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

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Important oils yield and composition

Among the many 14 seed pattern populations collected, the content material of EOs amongst populations ranged from 3.16 to five% (v/w). The bottom and highest EO content material was decided in Ghayen (P2) and Fars (P8) populations, respectively (Desk 1). Equally, the proportion 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 might fluctuate in vegetation relying on species, high quality (chemotype of the plant), situation (recent or dry), the structure of plant materials (e.g., leaf/stem ratio), harvest time, and likewise extraction technique15,16,36. The EO yield is a crucial high quality issue to carry medicinal vegetation to the pharmaceutical, and meals industries. Seed EO constituents of the 14 ajwain populations and chromatograms are proven in Desk 1 and Fig. S1. On this research, eleven constituents had been recognized in all 14 populations, and thymol was the key constituent starting from 59.92 to 96.4 p.c (Fig. S2). Different main constituents had been p-cymene (0.55–21.15%), γ-terpinene (0.23–17.78%), and carvacrol (0.41–2.77%) amongst populations studied. The very best content material of thymol (96.4%) and its structural isomer carvacrol (2.77%) had been discovered within the Ghayen inhabitants (P2). Moreover, the bottom thymol content material was detected within the Isfahan inhabitants (P13) (59.92%). The very best (17.78%) and lowest (0.23%) γ-terpinene content material was discovered within the Isfahan (P13) and Ghayen (P2) populations, respectively. The Birjand inhabitants (P3) displayed the best p-cymene content material (21.15%) and (P2) confirmed the bottom content material (0.55%).

Desk 1 The important oil composition of the fourteen Trachyspermum ammi populations.

The GC–MS spectra obtained from the Hamedan inhabitants (P7) are represented within the graphical diagram in Fig. 1. In keeping with our outcomes, the Ghayen inhabitants (P2) has the best ranges of thymol and carvacrol and lowest ranges of p-cymene and γ-terpinene. So, a better price of precursors (γ-terpinene and p-cymene) to remaining merchandise (thymol/carvacrol) will be transformed in remoted EO35. In keeping with the biosynthetic pathway, γ-terpinene precursor converts to thymol and carvacrol in the course of the developmental phases37.

Determine 1
figure 1

Characterize of graphical design of the current analysis.

On this context, EO compositions of ajwain have been reported from numerous geographical areas. In keeping with the chemical composition of ajwain oils, main constituents of thymol, γ-terpinene, and p-cymene11,12,33,35 carvone, limonene, and dillapiole13 and carvacrol and p-cymene14 have been documented. Thus far, the high-thymol content material populations from Iran had been between 34 to 55%33 48.8 to 61.435, and 65.411. Nevertheless, no chemotype of the plant EO has been reported with a really excessive share of thymol (> 90%). Thymol and carvacrol percentages of seed EO of 14 populations are proven in Fig. 2. As will be seen on this determine, populations P2 and P8 have the best thymol content material (greater than 90% of EO). The presence of a excessive share of thymol within the P8 and P2 will be industrially precious. Chemotypes are named based mostly on the primary constituents in EO inside single botanical species38. Usually ajwain oils available on the market are these wealthy in thymol and/or carvacrol with sturdy antibacterial properties and excessive antioxidant potential. Excessive purity thymol is available in the market and won’t have the following purification prices. Due to this fact, chemotypes P2 and P8 with a excessive share of thymol 96.4. 90.57% will be important respectively.

Determine 2
figure 2

Thymol + carvacrol (%) in EO in studied populations. Chemotype decided with hierarchical cluster evaluation (HCA).

Estimation of phyto-constituents of extract

Vital variations had been obtained among the many inhabitants for complete phenolic (TPC), complete flavonoid (TFC), and complete coumarin contents (TCC) (P ≤ 0.01) (Desk 2). Pure phenolic compounds are together with easy phenolics, phenolic acids, flavonoids, coumarins, tannins, stilbenes, curcuminoids, lignans, quinones, and others39. Phenolic compounds and flavonoids are main bioactive elements in medicinal vegetation and thus can comprise an important a part of the human weight loss plan40. The current research assessed the whole phenolic, flavonoid, and coumarin contents of ajwain populations, and the outcomes are offered in Fig. 3A–C. Thus far, no research have reported complete phenol, flavonoid, and coumarin contents of Iranian ajwain populations.

Desk 2 Evaluation of variance for 9 phytochemical traits in fourteen populations of Trachyspermum ammi.
Determine 3
figure 3

Phyto-constituents evaluation of seed samples of 14 studied populations of Trachyspermum ammi (A); complete phenolic content material (TPC) as mg Gallic acid/g DW equal. (B) Complete flavonoid content material (TFC) quantified based mostly on mg Quercetin/g DW. (C) Complete coumarin (mg Coumarin E/g DW).

Complete phenol content material (TPC)

The full phenolic content material within the evaluated extracts different from 26.91 (P13) within the Isfahan inhabitants to 43.20 (P2) mg GAE/g DW within the Ghayen inhabitants, Outcomes demonstrated that TPC within the populations different as the next the order P2 > P10 > P8 > P1 > P11 > P14 > P6, P9 > P3, P5 > P4 > P7 > P12 > P13 (Fig. 3A). Within the few evaluable sources, the whole phenolic content material of ajwain seeds extracted with CHCl3: MeOH (1: 2) solvent was 69 mg/g DW41. Within the current research, the best phenol content material (43.2 mg GAE/g DW) was recorded within the P2 inhabitants. The distinction in TPC with the out there report could also be attributable to genetic variety and variations in extraction strategies. In keeping with the presence of apolar thymol within the seed construction, a mix of polar and non-polar solvents to extract compounds might optimize the extraction efficiency. Numerous environmental circumstances in other places affect the content material and metabolic profile of phenolic compounds in plant populations. It appears that evidently excessive temperature and excessive UV radiation ranges, and variations in genotypes are the the explanation why the Isfahan inhabitants has a excessive content material of TPC15,16.

Complete flavonoid content material (TFC)

Evaluation of variance confirmed a big distinction in TFC content material at ranges P ≤ 0.01. The full flavonoid contents ranged from 4.45 (P7) within the Hamedan inhabitants to eight.03 (P8) mg QE/g DW within the Fars inhabitants. P6 and P10 with 7.38 mg QE/g DW had been additionally among the many excessive content material TFC populations (Fig. 3B). It appears that evidently the explanation for the dearth of complete flavonoids in Hamedan is due genetic variations and the low temperature of this area in comparison with different areas. Additionally, the explanation for the excessive stage of flavonoids within the Fars inhabitants could also be attributable to genetic variations and excessive temperatures in the course of the rising interval. It has been reported that seeds and spurts of ajwain include 0.58 and 1.15 mg/ g FW of TFC respectively42. Additionally, TFC of methanolic extract of Anethum graveolens L. (dill) seeds from the Apiaceae household have been reported to be 5.07 (mg QE /g)43. Flavonoid accumulation with many protecting roles could also be influenced by the mix of genetics (i.e., adaptation to native circumstances) and environmental results (i.e., phenotypic plasticity)44,45. Flavonoid accumulation charges amongst geographically totally different ajwain populations regarding local weather will be correlated positively with temperature and UV-B radiation and negatively with precipitation (Chalker-Scott, 1999; Koski and Ashman, 2015).

Complete coumarin content material (TCC)

The TCC content material of the T. ammi populations examined ranges from 0.079 (P12) to 0.26 (P1) mg coumarin equal to dry weight. The very best coumarin content material was obtained from the methanolic extract of Kalat (P1) (0.260 mg CE/g DW) and the bottom worth of coumarin was recorded for the inhabitants of Ardabil (Fig. 3C). Seed coumarin ranges in populations may result from genetic and environmental variations. It appears that evidently coumarin accumulation is decreased as a result of coolness situation in Ardabil metropolis in the course of the seed maturation stage. Ajwain is a coumarin-rich supply of coumarins (umbelliferone, scopoletin, xanthotoxin, bergapten) principally present in its sprouts46. Nevertheless, no literature supply was discovered to report the quantity of complete coumarin in ajwain seeds. These compounds have precious medicinal properties, together with edema discount and doable anticancer exercise47 Moreover, they’re extensively used as a flavoring in meals and pastries. Human publicity to coumarin from the weight loss plan has been calculated to be round 0.02 mg/kg/day and its most day by day consumption was estimated to be 0.07 mg/kg BW/day48.

Free radical scavenging results and antioxidant exercise of important oils and extracts

The antioxidant actions of EOs and extracts had been assessed utilizing the DPPH, FRAP free-radical scavenging, and complete antioxidant capability (TAC) assays (Fig. 4A–C).

Determine 4
figure 4

Antioxidant actions of methanolic extracts and important oils obtained from Trachyspermum ammi seed populations and 7 antioxidant requirements (A); Antioxidant exercise (DPPH) IC50 (µg/ml) (B); antioxidant exercise (FRAP) quantified by µmol Fe+2/g DW (C); complete antioxidant capability (TAC) quantified by mg Ascorbic acid equal (AAE).

Within the DPPH assay, the samples had been succesful to lower the DPPH free radical to guage their in vitro antioxidant exercise. Evaluation of variance on DPPH IC50 confirmed a big distinction in antioxidant exercise of EOs and extracts amongst populations (P < 0.01) (Desk 2). The worth of DPPH (IC 50) within the important oil different between (1.57–2.61 µg/ml). The very best price was associated to P13 and the bottom price was associated to P2 samples. Additionally, the DPPH IC50 within the extract was recorded within the vary of 8.06 to 31.95 respectively in P2 and P7. The antioxidant impact of Ajwain EO in comparison with ascorbic acid has been beforehand reported. In keeping with this supply, an quantity of 10 µg of important oil in comparison with the identical focus of vitamin C had a DPPH free radical scavenging impact of 76.4 to 97.2%49. DPPH radical scavenging actions of the methanolic extract of seeds of T. ammi within the vary of 30 to 240 µg/ ml have been reported to be 65–80%. Whereas with the identical concentrations ascorbic scavenging actions of DPPH had been within the vary of 90 to 100%50. Within the comparability of IC50 of EOs and extracts with frequent antioxidants; ascorbic acid (AA), BHT, PG and rutin (RU), quercetin (QU), gallic acid (GA), and thymol (TH) used within the current research, the next consequence was obtained from the bottom to the best. 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). Within the current research, a number of antioxidants had been used to raised comparability with important oils and extracts. BHT, PG, TH, and GA had been used as frequent artificial antioxidants. The explanation for utilizing artificial thymol was attributable to evaluating it with excessive thymol content material pure studied EO and extracts. Because the plant extract contained flavonoids and phenolic acids, it was tried to make use of pure phenolic and flavonoid antioxidants for comparability. Ascorbic acid was used within the current research as a result of it’s a identified and relevant antioxidant. Beforehand the antioxidant exercise of some extracts from the Apiaceae household has been reported. In keeping with these stories, IC50 of Heracleum persicum Desf., Prangos ferulacea (L.) Lindl, Chaerophyllum macropodum Boiss., Oliveria decumbens extracts had been 438, 242, 623, 98.5, and 86.1 (µg/ml), respectively37,51. Additionally based mostly on obtained outcomes, ajwain seed with notable important oil and extract will be launched as the brand new promising antioxidant supply from the Apiaceae household.

Within the current research, FRAP was used as one other technique to guage antioxidant exercise. Based mostly on the outcomes of the evaluation of variance, a big distinction was obtained among the many inhabitants’s EOs and extracts (Desk 2). Within the EO samples, the best decreasing energy was obtained in inhabitants 2 (P2), (10.31 mM Fe+2) and the bottom was obtained in P13 (6.23 mM Fe+2) (Fig. 4B). The decreasing energy of the extracts was obtained within the vary of 1.96 to three.68 mM Fe+2, by which the bottom was associated to P13 and the best to P2. Additionally, the ferric decreasing energy of the samples of important oils, extracts, and requirements used on this research had been 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 cut back the [Fe (TPTZ)2]3+ complicated to the ferrous state (Fe2+) normally entails the donation of electrons within the type of hydrogen ions and has been associated to the in vitro antioxidant exercise52.

The phosphomolybdenum assay is a quantitative technique to guage the whole antioxidant capability. Vital variations had been obtained within the EO samples in addition to the extract samples among the many populations (P < 0.01) (Desk 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). Outcomes demonstrated that ajwain seeds had notable complete antioxidant capability. The TAC worth amongst antioxidant requirements ranged from 11.4 to 34.08 within the following order: AA > GA > PG > TH > QU > BHT > RU. Additionally, this worth ranged from 8.3 to 16.6 amongst EO samples with the best worth in P2. TCA values in extracts had been recorded within the vary of 1.83–4.59 with the best worth obtained in P11. Different detailed data is proven in Fig. 4C.

Antibacterial exercise

The antibacterial exercise of ajwain EOs was evaluated towards two antibiotic resistance micro organism and their capacity was in contrast with Cefixime as a regular. Within the current research, we tried to make use of each gram-positive micro organism and gram-negative micro organism as samples. Staphylococcus aureus is a gram-positive pathogenic and antibiotic-resistant micro organism. It is usually one of the frequent causes of nosocomial infections. Additionally, Escherichia coli is offered and cheap, and simply cultured within the laboratory. It is among the most typical causes of urinary tract infections. Gram-negative micro organism are additionally proof against antibiotics and are an necessary species within the subject of microbiology. One of many essential issues within the subject of microbiology is the resistance of microbes to antibiotics and so introducing new antibiotics is important53. The explanations for utilizing Cefixime within the current research are attributable to its extensively used, nice therapeutic energy, and effectiveness towards a variety of microbes.

On this research, EOs exhibited bacteriostatic actions towards S. aureus (0.06–64 µg/mL) and E. coli (1–64 µg/mL) (Desk 3). Excessive thymol content material EO (P2) confirmed excessive antibacterial exercise (MIC = 0.06 µg/mL) towards S. aureus. Additionally, the EO from the Isfahan inhabitants (P13) confirmed the bottom antibacterial exercise with the best MIC worth (64 µg/mL). Within the current research, the imply MIC was not considerably totally different on gram-negative and constructive micro organism, and populations with excessive thymol had a excessive antibacterial capacity, indicating the antibacterial results of thymol. Some researchers have evaluated the antimicrobial exercise of ajwain oil14,54,55. Thymol and carvacrol had been discovered to be more practical in killing micro organism3,4,5,6,7,9. The antibacterial properties of pure merchandise, comparable to important oils and their elements, are extensively explored by each industrial and educational fields56. The antibacterial exercise of the EOs relies on the composition and focus, sort, and dose of the goal microorganism57. The excessive antibacterial potential of cumin important oil in comparison with Ferula important oil has already been recognized as a result of excessive ratio of phenolic monoterpene compounds to different monoterpenes58. It appears that evidently the antibacterial results of C. copticum are additionally primarily as a result of presence of phenolic monoterpenes comparable to thymol, carvacrol, p-cymene, and γ-terpinene. Due to this fact, ajwain EO can be utilized as a pure agent with antibacterial properties within the meals business and the therapy of infectious ailments, particularly antibiotic-resistant strains.

Desk 3 Minimal Inhibitory Concentrations (MIC) important oil Iranian 14 populations of Trachyspermum ammi towards Escherichia coli and Staphylococcus aureus.

Hierarchical cluster evaluation (HCA) of important oil constituents

HCA was carried out by utilizing the 11 recognized compounds and 14 populations (Fig. 5A). All used populations had been divided into two clusters; Cluster I included P4, P6, P7, P10, P11, P12, P13, and P14 and cluster II include P1, P2, P5, P8, and P9 samples. In cluster I the key constituents had been thymol (59.92–72.86), p-cymene (15.66–21.15), and γ-terpinene (10.22–17.78). Within the second cluster thymol (80.09–96.4) and carvacrol (0.5–2.77) had been the key constituents. Cluster evaluation can classify studied populations into a number of teams, in response to the chemical composition by ‘magnifying’ their similarities59. Forasmuch as, plant sources from environmentally totally different origins led to the emergence of recent chemotypes to baring domestication and cultivation to acquire uniform chemical vegetation together with applicable agricultural options60.

Determine 5
figure 5

(A) Warmth-map diagram of two-way hierarchical cluster evaluation (HCA) of fourteen Trachyspermum ammi populations based mostly on 11 important oil constituents quantified by GC and GC–MS. Blue colour with an awesome constructive share and purple colour with an awesome adverse share impacts cluster formation. (B) Principal element evaluation (PCA) based mostly on EO constituents. (C) PCA is predicated on all studied traits. (D) PCA is predicated on all studied traits in response to populations.

Principal element evaluation (PCA)

Principal element evaluation (PCA) is among the multivariate statistical methods used to clarify differentiation between populations and to acquire extra data on the variables that primarily affect the inhabitants’s similarities and variations61. The PCA was carried out to establish probably the most important variables within the information set (Fig. 5B). The identical information set (14 inhabitants × 11 elements) was used on this part. The PCA confirmed two elements with clarify 83.3% of the whole variance. The primary principal element (PC1) had probably the most portion of variance (74.5%) which was given by compounds comparable to γ-Terpinene, α-pinene, α-Thujene, p-cymene, and limonene. The second element (PC2), explaining 8.8% of the whole variance, consisted of compounds thymol, carvacrol, and 1, 8-cineol (Fig. 6). The outcomes of PCA agreed with these of the cluster evaluation the populations equally had been divided into two distinct teams together with excessive thymol/carvacrol and excessive thymol/p-cymene/γ-terpinene teams (Fig. 5B). Warmth map analyses had been drowned to find out how constituents impact on clustering. Based mostly on warmth map evaluation samples had been well-classified.

Determine 6
figure 6

Correlation between 24 traits on the studied Trachyspermum ammi populations: TPC: Complete phenolic content material, TFC: Complete flavonoid content material, TCC: Complete coumarin, EO: Important Oil yield, TSW: One thousand seed weight (g), MIC: minimal inhibitory focus, Ec: E. coli, MIC: minimal inhibitory focus, Sa: S. aureus, DPPH Ext.: DPPH assay Extract is expressed as IC50 index, DPPH EO: DPPH assay EO is expressed as IC50 index, FRAP Ext.: FRAP assay Extract, FRAP EO: FRAP assay Important oil, TAC Ext: The full antioxidant capability Extract, TAC EO: The full antioxidant capability Important oil.

Additionally, within the evaluation of the principal components (PCA) between all of the evaluated traits within the populations, the primary principal issue (PC1) confirmed 53.8% and the second principal issue (PC2) 14.7% of the variance. This evaluation decided the principal element, correlation of traits, and their relationship with populations. Accordingly, traits with constructive arrows present a constructive correlation and two traits with non-directional arrows present a adverse correlation. Accordingly, thymol and carvacrol have a excessive correlation with antioxidant properties and this property is correlated with populations of chemotype 1 (P1, P2, P5, P8, P9). Different relationships and particulars correlations are proven in Fig. 5C, D.

Correlation

Easy correlation estimated the connection between variables. Easy correlations between 24 studied traits within the current research are proven in Fig. 6. Thymol as the key constituent of EOs confirmed a excessive constructive correlation with TPC (0.71), carvacrol (0.64), FRAP EO (0.85), and FRAP ext. (0.66). Thymol additionally had a big adverse 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 constructive correlation with TFC, thymol, carvacrol, FRAP Ext., TAC Ext., and a big adverse correlation with DPPH Ext. The antioxidant strategies in extracts DPPH50 vs FRAP (-0.8), DPPH50 vs TAC (-0.67) and FRAP vs TAC (0.59) had been extremely correlated. Equally, in estimating the antioxidant exercise of important oil DPPH50 vs FRAP (-0.79), DPPH50 vs TAC (-0.48), and FRAP vs TAC Ext (0.55) had been extremely correlated. Additionally, the excessive correlation of all antioxidant strategies with thymol can clarify its constructive impact on the antioxidant exercise of the extracts and EOs. The correlations discovered between every of the traits will be crucial in breeding packages.

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