Some bioactivities of Limonium meyeri extracts and their separated compounds

Abstract

The antimicrobial and tyrosinase enzyme inhibition activities of various extracts and isolated compounds of Limonium meyeri were investigated. Apigenin-7-O-β-glucuronide and myricetin 3-O-α-rhamnoside were isolated from the ethyl acetate fraction using chromatographic methods. Methanol extract showed 62.0% and ethyl acetate extract showed 57.6% tyrosinase enzyme inhibition activities at 400 µg/mL concentration. Myricetin 3-O-α-rhamnoside (IC₅₀ = 27.7 ± 0.6 µg/mL) showed higher tyrosinase enzyme inhibitory effect than apigenin-7-O-β-glucuronide (IC₅₀ = 43.0 ± 0.9 µg/mL). n-Hexane extract was found to be effective against Enterococcus faecalis (MIC = 1 µg/mL), while methanol extract was effective against Pseudomonas aeruginosa (MIC = 16 µg/mL). Myricetin 3-O-α-rhamnoside showed antimicrobial effect against Enterococcus faecalis (MIC = 128 µg/mL). Thus, Limonium meyeri and its compounds may provide valuable insights for future studies as antityrosinase and antibacterial agents.

Full Article

Some Bioactivities of Limonium meyeri Extracts and their Separated Compounds

Masum Sami,^a Esen Sezen Karaoğlan ,^b,* Hayrunisa Hancı ,^c and Cavit Kazaz ^d

The antimicrobial and tyrosinase enzyme inhibition activities of various extracts and isolated compounds of Limonium meyeri were investigated. Apigenin-7-O-β-glucuronide and myricetin 3-O-α-rhamnoside were isolated from the ethyl acetate fraction using chromatographic methods. Methanol extract showed 62.0% and ethyl acetate extract showed 57.6% tyrosinase enzyme inhibition activities at 400 µg/mL concentration. Myricetin 3-O-α-rhamnoside (IC₅₀ = 27.7 ± 0.6 µg/mL) showed higher tyrosinase enzyme inhibitory effect than apigenin-7-O-β-glucuronide (IC₅₀ = 43.0 ± 0.9 µg/mL). n-Hexane extract was found to be effective against Enterococcus faecalis (MIC = 1 µg/mL), while methanol extract was effective against Pseudomonas aeruginosa (MIC = 16 µg/mL). Myricetin 3-O-α-rhamnoside showed antimicrobial effect against Enterococcus faecalis (MIC = 128 µg/mL). Thus, Limonium meyeri and its compounds may provide valuable insights for future studies as antityrosinase and antibacterial agents.

DOI: 10.15376/biores.20.1.2171-2182

Keywords: Limonium meyeri; Apigenin-7-O-β-glucuronide; Myricetin 3-O-α-rhamnoside; Antimicrobial; Antityrosinase

Contact information: a: Ataturk University, Faculty of Pharmacy, Department of Pharmacognosy, Erzurum, Turkey; b: Ataturk University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Erzurum, Turkey; c: Ataturk University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Erzurum, Turkey; d: Ataturk University, Faculty of Science, Department of Chemistry, Erzurum, Turkey; *Corresponding author: esen.karaoglan@atauni.edu.tr

Graphical Abstract

INTRODUCTION

Plants have been used for thousands of years to flavor and preserve food and to treat and prevent disease. Secondary metabolites produced by plants are recorded to have many biological effects, including the treatment of infectious diseases (Silva and Fernandes 2010; Qanash et al. 2024; Selim et al. 2024). It is important to develop technology for the isolation and extraction of bioactive compounds of medicinal value (Das et al. 2022). In this context, various traditional and non-traditional procedures such as maceration, infusion, digestion, percolation, decoction, ultrasound extraction, soxhlet extraction, turbo extraction, countercurrent extraction, ultrasound extraction, microwave-assisted extraction, supercritical fluid extraction, solid phase extraction, and column chromatography have been used. Ionic liquid extraction, high-performance gas phase chromatography, liquid chromatography, chiral phase chromatography are among the latest technologies (Kamil Hussain et al. 2019).

Plants secrete secondary metabolites such as alkaloids, flavonoids, terpenoids, and steroids. These secondary metabolites have various therapeutic activities. Flavonoids are molecules with activities such as antibacterial, antiviral, antioxidant, anti-inflammatory, antimutagenic, and anticarcinogenic (Roy et al. 2022). In addition, they are used in the food, cosmetics and pharmaceutical industries (Dias et al. 2021).

Antimicrobial research is being carried out using various methods to discover synthetic and plant-derived substances that are less harmful to humans, broad-spectrum, and effective at low doses against microorganisms that cause various infections (Abbasoğlu 1996). Drug-resistant bacteria pose a serious risk to human health, and new antimicrobial agents are needed. Natural products are an invaluable source of antimicrobial compounds (Álvarez-Martínez et al. 2020; Alghonaim et al. 2023; Al-Rajhi et al. 2023).

Tyrosinase contains copper. It is a key enzyme in the biosynthesis of melanin, which is found in microorganisms, plants, and animals and is involved in determining the color of mammalian skin and hair. Excessive melanin production causes various pigmentation disorders, such as melasma, age spots, and areas of actinic damage. Additionally, the tyrosinase enzyme causes undesirable browning in plant-based foods, a decrease in nutritional quality, and economic loss of food products. All these reasons increase the need for new potential tyrosinase enzyme inhibitors (Parvez et al. 2007). There are natural, synthetic or semi-synthetic compounds used to reduce melanogenesis activity. For example, kojic acid produced from various types of mushrooms, arbutin obtained from the bearberry plant, and vanillin obtained from vanilla beans are used for this purpose. Highly effective and less side-effect tyrosinase inhibitors are needed in the pharmaceutical, food, and cosmetic industries (Hassan et al. 2023).

Limonium is a genus belonging to the Plumbaginaceae family. Studies have shown that Limonium species are rich in flavonoids, which can be used in the treatment of some diseases such as high fever, hemorrhage, spasm, ulcer, and bladder stones (Lin et al. 2000; Temel and Ünver 2012; Kandil et al. 2020). Different Limonium species have been reported to have antioxidant, antimicrobial, anti-inflammatory, anticancer, antiobesity, antidiabetic, and enzyme inhibition activities (Sami 2022). Limonium meyeri is a perennial (rarely annual) herb or subshrub. The leaves are usually in basal rosettes, sometimes in axillary bundles on the stem branches. The spikelets are dense (sometimes capitate) or grouped in spikes, terminating the branches of the inflorescence. The fruits are utriculate or pyxidiate, narrowly ellipsoid and compressed (Doğan et al. 2020). As far as the authors have observed, the phytochemical and biological activity studies on L. meyeri are insufficient in the literature. Therefore, the antimicrobial and tyrosinase enzyme inhibition effects of methanol, n-hexane, dichloromethane, ethyl acetate, water extracts of L. meyeri and isolated apigenin-7-O-β-glucuronide and myricetin 3-O-α-rhamnoside were investigated in this study,

EXPERIMENTAL

Plant Material

Flowering aerial parts (flowers, leaves, branches) of L. meyeri was collected from the Erzurum-Dumlu road route (39°58.898’N, 041°18.182’E) in July 2018. The plant was identified by Esen Sezen Karaoğlan. The sample was kept in the Atatürk University Biodiversity Application and Research Center Herbarium (AUEF 1396). Harvested plants were transported with sealed bags. They were dried at room temperature in a dry, airy and clean room away from direct sunlight. After the plants were completely dried, they were stored with sealed bags until the beginning of the study.

Extraction Method

After the plant material (300 g) was dried and powdered, 1000 mL of methanol (Isolab, Wertheim, Germany) was added and left to macerate 24 h. It was filtered with pleated filter paper. Methanol was added to the remaining residual part and extracted (4 times, 800 mL) at 40 °C under reflux in a mantle heater (Barnstead Electrothermal EM5000/C, UK). When the extracts became lukewarm and clear, they were filtered with pleated filter paper. The filtrates were combined and concentrated using a rotavapor (Heidolph, Heizbad Hei-VAP, Germany). The main methanol extract (40 g) was obtained and named LMM. The concentrated extract was dissolved with a 9:1 water:methanol solvent system. Then, it was fractionated using a separating funnel with n-hexane (Sigma Aldrich, St. Louis, MO, USA), dichloromethane (Isolab, Wertheim, Germany), and ethylacetate (Sigma Aldrich, St. Louis, MO, USA) solvents, respectively. Thus, 4.8 g n-hexane (LMH), 1.1 g dichloromethane (LMD), and 3.5 g ethylacetate (LME) subextracts were ontained. The 25.9 g remaining part was used as the aqueous extract and named LMS (Ozgen et al. 2024).

Isolation and Structure Determination

About 3 g of ethyl acetate extract was added to the silica gel (Merck, Darmstadt, Germany) column. A solvent system consisting of dichloromethane:methanol:water (90:10:1→50:50:5, v/v) was used for elution. The fractions (120 fractions) were collected into tubes. The fractions in the tube were applied to thin layer chromatography (TLC) plate. The plates were evaluated at UV₂₅₄ / UV₃₆₆ nm wavelength. The spots were evaluated. Then, 1% vanillin/sulfuric acid was sprayed and heated at 110° C. The color of the spots was evaluated. Tubes with similar images were combined (Sami 2022).

Fractions 42 to 68: these fractions were added to the sefhadex (Sigma Aldrich, St. Louis, MO, USA) column. Water:methanol (95:5, 9:1, 5:1, 1:1, v/v) solvent systems were used as eluent. The fractions in the tube were applied to the TLC plate and evaluated. The images suggested that tubes 11 and 12 were pure and clean. These tubes were combined and named LME1 (Sami 2022).

Fractions 19 to 23: these fractions were applied to the sefhadex column. Methanol was used as the mobile phase. The fractions in the tube were applied to the TLC plate and evaluated. Tubes 28 to 35 were combined (383 mg) and applied to the silica gel column. Dichloromethane:methanol:water (90:10:1→50:50:5, v/v) was used as the mobile phase. The fractions in the tube were applied to the TLC plate and evaluated. The images suggested that tubes 51 to 58 were pure and clean. These tubes were combined and named LME2 (Sami 2022).

The structures of the pure substances (LME1 and LME2) were elucidated by 1D-NMR (¹H-NMR, ¹³C-NMR), 2D-NMR (HMQC and HMBC), and Q-TOF-MS measurements. The Q-TOF-MS spectra were recorded on an Agilent 6530 Accurate-Mass Q-TOF-LC/MS (Santa Clara, CA, USA) at Atatürk University Eastern Anatolia Advanced Technology Application and Research Center (DAYTAM), and NMR spectra were taken at Atatürk University Faculty of Science, Department of Chemistry. The NMR spectra were measured on Avance III 600 (Bruker, Karlsruhe, Germany) (400 MHz for ¹H-NMR and 100 MHz for ¹³C-NMR) spectrometers with tetramethylsilane (TMS) as an internal standard. LME1 was dissolved with DMSO, and LME2 was dissolved with CD₃OD.

Antimicrobial Activity Method

Standard bacterial strains of Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Proteus mirabilis (ATCC 29906), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 29213), and Enterococcus faecalis (ATCC 29212) were used. Antimicrobial activities of LMM, LMH, LMD, LME, LMS extracts and LME1 and LME2 compounds were determined by the microdilution method in accordance with the Clinical and Laboratory Standards Institute recommendations (Clinical and Laboratory Standards Institute 2017). Stock solutions were prepared with 10% dimethyl sulfoxide (DMSO) and cationized Mueller-Hinton broth medium (CAMHB). Sulfomerazine was used as standard antibiotic. Test substances and sulfomerazine were prepared in different concentration range by double dilution with CAMHB. Bacterial suspensions were prepared as 0.5 Mc Farland 1×10⁸ colony forming unit (CFU)/mL and then diluted 1/100 with CAMHB to ensure the concentration was 10⁵ CFU/mL. 50 µL bacterial suspensions were pipetted into plates containing 50 µL diluated extract. Thus, final concentrations in the wells were obtained. The plates were incubated at 35 °C for 24 h. The minimun inhibitory concentration (MIC) values were evaluated based on turbidity in the wells (Ozgen et al. 2024).

Tyrosinase Enzyme Inhibition Method

The tyrosinase inhibitory effects of LMM, LMH, LMD, LME, LMS extracts and LME1 and LME2 compounds were determined following the reported method using 3,4-dihydroxyl-L-phenylalanine (L-DOPA) (Sigma Aldrich, St. Louis, MO, USA) as a substrate (Masuda et al. 2005). Kojic acid (Sigma Aldrich, St. Louis, MO, USA) was used as a positive control. A mixture consisting of 40 µL of test solution at different concentrations, 40 µL of tyrosinase solution, and 80 µL of phosphate buffer (pH = 6.8) were pipetted into a 96-well microplate (Group C). Group A contains phosphate buffer (120 µL) and enzyme (40 µL), group B contains only phosphate buffer (160 µL), and group D contains phosphate buffer (120 µL) and sample (40 µL). The groups were replicated three times for each test sample. It was incubated for 10 min at 23 °C. Then, 40 µL L-DOPA was added to all wells and incubated at 23 °C for 10 min, and the absorbance was measured at 490 nm with a microplate reader (Bio Tek EL X 800). Percentage of inhibition (%) was calculated with the formula [[(A − B) − (C − D)] / (A − B)] × 100 (Karaoglan et al. 2023).

RESULTS AND DISCUSSION

Isolation and Structure Determination Results

The compounds LME1 and LME2 appeared yellow on the TLC plate, as a stain at UV₂₅₄ nm, dark purple at UV₃₆₆ nm, and bright yellow when heated with vanillin/H₂SO₄ reagent at 110 °C, indicating that they may be flavonoids. The ¹H-NMR, ¹³C-NMR, HMQC, HMBC, and Q-TOFF results and literature comparison of results showed that LME1 was apigenin-7-O-β-glucuronide (Lin et al. 2001; Ma et al. 2017) and LME2 was myricetin 3-O-α-rhamnoside (Hwang and Chung 2018; Katlego el al. 2020). Phytochemical analysis results of LME1 and LME2 are given below.

Apigenin-7-O-β-glucuronide (LME1)

Q-TOFF-MS: (447 [M+H]⁺, C₂₁H₁₈O₁₁; ¹H-NMR Spectrum (δ, ppm): 6.78 (s, H-3), 6.38 (d, J = 1.8 Hz, H-6), 6.76 (d, J = 2.2 Hz, H-8), 7.86 (d, J = 8.8 Hz, H-2′, 6′), 6.87 (d, J = 8.8 Hz, H-3′, 5′), 5.03 (d, J = 7.3 Hz, H-1”), 3.15 to 3.60 (H-2”, 3”, 4”, 5”); ¹³C-NMR Spectrum (δ, ppm): 164.9 (C-2), 103.4 (C-3), 182.6 (C-4), 161.6 (C-5), 100.3 (C-6), 163.7 (C-7), 103.4 (C-8), 157.6 (C-9), 105.9 (C-10), 121.2 (C-1′), 129.1 (C-2′), 116.7 (C-3′), 162.4 (C-4′), 116.7 (C-5′), 129.1 (C-6′), 100.1 (C-1”), 73.5 (C-2”), 74.3 (C-3”), 72.6 (C-4”), 77.1 (C-5”), and 172.9 (C-6”). The molecular structure of the compound (LME1) is shown in Fig. 1.

Fig. 1. Apigenin-7-O-β-glucuronide (LME1)

Myricetin 3-O-α-rhamnoside (LME2)

Q-TOFF-MS: (465 [M+H]⁺, C₂₁H₂₀O₁₂; ¹H-NMR Spectrum (δ, ppm): 6.19 (d, J = 2.0 Hz, H-6), 6.36 (d, J = 2.0 Hz, H-8, 6.95 (s, H- 2′, 6′), 5.30 (d, J = 1.6 Hz, H-1”), 4.21 (m, H-2”), 3.78 (dd, J = 9.4 Hz, J = 3.5 Hz, H-3”), 3.23 to 3.52 (H-4”, 5”), 0.95 (d, J = 6.2 Hz, H-6”); and ¹³C-NMR Spectrum (δ, ppm): 158.3 (C-2), 135.1 (C-3), 178.5 (C-4), 162.0 (C-5), 98.7 (6), 164.8 (7), 93.5 (8), 157.3 (9), 104.6 (10), 120.7 (1′), 108.4 (2′), 145.7 (3′), 136.7 (C-4′), 145.7 (C-5′), 108.4 (C-6′), 102.4 (C-1”), 70.9 (C-2”), 70.9 (C-3”), 72.1 (C-4”), 70.7 (C-5”), and 16.5 (C-6”). The molecular structure of the compound (LME2) is shown in Fig. 2.

Fig. 2. Myricetin 3-O-α-rhamnoside (LME2)

As far as we have observed, the apigenin-7-O-β-glucuronide has not been commonly found in Limonium species in previous studies. It was recorded that myricetin 3-O-α-rhamnoside compound was isolated from Limonium species such as L. bicolor, L. sinense, L. gmelinii, L. myrianthum, L. leptophyllum, and L. aureum (Lin and Chou 2000; Ye and Huang 2006; Kozhamkulova et al. 2010; Chen et al. 2017; Gadetskaya et al. 2017). Apigenin-7-O-β-glucuronide is a compound with a flavone structure consisting of 21 carbon atoms. Myricetin 3-O-α-rhamnoside is a compound with a 21-carbon flavonol structure. Flavonoids are effective in the treatment of diseases due to their high antioxidant and free radical scavenging activities (Chen et al. 2023). The isolation studies related to L. meyeri are insufficient in the literature. In this context, we believe that isolating apigenin-7-O-β-glucuronide and myricetin 3-O-α-rhamnoside from L. meyeri in this study is valuable.

Antimicrobial Activity Results

The LME showed antimicrobial activity against E. coli (MIC= 256 µg/mL), P. aeruginosa (MIC= 256 µg/mL), E. faecalis (MIC= 512 µg/mL); LMH against P. mirabilis (MIC= 128 µg/mL) and E. faecalis (MIC= 1 µg/mL); LMD against P. aeruginosa (MIC= 128 µg/mL) and S. aureus (MIC= 64 µg/mL); and LMM against P. aeruginosa (MIC= 16 µg/mL), S. aureus (MIC= 512 µg/mL), and E. faecalis (MIC= 128 µg/mL). The LMS was not effective against any bacteria. Additionally, it was observed that the isolated LME1 was effective against S. aureus (MIC= 1024 µg/mL), and the isolated LME2 was effective against E. coli (MIC= 512 µg/mL) and E. faecalis (MIC= 128 µg/mL). No test material was found to be effective against K. pneumoniae. According to the antimicrobial activity results, LMH was the most effective against E. faecalis (MIC= 1 µg/mL). Then, LMM was found to be effective against P. aeruginosa (MIC= 16 µg/mL). The results are shown in Table 1.

Table 1. MIC Values of Test Substances (µg/mL)

MIC: Minimum inhibitory concentrations, LMS: Aqueous extract, LMH: n-Hexane extract, LMD: Dichloromethane extract, LME: Ethylacetate extract, LMM: Methanol extract, LME1: Apigenin-7-O-β-glucuronide, LME2: Myricetin 3-O-α-rhamnoside

Root extracts of L. meyeri have been found to show a higher antimicrobial effect than the aerial part, especially against S. aureus (Yurchyshyn et al. 2017). In the present study, it was observed that dichloromethane extract, methanol extract, and apigenin-7-O-β-glucuronide were effective against S. aureus. Antimicrobial activities of different Limonium species are also recorded in the literature. Different fractions of the rhizome of the L. brasiliense species were tested. It has been shown to be effective against Enterococcus faecium, S. aureus, and K. pneumoniae (Blainski et al. 2017). Ethyl acetate extract of L. pruinosum showed activity against E. coli and S. aureus (Sultan et al. 2024). Methanol extracts of L. effusum, L. lilacinum, and L. globuliferum species were effective against many bacteria except E. coli (Avaz 2010). In the present study, ethyl acetate extract and myricetin 3-O-α-rhamnoside were effective against E. coli. In a conducted study, it was observed that the dichloromethane extract of L. lopadusanum was effective against E. faecalis (Gargiulo et al. 2024). In the present study, the ethyl acetate extract, methanol extract of L. meyeri, and myricetin 3-O-α-rhamnoside were found to be effective against E. faecalis. Moussaoui et al. (2010) reported that apigenin-7-O-β-glucuronide was moderately effective against E. coli, S. aureous, P. mirabilis, Proteus vulgaris, Klebsiella oxytoca, K. pneumonia, Morganella morgani, Enterobacter sp., Streptococcus sp., and Seratia sp. by the disk diffusion method. Additionally, they detected susceptibility only against S. aureus by the microdilution method. Aderogba et al. (2013) showed that myricetin 3-O-α-rhamnoside was effective against E. coli, K. pneumonia, and S. aureus. Myricetin 3-O-α-rhamnoside was found to be effective against E. coli and E. faecalis in this study.

Tyrosinase Enzyme Inhibition Results

In the research conducted with extracts, LMM, LME, LMS, LMD, LMH extracts showed tyrosinase inhibitory effects at 400 µg/mL concentration of 62.0%, 57.6%, 46.5%, 30.6%, 7.5%, respectively (Kojic acid, 94.75%). Then, the tyrosinase inhibitory effects of the compounds isolated from the ethyl acetate extract were investigated. LME1 and LME2 showed tyrosinase inhibitory activity (IC₅₀ = 43.0 ± 0.9 and 27.7 ± 0.6 μg/mL, respectively). These results are shown in Table 2.

Table 2. Tyrosinase Enzyme İnhibitory Effects of LME1 and LME2

IC₅₀: Half-maximal inhibitory concentration (mean ± SD of three experiments), LME1: Apigenin-7-O-β-glucuronide, LME2: Myricetin 3-O-α-rhamnoside

Tyrosinase enzyme inhibitory activities of various fractions of L. tetragonum were previously investigated, and 85% aqueous methanol and n-butanol extracts were found to be effective. In addition, isolated myricetin 3-galactoside (65%) and quercetin 3-O-β-galactopyrroloside (63%) were effective (Lee et al. 2017). The finding that methanol extract of L. delicatulum leaves and roots was effective supports the current study (Bakhouche et al. 2021). In related work, the ethanol extract of L. spathulatum leaves showed tyrosinase inhibitory (IC₅₀ = 0.34 mg/mL) effect (Youssef et al. 2023). Additionally, ethyl acetate fractions of L. effusum and L. sinuatum and n-hexane fraction of L. effusum showed antityrosinase activity (IC₅₀= 245.56±3.6, 295.18±10.57 and 148.27±3.33 g/mL, respectively) (Baysal et al. 2021). In the present study, the main methanol extract (62.0%), and ethyl acetate subfraction (57.6%) of L. meyeri showed the highest percentage tyrosinase inhibitory effect. In addition, myricetin 3-O-α-rhamnoside (27.7 ± 0.6 μg/mL) showed a higher tyrosinase inhibitory effect than apigenin-7-O-β-glucuronide (43.0 ± 0.9μg/mL). It is known that phenolic compounds and flavonoids are effective in inhibiting tyrosinase and melanin synthesis (Zolghadri et al. 2019). The high effects of methanol and ethyl acetate extracts may be due to their rich content of phenolic compounds and flavonoids.

CONCLUSIONS

1. Myricetin 3-O-α-rhamnoside and apigenin-7-O-β-glucuronide were isolated from L. meyeri. Various extracts and isolated compounds of L. meyeri exhibited antibacterial effects against different bacteria. Particularly, the n-hexane extract showed significant activity against E. faecalis.
2. Among the extracts, methanol and ethyl acetate extracts had a higher tyrosinase inhibitory effect, while both of the isolated compounds exhibited anti-tyrosinase activity.
3. It was observed that the some extracts and isolated compounds had tyrosinase inhibitory and antimicrobial effects. These findings may guide further research.

ACKNOWLEDGMENTS

This study was presented as a Master’s thesis at Ataturk University Health Sciences Institute.

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Article submitted: October 14, 2024; Peer review completed: December 9, 2024; Revised version received: December 26, 2024; Accepted: January 1, 2025; Published: January 22, 2025.

DOI: 10.15376/biores.20.1.2171-2182