Introduction

Medicinal herbs have been seen as a source of therapeutic aids in healthcare systems worldwide1,2. Their therapeutic effect is related to their antioxidant, anti-aging, anti-cancer, anti-atherosclerotic, antibacterial, and anti-inflammatory activities. Several studies have been undertaken to find natural compounds extracted from herbs that have the potential to cure a variety of health conditions. Among these herbs are thyme, rosemary, and oregano, mainly used for their antioxidant properties3,4,5.

Thymus capitatus (T.capitatus) is known in Palestine as “Zetmane.” It’s a common type of thyme with various biological effects, including antimicrobial and antioxidant activities6,7. T.capitatus grows in different regions in Palestine under variable environmental conditions. Since ancient times, people have turned to T. capitatus for various medical purposes, including anthelmintic, carminative, antispasmodic, expectorant, sedative, stimulant, tonic, anti-inflammatory, and analgesic. T. capitatus EO has been certified for use due to its pharmacological effect, which includes antioxidant and antibacterial properties8,9. Moreover, this herb was used in the past to flavor meats, stews, and soups and was also used as raw material without any preliminary preparation10,11.

EOs of Thymus capitatus are mainly of two chemical groups: terpenoids (sesquiterpenes of low molecular weight and monoterpenes) and less commonly phenylpropanoids12,13,14.

T. capitatus belongs to the Lamiaceae family and is native to the eastern Mediterranean region. It is a perennial shrub with fragrant flowers and leaves that grows in many places in the mountains of Palestine.

The antimicrobial activity of T. capitatus EO has been tested against a wide range of bacteria and fungi, including gram-negative bacteria as Escherchia coli and Klebsiella pneumoniae, gram-positive bacteria as Salmonella analum and Listeria monocytogenes, fungi as Mucorramamnianus and Aspergillus ochraceus, and yeast species like Saccharomyces cerevisiae and Candida albicans. The results showed that E. coli, L. monocytogenes, and K. pneumonia bacteria were suppressed by the tested T. capitatus EOs. Furthermore, considerable efficacy against fungi and yeasts was observed15. The radical cation 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonate) and the free radical 2,2-diphenyl-1-picrylhydrazyl were examined in antioxidant activity experiments of T. capitatus EO, and the findings revealed potent inhibitory concentration values16,17. Tepe et al. researched the in vitro antioxidant properties of EOs of two Thymus species extracts and presented their findings. About 71 compounds were identified and characterized by high monoterpene, especially phenolic carvacrol, thymol, p-cymene, and γ-terpinene. The oils were tested for their possible antioxidant activity by using (DPPH), and β-carotene/linoleic acid assays, and both of them showed high antioxidant activity in different proportions16,17,18,19.

Obesity, diabetes, and cancer remain significant global health challenges due to their prevalence, complex underlying mechanisms, and limited effectiveness of current treatments. The enzymatic inhibition of lipase has shown promise in managing obesity by reducing the absorption of dietary fats through inhibition of fat digestion in the small intestine20,21. Similarly, the inhibition of pancreatic α-amylase has emerged as a strategy for diabetes management, as it delays carbohydrate digestion, reduces the glucose absorption rate, and mitigates postprandial plasma glucose spikes22. Medicinal plants, such as Thymus species, have garnered interest for their ability to inhibit α-amylase due to their bioactive chemical constituents, offering potential antidiabetic effects23,24,25.

Thymus essential oils (EOs) have been extensively studied for their anticancer activities, attributed to key components such as carvacrol, thymol, terpinene, and p-cymene26,27.

In this research, T. capitatus dried aerial parts from three regions in Palestine were extracted using hydrodistillation to evaluate the efficiency of this extraction method. Gas chromatography-mass spectroscopy was employed to characterize the chemical components of T. capitatus EOs. This study explores the potential of T. capitatus as a source of bioactive phytochemicals with therapeutic properties, including antioxidant, antimicrobial, anti-obesity, antidiabetic, and anticancer activities. Lastly, the essential oils (EO) from three regions were analyzed to assess how variations in chemical composition influence biological activities. The study also explored the effects of environmental conditions on the chemical composition of T. capitatus post-extraction and its impact on EO activity.

Materials and methods

Materials and chemicals

The present study employed a variety of chemical reagents sourced from reputable manufacturers to evaluate pharmacological activities. Dimethyl sulfoxide (DMSO) from Riedel-de Haën, Germany, and high-purity methanol from Loba Chemie, India, were utilized as solvents. Pharmacological assays involved compounds such as 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), p-nitrophenyl butyrate (PNPB), and orlistat obtained from Sigma-Aldrich, USA, along with 2,2-Diphenyl-1-picrylhydrazyl (DPPH) from Sigma-Aldrich, Denmark, and α-amylase from Sigma-Aldrich, India. Porcine pancreatic lipase, type II, acarbose, and 3,5-Dinitrosalicylic acid (DNSA) were procured from Sigma-Aldrich, USA, facilitating specific enzymatic assays. Additionally, chloroform and Tween 40 from Loba Chemie, India, linoleic acid, and β-carotene from Sigma-Aldrich, USA, and potassium phosphate from Sigma-Aldrich, USA, were utilized for various experimental procedures. Microbiological assessments utilized analytical grade RPMI 1640 culture medium and essential supplements like glutamine, trypsin, amphotericin B, fetal calf serum, Hank’s balanced solution, Trypan blue solution, penicillin, and gentamicin. A diverse range of microbial strains, including Klebsiella pneumoniae (ATCC 13883), Proteus vulgaris (ATCC 8427), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), MRSA (Clinical sample) and Candida albicans were employed for evaluating antimicrobial activities.

Instrumentation

The following instruments were utilized in this research: An oven (Ari Levy, Inc, Israel) for controlled heating processes, a precision balance (AS 220/C/2, Radwag, Poland) for accurate mass measurements, a micropipette (MRC, Ltd., Israel) for precise liquid handling, a grinder (Uno Moulinex, China) for sample preparation, micropipettes (Macherey-Nagel, USA) for varied volumes, a vortex mixer (Heidolph, Germany) for efficient mixing, a microplate reader (Unilab, USA) for measurements, a water bath (BPXOP1001040, Lab Tech, South Korea) for controlled temperature applications, and a gas chromatography-mass spectrometry (GC-MS) system (Perkin Elmer, UK) for chemical analysis.

Collection of plant materials

T. capitatus aerial parts were collected in March (2021) from three cities that resembled three regions of Palestine: Jenin (north), Ramallah (middle), and Hebron (south). All necessary permissions and licenses for collecting T. capitatus have been obtained from the relevant authorities, including An-Najah National. Our collection follows all legal and ethical guidelines. Professor Nidal Jaradat, a pharmacognosist, recognized the plant samples in the Natural Products Laboratory, Faculty of Pharmacy at An-Najah National University. A voucher specimen was deposited in the abovementioned laboratory under the code (Pharm-PCT-2813).

Areal parts of the plant were carefully separated, washed twice with distilled water, dried for 15 days in the shade, grounded well, and stored in cloth bags until the extraction process began.

Extraction process from T. capitatus aerial parts

Hydro-distillation methods

Three T. capitatus aerial parts samples were dried and powdered. The extraction method for the EOs of this plant was utilized by hydro-distillation, which was used by Jaradat and colleagues28. The essential oil (EO) was extracted using a Clevenger apparatus at atmospheric pressure for 180 min, maintaining a temperature of 100 °C and a hydro-distillation rate of 0.54 mL/min. This process involved suspending 100 g of dry powder in 1 L of distilled water. After extraction, the EO was chemically dried with calcium carbonate and stored in the refrigerator at 4 °C until it was needed. The following formula was used to compute the yield of each sample.

$${\text{Yield percent }} = \left( {{\text{extract weight/dry weight of}}\;T.\; capitatus} \right) * 100$$

Chemical composition identification by gas chromatography/mass spectrometry

The chemical composition of the three EO samples examined was determined using the GC-MS method. Shimadzu QP-5000 GC-MS with Rtx-5ms column (30 m long, 0.25 m thickness, 0.250 mm inner diameter) was used to record GC-MS chromatograms. Helium was used as the carrier gas at a 1 mL/min flow rate. 220 °C was the injector temperature. The oven temperature was programmed to rise from 50 °C (1 min hold) to 130 °C at 5 °C/min, then to 250 °C at 10 °C/min, and kept isothermally for 15 min. The temperature on the transfer line was 290 °C. An electron ionization system with detector voltages of 1.7 KV was employed for GC-MS detection. The mass range was 38-450 M/Z, with a scan rate of 0.5 s and a scan speed of 1000 amu/Sect29.

NIST mass spectrometry data center and literature references were used to compare MS retention times and Kovats indices. This allowed us to determine which chemical constituents were present in the EOs. electronically produced quantitative data from integrated peaks30.

Antioxidant assay for samples of EOs from T. capitatus

DPPH assay

The methodology for antioxidant analysis followed the same approach as in our previous studies31,32; Methanol, a 100 µg/mL stock solution for the three T. capitatus EO samples, was produced. In addition, a Trolox and gallic acid solution of 100 µg/mL was produced (the reference standard). Each sample’s serial dilutions were made from the stock solutions, yielding (5,10,20,30,40,50,80, and 100 µg/mL). One milliliter of each sample dilution was combined with one milliliter of 0.002 g/mL DPPH in methanol. To make a final working volume of 3 mL, 1 mL of methanol was added. The DPPH solutions were made fresh because they are light-sensitive. A blank controlled with DPPH in methanol at a 1:2 ratio was also made without adding an extract. For around 30 min, all working solutions were incubated at room temperature (25⁰ C) in the dark. A spectrophotometer was used to detect optical densities at a wavelength of 517 nm. The following equation was used to compute the percent DPPH inhibition for three samples of EO from T. capitatus.

with trolox or gallic acid as the standard compound:

$${\text{DPPH inhibition \% = (ABl - Ats)/ABl}} \times 100$$

ABl: the absorbance measured for the blank solution, Ats: The absorbance was measured for the tested sample of T. capitatus solution.

Assay of β carotene-linoleic acid

The antioxidant activity is measured by the β-carotene-linoleic acid system model for the three EO samples from T. capitatus by Miller33. Based on the oxidative breakdown products of the linoleic acid, this assay measures the oxidation of β-carotene-linoleic acid. 1 mg of carotene was dissolved in 2mL chloroform with 20 mg of linoleic acid and 200 mg of Tween 40. Then, the chloroform was evaporated entirely with a rotary evaporator at a low temperature and lower pressure. Then, we added 200mL of distilled water saturated with oxygen, shaking vigorously for 30 min. (0.1 mL) of the EO samples from T. capitatus aerial parts and the positive control (α-tocopherol) were combined with aliquots (5mL) of these prepared solutions. A sample without antioxidants was also made as a control sample.

After an initial absorbance reading at 470 nm, the mixture was maintained in a thermostatic bath at 50 °C, and absorbance was recorded at 15- minutes to 120 min.

Pancreatic lipase Inhibition assay for T. capitatus EOs

The steps of the protocol for the porcine pancreatic lipase inhibitory assay were mainly the same as those described by Bustanji et al. (2010), with a few changes34,35.

The three samples of EOs from T. capitatus were used to make stock solutions of 500 µg/mL in 10% DMSO. From the stock solution, serial dilutions of five concentrations (50, 100, 200, 300, and 400 µg/mL) were made. Just before usage, a 1 mg/mL stock of porcine pancreatic lipase in Tris-HCl buffer was produced fresh. 20.9 mg of p-nitrophenyl butyrate (PNPB) was dissolved in 2 mL of acetonitrile to make the substrate.

Each working solution contained 0.1mL of 1 mg/mL porcine pancreatic lipase and 0.2mL of each dilution series member of the EO. Tris-HCL was added to produce the working solutions’ final volume of 1 mL, and they were incubated for 15 min at 37 °C. After incubation, each test tube received a 0.1 mL p-nitrophenyl butyrate solution. The mixture was then incubated at 37 °C for another 30 min. A UV spectrophotometer was used to measure the hydrolysis of PNPB into p-nitrophenolate at 410 nm, which was used to estimate pancreatic lipase activity. Using Orlistat as a standard reference chemical, the same technique was used. The following equation was used to compute the percentage of lipase inhibition by EOs in the three regions:

$${\text{Lipase inhibition }}\% = \left( {{\text{ABl }} - {\text{ Ats}}} \right)/{\text{ABl }} \times 100\%$$

ABl is the obtained absorbance of the blank solution, and Ats is the obtained absorbance of the tested sample solution.

In-vitro evaluation of α-amylase Inhibition

α-Amylase inhibitory activity of the three samples was assessed by the standard method of Wickramaratne, M.N., et al. (2015) with minor modifications36,37,38.

Each T. capitatus EO sample was diluted in a few milliliters of 10% DMSO, then further dissolved in (Na2HPO4 /NaH 2PO4(1:1) (0.02 M), NaCl (0.006 M) at pH 6.9) to yield stock solutions with 1000 µg/mL concentrations. The following dilutions were made from these: 50, 100, 200, 300, 400, and 500 µg/mL, using 10% DMSO as the diluent. A 0.2 mL amount of 2 units/mL porcine pancreatic amylase was mixed with 0.2 mL of EO-prepared solutions and incubated for 10 min at 30 °C. Following incubation, the tubes were given 0.2 mL of a freshly prepared 1% starch solution in water and incubated for at least three minutes more. The reaction was then paused by adding 0.2mL (3,5-dinitro salicylic acid (DNSA) reagent, diluted with 5 mL distilled water before being heated in a water bath at 90 °C for 10 min. The combination was then allowed to cool to the ambient temperature before being measured at 540 nm. The blank control was made using the identical ingredients as the above but with 0.2mL buffer instead of EOs. Following the process outlined above, acarbose was utilized as a standard reference. The -amylase inhibitory activity was estimated using the following equation for the three EO samples from T. capitatus:

$$\% {\text{ Of}} \; \upalpha{\text{-amylase inhibition }} = {\text{ }}\left( {{\text{ABl }}{-}{\text{ ATs}}} \right)/{\text{ABl }} \times 100\%$$

ABl: the absorbance of the blank sample.

ATs: the absorbance of the test sample.

Antimicrobial activity of T. capitatus EOs

Microorganisms and conditions for cultivation

EOs samples of T.capitate were tested against the following bacteria strains: Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 9027), MRSA (Clinical sample), Proteus vulgaris (ATCC 8427), Staphylococcus aureus (ATCC 6538), and Escherichia coli (ATCC 25922). The Antifungal activity of the EOs was examined against the growth of a diagnostically confirmed Candida albicans clinical isolate.

Antimicrobial assays for T. capitatus EOs for the three samples

The antimicrobial activity of the EO samples from three regions of Palestine was assessed by using the broth micro-dilution method according to the previous protocols by Balouiri, M. et al. (2016) and Bariş, Ö. et al. (2006) with some modifications39,40.

Bacteria stains developed in cultured broth for 18 h. 10% DMSO was used to dissolve each isolated T. capitatus EO. After filter sterilization, the T. capitatus EOs solutions were serially micro-diluted ten times in sterile nutritional broth before use. The dilution procedures were carried out in an aseptic environment in 96-well plates.

Antibacterial and anti-fungal assay for T. capitatus EOs for three samples

For the antibacterial assay, each 200 µL of the isolated EOs samples was dissolved in 150 µL of 10% DMSO, diluted with 150 µL distilled water, and left on UV for 15 min.

Bacterial suspensions were made; a swab was collected from the different types of bacteria and then placed in normal saline. Turbidity was measured using the UV at λ = 620. It should be between (0.08 and 0.12). If it was less than that, bacteria were introduced, and if it was more significant than this value, normal saline was added. Then, 50µL of the bacterial suspension was combined with 5mL of the media to obtain the final bacterial suspension. The prepared T. capitatus EOs solutions were filtered, sterilized, and then micro-diluted serially 10 times, starting with 50 µL of the EOs solutions added to a sterile nutrient broth containing 50 L of the media. The initial obtained concentration was 20% v/v of the extracted EOs at the first line of the 96 well plates. This process was repeated until plate 10, at which 50 µL taken from it and removed. Then, 50µL of the prepared bacterial or fungal suspensions were added (one type of the mentioned bacterial strains for each line) to each plate, except the 12th vertical line of the 96 well plates, so that the final concentration obtained at the first vertical line of the EO became 10% v/v, equal to 100 µg of the EO per ml. The EOs-free nutrient broth on vertical line 11 was used as a positive control for bacterial growth. Vertical line number 12, on the other hand, contained EOs-free nutrient broth that had not been inoculated with any of the tested bacterial cells and served as a negative control for the media. We also had a compound control (compound + media) to ensure that there was no contamination or turbidity and that the change at the last horizontal line of the 96 well plates was not due to the compound itself.

The T. capitatus EO samples were tested in triplicate on each bacterial cell included in this investigation. At 35 °C, all of the inoculation plates were incubated. The incubation period was around 18 h long. The minimum inhibitory concentration of T. capitatus EOs was defined as the lowest concentration at which no observable bacterial growth in that micro-well was observed.

Anticancer activity

Numerous forms of cancer are classified according to the site of infection or the underlying biological process41. In our study, we used hepatic G2 cancer cells (ATCC, Rockville, MD, USA) to study the anticancer activity of the T. capitatus EOs.

Cytotoxicity assay: RPMI 1640 media supplemented with 10% heated fetal bovine serum, 1% of 2 mM l-glutamine, 50 IU/mL penicillin, and 50 µg/mL amphotericin B was used to culture hepatic G2 carcinoma cells. Once mycoplasma and bacteria were ruled out, cells were cultured in RPMI 1640 media with 10% calf serum as a monolayer confluent at 35 °C. As a precaution, antibiotics were not administered to avoid sensitizing the cell membranes. Phosphate buffer saline (PBS) was used to wash cells three times for the experiment. PBS was decanted, and cells detached with 0.025% Trypsin–EDTA and RPMI 1640 medium were added to make up a volume of 10 mL. To make a single-cell suspension, the cell suspension was centrifuged at 1000 xg for 10 min, and the pellet was resuspended in 10 mL of media. Trypan blue exclusion was used to measure cell viability more significantly than 96% in a hemocytometer. After inoculation, stock cultures were duplicated weekly. The cell line was grown in 6-well tissue culture plates (9.8 cm2) at 35 degrees Celsius in a humidified atmosphere containing 5% CO2. The cells were treated with the isolated EOs after 24 h. 0.1 mL of each EO extract from the three districts was serially diluted to 1000, 500, 250, 125, and 62.5 µg/mL.

Statistical analysis and IC50 calculations

The tests were carried out in triplicate. The results were reported as means ± standard deviation (SD). The means of the different districts were compared using an unpaired one-way ANOVA test. The statistical significance was established based on a p-value of less than 0.05. The IC₅₀ value of the biological tests was calculated based on the inhibition percentage and the test material concentration. These values were plotted against the corresponding concentrations to create a dose-response curve. The IC₅₀ value is obtained through nonlinear regression analysis or interpolating from the curve.

Results and discussion

Extraction yield of the collected EOs from T. capitatus

The dried aerial parts of T. capitatus from three districts in Palestine were grinded and subjected to extraction by hydrodistillation. Ramallah district, which has an average elevation of 880 m above sea level and an average humidity and average temperature of (47%), (40 °F to 84 °F) respectively, gave the highest yield in both methods (2.8), followed by Hebron, which has an average elevation of 930 m above sea level and an average humidity (62%) and an average temperature of (27 °F to 78 °F), gave a yield of (1.26% ). Jenin, located 250 m above sea level with an average temperature of (52 °F to 89 °F) and an average humidity of 69%, gave a yield of 1.15%. In addition to the varied geographical locations and environmental conditions, the three districts have different soil types, components, and pH. All these factors worked together, not separately, to cause the quantity changes seen between the three samples.

Our results are consistent with other studies on different plant types, emphasizing the effect of environmental factors on the chemical composition and amount of EOs42,43.

Components of the essential oils

GC-MS was used to identify all the EO components of T. capitatus, their concentrations, and output orders of the EOs. An EO chromatogram and the reference substances in a spectrum library with a computerized data bank were compared. The GC-MS method was used to get the different mass spectra and retention indices of compounds in this extract40,41. T. capitatus EO has been meticulously investigated, and the compositional diversity of plants growing in different nations and even in other regions of the same country has produced numerous chemotypes44.

Table 1 shows the GC-MS analysis of EO components from three districts in Palestine (Ramallah, Jenin, Hebron). It showed more than 21 compounds were separated and identified; the EOs percentages yield of T.capitatus were (99.73%, 99.46%, and 94.41%) for Ramallah, Jenin, and Hebron, respectively. Components identified included sesquiterpenes, monoterpenes, and other compounds like alcohols, phenols, and organic acids.

Table 1 GC-MS analysis results.
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Differences in the chemical composition of essential oils arise from their geographical origins. This variation, known as chemotypic variation or chemical polymorphism, is influenced by environmental factors such as climate, soil type, and altitude. The detailed results are described in Table 1. In the Ramallah district, carvacrol (31.25%), γ-terpinene (30.94%), o-cymene (16.84%), and linalool (6.19%) were the main components (Supplementary Figure S1). While, γ -terpinene (67%), cis-b-terpineol (12.91%), carvacrol (6.44%), and thymol (5.51%) were the main components in Jenin (Supplementary Figure S2). On the other hand, thymol (40.35%), b-caryophyllene (13.23%), (carvacrol, methyl ether) (10.7%), p-cymene (8.41%), and camphene (5.56%) were the main components in Hebron sample (Supplementary Figure S3). The comprehensive results and mass spectrometry graphs are presented in the supplementary documents. The results indicate the presence of three key components in the essential oil extracted from the three districts. However, significant variations in the levels of γ-terpinene, carvacrol, and thymol were observed across the regions. These differences are likely influenced by geographical factors, which play a crucial role in determining their relative proportions.The variation of chemical components of the EO samples for the chosen districts can be explained by the plant’s origin, harvest period, soil components, genetic variables, and climatic conditions on the chemical structure of EOs45,46. In their research, Vaičiulytė V et al.44 studied the effects of soil PH and 14 chemical components of the soil on the chemical composition of Thymus pulegioides. Results showed that the amount of aluminum, copper, iron, potassium, and manganese in soil increased, leading to decreased EOs in the raw material of T. pulegioides. Also, an abundance of higher amounts of phosphorus in the soil led to increased biosynthesis of α-terpinyl acetate. This concludes that soil chemical compositions of the EO of the plants have a significant effect. Environmental factors, including soil composition and climate, significantly influence the chemical profile of Thymus capitatus essential oil. Variations in soil mineral content, such as calcium and potassium, and organic matter levels directly affect essential oil biosynthesis. Temperature and altitude are also crucial; warmer climates can enhance the production of phenolic monoterpenes like carvacrol and thymol. Studies show that both altitude and temperature affect the essential oil yield in related species, emphasizing the importance of these factors in oil composition43,47. Furthermore, research is needed to study the influence of each factor separately on the chemical composition of the EOs.

Antioxidant activity

DPPH assay

Antioxidant activity was assessed using DPPH (2,2-diphenyl-1-picrylhydrazil), one of the earliest free radicals utilized in research on antioxidant activity48. Table 3 shows the percent inhibition of EOs of the districts at different concentrations ranging from (0–100 g/mL), in addition to the values of IC50.

Analysis of Table 2 showed that the three districts had nearly the same IC50. However, the antioxidant activity of gallic acid and Trolox was less potent.

Table 2 Percent inhibition and IC50 of EO’s of the districts.
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Statistical results were analyzed using SPSS software and the Kruskal-Wallis Test. The results showed a significant value (P < 0.05), which concludes that there are significant differences between districts according to the DPPH assay.

β-Carotene assay

The β-carotene linoleic acid test is another antioxidant test performed on T. capitatus. To evaluate the antioxidant activity of the EOs, an emulsion system consisting of β-carotene and linoleic acid was used. Figure 1 shows the behavior of essential oils (EOs) from three districts over two hours. All samples exhibited higher antioxidant efficiency than the water control and the synthetic antioxidant α-tocopherol, which caused the highest β-carotene degradation. Higher absorbance values indicate greater β-carotene protection and antioxidant activity. After two hours, the absorbance values for Ramallah, Jenin, and Hebron were 0.79, 0.744, and 0.723, respectively, all exceeding the positive control and demonstrating strong antioxidant activity.

Numerous studies have found a correlation between monoterpenes and their oxygenated monoterpenes, which include phenols and alcohols, and the presence of antioxidant activity. Carvacrol (the main component in Ramallah EO sample) was discovered to have the most significant antioxidant activity. As the results of β the carotene assay showed, the Ramallah sample gave the highest antioxidant activity49,50. Other components of EOs, such as α-terpinene and γ-Terpinene, are also responsible for antioxidant activity51. The second potent anti-oxidant activity was the Jenin sample rich in γ-Terpinene (67%). Sabinene and other non-phenolic terpenoids have been shown to have significant antioxidant properties52.

Fig. 1
figure 1

β -Carotene assay of T. capitatus EOs in three districts.

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Anti-lipase activity

In terms of metabolic illnesses, obesity is one of the fastest-growing. One way to treat obesity is to limit the number of calories consumed and to take medicines that stop the body from absorbing fat from food, speed up metabolism, and prevent it from storing fats53.

Lipases cannot hydrolyze dietary fat in the presence of orlistat. It forms a covalent bond with the active serine site, rendering them ineffective. Flatulence, fecal urgency, deficiency in fat-soluble vitamins, steatorrhea, and abdominal cramping are possible side effects of the drug54.

The anti-lipase activity of T. capitatus against the positive control anti-lipase commercial medication Orlistat was evaluated using the porcine pancreatic lipase inhibitory assay.

Orlistat was used as an active, positive control; the results shown in Fig. 2 revealed that all tested extracts have anti-lipase action at a concentration of > 200 µg/mL. The three district EOs demonstrated anti-lipase activity greater than Orlistat.

The Kruskal-Wallis statistical result for the anti-lipase activity of the EOs in the three districts was (P = 0.946), which is higher than (0.05), indicating no significant differences between the districts.

Fig. 2
figure 2

Anti-lipase activity of EOs in three districts.

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literature on the anti-lipase activity of Thymus species is limited. One study on Thymus serpyllum indicated potential benefits in managing obesity parameters in mice on a high-fat diet55. Recent studies have also highlighted the anti-lipase activity of various plant extracts, with aqueous extracts of Vitis vinifera and Rhus coriaria showing significant effects, demonstrating IC₅₀ values of 14.13 and 19.95 µg/mL, respectively56.

α-Amylase

A diabetic is a metabolic condition due to insulin resistance or insulin shortage. Chronic hyperglycemia can create a wide range of problems that affect numerous cells and organs, resulting in several fatal disorders57. Oral anti-diabetic medicines and/or parental insulin are used to treat diabetes. A significant number of these pharmaceuticals are associated with a variety of significant adverse effects and potentially hazardous contraindications. Patients have strongly preferred herbal supplements and pharmaceuticals combining high therapeutic efficacy with low adverse effects in recent years. Anti-diabetic medicines derived from herbs show great promise, and the anti-diabetic potential of plants that have been used in traditional medicine for a long time is currently the subject of research58. Numerous plants have been confirmed to exhibit hypoglycemic capabilities, as evidenced by literature sources from multiple databases; these planets tend to reduce blood glucose either as an insulin mimetic or through insulin secretory activity. Most hypoglycemic plants are found in the following families: (Leguminoseae, Lamiaceae, Liliaceae, Cucurbitaceae, Asteraceae, Moraceae, Rosaceae, and Araliaceae). Anti-diabetic properties are attributed to polyphenols, flavonoids, terpenoids, coumarins, and other components in these medicinal plants59.

Figure 3 demonstrates α-amylase inhibitory activity for the EOs of the three districts contrasted with Acarbose, which is utilized therapeutically in a concentration-dependent manner for its inhibitory action. Samples at a 400 µg/mL concentration showed a 50% inhibition compared to Acarbose.

Fig. 3
figure 3

Alfa amylase percent inhibition of EOs in three districts.

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Statistical analysis tests of the three districts’ results showed a significant value (p = 0.041). The effect is ranked according to the following sequence: Jenin 11.3, Ramallah 12.21, Hebron 12.14, Acarbose 22.21. The results confirmed differences in the EO activities between the districts. Research on aqueous extracts from thyme leaf powder showed that higher concentrations increased inhibition of α-amylase in Trogoderma granarium larvae. A 10% extract concentration resulted in an 87 mg/mL inhibition rate, demonstrating a dose-dependent effect60.

Antimicrobial assay

Microbial infections are a global concern that poses a life-threatening threat to humanity. Owing to the abuse of antibiotics, antibacterial and antifungal resistance has emerged as an urgent global health issue. With an estimated 2 million patients afflicted with medication-resistant germs each year, an alternate strategy to combat drug resistance is required. As a result, there is a proclivity to employ traditional or unusual approaches to solve the problem and prevent the spread of infectious diseases27.

Gram-negative and gram-positive bacteria were both shown to be highly susceptible to T. capitatus EO, even though it was previously stated that EOs are less effective against gram-negative bacteria61. A possible explanation for this is that the EOs contain hydrophobic components, which may damage the cell membranes of bacteria and hence impede their ability to operate51. Further research has explained that T. capitatus EOs effectiveness against fungus may be due to the quantity of phenolic chemicals (carvacrol and thymol), which may interfere with enzymes involved in cell wall synthesis, such as chitin synthase and chitinase, and with α and ß-glucanases. The antifungal impact of T. capitatus was previously shown to involve telomerase suppression as it increased the rate of cell senescence and apoptosis, as previously reported. (γ-terpinène and p-cymene) have also been implicated in antifungal action61.

In this research, the antimicrobial activity of EO samples from three regions of Palestine was assayed by using the broth micro-dilution method according to a previous protocol (Balouiri M. et al.2016; Bariş, Ö. et al.2006) with some modifications.

Figure 4 demonstrates the antimicrobial activities of the EOs on different types of bacteria. EOs from all districts showed MIC values between (0.1953 and 1.5625 µg/mL) except for pseudomonas. The results also showed that the Hebron EO sample had the highest activity of 0.1953 g/mL against all types of bacteria, which was explained by the high percentage of thymol (40.35%). Moreover, the results demonstrate the efficiency of the EOs against the MRSA strain, which is resistant to most antibiotics. Similar work testing the antimicrobial efficacy of Thymus vulgaris essential oil showed significant effects in combating various bacterial strains62.

Fig. 4
figure 4

MIC values of T. capitatus EOs in three districts.

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Anticancer activity

Cytotoxic chemotherapy refers to chemicals and medicinal plants that kill various cancer cells. Cytotoxic medications include both plants and pharmaceuticals. They prevent the division of cells, which ultimately results in the death of cancer cells. Additionally, they can improve the results of radiotherapy and surgery and decrease the number of metastases63. The cytotoxic and anticancer activities of Thymus EOs and monoterpenes have been extensively studied. Carvacrol, thymol, terpinene, and p-cymene are the main parts of Thymus EOs that give them their healing effects64.

In this research, HepG2 cells were used to predict the anticancer assay of T. capitatus EO.

For a day, the Hep-G2 cells were exposed to increasing quantities of the investigated samples from three districts (1000, 500, 250, 125, and 62.5 µg/mL). The MTS test was used to obtain a quantitative reading on the cell viability.

Table 3 shows the percent inhibition of thymus EOs from three sites in Palestine on Hep-G2 cells. They showed almost similar cytotoxic activity with more than 85% inhibition at concentrations greater than 62.5 µg/mL. Hoverer, Ramallah EO samples showed 82.5% inhibition at 62.5 µg/mL. The results demonstrate that the cytotoxic effects against Hep G2 cells are different in the districts with superiority in Hebron and Jenin. More phytochemical and in vivo pharmacological research is required to validate these exceptional results.

Table 3 % Inhibition of T. capitatus EOs in three districts against hepatic G2 cancer cells.
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Many researches on Thymus species exhibit potential anticancer properties due to compounds like thymol and carvacrol. Research shows significant antitumor effects in mammary carcinoma models, with in vitro studies on MCF-7 and MDA-MB-231 breast cancer cell lines supporting these findings65.

Conclusion

Tested samples of T. capitatus were collected from three regions of Palestine (Hebron, Jenin, and Ramallah). T. capitatus extraction was performed using hydrodistillation. About 21 compounds were separated and identified by GC-MS analysis of EO components from the three samples of three districts. Apparent variations in their percentage between districts, mainly (γ-Terpinene, carvacrol, and thymol), were noticed due to environmental factors.

In vitro assessment of the antioxidant activity of the EOs was carried out using the β-Carotene assay and DPPH assay. The samples showed potent antioxidant activity. Similar results were demonstrated for Alfa amylase and Anti-lipase, showing the activity of EOs against both enzymes when compared with the positive control. The antimicrobial activity of T. capitatus EO was effective against all tested bacteria and fungi except pseudomonas. The Hebron sample gave distinguishable results at low concentrations against all bacterial and fungal strains put to the test. Moreover, the cytotoxic activity against Hep G2 cells showed more than 85% inhibition at concentrations greater than 62.5 µg/mL for Hebron and Jenin samples. Differences in the active consistency in different districts explain the variation in results.