New compound from the aerial parts of Achillea millefolium

ABSTRACT

Essential oils extracted by hydrodistillation from the aerial parts of Achillea millefolium was characterized by means of gas chromatography and nuclear magnetic resonance. A new compound, dihydro-a-cyclogeranyl hexanoate, along with 24 known were isolated from the essential oils A.millefolium. The new compound and borneol (known main component) were also subjected to antimicrobial, anti-inflammatory, and antioxidant activities. The new compound was particularly active against Escherichia coli, with the lowest minimum inhibitory concentration and minimum bactericidal/fungicidal concentration value. The new compound exhibited a higher activity in each antioxidant system with a special attention for β-carotene bleaching test, lipid peroxidation inhibition and reducing power. The new compound exhibited a potent nitric oxide scavenging effect and inhibited the expression of inducible nitric oxide synthase. These results indicated that dihydro-a-cyclogeranyl hexanoate might be applicable in natural medicine and healthy food.

KEYWORDS:

• New compound
• Essential oil
• Achillea millefolium
• NO scavenging

Introduction

Aromatic plants are potential natural sources of novel antibiotics and particular interest has focused on their essential oils (EOs) as main sources of potent antimicrobial and antifungal compounds classified as terpenoids, flavonoids, and phenolics.^[1,2] Biological activity of EOs depends on their chemical composition which is determined by the genotype and influenced by environmental and agronomic conditions. Aromatic and medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment, inhibiting bacterial or fungal growth.^[3] Indeed, natural crude extracts and biologically active compounds from plant species used in traditional medicine may represent valuable sources for such new preservatives.^[4] Application of plant materials as dietary regimens and preservatives is mainly due to their antioxidant, antimicrobial, and other biological potentials. Achillea, is one of the most important genera of the Compositae family. Achillea millefolium is one of the native species which grows wildly in different regions of Iran. This plant has been used as a medicinal herb for a long time and it now is an important drug used both in folk and official medicines.^[5] However, studies to support anti-inflammatory properties and lipid peroxidation inhibition of the volatile oil have not yet been reported. The objectives of this work were: To study the chemical composition and exploration of novel compounds from A.millefolium and to explore antimicrobial, anti-inflammatory, and antioxidant activity (AA).

Materials and methods

Plant materials and nano-Zn oxide treatments

The plant was identified by Mr. Esmaeili, and the voucher specimen was deposited at private herbarium of Dr, F. Esmaeili (voucher no. 158). Seeds of Achillea millefolium were sown in Jefe pot in experimental greenhouse, Iran (elevation 1339 m, latitude east 33.638, longitude north 46.431). Plants at two and four leave stage were sprayed with distilled water as a control and nano-Zn oxide at 2 and 4 mM. All sprays solution were sprayed to the point of run-off. The experiment was arranged in completely randomized block design with three replications for each treatment. The temperature conditions were 24 ± 5°C and 15 ± 4°C, during days and nights, respectively; with relative humidity of 70%. Aerial parts of A.millefolium were harvested and air-dried at ambient temperature in the shade.

General experimental procedures, isolation procedure of new compound

¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a 400 (100)-MHz Varian spectrometer, d in parts per million (ppm), using CDCl₃ as solvent and tetramethylsilane (TMS) as internal standard. High-performance liquid chromatography (HPLC) analysis was performed using a 250 mm × 20 mm, 5 µm, Kromasil 100-10-C₁₈ column on an Agilent 1260 HPLC system. Dried and powdered aerial parts of A.millefolium were hydrodistilled in a Clevenger-type apparatus for 3 h. The oil of A.millefolium was subjected to silica gel column chromatography (silica gel 60, 180 g, 70–230 mesh) using a solvent mixture n-hexane-ethyl acetate (95:5, 90:10, 85:15, 80:20, 75:25) to isolate dihydro-a-cyclogeranyl hexanoate. The resulting fractions were concentrated under reduced pressure and examined by Thin-layer chromatography (TLC) to offer three main fractions. Its chemical structure was determined by mass spectrometry (MS), ¹H NMR and ¹³C NMR spectroscopic methods and by comparing with previously reported spectral data^[6,7] as hexyl ester of dihydro-a-cyclogeranic acid.

Oil isolation, isolate borneol, and identification of known compounds

The aerial parts of A.millefolium were ground and the resulting powder was subjected to hydrodistillation for 3 h in an all glass Clevenger-type apparatus according to the method recommended by the European Pharmacopoeia.^[8] The obtained EOs were dried over anhydrous sodium sulphate and after filtration, stored at +4ºC until tested and analyzed. The gas chromatography (GC)/MS analyses were executed on a Hewlett–Packard 5973N gas chromatograph equipped with a column HP-5MS (30 m length × 0.25 mm i.d., film thickness 0.25 lm) coupled with a Hewlett-Packard 5973N mass spectrometer. The column temperature was programmed at 50ºC as an initial temperature, holding for 6 min, with 3ºC increases per minute to the temperature of 240ºC, followed by a temperature enhancement of 15ºC per min up to 300ºC, holding at the mentioned temperature for 3 min. Injector port temperature was 290ºC and helium used as carrier gas at a flow rate 1.5 mL/min. Ionization voltage of mass spectrometer in the EI-mode was equal to 70 eV and ionization source temperature was 250ºC. Linear retention indices for all components were determined by coinjection of the samples with a solution containing homologous series of C8-C22 n-alkanes and comparing them and their mass spectra with those of authentic samples or with available library data of the GC/MS system (WILEY 2001 data software) and Adams libraries spectra.^[9] The oil of A.millefolium was subjected to silica gel column chromatography (silica gel 60, 180 g, 70–230 mesh) using a solvent mixture n-hexane-ethyl acetate (95:5, 90:10, 85:15, 80:20, 75:25) to isolate borneol.

Total phenolic determination

Total phenolic contents in aerial parts of A.millefolium were determined by Folin–Ciocalteu method.^[10] The total phenolic content was expressed as gallic acid equivalents (GAE; mg g^_1).

Total flavonoid determination

Total flavonoid contents in aerial parts of A.millefolium were measured as previously described.^[11] The total flavonoid content was calculated as rutin equivalents (mg g⁻¹).

Microorganisms

Gram-positive bacteria: Bacillus cereus (ATCC 10876), Enterococcus faecalis (ATCC 49452), Staphylococcus aureus (ATCC 25923). Gram-negative bacteria: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 35659), Salmonella typhimurium (ATCC 13311), Citrobacter freundii (ATCC 8090). Fungal strains: Candida albicans (ATCC 10231) and Aspergillus fumigatus (ATCC 46645). The bacteria species were maintained in Mueller Hinton Agarand Tryptic Soy Agar. Strains of Candida spp. and Aspergillus spp. were maintained on Sabour and Dextrose Agar.

Antimicrobial activity

Minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC) concentrations were determined by microdilution method in 96-well microtitre plates, described by Douk et al.,^[12] and EUCAST.^[13] Briefly, fresh overnight cultures of bacteria were adjusted with sterile saline to a concentration of 1.0 × 10⁵ CFU per well, and 1.0 × 10⁴ CFU per well for fungi. EOs were added in Tryptic Soy Broth (TSB) medium for bacteria, and Sabouraud dextrose broth (SDB) medium for fungi. The microplates were incubated for 24 h at 37°C for bacteria, and 48 h at 37°C for fungi. The MIC was defined as the lowest concentration of EO inhibiting the visible growth of the test strain. However, the MIC/MBC values for bacteria and fungi were detected following the addition of 40 µL of piodonitrotetrazoliumviolet (INT) 0.2 mg/mL and incubation at 37°C for 30 min.^[14] The MBCs/MFCs were determined by serial subcultivations of 10 µL into microtiter plates containing 100 µL of broth per well and further incubation for 24 h at 37°C. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum. Following positive controls were used in both experiments: antibiotics (Streptomycin) and mycotic (Fluconazole). Each test was carried out in triplicates and repeated three times.

AA

The efficacy of the EOs to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was evaluated using a spectrophotometry method.^[15,16] On the basis of bleaching of the bluish-red or purple color of DPPH solution as a reagent. Briefly, a 50 µL volume of various dilutions of each samples was mixed with 5 mL of 0.004% methanol solutions of DPPH followed by 30 min incubation at ambient temperature. Thereafter, the sample absorbance was recorded against control at 517 nm. The inhibition percentages were measured using Eq. (1). The antioxidants activity of the test samples in concentration providing 50% inhibition, were considered as IC50 (µg/mL).

(1)

Butylhydroxyanisole (BHA) and ascorbic acid were used as positive controls. All experiments were repeated three times, the results were averaged, and standard deviations were calculated.

Rapid screening for antioxidants

For screening of antioxidant compounds in aerial parts of A.millefolium EO, the TLC-bioautography method was carried out.^[17,18] The diluted oil (1:20 in methanol) was spotted on silica gel sheets (silica gel 60 F254 TLC plates) and developed in n-hexane-ethyl acetate (9:1). Plates were sprayed with the methanolic solution of DPPH (0.2%). The active constituents were detected as yellow spots on a violet background. Only zones where their color turned from violet to yellow within the first 30 min (after spraying) were taken as positive results.

Activity guided fractionation of the EO for antioxidants

For the isolation and identification of the active compounds in the EO, TLC was performed using the conditions previously described.^[17] The regions showing DPPH scavenging activity were scrapped off then, they were eluted with chloroform. All resulting constituents were analyzed by GC/MS and also tested for their antioxidant activities.

β-carotene-linoleic acid model system (β-CLAMS)

The β-CLAMS method by the peroxides generated during the oxidation of linoleic acid at elevated temperature.^[19] The AA of the extracts was evaluated in term of β-carotene blanching using the following formula: AA (%) = [(A₀ – A₁)/A₀] × 100. where A₀ is the absorbance of the control at 0 min, and A₁ is the absorbance of the sample at 120 min. The results are expressed as IC₅₀ values (µg/mL). All samples were prepared and analyzed in triplicate.

Reducing power and lipid peroxidation inhibition

The ability of the extracts to reduce Fe³⁺ was assayed by the method of Oyaizu.^[20] One milliliter of aerial parts of A.millefolium EO and new component were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% K₃Fe (CN) 6. After incubation at 50°C for 25 min, 2.5 mL of 10% trichloroacetic acid was added and the mixture was centrifuged at 650 g for 10 min. Finally, 2.5 mL of the upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% aqueous FeCl₃. The absorbance was measured at 700 nm. The mean of absorbance values were plotted against concentration and a linear regression analysis was carried out. Increase absorbance of the reaction mixture indicated increased reducing power. EC₅₀ value (µg/mL) is the effective concentration at which the absorbance was 0.5 for reducing power. Ascorbic acid was used as positive control. Lipid peroxidation inhibition was determined by Shirwaikar et al.^[21] Ascorbic acid and Trolox was used for comparison.

Anti-inflammatory activity

To evaluate the anti-inflammatory potential of the oils, NO production in lipopolysaccharide (LPS)-stimulated macrophages was used. Exponentially growing macrophages (RAW 264.7 cells) were plated in 24-well microplates at a density of 2 × 10⁵ cells per well in 400 μL of culture medium and were allowed to adhere for 24 h at 37°C under 5% CO₂. Cells were then treated with increasing concentrations of EO and pure compounds dissolved in Dimethyl sulfoxide (DMSO). The final concentration of solvent in the culture medium was maintained at 0.5% (v/v) to avoid solvent toxicity. Cells were then stimulated with 100 μg/mL LPS and incubated at 37°C under 5% CO₂. After 24 h, cell-free supernatants were collected and NO was measured using the modified method of Green et al.^[22] Griess reagent (50 μL of 1% sulphanilamide and 50 μL of 0.1% N-1-naphtylethylenediamine dihydrochloride in 2.5% H₃PO₄) was added in equal volume (100 μL) to cell supernatant and incubated at room temperature for 30 min. N(G)-nitro-L-arginine methyl ester (L-NAME) was used as a positive control. Absorbance was measured using an ELISA automatic microplate reader at 550 nm and the nitrite concentration determined from a regression analysis prepared with serial dilutions of sodium nitrite.^[23]

Statistical analysis

The results are presented as mean±S.D and statistically analyzed by oneway analysis of variance (ANOVA) followed by Duncan test.

Results and discussion

Identification of new compounds

C₁: Isolated compounds were identified by UV, MS, and NMR instruments. Identification of dihydro-a-cyclogeranyl hexanoate (hexyl 2,2,6-trimethylcyclohexylcarboxylate): molecular formula C₁₆H₃₀O₂, MW 254. EIMS 70 eV, m/z (rel. int): 254 [M‏⁺] (0.8), 253 [M^‏+- H] (5), 208 (8), 169 [C₁₀H₁₇O₂‏⁺] (15), 168 [C₁₀H₁₆O₂⁺‏] (72), 154 (26), 153 [C₁₀H₁₇O⁺‏] (100), 124 (C₉H₁₆] (22), 109 (18), 108 (12), 85 [C₆H₁₃⁺] (41), 57 [C₄H₉^+‏] (43; Fig. 1).

Figure 1. Structure new compound isolated from aerial parts of A.millefolium.

Identification of known compounds

The constituents of the obtained Eos of A.millefolium treated with nano Zn oxide are presented in Table 1. Twenty-four components were identified in nano Zn oxide-treated plants (Table 1). The differences were supposed to be the effects of nano Zn oxide on chemical composition of A.millefolium EO. α-Pinene, camphene, limonene, borneol, and carvacrol were increased with nano Zn oxide-treatment (Table 1). The yield of the A.millefolium oil was 1.54% v/w (2 mM) and 2.01% (4 mM). Nano Zn oxide significantly increased the yield of EO (Table 1). It seems that the use of nano-particles causes increasing in pod and dry leaf weight and finally will increase total yield.^[24] Lu et al.^[25] showed that application of nano fertilizers could increase the nitrate reductase enzyme in soybean (Glycine max L.), increase its abilities of absorbing and utilizing water and fertilizer, promote its antioxidant system, and, in fact, accelerate its germination and growth. A previous report by Afsharypour et al.^[26] indicated the major constituent of the EO of Achillea tenuifolia was caryophyllene oxide and in other studies, borneol was the second most abundant constituent of oil.^[27]

Table 1. New compounds and known from the aerial parts of A.millefolium.

	Treatment
Compound	Nano Zn oxide (2 mM)%	Nano Zn oxide (4 mM)%	RI	Identification methods
Known compounds
α-Pinene	6.45	7.45	932	MS, RI
Camphene	2.14	4.65	950	MS, RI
Sabinene	1.02	1.87	975	MS, RI
α-Phellandrene	0.07	0.41	1000	MS, RI
Limonene	2.36	4.53	1020	MS, RI
Trans-limonene oxide	0.25	1	1140	MS, RI
Borneol	20.14	36.35	1160	MS, RI
γ-Terpinene	1.14	1.45	1195	MS, RI
Carone	1.54	1.1	1200	MS, RI
Carvone	2.35	1.45	1240	MS, RI
Bornyl acetate	2	1.74	1281	MS, RI
Thymol	10.14	10.01	1300	MS, RI
Carvacrol	8.14	10.14	1308	MS, RI
β-caryophyllene	1.25	1.74	1418	MS, RI
γ-Elemene	1.08	1.01	1430	MS, RI
γ-Muurolene	0.74	0.1	1476	MS, RI
β-Selinene	0.87	0.41	1490	MS, RI
β-Guaiene	0.7	0.98	1496	MS, RI
α-Selinene	0.35	0.11	1490	MS, RI
Calarene	0.47	0.14	1500	MS, RI
Calacorene	0.74	0.35	1529	MS, RI
Ccaryophyllene oxide	1.05	0.21	1579	MS, RI
Torreyol	1.45	0.78	1640	MS, RI
Cadalene	2.14	0.61	1670	MS, RI
Yield	1.54	2.01
Total	63.67	83.15
New compound
Dihydro-a-cyclogeranyl hexanoate				UV, MS, MPLC,NMR

^aPercentage composition determined on column HP 5.

^bThe retention Kovats indices were determined on HP 5 capillary column in reference to n-alkanes.

MS: mass spectroscopy, RI: retention index.

Extraction yield, total phenolic contents, and total flavonoid contents

The AA of plant extracts has been correlated to their total phenolic content due to their property of scavenging free radicals.^[28] It is well-known that phenolic compounds contribute to quality and nutritional value in terms of modifying color, taste, aroma, and flavor and also in providing health-beneficial effects.^[29] They also serve in plant defence mechanisms to prevent damage by microorganisms, insects, and herbivores.^[29] Moreover, a few studies on the A.millefolium revealed that they are good dietary sources of antioxidants. Thus, we determined the total phenolic and flavonoid contents of the methanol extracts of A.millefolium wild vegetables. As shown in Table S2, the extraction yield of A.millefolium ranged from lowest 66.14 ± 24 mgg^_1 (control) to highest 100.14 ± 55 mgg^_1 (Nano Zn oxide [4 mM]). Among the three A.millefolium extracts, A.millefolium treated with nano Zn oxide at 4mM showed the highest total phenolic content (162.14 ± 57 mgg⁻¹) and showed the highest total flavonoid content (121.47 ± 64 mg g⁻¹). These results showed that the total phenolic and total flavonoid contents have an obvious variation in various concentrations.

Antimicrobial activity

The in vitro antimicrobial activities of new compound and borneol against the studied microorganisms were assessed by the MIC and MBC/MFC (Table 3). According to the results given in Table 3, new compound and borneol exhibited significant antimicrobial activity against all tested strains. Inhibition values were in the following range: MIC 2.5 ± 0.05 (Escherichia coli) to 25.0 ± 0.11µg/mL (Pseudomonas aeruginosa) and MBC 2.5 ± 0.08 µg/mL (E.coli) to 20.0 ± 0.65 µg/mL (Staphylococcus aureus) for bacteria, and MIC 3.0 ± 0.35 (Candida albicans) to 4.5 ± 0.65 µg/mL (Aspergillus fumigatus) and MFC 3.0 ± 0.21 (C.albicans) to 4.5 ± 0.84 µg/mL (A.fumigatus) for fungi. Results obtained from MIC and MBC/MFC indicated that the antimicrobial activity of the new isolated compound against E.coli and C. albicans was greater than those of borneol. Among the individual constituents of EOs, carvacrol, isoeugenol, nerol, citral, and sabinene exhibited potent anti-H. pylori effects.^[30] The major components of thyme and oregano EOs, thymol, and carvacrol, inhibited pathogenic bacterial strains, such as E. coli, Salmonella enteritidis, S.choleraesuis, and S. typhimurium.^[31] Eugenol, terpenen-4-ol, and carvacrol showed an inhibitory effect against the growth of four strains of E. coli O157:H7 and L. monocytogenes,^[32] but results obtained indicated that three isolated compounds was greater than those of monoterpenes (benzyl alchol, camphor, cinnamaldehyde, borneol, and cineole) but weaker than those of monoterpenes (thymol, carvacrol, carveol, and geraniol). Comparing the results of compounds C₁ with that of standard, streptomycin, and fluconazole, it was concluded that the oils possess more potent anti-oral-pathogen activity. The compounds C₁ expressed higher antibacterial activity than borneol and both antibiotics tested. In our study, most of the antimicrobial activity in EOs from A.millefolium appears to be associated with compounds C₁ and borneol (Table 3).

Table 2. Effect of nano Zn oxide on extraction yields, total phenolic contents and total flavonoid contents of A.millefolium extracts.

	Extract	Extraction yield^a	Total phenolic^b	Total flavonoid^c
1	Control	66.14 ± 24	78.14 ± 32	80.24 ± 68
2	Nano Zn oxide (2mM)	68.47 ± 21	80.14 ± 78	84.24 ± 19
3	Nano Zn oxide (4mM)	100.14 ± 55	162.14 ± 57	121.47 ± 64

The data are expressed as mean ± SD.

^aExpressed as milligram of extract per gram dry material.

^bExpressed as milligram of gallic acid per gram dry extract.

^cExpressed as milligram of rutin per gram dry extract.

Table 3. Antimicrobial activity of the new compound and borneol (µg/mL) from A.millefolium using minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC) test.

	Borneol	C1	Antibiotic (Streptomycin)
	MIC	MIC	MIC
Microorganisms	MBC	MBC	MBC
Bacillus cereus	7.0 ± 0.12	3.5±0.27	10±0.70
	7.0 ± 0.21	3.5 ± 0.41	15 ± 0.32
Enterococcus faecalis	7.5 ± 0.21	4.0 ± 0.28	10 ± 0.59
	7.0 ± 0.74	4.0 ± 0.17	10 ± 0.52
Staphylococcus aureus	20.0 ± 0.14	3.5 ± 0.01	250 ± 0.0
	20.0 ± 0.65	3.5 ± 0.05	250 ± 0.0
Escherichia coli	4.5 ± 0.31	2.5 ± 0.05	25 ± 0.5
	5.0 ± 0.52	2.5 ± 0.08	25 ± 0.5
Pseudomonas aeruginosa	25.0 ± 0.11	10 ± 0.74	100 ± 0.34
	20 ± 0.05	10 ± 0.24	150 ± 0.87
Proteus mirabilis	10.0 ± 0.54	4.5 ± 0.61	20 ± 0.54
	8.0 ± 0.12	4.0 ± 0.08	15 ± 0.5
Salmonella typhimurium	5.5 ± 0.24	4.5 ± 0.06	15 ± 0.05
	4.5 ± 0.14	4.5 ± 0.11	15 ± 0.02
Citrobacter freundii	6.5 ± 0.08	4.5 ± 0.17	15 ± 0.87
	6.0 ± 0.14	4.5 ± 0.20	20 ± 0.56
Microorganisms	Borneol	C1	Mycotic (fluconazole)
	MIC	MIC	MIC
	MFC	MFC	MFC
Candida albicans	4.5 ± 0.21	3.0 ± 0.35	4.5 ± 0.00
	4.5 ± 0.24	3.0 ± 0.21	5 ± 0.00
Aspergillus fumigatus	4.5 ± 0.65	3.5 ± 0.28	5 ± 0.9
	4.5 ± 0.84	3.0 ± 0.47	3.5 ± 0.5

All tests were performed in triplicate, repeated three times twice (means ± S.D). Statistically, the differences were significant at p ≤ 0.01 or at least at p ≤ 0.05 (in few cases), using Duncan test. C1: dihydro-a-cyclogeranyl hexanoate.

AA

AA is a complex process usually occurring through several mechanisms. Due to its complexity, the evaluation of the AA for pure compounds or extracts should be carried out by more than one test method.^[33] The lower IC₅₀ value indicates a stronger ability of the extract to act as a DPPH scavenger while the higher IC₅₀ value indicates a lower scavenging activity of the scavengers as more scavengers were required to achieve 50% scavenging reaction. The results presented in Table 4 revealed that compound C₁ and borneol exhibited a remarkable activity. In particular, C₁ exhibited clearly a higher activity (11.79 ± 0.21 µg/mL) followed by borneol (12.11 ± 0.60 µg/mL) and A.millefolium EO (12.97 ± 0.14 µg/mL; Table 4).The positive controls BHT and ascorbic acid exhibited IC₅₀ values equal to 13.87 ± 0.22 µg/ml and 12.97 ± 0.11 µg/mL, respectively. Table 4 depicts the inhibition of β-carotene bleaching by the new compound and borneol. As shown in Table 4, the reducing power of new compound, expressed as CE₅₀, was clearly more significant than that of the positive BHA and AA. Because of high antioxidant and free radical-scavenging activities of new compound, further investigation was carried out to identify its active constituents. Therefore, a preliminary screening was initially carried out using the dot-blot DPPH staining method on TLC. As the new compound and borneol presented a significant AA in the assays and bioautography test, it was subjected to the TLC for isolation of the active compounds. Components identified and their AA relative percentages have been shown in Table 5. The major compound found in the active band was dihydro-a-cyclogeranyl hexanoate (45.14%; Table 5). Many aroma components of EOs, such as terpenes and terpenoids, were proposed to contribute to the AA of EOs; including thymol and eugenol, linalool and 1,8-cineole, results obtained indicated that two isolated compounds was greater than borneol. According to these results, there is a relationship between total phenolic contents and AA.

Table 4. Antioxidant activity new compound and borneol: scavenging activity (expressed as IC₅₀ values: µg/mL), and b-carotene bleaching test. Reducing power was expressed as EC₅₀ values (µg/mL). Butylhydroxyanisole (BHA) and ascorbic acid were used as positive controls.

Tested compounds	IC50 (μg/mL)
A.millefolium EO	12.97 ± 0.14 µg/mL
c1	11.79 ± 0.21 µg/mL
borneol	12.11 ± 0.60 µg/mL
A.millefolium EO (b–Carotenes IC50 µg/ml)	12.47 ± 0.28 µg/mL
c1	11.94 ± 0.74 µg/mL
A.millefolium EO (PR EC50 µg/ml)	12.97 ± 0.21 µg/mL
c1	11.65 ± 0.05 µg/mL
BHA	13.87 ± 0.22 µg/mL
AA	12.97 ± 0.11 µg/mL

Values are mean ± SD of three replications.

*IC₅₀ values have been presented with their respective 95% confidence limits.

Table 5. Components identified and their antioxidant activity relative percentages.

Compounds	%
c1	45.14
Borneol	21.11
Other components	36.68

Results are expressed as a percentage of antioxidant activity relative. Experiments were carried out in triplicate.

Lipid peroxidation inhibition

According to the results obtained, new compound and borneol significantly inhibited the formation of Thiobarbituric acid reactive substance (TBARS) in brain homogenates in a concentration-dependent manner (Table 6). The suppressive power on the lipid peroxidation of new compound were found to be the most potent (C1: 80.41 ± 0.24), followed by A.millefolium EO (78.54 ± 0.21 µg/mL). Ascorbic acid and Trolox showed significant suppressive power on lipid peroxidation in mice brain homogenate with IC₅₀ value of 75.61 ± 0.21 and 72.18 ± 0.35 µg/mL (Table 6). Phenolic compounds exist in most plant tissues as secondary metabolites, i.e., they are not essential for growth, development, or reproduction but may play roles as antioxidants and in interactions between the plant and its biological environment. Phenolics are also important components of the human diet due to their potential AA, their capacity to diminish oxidative stress induced tissue damage resulted from chronic diseases and their potentially important properties such as anticancer activities.^[34] These results indicated that new compound displayed significant AA in inhibiting peroxidation of rat brain homogenate in vitro.

Table 6. Lipid peroxidation inhibition of new component and borneol (expressed as IC₅₀ values: µg/ml). Trolox and ascorbic acid (AA) were used as positive controls.

Tested compounds	IC50 (μg/mL)
A.millefolium EO	78.54 ± 0.21 µg/mL
C1	80.41 ± 0.24 µg/mL
Borneol	75.60 ± 0.34 µg/mL
Trolox	72.18 ± 0.35 µg/mL
AA	75.61 ± 0.21 µg/mL

Experiments were carried out in triplicate and the results are expressed as mean ± SD.

Anti-inflammatory activity

The traditional use of EOs as anti-inflammatory agents suggests that they possess potent anti-inflammatory activity. The anti-inflammatory activity of new compound and borneol was evaluated on RAW 264.7 macrophages which were stimulated to induce an overproduction of NO. As presented in Supplementary Table 7, the new compound exhibited a strong inhibitory effect on LPS-induced NO secretion with C1: 88 ± 0.05 and inhibition observed at 45.0 μM. Comparatively, the L-NAME, used as positive control inhibited NO release by 73 ± 0.05% (Table 7). New compound was found to be the most active compound, inhibiting NO production by 88 ± 0.05% at 45.0 μM (Table 7). Therefore, this compound may be responsible for the anti-inflammatory activity of the oil. The anti-inflammatory potential of the new compound and borneol may be directly related to its scavenging ability and/or capacity to inhibit inducible nitric oxide (NO) synthase expression, the enzyme responsible for the release of high amounts of NO, during inflammatory conditions. Indeed, inflammatory mediators, such as NO have been reported to contribute to mutagenesis.^[35] This radical is an important regulator of physical homeostasis, whereas large amounts have been closely correlated with the pathophysiology of a variety of diseases and inflammations.^[35] EOs seem to be a good source of antioxidant and anti-inflammatory natural products.

Table 7. Effects of A.millefolium EO (45.0 μg/mL), new component and borneol (45.0 μM) on NO production in LPS-stimulated RAW-264.7 macrophages.

Tested compounds	NO inhibition (%)
A.millefolium EO	78 ± 0.21
C1	88 ± 0.05
Borneol	75 ± 0.04
L-NAME	73 ± 0.05

Results are expressed as a percentage of nitrite production by control cells maintained in culture medium. Experiments were carried out in triplicate and the results are expressed as mean ± SD.

Conclusions

Our data indicate that the new compound extracted from A.millefolium exhibits potent biological activities, which support their use in traditional medicine. There was a good correlation between new compound and antimicrobial, anti-inflammatory, and antioxidant capacity of the extracts. In conclusion, A.millefolium extracts appear to contain new compound with antimicrobial, anti-inflammatory, and antioxidant activities.