ANTIDIABETIC AND ANTI-ALZHEIMER POTENTIAL OF THREE TYMBRA SPECIES RICH IN HESPERIDIN AND ROSMARINIC ACID

The use of Thymus species amongst the public and their importance in the scientific world is increasing day by day. In addition to being consumed as tea and spice, Thymus species are used as folk medicine for respiratory, digestive, skin, circulatory, genital, nervous, visual and urinary diseases. In this study, it was aimed to examine the essential oil and ethanol extract of the root and aerial parts of Thymus pubescens Boiss. et Kotschy ex Celak in the terms of their biological activity and chemical content. The phenolic content of the species was determined by LC-MS/MS, while triterpenoid content, the chemical composition of the essential oil as well as flavour was determined by GC–MS. In addition, their antioxidant and cytotoxic activities, as well as acetyl- (AChE) and butyrylcholinesterase (BChE), urease, tyrosinase, elastase, collagenase, HMG-CoA reductase and angiotensin-converting enzyme (ACE) inhibitory activities were studied. Thymol (53.33%) was determined as the major component in the essential oil, while β-fenchyl alcohol (43.87%) was determined as the major component of the flavour. According to the LC-MS/MS and GC–MS results, it was found that especially the aerial parts of the species have a high amount of rosmarinic acid (3875.76 µg analyte/g extract), quinic acid (2392.55), naringenin (970.39), oleanolic (92785.96) and ursolic (63373.32) acids. The essential oil of T. pubescens species was observed to show high activity in four antioxidant assays, e.g. inhibition of lipid peroxidation, DPPH and ABTS radical scavenging activity as well as CUPRAC, while the ethanol extracts showed moderate antioxidant activity. In enzyme inhibition assays, the aerial parts exerted marked BChE, elastase, and collagenase inhibitory activities (92.43±1.28%, 42.59±0.56, and 48.61±0.39 at 100 µg/mL, respectively). On the other hand, AChE, urease, tyrosinase, HMG-CoA reductase and ACE inhibitory activities of all extracts were from low to moderate levels. In particular, the aerial parts of the species displayed a high cytotoxic effect (vitality%: 6.82±0.01 at 200 µg/mL) in breast cancer (MCF-7) cell line. Due to its remarkable antioxidant capacity, high content of rosmarinic, oleanolic, and ursolic acids and especially BChE, elastase, and collagenase inhibitory activity, T. pubescens has the potential for using in food supplements, food preservatives, cosmetics, and pharmaceutical industries.

Species belonging to the Zingiberaceae family are of high nutritional, industrial, and medicinal values. In this study, we investigated the effect of processing steps (fresh vs. dried milled rhizomes) and extraction methodologies (hydrodistillation vs. hexane extraction) of curcuma essential oil on its chemical content (using GC-MS analysis), its antioxidant behavior (using in vitro assays such as DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelation), and its enzyme inhibitory activities (on tyrosinase, acetylcholinesterase, butylcholinesterase, α-amylase, and α-glucosidase) supported by multivariate analysis, in silico studies, and molecular dynamics. The GC-MS investigations revealed a high degree of similarity in the chemical profile of fresh hydrodistilled and hexane-extracted essential oils with tumerone and curlone being the major metabolites. The extraction techniques affected the concentrations of other minor constituents such as terpinolene, caryophylla-4(12), 8(13)-dien-5α-ol, and neo-intermedeol, which were almost exclusively detected in the hydrodistilled fresh essential oil; however, zingiberene and β-sesquiphellandrene were predominant in the hexane-extracted fresh essential oil. In the dried curcuma rhizomes, tumerone and curlone contents were significantly reduced, with the former being detected only in the hydrodistilled essential oil while the latter was doubly concentrated in the hexane-derived oil. Constituents such as D-limonene and caryophyllene oxide represented ca. 29% of the dried hydrodistilled essential oil, while ar-turmerone was detected only in the dried hydrodistilled and hexane-extracted essential oils, representing ca. 16% and 26% of the essential oil composition, respectively. These variations in the essential oil chemical content have subsequently affected its antioxidant properties and enzyme inhibitory activities. In silico investigations showed that hydrophobic interactions and hydrogen bonding were the characteristic binding modes of the bioactive metabolites to their respective targets. Molecular dynamics revealed the stability of the ligand-target complex over time. From the current study we conclude that fresh hexane-extracted essential oil showed the best radical scavenging properties, and fresh rhizomes in general display better enzyme inhibitory activity regardless of the extraction technique.

Centaurea lycaonica is a local endemic species from the Centaurea L. genus. The Centaurea species has a wide range of usage in treating diseases in folk medicine. There are limited biological activity studies on this species in the literature. This study investigated enzyme inhibition and antimicrobial activity, antioxidant effect, and chemical content of extract and fractions of C. lycaonica. Enzyme inhibition activity was tested by α-amylase, α-glucosidase, and tyrosinase enzyme inhibition methods and antimicrobial activity by the microdilution method. The antioxidant activity was investigated using DPPH•, ABTS•+, and FRAP tests. The chemical content was determined by LC-MS/MS. The methanol extract showed the highest activity for α-glucosidase and α-amylase, even surpassing the positive control acarbose, with IC50 values of 56.333 ± 0.986 and 172.800 ± 0.816 μg/mL, respectively. Additionally, the ethyl acetate fraction also exhibited high activity for α-amylase with an IC50 value of 204.067 ± 1.739 μg/mL and tyrosinase with an IC50 value of 213.900 ± 1.553 μg/mL. Moreover, this extract and fraction were found to have the highest total phenolic and flavonoid contents and antioxidant activity. Additionally, LC-MS/MS analyses of active extract and fraction revealed mainly the presence of phenolic compounds and flavonoids. In silico molecular docking and molecular dynamics simulation studies of determining compounds apigenin and myristoleic acid, common in CLM and CLE extracts and active against α-glucosidase and α-amylase, were performed. In conclusion, methanol extract and ethyl acetate fraction showed potential enzyme inhibition and antioxidant activity as a natural agent. Molecular modeling studies corroborate the findings of in vitro activity analyses.

The Ziziphora species, classified under the Lamiaceae family, have a strong aromatic property. Ziziphora species have been used in folk medicine as sedative, gastric, aphrodisiac, bloating, and degassing. In the current study, the phenolic and flavanoid content of ethanol extracts of Ziziphora capitata L. species of flower, leaf, branch, mixed, and root parts was determined by the LC-MS/MS device. In addition, the antioxidant and cytotoxic activities of the extracts, as well as their inhibitory effects on enzymes (antihypertensive, AchE (acetylcholinesterase), BchE (butyrylcholinesterase), elastase, tyrosinase, collagenase and urease), were determined. The LC-MS/MS results showed that quinic acid (25578, 5842, 25171, 14055, 10597 µg g-1, respectively) was found in higher amounts in flower, leaf, branch, mixed, and root extracts of Z. capitata species compared to other components. Additionally, rosmarinic acid (17097 µg g-1), cynaroside (8432), and hesperidin (8067) were found to be major components. It was observed that the flower extract of the species exhibited strong antioxidant activity (IC50: 37.18±1.36 µg mL-1, 9.89±0.45, A0.5:16.27±0.02, respectively) in DPPH, ABTS and CUPRAC methods. It was concluded that the leaf extract of Z. capitata species had a strong cytotoxic effect on HT-29 (colon cancer cell line) (viability %: 9.26±0.69). It was observed that the root part of the species exhibited higher activity in butyrylcholinesterase (BChE) enzyme inhibition activity (inhibition %: 40.56±0.88) than other parts. It was determined that Z. capitata extracts did not show acetylcholinesterase, urease, tyrosinase, elastase, collagenase, and antihypertensive enzyme activity or showed low activity. As a result, it is thought that the flower extract of the Z. capitata species has better results in terms of the examined parameters, whereas the leaf extract needs to be subjected to more detailed in vitro and in vivo research conducted to be used in the pharmaceutical industry as a result of its cytotoxic effect against colon cancer cell lines.

Lavandula stoechas subsp. luisieri is a Spanish subspecies from the Lamiaceae family. Its essential oil has been traditionally used for several medical applications though little is known about other extracts. Similar to many other studies aiming to obtain traditional plant extracts to be used in different applications, this work evaluated the antioxidant and antimicrobial activities of Lavandula luisieri extracts and the correlation with their composition. Traditional hydrodistillation and ethanolic maceration were used to obtain the essential oil and the maceration extract, respectively. A green and sustainable methodology was applied to the maceration extract that was under a Supercritical Antisolvent Fractionation process to obtain a fine solid enriched in rosmarinic acid and the terpenes oleanolic and ursolic acids. Antimicrobial activities of all extracts and pure identified compounds (rosmarinic and ursolic acids) were evaluated against five bacterial strains; Listeria monocytogenes, Enterococcus faecium, Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli and were compared with the pure compounds identified, rosmarinic and ursolic acids. All strains were sensitive against L. luisieri essential oil. The solid product obtained from the supercritical process was concentrated in the identified actives compared to the maceration extract, which resulted in higher antimicrobial and DPPH scavenging activities. The supercritical sustainable process provided L. luisieri compounds, with retention of their antimicrobial and antioxidant activities, in a powder exemptof organic solvents with potential application in the clinical, food or cosmetic fields.

The Caatinga is an exclusively Brazilian biome where semiarid climatic conditions promote singularities in adaptive biodiversity. Many aromatic species are found in this region possessing antifungal properties, which are attributed to their essential oils. Thus, we questioned whether essential plant oils found in the Caatinga present anti-dermatophytic potential. Dermatophytes are keratinophilic fungi that cause one of the most prevalent mycoses globally, skin infections known as dermatophytoses (tineas). Here, we provide a comprehensive report of the available published information, analyzing the methods used to evaluate the antifungal activity, verifying the quality of the evidence and possible clinical applications, and discussing research trends in this area. The plants studied concentrated in the genera Croton (Euphorbiaceae), Lippia (Verbenaceae), Piper (Piperaceae), and Mentha (Lamiaceae). All of the studies used in vitro tests to analyze antifungal potential, and little evidence was ascertained concerning the mechanism of antifungal action. In addition, the essential oils also evidenced drug modifying activity of conventional antifungal drugs (Ketoconazole and Terbinafine). We believe that the anti-dermatophyte potential of plant essential oils occurring within the Caatinga is underestimated and that this review will encourage future chemical-pharmacological investigations into the plants within this biome.Key points• The essential oils from plants occurring in the Caatinga Biome present unknown anti-dermatophyte potential.• The studies against dermatophyte fungi concentrate on the families Lamiaceae and Verbenaceae.• In vitro assays were used to assess the anti-dermatophyte potential of the essential oils.

General background: Aromatic herbs from the Lamiaceae family represent valuable sources of rosmarinic acid (RA), a polyphenolic compound with notable antioxidant, anti-inflammatory, antimicrobial, and neuroprotective properties. Specific background: Traditional extraction methods such as Soxhlet and maceration consume excessive solvents and time while potentially degrading thermolabile compounds. Knowledge gap: Despite growing interest in green extraction technologies, limited validated protocols exist for combining ultrasound-assisted extraction (UAE) with high-performance liquid chromatography (HPLC) for efficient RA quantification across multiple aromatic species. Aims: This study developed and validated a sustainable UAE-HPLC method for extracting and quantifying RA from six aromatic herbs: Laurus nobilis, Thymus vulgaris, Salvia rosmarinus, Anethum graveolens, Mentha viridis, and Salvia officinalis. Results: Using ethanol:water (70:30 v/v) at 40 kHz and 250 W, optimal extraction occurred within 2.5-10 minutes, with Thymus vulgaris yielding the highest RA concentration (170,500 µg/mL). HPLC analysis demonstrated excellent linearity (R² > 0.998) and reproducibility. Novelty: This approach significantly reduces solvent consumption and extraction time while maintaining analytical precision. Implications: The validated UAE-HPLC platform provides a robust, environmentally friendly analytical framework for sustainable utilization of RA-rich herbs in nutraceutical and pharmaceutical applications.Highlight : UAE offers a rapid, low-solvent, and environmentally friendly approach for extracting RA. Variation in RA yields reflects differences in plant tissue structure and extraction conditions. HPLC provides reliable quantification, supporting accurate and consistent analysis of RA. Keywords : Rosmarinic Acid, Aromatic Herbs, Ultrasound-assisted Extraction, HPLC, Green Extraction

Identification and quantification of phenolic and volatile constituents in five different Anatolian thyme species using LC–MS/MS and GC-MS, with biological activities

In this study, essential oil from the aerial parts of Gelasia sericea (Aucher ex DC.) Zaika, Sukhor. & N. Kilian was extracted via hydrodistillation using a Clevenger apparatus. GC-MS/FID analysis determined aldehydes (28.31%) as the dominant group, with eucalyptol (8.71%) as the main constituent. Antioxidant activities were exhibited using DPPH, ABTS, and FRAP assays, with the essential oil demonstrating relatively higher antioxidant properties. The essential oil also showed higher levels of bioactive components, including total antioxidant capacity (160.26 mg AA eq./100 mL), total phenolic content (21.57 mg GA eq./100 mL), and total flavonoid content (14.85 mg QE eq./100 mL). While G. sericea essential oil demonstrated some antioxidant activity, it was significantly lower compared to the reference standard ascorbic acid (AA). The antimicrobial results showed relatively higher antimicrobial activity compared to most bacterial and fungal strains, as evidenced by lower MIC and MBC values and larger inhibition zones. Nevertheless, chloramphenicol (CH) and nalidixic acid (NA) continually showed superior antimicrobial efficacy, with the lowest MIC and MBC values and the largest inhibition zones. The essential oil also demonstrated potent enzyme inhibitory activity, especially against CA-II, AChE, and BChE, with the lowest IC50 value observed for CA-II, highlighting its potential as a potent enzyme inhibitor. However, the essential oil exhibited comparatively greater inhibition against AChE, BChE and α-amylase but was less effective overall. These findings suggest that the G. sericea essential oil has important antioxidant potential and enzyme inhibitory activity, while the essential oil shows varying efficacy across different enzymes.

The genus Salvia is regarded as an important medicinal plant and also as spice worldwide. Current report might be counted as one of the first investigations to reveal the enzyme inhibitory and antiglycation activities, together with phenolic and antioxidant profiles of 80% methanolic extract prepared from the aerial parts of S.virgata (SVM). Furthermore, the bioavailability parameters of major phenolic compounds in SVM were evaluated. An in vitro human digestion simulation model composed of both gastric and intestinal phases was used for this purpose. Total phenolic, phenolic acid and flavonoid components of each of these phases were calculated in order to assess the phenolic profile of the samples. In addition, quantitative analysis of major bioactive compounds (rutin and rosmarinic acid), before and after the simulation, was practiced by employing High Performance Thin Layer Chromatography (HPTLC) system. Moreover, the potential of non-digested (ND) and bioavailable (IN) phases on the inhibition of diabetes-related enzymes (α-amylase and α-glucosidase) was determined. Also, the inhibitory effect of the samples on the advanced glycation end products (AGEs), formed due to hyperglycemia, was detected. In addition, cholinesterase enzyme inhibition activity of both ND and IN samples was determined. Since, the role of oxidative stress on diabetes and neurodegenerative diseases is well-known, the antioxidant potential of the samples was also estimated by different methods: DPPH and DMPD tests for free radical scavenging activity, FRAP and CUPRAC tests for metal reducing activity and total antioxidant capacity test, as well. The results showed that total phenolic contents, antioxidant potential and diabetes-related and cholinesterase enzymes inhibition of the bioavailable phase were lower than non-digested sample. Besides, in vitro human digestion simulation system had a declining influence on the concentrations of the major bioactive metabolites (rutin and rosmarinic acid).

In this study, the fatty acid, essential oil and aroma composition of Thymbra sintenisii Bornm. & Aznav. subsp. sintenisii Bornm. & Aznav (TSS) and Thymbra sintenisii Bornm. & Azn. subsp. isaurica P.H.Davis (TSI) were determined by GC-MS. The cytotoxic activity was determined with MTT assay and antioxidant activities were determined with DPPH, ABTS, CUPRAC methods. Urease, acetyl, butyryl, angiotensin, α-amylase, tyrosinase, elastase, and collagenase enzyme inhibition activities of these species were also determined. The acaricidal effects of essential oils from aerial parts of TSS and TSI on ticks and the nanoparticle formation potential of their essential oils with AgNO3 were determined and compared. The main components of TSS essential oil and aroma were determined as carvacrol and o-cemen (66.78 and 35.44%, respectively), the major components of TSI essential oil and aroma were carvacrol and γ-terpinene (63.78 and 31.44%, respectively). The species exhibited remarkably high antioxidant activity, particularly in the ABTS and CUPRAC methods. It was also determined that the butyrylcholinesterase and acetylcholinesterase enzyme inhibition activities (Inhibition%: 76.12±0.83 and 74.13±0.33, respectively) of the species were quite high. Moderate antimicrobial activity against the bacteria was observed for both essential oils, with MIC values ranging from 312 to 1250 μg/mL. Moreover, essential oils of these species showed good acaricidal activity on Hyalomma marginatum (LC50: 11.82 μg/mL for TSS; LC50: 57.64 μg/mL for TSI). The high antioxidant, anticholinesterase, and acaracidal activities observed in both species are attributed to the presence of carvacrol in their essential oils. The species have potential to be used in food supplements-preservatives, pharmaceutical industry.

M. myrtifolia Boiss. & Hohen. is a strongly aromatic, endemic plant from the Lamiaceae family. The main compounds in the volatile fraction of M. myrtifolia are β- caryophyllene and caryophyllene oxide. Regarding the non-volatile fraction, the main compounds detected in the aqueous and methanol extracts are rosmarinic, syringic, chlorogenic, and caffeic acids, while the main compounds in the ethyl acetate extract are rosmarinic acid and apigenin. The essential oil of M. myrtifolia shows moderate antioxidant activity, while the aqueous and methanol extracts are characterized by extremely good antioxidant activity. The methanolic extract of M. myrtifolia and its phenolic compounds show remarkable antidepressant activities in in vivo and in vitro tests. It was found that the extracts, as well as the essential oil, can significantly inhibit the activity of α-amylase and tyrosinase. Also, it was confirmed that M. myrtifolia decoctions have antifungal activity against Penicillium digitatum and Botritis cinerea.

Enzyme inhibition activities, phenolic compounds, antioxidant activities, bioactive compounds, antimicrobial activities, and chemical components of essential oil and methanol extracts obtained from the aerial parts of S. cretica subsp. anatolica were investigated. The main phenolic compounds of aerial parts were catechin, oleuropein, and epicatechin. The determined enzyme inhibitor activities highlight the potential of S. cretica subsp. anatolica as a source of bioactive compounds, particularly for carbonic anhydrase and cholinesterase inhibition. The essential oil and methanol extract exhibited remarkable activities against CA-II, AChE, and BChE, although they were less potent than standard inhibitors. The essential oils generally showed stronger antimicrobial activity compared to the 30% methanol extracts across most bacterial and fungal strains, as evidenced by minimum lethal concentration (MLC) and lower minimum inhibitory concentration (MIC) values and larger inhibition zones. Chloramphenicol used alone exhibited the highest antimicrobial efficacy, with the lowest MIC and MLC values and the largest inhibition zones. The essential oils of S. cretica subsp. anatolica were determined as esters, oxygenated sesquiterpenes, and aldehydes in aerial parts. The main components were found to be hexahydrofarnesyl acetone in the aerial parts.

The rationale for this research is the investigation of the potential health benefits as well as the antibacterial and antifungal properties of selected aromatic plants from the Lamiaceae family, which may lead to the development of improved functional foods. The present study investigated the effects of incorporating dried aromatic plants Thymus vulgaris, Thymus serpyllum, Thymus × citriodorus, Origanum vulgare and Rosmarinus officinalis at a concentration of 1% in refined wheat flour and wholemeal flour on the production of functional tortillas. Sensory analysis was employed to identify the optimal 1% addition, with the objective of achieving a favorable flavor and aroma profile. It was hypothesized that this addition would affect water activity, moisture, texture, color, antioxidant content and phenolic content, thereby enhancing the tortillas as a source of bioactive compounds. The results indicated that the type of flour used had a significant impact on the water activity of the tortillas, with wholemeal flour resulting in higher water activity than refined flour. The water activity ranged between 0.735 and 0.821, while the water content remained relatively stable. The water activity in whole-grain tortillas was significantly higher than that of refined flour tortillas, with a value exceeding 0.8, which makes them susceptible to mold growth and the production of mycotoxins. The sensory evaluations indicated that the enriched refined flour tortillas with common thyme (Thymus vulgaris), lemon thyme (Thymus × citriodorus) and rosemary (Rosmarinus officinalis) were rated highly; a similar result was observed for the whole-grain tortillas enriched with wild thyme (Thymus serpyllum) and lemon thyme. The whole-grain tortillas with rosemary were rated the highest of all the tortillas. The addition of aromatic plants increased the phenolic content and the antioxidant potential, depending on the flour type and the plant used. The addition of wild thyme and rosemary resulted in a significant increase in the phenolic content of wheat tortillas, while all enriched whole-grain tortillas exhibited a higher phenolic content than the control samples. The highest phenolic content in whole-grain tortillas was found in those fortified with rosemary, oregano and wild thyme. The highest antioxidant content was recorded in tortillas prepared with rosemary, irrespective of whether the flour used was refined or wholemeal. Fourteen phenolic compounds were tentatively identified in aromatic plants tested. The main phenolic compounds in Origanum vulgare were flavonoids. Rosmarinic acid was the dominant phenolic compound in rosemary and all thyme species, reaching the highest level in rosemary. Such high levels of rosmarinic acid may be responsible for the high antioxidant and total phenolic contents observed in rosemary extracts and also in tortillas when this plant is included in the recipe. The results of this study indicate that selected aromatic plants, particularly rosemary, have the potential to be utilized as functional ingredients in bakery products. By incorporating dried aromatic plants from the Lamiaceae family into wheat flour tortillas, food manufacturers can create products that not only taste better but also provide added health benefits. The use of selected herbs can improve the nutritional profile of tortillas by increasing antioxidant properties and, due to the properties of herbs, extend the shelf life of the product.

In the present work, chemical composition, enzyme inhibitory activities, and molecular docking studies of essential oil (EO) of Knema globularia leaves collected from Vinh Phuc Province, Vietnam, were investigated. The EO from the leaves of K. globularia was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS) analysis. The leaf EO yield was 0.14 ± 0.01% (w/w), comprising 39 identified components, constituting 96.77% of the EO content. Notable constituents included β-caryophyllene (54.11%), α-humulene (12.67%), and (E)-β-ocimene (8.82%). Enzyme inhibitions were assessed via the α-amylase inhibitory assay (IC50 = 282.71 ± 10.06 μg/mL) and tyrosinase inhibitory assay (IC50 = 993.92 ± 37.40 μg/mL). The molecular docking method has been employed to observe valuable binding interactions and binding energy with the main compounds on the target enzymes α-amylase and tyrosinase. Caryophyllene oxide exhibits the strongest affinity with α-amylase among the other major compounds. Meanwhile, viridiflorene shows the best binding energy with the tyrosinase enzyme. This is the first study providing valuable scientific data on the in vitro inhibitory activities of α-amylase and tyrosinase enzymes of the leaf EO of K. globularia and evaluating its main compounds through a molecular docking approach on these enzyme targets.