Antimicrobial and antioxidant study of combined essential oils of Anethum Sowa Kurz. and Trachyspermum ammi (L.) along with quality determination, comparative histo-anatomical features, GC‒MS and HPTLC chemometrics | Scientific Reports

Scientific Reports volume 14, Article number: 27010 (2024) Cite this article

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Spices played crucial and variable roles in traditions, culture, history, religious ceremonials and festivals along with fetching food flavor and microbial protection globally due to presence of structurally unique and multi-natured chemotypes. Their existence in dishes portrayed key roles in improving shelf life by regulating spoilage of cuisine with different synergistic mechanism. Histo-anatomically (A) sowa exhibited distinguished cellular attributes which created remarkable differences with T. ammi. HPTLC chemometrics of both fruits revealed several peaks for active metabolomics with unique isocratic combination of menstruum. GC-MS study of hydro-distillate exhibited presence of monoterpenic cyclic and aromatic hydrocarbons, alcoholic and ketonic groups along with phenolic derivative that covers majorly 90% of total metabolites. Combined essential oils (EOs 1 + 2) of both fruits showed excellent antimicrobial activity against various clinical pathogenic strains such as K. pneumoniae at 10 µL/mL, S. aureus at 2.5 µL/mL, E. coli and E. faecalis at 1.25 µL/mL, and MRSA and Bcereus at 0.625 µL/mL and (C) albicans at 0.312 µL/mL as the MIC. The antioxidant study of (EOs 1 + 2) with maximum inhibition percentage to DPPH assay was 84.02 ± 1.05 at 100 µg/mL, and minimal inhibition was 72.31 ± 0.63 at 5 µg/mL with IC50 value 4.69 ± 0.22 µg/mL, while ABTS assay extreme was 79.15 ± 2.14 µg/mL and minimal was 67 ± 1.34 with the IC50 value of 18.37 ± 0.15 µg/mL, in superoxide assay uppermost inhibition was 81.03 ± 0.27 µg/mL and lowest was at 65.16 ± 3.15 with the IC50 value 16.46 ± 0.54, and H2O2 radical scavenging activity, predominant value was 78.01 ± 0.47 and least was 64.1 ± 2.01 with IC50 15.58 ± 0.34. These comparative key diagnostic features and synergistic effect of multicomponent natural antimicrobials will provide profound intellect of ancient utility and further scientists to explore their multiple mechanistic modality and application in food and beverages industry.

Along with traditional, cultural, and ethnic values, spices are becoming more widely accessible because they have biotherapeutic effects due to presence of their essence1,2. These essence are reservoirs of remedies because they potentially mitigate microbes and have been explored in aromatherapy, cosmetics and food industries. They are also enticing global interest for capital generation3 and are better alternatives than synthetic drugs due to their low toxicity and high volatility4. Their easy access and affordability5, people around the globe have developed faith despite the tranquil approachability of allopathic drugs6. Thousands of ethnic groups worldwide are using herbal medicines7 for their illness because of their vital and safer therapeutic potential8,9.

Trachyspermum ammi L., Sprague, (Apiaceae) Ajwain10, has astonishing and miraculous pharmacological and biological properties for combating various disorders11. It has rich cultural heritage and been explored for its flavor in food and medicine in Asian and Gulf countries. Fruit’s decoction is employed for the treatment of indigestion, coughing, and other gastrointestinal disorders in the traditional Ayurveda, Unani, Siddha, Homeopathy and Yoga (AYUSH) system12. It has anticholinergic, antihistaminic, and analgesic properties13,14,15 and also inhibits gut-associated bacteria, enteropathogenic bacteria, multidrug-resistant microbes16,17, nematodes and human Ascaris lumbricoides18,19. Its EOs may be applied as preservatives for enhancing the safety of food items56.

Similarly, Anethum sowa Kurz. (Family Apiaceae) Dill, Sowa in Hindi, has a very long history of medicinal use since Ebbers papyrus (1500 BC), exhibited antioxidant, antimicrobial, anti-inflammatory, anti-Alzheimer, antitumor, antidiabetic, antiparasitic, and insecticidal properties20,21,22,23,24,25,26. It is used in Unani medicine for the treatment of colic, digestive problems and gripe water50. Analytical techniques such as HPTLC and GC-MS studies predicts intensity of their biological roles by assuring nature and concentration of chemotypes in fruits because their generation is depending on influence of geo-climatic stress and growing conditions27,28,29,30,31.

The aim of this study was to establish comparative quality standards and histo-anatomical characteristics of two fruits and evaluate the antimicrobial and antioxidant properties of combined essential oils (EO1 + 2) for the first time along with those of individual EOs. This study will provide a strong understanding of the key features for identification, the combined effect of metabolomics on the shelf life of food products and their applications in the future (graphical abstract).

Fruits, Trachyspermum ammi L., Sprague and Anethum sowa Kurz. were collected from the North Tamil Nadu region of southern India and taxonomically identified by Pharmacognosist at the Regional Research Institute of Unani Medicine (Drug Standardization Research Unit) Chennai, and their voucher specimens for T. ammi [13862] and A. sowa [12589] were preserved in the museum of the Department of RRIUM Chennai for future reference.

The essential chemicals and reagents used for the experiments included vanillin (Merck Life Sciences Private Limited, Mumbai), toluene and ethyl acetate (Merck, KGaA, Darmstadt, Germany), ethanol, L-Ascorbic acid, Gallic acid (Sigma‒Aldrich Steinheim, Germany), Formic acid (Kemika, Zagreb, Croatia), and dimethyl sulfoxide (DMSO, Merck, KGaA, Darmstadt, Germany).

The attributes of A. sowa and T. ammi fruits were explored in detail by performing macroscopical descriptions32. Slides of fruit samples were prepared according to standard histological inspection protocols33. A Nikon Eclipse Ci Microscope was used to obtain the photomicrographs. The tissues were separated by using Schultz’s maceration method or by boiling in 5% KOH34, and the various elements were measured by Stage and ocular micrometers35. The microscopic diagnostic properties of the powder were investigated by using standard reagents and a Nikon Eclipse Ci Microscope36.

Dried fruits (100 g each) were pulverized into a fine powder and subjected to Clevenger apparatus assembly for extraction with a heating mantle. Repeated cycles (3 h × 3) of heating at ≤ 100 °C yielded light yellow colors of 1.5 mL and 2 mL of volatile oils from Sowa and Ajwain fruits, respectively. The oils were transferred to glass vials and kept in the dark at 30 °C until further use79,80.

Bacterial and fungal strains, viz. Bacillus cereus (Clinical Cochin University), Staphylococcus aureus (ATCC-29213), methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis (Clinical Cochin University) (gram-positive), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC-700603) (gram-negative), and Candida albicans (Clinical Cochin University) were obtained from the Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India.

Nutrient agar medium (NA, Hi Media) and Sabouraud dextrose agar (SDA, Hi Media) were used for activity analysis via the well diffusion method. Precultured bacterial strains were transferred to autoclaved medium, 100 mm Petri plates (25–30 mL/plate), 10 mm in diameter and filled with 1 mL of essential oils in DMSO. The combined essential oils Sowa (EO1), Ajwain (EO2), and EO1 + 2 were tested at concentrations of 5, 10, 50, and 100 µL/mL, respectively along with the DMSO as control. The plates were then incubated at 37 °C for bacteria and 30 °C for fungi for 24 to 48 h. The size of the prevented zone, the growth of the organisms surrounding the well, was used to measure the antibacterial activity, which was repeated twice37.

The minimal inhibitory concentration was determined by using the broth microdilution susceptibility method in 96-well trays by following the standard protocol of the clinical laboratory with notable modifications (three replications) of the experiment. Samples with different concentrations of 0.312, 0.625, 1.25, 2.5, 5.0 and 10 µg/mL along with 95 µL of culture media and 5 µL of bacterial suspension were added and incubated at 37 °C. After 24–48 h of incubation, changes in color or improvements in opacity were observed38.

The same microdilution procedure was employed to determine the minimal lethal concentration for the bacterial or fungal strains. Following a 24-hour incubation period, bacteria were exposed to various concentrations of essential oils, and 5 µL of contents from each well, was placed on nutritional agar media and incubated for 24–48 h. The colony-forming units (CFU/mL) were counted after incubation. The minimal bactericidal concentration, MBC, or minimal fungicidal concentration, MFC, refers to the lowest concentration at which microorganism growth is successfully reduced by 99%38.

A standard stock solution of DPPH was prepared39 along with EOs in DMSO. Different concentrations of EO1 (A. Sowa), EO2 (T. Ammi) and an equal ratio of combined EOs (1 + 2) were added at 5, 10, 50 and 100 µL in DMSO, and the volumes were adjusted to 1000 µL. In a 96-well plate, 5 µL of sample and 195 µL of 0.01 M DPPH were added to each well and incubated in the dark for 20 min for completion of the reaction at 28 ± 1 °C. The absorption was recorded at 517 nm using the UV‒Vis spectrophotometer40. The ability of each fraction to eliminate free radicals was determined by comparing its absorption to that of a blank solution and percentage inhibition and was calculated using the following equation:

where A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of the test or standard sample.

The radicals were generated by amalgamation of two stock solutions of 7 mM ABTS and 2.4 mM potassium persulphate in the equal proportion and allowed to react in the dark for 12 h at 28 ± 1 °C. This stock solution was re-diluted in ethanol for absorbance to 0.70 ± 0.01 at wavelength 734. The diluted ABTS+ solution (3 mL) was reacted with 20 µL different concentration EO1, EO2 and EO (1 + 2) and measured absorbance at 734 nm using ethanol as blank. The scavenging activity of EOs with ABTS radical was expressed using a standard calibration curve constructed by plotting percentage inhibition against concentration of trolox. The results were reported as mM eq trolox/Kg sample81,82.

Equivalent strength of Potassium dihydrogen and phosphate sodium hydroxide (0.2 M) were prepared. In a 200 ml volumetric flask, 50 ml potassium dihydrogen phosphate solution was put and added 39.1 ml of sodium hydroxide solution and made up 200 ml with distilled water to prepare phosphate buffer (pH 7.4). Phosphate buffer solution, 50 ml of was added to equal amount of hydrogen peroxide to generate the free radicals and solution was kept aside at room temperature for 5 min to finish the reaction. Different strength of EO1, EO2 and EO (1 + 2) (100µL) in 10% DMSO were added to 0.6 ml hydrogen peroxide solution and the absorbance was estimated at 230 nm in a spectrophotometer against a blank solution containing phosphate buffer solution without hydrogen peroxide. The percentage of scavenging of H2O2 by EO1, EO2 and EO (1 + 2) was determines by using the following equation:

where A0 is the absorbance of the control and A1 is the absorbance in the presence of the extract and standard83,84.

The reducing Nitroblue Tetrazolium (NBT) to formazan dye, which may be detected at 560 nm. To make a final volume of 1.4 mL, 0.1 mL NBT (1 mg/mL) was added to a reaction mixture containing 100µL alkaline DMSO (1 mL DMSO containing, 5 mM NaOH in 0.1 mL water) and 100 µL of different strength of EO1, EO2 and EO (1 + 2) in 10% DMSO85.

Accurately 2 g of each fruit powders were extracted with 20 mL of ethanol and hexane in an ultrasonicator for 30 min in duplicate at 35 °C followed by filtration of extracts were made through Whatman’s No. 1 and 41 filter papers and transferred to sample in vials for further analysis.

Two microliters of the EOs in hexane extracts and 8 µL of the ethanol and hexane extracts of each fruit were applied to 20 cm × 10 cm silica gel 60 F254 HPTLC plates (1.05554.0007, Merck, Darmstadt, Germany) as an 8 mm band by using an automatic TLC sampler 4 (ATS4, CAMAG, Muttenz, Switzerland) with a solvent combination (toluene - ethyl acetate - formic acid, 8:2:0.1, v/v/v) for 20 min saturation in a vertical twin trough chamber and observed at 254 nm and 366 nm with a TLC Visualizer (Serial No. 708690714, CAMAG, Muttenz, Germany). The plate was derivatized with vanillin–sulfuric acid reagent, and scanning was performed at 254 nm and 366 nm UV with absorption and fluorescence modes, respectively (CAMAG TLC SCANNER 3). The retention factors (Rf) and % area were calculated by WinCats software41.

The analysis was carried out (Win CATS version 1.4.9.2001) under high-pressure conditions, and nitrogen was utilized as the carrier gas. Mercury and deuterium-tungsten lamps were utilized to measure the absorbance at 254 nm, 366 nm and 520 nm.

A fused silica column packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250 μm df) and a Clarus 680 GC were utilized in the study. The components were separated using helium gas as the carrier at a constant flow rate of 1 mL/min. During the chromatographic run, the injector temperature was maintained at 260 °C. The temperature was gradually increased from 60 °C to 300 °C, with a ramp of 10 °C each minute, and then maintained at 300 °C for 6 min. The parameters used in the mass detector were 240 °C ion source temperature, 70 eV ionization mode electron impact, 0.2 s scan time, and 0.1 s scan interval. The component spectra were cross-referenced with those from the GC‒MS NIST (2008) library, which contains spectral databases for recognized components37.

Characteristic morpho-anatomical differences have been established between two fruits for clear-cut refinements. The comparative results are described in Table 1; Fig. 1.

Comparative macro-morphology of Anethum sowa Kurz. and Trachyspermum ammi (L.) Sprague (A) Anethum sowa Kurz. (B,C) Trachyspermum ammi (L.) Sprague.

Each mericarp has a smooth commissural exterior and a rounded dorsal surface. The dorsal surface has five obvious secondary ridges, and the lateral ridges branch out like a wing; the commissural surface has two primary ridges and four hidden primary ridges; and the primary crests contain six cup-shaped vittae located below. The dorsal crests have four vittae, and the commissural surface has two. The vittae are lined with parenchymatous epithelial cells and extend from the base of the mericarp to the apex, close to the stylopodium. The vittae are multicellular and uniseriate and are filled with yellowish cellular contents and oil globules. The secondary crests have vascular tissues. The mericarp has a generally plano-convex shape, the epicarp is made up of a single layer of colorless cells with consistently well-pronounced cuticular striations, and the mesocarp is made up of several layers of thick-walled cells with vascular tissues and six vittae. The innermost layer of the mesocarp is a layer of yellowish brown cells with thick walls (usually lignified) and a few unclear pits; this layer is often found attached to the endocarp. The endocarp is composed of a layer of thick-walled polygonal parenchyma cells arranged in groups, with the long axes of adjacent groups roughly parallel to each other. The endosperm cells are packed with aleurone grains and oil globules. Microspherioidal crystals are abundant in the endosperm52,53 (Table 2; Fig. 2).

Comparative micro-morphology of Anethum sowa Kurz. and Trachyspermum ammi (L.) Sprague (A) Anethum sowa Kurz.; (B). Anethum sowa – a portion enlarged; (C) Trachyspermum ammi (L.) Sprague PR- Primary ridge; SR- Secondary ridge; EP- Epicarp; EPD – Epidermis; VT- Vascular tissues; VI – Vitae; MS- Mesocarp; EN- Endosperm.

The fruit transverse sheath (cremocarp) is made up of two hexagonal structures that are joined by carpophores. The epicarp is composed of one layer of obliquely elongated tubular cells, which is externally covered by thick striated cuticles at some locations. There are also thick-walled, unicellular trichomes as protrusions with a serrate wall, and there are a few anomocytic stomata. Mesocarp cells are moderately thick-walled, rectangular to polygonal, and tangentially elongated. Similar transverse credentials are also exhibited in Ayurvedic Pharmacopoeia of India86. Six large vittae; four on the dorsal side between the ridges and two on the commissural surface, five vascular bundles are located beneath each ridges. Presence of thick-walled cluster of cells with radial extensions, are constituting carpophores. Layers of closely packed, lengthy, elongated inner epidermal cells that are adhere to the seed coat to form endocarp. Existence of an unseen layer of compressed cells, extending up to 8 μm, follows a layer of slightly expanded cells are in testa. Aleuronic grains and oil globules are packed into thick-walled polygonal parenchymatous cells that make up the endosperm54,55. The differences are established in Table 2; Fig. 2.

Powder microscopic descriptions of both fruit samples are compared, and differences are observed. A. sowa, brown; fibrous; pigmented cells in surface view; parenchyma cells; elongated sclereid cell length 350 μm and breadth up to 6 μm; sclerenchyma fiber length up to 450 μm and breadth up to 8 μm; four vittae occur on the upper side, and two vittae occur on the lower side; endosperm with microspheroidal calcium oxalate crystals up to 7 μm; and spiral vessels up to 8 μm are observed. T. ammi has oil globules and clusters of endosperm cells; oily and grayish-brown in color; endosperm with microspheroidal crystals up to 7 μm; three vittae occur on the upper side, and three vittae on the lower side; epidermal cells with unicellular bulbous trichomes and stomata; sclerenchyma fiber length up to 350 μm and breadth up to 10 μm; and spiral vessels up to 8 μm in diameter55. The marked differences are described in Table 3; Figs. 3 and 4.

Powder microscopic studies of Anethum sowa Kurz. – (A) Inner layer of mesocarp; (B) Parenchyma cells; (C) Reticulate part of mesocarp; (D) Elongated Parenchyma cells; (E) Endosperm with microspheroidal crystals of ca. ox.; (F) Epidermal hairs; (G) Elongated sclereid; (H) Fibres; (I) Spiral Vessels.

Powder microscopic studies of Trachyspermum ammi (L.) Sprague (A) Epidermal cells with unicellular bulbous trichomes and stomata; (B) Endosperm; (C) Parenchyma cells with trichome base; (D) Endosperm with microspheroidal calcium oxalate Crystals; (E) Spiral vessels; (F) Tracheids; (G) Fibres; (H) Vittae.

A total of seven compounds, major monoterpene, 90% of the total metabolites, are identified from the hydrodistillates of both fruits via GC‒MS analysis. They are crucial for activity and utility, even though some level of variation is observed in their percentage due to the influence of some physical and environmental stresses. Anethum sowa essential oil contains high concentrations of D-limonene, carvone, apiol, α thujene, β phallendrene, thujyl alcohol and cyclohexane24,51,57, while T. ammi contains thymol, cymene, DLl-limonene, 1,8-cineole, γ-terpinene, p-cymene, and - pinene47,56,58,64. This analysis has been validated by many previous studies, and their structures and GC-MS chromatograms are presented in Fig. 5a and b respectively.

Key compounds of both fruits. (a) GC-MS chromatogram for A. sowa. (b) GC-MS chromatogram for T. ammi.

The antimicrobial activity of the essential oils of fruits EO1 and EO2 and (EO1 + 2) against six pathogenic bacterial strains and one fungal strain were tested. The presence and absence of inhibitory zones have been quantified by varying concentrations of the oils, which shows antimicrobial activity toward both gram-positive and gram-negative organisms51,56,59. Thymol is one of the key ingredient of EO responsible for antimicrobial activity44 and might be used as a safer plant-based preservative for enhancing quality and shelf life of food products in future as an aletrnative56. Antimicrobial effects of T. ammi is also reported due presence of carvacol and thymol. Some studies supporting that A.sowa extracts which posses good antibacterial activity towards many pathogenic bacteria at the concentration of 400 µg/mL87,88. In present study combined outcome of essential oil is observed as it exhibited excellent inhibition of all the bacterial and fungal strains in lieu of individual EOs. This is the first study of EOs combination and acquired ashtonishing results. Their results are described in Table 4 and and Fig. 6 bar diagram of antimicrobial activity against individual and combined essential oils is shown in Fig. 7a, b and c, respectively.

Imgaes of antimicrobial activity of essential oil in combination (1:1) of A.sowa and T.ammi.

(a) Essential oil 1 against various pathogens. (b) Essential oil 2 against various pathogens. (c) Essential oil 1 + 2 against various pathogens. (d) Minimal inhibitory concentration (MIC) of various pathogens.

The activity of the combined EOs toward C. albicans and MRSA is twofold greater than other bacterial species (Table 5) and observed results are also inconsistent with a previous study42. The MBC and MFC, 100 µL of wells with no visible growth are cultured on nutrient agar and SDA media after incubation43. A substantial array of antibiotics have been identified in spices and play a decisive role in treating infectious diseases. Among all thymol has exhibited very good activity against both C. albicans and MRSA and had identical effects on Candida spp44. Essential oil of T. ammi is very effective in eliminating Gram-positive bacteria than gram negative with the MIC value of 10,000 µg/mL while present study surpassing the previous one by proving their vast range of effectiveness towards gram positive, gram negative and fungal strains from 0.1 to 10 µL/mL89. In another study revealed that MIC is observed towards S. aureus and E. coli at 150–500 µg/mL concentration and they used high concentration of essential oil and the observed values also have very higher than present90. Ajwain oil outperformed the standard antimicrobial compound nystatin by 125 times, at a minimum inhibitory concentration of 500 µg/mL against Candida species is also supporting to our findings42. Combinations of oils are evaluated first time for potential synergistic antibacterial effects and their MIC ranges were seen in Fig. 7d; Table 5. The latest results indicated that the EOs, have a plethora of bioactive compounds that can also be used as precursors for the semi-synthesis of therapeutic drugs to alleviate future challenges.

Antioxidant protects cell damage and destruction by eliminating free radicals and many studies have investigated that essential oil of T. ammi (64.1 ± 0.8%) at 100 µg/mL45 and A. sowa (49.23 ± 3.04%) at 200 µg/mL46 also have eradication power individually. This is first study where combination of both essential oils in equal proportions (EO 1 + 2) and their antioxidant activity provides extraordinary results compared with those of the individual oils. The maximum inhibition percentage observed for the combined essential oils with DPPH assay is 84.02 ± 1.05 at 100 µg/mL, and the minimum inhibition percentage is 72.31 ± 0.63 at 5 µg/mL and IC50 value 4.69 ± 0.22 µg/mL, whereas in ABTS assay the maximum inhibition is observed as 79.15 ± 2.14 µg/mL the minimal inhibition 67 ± 1.34 and with the IC50 value of 18.37 ± 0.15 µg/mL. In superoxide assay the highest inhibition was expressed as 81.03 ± 0.27 µg/mL and lowest was exhibited as 65.16 ± 3.15 with the IC50 value of 16.46 ± 0.54 and in hydrogen peroxide radical scavenging activity predominant value was expressed as 78.01 ± 0.47 and minimal value was expressed as 64.1 ± 2.01 with the IC50 value of 15.58 ± 0.34 and results are shown in Fig. 8a, b, c, d. Based on these data, essential oils from these umbelliferous fruit can be introduced as a new alternative for unnatural antioxidants. These factors might be useful in safeguarding foodstuff quality, taste and color and allowing foodstuffs to remain edible over a longer period of time by preventing biological or microbial spoilage.

(a) DPPH radical scavenging. (b) ABTS radical scavenging. (c) H2 O2 radical scavenging. (d) Superoxide radical scavenging activity of EO1 + EO2.

Activity of spices is due to presence of diverse active molecules60. Their usage either as such, extract or aroma in various occasions have attained significant thrust61,62. However, stabilizing scientific knowledge by identifying and revealing adulterants and performing qualitative and quantitative validation are crucial step for ensuring the safety, and efficacy of them65. Their quality values with diverse metabolomics and their accountability with HPTLC analytical technique may be very vital for determination of merits63. Significant pattern of chemotypes of two fruits of T. ammi and A. sowa treated with toluene: ethyl acetate: formic acid (8:2:0.1, v/v/v) as mobile phase has been established at three different wavelengths, viz., UV-254, 366 and 520 nm for the first time for identification of genuine material. Chromatograms are shown in Fig. 9A and B for clear understanding for determination of origin of botanicals and active biomarkers, key Rf values are mentioned in Table 6. After derivatization, Fig. 9C, again shows the presence of metabolites with various functional groups in different color. Overall this study might be useful for enunciation of biochemical patterns to test a crude drug and for the identification of substandard, spurious and exhausted drugs.

(A) HPTLC Photo documentation of seeds of T.ammi and A.sowa in alcohol and hexane extract obtained at UV 254 nm. (B) HPTLC Photodocumentation seeds of T.ammi and A.sowa in alcohol and hexane extract obtained at UV 366 nm. (C) HPTLC Photodocumentation seeds of T.ammi and A.sowa in alcohol and hexane extract obtained at UV 366 nm.

Plant-based medicines are developing contemporarily with humanity since evolution along with their traditions and culture. Ability of these medicines are to provide nutrition, mitigation of foodborne microorganisms and spoiling of eatables66, gained substantial momentum during global sentience67 of artificial preservatives that triggering significant apprehension and stress, gastrointestinal disorders, disruption of the gut microbiota, allergic reactions, respiratory complications, formation of carcinogenic and toxicological radicals in the body68,69. In Egyptian, Chinese, and Indian traditional system spices have been used for centuries for food preservation and flavors70 but these blessed heritagic traditional medicine are needed quality validation for reliance and ascertaining therapeutic values71. Ajwain and Sowa along with many more fruits are used extensively in diet72 because they have naturally generated many potential antimicrobial chemotypes, utilized in food preservation73. They are extending the shelf life of foods by restricting infectious growth through their free radical scavenging mechanisms, bacteriostatic and bactericidal actions74, as explored in HPTLC and GC-MS analysis. These chemotypes including phenolic, alcoholic, aldehydic, ketonic, ether groups, and hydrocarbons have various nature and mode of actions75, while synthetic antibiotics have a particular mechanisms and microbes have developed resistance against them76. Therefore, an alternative with multiple pharmacological routes are needed to encounter these challenges for improved shelf life of eatables with health safety77.

Spices have great scope in traditional culture, food and beverages industries and aromatherapy due to the presence of crucially unmated structurally diverse and therapeutic metabolomics. They have evolved naturally in response to physical, chemical, and ecological stresses48,49. Due to their remedial potential, these plants have had a profound therapeutic legacy from herbal ancestry since ancient times for various ailments78. They are now grabbing recognition to traders and the general public and embroidering global market values. These abilities have established a legal distinction of their substantial opportunity. Comparative histo-anatomical, HPTLC chemometric and GC-MS studies will significantly explore the quality endorsement of genuine material for subsequent global acceptability. The combination of EOs that demonstrates outstanding antioxidant activity and excellent antimicrobial efficacy supports the idea that foods are medicinal and medicines are food. This study will provides a foundation for understanding synergistic effect of multicomponent natural antimicrobial evolution and reinforced paraphernalia for researchers and industrialists. Further this study will also establish a rationale for molecular mechanisms involved in, needed to explore the combined effects of EOs on emerging microbial challenges.

The datasets used during the current study available from the corresponding author on reasonable request.

Essential oils

High-Performance Thin-Layer Chromatography

Thin Layer Chromatography

Gas Chromatography‒Mass Spectrometry

Inhibitory concentration 50

Sabouraud Dextrose Agar

Minimal inhibitory concentration

Minimum bactericidal concentration

Minimum fungicidal concentratio

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Authors are very thankful to Deputy Director, RRIUM Chennai and DG, Central Council for Research in Unani Medicine New Delhi for providing research facilities.

Regional Research Institute of Unani Medicine, Royapuram, Chennai, 600013, India

S.A. Wasim Akram, Mary Shamya Arokiarajan, J. John Christopher, Mohammad Jameel, Mohd Saquib, Tirumala Santosh Kumar Saripally, Noman Anwar, Mohd Asif & Kabiruddin Ahmed K

Central Council for Research in Unani Medicine, Ministry of AYUSH, Govt of India, New Delhi, 110025, India

Mary Shamya Arokiarajan

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SAWA: Executed histo-anatomical study & draft writing. MSA: Performed antioxidant, antimicrobial, drafting, review & editing. JJC: HPTLC chemometric study and draft writting. MJ: Methodology, Project administration, Supervision, Validation, Visualization, and draft writing – review & editing. MS: Analysis of literatures, TSKS: Visualization, Formal analysis. NA: Formal analysis, Resources, Visualization. MA: Visualization, and draft editing, KKA: Project administration, Formal analysis. ZA-Formal analysis, RM-Visualization, Formal analysis.

Correspondence to Mohammad Jameel.

The authors declare no competing interests.

The experimental procedures were carried in vitro studies only. Ethical approval not applicable.

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Wasim Akram, S., Arokiarajan, M., Christopher, J. et al. Antimicrobial and antioxidant study of combined essential oils of Anethum Sowa Kurz. and Trachyspermum ammi (L.) along with quality determination, comparative histo-anatomical features, GC‒MS and HPTLC chemometrics. Sci Rep 14, 27010 (2024). https://doi.org/10.1038/s41598-024-75773-8

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Received: 14 June 2024

Accepted: 08 October 2024

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DOI: https://doi.org/10.1038/s41598-024-75773-8

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