Abstract
Vitamin C, enzyme activities, phenolic compounds, antioxidant capacity, antimicrobial activity, and essential oil analyses of ripe and unripe fruits of P. terebinthus were investigated. Vitamin C amounts of ripe and unripe fruits were 63.2 and 15.4 mg/100g, respectively. The main phenolic compounds of unripe and ripe fruits are rutin, syringic acid, and gallic acid. It was determined that the enzyme inhibitor activities in the ripe and unripe fruits were 0.136 mg/mL and 2.14 mg/mL. In all of the free radical scavenging (DPPH and ABTS) activity, ferric (III) ion reducing antioxidant power (FRAP) capacity, total phenolic substance amounts (TPC), total flavonoid substance amounts (TFC), and total antioxidant activity (TAC) antioxidant methods analyzed with plant parts, the methanol extracts obtained from the ripe fruits of the P. terebinthus showed higher antioxidant properties than the methanol extracts obtained from the unripe fruits. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 showed antimicrobial activity against microorganisms, while methanol extracts obtained from unripe fruit samples did not show antimicrobial activity against the microorganisms used. The chemical grade with the most compounds in the essential oils of P. terebinthus were monoterpenoids, sesquiterpenes, and monoterpenes in unripe and ripe fruits, respectively. The main components were α-pinene with 22.8% and 27.3% ratios in unripe and ripe fruits, respectively.
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Chemical Composition of Pistacia terebinthus L. and its Phytochemical and Biological Properties
Muhammed Said Fidan,a,* Cemalettin Baltacı,b Mehmet Öz,c and Zeynep Akar d
Vitamin C, enzyme activities, phenolic compounds, antioxidant capacity, antimicrobial activity, and essential oil analyses of ripe and unripe fruits of P. terebinthus were investigated. Vitamin C amounts of ripe and unripe fruits were 63.2 and 15.4 mg/100g, respectively. The main phenolic compounds of unripe and ripe fruits are rutin, syringic acid, and gallic acid. It was determined that the enzyme inhibitor activities in the ripe and unripe fruits were 0.136 mg/mL and 2.14 mg/mL. In all of the free radical scavenging (DPPH and ABTS) activity, ferric (III) ion reducing antioxidant power (FRAP) capacity, total phenolic substance amounts (TPC), total flavonoid substance amounts (TFC), and total antioxidant activity (TAC) antioxidant methods analyzed with plant parts, the methanol extracts obtained from the ripe fruits of the P. terebinthus showed higher antioxidant properties than the methanol extracts obtained from the unripe fruits. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 showed antimicrobial activity against microorganisms, while methanol extracts obtained from unripe fruit samples did not show antimicrobial activity against the microorganisms used. The chemical grade with the most compounds in the essential oils of P. terebinthus were monoterpenoids, sesquiterpenes, and monoterpenes in unripe and ripe fruits, respectively. The main components were α-pinene with 22.8% and 27.3% ratios in unripe and ripe fruits, respectively.
DOI: 10.15376/biores.18.4.6862-6881
Keywords: Antioxidant-antimicrobial-enzyme activities; Phenolic compounds; Vitamin C; P. terebinthus; Essential oil; Chemical composition
Contact information: a: Bursa Technical University, Department of Forest Industry Engineering, Bursa 16310 Türkiye; b: Gümüşhane University, Department of Food Engineering, Gümüşhane 29100, Türkiye; c: Gümüşhane University, Department of Forestry, Gümüşhane 29100, Türkiye; d: Gümüşhane University, Department of Genetics and Bioengineering, Gümüşhane 29100, Türkiye;
*Corresponding author: said.fidan@btu.edu.tr
INTRODUCTION
Pistacia terebinthus, belonging to the family of Anacardiaceae, is a long-lived plant (Özcan et al. 2020; Akpulat et al. 2021). It is one of the 20 Pistacia species extensively distributed in the United States, Asia, and the Mediterranean (Couladis et al. 2003; Kavak et al. 2010; Bozorgi et al. 2013; Özcan et al. 2020). It is popularly known as ‘menengic’ in Türkiye (Bozorgi et al. 2013; Uysal et al. 2022). Terebinth coffee is one of the most popular herbal coffees in Türkiye. This coffee is commonly known as ‘‘menengic coffee’’ in Türkiye (Orhan et al. 2012). The appearance of the P. terebinthus plant is shown in Fig. 1. Medicinal plants, because of their bioactivity and nourishment features, arise as an alternative to synthetic products. They are used not only in conventional medicine but also in several pharmaceutical, ornamental, and food products (Foddai et al. 2015; Rauf et al. 2017).
Fig. 1. P. terebinthus plants (Photo: Mehmet ÖZ, 19.09.2021)
Most of the plant parts with the inclusion of resin, fruit fatty oil, and fruits are used as traditional medicine and food in the area (Baytop 1999; Stanojević et al. 2021). Plants are significant resources of potential phytochemicals, for instance, vitamins and polyphenols (Uysal et al. 2022). The small fruits of P. terebinthus are spherical nuttlets that are dark greenish when ripe. Its fresh fruits and shoots are used for human nutrition (Özcan et al. 2009). P. terebinthus is consumed as a snack food, used in soaps, or in making a coffee-like drink after roasted and ground (Topcu et al. 2007; Kavak et al. 2010; Durmaz and Gökmen 2011; Gogus et al. 2011; Orhan et al. 2012). The fruits are also used in the baking of a coffee substitute and as a specialty village bread (Özcan et al. 2009). P. terebinthus fruits are important sources of fiber, minerals, oil, and protein. The oil extracted from terebinth fruits is an alternative to herbal oils because it includes high amounts of omega-3 (linoleic acid) fatty acids and mono-unsaturated (oleic acid), and it has desirable taste and odor (Bozorgi et al. 2013; Özcan et al. 2020).
Moreover, the fruits of P. terebinthus are used in traditional medicine for diarrheic, throat infections, gastralgia, antiseptic for bronchitis, stimulant, rheumatism, diuretic, antitussive, wounds, urinary inflammations, burns, cough, stomachache, asthma, and eczema (Topcu et al. 2007; Özcan et al. 2009; Kavak et al. 2010; Durmaz and Gökmen 2011; Gogus et al. 2011; Orhan et al. 2012; Özcan et al. 2020).
Pistacia species have antioxidant activity as well as anti-inflammatory, cytotoxic, and antimicrobial properties due to their high phenolic, tocopherols, and flavonoids contents (Kavak et al. 2010; Durmaz ve Gökmen 2011; Durak and Uçak 2015; Özcan et al. 2020).
Many studies on the volatile oil constituents of P. terebinthus have been made and results have been observed in literature. These studies found α-pinene, β-pinene, sabinene, limonene, p-cymen-8-ol, terpinen-4-ol, and caryophyllene as major compositions (Couladis et al. 2003; Usai et al. 2006; Özcan et al. 2009; Orhan et al. 2012).
Phenolic compounds, bioactive properties, fatty acids, physicochemical features, essential oils, mineral compounds, antioxidant and antimicrobial features, biological activities, and phytochemicals of terebinth plants have been studied by various researchers (Ozcan et al. 2009; Kavak et al. 2010; Durmaz and Gökmen 2011; Gogus et al. 2011; Orhan et al. 2012; Rauf et al. 2017; Mollica et al. 2018; Zengin et al. 2018; Uysal et al. 2022). Nonetheless, there have been no studies that compare the features, antimicrobial properties, phenolic compounds, enzyme activity, and vitamin C profiles of different ripe and unripe fruits of P. terebinthus.
In this study, the effects of vitamin C, enzyme activities, phenolic compounds, antioxidant capacity, antimicrobial activity, and essential oil in ripe and unripe fruits of terebinth plants were determined. It is the first study that has been done with the terebinth plants located in the north of Türkiye. In terms of chemistry, it is a combined study in which enzyme inhibition, phenolic and volatile compounds, vitamin C, and antimicrobial-antioxidant analysis are performed.
EXPERIMENTAL
Plant Material
In this study, P. terebinthus unripe and ripe fruit (1000 g) samples were gathered in Torul-Köstere Village (40°36ꞌ28ꞌꞌN, 39°19ꞌ18ꞌꞌE, Altitude: 1045 m) located within the borders of Gümüşhane Province, Türkiye. The locations where the samples were taken are shown in Fig. 1. The taxonomic diagnosis of plant samples was determined by Assoc. Prof. Mutlu Gültepe, in the Department of Forestry, Dereli Vocational School, Giresun University, Giresun, Türkiye. The plant was saved with the number KTUB Gültepe 718 with identification from the Herbarium of Karadeniz Technical University, Faculty of Science, Department of Biology. Fruit samples were collected by hand on 19.09.2021 (autumn season) from the specified region and dried in the shade by mixing at regular intervals. Samples were stored in a cool, dry place out of the sun until analysis. For analysis, the fruits were ground and not treated differently.
Extraction Procedure
Essential oil extraction
The essential oils were obtained by hydrodistillation method in the Clevenger device. Sufficient amounts (100 g) of plant parts from unripe and ripe fruit samples were cut into pieces and placed in skewered balloons and distilled for 4 h by adding 500 mL of distilled water (Küçük et al. 2006). The essential oils acquired were dissolved in 1 mL of high performance liquid chromatography (HPLC) grade hexane, dried with anhydrous sodium sulfate, filtered, and stored in an indoor brown bottle at -18 °C until the analysis was completed.
Extraction of methanol
The extraction process was performed using an ultrasonic bath (3 L 320 W Bandelin Ultrasonic Bath). After the fruit parts were ground and 10 g were taken, 50 mL of 80% aq. MeOH was added, and then an ultrasound-assisted extraction process was applied at 60 min and 40 °C. At the end of 60 min, it was filtered 2 times through Whatman 1 filter and centrifuged at 4000 rpm for 10 min and plant extracts were obtained. At the end of centrifugation, the upper part was taken into a beaker, and the extracts were obtained by completely evaporating the methanol at 40 °C (Dranca and Oroian 2016).
Analysis of Vitamin C in Extracts
Vitamin C analyses of the samples were performed using an HPLC-UV device with a UV 1000 detector according to high pressure liquid chromatography UV detector method. The analytical column RP C18 (250 x 4,6 mm, 5 µm) was used with the mobile phase: methanol: water (5:95, v/v) pH= 3 (H3PO4), flow 1 mL/min, injection volume 20 µL, with detection of UV at 254 nm. For the calibration curve, standard solutions of 10, 30, 60, 90, and 120 mg/L concentrations were prepared from L-ascorbic acid. A total of 10 g of the ripe and unripe fruits of terebinth plants were taken and divided into pieces in a shredder. Then, 70 mL sufficient amount of metaphosphoric acid (15% m/m) was added to the smashed fruits and mixed in the homogenizer. The homogenized samples were completed to 100 mL and filtered through filter paper. After the filtrates were passed through a 0.45-micron filter, they were taken into vials and given to the HPLC device. The amount of analyzed vitamin C in the sample was calculated using the calibration graph method (y = 9498.7 x – 4236) (Öz et al. 2018).
Enzyme Inhibitory Activities of the Extracts
The α-glucosidase inhibitory activity of the samples was studied through modification (Yu et al. 2012). In the study, first, 650 μL of phosphate bumper (pH: 6.8 and 0.1 M) was added to the test tubes. Then, 20 μL of sample and 30 μL of α-glucosidase enzyme (Saccharomyces cerevisiae, lyophilized powder ≥ 10 units/mg protein) prepared in phosphate buffer were added. After the mixture was incubated at 37 °C for 10 min, 75 μL of substrate (4-nitrophenyl-α-D-glucopyranoside) was added. After the mixture was kept at 37 °C for 20 min, 650 μL of 1 M Na2CO3 was added to all tubes and the reaction was stopped. The absorbance value was read at 405 nm in an ultraviolet/visible (UV/VIS) spectrophotometer. Different densities of acarbose (positive control) were studied as the standard inhibitor. The study was performed in three parallel and reagent-sample blanks. The IC50 values of acarbose and samples (sample concentration that halves the enzyme activity present in the environment) were calculated.
Determination of Phenolic Compounds
All samples were ultrasonically bathed for 20 min and filtered through a syringe filter (0.45 µm) before analysis. Chromatographic analysis of dry methanol extracts of unripe and ripe samples was used Agilent 1260 Infinity HPLC-DAD system (Agilent Technologies, Waldbronn, Germany) device. The gallic acid, 4-hydroxybenzoic acid, caffeic acid, sesamol, paracoumaric acid, benzoic acid, protocatechuic acid, catechin, syringic acid, vanillin, syringaldehyde, rutin, protocatechuic aldehyde, vanillic acid, rutin, ferulic acid, coumarin, epicatechin, rosmarinic acid, t-cinnamic acid, quercetin, kaempferol, and chyricin were used as standards for the analysis. The analysis method of the phenolic compounds of the samples was studied by modifying the gradient flow of the mobile phase with some changes (Paje et al. 2022). Chromatographic separation of individual components was performed using a Hypersil HPLC Column (250 x 4.6 mm2, 5µm). Mobile phase solvent A was used as mixture 0.5% acetic acid in water (0.5: 95.5, v/v) and ACN (solvent B). The gradient elution was started with 95% of solvent A and reduced to 75% after 20 min. Solvent A was reduced to 50% at 45 min and to 10% at 55 min. It was then increased to 65% at 65 min and continued for up to 70 min. The injection volume was 10 µL, and the flow rate was 1.0 mL/min. The wavelengths used in the DAD detector were 240, 250, 254, 280, and 324 nm.
Determination of Antioxidant Capacity
The antioxidant capacities of the attained methanol extract were detected according to free radical scavenging (2,2-diphenyl-1-picrylhydrazil (DPPH) and ABTS) activity and ferric (III) ion reducing antioxidant power (FRAP) capacity methods. Additionally, some bioactive component amounts were shown by total phenolic substance amounts (TPC), total flavonoid substance amounts (TFC), and total antioxidant activity (TAC) works.
Free radical scavenging activity (DPPH)
The DPPH of unripe and ripe fruit methanol extract was obtained using DPPH according to the Sanchez-Moreno method (Sağdıç et al. 2011). The method was applied by mixing the methanol extract and DPPH solutions with specific concentrations by vortexing and keeping them at room heat and in the dark for 30 min. In the end of the period, the absorbance of the samples at 517 nm was read and the amount of DPPH remaining in the reaction medium was calculated according to the formula below. Results are given as mg AA eq./g, mg Trolox eq./g, and % free radical removal. The % inhibition was calculated using Eq. 1,
%Inh.=(absorbance of control – absorbance of sample / absorbance of control) x 100 (1)
where Inh. means inhibition. Three measurements for each combination were taken, and the mean values were reported.
Radical cation removal activity (ABTS)
The ABTS radical cation scavenging activity analysis was made using ABTS solution according to the method (Ahmed et al. 2015). First, 150 µL of methanol was utilized as the blank. Then, 150 µL of standards (ascorbic acid) were taken and the same procedures were performed. The obtained solution was then read at the spectrophotometer absorbance at 734 nm. The ABTS cation removal activity amounts in the samples were calculated following Ahmed et al. (2015). Results are given as mg AA eq./g, mg Trolox eq./g, and % free radical removal. The % inhibition was calculated using Eq. 2,
%Inh.=(absorbance of control – absorbance of sample / absorbance of control) x 100 (1)
where Inh. is Inhibition. Three measurements for each combination were taken, and the mean values were reported.
Ferric (III) ion reducing antioxidant potency (FRAP)
Analysis of the FRAP of methanol extract was made according to the method of Ahmed et al. (2015) using FRAP solution. First, 500 µL of distilled water was utilized as blank. Then, 250 µL of the standards were taken and the same procedures were performed. The FRAP amounts in samples were determined as mg FeSO4 equivalent/g using the correct equation of the calibration graph obtained with the FeSO4 solution (Ahmed et al. 2015).
Total phenolic substance amount (TPC)
Analysis of the total phenolic content of methanol extract was realized regarding the Kasangana method using Folin-Ciocalteu reagent (Kasangana et al. 2015). Then, the prepared mixture was vortexed. Then it was incubated in the dark at room temperature for 120 min. At the end of the incubation period, the absorbance of the mixture at 760 nm was read. A total of 3.7 mL water, 500 µL methanol + 100 µL Folin–Ciocalteu reagent + 600 µL 10% Na2CO3 mixture was used as blank. The amounts of phenolic substances in the samples were expressed as mg GA eq./g using the correct equation of the calibration graph obtained with the gallic acid solution.
Total flavonoid substance amount (TFC)
The total flavonoid content of unripe and ripe fruit methanol extract was determined according to the Kasangana method. The absorbance of the resulting mixture was measured in the spectrophotometer at 506 nm. First, 500 µL of distilled water was utilized as blank. Then, 500 µL of the standards were taken and the same procedures were performed. The total amount of flavonoid substances in the samples was made as mg QE eq./g using the equation of the calibration graph obtained with an ethyl alcohol solution of Catechin or Quercetin standards (Kasangana et al. 2015).
Total antioxidant amount (TAC)
Analysis of the total antioxidant content of ripe and ripe fruit methanol extract was performed using molybdate reagent according to the Kasangana method. A total of 250 µL of distilled water was used instead of the sample as a blank. The absorbance of the resulting reaction mixtures was read in a 695 nm spectrophotometer. Then, 500 µL of the standards were taken and the same procedures were performed. The total amount of antioxidant substances in samples was given as mg GA eq/g using the correct equation of the calibration graph obtained with the solution of ascorbic acid (Kasangana et al. 2015).
Determination of Antimicrobial Activity
Microorganisms utilized in the work were obtained from the laboratory of Gümüşhane University, Department of Food Engineering. The antimicrobial activities of the essential oil and methanol extracts were detected by disk diffusion method against 13 microorganisms, including 10 bacteria and 3 yeast-molds (Matuschek et al. 2014). Antimicrobial activity was realized in two phases: preparation of bacteria and yeasts and preparation of examples. Bacteria were used in a Nutrient Broth medium after 24 h of the first activation at 36 °C and after 18 h of the second activation at 36 °C. Then, 1% of the microorganisms to be used in the study were added to the prepared sterile solid media and they were poured into petri dishes and allowed to solidify. Then, 5-mm diameter wells were opened on the solidified media. The incubation process was carried out by adding the solutions of the essential oil prepared with hexane to the opened wells. Petri dishes containing bacteria were incubated for 24 h at 36 °C, and petri dishes containing mold and yeast were incubated for 48 h at 27 °C. In the end of the determined period, the results were found by measuring the transparent zones around the discs.
GC-MS/FID Conditions for Essential Oil Analysis
The essential oil obtained by the hydrodistillation method in the Clevenger system was dissolved in hexane, passed through a 0.45-micron filter, placed in amber colored vials, and placed in the autosampler. After the volatile compounds were separated on the gas chromatography column, the mass spectra of each were individually taken in the mass spectrophotometer, and their structures were elucidated by comparing the mass spectra of each component with the reference components of the Willey and NIST libraries. To confirm the detected compounds, the Kovats indices of the compounds were compared with the literature data (Adams 2007). The measurement of the essential oil was made with the gas chromatography flame ionization detector (Agilent Technologies Inc, Santa Clara, CA, USA). For GC, the split ratio was adjusted as 1:5 by injecting 1 µL of essential oil in hexane into the same column. The GC-MS/FID analyses were performed on Agilent-7890 model device and an HP-5 model apolar capillary column (30 m x 0.32 mm, film thickness 0.25 µm) was used for analysis. The injector, ion source and quadrupole rod temperatures were 250 °C, 230 °C, and 150 °C, respectively. Injections were applied in split (25:1) mode using helium (>99.999%) as the carrier gas with a flow rate of 1 mL/min. Then, 1 µL of essential oil solution in hexane (GC class) was injected and initially the GC oven temperature program kept at 60 °C for 2 min, increased to 240 °C with a rise of 3 °C/min, and spectra were obtained. Mass spectra were acquired at a scan speed of 2 spectra per second after a solvent delay of 3.8 min, and the mass scan range was set at m/z 45–450. The FID detector temperature was maintained at 250 °C with a hydrogen flow of 35 mL/min and air flow of 350 mL/min.
RESULTS AND DISCUSSION
The Analysis of Vitamin C in Extracts
The amount of vitamin C in the unripe fruit was 63.15 ± 0.45 mg/100 g, and the amount of vitamin C in the ripe fruit was 15.44 ± 0.13 mg/100 g. The amount of vitamin C in unripe fruits was higher than the amount of vitamin C obtained from ripe fruits. In terms of vitamin C, there is no study in the literature on the P. terebinthus plant. In the vitamin C analysis performed on unripe and ripe fruits of Pistacia lentiscus belonging to the same family, Ayad et al. (2023) reported 1.23±0.03 in red berries and 1.25±0.01g AA/100 g in black berries.
The amount of vitamin C detected in fruits decreased in ripe fruits. The content of vitamin C in vegetables and fruits can be influenced by various factors such as preharvest climatic conditions and cultural practices, maturity and harvesting methods, and postharvest handling, genotypic differences procedures (Medveckienė et al. 2021). Medveckienė et al. (2021) stated that the amount of vitamin C in rosehip fruits tended to decrease with the ripening stage.
Enzyme Inhibitory Activities of the Extracts
Enzyme inhibitor activity was 0.136 mg/mL in unripe fruits of P. terebinthus, and enzyme inhibitor activity was 2.14 mg/mL in ripe fruits. It has been determined that unripe fruits are more effective in enzyme inhibition.
Even though the terebinth plant was considered a significant plant, there was no study on the extensive enzyme inhibitory activity. In terms of the foregoing expressions, the authors determined enzyme inhibitory activity.
Yu et al. (2012) stated that a lower IC50 value of the sample results in more effective enzyme inhibition. The methanol extract also analyzed remarkable enzyme inhibitory activity. The outcomes suggest that the extracts in leaves of P. terebinthus have for use as a good nominee of native enzyme inhibitors and antioxidants (Uysal et al. 2022). In α-glucosidase inhibition assay, water (14.5 mmol ACAE/g) and methanol (14.6 mmol ACAE/g) extracts showed parallel activity. The enzyme inhibition outcomes of samples may be noticeable by flavonoid and phenolics constituents. Similarly, they determined the maximum BChE activity in the methanol extract of P. terebinthus leaves (Uysal et al. 2022). In recent years, many studies demonstrated phenolic constituents in plants play a significant role in enzyme inhibition (Khan et al. 2018; Kim et al. 2020; Yang et al. 2021). Some reports demonstrated distinct parts of terebinth fruits that indicated enzyme inhibitory activity. For instance, methanol fruit extract indicated the finest BChE inhibitory effect (45.74% at 200 μg/mL) (Orhan et al. 2012). Additionally, fruits of P. terebinthus indicated anticholinesterase activity (Hacıbekiroğlu et al. 2015). The oil of terebinth plants displayed antidiabetic activities in diabetic rats (Uyar and Abdulrahman 2020). Enzyme inhibitory activities in the extracts of unripe and ripe terebinth fruits are demonstrated in Table 1.
Table 1. Amount of Enzyme Inhibitory Activities in Fruit Samples of P. terebinthus
Table 2. Phenolic Compounds in Fruit Samples of P. terebinthus
*LoQ (limit of quantitation) value: 0.1 mg/kg
Determination of Phenolic Compounds
The major phenolic components of unripe fruit samples were rutin (4530 mg/kg), syringic acid (466 mg/kg), and gallic acid (4200 mg/kg). The main phenolic compounds of ripe fruit samples were rutin (10700 mg/kg), gallic acid (2050 mg/kg), and syringic acid (1670 mg/kg). The main phenolic compounds of unripe and ripe fruits are rutin, syringic acid, and gallic acid. Phenolic components of unripe and ripe terebinth fruits are shown in Table 2. In the present study, the amounts of rutin, syringic acid, and gallic acid were found to be high in ripe fruits. It can be thought that the antimicrobial properties of the ripe fruit may be due to these substances. Dubey et al. (2013) declared that rutin is a good antibacterial and antifungal agent as a result of their studies. Gallic acid may play a protective role in healthy individuals by inhibiting apoptosis and a naturally occurring gallic acid is highly antioxidant (Zahrani et al. 2020). Lima et al. (2016) reported that gallic acid and its methyl ester showed antibacterial activity on Bacillus subtilis, Staphylococcus aureus, and Escherichia coli.
Determination of Antioxidant Capacity
When the antioxidant capacity amounts are examined, the DPPH amount was 73.1 mg AA eq./g in P. terebinthus unripe fruit methanol extract and 133.2 mg AA eq./g in ripe fruit methanol extract. The DDPH % inhibition rates of the same samples were between 89.7% and 71.7%. When the DPPH amount and DPPH % inhibition rates of unripe and ripe fruit methanol extracts are compared, it can be said that the DPPH amount of ripe fruit methanol extract was higher than that of unripe fruit methanol extract and the DPPH % inhibition rates of unripe fruit methanol extract was higher than that of ripe fruit methanol extract.
When the DPPH amount and DPPH % inhibition rates of unripe and ripe fruit methanol extracts are compared, it can be said that methanol extract of ripe fruit was higher DPPH amount and methanol extract of unripe fruit was higher DPPH % inhibition rate.
In the authors’ study, ABTS amounts of the samples were 81.5 mg AA eq./mg in unripe fruit methanol extract and 134.26 mg AA eq./mg in ripe fruit methanol extract. ABTS % inhibition rates were 97.2% in unripe fruit methanol extract and 71.1% in ripe fruit methanol extract. The ABTS capacity of ripe fruit methanol extract was higher than that of unripe fruit methanol extract. The amount of FRAP in unripe and ripe fruit methanol extract was 18.4 mg FeSO4 eq./g and 18.7 mg FeSO4 eq./g, respectively (Table 3).
The authors proposed that some flavonoid derivatives can account for the high antioxidant capacity of the fruit extracts (Orhan et al. 2012). In a work on acetone and methanol extracts acquired from the fruits of terebinth plants, the extracts were analyzed for their antioxidant capacity and both extracts demonstrated a high DPPH and superoxide radical scavenging activity (Topçu et al. 2007). The antioxidant content outcomes demonstrated that methanol extract (DPPH: 2.06 mmol TE/g, ABTS: 3.29 mmol TE/g, FRAP: 1.62 mmol TE/g) exhibited promising antioxidant capacity (Uysal et al. 2022). DPPH of the extracts varied over a wide range (8.86% to 64.43%) and were correlated with TPCs (Durak and Uçak 2015). This study is in compliance with the authors’ data as the extracts attained in the work have a notable scavenging effect against DPPH radical. The outcomes suggest that the extracts of terebinth plant leaves have the potential to be utilized as a good nominee of natural antioxidants.
Examining the bioactive compounds
In this study, while the total phenolic content of unripe fruit methanol extract was 41.5 mg GA eq./g, it was 47.3 mg GA eq./g in ripe fruit methanol extract. P. terebinthus total phenolic content was higher in ripe fruit methanol extract than in unripe fruit samples.
In this work, the total flavonoid substance amount of unripe fruit methanol extract was 88.3 mg QE eq./g, while it was 169 mg QE eq./g in ripe fruit methanol extract. The total flavonoid content of the samples was high. In the authors’ research, the total antioxidant content of unripe and ripe fruit methanol extract in the samples was 45.9 mg GA eq./g and 72.8 mg GA eq./g, respectively. As shown in Table 3, the amount of total antioxidant substance was higher in ripe fruit methanol extract.
It is common for ultrasonic extraction to enhance the quantities of phenolic constituents in terebinth fruit extracts. Total phenolic quantities of examples ranged from 84.0 to 87.3%. Among the sonicated samples, the highest total phenolic (251 mg/100 g) amounts were obtained in examples sonicated for 30 min (Özcan et al. 2020). The increased amounts of phenolic constituents in sonicated P. terebinthus samples over the control could be due to the deterioration of the cell wall by sonication breakdown of the material, that leads to the release of more phenolic constituents (Abid et al. 2013). Additionally, Ma et al. (2009) demonstrated that ultrasonic treatments greatly enhanced the extraction of various phenolic constituents from citrus peels.
Total flavonoid and phenolic amounts in methanol extracts of the terebinth plants were 123 µg PEs/mg extract and 22.6 µg QEs/mg extract, respectively (Topçu et al. 2007). Total flavonoid and phenolic amounts of P. terebinthus fruit were 47.0 mg quercetin/g and 241 mg GAE/g, respectively (Orhan et al. 2012). Total flavonoids and phenolics amounts of the extracts were 65.5 to 211 mg GAE/g and 54.9 to 170 mg RE/g, respectively. The maximum level of TPC was detected in the methanol extract. The methanol extract (211 mg GAE/g) had the highest content TPC compared with other extracts (Uysal et al. 2022). The difference between this article and the literature studies may be because of the distinction in the phenolic profiles of the fruits. The previous and current studies clearly demonstrate that TPCs of P. terebinthus extracts differ greatly from each other. These differences can be due to factors such as fruit species and gathering sites (Farhat et al. 2013). Environmental and climate circumstances are also substantial factors affecting the TPCs of examples. Furthermore, a higher phenolic amount was demonstrated in unripe fruits than in ripe ones (Costa et al. 2013).
When the antioxidant analyses of unripe and ripe fruit methanol extracts were examined, it was observed that DPPH, FRAP, TPC, TFC, and TAC antioxidant values of fruit methanol extract were higher in ripe fruit methanol extract, except for ABTS. In this study, when the antioxidant capacities of the authors’ examples are estimated, it is seen that they are compatible with the literature. This situation shows parallelism with antimicrobial activities.
The extract attained from the P. terebinthus leaf has noticeably higher antioxidant activity when compared with the other natural and synthetic antioxidants. This may conceivably be because of the high phenolic and flavonoid compounds content of crude extract (Kavak et al. 2010). Between the extracts, methanol extract had the most powerful antioxidant capacity. These observations suggest that the phytochemical amounts in the methanol extract can be accountable for the major part of antioxidant capacity, as described in former studies (Kavak et al. 2010; Uysal et al. 2022). Former studies indicated that the antioxidant activity of terebinth plant fruits may be affected by main flavonoids and phenolics for example quercetin (Topçu et al. 2007; Uysal et al. 2022). Antioxidant analysis results of methanol extracts of ripe and unripe fruits of P. terebinthus plant are given in Table 3.
Table 3. Antioxidant Activity Contents and Bioactive Compounds of Essential Oil Attained from Fruit Samples of P. terebinthus
*: Means, ** ± : Standard deviation
Determination of Antimicrobial Activity
The results demonstrate that P. terebinthus ripe fruit methanol extracts showed antimicrobial activity. However, the essential oil samples of the unripe and ripe fruits of the terebinth plant did not show any antimicrobial activity against the microorganisms used.
It was determined that the ripe fruit of P. terebinthus formed zones of 4.24 mm diameters against Escherichia coli ATCC 25922 and 4.20 mm diameters against Staphylococcus aureus ATCC 25923. The methanol extract of the unripe fruits of the terebinth plant did not show any antimicrobial activity against the microorganisms used. Chemical variability may be due to genetic or environmental factors dependent on geographical and edaphoclimatic conditions, leading to the occurrence of different chemotypes as infraspecific chemical races (Llorens-Molina and Vacas 2015).
This study demonstrates the antimicrobial effects of methanol extract samples in ripe fruits of terebinth plant against Escherichia coli and Staphylococcus aureus. Of these, methicillin-resistant Staphylococcus aureus is known to be the cause of hospital-acquired infections and the main factor in community-acquired infections. This feature has been understood as a result of the emergence and increase in the frequency of infections acquired from the community.
Hospital infections are difficult to treat, expensive, and can involve drug-resistant microorganisms (Öztürk 2008). Therefore, it is not easy to treat a nosocomial infection, and antibiotics that counteract this infection have not been strong in the face of a nosocomial infection. The extract of the Pistacia terebinthus plant inhibited Staphylococcus aureus bacteria causing this infection with a zone diameter of 14 mm. For this reason, Akpulat et al. stated that Pistacia terebinthus plant can be used in the treatment of hospital infection by conducting necessary studies on its galls (Akpulat et al. 2021). The results of the essential oil samples showing antimicrobial activity are given in Table 4.
Table 4. Antimicrobial Activity of Crude Extract of P. terebinthus Ripe Fruits
*expressed as inhibition zone in mm
GC-MS/FID Conditions for Essential Oil Analysis
After analysis of essential oils by GC-MS/FID methods, the structure of a total of 98 components was detected in the unripe fruits of P. terebinthus, but the structure of 4 components could not be identified. The highest percentages of essential oils isolated from unripe fruits were α-pinene (22.81%), α-terpinolene (20.79%), and cis-β-ocimene (9.41%). The most abundant parent compound in unripe fruit samples is α-pinene. In the essential oil of the ripe fruits of terebinth plants, the structure of a total of 71 compounds was elucidated. The highest α-pinene (27.27%), α-terpinolene (17.11%), and trans-β-ocimene (10.73%) were the main compounds in ripe fruit samples. It is understood that the most common main compound in ripe fruit samples is α-pinene. It is seen that the main constituents of the essential oil in the ripe and unripe fruits are the same. Because of its antioxidant properties, α-pinene and β-pinene are widely used in the pharmaceutical industry, as well as in the food, perfumery and cosmetic industries (Kılıç Pekgözlü and Ceylan 2021). Puvača et al. (2019) reported that bioactive compounds such as α-terpinene, α-terpinolene and γ-terpinene showed high antioxidant activity. Terpinolene is also used as a synthetic flavoring additive and scent enhancer (Okumura et al. 2012).
α-pinene (51.3%), caryophyllene oxide (51.0%), p-cymen-8-ol (40.0%), and limonene (39.0%) were the main constituents for distinct places in Türkiye. The main compounds in the examples were caryophyllene oxide, α-pinene, p-cymen-8-ol, limonene, sabinene. Twenty-eight constituents representing 92.3 to 100.0% of the oils were defined (Özcan et al. 2009). α-pinene (26.31%) was dominant in the volatile oil, cis (9.34 ± 0.03%) and trans (15.88 ± 0.13%) isomers of β-ocimene as well as D,L-limonene (14.06 ± 0.21%) were other main compounds in the volatile oil (Orhan et al. 2012). Young ripe and unripe fruit oils resulted in the determination of 48 and 51 constituents, respectively. Limonene, β-pinene, α-phellandrene, and α-pinene were identified as the major compounds of ripe fruit oil. α-Pinene, β-pinene, limonene, α-phellandrene, and terpinolene were the important constituents of unripe fruit oil. β-Pinene and limonene were determined as the most abundant major components of ripe and unripe fruit oils (Couladis et al. 2003). When the results are examined, it can be emphasized that the plant analyzed in this study is a new chemotype of P. terebinthus due to the main component differences.
Previous studies realized the fruit volatile oil of P. terebinthus underlined the existence of some variations between the authors’ findings and those studies. For example, limonene was the main constituent in unripe (34.2%) and ripe (32.8%) fruits of P. terebinthus of Türkiye origin (Couladis et al. 2003), whereas α-pinene was the major constituent in unripe (22.8%) and ripe (27.3%) in the current study. In another study, the fruits of P. terebinthus included α-pinene (26.31%) as the major constituent, which is more similar to the authors’ results (Orhan et al. 2012). The fruitful twigs of terebinth plants collected from Sardinia included α-pinene (54.8%) as the major constituent, which is more analogous to the current study’s outcomes (Usai et al. 2006).
Certainly, remarkable variations in the oil constituents might be possibly resulting from climate and locality differences even within the same country, which was also supported by a work studying the effect of locality on constituents, the method of distillation, and oil yields of P. terebinthus (Özcan et al. 2009). Some variations may be due to the different handling and climatological factors. The volatile oil constituent also varies quantitatively and/or qualitatively with ripening and collection times (Couladis et al. 2003). GC/MS-GC/FID analysis outcomes of essential oils attained from ripe and unripe fruits of P. terebinthus are shown in Table 5.
Table 5. Percentage Component of Essential Oils from Ripe and Unripe Fruits of P. terebinthus