BIBECHANA Vol. 22, No. 1, April 2025, 15-21 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Chemical profiling, antioxidant, and antimicrobial activities of the essential oil of Matricaria recutita (Chamomile) Ram Prasad Baral1, Rameshwar Adhikari2, Achyut Adhikari1,∗ 1Central Department of Chemistry, Tribhuvan University, Kathmandu, Nepal 2Research Center Applied Science and Technology (RECAST), Kathmandu, Nepal ∗Corresponding author. Email: achyutraj05@gmail.com Abstract Matricaria recutita, commonly known as chamomile, is extensively utilized in the pharmaceu- tical, cosmetic, and food industries for its medicinal and essential oil properties. Local people from the Karnali Province of Nepal use this plant against skin and vaginal infection by bac- teria and fungi. This research focuses on extracting essential oil through hydro-distillation, followed by Gas Chromatography-Mass Spectroscopy (GC-MS) to identify its chemical con- stituents and antioxidant and antimicrobial activities against bacteria and fungi responsible for common skin infections. The GC-MS analysis of the essential oil from M. recutita identi- fied eleven chemical components. The major constituents, with respective area percentages at retention times 37.16, 11.79, and 7.88 in the GC chromatogram, were -farnesene (46.56%), - bisabolol oxide-A (11.79%), and menthol (7.88%). Limonene, methyl salicylate, and -bisabolol oxide-B also constituted the lowest area percentages at 1.36%, 1.59%, and 1.62%, respectively. The essential oil exhibited significant antioxidant activity with IC50 = 0.1924 µL/mL. Addi- tionally, the essential oil exhibited notable antibacterial activity against Staphylococcus aureus ATCC6538P and Candida albicans, displaying zones of inhibition measuring 10.64 mm and 14.44 mm, respectively. The broth method also revealed that the MIC is above the 250 mg/mL range and has more potential for inhibition. Keywords Matricaria recutita, essential oil, GC-MS analysis, antibacterial, antifungal, antioxidant, MIC and MBC . Article information Manuscript received: June 5, 2024; Revised: December 25, 2024; Accepted: January 2, 2025 DOI https://doi.org/10.3126/bibechana.v22i1.66431 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Nature has shown to be a promising source of ther- apeutic compounds, with many modern medica- tions derived from medicinal plants found in na- ture [1]. Herbal medicine has recently received much attention from scientists as a supplemental or replacement therapy [2]. The World Health Or- ganization (WHO) reports that herbal medicines are the primary source of medication for 70 – 80 % of the people in developing countries [3]. Among the broad assortment of plant products, essential 15 http://nepjol.info/index.php/BIBECHANA achyutraj05@gmail.com https://doi.org/10.3126/bibechana.v22i1.66431 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Ram Prasad Baral et al./ BIBECHANA 22 (2025) 15-21 16 oils (EO) have gotten much attention [4]. Es- sential oils are made up of highly volatile chemi- cals that are separated by a physical process. Es- sential oil combines oily fragrant volatile chem- icals such as monoterpenes, sesquiterpenes, aro- matic compounds, and their derivatives employed as antimicrobials, anti-inflammatory, sedatives, ex- pectorants, and diaphoretics, among other things [5]. German chamomile (Matricaria chamomilla) is a plant find in Western Europe, West Asia, India, and North America is a source of chamomile essen- tial oil, which has been used for its relaxing and restorative properties for a very long time [5]. It was historically used for anxiety and digestive prob- lems in traditional medicine in Europe and the Mid- dle East [6]. Because of its anti-inflammatory, an- tioxidant, and antibacterial qualities, a recent study emphasizes its potential in contemporary medicine and cosmetics [7]. Some of its constituents, such as chamazulene and bisabolol, are responsible for its medicinal properties [8]. Investigating its chemical makeup and its uses in medicine, cosmetics, and holistic well-being are the goals of this work [9–11]. It is the common name for various daisy-like plants in the Asteraceae family with yellow centers (about 1 - 1.5 cm in diameter) and white petals (be- tween 12 - 20 in number) [12]. The essential oils are extracted from the flowering tops. Chamomile has been widely used to treat wounds, skin irritation, burns, chickenpox, ear and eye infections, and nasal inflammation [9, 12, 13]; [14]. According to scien- tific analysis, extracted essential oils of chamomile were discovered to include - bisabolol oxide A and B and other flavonoids with anti-inflammatory and antiphlogistic activities [8]. These plants are most recognized for their calming properties when eaten as tea, frequently served with honey or lemon [6]. Local people from the Karnali Province of Nepal use this plant against skin and vaginal infection [15]. Much chemical and biological work has been re- ported in this plant but chemical and biological ac- tivities of plants from Karnali Province of Nepal have not been reported so far [?, 12, 13,16]. This research fulfills the above gap, and we have reported chemicals from the essential oil of chamomile, antibacterial and antifungal activity of microorganisms responsible for skin infection, and antioxidant activity. 2 Materials and Methods 2.1 Materials Plant Material: The German chamomile (Ma- tricaria recutita) plants were collected from the Surkhet district and the plant was identified in the National Plant Herbarium, Godawari, Lalitpur, and the collection number assigned to the herbarium (Collection No: 79). Chemicals: Anhydrous Na2SO4 and media for antibacterial activity test were bought from the lo- cal suppliers of Himedia, India, in Kathmandu. The DPPH and other chemicals used in GC were pur- chased from Merck. 2.2 Methods 2.2.1 Extraction of essential oil from chamomile Fresh flowers were collected and chopped into pieces smaller than 10 × 10 cm, then boiled in a Clevenger apparatus unit with 200 L of distilled water. After 5 – 6 hours, the oil distillation stopped and a cal- ibrated trap was used to determine the volume of essential oils [17, 18]. The distillate's essential oils were dried over anhydrous Na2SO4 and stored in the freezer. 2.3 Gas Chromatography-Mass spectrom- etry (GC-MS) analysis The GC-MS technique was used to identify chem- ical compounds present in the essential oil. The GC-MS was performed on a Shimadzu GCMS-QP 2010 plus equipped with a Rtx-5MS capillary col- umn with a column dimension of 60 m × 0.32 mm × 0.25 um. A capillary column that has a non- polar stationary phase (DB-5 or HP-5, f) was used for GC. The EO sample was diluted with hexane and 1 L of the essential oil sample was injected in split mode (split ratio 10:1), the injector tempera- ture was set at 250°C [10]. Helium gas was used as a carrier gas at a constant 4 mL/min flow rate. At first, the temperature was set at 50°C for two min- utes, then ramped up to 200°C at a pace of 10°C per minute, and then to 250°C at a rate of 5°C per minute, total run time was 70 minutes. To be- gin scanning, the detector's temperature was set at 280°C and mass range to m/z 40–400 [19]. The ion- ization mode of the mass was 70 eV electron impact (EI). 2.3.1 Antibacterial activities The Mueller-Hinton agar was used to prepare agar plates. Chamomile EO was tested against seven bacterial strains, utilizing the well-diffusion method. Overnight cultures of the respective bac- terial strains, obtained from the American Type Culture Collection (ATCC), were lawn cultured on Mueller Hinton Agar (MHA). Before swabbing, the indicator strains were standardized to a 0.5 McFar- land solution. Using a cork borer, wells of 5 mm di- ameter were created on the agar plates, into which 50 µL of chamomile essential oil (prepared in 50% Ram Prasad Baral et al./ BIBECHANA 22 (2025) 15-21 17 DMSO) was introduced, alongside positive (antibi- otic) and negative (DMSO) controls. The MHA plates were then incubated overnight at 37°C, and the subsequent zones of inhibition were measured the following day [20] 2.3.2 Antioxidant activity The DPPH assay was conducted following a version of a previously established protocol [5]. Specifically, 100L of chamomile essential oil at concentrations of (0.01, 0.005, 0.001, 0.0005, 0.0001, and 0.00005 g/mL) was mixed with 100L of DPPH solution (0.1mM) in methanol. The reaction mixture was thoroughly shaken and incubated in darkness for 30 minutes. Following incubation, the absorbance of triplicate readings was measured at 517 nm us- ing a microplate reader (Epoch TM 2 Microplate Spectrophotometer, Bio Tek Instruments, USA). S.activity % = (A sample−A control) A control × 100 where S. activity is Scavenging activity, A con- trol is the absorbance of the control (DPPH solu- tion without essential oil), and A sample is the ab- sorbance of the sample (essential oil with DPPH). The IC50 value, representing the concentration at which the essential oil scavenged 50% of DPPH radicals, was determined using Graph Pad Prism 8 software (Graph Pad Prism software, California, USA). 3 Results and Discussion 3.1 Gas Chromatography-Mass Spectrom- etry (GC-MS) analysis The essential oil was discovered as a light blue liquid with a pleasant, herbaceous aroma that smelt like straw. The components in the oil were examined using instrumental conditions set on the GC-MS and identified using direct mass spectral compari- son. Figure 1 shows chromatograms of chamomile extract with varied retention times. Figure 1: GC- chromatogram of essential oil extracted from chamomile (X-axis: time, Y-axis: abundance). The chromatogram abundance v/s retention time plot revealed 11 peaks, indicating the pres- ence of 11 phytoconstituents. Table 1 shows the chemicals discovered based on retention duration and area (%). The retention time was compared with the compounds present in the NIST library to identify the chemicals present in the essential oil. The Mass spectra of major compounds were com- pared with the mass spectra of compounds available in the NIST library. The principal components were discovered to include -farnesene (46.56 %), -bisabolol oxide-A (11.79 %), and menthol (7.88 %), as well as other minor components. The results revealed that es- sential oils isolated from Matricaria recutita include -Farnesene, -Bisabolol oxide-A, menthol, and nine others [20,21]. The following is the molecular structure of the critical components found in Matricaria recutita es- sential oils: Figure 2: Structure of major compounds of EO of Matricaria recutita Various compounds found in essential oil ex- tracts of Matricaria recutita from GC-MS analysis exhibit diverse pharmacological activities. - Farnesene, for instance, functions as an anti- carcinogenic, antibacterial, and anti-fungal agent while stimulating gastrointestinal tract receptors [21, 22]. -Bisabolol Oxide-A showcases anti- inflammatory, anti-irritant, antibacterial, and non- allergenic properties [22, 23]. Menthol, commonly used in ointments, cough drops, and nasal inhalers, is medicinal and acts as an anti-fungal agent [24]. Bisabolol is known for its anti-inflammatory and soothing properties, along with antimicrobial and antioxidant activities [22]. Chamazulene, another Ram Prasad Baral et al./ BIBECHANA 22 (2025) 15-21 18 compound, exhibits anti-inflammatory and antiox- idant properties, useful in reducing inflammation and oxidative stress [17, 22]. (-)--bisabolol also known for its antioxidant, anti-inflammatory, and anticancer properties, may also promote relaxation and reduce anxiety [25]. Luteolin, with strong antioxidant properties, is studied for neuroprotec- tion and reducing oxidative stress in the brain [26]. Lastly, Farnesene, a sesquiterpene hydrocar- bon, contributes to antimicrobial activity [27]. The bioactive compounds present in Matricaria recutita support its medicinal values and protect against various diseases. Thus, Matricaria recutita plants are considered valuable medicinal plants [20,28]. 3.2 Antioxidant activity Chamomile essential oil exhibited promising antiox- idant activity, as evidenced by its low IC50 value of 0.1924 µl/mL compared to the standard ascorbic acid, which had an IC50 of 0.0187 mg/mL. The in- hibitory percentage against the concentration graph is shown in Fig. 3. Figure 3: Antioxidant Activity of Chamomile (Ma- tricaria recutita) Essential Oil 3.2.1 Antibacterial and antifungal activi- ties The antibacterial efficacy of the isolated Chamomile (Matricaria recutita) essential oil was assessed against Staphylococcus aureus ATCC6538P, Pseu- domonas aeruginosa, Bacillus subtilis, Enterococcos faecalis, Protecus vulgaris, Shigella dysenteries, Es- cherichia coli, Klebsiella pneumonia, Staphylococ- cus epidermidis, and Salmonella typhi and exhib- ited noteworthy antibacterial activity for Staphylo- coccus aureus with a zone of inhibition measuring 14.44 mm, as compared to the positive control 27.00 mm, no significant activity was shown against other bacteria. Antifungal activity was tested for C. albi- cans and showed the zone of inhibition 10.64 mm, as compared to the positive control 21.11 mm (Ta- ble 2). Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC): The broth dilution method is a common approach to ascertain the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentra- tion (MBC) of antibiotics or other antimicrobial agents against specific bacterial strains. In this method, a range of concentrations of the antimi- crobial agent is tested against the bacteria to find the lowest concentration that inhibits visible growth (MIC) and the lowest concentration that kills the bacteria (MBC). In this particular study, the antibacterial activ- ity of a 100% essential oil was evaluated against Candida albicans and Staphylococcus aureus using the broth dilution method. Despite demonstrating antibacterial properties, the exact MIC of the essen- tial oil could not be determined. The essential oil exhibited significant inhibition at a concentration of 500 mg/mL against both Candida albicans and Staphylococcus aureus. However, when the concen- tration was reduced to 250 mg/mL, no significant inhibitory effect was observed. This suggests that the MIC for the essential oil is somewhere above 250 mg/mL, indicating that concentrations below this level are not effective for antibacterial activity. These findings are visually represented in Figure 4, which shows the difference in bacterial growth in- hibition at various concentrations of the essential oil. Figure 4: A) Minimum Inhibitory Concentration (MIC) and B) Minimum Bactericidal Concentra- tion (MBC) for Staphylococcus aureus and C) Min- imum Bactericidal Concentration (MBC) Candida albicans. Ram Prasad Baral et al./ BIBECHANA 22 (2025) 15-21 19 Chamomile essential oil's composition varies ac- cording to species, location, extraction techniques, collection time etc. In this research, we iden- tified the major components of chamomile es- sential oil, which include -Farnesene (46.56%), - Bisabolol oxide-A (11.79%), and menthol (7.88%). Compared to other research papers, our find- ings revealed a higher percentage of -Farnesene [29]. The obtained results from Stanojevic et al. (2016) showed the presence of 52 com- ponents, with the highest content of -farnesene (29.8%), -farnesene (9.3%), -bisabolol and its ox- ide (15.7%), chamazulene (6.4%), germacrene D (6.2%), and spiroether (5.6%) [30]. Additionally, the study by Kazemi (2015) reported the major compounds and their percentages in chamomile es- sential oil as follows: -bisabolol oxide (38%), cam- phene (9.11%), sabinene (4.87%), limonene (6%), 1,8-cineole (7.12%), camphor (6.54%), and -pinene (6%) [20]. Amiri et al. identified the major com- ponents in chamomile essential oil as -Bisabolol oxide A (17.14%), chamazulene (15.12%), En-in- dicycloether (6.22%), -Bisabolone oxide (6.15%), n- Octanal (6.00%), -Bisabolol oxide B (5.17%), 1,8- Cineole (3.86%), -Terpineol (3.11%), and Germa- crene D (3.02%) [21]. -Farnesene has shown an- timicrobial properties, which could lead to its use in developing new antimicrobial drugs or as an in- gredient in topical antimicrobial formulations [30]. The essential oil of chamomile from the Repub- lic of Srpska's northwest exhibits significant antioxi- dant and antibacterial properties when compared to previous studies. In a DPPH assay, the oil showed strong antioxidant activity after 90 minutes of in- cubation, with an IC50 value of 2.07 mg/ml [30]. Our studies show much better antioxidant activ- ity IC50 = 0.1924 µl/mL. The bacteria Staphylococ- cus aureus is responsible for skin infection and the fungi Candida ablicans for skin and vagina infec- tion, so inhibition of this bacteria and fungi by EO of chamomile validated the ethnomedicinal uses of this plant by local people. 4 Conclusion The essential oil of Matricaria recutita was suc- cessfully extracted using the steam distillation method. The Gas Chromatography-Mass Spectro- scopic (GC-MS) technique revealed the 11 different chemical constituents including -farnesene (46.56 %), -bisabolol oxide-A (11.79 %), and menthol (7.88 %) as major compounds. The EO of M. recu- tita showed significant antioxidant potential, with an IC50 value of 0.1924 µl/mL. Significant antimi- crobial activities against C. albicans and Staphy- lococcus aureus ATCC6538P were with inhibition zones measuring 10.64 mm and 14.44 mm, respec- tively. It was able to efficiently inhibit Candida albicans and Staphylococcus aureus at 500 mg/mL. 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Introduction Materials and Methods Materials Methods Extraction of essential oil from chamomile Gas Chromatography-Mass spectrometry (GC-MS) analysis Antibacterial activities Antioxidant activity Results and Discussion Gas Chromatography-Mass Spectrometry (GC-MS) analysis Antioxidant activity Antibacterial and antifungal activities Conclusion