Thymol screening, phenolic contents, antioxidant and antibacterial activities of Iranian populations of Trachyspermum ammi (L.) Sprague (Apiaceae)

Essential oils yield and composition

Among the 14 seed sample populations collected, the content of EOs among populations ranged from 3.16 to 5% (v/w). The lowest and highest EO content was determined in Ghayen (P2) and Fars (P8) populations, respectively (Table 1). Similarly, the percentage of EO in ajwain samples has been reported from Pakistan 3.5–5.2%³¹, India 2–4%^4,32, and Iran 2–6%^5,33,34,35. EO yield may vary in plants depending on species, quality (chemotype of the plant), condition (fresh or dry), the layout of plant material (e.g., leaf/stem ratio), harvest time, and also extraction method^15,16,36. The EO yield is an important quality factor to bring medicinal plants to the pharmaceutical, and food industries. Seed EO constituents of the 14 ajwain populations and chromatograms are shown in Table 1 and Fig. S1. In this study, eleven constituents were identified in all 14 populations, and thymol was the major constituent ranging from 59.92 to 96.4 percent (Fig. S2). Other major constituents were p-cymene (0.55–21.15%), γ-terpinene (0.23–17.78%), and carvacrol (0.41–2.77%) among populations studied. The highest content of thymol (96.4%) and its structural isomer carvacrol (2.77%) were found in the Ghayen population (P2). Additionally, the lowest thymol content was detected in the Isfahan population (P13) (59.92%). The highest (17.78%) and lowest (0.23%) γ-terpinene content was found in the Isfahan (P13) and Ghayen (P2) populations, respectively. The Birjand population (P3) displayed the highest p-cymene content (21.15%) and (P2) showed the lowest content (0.55%).

The GC–MS spectra obtained from the Hamedan population (P7) are represented in the graphical diagram in Fig. 1. According to our results, the Ghayen population (P2) has the highest levels of thymol and carvacrol and lowest levels of p-cymene and γ-terpinene. So, a higher rate of precursors (γ-terpinene and p-cymene) to final products (thymol/carvacrol) can be converted in isolated EO³⁵. According to the biosynthetic pathway, γ-terpinene precursor converts to thymol and carvacrol during the developmental stages³⁷.

In this context, EO compositions of ajwain have been reported from various geographical areas. According to the chemical composition of ajwain oils, major constituents of thymol, γ-terpinene, and p-cymene^11,12,33,35 carvone, limonene, and dillapiole¹³ and carvacrol and p-cymene¹⁴ have been documented. Up to now, the high-thymol content populations from Iran were between 34 to 55%³³ 48.8 to 61.4³⁵, and 65.4¹¹. However, no chemotype of the plant EO has been reported with a very high percentage of thymol (> 90%). Thymol and carvacrol percentages of seed EO of 14 populations are shown in Fig. 2. As can be seen in this figure, populations P2 and P8 have the highest thymol content (more than 90% of EO). The presence of a high percentage of thymol in the P8 and P2 can be industrially valuable. Chemotypes are named based on the main constituents in EO within single botanical species³⁸. Normally ajwain oils on the market are those rich in thymol and/or carvacrol with strong antibacterial properties and high antioxidant potential. High purity thymol is interested in the market and will not have the subsequent purification costs. Therefore, chemotypes P2 and P8 with a high percentage of thymol 96.4. 90.57% can be significant respectively.

Estimation of phyto-constituents of extract

Significant differences were obtained among the population for total phenolic (TPC), total flavonoid (TFC), and total coumarin contents (TCC) (P ≤ 0.01) (Table 2). Natural phenolic compounds are including simple phenolics, phenolic acids, flavonoids, coumarins, tannins, stilbenes, curcuminoids, lignans, quinones, and others³⁹. Phenolic compounds and flavonoids are major bioactive components in medicinal plants and thus can comprise an essential part of the human diet⁴⁰. The present study assessed the total phenolic, flavonoid, and coumarin contents of ajwain populations, and the results are presented in Fig. 3A–C. Up to now, no studies have reported total phenol, flavonoid, and coumarin contents of Iranian ajwain populations.

Total phenol content (TPC)

The total phenolic content in the evaluated extracts varied from 26.91 (P13) in the Isfahan population to 43.20 (P2) mg GAE/g DW in the Ghayen population, Results demonstrated that TPC in the populations varied as the following the order P2 > P10 > P8 > P1 > P11 > P14 > P6, P9 > P3, P5 > P4 > P7 > P12 > P13 (Fig. 3A). In the few evaluable sources, the total phenolic content of ajwain seeds extracted with CHCl₃: MeOH (1: 2) solvent was 69 mg/g DW⁴¹. In the present study, the highest phenol content (43.2 mg GAE/g DW) was recorded in the P2 population. The difference in TPC with the available report may be due to genetic diversity and differences in extraction methods. According to the presence of apolar thymol in the seed structure, a combination of polar and non-polar solvents to extract compounds may optimize the extraction performance. Various environmental conditions in different places influence the content and metabolic profile of phenolic compounds in plant populations. It seems that high temperature and high UV radiation levels, and differences in genotypes are the reasons why the Isfahan population has a high content of TPC^15,16.

Total flavonoid content (TFC)

Analysis of variance showed a significant difference in TFC content at levels P ≤ 0.01. The total flavonoid contents ranged from 4.45 (P7) in the Hamedan population to 8.03 (P8) mg QE/g DW in the Fars population. P6 and P10 with 7.38 mg QE/g DW were also among the high content TFC populations (Fig. 3B). It seems that the reason for the lack of total flavonoids in Hamedan is due genetic differences and the low temperature of this region compared to other regions. Also, the reason for the high level of flavonoids in the Fars population may be due to genetic differences and high temperatures during the growing period. It has been reported that seeds and spurts of ajwain contain 0.58 and 1.15 mg/ g FW of TFC respectively⁴². Also, TFC of methanolic extract of Anethum graveolens L. (dill) seeds from the Apiaceae family have been reported to be 5.07 (mg QE /g)⁴³. Flavonoid accumulation with many protective roles may be influenced by the combination of genetics (i.e., adaptation to local conditions) and environmental effects (i.e., phenotypic plasticity)^44,45. Flavonoid accumulation rates among geographically different ajwain populations concerning climate can be correlated positively with temperature and UV-B radiation and negatively with precipitation (Chalker-Scott, 1999; Koski and Ashman, 2015).

Total coumarin content (TCC)

The TCC content of the T. ammi populations examined ranges from 0.079 (P12) to 0.26 (P1) mg coumarin equivalent to dry weight. The highest coumarin content was obtained from the methanolic extract of Kalat (P1) (0.260 mg CE/g DW) and the lowest value of coumarin was recorded for the population of Ardabil (Fig. 3C). Seed coumarin levels in populations can result from genetic and environmental differences. It seems that coumarin accumulation is decreased due to the coolness condition in Ardabil city during the seed maturation stage. Ajwain is a coumarin-rich source of coumarins (umbelliferone, scopoletin, xanthotoxin, bergapten) mostly found in its sprouts⁴⁶. However, no literature source was found to report the amount of total coumarin in ajwain seeds. These compounds have valuable medicinal properties, including edema reduction and possible anticancer activity⁴⁷ Furthermore, they are widely used as a flavoring in foods and pastries. Human exposure to coumarin from the diet has been calculated to be around 0.02 mg/kg/day and its maximum daily intake was estimated to be 0.07 mg/kg BW/day⁴⁸.

Free radical scavenging effects and antioxidant activity of essential oils and extracts

The antioxidant activities of EOs and extracts were assessed using the DPPH, FRAP free-radical scavenging, and total antioxidant capacity (TAC) assays (Fig. 4A–C).

In the DPPH assay, the samples were capable to decrease the DPPH free radical to evaluate their in vitro antioxidant activity. Analysis of variance on DPPH IC50 showed a significant difference in antioxidant activity of EOs and extracts among populations (P < 0.01) (Table 2). The value of DPPH (IC 50) in the essential oil varied between (1.57–2.61 µg/ml). The highest rate was related to P13 and the lowest rate was related to P2 samples. Also, the DPPH IC50 in the extract was recorded in the range of 8.06 to 31.95 respectively in P2 and P7. The antioxidant effect of Ajwain EO compared to ascorbic acid has been previously reported. According to this source, an amount of 10 µg of essential oil compared to the same concentration of vitamin C had a DPPH free radical scavenging effect of 76.4 to 97.2%⁴⁹. DPPH radical scavenging activities of the methanolic extract of seeds of T. ammi in the range of 30 to 240 µg/ ml have been reported to be 65–80%. While with the same concentrations ascorbic scavenging activities of DPPH were in the range of 90 to 100%⁵⁰. In the comparison of IC50 of EOs and extracts with common antioxidants; ascorbic acid (AA), BHT, PG and rutin (RU), quercetin (QU), gallic acid (GA), and thymol (TH) used in the present study, the following result was obtained from the lowest to the highest. GA < EO (P2) < Qu, PG, EO (P8) < EOs (P9 < P3 < P1 < P10, P12 < P7 < P6 < P4, P11, P14 < P13) < AA < TH < RU < BHT < EXs (P2 < P8 < P10 < P11 < P6 < P3, P5 < P1 < P13 < P9, P14 < P4 < P12 < P7) (Fig. 4A). In the present study, several antioxidants were used to better comparison with essential oils and extracts. BHT, PG, TH, and GA were used as common synthetic antioxidants. The reason for using synthetic thymol was due to comparing it with high thymol content natural studied EO and extracts. Since the plant extract contained flavonoids and phenolic acids, it was tried to use natural phenolic and flavonoid antioxidants for comparison. Ascorbic acid was used in the present study because it is a known and applicable antioxidant. Previously the antioxidant activity of some extracts from the Apiaceae family has been reported. According to these reports, IC₅₀ of Heracleum persicum Desf., Prangos ferulacea (L.) Lindl, Chaerophyllum macropodum Boiss., Oliveria decumbens extracts were 438, 242, 623, 98.5, and 86.1 (µg/ml), respectively^37,51. Also based on obtained results, ajwain seed with notable essential oil and extract can be introduced as the new promising antioxidant source from the Apiaceae family.

In the present study, FRAP was used as another method to evaluate antioxidant activity. Based on the results of the analysis of variance, a significant difference was obtained among the population’s EOs and extracts (Table 2). In the EO samples, the highest reducing power was obtained in population 2 (P2), (10.31 mM Fe⁺²) and the lowest was obtained in P13 (6.23 mM Fe⁺²) (Fig. 4B). The reducing power of the extracts was obtained in the range of 1.96 to 3.68 mM Fe⁺², in which the lowest was related to P13 and the highest to P2. Also, the ferric reducing power of the samples of essential oils, extracts, and standards used in this study were as follows. GA > PG > QU, AA > BHT > TH > EO [P2 > P8) > RU > P5 > P1 > P12 > P14 ≥ P4 ≥ P3 > P9 > P6 > P7 > P13] > EX [P2 > P10 > P8 > P1, P6, P11 > P9 ≥ P14, P3 ≥ P5 > P12 > P4 > P13]. The mechanism by which extracts and EO reduce the [Fe (TPTZ)₂]³⁺ complex to the ferrous state (Fe²⁺) usually involves the donation of electrons in the form of hydrogen ions and has been related to the in vitro antioxidant activity⁵².

The phosphomolybdenum assay is a quantitative method to evaluate the total antioxidant capacity. Significant differences were obtained in the EO samples as well as the extract samples among the populations (P < 0.01) (Table 2). The values ranged from 8.30 (P9) to 16.61 (P2) (mM AAE/mg EO) in EO samples and from 1.84 (P13) to 4.59 (P11) (µM AAE/g DW) in samples of extracts (Fig. 4C). Results demonstrated that ajwain seeds had notable total antioxidant capacity. The TAC value among antioxidant standards ranged from 11.4 to 34.08 in the following order: AA > GA > PG > TH > QU > BHT > RU. Also, this value ranged from 8.3 to 16.6 among EO samples with the highest value in P2. TCA values in extracts were recorded in the range of 1.83–4.59 with the highest value obtained in P11. Other detailed information is shown in Fig. 4C.

Antibacterial activity

The antibacterial activity of ajwain EOs was evaluated against two antibiotic resistance bacteria and their ability was compared with Cefixime as a standard. In the present study, we tried to use both gram-positive bacteria and gram-negative bacteria as samples. Staphylococcus aureus is a gram-positive pathogenic and antibiotic-resistant bacteria. It is also one of the most common causes of nosocomial infections. Also, Escherichia coli is available and inexpensive, and easily cultured in the laboratory. It is one of the most common causes of urinary tract infections. Gram-negative bacteria are also resistant to antibiotics and are an important species in the field of microbiology. One of the main problems in the field of microbiology is the resistance of microbes to antibiotics and so introducing new antibiotics is necessary⁵³. The reasons for using Cefixime in the present study are due to its widely used, great therapeutic power, and effectiveness against a wide range of microbes.

In this study, EOs exhibited bacteriostatic activities against S. aureus (0.06–64 µg/mL) and E. coli (1–64 µg/mL) (Table 3). High thymol content EO (P2) showed high antibacterial activity (MIC = 0.06 µg/mL) against S. aureus. Also, the EO from the Isfahan population (P13) showed the lowest antibacterial activity with the highest MIC value (64 µg/mL). In the present study, the mean MIC was not significantly different on gram-negative and positive bacteria, and populations with high thymol had a high antibacterial ability, indicating the antibacterial effects of thymol. Some researchers have evaluated the antimicrobial activity of ajwain oil^14,54,55. Thymol and carvacrol were found to be more effective in killing bacteria^3,4,5,6,7,9. The antibacterial properties of natural products, such as essential oils and their components, are widely explored by both industrial and academic fields⁵⁶. The antibacterial activity of the EOs is dependent on the composition and concentration, type, and dose of the target microorganism⁵⁷. The high antibacterial potential of cumin essential oil compared to Ferula essential oil has already been identified due to the high ratio of phenolic monoterpene compounds to other monoterpenes⁵⁸. It seems that the antibacterial effects of C. copticum are also mainly due to the presence of phenolic monoterpenes such as thymol, carvacrol, p-cymene, and γ-terpinene. Therefore, ajwain EO can be used as a natural agent with antibacterial properties in the food industry and the treatment of infectious diseases, especially antibiotic-resistant strains.

Hierarchical cluster analysis (HCA) of essential oil constituents

HCA was performed by using the 11 identified compounds and 14 populations (Fig. 5A). All used populations were divided into two clusters; Cluster I included P4, P6, P7, P10, P11, P12, P13, and P14 and cluster II consist of P1, P2, P5, P8, and P9 samples. In cluster I the major constituents were thymol (59.92–72.86), p-cymene (15.66–21.15), and γ-terpinene (10.22–17.78). In the second cluster thymol (80.09–96.4) and carvacrol (0.5–2.77) were the major constituents. Cluster analysis can classify studied populations into several groups, according to the chemical composition by ‘magnifying’ their similarities⁵⁹. Forasmuch as, plant sources from environmentally different origins led to the emergence of new chemotypes to baring domestication and cultivation to obtain uniform chemical plants along with appropriate agricultural features⁶⁰.

Principal component analysis (PCA)

Principal component analysis (PCA) is one of the multivariate statistical techniques used to explain differentiation between populations and to obtain more information on the variables that mainly influence the population’s similarities and differences⁶¹. The PCA was performed to identify the most significant variables in the data set (Fig. 5B). The same data set (14 population × 11 components) was used in this section. The PCA showed two components with explain 83.3% of the total variance. The first principal component (PC1) had the most portion of variance (74.5%) which was given by compounds such as γ-Terpinene, α-pinene, α-Thujene, p-cymene, and limonene. The second component (PC2), explaining 8.8% of the total variance, consisted of compounds thymol, carvacrol, and 1, 8-cineol (Fig. 6). The results of PCA agreed with those of the cluster analysis the populations similarly were divided into two distinct groups including high thymol/carvacrol and high thymol/p-cymene/γ-terpinene groups (Fig. 5B). Heat map analyses were drowned to determine how constituents effect on clustering. Based on heat map analysis samples were well-classified.

Also, in the analysis of the principal factors (PCA) between all the evaluated traits in the populations, the first principal factor (PC1) showed 53.8% and the second principal factor (PC2) 14.7% of the variance. This analysis determined the principal component, correlation of traits, and their relationship with populations. Accordingly, traits with positive arrows show a positive correlation and two traits with non-directional arrows show a negative correlation. Accordingly, thymol and carvacrol have a high correlation with antioxidant properties and this property is correlated with populations of chemotype 1 (P1, P2, P5, P8, P9). Other relationships and details correlations are shown in Fig. 5C, D.

Correlation

Simple correlation estimated the relationship between variables. Simple correlations between 24 studied traits in the present study are shown in Fig. 6. Thymol as the major constituent of EOs showed a high positive correlation with TPC (0.71), carvacrol (0.64), FRAP EO (0.85), and FRAP ext. (0.66). Thymol also had a significant negative correlation with Mic EO (-0.74), Mic Sa (-0.69), α-Thujene (-0.84), α-Pinene (-0.77), β-Pinene (-0.75), β-Myrcene (-0.9), α-Terpinene (-0.85), p-Cymene (-0.98), Limonene (-0.89), γ-Terpinene (-0.97). TPC had a positive correlation with TFC, thymol, carvacrol, FRAP Ext., TAC Ext., and a significant negative correlation with DPPH Ext. The antioxidant methods in extracts DPPH50 vs FRAP (-0.8), DPPH50 vs TAC (-0.67) and FRAP vs TAC (0.59) were highly correlated. Similarly, in estimating the antioxidant activity of essential oil DPPH50 vs FRAP (-0.79), DPPH50 vs TAC (-0.48), and FRAP vs TAC Ext (0.55) were highly correlated. Also, the high correlation of all antioxidant methods with thymol can explain its positive effect on the antioxidant activity of the extracts and EOs. The correlations found between each of the traits can be very important in breeding programs.

Source: Ecology - nature.com