Antioxidant effect of aromatic volatiles emitted by Lavandula dentata, Mentha spicata, and M. piperita on mouse subjected to low oxygen condition

Abstract

This study aims to investigate the antioxidant effect of aromatic volatiles of three common aromatic plants, Lavandula dentata, Mentha spicata, and M. piperita. In this study, kunming mice subjected to low oxygen condition were treated with the volatiles emitted from these aromatic plants through inhalation administration. Then the blood cell counts, and the activities and gene expressions of antioxidant enzymes in different tissues were tested. The results showed that low oxygen increased the counts of red blood cells, white blood cells, and blood platelets of mice, and aromatic volatiles decreased their counts. Exposure to aromatic volatiles resulted in decreases in the malonaldehyde contents, and increases in the activities and gene expressions of superoxide dismutase, glutathione peroxidase, and catalase in different tissues under low oxygen. In addition, as the main component of aromatic volatiles, eucalyptol was the potential source that imparted positive antioxidant effect.

Graphical abstract

Treatment with the volatiles emitted from aromatic plants through inhalation administration improving the antioxidative capability in mouse.

Key words:

• antioxidant effect
• aromatic plants
• aromatic volatiles
• eucalyptol
• low oxygen

Free radicals are the highly reactive metabolic by-products in organism. Reactive oxygen species (ROS), the most common free radicals,^1,2) are accumulated excessively resulting in membrane lipid peroxidation, cell inactivation, and denaturation of deoxyribonucleic acid, which in turn accelerates senescence and causes cancer and cardiocerebral vascular diseases.^2–4) Therefore, scavenging free radicals plays an important role in treatment of some diseases and delaying aging. The plant secondary metabolites with high antioxidant activity have been extracted and used in cosmetics, health care products, and medicines.

Essential oils of aromatic plants have been shown to comprise a class of natural antioxidants. The essential oil of Lavandula is capable of scavenging hydroxyl radicals and superoxide radicals effectively.^5,6) The essential oil of Mentha piperita possesses a strong antioxidant property against DPPH and •OH radicals.⁷⁾ The extracts of nine Mentha species are found to have high DPPH free radical scavenging activity.⁸⁾ Methanol extracts of nine Mentha species have been shown to have high DPPH free radical scavenging activity.⁹⁾ The antioxidant properties of essential oils have promoted the application of aromatic plants in food, cosmetics, health products, and medicines.⁹⁾ In recent years, aromatic plants have been used in urban greening of fragrant landscapes, including vanilla gardens such as the Xinjiang Lavandula Manor and the Beijing Landiao Manor in China. In China, the first aromatic health care garden is constructed to facilitate in the improvement of sleep as well as lowering blood pressure in Shanghai. Although essential oils exhibit high antioxidant activities, information on the antioxidant properties of volatiles emitted from aromatic plants is limited due to difference in components. The emission of aromatic volatiles is also influenced by plant species and environmental factors.^10–12) Moreover, different aromatic volatile components may possess various functions.

In the present study, Kunming mouse which is the model animal in biomedical experiments in China was used as animal material. Kunming mouse originates from Swiss mouse and has high disease resistance, reproduction rate, survival rate, and adaptability. The mice were subjected to low oxygen condition, and then were treated with volatiles emitted by L. dentata, M. spicata, and M. piperita via inhalation administration. The contents of malonaldehyde (MDA) in the liver, kidney, brain, and whole blood of the mice were then measured to investigate the antioxidant effect of the aromatic volatiles. The activities and gene expressions of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) were also detected to explore the mechanism underlying the observed antioxidant effects. The volatile components of these three aromatic plants were then collected and detected to identify its bioactive components.

Materials and methods

Plant and animal materials

L. dentata, M. spicata, and M. piperita were cultured in plastic pots (20-cm diameter, 20-cm height) containing a medium composed of peat and vermiculite at a ratio of 2:1. The seedlings were placed in the greenhouse of the Science Park of Beijing University of Agriculture under 16 h/8 h light/dark 28 °C/22 °C cycle. They were irrigated every four days and supplied with a full Hoagland nutrient solution every two weeks.

Five-week-old male Kunming mice (20–25 g) were obtained from the Central Breeding Department of the Military Medical Sciences Hospital and used as animal materials. The mice were cultured at 22 ± 2 °C and a 12 h light/dark cycle in the laboratory. During the whole study, the principles of Laboratory Animal Ethics Committee of Beijing University of Agriculture for the protection of animals were strictly observed.

Low oxygen treatment

Ten mice were placed in a closed glass treatment box (50 cm × 50 cm × 40 cm) that was equipped with an inlet and an outlet. A portable air sampler (QC-1; Beijing Municipal Institute of Labour Protection, China) was connected to the inlet. First, the inlet and outlet were closed, thereby eventually depleting the oxygen in the treatment box due to mice respiration. When the oxygen concentration decreased to approximately 5%, the sampler was then turned on to pump air and maintain this particular concentration. The mice were treated for 6 days with 4 h per day (6: 00-10: 00). The control group consisted of mice in a treatment box with 21% oxygen concentration. Each treatment was performed using 3 independent replicates, and each replicate contained 10 mice.

Volatile inhalation experiment

After low oxygen treatment, the volatile inhalation experiment was conducted in the same closed glass box. The plants of L. dentata, M. spicata, and M. piperita were placed in three glass boxes, respectively. Ten mice under low oxygen condition were exposed to aromatic volatiles for 4 h per day (10: 00-14: 00). After 6 days, the antioxidant effects on the mice were evaluated. To maintain the same concentration of aromatic volatiles, a small vent was placed in each box, and a pump was connected to the inlet to provide fresh gas, which was filtered by activated carbon at a flow rate of 500-mL min⁻¹. After the volatile inhalation experiment, the liver, kidney, brain, and whole blood of each mouse were immediately extracted. Each treatment was performed using 3 independent replicates, and each replicate contained 10 mice.

Measurement of blood cell counts

Blood was collected from the mice by using the tail-cutting method. Using a CD-3700 automatic blood cell analyzer (ABBOTT, USA), the numbers of white blood cells (WBCs), red blood cells (RBCs), and blood platelet (PLTs) were determined and expressed as cell number per liter.

Measurement of MDA content

MDA content was measured using spectrophotometry kits (Nanjing Jiancheng Bioengineering Institute). The samples were homogenized and extracted with a trichloroacetic acid (TCA) solution, and then centrifuged for 10 min at 3500 rpm. The supernatant was mixed with 0.6% thiobarbituric acid to establish the reaction system and then heated in boiling water for 40 min, followed by being placed in a cold water bath to stop the reaction. The absorbance of the supernatant was measured at a wavelength of 532 nm. Then, the MDA contents of various organs and whole blood were calculated and expressed as nmol·mg⁻¹ protein and nmol·mL⁻¹, respectively.

Measurement of SOD, GPX, and CAT activities

SOD, GPX, and CAT activities were measured using spectrophotometry kits (Nanjing Jiancheng Bioengineering Institute). The samples were homogenized in a precooled normal saline solution consisting of 0.75% NaCl and 0.03% KCl. Then, the homogenate was centrifuged for 10 min at 10,000 rpm, and the supernatant was collected and used in the determination of enzyme activity. SOD activity was assessed by using the xanthine oxidase method through measuring change of nitrite content, which was based on the absorbance at a wavelength of 550 nm. GPX activity was assessed by the content change of GSH, which was based on the absorbance at a wavelength of 412 nm. CAT activity was assessed by change of H₂O₂ content, which was based on the absorbance at a wavelength of 405 nm. The units of U·mg⁻¹ protein and U·mL⁻¹ were used to describe the MDA contents in organs and whole blood, respectively.

Determination of gene expression

Total RNA was extracted from different tissues using TRIzol™ (Invitrogen, USA). First-strand cDNA was synthesized using a reaction system that included 3 μL of the RNA template, 1 μL of oligo(dT) primer, 0.8 μL of RNase, 4 μL of a 5 × RT reaction mix, and RNase-free H₂O at 42 °C for 45 min. The primers of each gene were designed using Primer 5.0 (Table S1). qRT-PCR was performed in triplicate. For one qRT-PCR reaction, 2 μL of a cDNA sample was used as reaction template. Other reagents were added to a total reaction volume of 25 μL, including 1.0 μL of the forward (F) primer, 1.0 μL of the reverse (R), primer, 12.5 μL of the PCR master mix, and ddH₂O. For the Mn-SOD gene, the PCR cycling conditions were as follows: 95 °C for 3 min, 40 cycles of 95 °C for 10 s, 59 °C for 30 s, and 72 °C for 50 s. For the Cu/Zn-SOD gene, the PCR cycling conditions were as follows: 94 °C for 5 min, 33 cycles of 94 °C for 30 s, 56 °C for 60 s, and 72 °C for 60 s. For the GPX gene, the PCR cycling conditions were as follows: 94 °C for 5 min, 33 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 50 s. For the CAT gene, the PCR cycling conditions were as follows: 94 °C for 5 min, 33 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 50 s. Relative mRNA levels of different samples were calculated using mouse NADPH gene as internal reference.

Collection and identification of aromatic volatiles

The aromatic volatiles emitted by L. dentata, M. spicata, and M. piperita were collected by dynamic headspace sampling.¹²⁾ The plants were placed in the glass box, and a stainless steel tube (0.25 × 3.5 inches containing Tenax-GR (60–80-mesh, Chrompack), which acted as a volatile trap, was connected to the outlet. A portable air sampler (QC-1; Beijing Municipal Institute of Labour Protection, China) served as a pump. The volatiles were collected for 15 min at a flow rate of 300 mL·min⁻¹. Afterwards, the stainless steel tubes were sealed and placed in a refrigerator. There were 3 independent replicates.

Then, automated thermal desorption-gas chromatography/mass spectrometry (ATD-GC/MS) was performed to analyze the volatiles. The volatiles collected in the stainless steel tube was desorbed by heating in ATD auto thermal desorber (TurboMatrix 650, PerkinElmer) at 260 °C for 10 min, and then cryofocused in a cold trap at −25 °C for 3 min. The cold trap was then rapidly heated to 300 °C for 5 min to transport the volatiles to the GC system (Clarus 600, Perkin Elmer). The GC was equipped with a capillary DB-5MS column (30 m × 0.25 mm i.d., with a 0.25-μm film thickness). Helium was used as the carrier gas. GC was performed at 40 °C for 2 min, 4 °C·min⁻¹ up to 160 °C, then 20 °C·min⁻¹ up to 270 °C, and held at 270 °C for 3 min. The MS (Clarus 600T, Perkin Elmer) was operated at an EI ionization mode of 70 eV, and a mass scan range of 29–600 amu was monitored. The temperatures of interface and ion source were 250 and 220 °C, respectively.

Preliminary identification of the compounds was performed by searching the NIST08 and WIELY library in the TurboMass Ver5.4.2 software and checked according to its retention index. To compare the release amounts of the volatile components, α-pinene (Fluka, USA) was used as the external standard. As previously described¹²⁾ and with minor modifications, α-pinene was dissolved in ethyl acetate at different concentrations. The unit of μg·h⁻¹ was used to describe the amounts of aromatic volatile compounds.

Statistical analysis

The data was statistically evaluated using Duncan by SPASS 17.0 at a significance level of p < 0.05.

Results

The effect of aromatic volatiles on the blood cell counts under low oxygen

Low oxygen treatment of mice resulted in increases in blood cell counts (Fig. S1). At the third and sixth day, the counts of RBCs (Fig. S1A), WBCs (Fig. S1B), and PLTs (Fig. S1C) were significantly higher than those of the control (p < 0.05). Especially after treatment with low oxygen for 6 days, the counts of RBCs, WBCs, and PLTs showed 0.72-fold, 3.20-fold, 1.61-fold increases, respectively. The results showed that the mice were in a stressed state, so the “6 days” was chosen as the treatment time of aromatic volatiles.

After inhalation of aromatic volatiles under low oxygen conditions for 6 days, decreases in blood cell counts were observed (Fig. 1). The volatiles of L. dentata, M. spicata, and M. piperita led to 26.61, 23.84, and 14.92% decreases in mouse RBCs counts, respectively, although these were not statistically significant (Fig. 1(A), p < 0.05). The volatiles of L. dentata resulted in 45.54% (Fig. 1(B)) and 48.62% (Fig. 1(C)) reductions in WBCs and PLTs counts, respectively (p < 0.05). Treatment with the volatiles of M. spicata and M. piperita under low oxygen also induced reductions in WBC and PLT counts, although no significant difference was observed (Fig. 1(B) and (C), p > 0.05). However, all three blood cell counts under low oxygen and treatment with the aromatic volatiles were higher than those of the mice in the box without plants, and the WBC counts showed statistical significance (Fig. 1(B), p < 0.05).

Fig. 1. The effect of aromatic volatiles on RBC (A) WBC (B) and PLT (C) counts in mice subjected to low oxygen.

Notes: Statistical significance of difference among various treatments is indicated by different small letters (p < 0.05). Standard errors are shown.

The effect of aromatic volatiles on the MDA content of different mouse tissues under low oxygen

Fig. 2 showed that low oxygen resulted in increases in MDA contents in different tissues. The significant increases were found in liver, kidney, and whole blood with 36.13, 113.13, and 92.31% respectively (p < 0.05). Under low oxygen after inhalation of the aromatic volatiles, though the MDA contents in different tissues were higher than those of the control, the MDA contents were lower than those of the mice in the box without plants, and significant difference was detected in liver, kidney, and whole blood (p < 0.05). In addition, although inhalation of aromatic volatiles did not cause significant difference in MDA content in the brain, a decreasing trend was observed (Fig. 2). Compared to M. spicata and M. piperita, the aromatic volatiles of L. dentata induced a greater decrease in MDA content than that observed in the mice in the box without plants, whereas in the kidney and whole blood, 33.33 and 32.51% reductions were detected, respectively.

Fig. 2. The effect of aromatic volatiles on the MDA content of different mouse tissues under low oxygen.

Notes: Statistical significance of difference in MDA contents among various treatments is indicated by different small letters (p < 0.05). Standard errors are shown.

The effect of aromatic volatiles on SOD, GPX, and CAT activities in different mouse tissues under low oxygen

The effect of aromatic volatiles on the activities of SOD, GPX, and CAT in the liver was measured under low oxygen (Table 1). We found that in response to low oxygen, the activities of SOD, GPX, and CAT obviously rose compared to the control (p < 0.05), especially CAT whose activity increased by 1.31 folds. Under low oxygen, the aromatic volatiles of these three plants did not result in a significant increase in liver SOD activity compared to that without plants (p < 0.05), but an increasing trend was observed. The application of the aromatic volatiles from L. dentata and M. spicata resulted in 7.43 and 4.33% increases in GPX activity, respectively (p < 0.05). CAT activity significantly increased after treatment with the aromatic volatiles of these three plants (p < 0.05), especially M. piperita whose volatiles resulted in a 0.50-fold increase. The activities of SOD, GPX, and CAT in liver after inhalation of aromatic volatiles were significantly higher than those of the control.

Table 1. The effect of aromatic volatiles on SOD, GPX, and CAT activities in different mouse tissues under low oxygen.

Tissue	Enzyme	Activity/U·mg^−1 protein
		Control	Low oxygen
			No plant	L. dentata	M. spicata	M. piperita
Liver	SOD	207.47 ± 4.06 b	324.95 ± 3.36 a	337.65 ± 4.41 a	328.77 ± 8.82 a	327.98 ± 2.07 a
	GPX	521.19 ± 26.98 c	886.07 ± 60.81 b	951.90 ± 46.05 a	924.42 ± 7.40 a	889.42 ± 19.21 b
	CAT	23.97± 3.60 d	55.49 ± 2.28 c	79.33 ± 7.43 a	69.60 ± 0.42 b	83.37 ± 5.57 a
Kidney	SOD	49.34 ± ± 2.06 c	158.43 ± 6.10 b	202.78 ± 3.83 a	206.90 ± 5.21 a	198.90 ± 7.68 a
	GPX	76.40 ± 26.98 b	279.64 ± 5.44 a	313.28 ± 25.66 a	294.79 ± 11.71 a	283.79 ± 22.34 a
	CAT	11.38 ± 0.87 c	22.23 ± 1.74 b	33.42 ± 0.58 a	25.30 ± 0.67 b	24.32 ± 0.39 b
Brain	SOD	58.29 ± 2.31 c	110.34 ± 2.17 b	125.23 ± 1.98 a	119.52 ± 3.25 a	120.81 ± 1.08 a
	GPX	167.34 ± 9.47 c	228.47 ± 10.86 b	264.97 ± 10.02 a	252.64 ± 7.85 a	231.31 ± 34.38 b
	CAT	0.55 ± 0.02 b	0.65 ± 0.02 a	0.73 ± 0.08 a	0.69 ± 0.06 a	0.71 ± 0.05 a
Whole blood	SOD	2629.42 ± 132.52 d	8525.76 ± 255.35 c	10,797.57 ± 264.14 a	10,961.61 ± 338.01 a	9230.37 ± 492.42 b
	GPX	167.37 ± 29.44 d	497.76 ± 37.08 c	597.64 ± 51.13 a	520.16 ± 46.72 bc	547.81 ± 39.94 b
	CAT	3.45 ± 0.22 c	10.82 ± 0.65 b	13.71 ± 0.58 a	11.12 ± 0.73 b	11.25 ± 0.43 b

Notes: Statistically significant difference in enzyme activities among various treatments in the same tissue is indicated by different small letters (p < 0.05). Standard errors are shown.

Treatment with aromatic volatiles also induced enhancements of SOD, GPX, and CAT activities in the mouse kidney under low oxygen (Table 1). It was found that low oxygen led to significant enhancements of SOD, GPX, and CAT with 2.21, 2.66, and 0.95 folds, respectively, compared to the control (p < 0.05). The CAT activity significantly increased by 50.33% after treatment with L. dentata volatiles compared to that in the mice without aromatic volatile treatment, whereas those of the two other plants did not cause significant change (p < 0.05). The inhalation of volatiles emitted from all the three plants resulted in a significant increase in SOD activity (p < 0.05). After treatment with the volatiles from L. dentata, M. spicata, and M. piperita, the SOD activities showed 27.99, 30.59, and 25.54% increases compared to those in mice without aromatic volatile treatment, respectively.

Under low oxygen, the activities of SOD, GPX, and CAT in the brain were higher significantly than the control, and inhalation of aromatic volatiles induced significant increases in SOD and GPX activities compared to that of mice without aromatic volatile treatment. (Table 1, p < 0.05). CAT activity increased to some extent but was not significantly different from that of mice without aromatic volatile treatment, which was higher obviously than the control (p < 0.05). Among these three plants, the volatiles of L. dentata induced the highest enzyme activities, with 13.49 and 15.98% increases in SOD and GPX activities, respectively.

Similar to in liver, kidney, and brain, low oxygen resulted in obvious increases in the activities of SOD, GPX, and CAT with 2.24, 1.97, and 1.14 folds in whole blood, respectively (Table 1, p < 0.05), and aromatic volatile treatment raised the activities. Inhalation of aromatic volatiles resulted in a significant increase in SOD activity of whole blood under low oxygen (p < 0.05). Compared to the mice without aromatic volatile treatment, whole blood SOD activities increased by 0.27, 0.29, and 0.08 folds after treatment with volatiles of L. dentata, M. spicata, and M. piperita, respectively. The volatiles of L. dentata and M. piperita also induced 20.06 and 10.05% increases in whole blood GPX activity, respectively. For whole blood CAT activity, only L. dentata volatiles led to a significant increase (26.71%) than that of mice in the box without plants (p < 0.05).

The effect of aromatic volatiles on gene expressions of SOD, GPX, and CAT in different mouse tissues under low oxygen

The gene expressions of SOD, GPX, and CAT were also measured in different mouse tissues in response to the aromatic volatile treatment by qRT-PCR, using the mouse housekeeping gene NADPH as reference. In the mouse liver, low oxygen caused significant increases in the gene expressions of SOD (including Mn-SOD and Cu/Zn-SOD), GPX, and CAT (Fig. 3(A), p < 0.05). The gene expression level of Cu/Zn-SOD was enhanced by 4.98 folds nearly compared to the control. Inhalation of aromatic volatiles induced increases in the gene expression levels of GPX and CAT. M. piperita volatile treatment led to 19.13 and 24.32% increases in the activities of GPX and CAT compared to those of mice in box without plants, respectively (Fig. 3(A), p < 0.05). But the gene expression of SOD (including Mn-SOD and Cu/Zn-SOD) decreased after inhalation of the aromatic volatiles under lox oxygen. The volatiles of L. dentata and M. piperita resulted in 28.61 and 41.23% reductions in Mn-SOD activity, respectively, compared to that of mice in box without plants, and Cu/Zn-SOD gene expression showed a 29.44% decrease after treatment with M. spicata volatiles.

Fig. 3. The effect of aromatic volatiles on the gene expressions of SOD, GPX, and CAT in different mouse tissues under low oxygen.

Notes: (A) liver; (B) kindney; (C) brain. Statistical significance of difference in gene expressions among various treatments is indicated by different small letters (p < 0.05). Standard errors are shown.

In the kidney, in response to low oxygen the gene expressions of SOD, GPX, and CAT also rose significantly compared to the control (Fig. 3(B), p < 0.05). The expression level of CAT increased to 2.50-fold of the control after low oxygen treatment. The inhalation of aromatic volatiles induced increases in the gene expressions of Mn-SOD, Cu/Zn-SOD, and GPX in the kidney under low oxygen. After treatment with L. dentata volatiles, the Cu/Zn-SOD gene expression exhibited the highest increase, which was 1.32-fold higher than that of the mice in the box without plants, whereas the Mn-SOD and GPX gene expression levels showed 1.87- and 1.52-fold increases, respectively. In response to the volatiles of M. spicata, and M. piperita, the Mn-SOD gene expression level also increased by 61.42 and 73.24%, respectively (p < 0.05). The Cu/Zn-SOD gene expression levels after inhalation of volatiles of M. spicata and M. piperita were lower than the treatment by L. dentata volatiles, but significantly increased compared to that of the mice in the box without plants (p < 0.05). M. piperita volatiles caused a 41.31% increase in GPX gene expression, whereas those of M. spicata did not lead to a significant increase (p < 0.05). For CAT gene expression, inhalation of aromatic volatiles also did not cause a significant increase than that without aromatic volatile treatment under low oxygen (p < 0.05). But the gene expression levels after treatment with different aromatic volatiles under low oxygen were all obviously higher than the control (p < 0.05).

Similar to in liver and kidney, low oxygen also resulted in significant increases in the gene expression levels of SOD, GPX, and CAT in brain (Fig. 3(C), p < 0.05), especially Cu/Zn-SOD whose expression level rose by 95.52%. The gene expressions of the Mn-SOD, Cu/Zn-SOD, GPX, and CAT in the brain after inhalation of aromatic volatiles showed different change patterns (Fig. 3(C)). The volatiles of L. dentata induced significant increases in gene expressions of Mn-SOD, Cu/Zn-SOD, and GPX (p < 0.05), especially Cu/Zn-SOD, which showed a 2.33-fold increase compared to that of the mice in the box without plants. M. piperita volatiles also resulted in marked increases in the expression levels of these three genes (p < 0.05), although the increases were not as high as those after treatment with L. dentata volatiles, and significant difference in the gene expressions of Cu/Zn-SOD was observed. The volatiles of M. spicata also caused 63.19 and 41.34% increases in the expression levels of Mn-SOD and Cu/Zn-SOD genes, respectively (p < 0.05). Similar to the kidney, no significant difference in the expressions of CAT gene was observed after inhalation of aromatic volatiles, whereas slight increases were observed after treatment with the volatiles of L. dentata and M. spicata. Also similar to in liver and kidney, the expression levels of these genes after treatment with aromatic volatiles under low oxygen were significantly higher than the control (p < 0.05).

The main components and release amounts of aromatic volatiles

Table 2 showed that the main components and release amounts of the aromatic volatiles of the three plants. The components were classified into three categories, including aliphatic compounds, aromatic hydrocarbons, and terpenoids. A total of 25, 24, and 20 main components were detected in the volatiles of L. dentata, M. spicata, and M. piperita (Table S2). Aliphatic compounds were the predominant components in the volatiles of the three plants. Sixteen aliphatic compounds were identified in the volatiles of M. spicata, and only 11 and 12 aliphatic compounds were emitted from L. dentata and M. piperita, respectively. In addition, L. dentata emitted 6 aromatic hydrocarbons and 8 terpenoids, both of which were more than those in M. spicata and M. piperita.

Table 2. The main components and release amounts of aromatic volatiles emitted from L. dentata, M. spicata, and M. piperita.

Group of compound	Component	Key m/z value	Release amount (μg·h^−1)
			L. dentata	M. spicata	M. piperita
Aliphatic compound	1,2-Dichloroethane	27, 49, 62, 64, 98	—	2.84 ± 0.08	—
	1-Butanol	27, 31, 41, 56, 74	4.12 ± 0.76	4.32 ± 1.48	6.52 ± 1.92
	Cyclobutanemethanol	29, 39, 44, 57, 67	4.6 ± 0.68	—	—
	2-Pentanone	27, 43, 58, 71, 86	—	—	5.88 ± 0.20
	1-Penten-3-ol	29, 71, 57, 86	—	5.08 ± 0.64	—
	Valeraldehyde	29, 44, 58, 71, 86	7.32 ± 1.60	10.44 ± 3.88	12.36 ± 1.56
	Methyl butyrate	43, 59,71, 74, 102	—	2.96 ± 0.40	—
	1,1-Diethoxyethane	31, 43, 59, 75, 90	2.60 ± 0.44	—	—
	1-Pentanol	31, 42, 55, 70	18.20 ± 7.12	16.28 ± 3.84	24.84 ± 1.40
	3-Ethyl-4,4-dimethyl-2-pentene	29, 41, 55, 69, 97, 111, 126	6.88 ± 4.16	—	—
	Butyl acetate	29, 43, 56, 61, 73	3.32 ± 0.12	—	—
	2,4-Dimethyl-heptane	43, 57, 71, 85, 128	—	3.36 ± 0.16	2.92 ± 0.16
	Hexyl alcohol	31, 43, 56, 69, 102	17.04 ± 4.56	15.88 ± 2.76	21.68 ± 1.04
	Cyclooctatetraene	27, 39, 51, 78, 104	—	6.68 ± 0.84	—
	2-Pentanone	27, 43, 58, 71, 86	—	2.92 ± 0.12	—
	Heptanal	29, 41, 55, 70, 81, 86, 96, 114	—	3.00 ± 0.48	3.04 ± 0.04
	Pentyl ester acetic acid	29, 43, 55, 61, 70	3.80 ± 0.40	6.92 ± 1.24	20.56 ± 0.64
	2-Pentylfuran	27, 41, 53, 81, 138	—	10.28 ± 2.88	18.72 ± 3.68
	(Z)-3-Hexen-1-ol acetate	15, 27, 43, 67, 82	—	3.20 ± 1.32	5.40 ± 0.16
	Hexyl acetate	43, 56, 61, 69, 84	7.20 ± 1.80	3.88 ± 0.16	8.56 ± 3.36
	1-Nonanal	29, 41, 57, 70, 82, 98, 142	3.52 ± 0.16	2.88 ± 0.20	—
	2,5-Dimethyl-3-hexanone	27, 43, 57, 71, 85, 128	—	—	3.28 ± 0.04
Aromatic hydrocarbon	Toluene	39 51, 65, 91, 92	3.68 ± 0.20	3.16 ± 0.32	—
	Ethylbenzene	39, 51, 65, 77, 91, 106	3.64 ± 0.08	—	3.20 ± 0.40
	1,4-Xylene	39, 51, 65, 77, 91, 106	3.72 ± 0.12	—	3.96 ± 0.24
	Styrene	39, 51, 63, 78, 104	—	—	8.44 ± 1.40
	1-Methylethyl-benzene	39, 51, 63, 77, 105, 120	4.52 ± 2.20	3.88 ± 0.36	5.48 ± 3.40
	1,2,3-Trimethyl-benzene	39, 51, 65, 77, 91, 105, 120	6.60 ± 3.76	7.80 ± 1.40	—
	1-Ethyl-4-methyl-benzene	39, 65, 77, 91, 105, 120	—	—	9.80 ± 0.88
	1-Methyl-3-(1-methylethyl)-benzene	65, 77, 91, 119, 134	5.92 ± 2.28	—	—
Terpenoid	α-Phellandrene	41, 77, 93, 121, 136	5.12 ± 1.76	—	—
	α-Pinene	41, 77, 93, 105, 121, 136	38.88 ± 12.96	5.32 ± 0.16	—
	Camphene	27, 39, 67, 79, 93, 107, 121, 136	5.00 ± 0.40	2.56 ± 0.20	—
	3-Methyl-6-(1-methylethylidene)-cyclohexene	41, 53, 79, 93, 107, 121, 136	3.16 ± 0.40	—	—
	β-Pinene	27, 41, 53, 69, 77, 93, 121, 136	140.56 ± 50.28	19.04 ± 1.44	6.24 ± 0.56
	Limonene	27, 39, 53, 68, 79, 93, 107, 121, 136	—	12.24 ± 2.36	3.64 ± 0.28
	Eucalyptol	43, 55, 71, 81, 93, 108, 139, 154	1526.12 ± 82.24	35.72 ± 5.08	33.28 ± 10.92
	g-Terpinene	43, 77, 93, 105, 121,136	13.64 ± 9.48	—	—
	L-Fenchone	27, 41, 69, 81, 109, 152	4.96 ± 2.04	—	—

Note: “—” indicates that the component is not detected in the aromatic volatiles.

The total release amounts of aromatic volatiles were significantly different among the three plants (Fig. 4(A); p < 0.05), with L. dentata exhibiting the highest release amount. The total release amount of L. dentata was nearly 8.78- and 7.90-fold higher than M. spicata and M. piperita, respectively. L. dentata emitted a high amount of terpenoids, which was significantly higher than that of aliphatic compounds and aromatic hydrocarbons (Fig. 4(B); p < 0.05). The release amount of eucalyptol exceeded 1500 μg·h⁻¹, and accounted for nearly 82.82% of the total amount of aromatic voaltiles. On the other hand, M. spicata and M. piperita emitted relatively high amounts of aliphatic compounds (Fig. 4(B); p < 0.05), which accounted for about 52.23 and 64.42% of the total release amount, respectively. However, among the volatile components of these two plants, eucalyptol also showed the highest release amounts.

Fig. 4. The release amounts of aromatic volatiles of L. dentata, M. spicata, and M. piperita.

Notes: (A) the total release amounts; (B) the amounts of different volatile components. Statistical significance of difference in release amounts among different plants is indicated by different small letters (p < 0.05). Standard errors are shown.

Discussion

Plant essential oils have been reported to possess diverse bioactive functions¹³⁾ and are widely used in the fields of food, pharmaceutical, and cosmetic, particularly in aromatic therapy.¹⁴⁾ Antioxidation is an important function of essential oils in aromatic therapy and was first described by the French chemist Gattefosse in 1928, and later proven in mice through feeding experiment.¹⁵⁾ The essential oils of young leaves and shoots of Blumea balsamifera have been shown to have strong antioxidant activities, which are mainly imparted by dimethoxydruene, α-caryophyllene, and β-caryophyllene.¹⁶⁾ Sun et al. have also found that the essential oil from leaves of M. piperita (MEO) grown in China exhibited potent anti-inflammatory activities in a croton oil-induced mouse ear edema model, and effectively inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages.¹⁷⁾ The coriander/cumin seed oil combination might indeed be used as a potential source of safe and effective natural antimicrobial and antioxidant agents via microbroth dilution, checkerboard titration, and DPPH free radical scavenging method.¹⁸⁾

However, whether the volatiles emitted by aromatic plants possess a similar antioxidant effect is unclear. Along with the wide application in landscaping and fragrance gardens of aromatic plants, the bioactive functions of aromatic volatiles need to be explored. In the present study, the mice were subjected to low oxygen condition using inhalation experiment to determine the antioxidant effect of volatiles emitted from aromatic plants.

Low oxygen treatment resulted in increases in RBC, WBC, and PLT counts in mice, which was indicative of a hypoxia-induced stressed state. After exposure to aromatic volatiles, the level of MDA decreased in various mouse tissues. Previous studies have reported that the essential oils of aromatic plants possess antioxidant effect. Costa et al. found that the metabolites extracted from L. pedunculata subsp. lusitanica had high antioxidant and free-radical scavenging activities, as well as acted as inhibitors of MDA production.¹⁹⁾ Compared to the H₂O₂-treated PC12 cells, pretreatment with Salvia lavandulifolia essential oil samples attenuated the morphological change and loss of cell viability, as well as decreased the MDA level and intracellular ROS production.²⁰⁾ In the present study, the reduction in MDA content proved that the aromatic volatiles also raised the antioxidant level in mice.

Moreover, the activities of three antioxidant enzymes, SOD, GPX, and CAT, were measured. The enhancements of antioxidant enzymes activities in different tissues resulted in reductions in ROS levels leading to decreases in MDA contents. Hancianu et al. reported that the application of lavender oils resulted in significant increases in the activities of the antioxidant enzymes SOD, GPX, and CAT and decreases in the total content of reduced GSH and lipid peroxidation (MDA level) in rat temporal lobe homogenates, which was suggestive of antioxidant potential.²¹⁾ The essential oils of another Lamiaceae plant, oregano, exert a protective effect against diquat-induced oxidative injury in the intestines of rats through oral administration, and the protective mechanism results from an increase in antioxidant capacity, including the enhancements of GPX and SOD activities.²²⁾ Furthermore, oregano essential oils markedly raise the activities of serum GPX and liver SOD, thereby indicating an enhancement of antioxidative capacity in pigs.²³⁾ The essential oils of S. lavandulifolia also exhibit a potent antioxidant capacity by enhancing the endogenous antioxidant system and inhibiting lipid peroxidation in a H₂O₂-induced oxidative stress model in astrocytes.²⁴⁾ In the present study, the inhalation administration of aromatic volatiles to mice showed the similar antioxidant effect. In addition, the underlying mechanism of this effect was investigated by detecting the gene expressions of SOD, GPX, and CAT. Most of the gene expression levels significantly increased in response to aromatic volatiles exposure. Although the SOD (Mn-SOD and Cu/Zn-SOD) gene expression level decreased in the liver after volatile treatment, the activity was significantly higher than that of the mice in the box without plants. These findings indicated that aromatic volatiles not only enhanced the activities of antioxidant enzymes but also stimulated gene expressions, thereby increasing the antioxidant capacity of mice. It was possible that a regulatory mechanism governed the activities and expressions of antioxidant enzymes. Moreover, after exposure to aromatic volatiles, the blood cell counts of the mice subjected to low oxygen condition decreased, thereby illustrating that the aromatic volatiles relieved the stressed response through mechanisms such as antioxidation. Our findings of the present study revealed that the aromatic volatiles of L. dentata, M. spicata, and M. piperita imparted positive effect on human health.

We observed that the L. dentata volatiles induced greater increases in the activities of most antioxidant enzymes as well as in the gene expression levels compared to those of the other two plants. Further analysis showed that the release amount of volatiles from L. dentata was significantly higher than that in M. spicata and M. piperita, which coincided with the antioxidant enzyme activities and gene expression levels. The larger release amount resulted in a higher concentration of aromatic volatiles in the glass chamber, thus allowing the mice to absorb more aromatic volatiles, which resulted in higher antioxidant effect. Subsequent component analysis indicated that a terpenoid compound, eucalyptol, was the predominant component in the volatiles of the three aromatic plants, with L. dentata showing the highest release amount. Fig. 5 illustrated the MS spectrum and chemical structure of eucalyptol. The molecular weight of eucalyptol was 154, which was a monoterpenoid and also was a cyclic ether. The key m/z values of eucalyptol were 43, 55, 71, 81, 93, 108, 139, and 154, among which 43 exhibited the highest relative intensity. Previous studies have confirmed the antioxidant effect of eucalyptol. Porres-Martínez et al. showed that high-density essential oils of S. lavandulifolia were characterized by a high amount of eucalyptol, thus imparting a higher antioxidant effect than a low-density sample that mainly emitted camphor.²⁰⁾ Eucalyptol attenuates colonic damage in rats via the anti-inflammatory mechanism of reducing myeloperoxidase activity and causing repletion of glutathione.²⁵⁾ Mitić-Ćulafić also showed that eucalyptol had substantial protective effect against oxidant-induced genotoxicity in bacteria and cultured human cells, which was predominately mediated by its radical scavenging activity.²⁶⁾ The present study showed that eucalyptol was also the main bioactive component of aromatic volatiles and was responsible for the observed increases in the activities and gene expression levels of various antioxidant enzymes. Moreover, the large release amount of eucalyptol in L. dentata resulted in more inhalation, which was followed by higher antioxidant effect in mice.

Fig. 5. The MS spectrum and chemical structure of eucalyptol.

The present study showed that the inhalation of aromatic volatiles imparted strong antioxidant effect, which was similar to the oral administration of essential oils. The aromatic volatiles were absorbed through the respiratory system and entered different tissues through the circulatory system. A previous study reported that after the administration of essential oils of Ocimum formacitratum leaves, Cymbopogon citrates herbs, Litsea cubeba bark, and Alpinia malaccencis rhizomes to mice, the major volatile compounds including eucalyptol were identified in the plasma.²⁷⁾ Eucalyptol acts as an antioxidant of cigarette smoke-induced acute lung infection by decreasing oxidative stress, inflammation, and the expression of the NF-kappa B p65 subunit.²⁸⁾ We thereby proposed that the antioxidant effect of aromatic volatiles involved several mechanisms. First, the volatile components absorbed by the mice acted as the free-radical scavengers. Second, the aromatic volatile components may activate the antioxidation system including SOD, GPX, and CAT. Due to the limited inhalation amount of aromatic volatiles compared to that via oral administration, the second pathway may play a leading role. It was possible that volatile components binded to a specific receptor, triggering a signaling pathway downstream that ultimately activated the antioxidant system. Additionally, Wu et al. reported that pleasant and unpleasant gases stimulated the hippocampus and the nucleus medialis thalami.²⁹⁾ So by stimulating the perceptual center of brain such as the hippocampus, volatile components can regulate hormone signals that stimulate the antioxidant system.²⁹⁾

Conclusion

The results of the present study confirmed that the aromatic volatiles of L. dentata, M. spicata, and M. piperita had potential antioxidant effect on human health, and eucalyptol may be a key active component and play a central role in the activation of the antioxidant system. However, the underlying mechanisms need further investigation.

Author contribution

Zenghui Hu designed the experiments and wrote the manuscript. Chunling Wang performed the experiments and analyzed the results. Hong Shen and Kezhong Zhang gave advice and guidance for experiment. Pingsheng Leng provided the idea, supervised the research work.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality [grant number IDHT20150503]; the National Natural Science foundation of China [grant number 31071817]; and Key Project of Beijing Municipal Education Commission [grant number KZ201510020021].

Supplemental materials

The supplemental materials for this paper are available at https://doi.org/10.1080/09168451.2017.1385382.

Acknowledgments

We also thank LetPub for its linguistic assistance during the preparation of this manuscript.