Effects of Leaves and Roots Extracts of the Halophyte Limonium delicatulum. S. Afr. J. Bot. 2021, 139, 42–49. [Google Scholar] [CrossRef]
  8. Ruiz-Riaguas, A.; Zengin, G.; Sinan, K.I.; Salazar-Mendías, C.; Llorent-Martínez, E.J. Phenolic Profile, Antioxidant Activity, and Enzyme Inhibitory Properties of Limonium delicatulum (Girard) Kuntze and Limonium quesadense Erben. J. Chem. 2020, 2020, 1016208. [Google Scholar] [CrossRef]
  9. Medini, F.; Bourgou, S.; Lalancette, K.G.; Snoussi, M.; Mkadmini, K.; Coté, I.; Abdelly, C.; Legault, J.; Ksouri, R. Phytochemical Analysis, Antioxidant, Anti-Inflammatory, and Anticancer Activities of the Halophyte Limonium densiflorum Extracts on Human Cell Lines and Murine Macrophages. S. Afr. J. Bot. 2015, 99, 158–164. [Google Scholar] [CrossRef]
  10. Medini, F.; Megdiche, W.; Mshvildadze, V.; Pichette, A.; Legault, J.; St-Gelais, A.; Ksouri, R. Antiviral-Guided Fractionation and Isolation of Phenolic Compounds from Limonium densiflorum Hydroalcoholic Extract. Comptes Rendus Chim. 2016, 19, 726–732. [Google Scholar] [CrossRef]
  11. Tang, X.H.; Yan, L.F.; Gao, J.; Yang, X.L.; Xu, Y.X.; Ge, H.Y.; Yang, H.D. Antitumor and Immunomodulatory Activity of Polysaccharides from the Root of Limonium sinense Kuntze. Int. J. Biol. Macromol. 2012, 51, 1134–1139. [Google Scholar] [CrossRef] [PubMed]
  12. Rodriguez, S.A.; Murray, A.P.; Leiro, J.M. Xanthine Oxidase Inhibition by Aqueous Extract of Limonium brasiliense (Plumbaginaceae). Chem. Proc. 2021, 3, 123. [Google Scholar] [CrossRef]
  13. Blainski, A.; Gionco, B.; Oliveira, A.G.; Andrade, G.; Scarminio, I.S.; Silva, D.B.; Lopes, N.P.; Mello, J.C.P. Antibacterial Activity of Limonium brasiliense (Baicuru) against Multidrug-Resistant Bacteria Using a Statistical Mixture Design. J. Ethnopharmacol. 2017, 198, 313–323. [Google Scholar] [CrossRef] [PubMed]
  14. Hassan, A.R.; Sanad, I.M.; Allam, A.E.; Abouelela, M.E.; Sayed, A.M.; Emam, S.S.; El-Kousy, S.M.; Shimizu, K. Chemical Constituents from: Limonium tubiflorum and Their In Silico Evaluation as Potential Antiviral Agents against SARS-CoV-2. RSC Adv. 2021, 11, 32346–32357. [Google Scholar] [CrossRef] [PubMed]
  15. Gadetskaya, A.V.; Mohamed, S.M.; Tarawneh, A.H.; Mohamed, N.M.; Ma, G.; Ponomarev, B.N.; Zhusupova, G.E.; Cantrell, C.L.; Cutler, S.J.; Ross, S.A. Phytochemical Characterization and Biological Activity of Secondary Metabolites from Three Limonium Species. Med. Chem. Res. 2017, 26, 2743–2750. [Google Scholar] [CrossRef]
  16. Kozhamkulova, Z.A.; Radwan, M.M.; Zhusupova, G.E.; Abilov, Z.Z.; Rahadilova, S.N.; Ross, S.A. Gmelinoside I, a New Flavonol Glycoside from Limonium gmelinii. Nat. Prod. Commun. 2010, 5, 1061–1062. [Google Scholar] [CrossRef] [PubMed]
  17. Korul’kina, L.M.; Shul’ts, E.E.; Zhusupova, G.E.; Abilov, Z.A.; Erzhanov, K.B.; Chaudri, M.I. Biologically Active Compounds from Limonium Gmelinii and L. Popovii I. Chem. Nat. Compd. 2004, 40, 465–471. [Google Scholar] [CrossRef]
  18. Nurkenov, T.; Tsoy, A.; Olzhayev, F.; Abzhanova, E.; Turgambayeva, A.; Zhussupova, A.; Avula, B.; Ross, S.; Aituarova, A.; Kassymova, D.; et al. Plant Extract of Limonium gmelinii Attenuates Oxidative Responses in Neurons, Astrocytes, and Cerebral Endothelial Cells in Vitro and Improves Motor Functions of Rats after Middle Cerebral Artery Occlusion. Antioxidants 2021, 10, 1814. [Google Scholar] [CrossRef]
  19. Tuohongerbieke, A.; Li, J.; Sabir, G.; Xin, X.; Hu, M.; Duan, X.; Liu, L.; Tang, D.; Zhu, J.; Aisa, H.A. Lignanamides from the Roots of Limonium gmelinii (Willd.) Kuntze and Their Anti-Diabetic, Cytotoxic and Anti-Inflammatory Activities. Phytochemistry 2021, 184, 112648. [Google Scholar] [CrossRef]
  20. Jantan, I.; Ahmad, W.; Bukhari, S.N.A. Plant-Derived Immunomodulators: An Insight on Their Preclinical Evaluation and Clinical Trials. Front. Plant Sci. 2015, 6, 655. [Google Scholar] [CrossRef]
  21. Strzelec, M.; Detka, J.; Mieszczak, P.; Sobocińska, M.K.; Majka, M. Immunomodulation—A General Review of the Current State-of-the-Art and New Therapeutic Strategies for Targeting the Immune System. Front. Immunol. 2023, 14, 1127704. [Google Scholar] [CrossRef] [PubMed]
  22. Chen, L.; Deng, H.; Cui, H.; Fang, J.; Zuo, Z.; Deng, J.; Li, Y.; Wang, X.; Zhao, L. Inflammatory Responses and Inflammation-Associated Diseases in Organs. Oncotarget 2018, 9, 7204–7218. [Google Scholar] [CrossRef] [PubMed]
  23. Sheu, T.T.; Chiang, B.L. Lymphopenia, Lymphopenia-induced Proliferation, and Autoimmunity. Int. J. Mol. Sci. 2021, 22, 4152. [Google Scholar] [CrossRef] [PubMed]
  24. Zhusupova, G.E.; Rakhimov, K.D.; Abilov, Z.A. Method for Obtaining a Total Polyphenolic Complex from the Roots of Limonium gmelinii 2007. Method for Obtaining a Total Polyphenolic Complex from the Roots of Limonium gmelinii. RK Patent No. 14418 from 15.10.2007. bul. No. 10 (rus.). Available online: https://kzpatents.com/5-14418-sposob-polucheniya-summarnogo-polifenolnogo-kompleksa-iz-kornejj-kermeka-gmelina.html (accessed on 6 October 2021).
  25. Zhusupova, G.E.; Abil’kaeva, S.A. Flavanes from Limonium gmelinii. II. Chem. Nat. Compd. 2006, 42, 112–113. [Google Scholar] [CrossRef]
  26. Yahfoufi, N.; Alsadi, N.; Jambi, M.; Matar, C. The Immunomodulatory and Anti-Inflammatory Role of Polyphenols. Nutrients 2018, 10, 1618. [Google Scholar] [CrossRef] [PubMed]
  27. Medina-Torres, N.; Ayora-Talavera, T.; Espinosa-Andrews, H.; Sánchez-Contreras, A.; Pacheco, N. Ultrasound Assisted Extraction for the Recovery of Phenolic Compounds from Vegetable Sources. Agronomy 2017, 7, 47. [Google Scholar] [CrossRef]
  28. Gajic, I.S.; Savic, I.; Boskov, I.; Žerajić, S.; Markovic, I.; Gajic, D. Optimization of Ultrasound-Assisted Extraction of Phenolic Compounds from Black Locust (Robiniae Pseudoacaciae) Flowers and Comparison with Conventional Methods. Antioxidants 2019, 8, 248. [Google Scholar] [CrossRef] [PubMed]
  29. Surin, S.; You, S.G.; Seesuriyachan, P.; Muangrat, R.; Wangtueai, S.; Jambrak, A.R.; Phongthai, S.; Jantanasakulwong, K.; Chaiyaso, T.; Phimolsiripol, Y. Optimization of Ultrasonic-Assisted Extraction of Polysaccharides from Purple Glutinous Rice Bran (Oryza sativa L.) and Their Antioxidant Activities. Sci. Rep. 2020, 10, 10410. [Google Scholar] [CrossRef]
  30. Albuquerque, B.R.; Prieto, M.A.; Barreiro, M.F.; Rodrigues, A.E.; Curran, T.P.; Barros, L.; Ferreira, I.C.F.R. Catechin-Based Extract Optimization Obtained from Arbutus unedo L. Fruits Using Maceration/Microwave/Ultrasound Extraction Techniques. Ind. Crops Prod. 2017, 95, 404–415. [Google Scholar] [CrossRef]
  31. Ivan, M.A.; Oprică, L. Study of Polyphenols and Flavonoids Contents of Some Halophytes Species Collected from Dobrogea Region. Bull. Transilv. Univ. Bras. Ser. II For. Wood Ind. Agric. Food Eng. 2013, 6, 121–128. [Google Scholar]
  32. Grigore, M.N.; Oprica, L. Halophytes as Possible Source of Antioxidant Compounds, in a Scenario Based on Threatened Agriculture and Food Crisis. Iran. J. Public Health 2015, 44, 1153–1155. [Google Scholar] [PubMed]
  33. Yang, L.; Wen, K.S.; Ruan, X.; Zhao, Y.X.; Wei, F.; Wang, Q. Response of Plant Secondary Metabolites to Environmental Factors. Molecules 2018, 23, 762. [Google Scholar] [CrossRef] [PubMed]
  34. Herath, K.H.I.N.M.; Kim, H.J.; Lee, J.H.; Je, J.G.; Yu, H.S.; Jeon, Y.J.; Kim, H.J.; Jee, Y. Sargassum horneri (Turner) C. Agardh Containing Polyphenols Attenuates Particulate Matter-Induced Inflammatory Response by Blocking TLR-Mediated MYD88-Dependent MAPK Signaling Pathway in MLE-12 Cells. J. Ethnopharmacol. 2021, 265, 113340. [Google Scholar] [CrossRef]
  35. Uttra, A.M.; Alamgeer; Shahzad, M.; Shabbir, A.; Jahan, S.; Bukhari, I.A.; Assiri, A.M. Ribes orientale: A Novel Therapeutic Approach Targeting Rheumatoid Arthritis with Reference to pro-Inflammatory Cytokines, Inflammatory Enzymes and Anti-Inflammatory Cytokines. J. Ethnopharmacol. 2019, 237, 92–107. [Google Scholar] [CrossRef]
  36. George, G.; Shyni, G.L.; Abraham, B.; Nisha, P.; Raghu, K.G. Downregulation of TLR4/MyD88/P38MAPK and JAK/STAT Pathway in RAW 264.7 Cells by Alpinia galanga Reveals Its Beneficial Effects in Inflammation: Immunomodulatory Effect of Alpinia galanga. J. Ethnopharmacol. 2021, 275, 114132. [Google Scholar] [CrossRef]
  37. Chan-Zapata, I.; Canul-Canche, J.; Fernández-Martín, K.; Martín-Quintal, Z.; Torres-Romero, J.C.; Lara-Riegos, J.C.; Ramírez-Camacho, M.A.; Arana-Argáez, V.E. Immunomodulatory Effects of the Methanolic Extract from Pouteria campechiana Leaves in Macrophage Functions. Food Agric. Immunol. 2018, 29, 386–399. [Google Scholar] [CrossRef]
  38. Parveen, A.; Zahiruddin, S.; Agarwal, N.; Akhtar Siddiqui, M.; Husain Ansari, S.; Ahmad, S. Modulating Effects of the Synergistic Combination of Extracts of Herbal Drugs on Cyclophosphamide-Induced Immunosuppressed Mice. Saudi J. Biol. Sci. 2021, 28, 6178–6190. [Google Scholar] [CrossRef]
  39. Hamadou, M.H.; Kerkatou, M.; Zucal, C.; Bisio, A.; Provenzani, A.; Inga, A.; Menad, A.; Benayache, S.; Benayache, F.; Ameddah, S. Limonium duriusculum (de Girard) Kuntze Exhibits Anti-Inflammatory Effect Via NF-KB Pathway Modulation. Braz. Arch. Biol. Technol. 2021, 64, e21200179. [Google Scholar] [CrossRef]
  40. Kassymova, D.T.; Alieva, A.B.; Zhuzeeva, M.S.; Zhusupova, G.E. Ultrazvukovaia Ekstraktsiia Kak Sposob Optimizatsii Tekhnolohii Izvlecheniia Biolohicheski Aktivnykh Veshchestv Iz Rastenii Vida. [Ultrasound Extraction as a Method Optimization of Extraction Technology from Plant Species]. Limonium Gmelinii 2020, 69, 59–67. [Google Scholar]
  41. Kupina, S.; Fields, C.; Roman, M.C.; Brunelle, S.L. Determination of Total Phenolic Content Using the Folin-C Assay: Single-Laboratory Validation, First Action 2017.13. J. AOAC Int. 2018, 101, 1466–1472. [Google Scholar] [CrossRef]
  42. Zengin, G.; Locatelli, M.; Carradori, S.; Mocan, A.M.; Aktumsek, A. Total Phenolics, Flavonoids, Condensed Tannins Content of Eight Centaurea Species and Their Broad Inhibitory Activities against Cholinesterase, Tyrosinase, α-Amylase and α-Glucosidase. Not. Bot. Horti Agrobot. Cluj-Napoca 2016, 44, 195–200. [Google Scholar] [CrossRef]
  43. National Research Council (US) Institute for Laboratory Animal Research. Guide for the Care and Use of Laboratory Animals; National Academies Press (US): Washington, DC, USA, 1996. [Google Scholar] [CrossRef]
Plants 12 04019 g001
Figure 1. (a) LC-DAD chromatograms at 254, 280, and 330 nm of the above-ground parts of L. gmelinii; (b) LC-QToF-MS chromatograms in negative and positive modes along with DAD chromatograms.
Figure 1. (a) LC-DAD chromatograms at 254, 280, and 330 nm of the above-ground parts of L. gmelinii; (b) LC-QToF-MS chromatograms in negative and positive modes along with DAD chromatograms.
Plants 12 04019 g001
Plants 12 04019 g002
Figure 2. Effects of L. gmelinii extracts on the secretion of IL-6 by non-activated (a) and activated (b) macrophages. Controls were used in the experiment: macrophages without treatment or stimulus and macrophages activated with LPS. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Figure 2. Effects of L. gmelinii extracts on the secretion of IL-6 by non-activated (a) and activated (b) macrophages. Controls were used in the experiment: macrophages without treatment or stimulus and macrophages activated with LPS. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Plants 12 04019 g002
Plants 12 04019 g003
Figure 3. Effects of L. gmelinii extracts on the secretion of IL-6 by splenic T lymphocytes without treatment or stimulus (a) and by splenic T lymphocytes activated with ConA (b). Controls were used in the experiment: splenic T lymphocytes without treatment or stimulus and splenic T lymphocytes activated with ConA. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Figure 3. Effects of L. gmelinii extracts on the secretion of IL-6 by splenic T lymphocytes without treatment or stimulus (a) and by splenic T lymphocytes activated with ConA (b). Controls were used in the experiment: splenic T lymphocytes without treatment or stimulus and splenic T lymphocytes activated with ConA. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Plants 12 04019 g003
Plants 12 04019 g004
Figure 4. Effects of L. gmelinii extracts on TNF-α secretion by intact (a) and activated (b) macrophages. Controls were used in the experiment: macrophages without treatment or stimulus and macrophages activated with LPS. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Figure 4. Effects of L. gmelinii extracts on TNF-α secretion by intact (a) and activated (b) macrophages. Controls were used in the experiment: macrophages without treatment or stimulus and macrophages activated with LPS. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Plants 12 04019 g004
Plants 12 04019 g005
Figure 5. Effects of L. gmelinii extracts on the secretion of TNF-α by splenic T lymphocytes without treatment or stimulus (a) and splenic T lymphocytes activated with ConA (b). Controls were used in the experiment: splenic T lymphocytes without treatment or stimulus and splenic T lymphocytes activated with ConA. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Figure 5. Effects of L. gmelinii extracts on the secretion of TNF-α by splenic T lymphocytes without treatment or stimulus (a) and splenic T lymphocytes activated with ConA (b). Controls were used in the experiment: splenic T lymphocytes without treatment or stimulus and splenic T lymphocytes activated with ConA. Data represent mean ± S.D (n = 6). (*) indicates significant differences in comparison to control; p < 0.05 in comparison to control.
Plants 12 04019 g005
Plants 12 04019 g006
Figure 6. Effect of L. gmelinii extracts on the proliferation of splenic T lymphocytes without treatment or stimulus (a) and spleen splenic T lymphocytes activated with Con A (b). Controls used in the experiment: splenic T lymphocytes without treatment or stimulus and T lymphocytes activated with ConA. Data represent mean ± S.D (n = 6).
Figure 6. Effect of L. gmelinii extracts on the proliferation of splenic T lymphocytes without treatment or stimulus (a) and spleen splenic T lymphocytes activated with Con A (b). Controls used in the experiment: splenic T lymphocytes without treatment or stimulus and T lymphocytes activated with ConA. Data represent mean ± S.D (n = 6).
Plants 12 04019 g006
Table 1. Yield and content of secondary metabolites in L. gmelinii extracts (mean  ±  SD, n  =  3).
Table 1. Yield and content of secondary metabolites in L. gmelinii extracts (mean  ±  SD, n  =  3).
Extract 1 (UAE)Extract 2 (CE)
Yield a, %30.2 ± 1.633.5 ± 2.4
The total amount of polyphenols a, mg GAE/g378.1 ± 4.5382.2 ± 3.3
The total amount of flavonoids a, mg QE/g90.22 ± 2.894.61 ± 1.9
a Results are presented as mean ± standard deviation.
Table 2. Table 2. Tentative identification and characterization of natural compounds in extracts from aboveground parts of L. gmelinii using LC-QToF in positive and negative ionization modes.
Table 2. Table 2. Tentative identification and characterization of natural compounds in extracts from aboveground parts of L. gmelinii using LC-QToF in positive and negative ionization modes.
#RT
(Min)		Tentative Compound NameMolecular FormulaMass[M+H]+Fragment ions
(Positive ion Mode)		[M-H]−Fragment ions
(Negative ion Mode)		Above-the-Ground Parts
Macer-ationUAE
Sulfated phenolic acids
13.40SulfosalidrosideC14H20O10S380.0777--379.0704
(379.0704) *		96.9602++
23.71Sulphonic acidC8H10O5S218.0249--217.0184
(217.0176)		96.9605++
34.66p-Coumaroyl-(β-D-glucose-6-O-sulfate)C15H18O11S406.0570--405.0506
(405.0497)		247.0291, 241.0034, 163.0404, 96.9607 ++
47.09dihydromyricetin-3′-O-sulfateC15H12O11S400.0100--399.0032
(399.0028)		319.0450, 151.0035, 137.0245++
511.41Deoxy-dihydromyricetin-3′-O-sulfateC15H12O10S384.0151--383.0083
(383.0078)		151.0042++
612.70Myricetin-3′-O-
sulfate		C15H10O11S397.9944--396.9874
(396.9871)		317.0308, 151.0039, 137.0246++
713.09Pinoresinol-4-sulfateC20H22O9S438.0985--437.0920
(437.0912)		396.9873, 380.9929 ++
Phenolic acid/derivatives
82.04Gallic acidC7H6O5170.0215--169.0148
(169.0142)		125.0250++
92.90Protocatechuic acidC7H6O4154.0266--153.0202
(153.0193)		109.0299++
108.94Syringic acidC9H10O5198.0528--197.0459
(197.0455)		169.0148++
Flavonoids
112.84Gallocatechin/Epigallo-catechin/3,5,7,3′,4′,6′-hexahydroxyflavanC15H14O7306.074307.0812
(307.0812)		139.0382305.0669 (305.0667)137.0246++
123.24++
134.00++
144.93++
155.00(−)-Epigallocatechin-(4β→8)-(-)-epigallocatechin-3-O-gallateC37H30O18762.1432763.1508
(763.1505)		307.0817, 139.0392761.1358
(761.1359)		423.0715++
166.07Epigallocatechin-(2β→7,4β→8)-epigallocatechin-3-O-gallateC37H28O18760.1276761.1348
(761.1348)		287.0544, 139.0389759.1200
(759.1203)		453.1038++
176.593-O-Galloylepigallo-catechin (4β→8)-epigallocatechin-3-O-gallate/Theasinensin A/Theasinensin DC44H34O22914.1542915.1607
(915.1614)		745.1371, 595.1069, 457.0760, 287.0555, 153.0186 913.1477
(913.1469)		423.0729, 285.0405++
186.98++
199.75++
206.72Epigallocatechin gallate/Gallo-epicatechin gallateC22H18O11458.0849459.0926 (459.0922)289.0711, 139.0393457.0777 (457.0776)305.0681, 169.0157++
217.45++
227.83Myricetin 7-(6′/6″-galloylglucoside)C28H24O17632.1013633.1089
(633.1086)		481.0966, 319.0446, 287.0545, 153.0180631.0947
(631.0941)		559.0402, 479.0836, 317.0307++
238.13319.0455, 153.0182479.0832, 316.0226++
247.91Samarangenin BC44H32O22912.1385913.1449
(913.1458)		743.1225, 617.0932, 455.0596911.1305
(911.1312)		-++
258.67Myricetin 3-galactoside (Gmelinoside I)/
Myricetin 3-β-D-sorboside/Myricetin 3-glucoside		C21H20O13480.0904481.0974
(481.0977)		319.0451479.0836
(479.0831)		316.0235++
268.91++
279.10++
289.46Myricetin 3-xylosideC20H18O12450.0798451.0871
(451.0871)		319.0453449.0727
(449.0725)		316.0229++
2910.05++
3010.383,3′,4′,5,5′,7-Hexahydroxyflavone; 3-O-[rhamnopyranosyl-(1→2)-rhamnopyranoside]C27H30O16610.1534611.1607
(611.1606)		465.1043, 319.0461609.1461
(609.1461)		463.0893, 316.0233++
3110.60Quercetin glucosideC21H20O12464.0955465.1030
(465.1028)		303.0497463.0881
(463.0882)		301.0346++
3210.86++
3312.2MyricetinC15H10O8318.0376319.0453
(319.0448)		153.0183317.0310
(317.0303)		151.0041, 137.0246++
3412.50Quercetin rhamnoside (Quercitrin)C21H20O11448.1006449.1080
(449.1082)		303.0504447.0929
(447.0933)		301.0354++
3512.99Apigenin-7-glucuronideC21H18O11446.0849447.0925
(447.0922)		271.0611445.0780
(445.0776)		269.0456++
3619.37ApigeninC15H10O5270.0528271.0603
(271.0601)		153.0185, 119.0489269.0460
(269.0455)		117.0351++
Lignanamides
3714.92Cannabisin AC34H30N2O8594.2002595.2081
(595.2075)		---++
3814.95Limoniumin AC26H19NO7457.1162458.1236 (458.1234)287.0550--++
3915.14Cannabisin B/3,3′-Demethyl-heliotropamideC34H32N2O8596.2159597.2235
(597.2231)		295.0599, 187.0391,
153.0181		--++
4016.40Limoniumin BC27H21NO7471.1318472.1382
(472.1391)		287.0547--++
4116.80Limoniumin F/H/I/Cannabisin CC35H34N2O8610.2315611.2383
(611.2388)		287.0546--++
4217.10++
4317.73++
4418.52Cannabisin D/FC36H36N2O8624.2472625.2544
(625.2544)		367.2091--++
4518.81++
4619.20++
Others
471.11Choline-O-sulfateC5H13NO4S183.0565184.0638
(184.0638)		104.1072 (Choline)182.0487
(182.0493)		-++
481.20Leucine/Isoleucine
(Amino acids)		C6H13NO2131.0946132.1022
(132.1019)		86.0957--++
491.81++
501.32Citric acid/Isocitric acidC6H8O7192.0270--191.0197
(191.0202)		111.0089++
511.41++
523.102-Ethyl citrateC8H12O7220.0583--219.0520
(219.0513)		183.0305, 111.0086++
534.51Hydroxyquinoline
(Quinoline derivative)		C9H7NO145.0528146.0604
(146.0600)		128.0495, 91.0546, 77.0391--++
5415.95N-trans-feruloyl tyramine
(Alkaloid)		C18H19NO4313.1314314.1385
(314.1381)		177.0541, 145.0281, 117.0330--++
* theoretical exact mass; ‘+’ indicates presence of the compound and ‘ND’ indicates the compound not detected in respective plant part extract; ‘UAE’ indicates Ultrasound-assisted extraction.
Table 3. Coordinates of the collection of plant materials.
Table 3. Coordinates of the collection of plant materials.
SpeciesCollection AreaGPS CoordinatesPhenophaseCollected PartPhytomass, kg
Limonium gmelinii, aboveground partsTalgar region, Zhanalyk villageH = 1326 m above the sea level
N = 43°34′28″
E = 077°03′20″		budding, beginning of flowering. sp-sol, bloomingAbove ground part (fresh)10.5
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kassymova, D.; Zhusupova, G.; Ogay, V.; Zhussupova, A.; Katragunta, K.; Avula, B.; Khan, I.A. Phytochemical Profiles and In Vitro Immunomodulatory Activities of Extracts Obtained from Limonium gmelinii Using Different Extraction Methods. Plants 2023, 12, 4019. https://doi.org/10.3390/plants12234019

AMA Style

Kassymova D, Zhusupova G, Ogay V, Zhussupova A, Katragunta K, Avula B, Khan IA. Phytochemical Profiles and In Vitro Immunomodulatory Activities of Extracts Obtained from Limonium gmelinii Using Different Extraction Methods. Plants. 2023; 12(23):4019. https://doi.org/10.3390/plants12234019

Chicago/Turabian Style

Kassymova, Dariya, Galiya Zhusupova, Vyacheslav Ogay, Aizhan Zhussupova, Kumar Katragunta, Bharathi Avula, and Ikhlas A. Khan. 2023. "Phytochemical Profiles and In Vitro Immunomodulatory Activities of Extracts Obtained from Limonium gmelinii Using Different Extraction Methods" Plants 12, no. 23: 4019. https://doi.org/10.3390/plants12234019

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop