Chemical constituents, antioxidant and antibacterial activities of essential oil from the flowering aerial parts of Heracleum moellendorffii Hance

Abstract

The conventional use of Heracleum moellendorffii Hance is for wind dispelling and toxin removal. This research aims to enhance the understanding of the flowering aerial parts of this plant by analyzing its volatile oil. Hydrodistillation was employed to extract the essential oil, which was subsequently analyzed using gas chromatography coupled with mass spectrometry. The analysis revealed the presence of 50 compounds, which accounted for 92.67% of the oil’s composition. The major constituents include germacrene D (21.78%), n-octyl acetate (19.57%), β-caryophyllene (7.35%), and octyl butyrate (4.36%). The antioxidant potential of the volatile oil was evaluated through six separate experiments, demonstrating significant scavenging abilities against 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (IC₅₀, 62.7 μg/mL), hydroxyl radicals (IC₅₀, 1611.1 μg/mL), and superoxide radicals (16.8%). However, it exhibited weak scavenging activity against 2,2-diphenyl-1-picryl hydrazyl (IC₅₀, 5827.0 μg/mL), and had low FRAP values. No CUPRAC activity was observed. Additionally, the antibacterial properties of the volatile oil were assessed against four common pathogenic bacteria, namely Escherichia coli, Aerogenic bacterium, Listeria monocytogenes, and Bacillus subtilis). The findings exhibited potent antibacterial activity against Escherichia coli (MIC: 16 mg/mL) and Aerogenic bacterium (MIC: 1 mg/mL). However, the volatile oil exhibited weaker antibacterial activity against Listeria monocytogenes (MIC: 8 mg/mL) and Bacillus subtilis (MIC: 64 mg/mL).

Keywords:

• Heracleum moellendorffii Hance
• essential oil
• chemical composition
• biological activity

REVIEWING EDITOR:

• Escudero-Gilete M. Luisa, Senior Editor, Universidad de Sevilla, Spain

SUBJECTS:

• Pharmacology
• food chemistry
• medicine

1. Introduction

Most plants belonging to the Heracleum genus in the Apiaceae family possess significant medicinal properties (Bahadori et al., 2016). Two notable examples are Heracleum hemsleyanum Diels and Heracleum sphondylium L., which are commonly used in traditional Chinese medicine. These plants, particularly their roots, are known for their ability to expel wind and remove cold, clear dampness, and alleviate pain (Matarrese & Renna, 2023; Wagan et al., 2017). They have been extensively utilized in the treatment of conditions such as wind-cold-dampness arthralgia, low back pain, cold headache, and swelling and pain of ulcerative carbuncle. Studies have identified several phytochemicals in these plants, including coumarins, volatile oils, flavonoids, phenolics, polysaccharides, terpenoids, and saponins (Bahadori et al., 2016). These phytochemicals contribute to the plants’ medicinal properties. Coumarins and volatile oils, in particular, are found to be abundant in the genus.

Heracleum moellendorffii Hance (H. moellendorffii), also known as Heracleum dissectum, Heracleum microcarpum, or Heracleum morifolium (Flora of China Editorial Committee of Chinese Academy of Sciences, 1992). The entire plant of H. moellendorffii can be used medicinally, offering various benefits such as wind dispelling, toxin removal, blood circulation promotion, stasis removal, wind-cold headache relief, dampness arthralgia alleviation, tranquilization, and alleviation of excitement (Zhao et al., 2018). The roots of this plant are commonly employed in the treatment of carpopedal spasm, low back pain, and rheumatoid arthritis (Geum et al., 2021). Moreover, they exhibit properties like blood vessel dilation, blood pressure reduction, and the stems and leaves of this plant have shown the ability to lower blood pressure and blood sugar levels (Zhao & Zhang, 2010). Additionally, H. moellendorffii is rich in amino acids and dietary fiber, making it beneficial for gastrointestinal peristalsis. The tender seedlings of the plant are edible and renowned for their excellent color, aroma, and taste. They can be eaten through methods like frying, cold mixing, and pickling (Jiang et al., 2018).

However, although H. moellendorffii has been widely used in folk medicine and consumption, research on its chemical composition and biological activity is quite limited. Therefore, we intend to study the volatile components of this plant from the perspective of volatile oils, which have unique odors and flavors within the plant and also endow it with special biological activity. By analyzing the volatile oil components of plants, we can reveal their specific chemical composition and functions.

In the present study, we investigated the volatile oil obtained from the flowering aerial parts of H. moellendorffii. We conducted in vitro experiments to evaluate its antioxidant and antibacterial activities, aiming to identify the chemical constituents responsible for its pharmacological effects. These findings provide a foundation for further research on the plant and its potential applications in medicine.

2. Material and methods

2.1. Reagents and chemicals

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and p-nitroblue tetrazolium chloride (NBT) were purchased from Sigma-Aldrich. 2,2-Diphenyl-1-picryl hydrazyl (DPPH) was purchased from Alfa Aesar. Salicylic acid, L-ascorbic acid, 2,4,6-tri(2-pyridyl)-s-triazine, ammonium acetate (NH₄Ac), ferrous sulfate heptahydrate (FeSO₄·7H₂O), cupric chloride dihydrate (CuCl₂·2H₂O), ferric chloride, butylated hydroxytoluene (BHT), neocuproine, tween 80, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 2,9-dimethyl-1,10-phenanthroline, potassium persulfate, and sodium acetate were purchased from Energy Chemical. Methanol, dimethyl sulfoxide (DMSO), diethyl ether, anhydrous sodium sulfate, sodium hydroxide (NaOH), concentrated hydrochloric acid, acetic acid, and 30% hydrogen peroxide (H₂O₂) were purchased from Sinopharm. Chloramphenicol and streptomycin sulfate were obtained from Macklin. Escherichia coli (ATCC0157), Listeria monocytogenes (ATCC19115), Bacillus subtilis (CMCC63501), and Aerogenic bacterium (ATCC13048) were provided by the Microbiology Laboratory of Tonghua Normal University.

2.2. Plant material

In June 2023, the flowering aerial parts of H. moellendorffii were collected from their natural habitat in Tonghua, Jilin Province, China. The geographic coordinates of the collection site were recorded as latitude N 41°55′15.98′′, longitude E 126°6′39.6′′, and an altitude of 738.02 m. The botanical identification of the specimen was confirmed by Prof. Junlin Yu, a plant taxonomist. To ensure its preservation and accessibility, a voucher specimen (Number of Voucher specimen: PI20230615B) was deposited at the Herbarium of Tonghua Normal University in Tonghua, China.

2.3. Extraction of essential oil

To obtain the essential oil, a hydrodistillation process was employed using a Clevenger-type apparatus. Fresh H. moellendorffii material was chopped and added to the apparatus, with a ratio of material mass to water volume set at 1/7. The hydrodistillation process lasted for 6 h under a bath temperature of 120 °C. The resulting essential oil was then separated from the aqueous layer. To remove residual moisture, the oil was dried with Na₂SO₄. The Eppendorf tube, which had been zeroed on the balance, was used to add 100 μL of dried volatile oil. The weight of the oil was recorded on the balance, which represented the weight of the oil. The density of the volatile oil in g/cm³ was obtained by dividing this weight by 100 μL. The weight conversion of the total volume of volatile oil obtained (0.5 mL) to weight (0.432 g) was achieved through density conversion. Next, the essential oil yield as a percentage was obtained by dividing it by the total weight of fresh material (1600 g). Finally, the essential oil was stored in an amber glass bottle at 4 °C until further use.

2.4. Chemical composition analysis of essential oil

The essential oil of H. moellendorffii was subjected to chemical composition analysis at the Medicinal Chemistry Laboratory in Tonghua Normal University. The analysis utilized a Thermo Fisher TSQ 8000 EVO GC-MS/MS instrument, equipped with a TG-5 quartz capillary column (Thermo Fisher Scientific; 30 m × 0.25 mm ID × 0.25 µm film thickness). The GC oven temperature was initially set at 60 °C for 5 min, followed by a gradual temperature increase of 2 °C per min until reaching 150 °C. Subsequently, the temperature was ramped up at a rate of 10 °C per min until 200 °C, and then further increased by 20 °C per min until 250 °C. The final temperature of 250 °C was maintained for 5 min. Helium served as the carrier gas, flowing at a rate of 1.0 mL per min. Each analysis involved injecting 1.0 μL of the essential oil solution (volatile oil diluted 10 times with ethyl ether) using split mode (1:5), at a temperature of 280 °C. The mass spectrometry analysis was performed with specific settings: a MS transfer line temperature of 280 °C, an ion source temperature of 300 °C, and a mass spectrum resolution of 70 eV (EI mode). The scan rate was set at 0.3 scans per sec, and the mass range scanned was from m/z 50 to 550.

Identification of compounds present in the essential oil was accomplished by comparing their mass spectra with those stored in the NIST library. The concentrations of the compounds were reported as relative percentages of each peak area over the total area. To determine the retention index (RI) of each eluent, a series of n-alkanes (23 n-alkane mix in n-hexane, 500 μg/mL) purchased from Tan-Mo Technology Co., Ltd, were used under the same GC conditions. The RI values obtained were compared with literature values obtained using a similar column. The identification of volatile compounds was based on the combination of matching the detected compound mass spectra with those stored in the NIST library, as well as other published mass spectra.

2.5. Antioxidant activities

All antioxidant activity assays (including those using DPPH, ABTS, hydroxyl radicals, superoxide radicals, FRAP and CUPRAC) were performed in accordance with our previous methods (Guo et al., 2023; Zhang et al., 2020), the specific experimental steps are as follows.

2.5.1. DPPH assay

In the DPPH assay, 100 µL of volatile oil in methanol and 100 µL of DPPH in methanol (50 µM) were combined in a microplate. The mixture was then left at room temperature in the dark for 20 min. The absorbance of the sample was measured at 515 nm. For comparison, L-ascorbic acid, trolox, and BHT were used as positive references. The Half-maximal inhibitory concentration (IC₅₀) values were calculated and expressed as the mean ± standard deviation (SD) in µg/mL.

2.5.2. ABTS assay

In the ABTS assay, 190 μL of diluted ABTS solution and 10 μL of volatile oil in DMSO were mixed in a microplate. The mixture was incubated in the dark for 20 min. The absorbance of the sample was measured at 734 nm. L-Ascorbic acid, trolox, and BHT were used as positive references. The IC₅₀ values were calculated and expressed as the mean ± SD in µg/mL.

2.5.3. Hydroxyl radical assay

In the hydroxyl radical assay, 50 µL of volatile oil in DMSO, 50 µL of FeSO₄ solution (3 mM), and 50 µL of H₂O₂ solution (3 mM) were mixed in a microplate and incubated for 10 min. Then, 50 µL of salicylic acid solution (6 mM) was added, and the mixture was further incubated at room temperature for 30 min in the dark. The absorbance of the sample was measured at 492 nm. L-Ascorbic acid, trolox, and BHT were used as positive references. The IC₅₀ values were calculated and expressed as the mean ± SD in µg/mL.

2.5.4. Superoxide radical assay

In the superoxide radical assay, 45 µL of volatile oil in DMSO (10 mg/mL), 15 µL of NBT in DMSO (1 mg/mL), and 150 µL of NaOH in DMSO (50 μM) were mixed in a microplate. The absorbance of the sample was immediately measured at 560 nm against a blank sample (NBT was replaced with DMSO). Curcumin was used as a positive reference. The scavenging activity was expressed as a percentage of the scavenging rate and calculated accordingly. $$\%\mathit{\text{scavenging}}=\left(1-\frac{\Delta A_{\mathit{\text{sample}}}}{\Delta A_{\mathit{\text{control}}}}\right)\times 100\%$$

2.5.5. FRAP assay

In the FRAP assay, 20 µL of volatile oil in DMSO and 180 µL of FRAP reagent were combined in a microplate and incubated at 37 °C for 30 min in the dark. A calibration curve was prepared using FeSO₄ (0–600 mg/L) as a standard. The absorbance of the sample was measured at 595 nm. L-Ascorbic acid, trolox, and BHT were used as positive references. The FRAP was expressed as the Trolox Equivalent Antioxidant Capacity (TEAC_FRAP).

2.5.6. CUPRAC assay

In the CUPRAC assay, 20 µL of CuCl₂ solution (100 mM), 50 µL of neocuproine in 96% ethanol (7.5 mM), 50 µL of NH₄Ac solution, 20 µL of volatile oil in DMSO, and 30 µL of distilled water were mixed in a microplate. The mixture was incubated at 50 °C for 20 min and then left at room temperature for 10 min. The absorbance of the sample was measured at 450 nm. L-Ascorbic acid, trolox, and BHT were used as positive references. The CUPRAC was expressed as the Trolox Equivalent Antioxidant Capacity (TEAC_CUPRAC).

2.6. Antibacterial activities

The antibacterial activities were assessed using a previously established method (Shen et al., 2019). Briefly, a stock solution of volatile oil was prepared at a concentration of 128 mg/mL using a 4% Tween 80 solution. Test solutions with different concentrations were then prepared from the stock solution using the doubling dilution method in Mueller-Hinton broth culture medium (hopebiol, China). The purchased bacteria were inoculated onto Luria-Bertani nutrient agar culture medium (hopebiol, China). After 24 h of constant temperature cultivation at 37 °C, monoclonal colonies were selected and added to Mueller–Hinton broth culture medium. The culture medium was shaken at 37 °C for 12 h. The bacterial solution was diluted with Mueller-Hinton broth culture medium and the bacterial concentration was measured by turbidimetry to be approximately 5 × 10⁸ CFU/mL. The bacterial solution was further diluted 5000 times to obtain the test bacterial solution with a final concentration of 1 × 10⁵ CFU/mL. In each well of a microplate, 100 μL of the test solution and 100 μL of the test bacterial solution (1 × 10⁵ CFU/mL) were added. The plates were then incubated at 37 °C for 24 h, and the wells were observed for any visible changes. The minimum inhibitory concentration (MIC) was determined as the concentration at which the first transparent and clean well appeared. Using this MIC result, the clarified culture medium was transferred to Mueller-Hinton agar culture medium (hopebiol, China) and incubated at 37 °C for 24 h. If no bacterial growth was observed, the concentration at this point was considered the minimum bactericidal concentration (MBC). Chloramphenicol and streptomycin were used as positive controls.

2.7. Statistical analysis

Statistical analysis was performed using SPSS software (Version 22.0) and Origin software (Version 8.0). All experiments were conducted in triplicate. One-way analysis of variance (ANOVA) with post-hoc LSD test was used to determine significant differences between the essential oil and the standard controls (p < 0.05).

3. Results and discussion

3.1. GC-MS analysis of the essential oil

Through hydrodistillation, a yellow-green transparent liquid with an aromatic odor is obtained. The volatile oil obtained from the flowering aerial parts has a density of 0.864 g/cm³, and the essential oil yield is found to be 0.027%. Analysis using GC-MS identified a total of 50 compounds, which account for 92.67% of the essential oil composition (Figure 1; Table 1). These compounds can be broadly classified into three groups: monoterpenoids, sesquiterpenoids, and others. Their proportions are 37.20%, 44.91%, and 10.56%, respectively. The major components of the volatile oil include germacrene D (21.78%), n-octyl acetate (19.57%), β-caryophyllene (7.35%), and octyl butyrate (4.36%). In contrast, the essential oil from the same plant and part displays different predominant components, namely apiol (11.0%), β-pinene (9.2%), α-terpineol (7.5%), myristicin (7.1%), and osthole (6.1%) (Chu et al., 2012). This variation can be attributed to several factors as follows:

Figure 1. GC-MS analysis results of essential oil of Heracleum moellendorffii.

Table 1. Chemical composition of essential oil of Heracleum moellendorffii.

No	RT^a	RI^b	RI^c	Compound	Content (%)
1	7.92	933	931	α-Pinene	2.48
2	9.74	972	964	Sabinene	1.03
3	10.64	992	981	β-Myrcene	1.29
4	11.29	1005	1007	α-Phellandrene	0.29
5	11.97	1016	1017	α-Terpinene	0.24
6	12.4	1023	1025.4	o-Cymene	0.31
7	12.64	1027	—	D-Limonene	3.15
8	13.24	1037	1036	trans-β-Ocimene	1.21
9	13.83	1047	1041	β-Ocimene	1.51
10	14.42	1057	1053	γ-Terpinene	1.43
11	15.21	1070	1054	1-Octanol	0.37
12	16.2	1087	1080	α-Terpinolene	0.31
13	16.99	1100	1081	β-Linalool	0.28
14	18.84	1129	1131	(E,Z)-2,6-Dimethylocta-2,4,6-triene	0.69
15	21.84	1175	1175	(−)-4-Terpineol	3.32
16	22.74	1188	1192	L-α-Terpineol	0.36
17	23.04	1193	1176	1-Hexyl butyrate	1.02
18	23.45	1199	1194	(3Z)-3-Octenyl acetate	1.07
19	24.46	1215	1191	n-Octyl acetate	19.57
20	25.36	1229	1220	(R)-(+)-β-Citronellol	0.25
21	28.95	1284	1270	Bornyl acetate	0.74
22	32.2	1335	1343.7	Elemene isomer	0.34
23	33.42	1354	1331	β-Citronellyl acetate	0.29
24	34.6	1372	1376	α-Copaene	0.39
25	34.87	1377	—	(Z)-3-Octen-2-ol	0.39
26	35.14	1381	1386	β-Bourbonene	0.30
27	35.72	1390	1374	Octyl butyrate	4.36
28	37.27	1415	1421	β-Caryophyllene	7.35
29	37.87	1425	1428	β-Copaene	1.59
30	38.22	1431	1425	γ-Elemene	0.22
31	38.38	1433	1427	α-Bergamotene	1.29
32	38.8	1440	1431	Isogermacrene D	0.65
33	39.32	1449	1439	Aromandendrene	1.65
34	39.81	1457	1449	cis-β-Farnesene	1.15
35	41.08	1478	1480	Germacrene D	21.78
36	41.96	1493	1492	Valencene	1.06
37	42.86	1508	1499	α-Farnesene	0.28
38	43.04	1511	1503.9	Butylated hydroxytoluene	0.43
39	43.18	1513	1514	(Z)-γ-Bisabolene	1.19
40	43.32	1516	1508	β-Cadinene	0.59
41	43.61	1521	1537	δ-Cadinene	1.32
42	44.15	1530	1514	(Z)-γ-Bisabolene	0.47
43	44.54	1537	1535	Selina-3,7(11)-diene	0.28
44	46.89	1577	1575	β-Caryophyllene oxide	0.27
45	48.87	1615	1618	Junenol	0.22
46	50.28	1647	1628	T-Muurolol	0.76
47	50.87	1660	1641	α-Cadinol	1.04
48	52.03	1687	1694.5	4(15),5,10(14)-Germacratrien-1-ol	0.29
49	57.55	—	1997	(+)-Falcarinol	0.60
50	59.22	—	2700	Heptacosane	1.20
Monoterpenoids					37.20
Sesquiterpenoids					44.91
Others					10.56
Total					92.67

RT^a = Retention time. RI^b = retention index on TG-5 column in reference to n-alkanes. RI^c = retention index of literature.

(1) Genetic differences: Different individuals of the same plant exhibit genetic variations that can affect their response to environmental and external stimuli. These genetic differences can influence the synthesis pathways and rates of specific compounds in the volatile oil. (2) Environmental factors: Factors such as light, temperature, humidity, soil type, and nutrient supply significantly impact the growth and metabolic processes of plants. These environmental factors can affect the activity of plant metabolic pathways, thereby altering the synthesis of volatile components. For instance, light intensity and spectrum regulate plant photosynthesis and auxin synthesis, ultimately influencing the composition of volatile oils. (3) Developmental stage: Plants synthesize different types and amounts of compounds at different growth stages. This is because the biological needs and functions of plants vary throughout their development. In this study, the plant samples were collected in June, while Chu et al. collected them in August (Chu et al., 2012). The difference in collection time contributes to variations in the synthesis of volatile components. (4) External stimuli: Plants respond to external stimuli by initiating defense reactions and synthesizing specific volatile components. These stimuli can be biotic (e.g. insect bites, bacterial infections) or abiotic (e.g. drought, salinization) stresses. Consequently, the volatile components of the same plant may vary under different stimuli. In conclusion, the major components of volatile oils from the same part of a plant can differ due to a combination of genetic differences, environmental factors, developmental stages, and external stimuli. These differences reflect the adaptability of plants to their environment and their survival strategies.

Of course, our study still includes six compounds (α-pinene, β-myrcene, γ-terpinene, 1-octanol, β-caryophyllene, and germacrene D) that are consistent with previous studies (Chu et al., 2012). Except for 1-octanol, the content of all other compounds exceeds 1%. Compared to the reference, the content of germacrene D increased from 4.4% to 21.78%, while the content of β-caryophyllene increased from 3.4% to 7.35%. The major components of the essential oil from the seeds of H. moellendorfii were octyl acetate (63.80%), octyl butyrate (12.23%), and n-octanol (11.51%) (Li et al., 2013). The difference in major components between our research and the reference is even more significant. There are several reasons for this variation. Firstly, the accumulation of sufficient nutrients is crucial for the growth and development of seedlings, as seeds serve as the reproductive organs of plants. In addition to providing nutrient storage, volatile oil in seeds plays a significant role in seed defense and attraction. Moreover, stems and leaves serve as the photosynthetic tissues of plants, promoting overall health and growth. The presence of volatile oils in stems and leaves can potentially offer a wide range of benefits, including antibacterial properties and resistance against insects. By possessing these properties, volatile oils in stems and leaves contribute to the overall well-being and resilience of plants (Figure 2).

Figure 2. Structural formula of chemical composition with content greater than 0.5% in essential oil of Heracleum moellendorffii.

3.2. Antioxidant activities assays

In terms of antioxidant activity assays, six experiments were conducted to assess the antioxidant activity of the essential oil extracted from H. moellendorfii. The findings from these experiments are presented in Table 2. The IC₅₀ value of the volatile oil against DPPH was determined to be 5827.0 μg/mL. When compared to L-ascorbic acid (2.3 μg/mL), trolox (2.2 μg/mL), and BHT (8.7 μg/mL), the volatile oil exhibited relatively weak DPPH scavenging activity. On the other hand, the IC₅₀ values for ABTS and hydroxyl radicals were 62.7 μg/mL and 1611.1 μg/mL, respectively, indicating that the volatile oil demonstrated strong scavenging activity against ABTS, hydroxyl radicals, and superoxide radicals. Additionally, the volatile oil had low FRAP values. No CUPRAC activity was observed. Previous research on the major components of volatile oils, germacrene D, n-octyl acetate, and β-caryophyllene, confirmed our experimental findings by showing weak DPPH scavenging ability and strong ABTS scavenging ability. Meanwhile, octyl butyrate displayed weak scavenging ability against both free radicals (Casiglia et al., 2017; Maggi et al., 2014; Salleh et al., 2012). Moreover, the observed low FRAP values in the volatile oil indicate a limited capacity to reduce metal ions, and no CUPRAC values were detected. These results align with existing literature, which indicates weak FRAP values for germacrene D (Casiglia et al., 2017), further supporting our conclusion that the volatile oil has a limited ability to reduce metal ions.

Table 2. Antioxidant activity of essential oil of Heracleum moellendorffii.

Samples	DPPH (IC50, μg/mL)	ABTS (IC50, μg/mL)	Hydroxyl radicals (IC50, μg/mL)	Superoxide radicals (%, 2143 μg/mL)	TEACFRAP	TEACCUPRAC
Essential oil	5827.0 ± 0.6^c	62.7 ± 0.4^c	1611.1 ± 7.4^d	16.8 ± 0.4^b	0.04 ± 0.00^c	None
L-Ascorbic acid*	2.3 ± 0.2^a	3.0 ± 0.1^a	78.3 ± 0.3^b	N.T.	1.08 ± 0.01^a	1.39 ± 0.01^a
Trolox*	2.2 ± 0.3^a	4.5 ± 0.2^b	11.7 ± 0.8^a	N.T.	0.99 ± 0.02^a	1.01 ± 0.01^b
BHT*	8.7 ± 0.2^b	3.8 ± 0.1^b	197.0 ± 1.6^c	N.T.	0.30 ± 0.00^b	1.48 ± 0.01^a
Curcumin*	N.T.	N.T.	N.T.	59.1 ± 1.3^a	N.T.	N.T.

^a–dColumns with different superscripts indicate a significant difference (p < 0.05). *Used as a standard antioxidant. DPPH: 2,2-Diphenyl-1-picrylhydrazyl; BHT: Butylated hydroxytoluene; ABTS: 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt; Trolox: 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; IC₅₀: Concentration required to scavenge 50% of the radicals present in the test solution; FRAP: Ferric-reducing antioxidant power; CUPRAC: Cupric ion reducing antioxidant capacity; TEAC: Trolox equivalent antioxidant capacity; N.T. indicates not tested. Values are the mean ± standard deviation of three independent experiments.

The volatile oil exhibited notable scavenging ability when exposed to water-soluble free radicals such as ABTS, hydroxyl radicals, and superoxide radicals. This remarkable effectiveness can be primarily attributed to the presence of antioxidant components (germacrene D, n-octyl acetate, and β-caryophyllene) within the oil that possess strong properties. These components efficiently interacted with water-soluble free radicals, effectively neutralizing and scavenging them, thereby demonstrating excellent scavenging activity in the experimental setting. However, the volatile oil showed comparatively weak performance in scavenging lipid-soluble free radicals, specifically DPPH. This difference could be attributed to variations in properties and reaction conditions between DPPH radicals and their water-soluble counterparts, as well as the distinct selectivity and reaction mechanisms exhibited by the antioxidant components towards different types of free radicals.

The essential oil derived from the fruits of Heracleum sphondylium L. subsp. ternatum (Velen.) Brummitt (IC₅₀, 105.78 μg/mL) demonstrated good DPPH scavenging activity. Additionally, it exhibited a FRAP value of 0.49 μmol TE/g DW (Maggi et al., 2014). Heracleum persicum’s aerial parts produced an essential oil (IC₅₀, 1430 μg/mL) with moderate DPPH scavenging activity (Gharachorloo et al., 2017). On the other hand, the roots of Heracleum transcaucasicum (IC₅₀, 77 μg/mL) and Heracleum anisactis (54 μg/mL) displayed excellent DPPH scavenging activity (Torbati et al., 2014). However, the essential oils derived from the aerial parts of Heracleum pastinacifolium (IC₅₀, 7300 μg/mL), Heracleum persicum (7400 μg/mL), Heracleum rechingeri (11,600 μg/mL), and Heracleum transcaucasicum (16,300 μg/mL) exhibited weak DPPH scavenging activity (Firuzi et al., 2010). When summarizing the antioxidant capacity of volatile oils from plants of the Heracleum genus, two main observations were made. Firstly, there is a significant variation in antioxidant capacity among different plant parts, including roots, stems, leaves, and fruits. Secondly, even within the same plant part, such as Heracleum persicum and Heracleum transcaucasium, the antioxidant capacity of volatile oils can differ significantly. These disparities can be attributed to factors such as species differences, variations in plant parts, and environmental conditions. These variations offer valuable insights for future research and utilization of volatile oils derived from plants in the Heracleum genus.

3.3. Antibacterial activity assays

Moving on to antibacterial activity assays, our experimental studies revealed that the volatile oil exhibited effective antibacterial activity against Gram-negative bacteria, such as Escherichia coli (ATCC0157) (MIC: 16 mg/mL) and Aerogenic bacterium (ATCC13048) (MIC: 1 mg/mL). However, the volatile oil displayed relatively weaker antibacterial activity against Gram-positive bacteria, specifically Listeria monocytogenes (ATCC19115) (MIC: 8 mg/mL) and Bacillus subtilis (CMCC63501) (MIC: 64 mg/mL) (Table 3). Notably, previous literature reports indicate that the major components of the volatile oil, germacrene D and β-caryophyllene, exhibit significant inhibitory activity against Escherichia coli (Simic et al., 2002) and moderate inhibitory activity against Listeria monocytogenes (Demirci et al., 2008).

Table 3. Antibacterial activity of essential oil of Heracleum moellendorffii.

Samples	Escherichia coli (ATCC0157)		Listeria monocytogenes (ATCC19115)		Bacillus subtilis (CMCC63501)		Aerogenic bacterium (ATCC13048)
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
Essential oil	16	>128	8	>128	64	>128	1	>128
Chloramphenicol^a	0.002	0.004	0.008	0.008	0.004	0.008	0.004	0.004
Streptomycin^a	0.002	0.002	0.008	0.008	0.008	0.008	0.002	0.004

^aUsed as a standard antibacterial agent. MIC: Minimum inhibitory concentration (mg/mL); MBC: Minimum bactericidal concentration (mg/mL).

Furthermore, the essential oils extracted from the aerial parts of Heracleum pastinacifolium (inhibition diameter: 7.3 mm, 6.4 mm), Heracleum persicum (6.8 mm, 6.4 mm), Heracleum rechingeri (6.4 mm, 7.5 mm), and Heracleum transcaucasicum (7.1 mm, 6.4 mm) showed moderate antibacterial activity against Bacillus subtilis (PTCC 1023) and Escherichia coli (PTCC 1338) (Firuzi et al., 2010). However, the essential oil derived from the aerial parts of Heracleum sphondylium L. exhibited weak antibacterial activity against Escherichia coli (ATCC 25922) (MIC: >44.8 mg/mL) and Listeria monocytogenes (ATCC15313) (11.2 mg/mL) (Matejic et al., 2016). These findings align with the results obtained in our own experiments.

In theory, Gram-negative bacteria have higher resistance to volatile oils due to the composition of their extracellular membrane, which includes lipopolysaccharides, phospholipids, and proteins. On the other hand, Gram-positive bacteria lack this characteristic and are typically more sensitive to volatile oils. However, it is important to note that volatile oil is a complex mixture of multiple volatile components, and its mechanism of action on bacterial cells is multifactorial. These volatile components can impact bacterial cells in various ways, affecting different aspects such as the integrity of cell membranes and the activity of intracellular enzymes. While the increased resistance of Gram-negative bacteria’s extracellular membrane provides some protection against volatile oils, the components present in these oils may still exert antibacterial effects by disrupting bacterial membranes, interfering with cell respiration, or influencing cellular metabolic processes (Theuretzbacher et al., 2020). Moreover, Gram-positive bacteria exhibit relative sensitivity to volatile oils due to the absence of an outer cell membrane. This allows the components of volatile oils to more easily penetrate the cell wall and interact with intracellular biomolecules, leading to enhanced antibacterial effects (Yin et al., 2020). It is important to consider that the specific components present in the volatile oil will play a role in this analysis.

The experimental results revealed a substantial disparity in MIC between essential oils and positive controls. Nevertheless, volatile oils exhibit immense potential for utilization in multiple industries, particularly in the food industry. Firstly, the two positive controls used in the experiment are conventional antibiotics, which suffer from widespread usage and common challenges in clinical applications, such as microbial resistance. These issues are exceedingly difficult to resolve. Secondly, volatile oils demonstrate tremendous promise in the food industry. Due to stringent regulations, the use of antibiotics for preservation purposes is strictly prohibited. This restriction offers favorable opportunities for the application of volatile oils. By serving as natural and alternative preservatives, volatile oils have already found various applications in food preservation (Masyita et al., 2020). This, in turn, aids in addressing food safety concerns. Moreover, the antibacterial properties of volatile oils can be utilized to develop safer and more sustainable disinfectants, or even serve as alternative skincare ingredients. Lastly, future research can explore the combination of volatile oils with antibiotics to enhance their antibacterial effects or to tackle antibiotic resistance problems. Consequently, despite the notable difference in antibacterial effectiveness between volatile oils and positive controls, volatile oils possess significant advantages in the food industry and exhibit tremendous potential for practical applications.

4. Conclusion

This study utilized GC-MS analysis to comprehensively analyze the chemical components present in the volatile oils extracted from the flowering aerial parts of H. moellendorffii. This detailed identification of the compounds holds great potential for future drug development and natural product research. Furthermore, the experimental findings concerning the antioxidant activity of the volatile oil revealed its significant ability to scavenge ABTS, hydroxyl radicals, and superoxide radicals. This suggests that volatile oils may serve as promising candidates for antioxidant research. Moreover, the research also ascertained the potent antibacterial properties of the volatile oils, demonstrating their remarkable efficacy against Escherichia coli and Aerogenic bacterium. This discovery holds profound implications in the quest for novel natural antibacterial agents or food preservatives. However, it is important to acknowledge the limitations and challenges encountered in this study. Despite the strong antibacterial activity observed against certain strains, the effectiveness of the volatile oils against Listeria monocytogenes and Bacillus subtilis was comparatively weaker. Additionally, the volatile oil exhibited relatively low scavenging activity against DPPH and displayed a limited capacity to reduce metal ions. In conclusion, this study extensively investigated the chemical composition, as well as the antioxidant and antibacterial properties, of volatile oils derived from the flowering aerial parts of H. moellendorffii. The obtained results are of immense value to the scientific community, offering robust support for further research and potential applications in related fields.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available on request from the corresponding author.

Additional information

Funding

This work was supported by the Project of The Education Department of Jilin Province [No. JJKH20240652KJ]; Jilin Provincial Department of Human Resources and Social Security [No. 2023QN34]; and Administration of Traditional Chinese Medicine of Jilin Province [No. 2023091].

Notes on contributors

Hao Zang

Dr. Hao Zang is a full-time professor at Tonghua Normal University in the School of Pharmacy and Medicine, where he teaches multiple courses to students. His research interests include component analysis and pharmacological evaluation of medicinal and edible plants.